Lysozyme and complement concentrations in
horses, donkeys and mules
L. SOTIROV
Department of Animal Genetics, Faculty of Veterinary Medicine, Trakia University, 6000 Stara Zagora, Bulgaria
Address of author: Assoc. Prof. Dr. L. Sotirov, Department of Animal Genetics; Faculty of Veterinary Medicine, Trakia University, 6000 Stara Zagora, Bulgaria
SUMMARY
RÉSUMÉ
Sera from 143 horses belonging to six breeds (Thoroughbred, East
Bulgarian, Arabian, Hanover, Haflinger and Russian Risak), from 61 local
donkeys and from 12 mules have been tested for determining lysozyme
concentrations with agar diffusion and alternative pathway of complement
activation (APCA) through induction of haemolysis. The highest average
values of lysozyme concentrations and APCA were obtained in horses
(lysozyme concentration : 1,24 ± 0,12 mg/l and APCA : 68,92 ± 2,32
CH50), while donkeys presented intermediate results (1,86 ± 0,12 mg/l and
62,42 ± 2,32 CH50 respectively) and mules showed the lowest values (1,38
± 0,11 mg/l and 59,11 ± 1,49 CH50). Breed-related differences for the both
parameters were evidenced in horses, but environmental conditions would
a1so induce changes in lysozyme concentrations and APCA particularly in
donkeys and age tended to decrease the innate immunity.
Concentrations sériques du lysozyme et du complément chez les chevaux, ânes et mules. Par L. SOTIROV.
KEY-WORDS : horse - donkey - mule - lysozyme - complement.
MOTS-CLÉS : cheval - âne - mules - lysozyme - complément.
1. Introduction
viruses, virus-infected cells, and neoplasic cells etc. [10]. In
horses, HATZIPANAGIOTOU et al. [9] report that the
highest lysozyme concentrations in the milk of lactating
mares are observed between postparturient days 1 and 3 and
afterwards, they progressively decline to reach the 1owest
values by day 83.
The resistance of animals against infectious and parasitic
diseases is determined by the various factors of immune system. A particular interest among them is paid on lysozyme
and complement system as primary components of innate
immunity. Lysozyme is most effective against Gram-positive
bacteria but together with complement it is able to defeat
Gram-negative organisms too [1, 3, 4, 20]. Others [2, 8, 11]
report that this enzyme could protect the organism from the
fowl-pox virus in hens, equine herpes virus 4/1 and even the
HIV virus. The alternative pathway of complement activation is primarily effective against Gram-negative bacteria,
Revue Méd. Vét., 2004, 155, 4, 221-225
Les sérums de 143 chevaux de 6 races différentes (Pur sang anglais,
Bulgare de l’Est, Arabe, Hanovre, Haflinger et Risak russe), de 61 ânes et
de 12 mules ont été utilisés afin de déterminer les concentrations de lysozyme (par une technique de diffusion en gel d’agar) et l’activation du complément par la voie a1terne (par une technique hémolytique). Les concentrations moyennes de lysozyme et d’activation du complément les plus élevées ont été obtenues chez les chevaux (concentration en lysozyme : 1,24 ±
0,12 mg/l et activation du complément : 68,92 ± 2,32 CH50), tandis que les
ânes ont présenté des valeurs intermédiaires (respectivement 1,86 ± 0,12
mg/l et 62,42 ± 2,32 CH50) et les mules, les valeurs les plus faibles (1,38 ±
0,11 mg/l et 59,11 ± 1,49 CH50). L’influence de la race a été mise en évidence chez les chevaux pour ces 2 paramètres, mais les conditions environnementales, particulièrement étudiées chez les ânes, peuvent également
modifier les concentrations sériques de lysozyme et l’intensité de l’activation du complément par la voie alterne. L’immunité innée aurait aussi tendance à diminuer avec l’âge.
WISNIEWSKl and KUZMA [25] observed that the intravenous injection of E. coli lipopolysaccharide to Polish primitive horses increased serum lysozyme concentrations by
50 %. WATSON et al. [22] found out that complement activities were significantly elevated in mares with endometritis
compares to endometritis-resistant animals.
222
The bibliographic review evidences that those factors of
innate immunity play an important role in equine defense
against infectious agents. The present study was motivated
by the fact that this information is very limited, and in donkeys and mules almost lacking.
2. Material and methods
ANIMALS
The study was performed on animals as followed :
I. 143 mares from 6 breeds - 28 Thoroughbred horses
which 11 came from Balchik and 17 from Shoumen ; 38 East
Bulgarian horses horses which 23 came from Balchik and
15 from Shoumen ; 31 Arabian (Shoumen) ; 37 Hanoverian
(Experimental Farm of Trakia University, Stara Zagora) ;
5 Haf1inger (Shoumen) and 4 Russian Risak (Balchik). The
horses were bred in three farms : 1) Experimental Farm of
Trakia University, Stara Zagora; 2) the «Kabiuk» Equine
Farm, Shoumen and 3) the «Stefan Karadja» Equine Farm,
Balchik. The animals were aged between 3-5 years.
II. 61 donkeys, female, 5-20 year old, of 1ocal breed, from
three settlements in the Stara Zagora region : Elenino, Zagore
and Madjerito.
III. 12 mules female, 7-9 year old, from Gorna Kapina,
region of Kardjali.
The blood samples were taken from v. jugularis and then
were left to clot for an hour at room temperature (25°C) and
centrifuged (800 g) for 5 min.
METHODS
Sera were examined for lysozyme and complement activities [13, 16]. Briefly 20 ml of 2 % agarose (ICN, UK, Lot
2050) dissolved in phosphate buffer (7 mM Na2HPO4 and
NaH2PO4, pH : 6.2) was mixed with 20 ml suspension of
24 hours culture of Micrococcus lysodeicticus at 67°C. This
mixture was poured out in Petri’s dish (14 cm diameter).
After solidifying at room temperature 32 wells were made
(5 mm diameter). Fifty microliters of undiluted sera were
poured out in each well. Eight standard dilutions (from 0,025
to 3,125 mg/l) of lysozyme (Veterinary Research Institute,
Veliko Tirmovo) were used in the same quantity as well. The
samples were incubated for 20 hours at 37°C and lytic diameters were measured.
The alternative pathway of complement activation (APCA)
was studied by method of SOTIROV [16]. For this aim we
used veronal-veronal Na buffer (0.97 M NaCl, 12.13 mM
5,5-Diethylbarbiturate Natrium salt, 20.82 mM 5,5-Diethylbarbituric acid, 6.7 mM EGTA and 5.3 mM MgCl2, pH : 7.5).
450 µl from each serum sample (undiluted and diluted) were
poured in 0,5 ml tube. Seven dilutions were made : 8/10 ;
7/10 ; 6/10 ; 5/10 ; 4/10 ; 3/10 and 2/10 using U bottomed
microplates (Flow Laboratories, UK). Then 50 µl buffer were
added to each well and 100 µl of 1 % rabbit erythrocyte suspension (target cells) were dropped and were incubated at
37°C for 1 hour. Optical density were measured by «SumalPE2» ELISA reader (Karl Zeiss, Germany) at 540 nm.
SOTIROV (L.)
Lysozyme content, APCA activity (CH50 - complement haemolysis at 50 % lysis of target cells) and statistical analysis
(threshold of significance p < 0,05) were calculated using
special computer programs developed in Thrakia University.
3. Results
The average serum lysozyme concentrations for all horse
breeds was 1,24 ± 0,12 mg/l, but some discrepancies between
breeds occurred. The Hanoverian, Arabian, and Russian risak
horses presented the highest concentrations, then East
Bulgarian and Thoroughbred horses and final1y Haflinger
horses (Table I). The differences in serum lysozyme concentrations between Hanoverian breed and the others were not
statistically significant because of the very substantial phenotypic variation of this parameter, although all Hanoverian
horses included in this study came from the Trakia University
farm. Despite the sma11 number of sampled animals, serum
lysozyme concentrations of Russian risak horses were elevated in comparison with the other breeds (p < 0.01) except for
Hanoverian and Arabian horses, whi1e Haflinger horses showed the lowest serum lysozyme concentrations (p < 0.01).
For East Bulgarian and Thoroughbred horses, a great scattering of values was noticed, that could result from the origin of
animals. Indeed, the sampled horses came from 2 geographic
sittings : the «Kabiuk» equine farm in Shoumen and the
«Stefan Kardja» farm in Balchik. The serum lysozyme
concentrations obtained in Thoroughbred horses from the
Shoumen country were higher than those from horses originated from Ba1chik country (p < 0.01). A moderate but not
significant increase in lysozyme concentration was also noticed for East Bulgarian horses living in Shoumen country.
Consequently, the origin of horses had to be taken into
account for analysis of serum lysozyme concentrations and
comparisons between horse breeds was more relevant when
animals came from the same country. For example, in
Shoumen country, lysozyme concentrations were higher in
Thoroughbred and Arabian horses in comparison with East
Bulgarian horses (p < 0.05) and Haflinger horses (p < 0.001)
and these latter presented the lowest results (p < 0.05).
The results about a1ternative pathway of complement activation (APCA) in the various equine breeds are presented in
Table I. The highest values were obtained in Haflinger horses
(p < 0.05 vs. Arabian and Hanoverian horses), then APCA
appeared to be diminished in Russian Risak, Thoroughbred
and East Bulgarian horses, whereas Arabian and Hanoverian
horses presented the lowest values (p < 0.05 - p < 0.01).
Whereas APCA values were almost identical in East
Bulgarian horses from Balchik or from Shoumen countries,
the comparison of anima1s belonging to one breed
(Thoroughbred) revealed that the highest values were obtained in the Shoumen country («Kabiuk» farm) like serum
lysozyme concentrations. However, no positive correlation
was found between serum lysozyme concentrations and
APCA in horses of this study.
The average serum lysozyme concentrations and
Alternative Pathway Complement Activation (APCA) in
donkeys were presented in Table II and these 2 parameters
Revue Méd. Vét., 2004, 155, 4, 221-225
LYSOZYME AND COMPLEMENT CONCENTRATIONS IN HORSES, DONKEYS AND MULES
223
TABLE I. — Serum lysozyme concentrations (mg/l) and alternative pathway of complement activation (APCA - CH50) in different horse breeds. Results were expressed as mean ± Standard
error (X ± Sx) ; n : size.
TABLE II. — Effect of geographic region on lysozyme concentrations (mg/l) and alternative
pathway of complement activation (APCA - CH50) in donkeys. Results were expressed as
mean ± Standard error (X ± Sx) ; n : size; *** : p < 0.001.
were greatly influenced by the origin of animals. The highest
values for serum lysozyme concentrations and APCA were
obtained in donkeys originated from Elenino province
(p < 0.001 vs. the 2 others settlements), while the values in
the animals from Madjerito were the lowest. Those considerable differences were probably due to the different housing
of animals. The exterior of the donkeys from Elenino was
significantly superior to the other settlements. Because of the
minor variability of these parameters in the 3 donkey populations, the differences between the average values were highly
significant and consequently, these resu1ts could not be considered as occasional.
FIGURE 1. — Effect of age on serum lysozyme concentrations (mg/l) in donkeys. ** p < 0.01.
FIGURE 2. — Effect of age on Alternative Pathway of Complement Activation (APCA - CH50) in donkeys.
Revue Méd. Vét., 2004, 155, 4, 221-225
The influence of age upon lysozyme concentrations and
APCA were reported in Figures 1 and 2. Donkeys were divided into 5 subgroups according to their estimated age
through information provided by the owners and the incisor
224
wear. In the group I, the donkeys were approximately 5 years
old, in the group II 10 years o1d, in the group III 15 years old,
in the group IV 20 years old and in the group V they were
over 20 years old. A genera1 decline in serum lysozyme
concentrations was seen with the age although the differences in 10-20 years old donkeys were not significant.
However, when donkeys were over 20 years old, a sharp and
significant decrease of lysozyme concentrations was observed (p < 0.01 vs. 5- and 10- years old animals). Although the
differences were not statistically significant, the same tendency was noticed as far as APCA was concerned. Despite
the similar effects of geographic origin and of age on serum
lysozyme concentrations in donkeys, no positive and significant correlation was found between these 2 parameters.
In mules, the serum lysozyme concentration average
(1,38 ± 0,11 mg/l, n = 12) was not statistically significant
from horses and donkeys, but a lower result of APCA was
observed than in horses or in donkeys (59,11 ± 1.49 CH50,
vs. 68,92 ± 2,32 CH50 in horses, and 62,42 ± 2,32 CH50 in
donkeys). This difference was statistically significant (p <
0,001).
4. Discussion
This study shows that serum lysozyme concentrations and
APCA in Equidae were dependent on breed, on conditions of
breeding and on age, although the latter induces only minor
variations particularly on old animals. Nevertheless, even if
these 2 parameters for innate immunity were influenced in
the same way by the same factors, no positive correlations
between them was found. The breed effect was particularly
studied in horses using 6 different breeds. The highest
serum lysozyme concentrations were found in Arabian,
Thoroughbred and Hanoverian horses. But, in this last breed,
the heterogeneous distribution of values would result from
frequent crossbreedings of Hanoverian stallions with mares
from local breeds (Danubian, East Bulgarian and probably
Arabian). The breed-related differences in serum lysozyme
concentrations found out in our study were characteristic not
only for horses. Breed-related differences are established in
pigs, sheep and turkeys as well [17-19]. LIE et al. [12] reports
that in cattle, there is a primary gene that determines the high
lysozyme concentration in animal serum. This fact is further
confirmed by WALAWSKI et al. [21]. WEN and IRWIN [24]
point out that in bovine and ovine genome there are at least
10 genes coding lysozyme, and 4 of them are expressed in the
stomach. The cause for the appearance of this big number of
genes is the duplication of a single gene, that probably occurred 40-50 millions of years ago, prior to the divergence of
cattle and sheep. In the dog, two kinds of lysozyme are found
- in milk and in spleen [7]. Their biochemical analysis shows
that the milk lysozyme in the dog is very similar to those of
the horse and the donkey (83 % identity). Milk lysozyme in
the three animal species is able to bind Ca2+ whereas lysozyme synthesized in spleen does not possess such an active
center. In mice two genes coding lysozyme are found (in
intestines : P and macrophages : M). Both genes are expressed in mice but only one in rats [26]. Pigs have one conventional gene expressed in the stomach as well as in non-sto-
SOTIROV (L.)
mach tissues [14]. All those studies have been performed
with the aim to elucidate the genetic determination of lysozyme activity. According to us, the following stage of the studies has to associate the genetic polymorphism and the actual
lysozyme concentrations in body fluids. On the other hand,
Haflinger, Russian risak and Thoroughbred horses presented
the highest APCA values.
Besides the influence of breed, the origins and the conditions of life of animals would be considered. Serum lysozyme concentrations and APCA were significantly more
elevated in Thoroughbred horses from Shoumen country
than in those from Balchik country. In the «Kabiuk» farm
(Shoumen), horses were bigger and in better conditions than
those from «Stefan Kardja» farm (Balchik) and probably
had a better innate immunity. Furthermore, the average
serum lysozyme concentrations in mules were comparable
with horses and donkeys and the average APCA were even
lower, although hybrids are expected to exhibit a positive
heterosis effect. These unexpected results could result from
geographic conditions : horses and donkeys are bred in plane
whereas mules live in mountain regions where the conditions of breeding are harder. Finally, the environmental
effect was clearly demonstrated in donkeys. The animals
originated from Elenino province presented higher serum
lysozyme concentrations and APCA than those from the 2
other settlements and they enjoyed the best conditions of
breeding.
Old donkeys showed weak serum lysozyme concentrations
and APCA. These results suggest that the innate immunity
decreased with age and therefore old animals became more
sensible to infectious diseases. In swine we found out that
2 years old animals have significantly higher lysozyme and
complement concentrations than 2 months piglets [17]. The
same results we received in sheep (unpublished data).
WEKSLER et al. [23] used the B16 melanoma to explore the
relationship between age, T-lymphocyte function, and the
rate of tumor growth. According them increasing age is associated with a decrease rate of tumor growth and impaired Tcell function in C57BL/6 mice. FLOOD et al. [5] show that
ultraviolet light-induced tumors grow more slowly in 3- or 9month-old animals than in 15 month old mice. They assume
that the major cause of increased susceptibility to tumor
growth in older mice is a decline in antigen-specific CD8+
CTL. ORGEUR et al. [15] compared the behavior, growth
and immune response of piglets that were weaned early at
6 days of age (EW) vs. control Large White piglet’s (C) suckled by their mothers. In EW piglets, at 36 days of age, there
was a higher density of T- and B-lymphocytes in the gut epithelium and lamina propria, respectively, in relation to the
size of lymphoid follicles of Peyer’s paches. The results indicate great behavioral adaptation capacities of very early-weaned piglets, together with earlier maturation of their gut
immune system. All of these results indicate that immune
indices arise from born to mature age of the animals and
decrease when they are going older. Other investigators [6]
report that serum lysozyme concentration was influenced by
the season and the physiological condition of mares. They
state that lysozyme concentrations were higher in winter but
not in barren mares. In pregnant mares the moderate and insiRevue Méd. Vét., 2004, 155, 4, 221-225
LYSOZYME AND COMPLEMENT CONCENTRATIONS IN HORSES, DONKEYS AND MULES
gnificant elevation would result from pregnancy that diminishes the sensitivity of animals to season-related changes.
As a conclusion, average values of lysozyme concentrations and APCA in horses, donkeys and in mules were given
in this study. Moreover, the breed-related differences in
horses for the 2 parameters for innate immunity and the
influence of other factors, like environmental conditions and
age were outlined.
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