VOL.5 ISSUE 11 Editor Sub Editor MARCH 2012 : B.V. SHIV SHANKAR : R. SHAILESH NAATH CONTENTS 1. From the Editor's Desk 2. Antimicrobial Dosing Regimens: a Dynamic... 4-8 - Dr. Vijaykumar.M 3. Concepts of Bypass Protein Feeding in... - Dr. Nilufar Haque 9-13 4. A Few Nutritional Updates for Feeding... - Dr Trishna B. Kayastha 14-17 5. Common Diseases of Livestock CausedвЂ¦ 18-24 - Dr. Phaniraja.K.L 6. Heat Stress Takes Toll on Dairy Animal - Dr.Arindam Chatterjee 29-31 7. Livestock Improvement Strategies in... - Dr. Dibyendu Chakraborty 32-34 8. Importance of Biotechnology in Animal... - Dr. Dharmendra Vyas 35-36 9. Modifications of Polymerase Chain... - Dr. Arunkumar Patel 37-44 10. Mycotoxin: A Threat to Human and... - Dr. Mahipal Choubey 45-48 11. Impact of nanotechnology in veterinary... 49-50 - Dr. S. Ganguly 12. Press Release INDEX OF ADVERTISEMENTS 1. B.V. Bio-Corp. Pvt. Ltd. Title Cover I 2. DSM Nutritional Products India Pvt. Ltd. 3. Jefo Inside Colour 25 51-52 Title Cover II 4. Kemin Industries South Asia Pvt Ltd. 5. Polyglov Inside Colour 28 2 6. Trow Nutrition India Pvt. Ltd. Title Cover III 7. Vetoquinol India Animal Health Pvt. Ltd. Title Cover IV B.V. Shiv Shankar B. Kishore Kumar B. Shailajaa Sathyendranath B. L.Narasimham K. Raghuramaraju 3 - Managing Partner Media Executive Circulation Manager Marketing Manager Regional Representative Publication Consultant (09440231211) Printed, Published and Owned by B.V. Shiv Shankar, Printed at Venu Graphics, 2-1-392/1/3/8, Opp. Fever Hospital, Nallakunta, Hyderabad - 500 044. India. Published at 2-1-444/16, 1st Floor, O.U.Road, Nallakunta,Hyd-44. Editor: B.V. Shiv Shankar. LIVESTOCK LINE, MARCH 2012 1 LIVESTOCK LINE, MARCH 2012 2 From the Editor's Desk....вЂ¦.. OPERATION FLOOD The National Dairy Development BoardвЂ™s (NDDB) Annual Report for 2010-11 has conveyed that India continued to be the largest milk producing nation in 2010-11. The countryвЂ™s estimated milk production for 2010-11 is 121 million tonnes, close to 17 per cent of world milk production. This was possible with government schemes like Mahatma Gandhi National Rural Employment Generation Agency (MGNREGA) which aims at rural employment generation, milk production etc. The NDDB on its side offers technical assistance for balanced diet, increase in milk production etc and devised a plan known as National Dairy Plan (NDP) with a good span of 15 years hence. The National Dairy Plan aims at contributing to increasing milk production by increasing productivity in existing dairy animals through a focused and scientific process for breeding and feeding. Production of high genetic merit bulls, Production of disease free quality semen, Extension and demonstrations for fodder development, Interventions to strengthen village based milk procurement systems, Augmenting systems in the villages for procurement of milk in a fair and transparent manner, Project learning and monitoring and capacity building and training. The project is proposed to be carried out by State Cooperative Dairy Federations; District Cooperative Milk Producers Unions; Producer Companies and State Livestock development Boards that meet the criteria for each activity. Existing farmers and new entrepreneurs may take advantage of this programme and augment their personal wealth as well as nationвЂ™s wealth. - Editor LIVESTOCK LINE, MARCH 2012 3 Antimicrobial Dosing Regimens: a Dynamic Challenge Vijaykumar.M1, U.Sunilchandra2 and Meera V.C3 and Shrikant kulkarni 4 Department of Pharmacology and Toxicology 1 Department of Veterinary Physiology, KVAFSU, Veterinary College, Bidar.Karnataka. U Dosing regimens for antimicrobials exemplify the integration of pharmacokinetics (what the body does to the drug) and pharmacodynamics (what the drug does to the body). For antimicrobial therapy, the вЂњbodyвЂќ is the microbe. Integration is based upon what is needed to achieve the pharmacodynamic responseвЂ”in this case, the minimum inhibitory concentration of the drug of interest for the infecting microbeвЂ”and comparing it with what will be achieved at the chosen dose. For this discussion, we will build a dose around a desired pharmacodynamic index (PDI). If the MIC of the infecting is not known, then the MIC 90 is a reasonable surrogate. The MIC90 is the MIC at or below which 90% of the isolates is an sample population of the organism is inhibited. 1.Relationship Between MIC, Plasma and Tissue Drug Concentrations: The parameters that are most predictive of antimicrobial efficacy and lack of resistance are the ratio of Cmax / MIC, the area under the inhibitory curve (AUC/MIC); and the percent time that PDC are above the MIC [T> MIC]. Based on these relationships, two generally categories of drugs have been described. Exceeding the efficacy targets decreases resistance. Dosing regimens can be designed based on population statistics, using MIC 90 (e.g., packaged inserts) as a surrogate indicator of what is needed, or using MIC data from a culture report. The design of the dosing regimen depends upon the drug and its relationship between plasma drug concentrations, the MIC of the infecting organisms and whether or not the drug has a substantial post antibiotic effect (PAE). The PAE refers to the continued inhibition of microbial growth after a short exposure of the organisms to the drug. The impact is particularly profound for concentration-dependent drugs, and allows some drugs to be administered at long dosing intervals. The PAE may be absent for some organisms or some patients (e.g., some immunocompromised patients). In general, the PAE of concentration-dependent drugs is increased as the Cmax/MIC increases. 1,2,3 2.Time versus Concentration Dependent Drugs: The relationship between MIC and the magnitude and time course of PDC allows drugs to be categorized as to either concentration-dependent (sometimes referred to as dose dependent) or timedependent; these definitions are supported by primarily by in vitro but also in vivo studies. Concentration dependent drugs, best represented by the fluoroquinolones and aminoglycosides, are characterized by efficacy best predicted by the magnitude of plasma drug concentration (C max) compared to the MIC of the infecting organism. For such drugs, the magnitude of the ratio generally should be 10 to 12, but ideally is higher for more difficult infections (e.g., Pseudomonas aeruginosa, or infections caused by multiple organisms. The duration that PDC is above the MIC is not as important; in fact, efficacy may be enhanced by a drug-free period (i.e., a long interval between doses). For concentration dependent drugs, a dose that is too low is particularly detrimental. As such, concentration-dependent drugs generally can be administered at longer intervals, i.e., once a day. Package inserts can be used to demonstrate the design of a dose for a FQ. As such, theoretically the low dose would be appropriate for treatment of both. For Staph intermedius, with an MIC 90 of 0.25 mcg/ ml, 2.5 mcg/ml is the target. The higher dose of 5.5 mg/kg would be more prudent. For organisms with an MIC of 0.5 mcg/ml, the target of 5 mcg/ml could not be reached at 5.5 mg/kg. However, for the fluorinated quinolones (FQ), efficacy also is predicted in vitro by AUC/MIC: a ratio of < 60 renders the drugs bacteriostatic, whereas > 125 results in (slow) killing but also decreases the risk of resistance and > 250 causing more rapid bacterial killing. Thus, resistance might be less likely to develop for FQ characterized by longer half-lives (or for ENR, by the production of an active metabolite). Twice daily administration of an FQ might be indicated for organisms already characterized by low level resistance (see MPC below); however, the once daily dose should be given twice daily in such situations. Assistant Professor,Department of Pharmacology and Toxicology 4 Assistant Professor,Department of Veterinary Physiology LIVESTOCK LINE, MARCH 2012 4 The use of a second drug in combination with the FQ might also be considered for isolates whose MIC are sufficiently high that a Cmax/MIC >10 is difficult to achieve. 3. Time Dependent Drugs : In contrast to concentration dependent drugs, efficacy of timedependent drugs (e.g., Гў-lactams) is enhanced if PDC remain above the MIC for the majority (50 to 70%) of the dosing interval; efficacy is best predicted by percent time that PDC are above the MIC [T> MIC]. For such drugs, simply achieving the MIC (Cmax/MIC =1) is insufficient because PDC (and certainly tissue concentrations) fall below the MIC immediately. With time-dependent drugs, generally a Cmax/MIC of 4 is a good starting point because it assures 2 half-lives will lapse before T=MIC. Two more half-lives can then be added to the dosing interval before the next dose must be given if T>MIC 50%. However, while this sounds like a long time, for amoxicillin and cephalexin, with a half-life of about 1.5 hr, the dosing interval can only be 6 hrs if Cmax/ MIC = 4. For example, the MIC 90 for Staph intermedius and amoxicillin-clavulanic acid is < 0.5 mcg/ml. Cmax of 5.5 mcg/ml will be achieved at the labeled dose of amoxicillin-clavulanic. The duration of the dosing interval with this dose depends on the number of half-lives that can lapse as drug concentrations decline to the MIC. In one half-life, plasma drug concentrations will be 2.75 mcg/ml; in two half-lives, 1.35 and in three half-lives, 0.65, which is just above the target. The half-life of amoxicillin is at best 1.5 hrs resulting in 4.5 hrs of T>MIC. The dosing interval can be twice this long. Thus, the next dose should be administered at 9 hrs (8 hr). To reach a 12 hr dosing interval, 3 more hours, or two halflives are needed. T>MIC is needed for one more half-life; thus, the dose needs to be doubled. Thus, to treat an organism with an MIC of 0.5 mcg/ml with amoxicillin-clavulanic acid, a dose of 13.5 mg/kg every 8 hrs, or 27 mg/kg every 12 hrs must be given. The dose would need to be further modified for drug, microbial and host factors. Staphylococcus intermedius is characterized by a low MIC; if we repeated the process for Staph. aureus, its MIC 90 is 4 mcg/ml. At 13.5 mg/kg, drug concentrations will reach the MIC before one half-life lapses. Even for E. coli, with an MIC90 for amoxicillin-clavulanic acid at 1 mcg/ml, a dose of 26 mg/kg every 8 hrs is the minimum that should be considered. The process can be repeated for cephalexin, with a half-life of 1.3 hr. At 25 mg/kg PO, 15 mcg/ml is achieved. The LIVESTOCK LINE, MARCH 2012 MIC 90 for Staphylococcus intermedius is 2 mcg/ml; a dose of 25 mg/kg achieves 15 mcg/ml. The amount of time that can lapse can be calculated as follows: the Cmax/MIC (15/2)= 7.5. This equates to essentially 8, or 3 half-lives (2*2*2). Thus, 4 hours can lapse during T>MIC; a dosing interval of 8 hrs is indicated. (To check: 15 mcg/ml > 7.5 > 3.85 > 1.9 mcg/ml = 3 half-lives). For Staphylococcus aureus, the MIC90 is 8 mcg/ml; the Cmax/MIC = 15/8 = essentially 2. One half-life can lapse during T>MIC; the dosing interval can be 2 half-lives, or 3 hours long. Increasing the dose to treat St. aureus at a convenient dosing interval is not practical. For E coli, with an MIC 90 of 16 mcg/ml, not even one half-life can lapse. Cephalexin should not be used to treat E coli. In general, for time dependent drugs, especially if the half-life is short, adding an additional dose is more cost effective than increasing the dose. This is in contrast for time dependent drugs that have a long half-life. For example, once daily dosing may be appropriate for cefpodoxime, depending on the organism. According to the package insert, the MIC 90 for both Staph intermedius and E coli is 0.5 mcg/ ml. The Cmax at 10 mg/kg achieves 16 mcg/ml. The number of half-lives that can lapse is 16/0.5 = 32 = 5 half-lives (2X2X2X2X2), or 16 > 8 > 4 > 2 > 1 > 0.5. The half-life of cefpodoxime is 4.5 hrs, thus the dosing interval can be 25 hr X 2, or (theoretically) every 2 days. However, the variability in drug concentrations is marked, and prudence suggests that a 24 hour dosing regimen, as is indicated on the label, is appropriate. For Staph aureus, the MIC 90 is 2 mcg/ml. Three half-lives can lapse during T>MIC; 6 half-lives or essentially a day can lapse before the next dose. However, because this facilitates efficacy, but not necessarily avoids resistance, and because these calculations assume all drug in plasma makes it to the site of infection, a 12 hour dosing interval might be more prudent. For cefovecin, with a 133 hr half-life (due to protein binding which slowly releases the drug), for each 2X Cmax/MIC, 6 days can lapse (assuming time dependency is valid for periods beyond 24 hrs). However, the Cmax must be based on unbound, not bound drug. The Cmax of unbound drug in dogs approximates 4.0 mcg/ml. The MIC 90 for Staph intermedius is 0.25 mcg/ml; approximately 4 half-lives can lapse during T>MIC; approximately 8 half-lives (40 days) can lapse before the next dose is given. However, if the target organism is Staph aureus, with 5 an MIC of 2 mcg/ml, T>MIC for only one half-life and dosing should occur (if indicated) in one week or less. Constant rate infusion or slow release products might be ideal for time dependent drugs with shorthalf-lives in the critical patient. Slow release products might be considered for time dependent drugs; however, the dose must be designed to assure that the MIC is achieved for the older slow release products because MIC have changed through the years. Azithromycin is another example of a drug with a very long half-life (72 hr) because of tissue distribution and accumulation. Although the drug can be administered at 2 day intervals. depending on the target MIC, note that the drug may not reach steady-state concentrations for 6 to 14 days. Indeed, care must be taken to remember that the maximum effect of any drug with a long half-life will not be achieved for 3 to 5 half-lives and a loading dose might be indicated for such drugs. Finally, some time dependent drugs have a very long half-life. Based in the Pseudomonas aeruginosa, the dose for amikacin should be sufficient to achieve Cmax/ MIC = 10 or 10 X 16 mcg/ml = 160 mcg/ml. Table 1 indicates that 22 mg/kg achieved 64 mcg/ml in the blood stream. One could calculated the dose needed to achieve 160 mcg/ml based on either a proportion of : (160 mcg/ml)/(64 mcg/ml) = 2.5 X 22 mg/kg = 55 mg/kg; or one could calculate the dose based on target (160 mcg/ml) X Vd = 37 mg/kg. Either calculation is likely to result in the same conclusion: the drug may not be safe at this high dose, even with once daily dosing, and the addition of a second drug is indicated. For enrofloxacin, the target is 1 mcg/ml * 10 = 10 mcg/ml. At 20 mg/kg, enrofloxacin achieves 4 mcg/ml and its active metabolite ciprofloxacin, achieves 2.9 mcg/ml for a total bioactivity of approximately 7 mcg/ml. Interestingly, despite an вЂњIвЂќ designation, enrofloxacin comes closer to achieving the target Cmax/MIC of 10 (7 is achieved). For enrofloxacin, a second 20 mg/kg dose could be added. However, the combination of enrofloxacin and amikacin would be a wiser choice. For the MRSA, note that chloramphenicol, despite an вЂњSвЂќ designation, requires an MIC of 8 mcg/ml. The dosing table indicates that 55 mg/kg PO will achieve a Cmax of 10 mcg/ml. Thus, not even one half-life can lapse before T>MIC is reached. A dose of 80 mg/kg would achieve approximately 16 mcg/ ml, which would allow one half-live of T>MIC, or a 2 half-life dosing interval. The reported half-life is LIVESTOCK LINE, MARCH 2012 variable; we will us an average of 4 hours. Thus, if the dog could tolerate it (unlikely), a dose of 80 mg/ kg every 8 hours would be indicated. However, this would only result in bacteriostatic concentrations. The combination with rifampin would at least increase the changes of therapeutic success. The previous demonstrations have been based on the assumption that all drug in plasma makes it to the site of infection. However, a variety of host, drug and microbial factors should cause the dosing regimen to be modified even further. 4.Host Factors: The impact of host response to infection can be profound. Problems contributing to therapeutic failure include immunocompromise (design a dosing regimen that will assure bactericidal concentrations of the chosen drug reach the site of infection), inflammatory response (debride or otherwise appropriate clean/drain accessible infections, select a drug that distributes into tissues well and ideally accumulates in phagocytes and increase the dose appropriately). Interpretation of C&S is based on the assumption that the MIC should be achieved in plasma. Basing MIC interpretation on plasma drug concentrations (PDC) might result in over or under estimation of drug efficacy. For tissues which concentrate the drug (or if the drug can be applied topically), and for drugs which can be concentrated by phagocytes and thus transported to the site of infection, concentrations may markedly exceed PDC, resulting in underestimation of efficacy for several reasons. Much of the data for water soluble drugs (volume of distribution [Vd] generally < 0.3 L/kg) suggests antimicrobial concentrations may be 30% or less of PDC in some tissues, particularly those characterized as sanctuaries, i.e., non-fenestrated capillaries. In humans, recommended doses of beta-lactams drugs (water soluble) are increased 5 to 10 fold when treating infections of the central nervous system. Even tissues traditionally considered вЂњwell perfusedвЂќ might be of concern. For example, drugs do not penetrate bronchial secretions well, despite the fact that the lungs are well perfused. Amoxicillin is often used to treat respiratory tract infections. Yet, only 30% of the amoxicillin that is in plasma is distributed to bronchial secretions. Theoretically, one must dose amoxicillin 3X the recommended dose to achieve targeted PDC in bronchial secretions. Most water soluble drugs (beta-lactams and aminoglycosides) reach only 20 to 25% of PDC in bronchial secretions 6 whereas over 50% of lipid soluble drugs reach bronchial secretions. Dosing adjustments also are necessary for those infections that are intracellular or complicated by host response to infection. In the presence of marked inflammation, use of a drug that accumulates in phagocytes (e.g., FQs, macrolides, lincosamides) is likely to increase distribution of the drug to the site. prevention concentration (MPC) is defined as the highest MIC identified in a population (> 107) infecting the patient. The MPC, rather than the MIC, should be the targeted concentration of drug at the site of infection if resistance is to be avoided. Unfortunately, determining the MPC of an isolate cultured from a patient requires culture techniques based on > 107 organisms, which currently is not possible. 5.Microbial Factors: Materials released from microbes facilitate invasion, impair cellular phagocytosis, and damage host tissues. Most staphylococci associated with canine pyoderma produce вЂњslime,вЂќ a material that facilitates bacterial adhesion to cells. Soluble mediators released by organisms (hemolysin, epidermolytic toxin, leukocidin) may damage host tissues or alter host response. Staphylococcal organisms contain protein A, which impairs antibody response, activates complement, and causes chemotaxis. Nocardia stimulates the formation of calcium-containing вЂњsulfur granulesвЂќ that impair drug penetration to the organisms. Pseudomonas and other gram-negative organisms produce a glycocalix, or biofilm, that protects the organism. Biofilms are microcolonies of pathogenic and host microbes embedded in a polysaccharide matrix (вЂњslimeвЂќ or вЂњglycocalyxвЂќ) produced by the bacteria; dental plaque is the prototypic example. Normal microflora of the skin or mucous membranes in the biofilm are lost with shedding of the skin surface or by the excretion of mucus; new cells and mucus are rapidly colonized by biofilm forming bacteria. Translocation of the normal microflora to otherwise sterile tissues (which can be facilitated by the presence of foreign bodies) may lead to acute infections (again, associated with biofilm) and accompanying inflammatory response. Persistent, chronic bacterial infections may reflect biofilm producing bacteria; persistent inflammation associated with immune complexes contributes to clinical signs. Unfortunately, bacteria growing in biofilms more easily resist antimicrobial killing and immune defenses of the host. In addition to debridement or other methods of cleansing should facilitate antimicrobial penetration; dose modification (increase) may be indicated to compensate for debris. Attention to PDC is important not only for efficacy, but also in order to reduce the risk of resistance. For drugs in which resistance emerges as a result of point mutations, dosing regimens should be designed to target the MPC. The mutant 6. Drug Factors : In addition to drug characteristics previously addressed (e.g., concentration versus time dependent, static versus cidal, drug distribution), pharmaceutical manufacturers have been able to manipulate antimicrobial drugs in a variety of ways such that efficacy and thus bacterial killing is enhanced such that resistance might be reduced. For example, efficacy has been decreased by synthesizing smaller molecules that can penetrate smaller porins (e.g., the extended spectrum penicillins ticarcillin and piperacillin); вЂњprotectingвЂќ the antibiotic (e.g., with clavulanic acid, which вЂњdrawsвЂќ the attention of the Гў-lactamase away from the penicillin); modifying the compound so that it is more difficult to destroy (e.g., amikacin, which is a larger and more difficult to reach molecule than gentamicin); and developing lipid-soluble compounds that are more able to achieve effective concentrations at the site of infection (e.g., doxycycline compared with other tetracyclines). However, with each innovative approach to reducing resistance, microbes are able to circumvent the drug in a disconcertingly short time. The use of pro-biotics or pre-biotics to minimize emergence of resistance in the gastrointestinal tract is controversial and requires additional scientific evidence. LIVESTOCK LINE, MARCH 2012 7.Duration of Dosing : Increasingly, in an effort to reduce antimicrobial resistance, the duration of dosing in human medicine is being limited. Durations of 5 days or less are recommended for noncomplicated infections. High and/or frequent dosing is intended to result in rapid kill. For slow growing organisms, or infections complicated by poor local immunity or prolonged healing, longer durations are indicated. Note that pulse dosing at high, frequent doses intermittently might be preferred to long term dosing at lower doses. The former should be approached such that mutants are killed. In such cases, recurrent infection might be caused by organisms that are not resistant. References: 7 1. Papich, M. G. Tissue concentrations of antimicrobials: the site of action. Problems in Veterinary Medicine 1990; 2: 312. 2.Ling, G. V. Therapeutic strategies involving antimicrobial treatment of the canine urinary tract. Journal of the American Veterinary Medical Association 1984; 185: 1162. 3.Klausner, J. S. Management of canine bacterial prostatitis. Journal of the American Veterinary Medical Association 1983; 182: 292. 4. Bemis, D. A., Appel, M. J. G. Aerosol, parenteral and oral antibiotic treatment of Bordetella bronchiseptica infection in dogs. Journal of the American Veterinary Medical Association 1977; 170: 1082. 5.Pennington, J. E., Reynolds, H. Y. Concentration of gentamicin and carbenicillin in bronchial secretions. Journal of Infectious Diseases 1973; 128: 63. 6.Hall, B. B., Fitzgerald, R. H., Kelly, P. J., Washington J. A. Pharmacokinetics of penicillin in canine osteomyelitic bone. Orthopaedic Transactions 1980; 4: 175. 7.Wiggins, C. E., Nelson, C. L., Clarke, R., Thompson, C. H. Concentration of antibiotics in normal bone after intravenous injection. Journal of Bone and Joint Surgery 1978; 60A: 93. 8.Johnson, K. A., Watson, A. D. J., Page, R. L. Skeletal diseases. In: Ettinger, S. J., Feldman, E. C. eds. Textbook of Veterinary Internal Medicine. Philadelphia, W. B. Saunders Co., 1995: 2077. 9.Dwozack, D. L. Emergence of resistance in Gramnegative bacteria: a risk of broad-spectrum betalactam use. Drug Intelligence and Clinical Pharmacy 1986; 20: 562. 10.Saunders, W. E., Saunders, C. C. Inducible blactamases: clinical and epidemiologica1 implications for use of newer cephalosporins. Reviews in Infectious Disease 1988; 10: 830. 11.Marcellin-Little, D. J., Papich, M. G., Richardson, D. C., DeYoung, D. J. Pharmacokinetic model for cefazolin distribution during total hip arthroplasty in dogs. American Journal of Veterinary Research 1966; 57: 720. d its relationship to pyogenic vertebral osteomyelitis. Journal of Bone Joint and Surgery 1959; 41B: 797-809. LIVESTOCK LINE, MARCH 2012 8 Concepts of Bypass Protein Feeding in Ruminants Nilufar Haque and Sk. Asraf Hossain, National Dairy Research Institute, Karnal, Haryana-132001 Email ID of corresponding author: [email protected] U In the developing countries of Asia and Africa, the feed inadequacy is the major impediment coming in the way of development of livestock sector. The problem is really acute in India, where the bovine population is the largest (185.2 million cattle and 97.9 million buffaloes according to 17th Livestock Census, 2003), which is increasing @ 1% annually. Thus, the constant increase in bovine population in India dilutes any effort made in increasing the feed supply to these animals, through non-conventional feed resources. However, there is also an alternative way of increasing the nutrient supply to bovines in these countries, and that is, by modifying the feeds and the feeding conditions, and also by manipulating the digestive tract, or through better feeding management. Such an approach can result in increasing the feed conversion efficiency of feeds within the animal system. Times when AA balance is critical: 1. When attempting to reduce the amount of protein fed, thereby reducing the cost of the diet and increasing the space in the diet for higher energy feeds. For the high producing dairy cow, energy is usually more in deficit than AA. Lowering protein in the diet will also reduce the spilling of nitrogen into the environment and lower the threat of regulation. This becomes an extra bonus and for herds under environmental regulation may be a primary use of AA balance. 2. For fresh cows, both to minimize body condition loss as well as increase milk production. 3. To increase milk protein (Patton, 2009). Bypass protein supplement On account of shortage and to exploit milk production of dairy animals, limited feed ingredients available in India should be utilized efficiently with value addition. The total annual availability of protein meals in India is approximately 19-20 MMT, against a requirement of about 30-35 MMT. Out of 20 MMT protein meals produced in the country, approximately 4-5 MMT are exported, which further LIVESTOCK LINE, MARCH 2012 increases the gap between the requirement and the availability. Protein is usually the first limiting nutrient for cattle fed low-quality forages. Most of the farmers in India feed regionally available protein meals to the dairy animals, along with other ingredients. A significant part of these protein meals is broken down to ammonia in first stomach of ruminants; therefore, net availability of amino acids per unit of feed for growth and milk production is low. However, if these meals are subjected to suitable chemical treatment вЂ“ termed as вЂњbypass protein technologyвЂќ, then their efficiency of utilization can be significantly improved. When chemically treated protein meals replace untreated one, then due to less degradability of the protein, excessive loss of both nitrogen and energy could be avoided, resulting in an increased energy and nitrogen balance and causing increase in milk yield and different milk constituents. In a typical diet, approximately 40% of the protein eaten must be true protein that escapes degradation, whereas 60% of the protein value can be a mixture of protein and non-protein nitrogen that is degraded and incorporated into the rumen microbes (Tarique et al., 2010). Usually, protein meals are degraded in the rumen to the extent of 65-70 per cent, leading to wastage of nitrogen by its excretion through dung and urine. These protein meals are treated suitably, so as to reduce their degradability in the rumen from 60-70% to 25-30%, in a specially designed airtight plant. Cost of treatment of protein meals is less than a rupee per kg and on feeding one kg treated meal in comparison to untreated; there is increase in milk production by more than a liter. Bypass protein technology is being provided to the dairy cooperatives and private agencies (Gulati et al., 2001). Desirable characteristics for bypass protein supplements High level of crude protein Optimal essential amino acid profiles About 70-80% of the protein to be in 9 a rumen undegradable form time and heat which decreases the solubility of proteins by creating cross linkages both within or among peptide chains and to carbohydrates. It is done by two methodsJet sploding method and Extrusion method .In jet sploding method, high temperature treatment of protein is done at 315oC for short time. In extrusion method, heat treatment is done along with steam. But it has disadvantage that, it causes mallard reaction and produces melanoidins. Approximately about 80% of the rumen undegradable protein to be digestable in the small intestine Advantages of bypass protein feed technology вЂў Higher availability of amino acids per unit of feed вЂў Better utilization of those protein meals having higher rumen protein degradability вЂў Judicious utilization of protein meals, available in limited quantity вЂў Improves growth and milk production вЂў Improves protein percent in milk, hence, improves SNF content of milk вЂў Oesophageal groove closure: Oesophageal groove is functional in young animals and nonfunctional in adult ruminants. Certain chemicals activate oesophageal groove in adult animals like salts of Copper, Silver, Zinc, Sodium etc. вЂў Tannic acid Treatment: Tannins are polyphenolic compounds. They have greater affinity towards proteins. It has been found that 4% tannin content in diet has increased the protein and amino acid flow to lower GIT and are absorbed in lower GIT and improved the nitrogen retention in animals, thus concluding 4% tannin can be used as protein protectant. Condensed tannin protein complex is insoluble even under acidic conditions. вЂў Use of analogs and derivatives of methionine: Amino acid derivatives are free amino acids to which a chemical blocking group has been attached to вЂќвЂќ-amino group or acyl group e.g. Isopropyl DL-methionine, t-butyl DL- methionine, N-stearoyl DLmethionine, Capryl-caproylic DLmethionine. Amino acid analogs are produced by substitution of вЂќвЂќ-amino group of amino acid with hydroxyl group. Most commonly studied analog is methionine hydroxyl analog of 2-hydroxyl-4methyliobutanoic acid (HMD) вЂў Post ruminal Infusion: Proteins and amino acids are directly infused into abomasum or duodenum instead of being a part of diet. вЂўImproves fat percent in milk вЂў Better economic returns, for same input cost вЂў Positively influenced wool growth and quality вЂў Useful for low and high yielding animals, very relevant to Indian conditions of feeding and management An additional output has been the development of a slow-release NH3 source, which when used in combination with by-pass protein feed supplements lifts milk production a further 5-10 %. Methods tried for enhancing bypass protein value of protein meals: вЂў Formaldehyde Treatment вЂў Alkali Treatment (NaOH): 1 %, 2 % and 3% supplementation may increase in rumen bypass ability by 4-5 %. вЂў Alkali Treatment (NH4OH): 0.3%, 0.5% and 1.0% supplementation may increase in rumen by pass ability by 7-8 %. вЂў Heat Treatment: It is the combination of LIVESTOCK LINE, MARCH 2012 Among these, formaldehyde treatment is most commonly used and economically feasible. 10 Advantages of formaldehyde treatment in the production of bypass protein: Protein sources differ in their rumen degradability. Some protein meals contain naturally available rumen bypass protein (30 to 50 % of total CP) viz. cottonseed meal, toasted soybean, toasted groundnut meal, maize gluten etc., which can be used in bypass protein feeds. The cost of these ingredients is high, whereas, rapeseed meal, sunflower meal, guar meal etc. are available at cheaper rate but rumen protein by-pass content in these meals is low. Such protein meals having high rumen degradability can be subjected to heat or chemical treatment for increasing the level of rumen by-pass occurring. These by-pass protein meals can enhance the post ruminal supply of critical amino acids (Prasad and Reddy, 1998). Protein meals treated with formaldehyde in sealed chambers, where these undergo formation of complexes resist degradation in the rumen (Ashes et al., 1995). The process occurs under occupational health and safety procedures (Owens et al. 1990). This attributes to HCHO-binding to the proteins by formation of methylene bridges (Fraenkel-Conrat and Olcott, 1948), which makes them resistant to microbial attack (Walker, 1964). Different protein meals could be tested for degree of protection using in vitro procedure to measure the degree of protection (Ashes et al. 1995, Gulati et al., 2002; Garg et al.,2004). Treating protein meals with formaldehyde has the following advantages: вЂў Desired level of protein protection can be achieved. вЂў Under and over protection of proteins can be eliminated. вЂў The bio-availability of the essential amino acids can be maximized. вЂў It does not increase the proportion of ADN and NDN contents. вЂў Less expensive than heating. вЂў Helps to control salmonella and reduce mould growth in feedstuffs. LIVESTOCK LINE, MARCH 2012 Protein meals treated with formaldehyde to produce bypass protein should have the following characteristics: proportion of UDP 70-80%, bioavailable lysine 80-85%, unchanged levels of acid detergent insoluble nitrogen (ADIN) 2.4-3.0% and neutral detergent insoluble nitrogen (NDIN) 4.05.0%. These bypass protein supplements could be included in the diets of lactating cows and buffaloes for improving milk yield and composition. Optimization of treatment for protein meals To avoid over or under protection, protein meals need to be given optimum chemical treatment, so that their digestion in the intestine can be maximized. Maximum protection of protein meals was obtained at 9-10 days of incubation in airtight conditions. Lysine and methionine are reported to be the most limiting amino acids for milk production (Schwab, 1995; Xu et al., 1998).On protection, availability of limiting amino acids increased significantly. Operational health and safety aspects Formaldehyde is widely used in industry and occurs naturally as a constituent of many foods including dairy and meat products, coffee, fruits, smoked fish e.g. 0.2 Г¬g/g in meat; 0.1 Г¬g/g in milk; 10 Г¬g/g in cheese; 180 Г¬g/g in fish (Owens et al, 1990). Formaldehyde is converted to formic acid by the action of the formaldehyde dehydrogenase enzyme; formic acid is metabolized to carbon dioxide and water, or incorporated into the one carbon pool or excreted in the urine as a sodium salt (Owens et al, 1990). Hence, mammalian systems have the biological pathways to effectively metabolize ingested formaldehyde and there is no evidence to suggest that formaldehyde is a carcinogen when consumed orally (FDA, 1998). The formaldehyde present in treated feedstuffs is metabolized by ruminants and does not significantly change the naturally occurring levels of formaldehyde in meat and milk (Atwal and Mahadevan, 1997). Formaldehyde is approved for use as a feed additive to protect proteins from ruminal degradation, to preserve silages, to maintain animal feeds or feed ingredients free of salmonella, to control fungi and to improve the handling characteristics of oilseeds and meals, and animal fat pre-mixes (FDA, 2004). For treatment of protein meals, level of 11 formaldehyde used is not more than 0.8 per cent. After two days of incubation, formaldehyde level in protein meal is detected below 2 ppm. So, handling of treated protein meals is not a serious problem from animal and consumer health hazard point of view. Table 1: Level of essential amino acids available for absorption in unprotected and protected protein meals: Source: Gulati et aal., 2002 LIVESTOCK LINE, MARCH 2012 12 Reference Ashes, J.R., Gulati, S.K., and Scott, T.W. (1995). The role of rumen protected proteins and energy sources in the diet of ruminants. In: Animal Science Research and Development (Ed. Ivan, M). Centre for Food and Animal Research Agriculture and AgriFoods, Canada, pp.177. Atwal, A. S. and Mahadevan, S. (1997). Formaldehyde in milk not affected by feeding soybean meal coated with chemically treated zein. Canadian Journal of Animal Science, 74: 715-716. FDA. (2004).вЂ�Food Additives Permitted in Feed and Drinking Water of Animals: Formaldehyde.вЂ™ Food and Drug Administration, Department of Health and Human Services, Washington, DC. 21CFR Part 573 (Docket No. 1998F-0552). Gulati, S.K., Scott, T.W., Garg, M.R. and Singh, D.K. (2002). An overview of rumen protected or by-pass proteins and their potential to increase milk production in India. Indian Dairyman, 54: 31-35. Owens, B.A., Dudney, C.S., Tan E.L. and Easterly, C.E. (1990). Formaldehyde in drinking water: Comparative hazard evaluation and approval to regulation. Regulatory Toxicology and Pharmacology, 11: 220-236. Patton, R.A. 2009. The Strategic Use of Ruminally Protected Amino Acids in Dairy Nutrition. d. ifas.ufl.edu Prasad, P.E. and Reddy, R.R. (1998). Effect of formaldehyde treated groundnut cake on in vitro and in sacco protein degradability. Indian Journal of Animal Nutrition, 15: 52-54. Fraenkel-Conrat, H. and Olcott, H.S. (1948). Reaction of formaldehyde with proteins. Cross linking amino groups with Phenol, Imidazole or Indole group. Journal of Biological Chemistry, 174:827. Schwab, C.G. (1995). Rumen protected amino acids вЂ“ their role in nutrition of high producing dairy cows. In: Animal Science Research and Development: Moving towards New Century. (Ed. Ivan, M.) Ottawa, Canada. Garg, M. R., Sherasia, P. L, Bhanderi, B. M., Gulati, S. K. and Scott, T. W. (2002). Effect of feeding rumen protected nutrients on milk production in crossbred cows. Indian Journal of Animal Nutrition, 19 (3):191-198. Taquire N.A., Shahzad M.A., Nisa M., Sarwar M. and Fayyaz M. 2010. Influence of bypass protein on Buffalo productivity. Proceedings 9th world buffalo congress. New Delhi, India. Garg, M. R., Sherasia, P. L., Bhanderi, B. M., Gulati, S. K. and Scott, T. W. (2004). Effect of feeding protected protein on milk production and composition of lactating cows. Indian Veterinary Journal, 81(1): 48-50. Garg, M.R. (1998). Role of bypass protein in feeding ruminants on crop residue based diets. AsianAustralasian Journal of Animal Sciences, 11: 107116. Gulati. S.K., Ryde. I., Kaur. R., Scott. T.W., Garg. M.R., Serasia P.L. and Singh D.K. (2001). Role of protected nutrients in sustainable milk production., In Proc. X Animal nutrition conference. Karnal, India. Walker, J.F. (1964). Formaldehyde. 3rd Ed. Reinhold Publication, New York (Fide McDonald, I.W. 1968). Nutritional Aspects of Protein Metabolism in Ruminants. Australian Veterinary Journal, 44:145. Walli, T.K. (2005). Bypass protein technology and the impact of feeding bypass protein to dairy animals in tropics: A review. Indian J. Anim. Sci. 75 : 135142. Xu, S., Harrison, J.M., Chalupa, W., Sniffen, C., Julien, W., Sato, H., Fuvieda, T., Watanabe, K., Veda, T. and Suzuki, H. (1998). The effect of ruminal bypass lysine and methionine on milk yield and composition of lactating cows. Journal of Dairy Science, 81: 1062-1077. U LIVESTOCK LINE, MARCH 2012 13 A Few Nutritional Updates for Feeding Dairy Animals Dr Trishna B. Kayastha & Dr Sanjeeb Dutta Department of Animal Nutrition & Livestock Production and ManagementApollo College of Veterinary Medicine, Jaipur-31 Mobile phone no 09602564270. U India is the highest milk production country in the world and by the end of 2022, India aims at producing 172 MMT milk at an annual growth rate of 4 per cent, but its cattle feed industry has not kept pace ahead. There is a need to change the conventional system of preparing cattle feed so as to meet the current challenge of high yielding cross bred cows and improved buffaloes. The bio-mass resources is very limited and there is shortage of feed and fodder resources in the country. So the available feed resources would need to be utilized judiciously and with value addition. Farmers therefore need to be encouraged to adopt improved and balanced feeding practices so that they could improve yields with available feed resources in a cost effective manner. In this way milk out put could be doubled. Feeding plays vital role in exploiting the genetic potential of dairy animals. Feeding is also considered critical in the overall success of dairy development program, as feeding alone consists more than 70% of the total cost of milk production. Indian cattle industry is an integral part of Indian agriculture and contributes to the well being of its people. Over 80% of these livestock unit is ruminants and can be largely described as grass eater while the rest is monogastric grain eaters. Presently India is bestowed with a huge livestock population comprising 222 million cattle, 98 million buffaloes, 124 million goats, 61 million sheep and 489 million poultry. Animal Husbandry, Dairy and Fisheries generate supplementary incomes and gainful employment for rural house holds, particularly among landless, marginal or small farmers, as well as women in one hand and the products obtain from these sectors are also a source of valuable nutrients to millions of people in India. Dairying only provides nearly 2/3 of the total livestockвЂ™s contribution to GDP with an encouraging growth rate of 5 percent. Agriculture and allied sectors account for about 24% of GDP. Of this, animal husbandry and dairy accounts for about LIVESTOCK LINE, MARCH 2012 25%. More than 70 million rural families are engaged in milk production in India. Landless, small and marginal farmers with limited resources account for 65% of the total milk production in the country. Dairy cattle production is mostly based on crop residues such as straws, stovers and agroindustrial byproducts. Over the years there has been a perceptible change in total livestock population. The milch buffaloes and crossbred cattle population has been increasing gradually, while the male population of cattle and buffaloes have been decreasing due to mechanization in agriculture and shortage of feed resources for feeding unproductive animals. Feed resources can be broadly categorized into dry fodder (crop residues), green fodder and concentrates. Crop residues include mostly wheat, paddy, sorghum and millet straws, kabdies etc; green fodder include cultivated legumes and non-legumes, pastures, sugarcane tops etc and concentrates include grains, oil cakes/meals, brans, chunnies, and agroindustrial by-products. There always exist a huge shortage of concentrate and green fodders that has been calculated using appropriate grain to straw ratios for crop residues, extraction rates for concentrates and average green bio mass production potential for different categories of land. To minimize the gap between requirement and availability of feed resources, some of the technologies that could be used in utilizing feed resources judiciously with value addition are briefly describe below. 1. Implementation of Ration balancing program. 2. Feeding of Compound Cattle Feed. 3. Supplying protein source of feed in the form of by pass protein. 4. Supplementation of area specific mineral mixture. 5. Feeding of Urea Molasses Mineral Block Lick. 6. Enrichment and Densification of Crop Residues. 7. Enhancement of Green Fodder Production. Implementation of Ration balancing program 14 In Indian dairy animal ration only 10% of total feed ingredients are provided in the form of compounded cattle feed. The rest of the feed ingredients in the ration comprise with locally available or home grown feed ingredients as such or some farmers used only brans, grains or cakes to feed their animal. These ingredients are rich in only one or two nutrients. For eg brans are rich source of Phosphorus and low in Calcium. But animals respond better in terms of growth and milk production when they receive all the major as well as minor nutrients in right proportion вЂњso called balance rationвЂќ as per their bodyвЂ™s requirement that can only be obtain by preparing ration with ingredients like energy rich eg;- cereal grains; medium energy & medium protein eg: choker churies and brans; protein rich eg: oil cakes and other agro-industrial byproduct that are locally available with more or less similar or lower input cost. These are mixed in right proportion and fortified with small quantity of mineral and vitamin. So, it is very much essential to implement the ration balancing program at the farmers door step so that such type of balance feeding can fully exploit the genetic potentiality of the dairy animal. NDDB has developed computer software for ration balancing program. In this training program farmers are advised to feed their animals a balance ration which is computed by computer based least cost computation and which takes into account the animals nutrient requirement according to different physiological conditions as well as the nutrient available to the animal from the prevailing feeding practices. Introducing Compounded Cattle feed for dairy animals The compounded feed is a blend of all feed stuffs ie fodder, cereal grains, oil seed cakes, brans, churies, agro-industrial byproducts, mineral and vitamins that are thoroughly mixed in suitable proportion which provide adequate nutrients to meet the needs of dairy animals. Each bite consumed provides the required level of nutrients needed by the animals. With the feeding of compounded feed along with basal diet, nutrient requirement of animal can be met more efficiently and economically, but this needs proper grouping of animal according to physiological stage and production level. For example high energy ingredients can be liberally fed to high producers kept in the separate group LIVESTOCK LINE, MARCH 2012 without overfeeding to the late-lactation. It provides greater flexibility in feeding exact amounts of nutrients as per level of milk production. Thus compounded cattle feed system of feeding can save labour and reduce overall feeding cost. The Bureau of Indian Standards (BIS) have been prescribe two categories of compounded cattle feed Type I (containing min 22% CP, min 3% EE, max7% CF & max 3% AIA) and Type II (containing min 20% CP, min 2.5% EE, max 12% CF & max 4% AIA) which are suitable to feed the cattle or buffalo yielding more than 10 liters of milk per day. But a majority of dairy farmers in India possess two or three cows/buffaloes yielding about 2 to 4 liters of milk per day. The nutritional parameter prescribed for these two type of cattle feed do not allow the incorporation of abundantly available agro-industrial by product so these are cost effective. In order to encourage the majority of the farmers to use compounded cattle feed CLFMA(Compounded Livestock Feed Manufacturer Association ) felt necessity to introduce additional categories to feed animals of different production capacities based on milk yield and body weight as for High yielder Type I (containing min 20% CP, min 2.5% EE, max 7% CF & max AIA 4%), for medium yielder Type II (containing min 18% CP, min 2.5% EE, max 12% CF & max AIA 4.5%) and for low yielder Type III (containing min 16% CP, min 2% EE, max 14% CF & max AIA 5%). Practice of Feeding Bypass protein supplements The availability of protein source feed for animal in India is very limited. Usually about 70% proteinacious feed is degraded in the rumen to ammonia by the ruminal microbes and significant portion of it is excreted out in the form of urea through urine. However, if such type of proteinacious feed is subjected to suitable chemical treatment termed as вЂњbypass protein technologyвЂќ, their efficiency of utilization can be significantly improved. NDDB has standardized and commercialized bypass protein technology, using locally available proteinacious feed such as groundnut cake, mustard oil cake, sunflower cake, guar meal etc. These proteinacious feeds are treated suitably, so as to reduce their degradability in the rumen from 60-70% to 25-30% in a specially designed airtight plant. Cost of bypass protein supplement is less than a rupee per kg and on feeding one kg treated bypass protein supplement; there is increase in 15 milk production by more than a liter in comparison to untreated proteinous feed, which is well established after several feeding trial in dairy animals. The specifications of bypass protein feed produced in NDDB on dry matter basis is min 30% CP, min 3.5% EE, max 8% CF, max 2.5% AIA, min 20% UDP & max 9% RDP. The overall benefits of feeding bypass protein to the dairy animals are summarized as follows. 1) Higher availability of amino acids per units of feed. 2) Better utilization of proteinacious feed having higher rumen protein degradability. 3) Judicious utilization of protein source available in limited quantity. 4) Improves growth and milk production (0.8-1.2lit/day) 5) The percentage of protein in the milk increases (0.1-0.3%), hence improves SNF content of milk. 6) The percentage of fat in the milk increases (0.2-0.8%). 7) Better economic returns from same input cost. 8) Useful for low and high yielding animals, relevant to Indian condition of feeding and management. Supplementation of Area Specific Mineral Mixture In India, crop residues are used as staple feed for dairy animals which are very poor in essential minerals as it contains several anti-nutritional factors like silicate, oxalate, phytic acid etc that further inhibit the utilization of several minerals. As animals can not synthesize minerals inside their body, supplementation of mineral mixture with their ration is outmost important. Minerals both macro (Ca, P, Na, K, Cl, S, Mg) as well as micro (Zn, Fe, Cu, Co, I, Se, Mn etc) are equally play important role in growth, production, reproduction and many metabolic activities of the body. In India BIS has recommended two types of mineral mixture Type I (Containing salt) and Type II (without salt) for supplementing cattle feed, the characteristic of which are as follows:- Implementation of Urea Molasses Mineral Block lick (UMMB) UMMB are the lick blocks containing urea, molasses, vitamins, minerals and other multinutrients. The feeding of the block is a convenient and inexpensive method of providing all the nutrients required by both the rumen microbes and the animal which may be deficient in the diet. A standard UMMB consists of molasses (30-50%), urea (5-10%), a cereal bran such as rice, wheat or maize bran (15-25%), an oil cake (10-12%), salt (5-7%), lime or calcium carbonate (5-10%), bone meal (5-7%) and trace mineral mixture (1-2%). Urea is only source of nitrogen (46%) which readily hydrolyzed in the rumen producing ammonia. So readily available source of energy such as molasses (cheap source must be given in urea LIVESTOCK LINE, MARCH 2012 16 supplemented feed so that the rate of hydrolysis of ammonia from urea and release of energy from the molasses remain constant. As a result the rumen microbes can easily form the microbial protein for the host animal without causing ammonia toxicity. Such multinutrient block not only provides overall nutrient requirement of ruminant animal but also these are more convenient for packaging, storing and transportation. However the consistencies of the molasses play an important role in the successful manufacture of UMMB which depend upon the quantity of sugar in the molasses. This sugar quantity, expressed as a percentage of total weight in the molasses is called the BRIX value. To ensure good hardening the BRIX value should be 80 or more that can be checked with a small pocket refractometer. Enrichment and Densification of Crop Residues The availability of crop residue all over India throughout the year is not uniform, with some areas having a surplus and others facing a perennial shortage of dry fodder. Regional imbalances and shortages of crop residues lead to Sub-optimal livestock productivity due to imbalance feeding and significant cost on account of transportation. In India, major part of basal ration of ruminants constitute crop residue. So, there is a need to manage feed and fodder resources efficiently with value addition. In areas where crop residues are deficit are sold @ Rs 4/kg, on the other hand they are often burnt in surplus areas. If crop residues are enriched and densified in the form of blocks, pellets, briquettes etc, these can be transported at lower cost from the surplus to deficit regions. Some examples of Straw-based pellet and Strawbased block produced by NDDB are respectively as follows Wheat straw-40%, Deoiled rice bran-37%, Mineral mixture-1%, Common salt-1%, Rice polish fine-5%, Urea-1%, Molasses-15%. & Straw-64%, Deoiled rice bran- 20%, Urea-1% and Molasses-15%. Such multinutrient UMMB provide approximately 810% CP and 55% TDN. Enhancement of Green Fodder Production The fodder based systems of feeding help to lower feed costs, but the scope for such system is limited LIVESTOCK LINE, MARCH 2012 in India because of the need to give priority to food crop. The average cultivated area under fodder crop is estimated as 4.4% ie around 9.38 million hector. In areas with better irrigation facilities, intensive fodder production is practiced in the Northern Region particularly Punjab and Haryana where 10% of the irrigated land is allocated for fodder cultivation (mainly Lucerne, berseem, maize, oat, sorghum etc). As limited land is available for green fodder production, fodder yield per hector can be improved by (a) Supplying farmers certified fodder seeds, to cover at least 10% of the total area for fodder production, (b) Efforts should be made to develop wasteland through watershed management for green fodder production, so that the gap between the requirement and the availability could be minimized. NDDB has designed and implemented fodder seed multiplication and distribution project through dairy co-operatives. NDDB is assessing milk co-operatives in the procurement of breeder seeds, its multiplication by the farmers and establishing fodder seed processing plants. Conclusion Keeping in view the wide gape of the availability of feeds and fodder for the livestock as per their requirements, nutrition research should emphasize the development of feeding systems based on existing feed resources under farm conditions. There should be a feed security system for animals needs to be developed to meet the requirements of livestock in famine and for draught or flood prone areas. Further introduction of cultivated fodders having high yield and identification of nonconventional feeds for livestock and developing processes for improving their nutritive values needs to be undertaken on large scale. References Dr D.D.Sharma. (2002). Scope for using complete feed blocks for increasing dairy production in India. All India Dairy Husbandry OfficersвЂ™ Workshop (2627) th November, 55-61. David J. Schingoethe. (2010). Feeding dairy cows. Livestock Feeds and Feeding. 306-322. Dr D.V. Reddy, Nutritional Requirement of Indian Cattle and Buffalo. Applied Nutrition. Pp 53-104. Richard O., Kellems and D.C. Church. (2010) Supplemental Protein Sources. Livestock Feeds and Feeding. 84-113. 17 COMMON DISEASES OF LIVESTOCK CAUSED BY BACTERIA AND THEIR DIAGNOSIS * * Phaniraja.K.L., Prasanna.S.B and Ravikumar .C Dept. of Veterinary Microbiology, Veterinary College, KVAFSU, HASSAN- 573 118 TUBERCULOSIS U Tuberculosis (TB) remains as a world wide public health concern despite its agent was established 100 yrs ago and efficient drugs and vaccines are available since then. Even today the world records an occurrence of 7.25 million new cases and 3 million deaths every year due to TB. It is a chronic infectious disease of animals and also Humans caused by an acid fast bacilli Mycobacterium bovis, a facultative intracellular pathogen with an ability of resisting intracellular killing by phagocytes. Bovine TB organism is closely related to Mycobacterium tuberculosis in nucleotide sequence, with a high degree of antigenic relatedness. . Factors contributing spread of bovine TB: Bovine TB gains its utmost importance because of its relatedness to Mycobacterium tuberculosis and its ability to cause similar disease in human beings causing a significant public health crisis. TB caused by M.bovis is clinically indistinguishable from that of M.tuberculosis. Information on human disease by M.bovis in developed and developing countries is scarce. However, the abstract of several Zoonotic TB studies carried out around the world indicate the proportion of human cases due to M.bovis accounted for 3.1% of all forms of TB; 2.1% of pulmonary forms and 9.4% of non pulmonary forms. Since the disease is zoonotic, risk factors contributing zoonosis log on to both animals and human population.. A. Animal risk factors: 1. Constant source: The distribution of M.bovis in domestic and wild animal population represents a huge pool of infectious agent, even in countries which had totally eradicated the disease; it is again on a rise because of wild reservoirs, where in U.K is a great example with badges being the source of reinfection after almost eradication. 2. Milk production and animal husbandry: Introduction of more productive cross breed /exotic animals leading to white revolution has made India today the highest milk producer in the world. It is a well known fact that indigenous animals are resistant to tuberculosis. Bovine TB is most common in organized dairy herds especially with more numbers of crossbreeds/exotic animals (in urban areas).The LIVESTOCK LINE, MARCH 2012 intensive husbandry activities with poor management and overcrowding are playing a major role in the spread of the disease. 3. Inadequate control programmes: The basic strategies required for control and elimination of Bovine TB are well established however due to financial constraints, scarcity of trained professionals, lack of political will, as well as the underestimation of importance of TB in both animal and public health by successive Governments., control measures are applied inadequately or not at all put into practice . Presently we have got a вЂњtest and isolateвЂќ policy against the global policy of вЂњtest and slaughterвЂќ which itself is creating a big window for disease spread. B. Risk factors from Human population: 1. Physical contact : In India cattle are very much an integral part of human social life, hence there is close contact between man and potentially infected animals, with a very high risk human infection. 2. Food hygiene practices : In principle, milk from affected animals has been regarded as principle source of M.bovis to humans. In areas where bovine TB is endemic, milk borne infection is the main cause of cervical lymph adenopathy and other forms of non pulmonary TB. In India, of late, a huge competition has been created by large scale state run, marketing enterprises and the informal sector. The informal sectors can ignore standards of hygiene and quality, which could be a direct source of infection to the consumers. 3. HIV /AIDS: It has been well established that TB is the most frequent opportunistic disease along with HIV infection. Persons with both infections have an annual risk of progression to active TB of 5% to15% depending on their level of immunosupression. LABORATORY DIAGNOSIS: A. Single Intra dermal PPD testing in cattle/ Tuberculin Test: involves injection of 0.1.ml of PPD at the neck or caudal fold region of the animal. If erythema and induration follow, the test is considered as positive. B. Demonstration of organisms by acid fast staining. 18 C. Postmortem and histopathology. D. Cultivation of the organisms on primary isolation medium. E. Molecular and Nucleic acid recognition methods: RFLP, IS- printing and PCR has been widely evaluated for the detection of M. tuberculosis complex in clinical samples. F. Serological Diagnosis by ELISA, IFN analysis. Control / Eradication : Bovine TB does not often justify the emergency measures required for other diseases (e.g., Rinderpest, Leptospirosis, and foot and mouth disease). The full economic implications of zoonotic TB are, however, overlooked in India where the overall impact of the disease on human health and animal production needs to be assessed. The present concept of TB control is by testing and isolating the positive animals in Goshalas. This concept, instead of reducing the disease incidence it facilitates increased disease spread. ItвЂ™s high time to strongly incorporate the policy of вЂњtest and slaughterвЂќ. In the name of ethics and socio religious reasons, that cattle should not be slaughtered, we are making the infected animal to suffer from the disease for a longer period which is also a cruelty in real sense. In an era, where Governments across the globe are permitting the human euthanasia in totally incurable conditions to avoid further suffering, we have to think seriously in these lines in order to eradicate the disease from our country. Following may be the practical and field oriented concept of Tuberculosis eradication. 1. Test by single intra dermal tuberculin test (or any other better tests, if feasible) 2. Immediate culling of the positive animals from the herd by humane slaughter. 3. Careful management of above listed risk factors. 4. Development of an Anti TB vaccine вЂ“ this is most relevant today because it was M.bovis strain that is used as BCG for human TB control with satisfactory results across the world. Research and development is the need of the hour in this direction to develop a better, new generation vaccine for animal use. Even if the cost of this is in terms of few crores it is much smaller when compared to total annual economic loss due to Bovine tuberculosis apart from immense zoonotic significance the bacteria posses which can never be assessed financially. JOHNEвЂ™S DISEASE / PARATUBERCULOSIS Paratuberculosis, popularly known as JohneвЂ™s disease, is an infectious disease caused by LIVESTOCK LINE, MARCH 2012 Mycobacterium avium sub species Para tuberculosis. It is primarily a disease of domestic and wild ruminants, the disease also been reported in horse, pigs, deer and recently in rabbits and fox. The disease causes heavy economic losses to the dairy industry in terms of reducing milk loss and treatment. The disease is characterized by dehydration, emaciation, chronic diarrhea and thickening of the intestine (corrugation). Under natural conditions, the disease in cattle spreads by ingestion of M.paratuberculosis from the contaminated environment. The disease persists after the introduction of infected animals. Infection can be spread vertically to the fetus and semen can be infected with the organism. The primary source of infection in claves is milk from infected cows or milk that is contaminated with the feces of diseased cattle. Transmission and pathogenesis: Feces containing the organisms are primary source of infection which is acquired by ingestion of contaminated feed and water. It has very long incubation period (15-18 months). The organisms have also been isolated from genitalia and semen of infected bulls. Following infection, organism penetrates the intestinal mucosa and sets up residence within macrophages. The organism multiplies intracellular without killing host cells and are resistant to intracellular digestion. They grow inside macrophages and distributed throughout the body. The primary site for bacterial multiplication is terminal ileum and the large intestine leading to decreased absorptive surface, chronic diarrhea and mal absorption. Early lesions occur in the walls of the small intestine and the draining mesenteric lymph nodes, and infection is confined to these sites at this stage. As the disease progresses, gross lesions occur in the ileum, jejunum, terminal small intestine, caecum and coon and in the mesenteric lymph nodes. Mycobacterium paratuberculosis is present in the lesions and terminally, throughout the body. The intestinal lesions are responsible for a protein leak and a protein mal absorption syndrome, which lead to muscular wasting. Clinical sings usually first appear in young adulthood, but the disease can occur in animals at any age over 12 years. The infection progress and the animal still does not show any clinical sings. Nevertheless, the organisms are being excreted in very high numbers, probably enough to infect other animals in contact. Infection is detectable by fecal culture techniques but not often blood tests. In later stage the animal show early signs of disease and most diagnostic tests can detect the infection. 19 LABORATORY DIAGNOSIS: A. The cultural examination of feces and direct microscopic examination of acid fast stain reveals the presence of organisms like clumps (three or more organisms) of small (0.5-1.5Г¬m), strongly acid fast bacilli are found. The presence of single acid fast bacilli in the absence of clumps does not indicate definitive diagnosis. The disadvantage of this test is that only about one вЂ“third of cases can be confirmed of microscopic examination of a single faecal sample. Mycobacterium paratuberculosis infection mainly involves the lower small intestine and adjacent caecum. Mycobacterium paratuberculosis organisms are vastly outnumbered by other bacteria in faecal and intestine tissue specimens. The commonly using medias are HerroldвЂ™s egg yolk medium with mycobactin and modified DuboвЂ™s medium. B. Using single intradermal PPD Johnin has been used for many years for screening of animals in herds. After 72 hrs of infection, it is examined for the presence in thickness 4mm or above for positive. C. ELISA is reported to be most sensitive and specific test for serum antibodies to M. paratuberculosis. Its sensitivity is comparable with that of the CF test in clinical cases, but is greater than that of the CF test in sub clinically infected carriers. D. CFT: the CF test has been the standard test used for cattle for many years. The CF test works well on clinically suspect animals, but does not have sufficient specificity to enable its use in the general population for control purposes. Control: Following may be the practical and field oriented concept of JohneвЂ™s disease eradication. 1. Test by single intra dermal Johnin test (or any other better tests, if feasible) 2. Immediately cull the positive animals from the herd by humane slaughter. BRUCELLOSIS Brucellosis is an economically important reproductive disease of livestock including cattle, buffalo, sheep , goats and pigs. The disease induces infertility, delayed heat, interrupted lactation, loss of calves, wool, meat and milk production and is of zoonotic importance in developing countries, including India. In India, the abortions in livestock due to brucellosis was first reported as early as in 1918. Subsequently, the serological and cultural evidence of infection in livestock and human beings have been reported from LIVESTOCK LINE, MARCH 2012 various states in the country, including Karnataka. There is concern that the disease may further flare up due to intensive dairy development programmes. In this context, an indepth understanding of the epidemiology of brucellosis is required in view of itвЂ™s trans host transmission and institution of practical strategies to control the disease. This paper highlights the aspects of epidemiology and control of bovine brucellosis in Karnataka. Serological survey in bovines: Seroprevalence studies form the backbone of epidemiological investigations and have been used to identify brucellosis-infected herds. Generally, the cases of reproductive failure and abortion are screened for brucellosis by RBPT and SAT. During late 1990s, serum and milk based ELISA kits developed at Project Directorate on Animal Disease Monitoring and Surveillance (PD_ADMAS), Bangalore found wide acceptance. Milk based ELISA was preferred for screening pooled milk samples at village based milk co-operative societies. The details of brucellosis survey is dealt elsewhere. Brucellosis control: The Govt. of India has a centrally sponsored scheme on brucellosis control in most of the states including Karnataka, in the name of Systematic Control of Livestock Diseases of National Importance. The functioning of this programme are mostly restricted to serological survey and suggesting the livestock owners to send the sero-positive animals to Goshalas. The cost involvement in maintenance of such infected and unproductive animals is high and is economically not feasible. However, due to lack compensation, such brucellosis positive animals are sold due to distress instead of being sent to Goshalas. On the other hand, the ongoing individual animal screening and declaration of diseased animals could be partially responsible for spread of disease. Thus this programme appears to be counter-productive and has hardly contributed much to any approach towards disease control. Furthermore, cow slaughter is banned on religious basis. In addition, the commercialization of dairy sector provides for frequent movement of animals. However, it is practically difficult to restrict the movement of such animals not only within the state but also at the inter state level. Recently, Govt. of India has directed that all the bulls used in the production of semen for artificial insemination purpose should be regularly tested and strictly brucellosis free bulls to be used for semen collection. It has also identified Regional Disease 20 Diagnostic Laboratory for this purpose (Southern Regional Disease Diagnostic Laboratory, SRDDL in Karnataka). With this directive, practically all the bull farms in the Govt. and Co-operative sectors are subjected to regular testing and affected bulls are being culled. Due to this initiation, breeding bulls in these semen stations may no longer serve as source of infection to inseminated cows. In Karnataka, vaccination against bovine brucellosis is not generally practiced except in some infected, organized, private and military dairy farms. In general, Calf hood vaccination with B. abortus S-19 is practiced in a meager scale. Vaccination of calves, the elimination of reactors, improved herd management and zoosanitary measures are recommended for effective control of brucellosis. An awareness programme needs to be introduced to highlight the public health importance of this disease. Brucellosis should be controlled by vaccination of bovines together with other measures such as movement control and testing and isolation of infected animals. This eventually reduces the transmission to human beings. While the effective control measures are still need to be implemented, veterinarians and other workers may enlighten the public, villagers, risk groups to prevent brucellosis by boiling of milk, avoid consumption of unpasterurised milk. Farmers should know that vaccination of their animals for brucellosis is important. Vaccination of livestock is relatively cheap and will increase the value and productivity of their animals. Brucellosis in sheep and goats in the state, which is mostly responsible for brucellosis among veterinarians and Inspectors, is also reported by several researchers including both veterinary and Medical professionals. Generally it is believed that B. abortus infects bovines and B. melitensis infects small ruminants. However, isolation of B.melitensis from Jersey cross bred cows of the dairy farm belonging to a semi Govt. organization in 1987 was of major concern. The authors postulated that the infection from sheep and goats in the same farm was transmitted to cattle through contaminated fodder or some other vehicle, as livestock species such as cattle, buffalo, sheep and goats in the farm had access for common grazing. Thus, free grazing and movements with frequent mixing of flocks of sheep and goats with cattle also contribute to the high prevalence and wide distribution of brucellosis in these animals. This situation needs to be viewed seriously from public health point of view as B.melitensis infections, than B. abortus, are most commonly encountered in human patients. Therefore LIVESTOCK LINE, MARCH 2012 there is an urgent need for the strict implementation of a control policy not only for cattle but also for small ruminants. Unfortunately, non-availability of vaccine locally for controlling brucellosis in sheep and goats is a major limitation. The situation demands itвЂ™s immediate availability for the local use in view of controlling trans host transmission of brucellosis from small to large ruminants and also reducing the zoonotic impact of the disease. BLACK QUARTER (BLACK LEG / INFECTIOUS MYOSITIS) True Balckleg / Black Quarter is the Clostridial myositis of skeletal muscles caused by Clostridium chauvoei (Cl. feseri), a Gram positive, spore-forming rod-shaped bacterium. Disease is common only in cattle but occurs in other animals under traumatic conditions. Epidemiology: Cattle in age group of 6 months to 2 years that are rapidly growing with high nutritional status are affected. Occurs in warm wet months, spring to autumn, high rain fall, excavation of soil which exposes and activates spores of the causative agent and the mortality goes upto 100 per cent. It is a soil-borne infection. Portal of entry is probably through the alimentary mucosa by contaminated feed or during erupting teeth. Bacteria may be found in the spleen, liver and alimentary tract of normal animals. Contamination of soil and pasture occur from infected feces or carcasses of affected animals. Disease develops by invasion of tissues by organisms during trauma or anoxia. In cattle the disease occurs without history of trauma but in sheep it is always a wound infection during shearing, lambing, fighting, during vaccinations with tissue damage or fetus of infected ewes. Pathogenesis: Trauma or unnoticed trauma may trigger the disease. Toxins(ГЎ,Гў,ГЈ and Г¤) formed by the organism produces necrotizing myositis locally in skeletal muscles and systemic fatal toxaemia. In cattle and sheep atypical outbreaks of sudden death occurs in which the lethal lesion is a clostridial cardiac myositis. Clinical findings: Lameness, pronounced swelling of upper limb, myonecrosis of skeletal or cardiac muscles, high temperature (106o F) and pulse rate (100-120 / min), severe toxaemia, depression, ruminal stasis, anorexia, gaseous crepitating emphysema, discolored, dry and cracked lesions. Animals may be found dead with high mortality. Lesions may vary slightly in sheep and horses. Necropsy findings: Generally myositis, dark coloured, rancid odour, metallic sheen on the cut 21 surface of affected muscle are seen. Cattle are found dead lying on the affected side of the hind limb with stiffness. Bloating and putrefaction occur quickly. Blood-stained froth exudes from the nostrils and anus. Blood clotting occurs rapidly. In sheep lesions are not so marked as in cattle. LABORATORY DIAGNOSIS: Smears from affected tissues are collected for bacteriological examination as soon as possible. Isolation and identification of Cl chauvoei and Cl novyi is difficult due to their fastidious nature and due to clostridial post-mortem invaders from the gastro intestinal tract. 1. Muscle pieces collected under aseptic precautions are kept in sterile air-tight container and sent to laboratory as quickly as possible for anaerobic culture 2. Four air dried impression smears for FAT and microscopic examination. 3. Culture from needle biopsy or swabs from wounds. Serological test: By fluorescent antibody test (FAT) for tissue smears Control: Annual vaccination of all cattle between 3-6 months with 2 vaccinations given 4 weeks apart and annual booster vaccination done prior to risk period. In an outbreak all unaffected cattle should be vaccinated immediately and treated with 10,000 units / kg penicillin, I/M. Segregation, early treatment are beneficial. Clostridial vaccines have poorer antigenecity in sheep and goats than in cattle. Proper disposal and zoo-sanitary measures like deep burial of dead carcasses and Infected materials with maintenance of hygienic conditions of the premises. Inactivated alum-precipitated vaccine is widely used in the country with satisfactory results. ANTHRAX Anthrax is an important zoonotic disease primarily affecting large domesticated animals and infects man accidentally through contact with infected animals and animal products. In natural conditions, both wild herbivores and domestic animals are highly susceptible and birds are resistant to the disease. Bacillus anthracis is the causative organism, is of world wide distribution. Repeated outbreaks occur in Asia, Australia, Africa, Southern Europe & Southern America. It has roughly 1,200 various strains. It is a gram positive large rod, non motile, capsulated, non haemolytic bacillus, occurring in short or long chains.and can take two forms: the vegetative bacilli and the spore. B. anthracis is more dependent on sporulation for species survival LIVESTOCK LINE, MARCH 2012 making it an obligate pathogen. The spores are resistant to chemical disinfectants and heat. However, autoclaving at 121В° C at 15 lbs pressure destroys them in 15 minutes. The infection is transmitted by the spores of the bacillus, which are shed in large numbers in the terminal stages of infection. The disease is therefore unusual in that the infection is spread only from dying or dead host and that the causative organism may survive for a long time in the environment. Pathogenesis and Virulence determinants: The pathogenicity depends on two properties of the organism that are not found in saprophytic Bacillus species. The most easily demonstrated determinant is the capsule which is unusual in being a polypeptide, of D-glutamic acid which inhibits opsonophagocytosis. The other determinants are the potent exotoxins comprising of three proteins produced by the organism viz. Protective antigen ,lethal factor and edema factor. The protective antigen attach to the cell surface and they bind to one another in groups of seven forming a doughnut shaped space called вЂ�preporeвЂ™. Either the lethal factor or edema factor binds to the prepore and enters into the cell through endocytosis resulting in destruction of cells. The main effect of toxins is to increase the vascular permeability, which leads to shock. Clinical signs: Clinical signs in animals differ by the species, with ruminants being the most at risk. The peracute form most often affects ruminants, including cattle, sheep and goats. Sudden death may be the only clinical sign, so careful attention should be paid to the carcass. The toxins in Bacillus anthracis prevent the blood from clotting so animals will often have bloody discharge coming from orifices, including the mouth, nostrils, eyes, ears, vulva, and anus. The carcass will decompose fairly rapidly leading to bloating, but rigor mortis will not be complete. The course of an acute infection is usually 1-3 days but may take up to 7 days and will affect ruminants, as well as horses. An acute infection may manifest with a brief period of high fever (up to 107В° F), excitement initially followed by depression, muscle tremors, staggering, dyspneoa, cardiac distress, and disorientation prior to death. Sub acute to chronic infections occur in less susceptible species such as pigs, but is also seen in cattle, horses, dogs and cats. The main symptoms are pharyngeal and lingual edema with animals dying from asphyxiation. Extensive localized subcutaneous edema of the ventrum, including the neck, sternum, and flank can also be seen. The carcass will decompose fairly rapidly leading to bloating, but rigor 22 mortis will not be complete. Treatment with antibiotics can be successful if begun early in the course of the disease. Penicillin is the drug of choice for treating affected animals. Doxycycline and Ciprofloxacin are also very effective against anthrax. LABORATORY DIAGNOSIS: 1.Direct demonstration of the organisms in the blood smears: Blood smears are prepared from peripheral blood or oozing blood and stained with a special stain polychrome methylene blue. Wrights or Giemsa stains can also be used. When the blood smears are stained with polychrome methylene blue, organisms appear as large blue bacilli surrounded by a purplish granular stained capsule ( McFadyeansвЂ™ reaction). 2. Ascoli test- a thermoprecipitation test used to demonstrate the presence of antigen in tissues like ear piece or muzzle piece using standard hyperimmuneserum. 3. Animal inoculation test: Guinea pigs and mice are highly susceptible. Guinea pigs are inoculated subcutaneously with pathological material or pure culture.Animal dies within 2-3 days with marked inflammatory lesions at the site and extensive gelatinous oedema in the subcutaneous tissues. Organisms are demonstrated in large numbers in local lesions / heart blood / spleen. Annual vaccination of livestock in Vaccination: endemic areas is recommended. The most widely used vaccine is the Sterne-strain vaccine (Anthrax spore vaccine) produced from the non encapsulated Sterne strain. The live spores are suspended in 50% glycerol saline. All the susceptible animals should be vaccinated once in six months in endemic areas. Prevention and control: Anthrax is a notifiable disease, if anthrax is suspected the veterinarian and local health officials should be contacted. Do not open the carcass to perform a necropsy due to the potential for contamination and exposure, it is best to burn or bury the carcasses and all contaminated materials. The animal anthrax vaccine can be used on susceptible healthy livestock. Then decontaminate the soil and contaminated materials with 5% quicklime (anhydrous calcium oxide). Hydrogen peroxide, peracetic acid or gluteraldehyde may be good alternatives. Commercially available bleach or 0.5% hypochlorite solution (a 1:10 dilution of household bleach) may be used but it may be corrosive to some surfaces. ENTEROTOXAEMIA LIVESTOCK LINE, MARCH 2012 Enterotoxaemia is an acute toxaemia resulting sudden death of sheep caused by the proliferation of the gram-positive anaerobe, Clostridium perfringens type D, in the small intestine and the liberation of epsilon (ГҐ) toxin and causes severe vascular damage. C. perfringens is a normal intestinal inhabitant. Disease often follows upsets in the gut flora, which can result from sudden changes to a rich diet or continuous feeding of concentrates. Rapid multiplication of the organism and production of ГҐ вЂ“toxin. The effects of ГҐ вЂ“toxin on the CNS and other tissues cause sudden death, preceded in some cases by clinical signs such as opisthotonus and convulsions. Transmission can occur by fecal-oral route, or by ingestion of a large quantity of the bacteria through contaminated soil, water or feed. In healthy animals, most of the ingested C. perfringens type D are destroyed in the rumen and abomasum. Although the alkaline pH of the duodenum is quite favourable for multiplication of these bacteria, toxaemia does not occur, as continual movement of ingesta keeps the bacterial population and toxin contents low. Animals with high levels of epsilon toxin may move about without showing signs of illness until found dead or exhibiting the acute form of enterotoxaemia . Susceptible animals: The disease is prevalent in sheep and goats, with per acute cases occurring at 3-10 weeks of age , although both acute and chronic enterotoxaemia can occur in both young and adult sheep and goats. The tendency for chronic cases to occur is relatively higher in vaccinated adult goats, while acute enterotoxaemia usually occurs in unvaccinated young and adult goats. The epsilon toxin is produced as an inactive protoxin initially and activated by trypsin where in trypsin removes a 13-residue N-terminal peptide. Epsilon toxin is a potent toxin responsible for a rapidly fatal enterotoxemia in sheep. One of the main properties of epsilon toxin is the production of edema especially in brain, necrosis of brain tissue and death. The toxin is known to increase intestinal permeability, and can also cause liver damage, elevate blood pressure and cause an increase in vascular permeability. This can lead to vascular damage and edema in many organs including brain, heart, lung and kidneys. This disease is also known as lamb вЂ�overeating diseaseвЂ™ or Pulpy kidney diseases. The organism establishes in the gut and multiplies. The epsilon toxin produces a systemic toxemia. This leads to CNS lesions including opisthotonus, convulsions and 23 sudden death. Kidney lesions are also commonly associated with this form of the disease. epicardium and tissues with yellowish gelatinous exudates. LABORATORY DIAGNOSIS: It requires evaluation of clinical signs, gross and microscopic lesions, bacteriologic culturing of appropriate specimens (feces, intestinal contents) and typing of isolates. Clinical findings: Acute septicaemia characterized by sudden onset of fever (106o-107o F), profuse salivation, sub-mucosal petechiation, severe depression, death resulting in 24 hours. Animals may be found dead without any clinical signs. Localizations in subcutaneous tissue resulting in warm, painful swellings around the throat, dewlap, brisket, perineum and severe dyspnoea. In some cases in later stages animals develop pulmonary or alimentary involvement. Pasteurella can be isolated from saliva and blood. The disease in pigs is similar to that in cattle. Demonstration of toxins in the intestinal contents, feces or serum (trypsin treated or untreated, neat or mixed with antitoxins) are examined in mice(injected intravenously) or in guinea pigs(injected intra dermaly) PCR genotyping can be useful complement to other diagnostic methods. Genes for the major toxins (epsilon toxin) can be detected. Control: Formaldehyde Inactivated alumprecipitated vaccine is widely used in the country with satisfactory results. Biannual vaccination of all adult and young sheep of above 3months old should be followed in endemic areas . HAEMORRHAGIC SEPTICAEMIA (SEPTICAEMIC PASTEURELLOSIS/ BARBONE) The disease is caused by Pasteurella multocida type1 or B (6:B) and occasionally by type 4 (D) and type E (1&2) (6:E in Africa) and is characterized by per acute septicemia and high mortality rate. Epidemiology: The disease is seen in cattle, buffalo, sheep, goats, pigs, yaks, bisons, camels and horses. It occurs in Asian countries, Europe, Russia and Africa. Most affected animals are those exposed to chilly and inclement weather or exhausted by heavy work. Animals of all ages and breeds are susceptible but most susceptible age group is 6 months to 2 years. Both morbidity and mortality rates vary from 50 -100 per cent. Mortality depends on the immune status of the herd either acquired naturally or by vaccinations. After the outbreaks the causative agent persists on the tonsillar and naso-pharyngeal mucosae of carrier animals and as a commensal in healthy animals. Spread occurs by ingestion of contaminated food, carriers, clinical cases, ticks and insects. The saliva of infected animals contains large numbers of pasteurellae but the organisms do not survive on pasture for more than 24 hours. Pathogenesis: The portal of entry of infection is tonsils. The organisms multiply freely in tissues, respiratory tract, heart and gastro-intestinal tract and results in severe septicaemia. Death occurs within 24 hours from an overwhelming endotoxaemia resulting in extensive intra-vascular coagulation in tissues, petechial and ecchimotic haemorrhages on LIVESTOCK LINE, MARCH 2012 Necropsy findings: Generalized petechial haemorrhages under serosae, oedema of lungs and lymph nodes. Sub-cutaneous infiltrations of gelatinous fluid in some lesions of early pneumonia and haemorrhagic gastro-enteritis. In lungs congestion, consolidation, prominent thickening of inter lobular septa. Isolation of organisms is best from heart blood and spleen. LABORATORY DIAGNOSIS: 1. Direct Microscopic examination of blood smears or smears from exudates, tissues such as spleen, liver stained with Methylene blue or Geimsa, appearance of characteristic bipolar organisms confirms the disease. 2. Isolation and identification of the causative agent from blood or nasal swab within a few hours of death. During clinical phase, blood or nasal swabs are not reliable. 3. Long bones of carcasses are used for culturing. 4. Samples of blood are injected to mice which will die within 24-36 hours. Organisms can be isolated from mice. 5. Smears from saliva, blood and aspirated exudates from swellings reveal organisms. 6. Rapid ELISA to identify the serotype. Control: 1. Killed vaccine with oil adjuvant is highly effective when used prophylactically and also in the face of an outbreak. Solid immunity for at least 12 months is conferred. But the disadvantages are persistent subcutaneous swellings and anaphylactic shock. 2. Live vaccine of serotype B:3, 4 has been successful and is free of anaphylactic shock. It gives protection up to one year. Freeze-drying is necessary for large scale production which is very expensive. 3. Inactivated alum-precipitated vaccine is widely used in India and has given satisfactory results. Zoo-sanitary measures by treating the affected areas with disinfectants and proper disposal of dead carcasses and maintenance hygienic conditions. 24 LIVESTOCK LINE, MARCH 2012 25 LIVESTOCK LINE, MARCH 2012 26 LIVESTOCK LINE, MARCH 2012 27 LIVESTOCK LINE, MARCH 2012 28 U LIVESTOCK LINE, MARCH 2012 29 LIVESTOCK LINE, MARCH 2012 30 LIVESTOCK LINE, MARCH 2012 31 Livestock Improvement Strategies in Northern Hilly Regions of India Dibyendu Chakraborty1, A K Das2, N Kumar1 and D Kumar1 Division of Animal Genetics and Breeding, SKUAST-J, R S Pura, Jammu-181 102 U Being vast country India having different agroclimatic regions. The life-style of the people is largely influenced by the habitat. The main source of income for the people is agriculture-based. But, still there are some places in India where cultivation is not possible. At those places Livestock raring is the major source of income. Northern hilly region is one such agro-climatic region. Northern hilly region comprises of Jammu & Kashmir, Himachal Pradesh, Uttarakhand and hilly part of Uttar Pradesh. Main Livestock species of this region are cattle, buffalo, sheep, goat and horse. Beside these pigs, donkey, camel and yak are also available. Cattle Due to hilly regions the indigenous cattle breeds of this region are of small size. The cattle breeds of this region are Panwar breed (Small hill-type cattle found in foot-hills of Pilibhit district of UP); Pahari (local non-descriptive breed); Ladhakhi (Kashmir type of Indian hill cattle) (Maule, 1990). During the year 2006-07 the organized breedable population coverage was reached to 30.53% (17.58% through AI & 12.95% through natural breeding) which was only 5% at the inception of implementation of NPCBB in the state of Uttarakhand (Annual Report, 2006-07 ULDB). Crossbreed animals constitute only 6% of the total cattle population in HP (Negi, 1994). Similarly in Uttarkhand, the Central Indian Himalayas, crossbred cattle make up less than 2% of the total cattle population (Sherpa, 1997). According to breeding policy of the State, inheritance of exotic blood i.e. Jersey/Holstein is to be kept at 50% and remaining 50% inheritance will be contributed by Pahari /Hilly cattle (Dept. of Animal Husbandry HP, 2007). In J&K state the crossbreeding of cattle is going with Jersey and HF. The exotic inheritance of crossbred cattle is restricted to 50% only. Some local breeds are facing extinction due to different state livestock development policies. Pahari breed was vanishing as the department was promoting the Jersey, Holstein and Red Sindhi breeds in the state under its livestock development 1 policy (Chauhan, Mar. 24, 2009, Tribune news service). Under bull production program of NPCBB the ULDB has established one state of art Embryo Transfer Technology Laboratory at Animal Breeding Farm, Kalsi for the conservation & propagation of Red Sindhi breed of cattle. Buffalo In the mountain villages, buffalo milk contributes 98% of total milk (Singh, 1992). Livestock population trend in the Central Himalayan Uttrakhand hills of India shows that cattle population has declined by 5% while buffalo population has increased by 15% between 1978 and 1988 (Mehta, 1997). In Himachal Pradesh buffalo breeding was initiated in 1980s. Due to the lack of technical knowledge (like how in the preservation of buffalo semen) breeding was performed through natural services by locating 92 Murrah bulls in 1980 at different places in the state. Thereafter through artificial insemination facilities that had been extended to 190 extension stations (Dept. of Animal Husbandry Himachal Pradesh, http:/ /www.ahdhp.org). Upgradation of local buffalo breeds by using frozen semen of Murrah buffaloes is practiced in J&K state. Sheep This region is well known for good quality sheep breeds. Sheep breeds are extensively used for wool production. The sheep breeds of this region are Gaddi, Rampur Bushair, Bhakarwal, Poonchi, Karnah, Gurez and Changthangi (Fig.1). Kashmir Merino breed is originated from crosses of different Merino types (at first Delaine Merinos, and subsequently Rambouillet and Soviet Merinos) with predominantly migratory native sheep breeds, such as Gaddi, Bhakarwal and Poonchi. The level of inheritance in the cross-bred animals included in Kashmir Merino varies from very low to almost 100% Merino. The level of 50 to 75% exotic inheritance predominates (Handbook of Animal Husbandry, 2002). According to the National Commission on Agriculture (NCA, 1976) the breeding strategy in this region involves breeding for apparel wool through cross- Assistant Prof. , Div.- AGB, FVSc & AH, SKUAST-J, R S Pura, Jammu-181 102. (E-mail: [email protected]) 2 Associate Professor, Div.- AGB, FVSc & AH, SKUAST-J, R S Pura, Jammu-181 102 LIVESTOCK LINE, MARCH 2012 32 breeding indigenous breeds with exotic fine-wool breeds. The indigenous breeds are improved through selective breeding. Exotic breeds used for cross breeding in this region are Rambouillet and Merino. The native sheep breeds of J&K are facing extinction due to large scale introduction of Merino and Rambouillet (Nivsarkar et al., 1994). Sheep crossbreeding with Polled Dorset (Mutton breed) has remained confined to selected pockets in the Valley such as Hajan block. Corriedale breed has shown good adaptability and performance in the orchard belt of Kashmir, i.e., Shopian area. Nowadays efforts are made for crossbreeding with Garole sheep or introducing of high fecundity gene (Fec B gene) for production of twins and triplets to increase the sheep population. Goat The goat breeds Bhakarwal, Changthangi, Chegu, Gaddi, Kangan, White Himalayan are distributed in J & K, H.P., Northern Punjab, Uttarakhand and Hill districts of U.P (Fig. 2). Generally unrecognised crossing with dairy goats with Angora is adopted by HP and J & K states. No improvement programme of Changthangi, Chegu and Gaddi breeds is still initiated. Crossbreeding for pashmina fibre production in Changthangi breed in J&K with exotic breed Orenberg (Russia) is practiced (Source: Handbook of Animal Husbandry, 2002). There are over 150,000 pashima goats in Chanthang plateau in Ladakh region, which contribute to 90 per cent of pashima wool production in the country. (PTI, February 20, 2009). Ladakh has a large pool of genetic material within its indigenous goat population and produces some of the best pashmina fibre in the world. Thus, crosses with foreign breeds have been discouraged. Selective goat breeding is being conducted on government farms to select for pure white hair so as to raise the value of the pashmina and to improve pashmina production from 200 g per goat to 300 g by increasing secondary follicular hair density. Lambing and kidding occurs in February-March, the coldest months of the year, and mortality rates can be as high as 40-70%. So the optimum breeding time is from August to October so that green grass is available for milking dams in April and May and more milk will be available for newborns. Thus it will reduce kid mortality. Reduced kid mortality from 30 to 2% and increased adult survivability by changing goat breeding times on government goat farms were reported (Development of Pashmina in Ladakh, Jammu and Kashmir, 2009). Generally unrecognised crossing with dairy goats with Angora was adopted by HP and J & K. Crossbreeding for pashmina fibre LIVESTOCK LINE, MARCH 2012 production in Changthangi breed in J&K with exotic breed Orenberg (Russia) is practised. The CSK, Himachal Pradesh Agricultural University has been sanctioned a new research project worth Rs. 1.39 corer under the National Agricultural Innovations Project (NAIP) of the Indian Council of Agricultural Research (ICAR) to undertake research and developmental activities for promoting pashmina production and its efficient utilization in Himachal http:// Pradesh (February 11, 2009 www.fibre2fashion.com/). Now a days cloning of Pashmina goat is tried to increase the number of Pashmina goat in Jammu and Kashmir state (http:// www.dnaindia.com/report.asp?newsid=1232619). Horse The Spiti ponies have two strains Spiti pure and Konimare. Spiti show by HP government and Zanskari show by JK government helped in dissemination and improvement of these breeds. In Zanskari horse breeding farm at Padum Zanskar in Kargil district of Ladak selective breeding is practiced for breed improvement and conservation. Donkey, yak and double hump camel The modern domesticated asses have mainly descended from the Nubian race. The greatest contribution to animal husbandry that ass has made is the production of mules. Characterization of yak genetic resources was also considered, and a pilot study was done by the National Bureau of Animal Genetic Resources in the yak-raising areas (Pal, 2001). Doubled hump camel (Camelus bactrianus) found in Nubra valley in Ladakh area of J & K. Conclusions Parasitic diseases cause a huge loss in Animal Husbandry in Himachal Pradesh (Jithendran, 2000). Inspite of efforts in animal breed improvement, the bulk of livestock population in the Himalayan region remain local species. There is paucity of feed and fodder for the livestock, especially during winter months as a result productivity suffers (A quarterly news bulletin of SKUAST, Srinagar, January-march, 2007). So, attentions should be given to the Priority production system- Pastoral systems. Due to large introduction of exotic germplasm the indigenous sheep and goat breeds of this region are facing extinction. So, importance should be given on selective breeding and sustainable management to increase the productivity of these breeds as well as conservation. Attentions should be given in the following fields- 33 v To develop an appropriate institutional framework for the conservation, classification and utilization of animal genetic resources v Pasture management and additional feed resources v Disease control and herd management v To promote regional gene banks for specific livestock species v To support and stimulate scientific and sustained conservation work. References Domestic Animal Diversity Conservation & Sustainable Development, 2000. Handbook of Animal Husbandry. 2002. Indian Council of Agricultural Research, New Delhi. http://ahdhp.org/achievements.html http://ahdhp.org/breeding.html http://ahdhp.org/breeds_of_Sheep.html http://ahdhp.org/livestock.html http://ahdhp.org/schemes.html http://gbpihed.gov.in/envis/HTML/vol82/vol82-geeta njali.html h t t p : / / i n d i a . g o v. i n / g o v t / v i e w s c h e m e . php?schemeid=945 http://news.webindia123.com/news/Articles/India/ 20081019/1082519.html http://nird.ap.nic.in/clic/solan.html http://planningcommission.nic.in/plans/stateplan/ sdr_jandk/sdr_jkexecutive.pdf http://planningcommission.nic.in/plans/stateplan/ sdr_jandk/sdr_jkch3a.pdf http://www.dnaindia.com/report.asp? newsid=1232619 h t t p : / / w w w. i f a d . o r g / l r k m / r e g i o n / p i / I C I M O D / in_pashmina.html h t t p : / / w w w. i n d i a d a i r y. c o m / t e c h _ l i s t _ geniticsbreeding_sahiwal.html http://www.mapsofindia.com/jammu-kashmir/ economy/animal-husbandry.html h t t p : / / w w w. m e r i n e w s . c o m / c a t F u l l . j s p ? articleID=15709858 http://www.skyscrapercity.com/archive/index.php/t488675.html Jithendran, K.P. (2000). A brief profile of blood protista of domestic animals in Himachal Pradesh. Himachal Vet. J., 4: 19-21. Mehta D.S. 1997. Development experiences and options in a hill region: The case of Uttarakhand, U.P., India. Discussion Paper Series MEI 97/4. LIVESTOCK LINE, MARCH 2012 ICIMOD (International Centre for Integrated Mountain Development), Kathmandu, Nepal. NCA, New Delhi. 1976 Report of the National Commission on Agriculture, Part VII: Animal Husbandry. Govt. of India, Ministry of Agriculture and Irrigation, New Delhi. Negi G.C. 1994. Livestock development in Himachal Pradesh: Retrospect and prospect. MFS Series 7. ICIMOD (International Centre for Integrated Mountain Development), Kathmandu, Nepal. Pal, R. N. 2001. Yak husbandry in India. FAO corporate document repository. Sherpa. 1997. Proceedings and recommendations of the SHERPA seminar on вЂ�Fodder Problems in the Himalayan Region of IndiaвЂ™, held at Pashulok, Rishikesh, India, 22вЂ“23 December 1997. Singh V. 1992. Dynamics of unsustainability of mountain agriculture. Report of the MFS-ICIMOD commissioned Study in the Garhwal Himalaya, India. ICIMOD (International Centre for Integrated Mountain Development), Kathmandu, Nepal. Fig 1: Sheep breeds in the northern temperate region of India Fig. 2 Goat breeds in the northern temperate region of India (http://www.fao.org/docrep/004/X6532E/X6532E06.htm) 34 IMPORTANCE OF BIOTECHNOLOGY IN ANIMAL FEEDING Dharmendra Vyas And Ruchi Tripathi Apollo College of veterinary medicine Agra road Jaipur U Biotechnology is the application of technologies, such as recombinant DNA techniques, biochemistry, molecular and cell biology, genetics and genetic engineering, and cell fusion techniques etc. Using living organisms or its products, to manufacture industrial products including antibiotics, insulin, and interferon, to improve plants or animals, to develop microorganisms for specific uses, to identify targets for pharmaceutical development, to transform biological systems into useful processes and products or to develop organisms for specific uses. The largest impact of biotechnology on livestock production is increasing the livestock feeds through improving nutrient content as well as the digestibility of low quality feeds through use of efficient feed additives. Its attention in two areas, 01) Development of genetically modified feed ingredients in order to nutritionally enhance and improve the production capabilities. 02) Improve certain feed ingredients which have inherently low nutritional capabilities like high fiber, antinutritive factors, low protein, and deficiency of certain amino acids through the addition of feed additives Some of the limitations which the nutritionist face during feed formulation are the antinutritive factors like trypsin inhibitors, saponins, tannins, phytates, oxalates, high fiber, limitation of phosphorus content etc in feed. Developing genetically modified feed having improved nutritional values could solve these problems. Value added feed stuff are Low phytate corn, High oil corn, Low oligosaccharide soybean, Soybeans with high lysine, GM crops with improved amino acid profiles Low phytate corn: All plant feed ingredients contain natural phosphorus, which is only 30 % available, and the rest 70 % is in the form of phytate phosphorus. Grains with low phytate phosphorus and high available phosphorus were made High oil corn. This variety contains 87 % higher crude oil fat and 3.3% higher crude protein compared to typical corn. Feeding studies with high oil corn on broilers shows -Significant improvement in body weight & feed LIVESTOCK LINE, MARCH 2012 conversion. -Less abdominal fat -Better feed to egg ratio. -Egg yolks contained increased levels of linoleic acid and oleic acid. Low oligosaccharide soybean-: Soybeans contain raffinose and stachyose the oligosaccharides, which act as antinutritive factors. Genetically modified soybeans with low oligosaccharides gave an increased 3% in amino acid digestibility and 5 % increased in dry matter digestibility. Soybeans with high lysine: Increased lysine content from 3 % to 4.5 % It reduce the supplemental addition of lysine in diets. GM crops with improved amino acid profiles Great potential to decrease nitrogen excretion in poultry. Feed additives-: Adding specific nutrients to feed improves animal digestion and thereby reduces feed costs. A lot of feed additives are being currently used and new concepts are continuously developed. Enzymes Pro-biotic Pre-biotic Dietary amino acids Toxin Binders Minerals and Vitamins Bypass proteins Metabolic modifiers Enzymes-: are biological catalysts and they improve the nutrient availability from feedstuffs, lower feed costs and reduce out put of waste into the environment. 01) Microbial phytase as the result of biotechnology is an enzyme that breaks down the indigestible phytic acid (phytate) in cereals and oilseeds and releases digestible phosphorus. This reduces the use of expensive supplemental inorganic phosphorus like dicalcium phosphate. Phytase also releases minerals (Ca, Mg, Zn and K), amino acids and proteins, which are complied with the Phytate molecule. 02) Concentrate feed pellets could be made by incorporating cellulase,pectinace and xylanase with straw, bagasse and other agricultural waste. Probiotics-: Probiotics can help to build up the beneficial bacteria in the intestine and competitively exclude the pathogenic bacteria. These bacteria also release enzymes, which help in the digestion of feed. The common organisms in probiotic products are Aspergillus oryzae, Lactobacillus acidophilus, L. bulgaricus, L.plantarium, Bifidobacterium bifidium, Streptococcus lactis and Saccharomyces cerevisiae. 35 Can be administered through water or incorporated in the feed. Useful in the early stages of chick growth since the gut of the newly hatched chick is sterile Helps to build up beneficial bacteria much faster than the normal course. Prebiotics-: Some of the prebiotics, which are currently used in animal feed, are Mannanoligosaccharides (MOS), fructo-oligosaccharide and mixed oligo-dextran. Mannan-oligosaccharides are mainly obtained from cell walls of yeasts. Other sources of MOS are copra or palm kernel meal. The advantages of prebiotics are that it can stand high palletizing temperatures in the feed and also have a long shelf life. Amino acids-: Essential amino acids are added as supplement to the feed to get a balanced amino acid profile. The new trend is to formulate diets on digestible amino acid levels thereby reducing the requirement of protein. Lysine is produced by microbial fermentation and methionine is chemically synthesized to add as supplement . Genetically enhanced micro-organisms are being used to produce threonine and tryptophan on a commercial basis. Using all these amino acids it is possible to lower dietary crude protein level by 2 вЂ“ 3 %, which is a substantial saving for the farmer.. Toxin Binders Present day methods are generally use of organic acids and their salts like propionic acid or adsorbents like bentonites, zeolites, hydroxyl aluminosilicates. In the future, biotechnology based products like microbes, herbal extracts or esterified glucomannan could be used. Aqua extracts of garlic, onion, turmeric, neem have been shown to exert antifungal activity or inhibit aflatoxin production. Minerals and Vitamins-: The absorption and availability of inorganic trace minerals varies depending upon the nature of the minerals (sulphate, oxide or carbonate), their solubility, ionization etc. Trace minerals are now being attached to oligopeptides to make them more bioavailable. Commercial preparations of proteinated selenium and chromium are used in poultry. In the case of vitamins due to varying availability and stability of vitamins in ingredients supplemental vitamins are incorporated in diets. These vitamins are much more stable than naturally occurring forms. Metabolic modifiers-: Metabolic modifiers are a group of compounds that modify animal metabolism in specific and directed ways. They have the overall effect of Improving productive efficiency (weight gain LIVESTOCK LINE, MARCH 2012 or milk yield per feed unit) Improving carcass composition (lean:fat ratio) in growing animals, Increasing milk yield in lactating animals and Decreasing animal waste per production unit. Two classes of compounds have received major focus, 1. Somatotropins (STS) and 2. Adrenergic agonists. Commercially these compounds are produced by the use of recombinant DNA technology to selectively produce specific components for a species The most common somatotropin is the bovine somatotropin (bST) which is administered to dairy cows. Similarly somatotropins have also been used in pigs which resulted in greater nutrient use. Administering of Гў adrenergic agonist components lead to improved feed conversion ratio, daily weight gain and carcass leanness. These components induce changes in endocrine and cellular mechanism. Merits and demerits-: Increase the digestibility of low quality feeds through use of efficient feed additives. Developing genetically modified grains with improved nutritional values avoid limitations of antinutritive factors like trypsin inhibitors, saponins, tannins, phytates, oxalates and high fiber content and limitation of phosphorus content during feed formulation Less phosphorus would be thrown in the litter and manure, which would lead to the control of eutrophication. If phytase enzyme used in poultry, aquaculture integrated farmers are benefiting in controlling the algal bloom due to the reduction in ground water phosphorus levels. Also it reduces the use of expensive supplemental inorganic phosphorus such as dicalcium phosphate. Reduce the cost of animal feed. Increased milk yields, improved productive efficiency (milk/feed) and decreased animal waste. The inclusion of genetically modified feedstuffs in animal feed could also pose certain risks. GM plants are produced by transferring foreign genes of particular characteristics into feed grain crops. For example introducing antibiotic resistant marker genes may render common infectious diseases untreatable or certain proteins may cause allergic reactions to animals and humans. Hence proper lab, field assessments as well as health assessments have to be made before release of such plants for commercial cultivation. Due to enzymes attract import duty, making their usage expensive. There are very few companies producing enzymes due to lack of technology. 36 Modifications of Polymerase Chain Reaction and their Applications: An Overview Arunkumar Patel1, Satish Kumar*, Pradip Ranaware1, Manish Kumar2 INTRODUCTION U Polymerase Chain Reaction (PCR) is in vitro an enzymatic amplification of specific DNA sequences in exponential manners. Kary Mullis (1983) is credited for the invention of PCR assay. In general PCR method require suitable DNA polymerase which is able to withstand the high temperatures of >90В°C (>195В°F) required for separation of the two DNA strands in the DNA double helix after each replication cycle. The DNA polymerases initially employed for this was unable to withstand such high temperatures. So the early procedures for DNA replication were very inefficient, time consuming, and required large amounts of DNA polymerase and continual handling throughout the process. Therefore in each cycle it needs incorporation of fresh polymerase which it tedious in addition to time consuming. The discovery of Taq polymerase (a DNA polymerase purified from the thermophilic bacterium, Thermus aquaticus, which naturally occurs in hot spring environments) paved the way for dramatic improvements and thus wide acceptance of the PCR method. The DNA polymerase isolated from T. aquaticus is stable at high temperatures remaining active even after DNA denaturation, thus obviating the need to add new DNA polymerase after each cycle. This allowed an automated thermocycler-based process for DNA amplification. MODIFICATIONS OF PCR On basis of need various modifications were employed in basic PCR methods from time to time which can be briefly describe as follow. I. Basic modifications Often only a small modification needs to be made to the standard PCR protocol to achieve a desired goal. 1. Competitive PCRs This is a method used for quantifying DNA using realtime PCR. A competitor internal standard is coamplified with the target DNA and the target is quantified from the melting curves of the target and the competitor. 2. Long вЂ“PCR (LA-PCR) It is used for the amplification of long target DNA sequences. The key to LA-PCR is an enzyme, a thermostable DNA polymerase, which possesses 3вЂ™ to 5вЂ™ exonuclease activity, or proofreading activity. The efficiency drastically declines when incorrect bases are incorporated. The 3вЂ™to5вЂ™ exonuclease activity removes these misincorporated bases and makes the further reaction proceed smoothly. Therefore, the amplification of long DNA fragment can be achieved. Enzymes like pfu were employed for such PCR. 3. Multiplex PCR Multiplex PCR is the term used when more the one pair of primers is used in a PCR. The goal of Multiplex PCR is to amplify several segment of target DNA simultaneously. This PCR technique is used for genetic screening, micro-satellite analysis and other applications where it is necessary to amplify several products in a single reaction. This technique often requires extensive optimization because having multiple primers pairs in a single reaction increases the likelihood of primer-dimers and other non-specific product that may interfere with the amplification of specific products. In addition, the concentration of individual primer pair often needs to be amplified with differing efficiencies and multiple primer pair can compete with each other in reaction. When two pair of primers is used to amplify two segment of target DNA simultaneously it is called as DUPLEX PCR (e.g., HA and NA gene segment of orthomyxoviruses). When three pair of primers is used to amplify three segment of target DNA simultaneously it is called as TRIPLEX PCR (e.g., H, N, P gene segment of PPR virus) Advantages: Multiplex PCR has the potential to produce considerable savings of time and effort within the laboratory without compromising test utility. Since its introduction, multiplex PCR has been successfully applied in many areas of nucleic acid diagnostics, including gene deletion analysis *Corresponding author, M.V.Sc., Division of Livestock Products Technology, Indian Veterinary Research Institute, Izatnagar, Bareilly, UP -243 122, India., Phone: +91-9457917989 Email: [email protected] 1- Ph.D. Scholar, Division of Virology, Indian Veterinary Research Institute, Izatnagar, Bareilly, UP -243 122, India. 2- Ph.D. Scholar, Division of Physiology and Climatology, Indian Veterinary Research Institute, Izatnagar, Bareilly, UP -243122,India. LIVESTOCK LINE, MARCH 2012 37 mutation and polymorphism analysis quantitative analysis, and RNA detection. Disadvantages: The optimization of multiplex PCRs can pose several difficulties, including poor sensitivity or specificity and/or preferential amplification of certain specific targets. The presence of more than one primer pair in the multiplex PCR increases the chance of obtaining spurious amplification products, primarily because of the formation of primer dimers. These nonspecific products may be amplified more efficiently than the desired target, consuming reaction components and producing impaired rates of annealing and extension. Thus, the optimization of multiplex PCR should aim to minimize or reduce such nonspecific interactions. 4. Multiplex Ligation-dependent Amplification (MLPA) Probe It permits multiple targets amplification with only a single primer pair, thus avoiding the resolution limitations of multiplex PCR. 5. Multiplex RTвЂ“PCR: (also known as Relative RT-PCR) It is commonly used for the semi-quantitative analysis of gene expression levels. Typically, multiplex RTPCR is performed to determine the changes in expression levels of gene in a series of tissue types throughout stages of development or cellular differentiation, or after specific experimental treatment. Multiplex RT- PCR is also commonly used to examine the expression patterns of a series of related genes and to look at various regions of a large message for mutation analysis. 6. Nested PCR Nested PCR is used to increases the specificity of DNA amplification, by reducing background due to non-specific amplification of DNA. Two sets of primers are being used in two successive PCRs. In the first reaction, one pair of primers is used to generate DNA products, which besides the intended target, may still consist of non-specifically amplified DNA fragments. The product(s) are then used in a second PCR with a set of primers whose binding sites is fall inside the amplicon of first primer set. Nested PCR is often more successful in specifically amplifying long DNA fragments than conventional PCR and so it has greater specificity. Nested PCR reduce the contamination in product due to amplification of unexpected primer binding sites. When either primer of primary set were employed with corresponding new primer for secondary amplicon is known as semi- LIVESTOCK LINE, MARCH 2012 nested PCR and when cDNA that has been reverse transcribed from RNA before nested PCR then it is known as Nested RT-PCR. 7. Quantitative PCR (Q-PCR) It is used to measure the quantity of a PCR product (preferably real-time). It is the method of choice to quantitatively measure starting amounts of DNA, cDNA or RNA. Q-PCR is commonly used to determine whether a DNA sequence is present in a sample and the number of its copies in the sample. The method with currently the highest level of accuracy is Quantitative real-time PCR. It is often confusingly known as RT-PCR (Real Time PCR) or RQ-PCR. QRT-PCR or RTQ-PCR is more appropriate contractions. RT-PCR commonly refers to reverse transcription PCR which is often used in conjunction with Q-PCR. The commonly used methods for QPCR are use of fluorescent dyes, such as SYBR Green and fluorophore-containing DNA probes, such as TaqMan Probe, Molecular Beacons, Scorpion Probes etc. Type of real-time PCR includes: a. Real-time RT вЂ“PCR: This term refers to a realtime PCR that is initiated with cDNA that has been reverse transcribed from RNA. b. Real-time RT-asymmetric PCR: This term refer to an asymmetric PCR that is initiated with cDNA that has been reverse transcribed from RNA. c. Real-time RT- semi-nested PCR: This term refer to a semi-nested PCR that is initiated with cDNA that has been reverse transcribed from RNA. d. Real-time RT-nested multiplex PCR: This term refer to a nested PCR that is initiated with cDNA that has been reverse transcribed from RNA and includes multiple primer pairs at one or both of the consecutive PCRs. II. Pretreatments and extensions modifications 1. Colony PCR Colony PCR is useful to differentiate the recombinant colonies from non recombinant colonies. Primers for the specific sequences (antibiotic resistance or primers flanking a cloned region, specific gene primer) should be used when preparing reaction mixture and it allow rapid detection of transformants containing the desired sequence. 2. Hairpin PCR It is a method of error-free DNA amplification for mutation detection. In this method sequence was first 38 convert hairpin like structure. On amplification true mutations will maintain the hairpin structure while PCR error will disrupt the hairpin structure. 3. Hot-start PCR This is a technique that reduces non-specific amplification during the initial set up stages of the PCR. The technique may be performed manually by heating the reaction components to the melting temperature (e.g., 95ГљC) before adding the polymerase or specialized enzyme systems have been developed that inhibit the polymeraseвЂ™s activity at ambient temperature, either by the binding of an antibody or by the presence of covalently bound inhibitors that only dissociate after a hightemperature activation step. Hot-start/cold-finish PCR is achieved with new hybrid polymerases that are inactive at ambient temperature and are instantly activated at elongation temperature. Now a dayвЂ™s various real time PCR pre mix available with follow hot start procedures. 4. In-Situ PCR (IS-PCR) IS-PCR is performed on fixed cells. DNA or RNA is immobilized in their sub-cellular locations. In-Situ Hybridization (ISH) or IS-PCR has proven to be a very important molecular tool in diagnostic and research and has significantly advanced the study of gene structure and expression at the level of individual cells .This technique has result in an increased understanding of infectious and neoplastic diseases. More recently, an intracellular reverse transcription step to generate complimentary DNA from mRNA template prior to in-situ PCR has been for detection of low copy mRNA sequences. This modification of in-situ PCR has been termed as вЂњinsitu RT-PCRвЂќ or вЂњRT in-situ PCRвЂќ or вЂњin-situ cDNA PCRвЂќ. Utility of immuno-histochemistry, in-Situ Hybridization and in-situ PCR amplification in the surgical and cytopathology of viral infection has been reported. .Main advantages are its low background, high specificity, fast assay with shorter turn-around time and no need of radioactive chemicals. 5. Ligation-mediated PCR 6. This method uses small DNA linkers ligated to the DNA of interest and multiple primers annealing to the DNA linkers; it has been used for DNA sequencing, genome walking, and DNA footprinting. 7. Methylation-specific PCR (MSP) The MSP method was used to detect methylation of CpG islands in genomic DNA. DNA is first treated with sodium bisulfite, which converts unmethylated LIVESTOCK LINE, MARCH 2012 cytosine bases to uracil, which is recognized by PCR primers as thymine. Two PCRs are then carried out on the modified DNA, using primer sets identical except at any CpG islands within the primer sequences. At these points, one primer set recognizes DNA with cytosines to amplify methylated DNA, and one set recognizes DNA with uracil or thymine to amplify unmethylated DNA. MSP using qPCR can also be performed to obtain quantitative rather than qualitative information about methylation. 8. RT-PCR (Reverse Transcription PCR) RT-PCR is a method used to amplify, isolate or identify a known sequence from a cellular or tissue RNA. The PCR is preceded by a reaction using reverse transcriptase to convert RNA to cDNA. RTPCR is widely used in expression profiling, to determine the expression of a gene or to identify the sequence of an RNA transcript, including transcription start and termination sites and, if the genomic DNA sequence of a gene is known, to map the location of exons and introns in the gene. The 5' end of a gene (corresponding to the transcription start site) is typically identified by an RT-PCR method, named RACE-PCR, short for Rapid Amplification of cDNA Ends. AMV-RT and Mo-MLV-RT are commonly used enzymes in RT вЂ“PCR. 9. Touchdown PCR It is a variant of PCR in which degenerate primers are used to reduce nonspecific background by gradually lowering the annealing temperature as PCR cycling progresses. The annealing temperature at the initial cycles is usually a few degrees (3-5ГљC) above the Tm of the primers used, while at the later cycles, it is a few degrees (3-5ГљC) below the primer Tm. The higher temperatures give greater specificity for primer binding, and the lower temperatures permit more efficient amplification from the specific products formed during the initial cycle. III. Primer modifications 1. Alu вЂ“PCR This PCR is performed using the Alu primers designed to have recognitions sequences of Alu restriction enzyme. 2. Asymmetric PCR A PCR in which the predominant product is a single stranded DNA, as result of unequal primer concentrations. It finds use in some types of sequencing and hybridization probing where having only one of the two complementary strands is 39 required. PCR is carried out as usual, but with a great excess of the primers for the chosen strand. Due to the slow (arithmetic) amplification later in the reaction after the limiting primer has been used up, extra cycles of PCR are required. 3. LATE-PCR It is modification of asymmetric PCR. LATE stands for Linear-After-The-Exponential, LATE-PCR uses a limiting primer with a higher melting temperature (Tm) than the excess primer to maintain reaction efficiency as the limiting primer concentration decreasesmid-reaction.This type of PCR has been used for the detection of a target gene of Adenovirus. 4. Allele-specific PCR This is a selective PCR amplification of one of the alleles to detect single Nucleotide Polymorphism (SNP). Selective amplification is usually achieved by designing a primer such that the primer will match/ mismatch one of the alleles at 3вЂ™-end of the primer. This diagnostic or cloning technique that requires prior knowledge of a DNA sequence, including differences between alleles, and uses primers whose 3' ends encompass the SNP. PCR amplification under stringent conditions is much less efficient in the presence of a mismatch between template and primer, so successful amplification with an SNPspecific primer signals presence of the specific SNP in a sequence. 8. Degenerate PCR Degenerate PCR is in most respect identical to ordinary PCR, but with one major difference i.e. instead of using specific PCR primers with a given sequence, mixed PCR primers will be used .That is, вЂњwobbleвЂќ are inserted into the primers in case if the exact sequence of the gene is not known so that there will be more than one possibility for exact amplification. Degenerate PCR has proven to be a very powerful tool to find вЂњnewвЂќ genes or gene familiesвЂ™. To design primer conserved and variable parts is studied by alignment of various sequences of related proteins. 9. Differential Display PCR (DD-PCR): DD-PCR is used for cloning purpose; it combines the comparative analysis of several samples with the sensitivity of PCR. Recent studies shows that by modifying the primer design, sampling of differentially expressed genes can be greatly enhanced and relevant genes can be isolated. 10. Miniprimer PCR Assembly PCR is the artificial synthesis of long DNA sequences by performing PCR on a pool of long oligonucleotides with short overlapping segments. The oligonucleotides alternate between sense and antisense directions and the overlapping segments determine the order of the PCR fragments thereby selectively producing the final long DNA product. Miniprimer PCR uses a novel thermostable polymerase (S-Tbr) that can extend from short primers (вЂњsmalligosвЂќ) as short as 9 or 10 nucleotides, instead of the approximately 20 nucleotides required by other polymerase like Taq. This method permits PCR targeting smaller primer binding regions, and is particularly useful to amplify unknown, but conserved, DNA sequences, such as the 16S (or eukaryotic 18S) rRNA gene. 16S rRNA miniprimer PCR was used to characterize a microbial mat community growing in an extreme environment. Miniprimer PCR may reveal new dimensions of microbial diversity. By enlarging the вЂњsequence spaceвЂќ that may be queried by PCR primers, this technique may enable novel PCR strategies that are not possible within the limits of primer design imposed by Taq and other commonly used enzymes. 6. Box вЂ“PCR 11. Inverse PCR Box elements are repetitive sequences elements in bacterial genome such as streptococcus genome. Single PCRs targeting to the repeats can be used to fingerprint bacterial species. It is a type of standard PCR that is used to amplify the segment of DNA that lies between two inward вЂ“ pointing primers. Inverse PCR (also known as inverted or inside out) is used to amplify and clone unknown DNA that flanks one end of known DNA sequence and for which no primers are available. The technique involves digestion by a restriction enzyme of a preparation of DNA containing the known sequences and its flanking region. The individual restriction fragments are converted into circles by 5. Assembly PCR or Polymerase Cycling Assembly (PCA) 7. Consensus вЂ“PCR This PCR is carried out with flanking primer to amplify repeat regions from a number of species. In this case, degenerate /consensus primers may be used for amplified the flanking sequences. LIVESTOCK LINE, MARCH 2012 40 intermolecular ligation and circularized DNA is then used as template in PCR. The unknown sequence is amplified by two primers that bind specifically to the known sequence and point in opposite direction. The bacteriophage T4 DNA polymerase was also initially used in PCR. It has a higher fidelity of replication than the Klenow fragment, but is also destroyed by heat. 12. PCR-RFLP 3. Taq polymerase Restriction Fragment Length Polymorphism (RFLP) is a technique in which organism may be differentiated by analysis of patterns derived from cleavage of their DNA by a set of restriction enzyme (RE). If two organisms differ in the distance between sites of cleavage of a particular restriction endonuclease, the length of the fragment produced will differ when DNA is digested with a RE. The similarity of the patters generated can be used to differentiate species (and even strains) from one another. By designing primers that will introduce or destroy a restriction site for one of the alleles, the PCR product for SNP alleles can be distinguished by restriction fragment length analysis. The DNA polymerase from Thermus aquaticus, was the first thermostable polymerase used in PCR, and is still the one most commonly used. 13. Overlap-extension PCR It is a genetic engineering technique allowing the construction of a DNA sequence with an alteration inserted beyond the limit of the longest practical primer length. 14. Vectorette PCR Vectorette PCR is a method that enables the amplification of specific DNA fragment in situation where the sequence of only primer is known. Thus it extends the application of PCR to stretches of DNA where the sequence information is only available at one end. IV. Post PCR modifications PCR-ELISA The PCR product are labeled (digoxigenin) during amplification. This labeled amplicon is immobilize in immuno-well plate. Normal ELISA is then employed to quantitate PCR product. V. Modification in DNA Polymerases There are several DNA polymerases that are used in PCR viz. 1. Klenow fragment The Klenow fragment, derived from the original DNA Polymerase-I from E. coli, was the first enzyme used in PCR. Because of its lack of stability at high temperature, it needs be replenished during each cycle, and therefore is not commonly used in PCR. 2. Bacteriophage T4 DNA polymerase LIVESTOCK LINE, MARCH 2012 4. Stoffel fragment The Stoffel fragment is made from a truncated gene for Taq polymerase and expressed in E. coli. It is lacking 5'-3' exonuclease activity, and may be able to amplify longer targets than the native enzyme. 5. Faststart polymerase Faststart polymerase is a variant of Taq polymerase that requires strong heat activation, thereby avoiding non-specific amplification due to polymerase activity at low temperature. Currently using in most of real time PCR. 6. Pfu DNA polymerase Enzyme Pfu DNA polymerase, isolated from the Pyrococcus furiosus, has proofreading activity, and a 5-fold decrease in the error rate of replication compared to Taq. Since errors increase as PCR progresses, Pfu is the preferred polymerase when products are to be individually cloned for sequencing or expression. 7. Vent polymerase Vent polymerase is an extremely thermostable polymerase isolated from Thermococcus litoralis. 8. Tth polymerase Tth polymerase is a thermostable polymerase from Thermus thermophilus. It has reverse transcriptase activity in the presence of Mn2+ions, allowing PCR amplification from RNA targets. VI. Other modifications 1. Amplified Fragment Length Polymorphisms (ALFP) The use of randomly amplified polymorphic DNA (RAPD) markers in systematic studies has been reviewed. The use of randomly amplified polymorphic DNA (RAPD) markers in systematic levels of polymorphism and their low cost compared to other techniques, such as allozymes and Restricrion Fragment length polymorphism (RFPL). Two new marker methodologies appear to be supplanting RAPD analysis (AFLPs and simple sequence 41 Repeats).Whilst the RAPD technique is fairly simple, both AFLP and SSR protocols are technically demanding. 2. Forensic PCR Forensic PCR is a PCR which was normally employed in various vetro-legal cases to find the source of suspected samples. The VNTR (variable and frequently observed tandem repeats in human individual Genome) locus is amplified by PCR to compare DNA samples from different sources. 3. Helicase-dependent amplification This technique is similar to traditional PCR, but uses a constant temperature rather than cycling through denaturation and annealing/extension cycles. DNA Helicase, an enzyme that unwinds DNA, is used in place of thermal denaturation, it is very well exploited in LAMP system. 4. RACE-PCR Rapid Amplification of cDNA Ends PCR (RACE-PCR) is used to obtain the 3вЂ™ end of a cDNA; it requires some sequence information internal to the mRNA under study. The sequence information obtain from technique can be utilized to obtained full length cDNA clones using the 5вЂ™ RACE technique. 5. Solid Phase PCR It encompasses multiple meanings, including colony Amplification (where PCR colonies are derived in a gel matrix, for example), вЂ�Bridge PCRвЂ™ (the only primers present are covalently linked to solid support surface), conventional Solid Phase PCR (where Asymmetric PCR is applied in the presence of solid support bearing primer with sequence matching one of the aqueous primers) and Enhanced Solid Phase PCR (where conventional Solid Phase PCR can be improved by employing high Tm solid support primer with application of a thermal вЂ�stepвЂ™ to favor solid support priming). 6. Universal Fast Walking This method allows genome walking and genetic fingerprinting using a more specific вЂ�two-sidedвЂ™ PCR than conventional вЂ�one-sidedвЂ™ approaches (using only one gene-specific primer and one general primer - which can lead to artefactual вЂ�noiseвЂ™) by virtue of a mechanism involving lariat structure formation. Streamlined derivatives of UFW are LaNe RAGE(lariat-dependent nested PCR for rapid amplification of genomic DNA ends), 5вЂ™RACE LaNe and 3вЂ™RACE LaNe. LIVESTOCK LINE, MARCH 2012 APPLICATIONS OF PCR I. Medical applications PCR has been applied to a large number of medical procedures: 1. Genetic Testing -The first application of PCR was for genetic testing, where a sample of DNA is analyzed for the presence of genetic disease mutations. Prospective parents can be tested for being genetic carriers, or their children might be tested for actually being affected by a disease. DNA samples for prenatal testing can be obtained by amniocentesis, chorionic villus sampling, or even by the analysis of rare fetal cells circulating in the motherвЂ™s bloodstream. PCR analysis is also essential to Preimplantation genetic diagnosis, where individual cells of a developing embryo are tested for mutations. 2. Sensitive Test For Tissue Typing- PCR can also be used as part of a sensitive test for tissue typing, vital to organ transplantation. As of 2008, there is even a proposal to replace the traditional antibody-based tests for blood type with PCR-based tests.[2] 3. Oncogenes: Many forms of cancer involve alterations to oncogenes. By using PCR-based tests to study these mutations, therapy regimens can sometimes be individually customized to a patient. II. Infectious disease applications Characterization and detection of infectious disease organisms have been revolutionized by PCR: 1. Human Immunodeficiency Virus: The Human Immunodeficiency Virus (or HIV), responsible for AIDS, is a difficult target to find and eradicate. The earliest tests for infection relied on the presence of antibodies to the virus circulating in the bloodstream. However, antibodies donвЂ™t appear until many weeks after infection, maternal antibodies mask the infection of a newborn, and therapeutic agents to fight the infection donвЂ™t affect the antibodies. PCR tests have been developed that can detect as little as one viral genome among the DNA of over 50,000 host cells.[3] Infections can be detected earlier, donated blood can be screened directly for the virus, newborns can be immediately tested for infection, and the effects of antiviral treatments can be quantified. 2. Detection of Disease Organisms: Some disease organisms, such as that for Tuberculosis, are difficult to sample from patients and slow to be grown in the laboratory. PCR-based tests have 42 allowed detection of small numbers of disease organisms (both live and dead), in convenient samples. Detailed genetic analysis can also be used to detect antibiotic resistance, allowing immediate and effective therapy. The effects of therapy can also be immediately evaluated. 3. Monitoring Spread of A Disease: The spread of a disease organism through populations of domestic or wild animals can be monitored by PCR testing. In many cases, the appearance of new virulent sub-types can be detected and monitored. The sub-types of an organism that were responsible for earlier epidemics can also be determined by PCR analysis. III. Forensic applications The development of PCR-based genetic (or DNA) fingerprinting protocols has seen widespread application in forensics: 1.Genetic fingerprinting: In its most discriminating form, Genetic fingerprinting can uniquely discriminate any one person from the entire population of the world. Minute samples of DNA can be isolated from a crime scene, and compared to that from suspects, or from a DNA database of earlier evidence or convicts. Simpler versions of these tests are often used to rapidly rule out suspects during a criminal investigation. Evidence from decades-old crimes can be tested, confirming or exonerating the people originally convicted. 2. Parental testing: Less discriminating forms of DNA fingerprinting can help in Parental testing, where an individual is matched with their close relatives. DNA from unidentified human remains can be tested, and compared with that from possible parents, siblings, or children. Similar testing can be used to confirm the biological parents of an adopted (or kidnapped) child. The actual biological father of a newborn can also be confirmed (or ruled out). IV. Research applications PCR has been applied to many areas of research in molecular genetics: 1. Rapid Production of DNA: PCR allows rapid production of short pieces of DNA, even when nothing more than the sequence of the two primers is known. This ability of PCR augments many methods, such as generating hybridization probes for Southern or northern blot hybridization. PCR supplies these techniques with large amounts of pure DNA, sometimes as a single strand, enabling analysis even from very small amounts of starting material. LIVESTOCK LINE, MARCH 2012 2. Extract Segments From A Completely Unknown Genome: The task of DNA sequencing can also be assisted by PCR. Known segments of DNA can easily be produced from a patient with a genetic disease mutation. Modifications to the amplification technique can extract segments from a completely unknown genome, or can generate just a single strand of an area of interest. 3. DNA Cloning: PCR has numerous applications to the more traditional process of DNA cloning. It can extract segments for insertion into a vector from a larger genome, which may be only available in small quantities. Using a single set of вЂ�vector primersвЂ™, it can also analyze or extract fragments that have already been inserted into vectors. Some alterations to the PCR protocol can generate mutations (general or site-directed) of an inserted fragment. 4. Human Genome Project: A Sequence-tagged site is a process where PCR is used as an indicator that a particular segment of a genome is present in a particular clone. The Human Genome Project found this application vital to mapping the cosmid clones they were sequencing, and to coordinating the results from different laboratories. 5. Phylogenic Analysis: An exciting application of PCR is the phylogenic analysis of DNA from ancient sources, such as that found in the recovered bones of Neanderthals, or from frozen tissues of Mammoths. In some cases the highly degraded DNA from these sources might be reassembled during the early stages of amplification. 6. Gene Expression: A common application of PCR is the study of patterns of gene expression. Tissues (or even individual cells) can be analyzed at different stages to see which genes have become active, or which have been switched off. This application can also use Q-PCR to quantitate the actual levels of expression 7. Genetic Mapping: The ability of PCR to simultaneously amplify several loci from individual sperm has greatly enhanced the more traditional task of genetic mapping by studying chromosomal crossovers after meiosis. Rare crossover events between very close loci have been directly observed by analyzing thousands of individual sperms. Similarly, unusual deletions, insertions, translocations, or inversions can be analyzed, all without having to wait (or pay for) the long and laborious processes of fertilization, embryogenesis, etc. 43 V. Others PCR is also important in answering basic scientific questions. In the field of evolutionary biology, PCR has been used to establish relationships among species. In anthropology, it has used to understand ancient human migration patterns. In archaeology, it has been used to help identify ancient human remains. Paleontologists have used PCR to amplify DNA from extinct insects preserved in amber for 20 million years. The Human Genome Project, which had a goal of determining the sequence of the 3 billion base pairs in the human genome, relied heavily on PCR. The genes responsible for a variety of human diseases have been identified using PCR. For example, a PCR technique called multiplex PCR identifies a mutation in a gene in boys suffering from Duchenne muscular dystrophy. PCR can also be used to search for DNA from foreign organisms such as viruses or bacteria. CONCLUSION Polymerase Chain reaction is one of the most important molecular diagnostic tools. It is used in all the fields of biology as a diagnostic because of its high sensitivity and specificity. Theoretical consideration and practical applications indicate that PCR and RT-PCR assay system share several advantage over other quantitative molecular methodologies, thus suggesting that these technique are the methods of choice for the absolute quantitation of viral nucleic acid. The PCR is very promising to elucidate the etiological agent of which is present in too small numbers to be detected by traditional techniques, agents difficult or impossible to cultivate and in making distinction between infection and rejection in transplant recipients. Thus the advent of nucleic acid amplification techniques for the clinical laboratory provides not only new diagnostic opportunities but new challenges as well. REFERENCES: 1. Bell J. The polymerase chain reaction. Immunol. Today 1989; 10:351-4. 2. Boehnke M et.al. вЂњFine-structure genetic mapping of human chromosomes using the polymerase chain reaction on single sperm.вЂќ Am. J. Hum. Genet. Vol. 45(1) pp. 21-32 (1989). 3. Erlich HA, Gelfand DH, Saiki RK. Specific DNA amplification. Nature 1988; 331:461-2. 4. Frohman MA, Dush MK, Martin GR. Rapid production of fulllength cDNAs from rare transcripts: amplification using a single gene-specific LIVESTOCK LINE, MARCH 2012 oligonucleotide primer. Proc. Natl. Acad. Sci. USA 1988; 85:8998-9002. 5. Giulietti A,Overbergh L , Valckx D: An overview of real ime quantitation PCR: application to quantify cytokine gene expression. Methods 2001;25: 386401 6. Kwok S et.al. вЂњIdentification of HIV sequences by using in vitro enzymatic amplification and oligomer cleavage detection.вЂќ J. Virol. Vol. 61(5) pp. 1690-4 (1987). 7. Kwok S and Higuchi R. Avoiding false positives with PCR. Nature 1989; 339:237-8. 8. Maitland N. Report from 1st National Symposium on the Polymerase Chain Reaction, 9 May 1989, London. 9. Marx JL. Multiplying genes by leaps and bounds. Science 1988; 240: 1408-10. 10. Quill E вЂњBlood-Matching Goes GeneticвЂќ Science Magazine (14 March 2008) pp. 1478-1479. 11. Ririe KM; Rasmussen RP and Wittwer CT: product differentiation by analysis of DNA melting curve during polymerase chain reaction. Anal. Biochem. 1977;245:154-6 12. Saiki RK et.al., вЂњEnzymatic Amplification of Гўglobin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell AnemiaвЂќ Science vol. 230 pp. 1350-54 (1985). 13. Saiki RK, Gelfand DH, Stoffel S, et.al, 1988.. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 1988; 239:487-91. 14. Schochetman G, Ou C-Y, Jones WK. 1988. Polymerase chain reaction. J. Infect. Dis. 1988; 158:1154-7. 15. Triglia T, Peterson MG, Kemp DJ. 1988. A procedure for in vitro amplification of DNA segments that lie outside the boundaries of known sequences. Nucleic Acids Res. 1988; 16:8186. 16. White TJ, Arnheim N, Erlich HA. 1989. The polymerase chain reaction. Trends Genet. 1989; 5:185-9. 17. Wittwer C. 2001.Rapid cycle real time PCR: Method s and application, In Rapid cycle real time PCR (eds Meuer et.al.) 2001, pp1-11 Springer ,Heidelberg, Germany 2. Niesters H.G: Quantitation of viral load using real-time amplification. Methods 2001, 25:419-2. 44 LIVESTOCK LINE, MARCH 2012 45 LIVESTOCK LINE, MARCH 2012 46 LIVESTOCK LINE, MARCH 2012 47 LIVESTOCK LINE, MARCH 2012 48 Impact of nanotechnology in veterinary science вЂ“ a review S. Ganguly1*, A. Prasad2, I. Paul3, D. Seth4, S.K. Mukhopadhayay5 U will revolutionize animal health and help to boost up AbstractNanotechnology refers to the use of very tiny (nano-scale) materials in a range of novel ways. вЂ�NanoвЂ™ means tiny and nano-particles are tiny particles, more than 8000 times smaller than a human hair. The properties of nano-particles make them suitable for a range of environmental applications, both in terms of improving existing environmental problems or by anticipating and preventing future environmental problems. Some of the greatest potential uses or application of nanotechnology in the environment are as biosensors and in the sectors of treatment, agriculture, veterinary science, fisheries, bioremediation and for green nanotech manufacturing and engineering. The present article has been constructed considering the tremendous potential and application of nanoscience and nanotechnology in the concerned fields.Keywords: nanotechnology; nano-particles; veterinary science; nanoscience.IntroductionVeterinary health care is a highly responsible and growing concern not only for pet owners, but also for our nation and government. With an ever increasing pet population throughout the globe, along with higher costs for medications and veterinary care, the need for new solutions is urgent. At this period of time the main objectives of Veterinary Medicine is to excel according to the accepted standards of scientific excellence in the creation of new knowledge and its translation into improved health for the other species with which we share our world, to create more effective veterinary services and products and to strengthen the veterinary education system.Nanotechnology has a tremendous potential to revolutionize agriculture and livestock sector. It can provide new tools for molecular and cellular biology, biotechnology, veterinary physiology, animal genetics, reproduction etc. which will allow researchers to handle biological materials such as DNA, proteins or cells in minute quantities usually nano-liters or pico-liters. Nanotechnology tools like micro-fluidics, nano-materials, bio-analytical nano-sensors, etc. has the potential to solve many more puzzles related to animal health, production, reproduction and prevention and treatment of diseases. It is reasonable to presume that in the upcoming yearвЂ™s nanotechnology research will reform the science and technology of the animal health and will help to boost up the livestock production. Nanotechnology will have a profound impact, but not in the immediate future as it is in the early stages of its development and needs to equip scientists, engineers and biologists to work at the cellular and molecular levels for significant benefits in healthcare and animal medicine. But It is reasonable to presume that in the upcoming yearвЂ™s nanotechnology research 1,2 livestock production (Patil et al., 2009). Livestock and fisheries will be affected by the nanotechnology revolution. While the great hopes of nanomedicine are disease detection and new pharmaceuticals for humans, veterinary applications of nanotechnology may become the proving ground for untried and more controversial techniques - from nano-capsule vaccines to sex selection in breeding. Nanotechnology, dealing with functional structures and materials smaller than 100nm, is emerging as a truly interdisciplinary research area spanning several traditional scientific disciplines. In keeping with the growing trend, there is a strong need for a platform to share original research related to applications of nanotechnology in biomedical fields.In the era of new health related technologies, Veterinary Medicine will enter a phase of new and incredible transformations. The major contributor to those changes is our recent ability to measure, manipulate and organize matter at the nanoscale level. Our understanding of the principles that rule the nano-scale world will be of great impact on veterinary research leading to new discoveries never before imagined.Nanotechnology has the potential to impact not only the way we live, but also the way we practice veterinary medicine. Today scientists foresee that the progress in the field of nanotechnology could represent a major breakthrough in addressing some of our technical challenges not only in engineering but also in the fields of both human and veterinary medicine. Very soon engineers will develop tiny motors to power computers and appliances and doctors will have miniature devices that aim to fight cancer on the molecular level at their disposal.In the veterinary community, some of the principal areas of nanotechnology research are currently being undertaken in the world of medicine because of the vast scope of the medical applications of nanotechnology. Many discoveries of veterinary and allied professions in the field of nanotechnology have been made till date and it is needed to provide a glimpse of the potential important targets for nanotechnology in the field of veterinary medicine. However, nanotechnology is in its early stage of development and it may take several years to perform the necessary research and conduct clinical trials for obtaining meaningful results, but professionals should begin to take note of it (Feneque, 2003).Biochips - current and future industry applications Using biochips, biological samples such as blood, tissue and semen can be instantaneously analysed and manipulated. In fewer than five years, biochips have Department of Veterinary Microbiology, Faculty of Veterinary Science & Animal Husbandry, Birsa Agricultural University, Kanke, Ranchi - 834 006 (Jharkhand), India. 3Department of Veterinary Microbiology, WBUAFS, India. 4,5 Department of Veterinary Pathology, Faculty of Veterinary & Animal Sciences, West Bengal University of Animal & Fishery Sciences (WBUAFS), Kolkata - 700 037 (West Bengal), India. *Corresponding author, E-mail: [email protected] LIVESTOCK LINE, MARCH 2012 49 become a standard technology for genomics and drug discovery and they are now moving into commercial healthcare and food safety applications (ETC Group Report, 2004). Use of biochips (Microarrays) to study genetic sequencesA biochip (or microarray) is a device typically made of hundreds or thousands of short strands of artificial DNA deposited precisely on a silicon circuit. In DNA arrays, each DNA strand acts as a selective probe and when it binds to material in a sample (e.g. blood) an electrical signal is recorded. Rather like conducting a word search across a piece of text, the biochip is able to report back on found genetic sequences based on the DNA probes built into it. The best known biochips are those produced by Affymetrix, the company that pioneered the technology and was first to produce a DNA chip that analyses an entire human genome on a single chip the size of a dime (ETC Group Report, 2004). Using biochips in biowarfare agents and in disease detection applicationsIn addition to DNA biochips, there are other variations that detect minute quantities of proteins and chemicals in a sample, making them useful for detecting bio-warfare agents or disease. Biochip analysis machines the size of an inkjet printer are commercially available from companies such as Agilent (Hewlett-Packard) and Motorola - each able to process up to 50 samples in around half an hour (ETC Group Report, 2004). Using biochips for disease detection in animals and for tracing the source of foodsChips can be used for early disease detection in animals. Researchers at the University of Pretoria are developing biochips that will detect common diseases borne by ticks. Biochips can also be used to trace the source of food and feeds. For example, bioMГ©rieuxвЂ™s вЂњFoodExpert-IDвЂќ chip rapidly tests feed to detect the presence of animal products from forty different species as a means to locate the source of pathogens - a response to public health threats such as avian flu and mad cow disease (ETC Group Report, 2004). Use of biochips in animal breeding to remove genetic diseasesOne goal is to functionalise biochips for breeding purposes. With the mapping of the human genome behind them, geneticists are now rapidly sequencing the genomes of cattle, sheep, poultry, pig and other livestock hoping to identify gene sequences that relate to commercially valuable traits such as disease resistance and leanness of meat. By including probes for these traits on biochips, breeders will be able to speedily identify champion breeders and screen out genetic diseases (ETC Group Report, 2004). вЂ�MicrofluidicsвЂ™ and вЂ�NanofluidicsвЂ™Microfluidics is a newer technology platform on the same scale as biochips. Microfluidic and nanofluidic systems analyse by controlling the flow of liquids or gases through a series of tiny channels and valves, thereby sorting them, much as a computer circuit sorts data through wires and logic gates. Microfluidic channels, often etched into silicon, can be less than 100 nm wide. This allows them to handle biological materials such as DNA, proteins or cells in minute quantities - usually nano-liters or pico-liters (1000 times smaller than a nano-liter). Microfluidics not only enable very precise analysis, they also open up the potential for manipulation of living matter by mixing, separating and handling different components at the nanoscale (ETC Group Report, 2004). Use of microfluidics in livestock breedingMicrofluidics is being used in livestock breeding to physically sort sperm and eggs. Leader in this field is XY, Inc. of Colorado (USA), which is using a microfluidic technique called flow cytometry to segregate male and female sperm for sex selection. XY has successfully bred sex-selected horses, cattle, sheep LIVESTOCK LINE, MARCH 2012 and pigs and now provides its technology to commercial breeders. Nanotech startup Arryx, which has developed a new microfluidic system called MatRyx, uses a nanotechnique in which tiny laser tractor beams trap individual sperm and then sort them by weight. MatRyx can sort around 3,000 sperm per second, and aims for commercialisation in cattle breeding. вЂњThis way dairy farmers can have cows and beef farmers can have bulls that have more meat,вЂќ was explained by ArryxвЂ™s CEO, Lewis Gruber, with a goal to produce a simple one-button sex sorter (ETC Group Report, 2004). Uses of microfluidic devices in biomimeticsMatthew Wheeler, University of Illinois professor of animal science, has gone one further in developing a microfluidic device that not only sorts sperm and eggs but also brings them together in a way that mimics the movement of natural reproduction and then handles the resulting embryo. According to Dr. Wheeler, such a technique would make mass production of embryos cheap, quick and reliable. He and his colleagues have started a spin-off company, Vitaelle, to commercialise this technology (ETC Group Report, 2004). Nanotechnology, as an enabling technology, has the potential to revolutionize veterinary medicine. Examples of potential applications in animal, agriculture and veterinary medicine include disease diagnosis and treatment delivery systems, new tools for molecular and cellular breeding, identity preservation of animal history from birth to a consumerвЂ™s table, the security of animal food products, major impact on animal nutrition scenarios ranging from the diet to nutrient uptake and utilization, modification of animal waste as expelled from the animal, pathogen detection and many more. Existing research has demonstrated the feasibility of introducing nanoshells and nano-tubes into animals to seek and destroy targeted cells. Thus, building blocks do exist and are expected to be integrated into systems over the next couple of decades on a commercial basis. While it is reasonable to presume that nano-biotechnology industries and unique developments will revolutionize veterinary medicine in the future, there is a huge concern, among some persons and organizations, about food safety and health as well as social and ethical issues which can delay or derail technological advancements (Scott, 2007). The U.S. Food and Drug Administration (FDA) regulates a wide range of products, including foods, cosmetics, drugs, devices, veterinary products, and tobacco products some of which may utilize nanotechnology or contain nanomaterials. Nanotechnology allows scientists to create, explore and manipulate materials measured in nanometers (billionths of a meter). Such materials can have chemical, physical, and biological properties that differ from those of their larger counterparts. References ETC Group (Action Group on Erosion, Technology and Concentration) Report (November 2004) вЂ�Down on the Farm: the Impact of Nano-Scale Technologies on Food and AgricultureвЂ™ Jose Feneque (December 2003) Brief Introduction To The Veterinary Applications Of Nanotechnology, Nanotechnology Now N.R. Scott (2007) Nanoscience in veterinary medicine. Veterinary Research Communications, 31(Suppl. 1), 139вЂ“ 144 S.S. Patil, K.B. Kore, Puneet Kumar (2009) Nanotechnology and its applications in Veterinary and Animal Science. Veterinary World, 2(12), 475-477 U.S. Food and Drug Administration (FDA) Report (2010) Science and Research Special Topics 50 PRESS RELEASE DAIRY SEMINAR вЂ“ PUNE A one day Seminar on вЂњChallenges for Indian Dairy Sector in the Coming DecadeвЂќ was organised by CLFMA of India on 24th January 2012 at Pune. Shri. Gopal Rao Mhaske, Chairman, Pune Milk Co-operative Federation graced the occasion as Chief Guest. Dr. C. S. Prasad, Vice Chancellor, Maharashtra Animal & Fishery Science University, Nagpur was Guest of Honour. After Inauguration and the traditional lighting of Lamp, Mr. B. S. Yadav, Chairman, CLFMA of India delivered the welcome address. Dr. C. S. Prasad delivered the Keynote address. Mementos were presented to the Chief Guest and the Guest of Honour. Technical Sessions were chaired by Dr. C. S. Prasad and Co-chaired by Mr. Amit Saraogi. Dr. Dinesh Bhosale presented an overview on activities of CLFMA and set the tone for the presentations which followed. LIVESTOCK LINE, MARCH 2012 The first speaker Dr. S. Anandan, Senior Scientist, National Institute of Animal Nutrition and Physiology, spoke on Availability and Demand of Feeds and Fodders in the country. Mr. Vishvas Chitale, CEO, Chitale Dairy informed the gathering about Challenges faced by the Private Dairy Industry. Mr. Rahul Kumar, Managing Director, Kaira Dist. Co-op. Milk Prod. Union Ltd. presented the Challenges faced by Milk Cooperative Sector. Mr. Girish Sohani President, BAIF, spoke on the Challenges faced by NGOвЂ™s. As a token of appreciation, memento were presented to all speakers and sponsors of the event. Mr. S.V. Bhave who was convener of the seminar delivered Vote of Thanks. The response to this successful seminar was overwhelming with over 130 delegates in attendance. 51 LIVESTOCK LINE, MARCH 2012 52