Investigation of an outbreak of besnoitiosis in donkeys in northeastern Pennsylvania

SallyAnne L. Ness Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853.

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Jeanine Peters-Kennedy Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853.

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Gereon Schares Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Epidemiology, Seestrasse 55, D-16868 Wusterhausen, Germany.

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Jitender P. Dubey Animal Parasitic Diseases Laboratory, Animal and Natural Resources Institute, Agricultural Research Service, United States Department of Agriculture, Beltsville, MD 20705.

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Linda D. Mittel Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853.

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Hussni O. Mohammed Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853.

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Dwight D. Bowman Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853.

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M. Julia B. Felippe Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853.

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Susan E. Wade Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853.

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Nicole Shultz Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853.
Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853.
Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Epidemiology, Seestrasse 55, D-16868 Wusterhausen, Germany.
Animal Parasitic Diseases Laboratory, Animal and Natural Resources Institute, Agricultural Research Service, United States Department of Agriculture, Beltsville, MD 20705.
Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853.
Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853.
Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853.
Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853.
Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853.
Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853.

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Thomas J. Divers Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853.

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Abstract

Objective—To describe the clinical, endoscopic, and serologic features of an outbreak of besnoitiosis in 2 donkey operations in northeastern Pennsylvania and to report the outcome of attempted treatment of 1 naturally infected individual.

Design—Observational study.

Animals—29 donkeys (Equus asinus) in northeastern Pennsylvania.

Procedures—Donkeys were examined for lesions suggestive of besnoitiosis in an outbreak investigation. Information was collected regarding the history and signalment of animals on each premises. Rhinolaryngoscopy was performed to identify nasopharyngeal and laryngeal lesions. Serum samples were collected for immunofluorescent antibody testing and immunoblotting for Besnoitia spp. Skin biopsy samples were obtained from 8 animals with lesions suggestive of besnoitiosis for histologic examination. Quantitative real-time PCR assay for Besnoitia spp was performed on tissue samples from 5 animals.

Results—Besnoitiosis was confirmed in 6 of the 8 suspected cases. The most common lesion site was the nares, followed by the skin and sclera. Donkeys with clinical signs of disease had higher serum antibody titers and tested positive for a greater number of immunoblot bands than did donkeys without clinical signs of disease. All animals evaluated by PCR assay tested positive. Putative risk factors for disease included age and sex. Ponazuril was not effective at treating besnoitiosis in a naturally infected donkey.

Conclusions and Clinical Relevance—Knowledge of clinical and serologic features of besnoitiosis in donkeys will assist clinicians in the diagnosis and prevention of this disease in donkey populations. Besnoitiosis may be an emerging disease of donkeys in the United States.

Abstract

Objective—To describe the clinical, endoscopic, and serologic features of an outbreak of besnoitiosis in 2 donkey operations in northeastern Pennsylvania and to report the outcome of attempted treatment of 1 naturally infected individual.

Design—Observational study.

Animals—29 donkeys (Equus asinus) in northeastern Pennsylvania.

Procedures—Donkeys were examined for lesions suggestive of besnoitiosis in an outbreak investigation. Information was collected regarding the history and signalment of animals on each premises. Rhinolaryngoscopy was performed to identify nasopharyngeal and laryngeal lesions. Serum samples were collected for immunofluorescent antibody testing and immunoblotting for Besnoitia spp. Skin biopsy samples were obtained from 8 animals with lesions suggestive of besnoitiosis for histologic examination. Quantitative real-time PCR assay for Besnoitia spp was performed on tissue samples from 5 animals.

Results—Besnoitiosis was confirmed in 6 of the 8 suspected cases. The most common lesion site was the nares, followed by the skin and sclera. Donkeys with clinical signs of disease had higher serum antibody titers and tested positive for a greater number of immunoblot bands than did donkeys without clinical signs of disease. All animals evaluated by PCR assay tested positive. Putative risk factors for disease included age and sex. Ponazuril was not effective at treating besnoitiosis in a naturally infected donkey.

Conclusions and Clinical Relevance—Knowledge of clinical and serologic features of besnoitiosis in donkeys will assist clinicians in the diagnosis and prevention of this disease in donkey populations. Besnoitiosis may be an emerging disease of donkeys in the United States.

Besnoitiosis is caused by infection with protozoan parasites (Besnoitia spp), which are cyst-forming coccidians that affect multiple host species worldwide.1–4 Besnoitiosis in cattle, caused by Besnoitia besnoiti,5–9 was historically an economically important disease of cattle in subtropical Africa and Asia; however, recent outbreaks in Europe suggest that the disease may be spreading globally.6–9 Cutaneous besnoitiosis has been described in reindeer and caribou in Canada, Alaska, Finland, and Sweden,3,10,11 and Besnoitia tarandi has been isolated in vitro from a reindeer in Finland.3 Besnoitiosis in equids is caused by Besnoitia bennetti and was first described in horses and donkeys in Sudan in the 1920s.12,13 Phylogenically, B besnoiti, B bennetti, and B tarandi are very similar and differ primarily by biological traits such as intermediate host preference. There are only minor differences in the rRNA genes of these 3 Besnoitia spp, and there are currently no molecular markers to distinguish them on a genetic level3,8,14,15

Reported equid cases of besnoitiosis in North America have been limited to a few isolated cases and an outbreak in a Michigan herd of miniature donkeys in 2005.15–18 The endoscopic, serologic, and molecular features of donkeys affected by an outbreak of besnoitiosis have not been previously reported.

Clinical disease is characterized by the development of multifocal pinpoint parasitic cysts, approximately 1.0 mm in diameter, in the skin over the face and body, within the nares, on the internal and external pinnae, and on the limbs and perineum.15–18 Mucous membranes are also frequently affected, and one of the most unusual clinical features of besnoitiosis is the development of cysts along the limbal margin of the sclera (ie, scleral pearls).15,17 Some infected animals remain otherwise healthy, whereas others become cachexic and debilitated as a result of the disease.15,17 The reason for this difference in host response to infection is unknown, but similar clinical subtypes are observed with besnoitiosis in cattle.7 The current gold standard for diagnosing besnoitiosis in donkeys is histologic identification of Besnoitia cysts within the dermis of individuals with lesions, generally achieved via skin biopsy.15–18

There is currently a paucity of information regarding besnoitiosis in equids. The life cycle of Besnoitia spp involves both a definitive (predator) and intermediate (prey) host.1,2 A feline definitive host has been identified for Besnoitia oryctofelisi, Besnoitia darlingi, and Besnoitia neotomofelis, which affect rabbits, opossums, and southern plains woodrats, respectively.1,3,7,19 Attempts to demonstrate cats, or any other animal, as the definitive host for B bennetti have been unsuccessful, precluding researchers from elucidating the parasite's life cycle and mode of disease transmission in equine infection.15

The purpose of the study reported here was to describe the clinical, endoscopic, and serologic features of an outbreak of besnoitiosis in 2 donkey herds in the northeastern United States. We also report the outcome of 37 days of treatment with ponazurila,b in 1 donkey naturally infected with Besnoitia spp.

Materials and Methods

Study population—In the fall of 2010, individual donkeys from 2 herds (herds A and B) in Pennsylvania were diagnosed with besnoitiosis on the basis of evaluation of skin biopsy samples obtained by the farms' primary veterinarian (NS). The biopsies were performed to evaluate chronic dermatitis nonresponsive to various topical treatments. Subsequent diagnosis of besnoitiosis prompted the veterinarian to contact veterinarians at Cornell University to initiate a herd investigation.

Data collection—Both premises were visited by 1 of the authors (SLN) in October of 2010. Every donkey and horse on the property was examined for external lesions suggestive of besnoitiosis, including pinpoint, white, miliary papules on the face, nares, pinnae, and perineum. Vulvar mucous membranes were examined for lesions in female donkeys. Both eyes were inspected for the presence of scleral pearls. Location and distribution of lesions were recorded when observed. Upper airway endoscopy of all individuals in herd A (n = 15) was performed to identify animals with lesions involving the nasopharynx and larynx. Lesions visualized on rhinolaryngoscopy were recorded as present or not present. A questionnaire was used to obtain information regarding age, sex, breed, date of appearance of clinical signs, duration of time on the farm, contact with confirmed cases, and whether the donkey had been born on the premises.

Blood samples were collected from each animal from either the left or right jugular vein. Skin biopsy samples were obtained from all animals identified as having lesions suggestive of besnoitiosis. Biopsy sites were selected to maximize the number of lesions obtained within the sampled tissue. Biopsy samples were obtained from each of the affected donkeys with 4- to 6-mm disposable punch instrumentsc and preserved in neutral-buffered 10% formalin solution for histologic examination. Owner consent was obtained for all examinations and procedures.

Histologic examination—All formalin-fixed samples were hemisectioned, routinely processed, paraffin embedded, sectioned at a thickness of 4 μm, and stained with H&E stain for histologic examination performed by one of the authors (JP).

Case definition—Donkeys were classified as confirmed, suspected, or non–case animals. A confirmed case animal was defined as a donkey identified to have characteristic lesions suggestive of besnoitiosis with histologic confirmation of Besnoitia spp cysts on evaluation of skin biopsy samples. A suspected case animal was defined as a donkey identified to have characteristic lesions suggestive of besnoitiosis but no histologic evidence of Besnoitia spp cysts on skin biopsy sample evaluation. A non–case animal was defined as a donkey displaying no clinical signs of besnoitiosis.

Serology—Immunofluorescent antibody testing and immunoblotting were performed as previously described.20 Briefly, suspensions of B besnoiti Bb1Evora0321 tachyzoites (5 × 106 tachyzoites/mL) in PBS solution (pH, 7.2) were air-dried on glass slides and frozen at −20°C until used. The slides were fixed with ice-cold acetone prior to the examination. Sera were diluted in PBS solution. After serum incubation, the slides were rinsed with fluorescent antibody buffer (25mM Na2CO3, 100mM NaHCO3, and 35mM NaCl; pH, 9.0) and PBS solution prior to conjugate incubation. Fluorescein isothiocyanate–conjugate anti-horse IgG (H+L),d diluted 1:50 in PBS solution with 0.05% Evans blue, was added, and the slides were examined with a fluorescence microscope.e Only peripheral but not apical fluorescence was considered specific.

To prepare antigen-coated membranes by western blotting, samples containing 4 × 107 B besnoiti zoites (either cell culture–derived Bb1Evora0321 tachyzoites or bradyzoites released from the skin of infected cattle20) were treated for 10 minutes at 94°C with nonreducing sample buffer (2% [wt/vol] SDS, 10% [vol/vol] glycerol, and 62mM Tris HCl; pH, 6.8) and electrophoresed in an SDS-polyacrylamide minigel. Separated parasite antigens and marker proteins were electrophoretically transferred to polyvinylidene fluoride membranesf in a semidry transfer system.g The antigen-coated membrane was blocked with PBS-TG,h air-dried overnight, cut into strips, and stored frozen at −20°C until used.

Prior to immunoblot analysis, serum samples were diluted 1:200 in PBS-TG, and the strips were blocked again with PBS-TG. After washing in PBS solution with 0.05% (vol/vol) Tween 20, the strips were incubated with peroxidase conjugate solution (affinity-purified goat anti-horse IgG [H+L],d diluted 1:500 in PBS-TG). After washing in PBS-TG and distilled water, antibody reactions were detected by adding substrate solution (40 μL of H2O2 [30% {vol/vol}] and 30 mg of 4-chloro-1-naphtholi in 40 mL of PBS solution and 20% [vol/vol] methanol). Preinfection and postinfection sera from a heifer experimentally infected with Neospora caninum22 were used as negative controls, and sera of 3 cattle naturally infected with Besnoitia21,23 were used as positive controls. To detect antibodies against Besnoitia, affinity-purified goat anti-horse IgG (H+L)d diluted 1:500 in PBS-TG was used. In both the Besnoitia tachyzoite and bradyzoite immunoblotting, a diagnosis was made on the basis of 10 selected specific bands, which had been described previously.20

Quantitative real-time PCR assay—Quantitative real-time PCR assay for Besnoitia spp was performed on biopsy tissue from each of the 2 suspected cases and 3 of the confirmed cases as previously described.24 This PCR assay had previously been shown to be specific for Besnoitia spp from ungulates (ie, B besnoiti, B bennetti, and B tarandi).24

Deoxyribonucleic acid was extracted from tissue biopsy samples of confirmed cases by proteinase K digestion and purification by means of a kitj according to the manufacturer's protocol. Reactions were performed in a final volume of 20 μL with a commercial master mixk and instrument.l As primersm and probe,m the BbRT2 set was used including the primers Bb3 and Bb6 at a final concentration of 500nM and the probe Bb3-6 at a final concentration of 200nM.24 The cycling conditions were 95.0°C (initial denaturation, 5 minutes), followed by 45 cycles in which the samples were first incubated at 95°C for 10 seconds and then 62.5°C for 30 seconds. After each cycle, the light emission by the fluorophore was measured. Real-time PCR assay results were analyzed with a commercial software program.n

Sequencing of the ITS-1 region of the rDNA—Amplification of the ITS-1 region of the Besnoitia rDNA and sequencing of these amplified fragments was performed essentially as previously described by use of the primers TIM2 and TIM11.23 Forward and reverse sequences obtained from DNA of the tissues of 4 animals from farm A were assembled with computer softwareo and compared with sequences of other Besnoitia spp in GenBank via a bioinformatics search tool.p The obtained consensus sequence was deposited in GenBank (accession No. JQ013812).

Statistical analysis—Descriptive statistics on the occurrence of the disease in the 2 herds and the distribution of the putative risk factors were performed with statistical software.q A frequency distribution of the most common locations of lesions was reported. An epidemic curve was not constructed because of the small number of cases and potential for inaccuracy in owners' recollection of historical data. The association between each of the putative risk factors and the likelihood of besnoitiosis was assessed via either the χ2 test (for categorical variables) or the Student t test (for interval variables). For categorical variables, the risk was quantified with the odds ratio and 95% confidence intervals. For all the tested hypotheses, values of P < 0.05 were considered significant.

Attempted treatment of 1 naturally infected donkey—A confirmed case animal from herd A was donated to the Cornell University Hospital for Animals for attempted treatment with ponazuril. The animal had previously been treated by the owner with trimethoprim sulfamethozazole (30 mg/kg [13.6 mg/lb], PO, q 12 h) for approximately 60 days with no clinical signs of improvement, and skin biopsy samples obtained at initial evaluation were positive for besnoitiosis (histologic confirmation of the presence of Besnoitia spp cysts).

This donkey received 28 days of treatment with ponazuril (15 mg/kg [6.8 mg/lb], PO, q 24 h), followed by an additional 7 days of ponazuril at a higher dosage (50 mg/kg [22.7 mg/lb], PO, q 24 h). For the duration of the trial, daily physical and weekly endoscopic examinations were performed to evaluate clinical response to treatment. Skin biopsy samples were obtained on days 7, 14, 21, and 35 to assess histologic response to treatment. A CBC and serum biochemical analysis were performed on days 0 and 35. At the conclusion of the treatment period, the donkey was castrated and adopted back to the original owner. The testes were preserved in neutral-buffered 10% formalin solution and submitted for histologic examination. The study protocol and care of the donkey were approved by the Cornell University College of Veterinary Medicine Institutional Animal Care and Use Committee.

Results

Description of the outbreak—The besnoitiosis outbreak occurred in a 4-month period in 2010 in 2 donkey herds in rural northeastern Pennsylvania, approximately 22 km (15 miles) apart. The first affected herd (herd A) was composed of 15 miniature donkeys housed together in 1 barn directly adjacent to a heavily wooded area. The barn contained several indoor and outdoor runs, each with between 1 and 3 donkeys, but individuals were frequently switched between groups. All groups alternated through a common grazing paddock located adjacent to the barn. One adult male donkey was housed alone and had no contact with other donkeys except for breeding. All donkeys were fed locally grown grass hay and a commercially formulated concentrate and had a common water source. Management practices were similar for all donkeys. Two horses were kept on the property in a pasture approximately 150 m (500 feet) from the donkey barn and had no direct contact with the donkeys. Two feral barn cats roamed freely on the premises. Wildlife frequently observed in the adjacent wooded area included opossums, raccoons, deer, rodents, and various species of birds.

The second affected herd (herd B) was composed of 13 miniature donkeys and 1 mammoth donkey housed densely together in 1 common paddock. The property was surrounded predominantly by land used for pasture and crop cultivation. All animals had access to a shelter structure located within the paddock. Two horses were kept in the paddock among the donkeys. All animals were fed locally grown grass hay and shared a common water source. No barn cats were present on the property, but wildlife species frequently observed nearby were similar to those on farm A.

Twenty-nine donkeys on the 2 premises were examined. Lesions that were clinically evident and consistent with a diagnosis of besnoitiosis were identified in 8 donkeys. Of those, 6 donkeys were confirmed to be infected with Besnoitia spp on the basis of evaluation of skin biopsy samples (confirmed case animals). Examination of skin biopsy samples failed to demonstrate infection in 2 donkeys with clinical lesions (suspected case animals). The remaining 21 donkeys did not have visible or palpable lesions (non–case animals). Four confirmed case animals and 2 suspected case animals were from herd A, whereas 2 confirmed case animals were from herd B. There was no significant correlation between herd and prevalence of besnoitiosis (P = 0.12). No horses on either premises were found to have lesions suggestive of besnoitiosis.

Signalment and history—The 2 herds comprised 23 females, 5 sexually intact males, and 1 castrated male. Four females, 3 sexually intact males, and 1 castrated male were considered confirmed or suspected case animals. None of the 3 sexually intact males that were confirmed case animals were used for breeding. Males were significantly (P = 0.02) more likely than females to develop besnoitiosis (odds ratio, 9.5; 95% confidence interval, 1.3 to 70). Mean overall age was 62.1 months (median, 60 months; range, 6 to 192 months). The mean age of confirmed or suspected cases was 32.8 months (median, 18 months; range, 12 to 72 months), whereas the mean age of non–case animals was 73.2 months (median, 60 months; range, 6 to 192 months). The mean age of confirmed or suspected case animals was significantly (P = 0.01) less than that of non–case animals. All 8 of the confirmed or suspected case animals had a history of direct contact with a donkey confirmed to be infected with besnoitiosis. Three donkeys, all non–case animals, had no history of contact with an infected donkey. Three of the 8 confirmed or suspected cases had been born on the premises. The mean duration of residence on the farm for confirmed or suspected cases was 20.3 months (median, 18 months; range, 5 to 24 months), whereas the mean duration of residence on the farm for non–case animals was 36.1 months (median, 41 months; range, 2 to 96 months). There was no significant association between the risk of besnoitiosis and contact with an infected donkey, being born on the farm, or duration of residence on the farm.

Clinical signs—Mean time since owner observation of clinical signs in confirmed or suspected case animals was 11.9 months (median, 5 months; range, 0 to 48 months). The most common site of lesions in infected donkeys was the nares (Figure 1), identified in 6 of 6 confirmed case animals and 1 of 2 suspected case animals. Skin lesions and scleral pearls (Figure 2) were identified in 5 of 6 confirmed infected donkeys and 2 of 2 suspected infected donkeys. Affected regions were nonpruritic and variably alopecic. Vulvar lesions were identified in 2 of 4 infected female donkeys.

Figure 1—
Figure 1—

Photographs of nares lesions (A) and scleral pearls (B; arrow) in a donkey with besnoitiosis.

Citation: Journal of the American Veterinary Medical Association 240, 11; 10.2460/javma.240.11.1329

Figure 2—
Figure 2—

Photographs of nasopharyngeal lesions (A; arrows) and epiglottic lesions (B) in a donkey with besnoitiosis.

Citation: Journal of the American Veterinary Medical Association 240, 11; 10.2460/javma.240.11.1329

RhinolaryngoscopyBesnoitia lesions were identified endoscopically in the larynx and nasopharynx in 2 of 4 confirmed case animals and 2 of 2 suspected case animals from herd A (Figure 3). These lesions appeared as multifocal pinpoint papules, similar to those observed externally. Endoscopic lesions were not identified in any non–case animals from herd A.

Figure 3—
Figure 3—

Photomicrograph of a Besnoitia cyst within the dermis of a donkey infected with Besnoitia spp. The protozoal cyst is surrounded by moderate numbers of macrophages, eosinophils, lymphocytes, and plasma cells. The host cell nucleus is indicated (arrow). H&E stain; bar = 200 μm.

Citation: Journal of the American Veterinary Medical Association 240, 11; 10.2460/javma.240.11.1329

Histopathologic findings—Skin biopsy samples in 6 of the 8 donkeys evaluated contained multiple spherical protozoal cysts measuring 150 to 450 μm in diameter with 20- to 50-μm-thick walls, located within the superficial and deep dermis. These cysts contained myriads of tightly packed bradyzoites (Figure 3). The cyst walls were pale amphophilic to eosinophilic with a homogeneous dull appearance, and just inside the wall, there was ≥ 1 flattened peripherally displaced host nucleus. Most cysts were surrounded by small to sometimes moderate infiltrates of lymphocytes, plasma cells, eosinophils, and histiocytes. Some cysts had no associated inflammation. Occasionally, within the dermis, there were foci of central smudged eosinophilic material surrounded by multinucleated giant cells and variable numbers of lymphocytes, plasma cells, histiocytes, and eosinophils (presumed ruptured Besnoitia spp cysts). Other histopathologic changes included mild multifocal epidermal hyperplasia, spongiosis, lymphocytic exocytosis, mild lymphocytic mural folliculitis, and diffuse mild to moderate superficial to mid-dermal perivascular infiltrates composed of mixed mononuclear cells with variable numbers of eosinophils, mild compact orthokeratotic to parakeratotic hyperkeratosis, and occasional small cellular crusts. Occasional cysts appeared to be within or very closely associated with hair follicles. Histopathologic changes within the skin of the other 2 donkeys without protozoal cysts (suspected case animals) included mild diffuse lymphoplasmacytic superficial perivascular infiltrates and occasional multifocal lymphocytic exocytosis.

Serologic evaluation—The mean IFAT reciprocal titer for confirmed case animals was 1,200 (median, 800; range, 400 to 3,200). When confirmed and suspected case animals were grouped together, mean IFAT reciprocal titer was 931 (median, 600; range, 50 to 3,200). All non–case animals had reciprocal titers ≤ 200. Confirmed and suspected case animals combined were positive for a mean of 4.6 bands (median, 5 bands) and 5.9 bands (median, 6.5 bands) on the B besnoiti tachyzoite and bradyzoite immunoblot assays, respectively. Non–case animals were positive for a mean of 1.7 bands (median, 1 band) and 1.9 bands (median, 2 bands) on the B besnoiti tachyzoite and bradyzoite immunoblot assays, respectively.

Quantitative real-time PCR assay—All 5 animals evaluated by PCR assay (3 confirmed and 2 suspected case animals) tested positive for Besnoitia spp. The mean TC value for confirmed case animals was 15.5 (median, 15.2; range, 14.4 to 16.8), whereas the mean TC value for suspected case animals was 24.1 (median, 24.1; range, 19.6 to 28.6).

Sequencing of the ITS-1 region of the rDNA—The ITS-1 sequences obtained from the 4 animals from farm A showed an identity of 100% (344/344) with 2 sequences deposited for B bennetti (AY827839 and AY665399) and an identity of 99.7% (343/344) with sequences deposited for B besnoiti, Besnoitia caprae, and B tarandi (eg, JF314861, HM008988, and AY665400). With the sequences deposited for other Besnoitia spp (B darlingi [AF489696], B oryctofelisi [AY182000], Besnoitia akodoni [AY545987], B neotomofelis [HQ909085], and Besnoitia jellisoni [AF076860]) identities of 82.1% (299/364), 82.4% (295/358), 80.6% (279/346), 80.2% (275/343), and 75.8% (207/273) were observed, respectively.

Ponazuril treatment of a naturally infected donkey—The naturally infected donkey was an 18-month-old male miniature donkey in good body condition. Generalized dermatitis and white pinpoint papules in the nares had first been noticed by the owner 4 months prior to treatment with ponazuril. Initial physical examination revealed severe generalized besnoitiosis with lesions identified on the nares, pinnae, neck, trunk, medial aspect of the thighs, and perineum. Several scleral pearls were noted in both eyes. The remainder of the physical examination was unremarkable. Rhinolaryngoscopy revealed Besnoitia spp cysts covering the nasal mucosa, nasopharynx, and epiglottis. Xylazine hydrochlorider (0.3 mg/kg [0.14 mg/lb], IV) was administered prior to each endoscopic examination. A pronounced sneezing episode occurred each time following sedation, prior to passing the endoscope, and was assumed to be related to the large number of nasal mucosal cysts and nasal mucosal vasodilation following tranquilization. This donkey received 28 days of treatment with ponazuril (15 mg/kg, PO, q 24 h), followed by an additional 7 days of ponazuril at a higher dosage (50 mg/kg, PO, q 24 h).

Histopathologic findings on skin biopsy samples obtained on day 0 from this donkey included multiple 250- to 350-μm-diameter protozoal tissue cysts scattered in the superficial and deep dermis. Each cyst contained hundreds of tiny crescent-shaped basophilic bradyzoites. Cyst walls were 20 to 40 μm thick, were amphophilic to pale eosinophilic, and contained a single to sometimes multiple flattened nuclei. Cysts were generally surrounded by variable numbers of lymphocytes, plasma cells, histiocytes, and eosinophils. Some were associated with no inflammation. Focally in the deep dermis, there was a 200-μm area of smudged eosinophilic material surrounded by moderate numbers of lymphocytes, plasma cells, macrophages, and multinucleated giant cells. This area most likely represented a ruptured protozoal cyst. Other histopathologic changes included mild orthokeratotic hyperkeratosis and mild to moderate superficial to deep lymphoplasmacytic and eosinophilic perivascular inflammation. Throughout the 35-day treatment, no changes were observed in physical examination findings, lesion distribution, or overall lesion severity. Weekly endoscopic examination revealed no change in laryngeal or nasopharyngeal lesions. Similar histopathologic findings were noted on skin biopsy samples from days 7, 14, 21, and 35. No adverse effects of treatment or serum biochemical or hematologic changes were noted at any time.

Histopathologic examination of the testicles revealed a moderate multifocal to coalescing lymphocytic orchitis characterized by multifocal to coalescing lymphocytic infiltrates within the intertubular tissue, which occasionally formed small dense foci that expanded the intertubular space between seminiferous tubules. Multifocally, lymphocytes infiltrated seminiferous tubules, and this was associated with a mild degree of karyorrhectic debris. Multifocally, within the connective tissue and smooth muscle between the epididymal duct lumens, 2 Besnoitia spp tissue cysts were observed. These organisms were surrounded by moderate numbers of lymphocytes with fewer histiocytes, plasma cells, and multinucleated giant cells. Throughout the connective tissue of the epididymis, there were mild multifocal perivascular aggregates of lymphocytes.

Discussion

In the present study, a diagnosis of besnoitiosis was confirmed by histologic identification of Besnoitia spp cysts in the tissues of 6 donkeys on 2 farms in Pennsylvania in 2010. The histologic findings compatible with besnoitiosis were further confirmed by a real-time PCR assay previously shown to be specific for a Besnoitia sp from ungulates.24 Sequencing of the ITS-1 region of the recombinant DNA amplified from tissues of donkeys from farm A revealed a 100% identity with sequences deposited for B bennetti, further confrming our initial diagnosis. Additionally, several serologic tests (2 immunoblot tests and an IFAT) were used to identify serologic evidence of infection. Because neither B bennetti antigens nor control sera were available, we applied antigens obtained from B besnoiti tachyzoites and bradyzoites and control sera from B besnoiti–infected cattle.20 Because of the close phylogenetic relationship of B besnoiti and B bennetti, cross-reactions were expected. The concordance of the serologic and clinical findings in the cases described in the present report suggested that the use of B besnoiti–based tests for the serologic diagnosis of besnoitiosis in donkeys might be very valuable until tests applying B bennetti antigens are available.

In this outbreak, young animals appeared at greater risk for the development of besnoitiosis (mean age of confirmed or suspected case animals, 32.8 months; median, 18 months; mean age of non–case animals, 73.2 months; median, 60 months), which is similar to a previously described outbreak in a herd of miniature donkeys, in which all infected animals were < 6 years of age.17 The potential for resolution of clinical signs of besnoitiosis in infected donkeys is currently unknown, but it is possible that the development of acquired immunity after exposure or infection is a relevant factor in the observed age discrepancy. However the total number of animals in the present outbreak was small; therefore, definitive conclusions cannot be drawn. The duration of time the donkey had lived on the premises was not a significant risk factor for development of besnoitiosis in the population of the present study.

In the present study, the most common site of lesions in infected donkeys was the nares, identified in 6 of 6 confirmed case animals and 1 of 2 suspected case animals. A similarly high number of affected animals developed skin lesions and scleral pearls (5/6 confirmed case animals and 2/2 suspected case animals), which is consistent with previous descriptions.15–18 This is the first study describing endoscopic Besnoitia spp lesions, and we found rhinolaryngoscopy to be effective at identifying infection in only 3 of 6 confirmed case animals. In contrast to previous reports15–17 identifying occasional infected donkeys with signs of poor health such as weight loss, reduced appetite, and excessive hair loss, all infected animals in the present study were in generally good condition with no overt signs of ill thrift. Furthermore, focal areas of marked alopecia, which has been previously described as a clinical sign of besnoitiosis,15–17 were not observed in any of the affected animals in the present study.

Male donkeys were at an increased risk for infection, compared with female donkeys, in the present study. The reason for this difference is unknown, given that there were no appreciable differences in the management and housing of the infected male and infected female animals in either of the 2 herds and none of the infected male donkeys were used for breeding. Sex predisposition in B besnoiti infection in cattle has yet to be fully elucidated. Some studies have suggested that females are at higher risk for infection, whereas others have had similar findings for males.7

Although contact with an infected donkey was not identified as a risk factor for disease in this study, transmission between donkeys has been proposed17 and would suggest a similar pathogenesis to besnoitiosis in cattle, in which introduction of an infected animal into a naïve herd is a known risk factor for the development of disease.7,20 Arthropods have been previously reported as potential vectors in the transmission of besnoitiosis in cattle,7 but the potential for similar transmission in equids is currently unknown.

The current gold standard for diagnosis of besnoitiosis in donkeys is histologic identification of Besnoitia spp cysts in tissue. Two donkeys in the present report developed external and endoscopic lesions consistent with besnoitiosis but had no Besnoitia spp cysts detected on evaluation of skin biopsy samples. The effect of biopsy location on diagnosis is not currently known, but it is possible that Besnoitia spp cysts would have been identified had the biopsies been performed at other sites or had more than 1 biopsy sample been obtained. With multifocal or generalized dermatosis, it is generally recommended that clinicians obtain multiple samples and obtain specimens from a variety of lesions because histologic examination of the full spectrum of lesions gives more information than does the examination of 1 lesion.25 Therefore, we recommend that repeated skin biopsy samples should be obtained from animals with clinical evidence of infection but without Besnoitia spp cysts detected following initial biopsy. Although histologic examination, IFAT, and immunoblot results were unable to confirm besnoitiosis infection in either of the 2 suspected case animals, both animals had lesions and PCR results suggestive of disease. There is a report24 of similar findings in cattle, in which 2 clinically positive animals with IFAT titers < 50 tested positive for besnoitiosis by PCR assay. The reason for this observation is unknown.

In the present study, IFAT reciprocal titers of confirmed and suspected infected donkeys were higher than those of donkeys with no signs of besnoitiosis. The same was true for the number of positive bands on both the tachyzoite and bradyzoite immunoblot assays in confirmed and suspected case animals, compared with non–case animals. Both IFAT and immunoblot have been validated for the diagnosis of besnoitiosis in cattle, with B besnoiti used as an antigen.20 If criteria for serologic diagnosis in cattle by use of IFAT and immunoblot were applied to the donkey sera in these 2 herds (IFAT reciprocal titer, > 100; immunoblot, > 3 bands), some of the clinically negative animals (ie, non–case animals) tested serologically positive. This is similarly described for outbreaks of besnoitiosis in cattle, in which usually only a portion of serologically positive animals develop clinical signs.7,20

All 5 animals evaluated by PCR assay tested positive for Besnoitia spp. Lower TC values were observed in confirmed case animals than in suspected case animals. That is, fewer cycles were required to obtain positive results in animals in which Besnoitia spp cysts were observed histologically. Furthermore, fewer cycles tended to be necessary in individuals possessing the greatest concentration of antibodies. This observation has been made in cattle with besnoitiosis, leading to the hypothesis that higher parasite concentrations induce stronger antibody responses.24

A limitation of the present study is small sample size. Statistically significant findings may have been influenced by lack of power, and further analysis of the epidemiological and serologic features of infected donkeys on the basis of data from larger populations is needed. Although inadequate sample size precluded further analysis of serologic and molecular results in the present study, further studies are warranted to investigate the correlation between IFAT, immunoblot, and PCR assay results and clinical signs of disease for potential validation as diagnostic screening tests for besnoitiosis in donkeys.

Ponazuril is a triazine antiprotozoal agent approved for use in horses with cidal activity against Sarcocystis neurona, an apicomplexan protozoan parasite closely related to Besnoitia. It was not effective at treating besnoitiosis in 1 donkey, even at 3 times (15 mg/kg, PO, q 24 h for 28 days) and then 10 times (50 mg/kg, PO, q 24 h for 7 days) the recommended dose in horses. Reported attempts to treat infected animals with trimethoprim-sulfamethozazole and nitazoxinide have had a similar lack of success,15–17 but limited case numbers and previous lack of knowledge regarding management of clinically affected animals make drawing useful conclusions from the existing literature difficult.

Testicular and epididymal involvement in a donkey with besnoitiosis has been reported18 and is similar to the distribution of Besnoitia spp lesions observed in cattle, in which infected bulls can develop orchitis or epididymitis,26 resulting in temporary or permanent infertility.5,7 The resulting inflammation and parenchymal destruction cause abnormal sperm morphology as well as decreased fertility. The effect of besnoitiosis on fertility in donkeys is currently unknown. In the case described in the present report, the lack of active spermatogenesis reflects the young age of the donkey. Given the degree of orchitis and lymphocytic infiltrate into seminiferous tubules with tubular destruction, fertility would have likely been altered.

The present report may highlight the possible underreporting of besnoitiosis in the United States. The investigation of the 2 herds in the present study has subsequently led to tissue samples being sent to Cornell University over the past 20 months that have confirmed a diagnosis of besnoitiosis on the basis of histopathologic testing in donkeys in several states, including New York, Massachusetts, New Jersey, Montana, Minnesota, Vermont, Tennessee, Texas, Washington, and Oregon (Figure 4). Similar to the cases described in the present report, infected donkeys frequently have a history of chronic dermatitis that is unresponsive to routine topical and systemic treatments. Both male and female donkeys appear to be affected, and most are < 3 years old. In some instances, the diagnosis of one donkey has led to subsequent identification of other infected donkeys within a herd. Several donkeys have a history of being recently purchased and arriving at their new residence with clinical signs of besnoitiosis, including lesions on the nares, neck, and vulva and scleral pearls. This incoming information suggests that the disease is more prevalent than previously known and may be emerging as an important disease of donkeys in the United States. Further study of the pathogenesis and transmission of besnoitiosis as well as validation of diagnostic screening tests will assist clinicians in the diagnosis and prevention of this disease in donkey populations. Although besnoitiosis has not yet been reported in horses in North America, equine cases have been described in Africa,12,13,26 and the potential for similar infections in the United States cannot be ignored or discounted.

Figure 4—
Figure 4—

Geographic distribution (stars) of donkeys in which besnoitiosis was diagnosed on the basis of results of histologic testing in the United States from August 2010 to March 2012.

Citation: Journal of the American Veterinary Medical Association 240, 11; 10.2460/javma.240.11.1329

ABBREVIATIONS

IFAT

Immunofluorescent antibody test

ITS

Internal transcribed spacer

PBS-TG

Fish gelatine liquid containing 0.05% (vol/vol) PBS solution and 2% (vol/vol) Tween 20

TC

Threshold cycle

a.

Marquis, Bayer Animal Health, Shawnee Mission, Kan.

b.

Provided by Bayer Animal Health, Shawnee Mission, Kan.

c.

Robbins Instruments, Chatham, NJ.

d.

Jackson ImmunoResearch Laboratories, West Grove, Pa.

e.

Olympus Vanox AHBT3, Hamburg, Germany.

f.

Immobilon-P, Millipore, Germany.

g.

Pharmacia Biotech, Freiburg, Germany.

h.

SERVA, Heidelberg, Germany.

i.

Sigma, Deisenhofen, Germany.

j.

DNeasy Tissue Kit, QIAGEN, Hilden, Germany.

k.

iQ Supermix, Biorad Life Science Research, Hercules, Calif.

l.

CFX384, Biorad Laboratories, Munich, Germany.

m.

MWG Biotech, Ebersberg, Germany.

n.

CFX Manager Software, version 1.6, Biorad Laboratories, Munich, Germany.

o.

Lasergene, version 7.0 software 161, DNASTAR Inc, Madison, Wis.

p.

BLAST, National Center for Biotechnology Information, National Institutes of Health, Bethesda, Md. Available at: blast.ncbi.nlm.nih.gov/. Accessed Sep 11, 2011.

q.

Statistix, version 9.0, Analytical Software Inc, Tallahassee, Fla.

r.

Rompun, Bayer Animal Health, Shawnee Mission, Kan.

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