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Immunohistochemistry as a tool for the accurate diagnosis of diseases in
reptiles
J. Orós*,1, A. Arencibia1 and H. E. Jensen2
1
Department of Morphology, Veterinary Faculty, University of Las Palmas de Gran Canaria, Trasmontaña s/n, 35413
Arucas (Las Palmas), Spain
2
Department of Veterinary Disease Biology, University of Copenhagen, DK-1870 Frederiksberg C, Copenhagen,
Denmark
Increased efforts have been devoted in recent years to the study of reptile medicine and surgery, including medical
management, pathological studies, and diagnosis of diseases in reptiles. Immunohistochemistry has proved to be a
powerful tool for the accurate diagnosis of a number of important diseases in humans and domestic animals, depending on
the production of appropriate primary antibodies and the availability of monoclonal antibodies. Although molecular
techniques for pathogens identification have been recently developed, the diagnosis of pathogens in reptiles is usually
achieved by isolation, culture or histopathology. However, immunohistochemistry allows the demonstration of antigens of
specific pathogens in paraffin-embedded histological sections, being the major advantage the possibility to directly
associate a specific pathogen with histologic lesions. Using different clinical cases of bacterial, viral, mycotic and parasitic
diseases in reptiles we review the basics of the immunohistochemistry. Clinical cases include immunohistochemical
detection of Salmonella sp. in snakes, ophidian paramyxovirus in snakes, pathogenic fungi in sea turtles, and filarial
nematodes in lizards. Efforts should be made to develop standardized and commercially available specific polyclonal and
monoclonal antibodies against pathogens described in reptiles. We conclude that immunohistochemistry is an optimal tool
for the accurate diagnosis in reptiles, emphasizing the usefulness of the microscopic studies in reptile pathology.
Keywords: immunohistochemistry; pathology; reptiles
1. Introduction
The popularity of reptiles as pets has literally been exploding around the world in recent years. Concurrently, increased
efforts have been devoted to the study of reptile medicine and surgery, including medical management, pathological
studies, and diagnosis of diseases in reptiles. Several textbooks have compiled the last advances in reptile medicine and
surgery [1] and infectious diseases and pathology of reptiles [2]. Although molecular techniques for pathogens
identification have been recently developed, the diagnosis of pathogens in reptiles is usually achieved by isolation,
culture or histopathology. However, immunohistochemistry allows the demonstration of antigens of specific pathogens
in paraffin-embedded histological sections, being the major advantage the possibility to directly associate a specific
pathogen with histologic lesions [3,4].
Historically, after the development of the fluorescent-labeled antibody technique [5], the use of horseradish
peroxidase enzyme-labeling of antibodies was remarkable [6], allowing to counterstain the labeled cells. Since then,
several enzyme techniques were developed: peroxidase-anti-peroxidase (PAP) [7], alkaline phosphatase labeling [8],
avidin-biotin peroxidase labeling (ABC) [9, 10], and alkaline phosphatase-anti-alkaline phosphatase (APAAP) [11]. In
addition, the method for the production of monoclonal antibodies [12] was a very important step. Other significant
advances were the use of trypsin digestion on paraffin sections for unmasking antigens [13], and the heat treatment for
antigen retrieval [14].
Using different clinical cases of bacterial, viral, mycotic and parasitic diseases in reptiles we review the basics of the
immunohistochemistry.
2. Brief fundamentals of immunohistochemistry
Immunohistochemistry is a technique for identifying antigens by means of antigen-antibody interactions, the site of
antibody binding being identified either by direct labeling of the antibody, or by use of a secondary labeling method
[15]. Among immunoglobulins IgG is the most frequently used antibody for immunohistochemistry; the IgG molecule
is composed of two pairs of light and heavy polypeptide chains linked by disulphide bonds to form a Y-shaped
structure. The terminal regions (variable domains) vary in amino acid sequence providing specificity for a particular
epitope [15].
Polyclonal antibodies are produced by immunizing an animal with a purified specific molecule bearing the antigen of
interest [15]. Polyclonal antibodies are frequently contaminated with other antibodies due to impure antigen used to
immunize the host animal [16]. However, subjecting polyclonal antibodies to multiple adsorption protocols for a variety
of antigens will increase their specificity [3]. Hybridoma technique is a technology of forming hybrid cell lines by
fusing a specific antibody-producing B cell with a myeloma cell that is selected for its ability to grow in tissue culture
and for an absence of antibody chain synthesis. The antibodies produced by the hybridoma are all of a single specificity
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and are therefore monoclonal antibodies [12]. The original antigen need not be pure, for hybrids reacting to unwanted
antigens can be eliminated during screening, and the result is a constant reliable supply of one pure antibody with
known specificity [15]. The production of monoclonal antibodies has increased the number of antibodies available for
immunohistochemistry.
There are several enzymes and chromogens available for immunohistochemistry. Horseradish peroxidase is the most
widely used enzyme, and in combination with the chromogen 3,3’-diaminobenzidine tetrahydrochloride (DAB) it yields
a dark brown reaction end product [15]. Other alternative chromogen for demonstration of peroxidase is the 3-amino-9ethylcarbazole (AEC), providing a red color. Endogenous peroxidase activity is found in many tissues and can be
detected by reacting fixed tissue sections with DAB substrate. The solution for eliminating endogenous peroxidase
activity is by the pre-treatment of the tissue section with hydrogen peroxide prior to incubation of primary antibody.
Calf intestinal alkaline phosphatase is the most widely used enzyme alternative to horseradish peroxidase, particularly
since the development of the APAAP method [11]. Fast red TR used with naphthol AS-MX phosphate sodium salt
gives a bright red reaction end product [15]. Many tissues also contain endogenous alkaline phosphatase activity and
should be blocked by the pre-treatment of the tissue section with levamisole.
2.1 Direct method
The primary antibody is conjugated directly to the label [15]. This method is one step staining method, and involves a
labeled antibody (i.e. FITC conjugated antiserum) reacting directly with the antigen in tissue sections. This technique
utilizes only one antibody and the procedure is short and quick. However, it is insensitive due to little signal
amplification and rarely used since the introduction of indirect method.
2.2 Indirect method
Indirect method involves an unlabelled primary antibody and a labeled secondary antibody directed against the primary
antibody [15]; the secondary antibody must be against the IgG of the animal species in which the primary antibody has
been raised. This method is more sensitive due to signal amplification through several secondary antibody reactions
with different antigenic sites on the primary antibody. In addition, it is also inexpensive since one labeled second layer
antibody can be used with many first layer antibodies (raised from the same animal species) to different antigens. The
second layer antibody can be labeled with a fluorescent dye such as FITC, rhodamine or Texas red, and this is called
indirect immunofluorescence method. The second layer antibody may be labeled with an enzyme such as peroxidase,
alkaline phosphatase or glucose oxidase, and this is called indirect immunoenzyme method.
2.3
Peroxidase anti-peroxidase (PAP) method
This method is a further development of the indirect technique, involving a third layer formed by a soluble peroxidaseanti-peroxidase complex [7,15]. These complexes are formed by three peroxidase molecules and two anti-peroxidase
antibodies. They are bound to the unconjugated primary antibody by a second antibody (usually a swine or goat antirabbit) applied in excess so that one of its two identical binding sites binds to the primary antibody and the other to the
rabbit PAP complex [7,15]. The sensitivity is about 100 to 1000 times higher since the peroxidase molecule is not
chemically conjugated to the anti IgG but immunologically bound, and loses none of its enzyme activity. It also allows
for much higher dilution of the primary antibody, thus eliminating many of the unwanted antibodies and reducing nonspecific background staining.
2.4 Avidin-biotin complex (ABC) method
In this method, avidin, a large glycoprotein, can be labeled with peroxidase and has a very high affinity for biotin.
Biotin, a low molecular weight vitamin, can be conjugated to a variety of biological molecules such as antibodies. This
method involves three layers; the first layer is unlabeled primary antibody. The second layer is biotinylated secondary
antibody. The third layer is a complex of avidin-biotin peroxidase [9,10].
2.5
Labeled streptavidin biotin (LSAB) method
Streptavidin, derived from Streptomyces avidini, is an innovation for substitution of avidin. The streptavidin molecule is
uncharged relative to animal tissue, unlike avidin that has an isoelectric point of 10, and therefore electrostastic binding
to tissue is eliminated. In addition, the lack of oligosaccharide residues of the streptavidin gives it advantages over the
avidin eliminating possible binding to tissue lectins and background staining [15]. LSAB is technically similar to
standard ABC method. The first layer is unlabelled primary antibody. The second layer is biotinylated secondary
antibody. The third layer is enzyme-streptavidin conjugates (HRP-Streptavidin or AP-Streptavidin) to replace the
complex of avidin-biotin peroxidase. The enzyme is then visualized by application of the substrate chromogen solutions
to produce different colorimetric end products. Several reports suggest that LSAB method is about 5 to 10 times more
sensitive than standard ABC method [15].
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2.6
Polymeric methods
EnVision Systems are based on dextran polymer technology. This method permits binding of a large number of enzyme
molecules (horseradish peroxidase or alkaline phosphatase) to a secondary antibody via the dextran backbone. The
benefits include increased sensitivity, minimized non-specific background staining and a reduction in the total number
of assay steps as compared to conventional techniques [17].
ImmPRESS (polymerized reporter enzyme staining system) is based on a new method of polymerizing enzymes and
attaching these polymers to antibodies. The novel approach employed to form enzyme "micropolymers" avoids the
intrinsic shortcomings of using large dextrans or other macromolecules as backbones. Attaching a unique
"micropolymer” with a high density of very active enzyme to a secondary antibody generates a reagent that overcomes
steric interference and provides enhanced accessibility to its target. The benefits include outstanding sensitivity, signal
intensity, low background staining, and reduced non-specific binding [18].
3. Clinical cases
3.1
Salmonellosis in snakes
Salmonella arizonae is a well-recognized pathogen of mammals and birds, but its true role an aetiological agent of
disease in reptiles has been controversial, suggesting that some snakes can be natural reservoirs of Salmonella arizonae
[19, 20]. Despite the failure to produce experimental infections, and frequent suggestions that Salmonella arizonae is no
more than an opportunistic secondary invader in reptiles, reports continue to appear implicating it as a primary agent in
immunosuppressed reptiles [19, 20]. In addition, reptiles have been implicated in transmitting Salmonella arizonae to
man, livestock and companion animals [21]. The usual clinical signs of salmonellosis in reptiles are those associated
with acute enteritis, septicaemia, pneumonia, coelomitis and hypovolemic shock, followed by death [20].
Snake 1 was a 6-year-old male rosy boa (Lichanura trivirgata) submitted for necropsy. Clinical history included
anorexia, postprandial regurgitation and blood and mucus in the vomitus. Necropsy revealed a diffuse thickening of the
gastric wall, with fibrinonecrotic debris on the mucosal surface and multifocal yellowish necrotic plaque-like lesions.
Histologically, a severe, diphtheritic, necrotizing gastritis, characterized by a heterophilic fibrinonecrotic exudate
forming pseudomembranes over the damaged surface, was seen. Necrotic areas were also observed in the lamina
propria. In the necrotic and ulcerative areas, large numbers of Gram-negative coccobacilli were found, closely
associated with the primary lesion in the stomach. The only pathogenic microorganism isolated from the stomach was
identified as Salmonella arizonae from the biochemical profile after incubation for 24 h.
Snake 2 was a 7-year-old male double-headed Honduran milk snake (Lampropeltis hondurensis) also submitted for
necropsy. It was found dead, no clinical signs having been seen. At necropsy two tracheas and a single lung were
observed. The left trachea was of normal size with well-developed hyaline cartilaginous rings, but the right one was
considerably larger, with flat tracheal rings. The lumen of this abnormal trachea was full of yellow-green mucopurulent
material obstructing the airflow. Salmonella arizonae was isolated in pure culture from this material. Histological
examination of the right trachea revealed hyperplasia of the ciliated pseudostratified columnar epithelium and necrotic
epithelial debris in the tracheal lumen. Heterophils and mixed mononuclear leucocytes infiltrated the lamina propria and
respiratory epithelium. Degenerate heterophils were also seen in the tracheal lumen. Large numbers of Gram-negative
bacteria were observed in the necrotic epithelial debris in the tracheal lumen.
In both cases, an immunohistological study was carried out in order to directly associate the pathogen Salmonella
arizonae with histologic lesions. Monospecific rabbit antiserum to Salmonella arizonae (supplied by the Respiratory
Disease Research Unit, National Animal Disease Center, Ames, Iowa) was used in an immunoperoxidase technique.
Negative control procedures included replacing the primary antibody with non-immune rabbit serum. Tissues from a
non-infected snake and tissues from mammals naturally infected with Salmonella spp. other than Salmonella arizonae
were also used as negative immunoperoxidase controls.
Immunoreactivity was strong in the necrotic areas of the stomach of the rosy boa, being particularly intense around
the edges of the necrotic areas (Fig. 1A). Some mononuclear cells near these areas showed intense labeling of
Salmonella arizonae antigen. Intense immunolabelling was also detected in the necrotic debris of the abnormal trachea
of the Honduran milk snake (Fig. 1B). No immunoreaction was found when normal serum was used, or in the noninfected snake. These immunohistochemical results established a direct relation between Salmonella arizonae and
digestive and respiratory lesions in snakes, thus providing evidence to suggest pathogenicity [22, 23].
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Fig. 1 (A) Gastric mucosa of the snake 1 showing brown immunolabelling for Salmonella arizonae antigen around the necrotic
areas. Scale bar = 830 μm. (B) Red immunolabelling (arrow) for Salmonella arizonae antigen in the necrotic debris of the abnormal
trachea of the snake 2. Scale bar = 40 μm.
3.2 Ophidian paramyxovirus in snakes
Ophidian paramyxovirus (OPMV) has been identified as a very important pathogen in both viperid and non-viperid
snakes. In 1972, a respiratory epizootic spread through a collection of fer-de-lance snakes (Bothrops sp.) at a
serpentarium in Switzerland. Although the disease was originally thought to be bacterial in origin, a paramyxo-like
virus was isolated [24]. Since 1979, numerous outbreaks have been described in collections of snakes in the USA,
Mexico and Germany [25, 26]. The disease is characterized mainly by proliferative pneumonia, with or without
interstitial inflammation [26]. A transmission study was carried out at the University of Florida and Koch’s postulates
were fulfilled [27]. Diagnosis is based upon isolation of the virus in tissue culture, demonstration of the virus by
electron microscopy, demonstration of rising titres of antiviral antibodies by haemagglutination inhibition tests, and by
immunoperoxidase staining of viral antigen in sections of infected lungs [28].
In our study, tissues from 17 snakes belonging to several private collections in Gran Canaria that experienced
mortalities attributed to respiratory infection were selected. The species studied were: a western diamondback
rattlesnake (Crotalus atrox), pygmy rattlesnake (Sistrurus miliarius), rhinoceros viper (Bitis gabonica rhinoceros),
palm viper (Bothrops sp.), anaconda (Eunectes murinus), brown tree snake (Boiga irregularis), indigo snake
(Drymarchon corais), Asian rat snake (Gonyosoma oxycephala), emerald tree boa (Corallus canina), black rat snake
(Elaphe obsoleta), two ball pythons (Python regius), two Burmese pythons (Python molurus bivittatus) and three king
snakes (Lampropeltis getulus). At necropsy, a variety of pulmonary gross lesions were detected in all snakes, including
diffuse to focal accumulation of caseous necrotic cellular debris, diffuse haemorrhage of the lung and air sac system,
and edema and severe diffuse congestion of the lung. Moderate multifocal exudative to granulomatous pneumonia was
observed in five snakes. Histological changes in affected lungs included hyperplasia and hypertrophy of septal and
faveolar epithelial cells, loss of ciliated cells, mixed leucocytic interstitial infiltrates, and fibrinonecrotic exudate in the
lumen of proximal and distal faveolar compartments. Macrophages and Gram-negative bacteria were often detected
within this exudate. No intraepithelial intracytoplasmic inclusion bodies or epithelial syncytial cell formation were
observed.
For immunohistochemistry we used the LSAB method. A polyclonal antiserum raised in rabbits against an Aruba
Island rattlesnake (Crotalus unicolor) isolate of OPMV was applied at dilution 1:500 [29]. The chromogen was the
AEC. Paraffin-embedded normal tissues from two healthy boa constrictors and non-immune serum from a rabbit served
as negative controls. Tissues from an Aruba Island rattlesnake experimentally inoculated with OPMV [27] were used as
positive controls.
Viral antigen was immunohistochemically detected in the lungs of six snakes belonging to three different reptile
collections. There was strong multifocal to diffuse linear red labeling of the luminal surface of bronchial (Fig. 2A) and
faveolar epithelium (Fig. 2B). In cases where there was a hyperplastic epithelium, cytoplasmic labeling was observed
within the superficial cells lining air passageways. Immunoreactivity was also detected in some hepatocytes and
Kupffer cells of the liver of the western diamondback rattlesnake.
The pulmonary lesions associated with OPMV infection are highly suggestive of the infection but are not considered
pathognomonic [28]. In our study, approximately 40 per cent of the sections that had proliferative pneumonia did not
show immunoreactivity. Antigen denaturation associated with formalin fixation [30], and possible antigen variability
among strains of OPMV [28] should be considered.
In a previous study, the authors used the ABC method and DAB as chromogen [28]. We used the streptavidin
method because it has been described as being more sensitive than the ABC method. The use of AEC as the substrate of
the immunoreaction is considered more efficient than DAB because the strong red color provided by AEC facilitates the
interpretation of results.
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Fig. 2 (A) Immunoperoxidase red labeling for OPMV antigen of the luminal bronchial epithelium of a ball python (Python regius).
Scale bar = 60 μm. (B) Red immunolabelling for OPMV antigen of the faveolar epithelium of a western diamondback rattlesnake
(Crotalus atrox). Scale bar = 80 μm.
Although primary lesions of paramyxoviral infections in snakes are confined to the respiratory system, particularly
the lungs, lesions in other organs have been described. Moderate chronic hepatitis and diffuse hepatic necrosis are often
observed [25]. Immunoreactivity for OPMV was observed in the liver of the western diamondback rattlesnake, and
although isolation of OPMV from the liver has been described [25], there are no reports of immunohistochemical
detection of OPMV antigen in this organ. The viraemic status of the snake could explain this finding.
This study confirmed the usefulness of this polyclonal anti-OPMV antiserum to detect OPMV antigens in formalinfixed paraffin-embedded lung of infected snakes [31].
3.3
Mycotic infections in sea turtles
There are relatively few reports of mycotic diseases in sea turtles. Such infections have been described involving both
captive [32-34] and wild sea turtles [35, 36]. Because of their solitary habits, wild sea turtles seem far less vulnerable to
exposure to fungal pathogens, whereas crowded conditions in captivity predispose sea turtles to outbreaks of mycotic
infection [32]. Fungal skin diseases in captive marine turtles are thought to be of major importance [33]. Although
mycotic granulomas can be found in the liver and throughout the coelomic cavity of wild and captive sea turtles,
systemic mycotic infections occur primarily in the lungs [37].
Turtle 1 was a juvenile female loggerhead sea turtle (Caretta caretta) stranded in Gran Canaria. At necropsy the
small intestine was pleated and gathered around a linear foreign body identified as a fishing line. Histologically, severe
necrotic enteritis, multiple haemorrhages, and marked edema of the intestinal submucosa of the duodenum were
observed. Numerous yeast cells and fungal hyphae were observed in the lamina propria of the mucosa and in the
connective tissue of the submucosa of the duodenum.
Turtle 2 was a Kemp’s ridley sea turtle (Lepidochelys kempi) stranded in Portugal. Macroscopically, several firm
white granulomas, 1-2 mm in diameter, and bronchial deposits of yellow caseous material were observed multifocally
within the parenchyma of both lungs. Histologically, a severe multifocal granulomatous pneumonia was diagnosed, and
there were some foci of suppurative and necrotizing bronchopneumonia. In both types of lesions, numerous periodic
acid-Schiff (PAS)- and Grocott’s methenamine silver (GMS)-positive fungal hyphae were observed.
Turtle 3 was a subadult female olive ridley sea turtle (Lepidochelys olivacea) stranded in Gran Canaria. At necropsy
the liver was enlarged and covered by fibrin and numerous granulomas. The spleen was enlarged and covered partially
by fibrin and small granulomas. Notable amounts of fibrin and small granulomas were also detected on the coelomic
wall and the gastric serosa. Both lungs showed numerous, yellow-white, 2-6 mm in diameter, round foci scattered
randomly throughout the parenchyma of the caudal area. Firm deposits of fibrin were also observed covering caudally
the pleural surface of the right lung. Histologically, a severe fibrinous and granulomatous polyserositis (liver, spleen,
stomach, urinary bladder, coelomic wall) was diagnosed. Numerous round granulomas surrounded by multinucleated
giant cells were also observed in the serosa of these organs. Numerous PAS- and GMS-positive fungal hyphae were
observed associated with these lesions. Severe multifocal granulomatous pneumonia was also diagnosed in both lungs.
Severe fibrinous pleuritis was diagnosed in the right lung. In both types of lesions, numerous fungal hyphae were also
observed.
Because fungal cultures were not taken at the time of necropsies, several immunohistochemical studies were carried
out in order to determine the aetiological agents involved in the lesions of the turtles. In the tests, a panel of specific
monoclonal and heterologously absorbed polyclonal antibodies served as the primary reagents [38]. Fungal specificity
and dilutions of the antibodies are summarized in Table 1. Experimentally infected tissues containing reference fungi
were used as positive controls [38-40].
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Table 1
Antibodies used in the immunohistochemical studies of mycotic diseases in three sea turtles.
Antibody
Monoclonal
Fungal species
Aspergillus sp
Fungal specificity
Aspergillus spp
Dilution
1:2
Monoclonal
Mucorales (Zygomycetes)
Mucorales (Zygomycetes)
1:4
Polyclonal
Trichophyton mentagrophytes
Trichophyton spp
1:64
Polyclonal
Trichophyton verrucosum
Trichophyton spp
1:64
Polyclonal
Aspergillus fumigatus
Aspergillus spp
1:64
Polyclonal
Candida albicans
Candida spp
1:256
Polyclonal
Geotrichum candidum
Geotrichum candidum
1:32
Polyclonal
Fusarium solani
Fusarium spp
1:16
Polyclonal
Scedosporium apiospermum
Scedosporium spp
1:16
Fig. 3
(A) Specific immunostaining of Candida albicans in the intestinal lesions of the sea turtle 1 using and indirect
immunofluorescence staining technique. Scale bar = 50 μm. (B) Immunohistochemical labeling of fungal hyphae for Fusarium spp.
antigen in the lung of the sea turtle 2. Scale bar = 60 μm. (C) Immunohistochemical labeling of fungal hyphae for Trichophyton spp.
antigen in the lung of the sea turtle 3. Scale bar = 60 μm.
Identification of fungal elements in the sea turtle 1 was accomplished through specific immunostaining using an
indirect immunofluorescence staining technique (IIF); the fungal elements within the intestinal lesions were strongly
stained only by a polyclonal rabbit antibody against wall mannan of Candida albicans, and a monoclonal antibody
against Candida albicans whereas no reaction was obtained when other antibodies were applied (Fig. 3A).
For the sea turtle 2, the monoclonal antibodies were either used in a three-layer indirect enzyme
immunohistochemical technique using APAAP immunocomplexes, or an IIF technique. The polyclonal antibodies were
applied in a PAP technique. As a substrate, fast red was used in the APAAP technique, whereas AEC was used in the
PAP technique. All GMS-positive hyphae were identified as Fusarium spp because a strong and uniform reactivity was
obtained only with a heterologously-absorbed polyclonal antibody raised against somatic antigens of Fusarium solani
(Fig. 3B).
For the sea turtle 3 we used the detection system Power-vision+ Poly-HRP Histostaining Kit (Immunovision
Technologies Co., Burlingame, CA, USA), followed by incubation in AEC solution. The fungal elements within the
pulmonary and hepatic lesions were strongly stained only by the heterologously absorbed, dermatophyte specific
antibodies raised towards Trichophyton spp (Fig. 3C), whereas no reaction was obtained when other antibodies were
applied.
These cases of mycotic infections in sea turtles confirmed the usefulness of the immunohistochemistry in order to
know the aetiological agent in absence of fungal cultures [41-43].
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3.4
Filarial nematodosis in lizards
Reptiles serve as hosts for five subfamilies, as well as a large variety of genera, of filarial nematodes, some of which are
specific [44]. Susceptible host reptiles are infected when blood-sucking arthropods, primarily mosquitoes containing
infective third-stage larvae, transmit microfilariae during feeding [45]. Diagnosis of filariasis in reptiles is based upon
the presence of microfilariae in the blood. At necropsy, adult worms may be found in the lumen of great vessels or in
the subcutaneous tissues and free in the body cavities. Histologically the microfilariae may be detected in tissue sections
[46].
Lizard 1 was an adult male captive Oustalet’s chameleon (Furcifer oustaleti) submitted for necropsy. The reptile
exhibited anorexia and lethargy for one week before its death. At necropsy, five adult filarial nematodes were collected
from the subcutaneous tissues of the lateral abdominal and thoracic areas. Blood was collected from the ventricle by
cardiocentesis, and several air-dried blood films were made immediately following collection. Numerous microfilariae
were found in these blood films stained with routine Romanowsky’s stain. The worms were identified as Foleyella
species. Histological lesions included chronic interstitial pneumonia associated with numerous microfilariae in the
septal capillaries, mild diffuse membranoproliferative glomerulonephritis with microfilariae in the glomerular
capillaries.
Lizard 2 was an adult male captive Mexican beaded lizard (Heloderma horridum) submitted for necropsy. Grossly,
no parasites were detected. Histologically, an adult filarial nematode was observed in the lumen of a great pulmonary
vessel, as well as several microfilariae. A chronic interstitial pneumonia associated with numerous microfilariae in the
septal capillaries was diagnosed. No species identification of the filarial nematode was carried out.
For immunohistochemistry we used the LSAB method. A polyclonal antiserum raised in rabbits against excretorysecretory (ES) products of adult Dirofilaria immitis [47] applied at a dilution of 1:600 was used. The chromogen was
the AEC. Paraffin-embedded normal tissues from two healthy chameleons and non-immune serum from a rabbit served
as negative controls. Tissues from two dogs naturally infected with D. immitis [47] were used as positive controls.
There was intense red labeling of circulating microfilariae when the serum anti-ES products of D. immitis was used.
Immunoreactivity was detected in the adult filarid of the lizard 2 (Fig 4A). In addition, numerous immunopositive
microfilariae were also detected within pulmonary septal blood vessels, in the lumen of glomerular capillaries, in the
hepatic sinusoids, central veins and vessels of the portal areas, and in the splenic sinusoids of both lizards (Fig 4B).
None of the negative controls stained positively for filarial antigens.
Fig. 4 (A) Immunoperoxidase red labeling of an adult filarial nematode in the lumen of a pulmonary vessel of the lizard 2. Scale
bar = 120 μm. (B) Immunoperoxidase red labeling of Foleyella spp. microfilariae within the pulmonary septal blood vessels of the
lizard 1. Scale bar = 10 μm.
These cases showed the usefulness of this antiserum to detect several species of filarial nematodes in formalin-fixed,
paraffin-embedded tissues. The presence of common antigens in first-stage larvae of the filarial nematodes in the two
lizards and adult D. immitis worms could explain this immunoreaction [47]. Circulating microfilariae can be detected
histologically on tissue sections, particularly when they are cut longitudinally. Cross-sections of microfilariae can be
mistaken for mononuclear cells or nuclear debris. In these cases, the use of the antiserum allowed the detection of
numerous microfilariae, not observed previously using routine haematoxylin and eosin stain. Thus,
immunohistochemical detection of microfilariae can be useful in the diagnosis of filariasis in reptiles, especially when
the worm burden is low [48].
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4. Conclusions
Immunohistochemistry had been proved to be a powerful tool for the accurate diagnosis of a number of important
diseases in humans and domestic animals, depending on the production of appropriate primary antibodies and the
availability of monoclonal antibodies [38]. In this chapter, the clinical cases affecting several reptiles and the use of
immunohistochemistry as a tool for the diagnosis, demonstrate the usefulness of this technique for several approaches:
a) study of the pathogenesis of the disease (i.e. establishing a direct relation between Salmonella arizonae and digestive
and respiratory lesions in snakes), b) diagnosis of viral diseases in formalin-fixed, paraffin-embedded tissues in absence
of viral isolation (i.e. diagnosis of OPMV in snakes), c) diagnosis of the specific mycotic agent involved in the disease
in absence of fungal cultures (i.e. diagnosis of several mycosis in sea turtles), and d) detection of larval stages of
parasites not observed previously using routine haematoxylin and eosin stain (i.e. detection of microfilariae in lizards).
Acknowledgements The authors would like to thank P. Castro, Department of Morphology, University of Las Palmas de Gran
Canaria (ULPGC), for technical assistance.
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