Nematode-associated intramural alimentary nodules in pumas are histologically similar to gastrointestinal eosinophilic sclerosing fibroplasia of domestic cats.

Intramural alimentary nodules in the gastric pylorus and proximal duodenum are a common finding in free-ranging pumas (Puma concolor) in North America, and are often associated with the presence of an indwelling nematode (most commonly Cylicospirura spp.). This study compares the histological, histochemical and immunohistochemical appearance of three proximal gastrointestinal nodules in pumas with four cases of eosinophilic sclerosing fibroplasia in domestic cats. Histologically, the pattern of inflammation and repair was strikingly similar, consisting of lamillated anastomosing trabeculae of dense sclerotic collagen with interspersed inflammatory cells and reactive fibroblasts. The stromal trabeculae were histologically reminiscent of osteoid and were uniformly positive for collagenous protein by Masson's trichrome stain and negative for mineralized osteoid deposits with Von Kossa's stain. Trabecular cells expressed osteonectin, but not osteocalcin immunohistochemically. Collectively, these findings are most consistent with a stroma comprised of dense collagenous trabeculae that resembles, but is distinct, from osteoid. Both the puma and domestic cat lesions demonstrated an eosinophilic inflammatory component; however, eosinophils were present in small numbers in the puma nodules relative to the nodules in domestic cats. These entities likely represent a unique and stereotypic gastrointestinal repair response of felids, given their similar histological, histochemical and immunohistochemical profiles.

Characteristics of Thoroughbred and Quarter Horse racehorses that sustained a complete scapular fracture.

REASONS FOR PERFORMING STUDY: To determine if scapular fractures occur in racehorses with distinctive characteristics. OBJECTIVES: To test the hypothesis that Thoroughbred (TB) and Quarter Horse (QH) racehorses with a scapular fracture have similar characteristics that are different from those of their respective racetrack populations. METHODS: Necropsy findings, case details, last race information and career earnings for TB and QH racehorses that had a scapular fracture in California between 1990 and 2008 were retrospectively compared between breeds. Horse signalment, career earnings, career starts and race characteristics were obtained for all California racehorses. Comparisons were made between affected horses, other racehorses that died, and all horses that raced, in California during the 19 year period. RESULTS: Seventy-three TB and 28 QH racehorses had a similar, complete comminuted scapular fracture with an articular component, and right forelimb predilection. The QHs had a higher incidence of scapular fracture incurred during racing than TBs (0.98 vs. 0.39/1000 starters). The TB and QH incident rates for musculoskeletal deaths incurred racing were 20.5 and 17.5/1000 starters, respectively; however, a greater proportion of TB musculoskeletal deaths occurred training (40% vs. 8%). Horses with a scapular fracture were more likely to be male and aged 2 or ≥ 5 years than the racetrack population. Most affected QHs (64%) were 2-year-olds; most TBs (74%) were aged ≥ 3 years. Scapular fractures occurred more commonly during racing in QHs (70%) than TBs (44%). Race-related scapular fracture was more likely to occur in a Maiden race than in a non-Maiden race. Horses with a scapular fracture had fewer career starts than the racetrack population. CONCLUSIONS AND POTENTIAL RELEVANCE: Despite breed differences for signalment and exercise distances, both breeds incur a complete scapular fracture that is more likely to occur in the right scapula of young and older, male racehorses, early in their race career or after few races. Quarter Horses sustain a catastrophic scapular fracture more frequently than TBs.

Systemic Coxiella-like infection with myocarditis and hepatitis in an eclectus parrot (Eclectus roratus).

A multiorgan infection with a Coxiella-like organism was determined to be the cause of death of a female eclectus parrot (Eclectus roratus). The diagnosis was based on gross lesions, histopathology, Gimenez and Gram special stains, immunohistochemistry, electron microscopy, and polymerase chain reaction amplification and sequencing of a bacterial 16s rRNA gene fragment isolated from hepatic and cardiac tissue. Gross postmortem examination revealed multifocal to coalescing foci of hepatic necrosis. The most significant histologic lesions included multifocal lymphohistiocytic necrotizing hepatitis, locally extensive lymphoplasmacytic myocarditis, and myocardial degeneration and necrosis. Intralesional cytoplasmic organisms were identified in cardiomyocytes, biliary epithelium, and pancreatic exocrine cells. This is the first description of a Coxiella-like organism with wide-ranging cellular tropisms in a psittacine bird. In addition, lymphoplasmacytic neuritis, myositis, splenitis, airsacculitis, and enteritis were detected. It is also the first report of a Coxiella-like infection in an eclectus parrot.

Endogenous transplacental transmission of Neospora hughesi in naturally infected horses.

Over a 2-yr study period, we investigated possible endogenous transplacental transmission of Neospora hughesi in 74 mare and foal pairs following the diagnosis of neuronal neosporosis in a weanling foal. Presuckle and postsuckle serum of each foal, serum and colostrum of each periparturient mare, and serum of each mare and foal pair, collected at 3-mo intervals thereafter, were tested for N. hughesi using an indirect fluorescent antibody test (IFAT). Furthermore, whole blood and colostrum samples and placentae were tested for the presence of N. hughesi by real-time PCR. The mares' seroprevalence at foaling based on IFAT (titer ≥ 160) was 52 and 6% in 2006 and 2007, respectively. Colostral antibodies against N. hughesi were detected in 96 and 11% of the mares in the 2-yr study. With the exception of 3 foals, all remaining foals were born seronegative to N. hughesi. Passive transfer of colostral antibodies to N. hughesi was documented in 15 foals. Three foals born from 2 different mares had presuckle antibodies at a titer ranging from 2,560 to 20,480. All 3 foals were born healthy. Two foals were born to the same dam that also gave birth to the weanling diagnosed with neuronal neosporosis in 2005. The third foal was born to a second mare with no previous foaling history at the farm. Seroconversion was documented in 10 foals and 9 mares over the 2-yr study. All blood and colostrum samples tested PCR negative for N. hughesi. Only 1 placenta collected in 2007 from the mare with the 2 congenitally infected foals tested PCR positive for N. hughesi. In conclusion, N. hughesi persisted in this population via endogenous transplacental infection.

Lyme neuroborreliosis in 2 horses.

Lyme neuroborreliosis--characterized as chronic, necrosuppurative to nonsuppurative, perivascular to diffuse meningoradiculoneuritis--was diagnosed in 2 horses with progressive neurologic disease. In 1 horse, Borrelia burgdorferi sensu stricto was identified by polymerase chain reaction amplification of B burgdorferi sensu stricto-specific gene targets (ospA, ospC, flaB, dbpA, arp). Highest spirochetal burdens were in tissues with inflammation, including spinal cord, muscle, and joint capsule. Sequence analysis of ospA, ospC, and flaB revealed 99.9% sequence identity to the respective genes in B burgdorferi strain 297, an isolate from a human case of neuroborreliosis. In both horses, spirochetes were visualized in affected tissues with Steiner silver impregnation and by immunohistochemistry, predominantly within the dense collagenous tissue of the dura mater and leptomeninges.

Radiofrequency-induced thermal inactivation of Toxoplasma gondii oocysts in water.

Toxoplasma gondii, a ubiquitous parasitic protozoan, is emerging as an aquatic biological pollutant. Infections can result from drinking water contaminated with environmentally resistant oocysts. However, recommendations regarding water treatment for oocyst inactivation have not been established. In this study, the physical method of radiofrequency (RF) power was evaluated for its ability to inactivate T. gondii oocysts in water. Oocysts were exposed to various RF energy levels to induce 50, 55, 60, 70 and 80 degrees C temperatures maintained for 1 min. Post-treatment oocyst viability was determined by mouse bioassay with serology, immunohistochemistry and in vitro parasite isolation to confirm T. gondii infections in mice. None of the mice inoculated with oocysts treated with RF-induced temperatures of > or =60 degrees C in an initial experiment became infected; however, there was incomplete oocyst activation in subsequent experiments conducted under similar conditions. These results indicate that T. gondii oocysts may not always be inactivated when exposed to a minimum of 60 degrees C for 1 min. The impact of factors such as water heating time, cooling time and the volume of water treated must be considered when evaluating the efficacy of RF power for oocyst inactivation.

Ultrastructural and molecular confirmation of the development of Sarcocystis neurona tissue cysts in the central nervous system of southern sea otters (Enhydra lutris nereis).

In 2004, three wild sea otters were diagnosed with putative Sarcocystis neurona-associated meningoencephalitis by histopathology and immunohistochemistry. Schizonts, free merozoites and tissue cysts were observed in the brains of all three infected animals. Tissue cysts walls from sea otter 1 (SO1) stained positively using anti-S. neurona polyclonal antiserum. However, positive staining does not preclude infection by closely related or cross-reactive tissue cyst-forming coccidian parasites. Two immature tissue cysts in the brain of SO1 were examined using transmission electron microscopy. Ultrastructural features included cyst walls with thin villous projections up to 1 microm long with tapered ends and a distinctive, electron-dense outer lining layer composed of linearly-arranged, semi-circular structures with a "hobnailed" surface contour. Small numbers of microtubules extended down through the villi into the underlying granular layer. Metrocytes were short and plump with an anterior apical complex, 22 sub-pellicular microtubules, numerous free ribosomes and no rhoptries. Some metrocytes appeared to be dividing, with two adjacent nuclear profiles. Collectively these ultrastructural features were compatible with developing protozoal cysts and were similar to prior descriptions of S. neurona tissue cysts. Panspecific 18S rDNA primers were utilized to identify protozoa infecting the brains of these otters and DNA amplification and additional sequencing at the ITS1 locus confirmed that all three otters were infected with S. neurona. No other Sarcocystis spp. were detected in the brains or skeletal muscles of these animals by immunohistochemistry or PCR. We believe this is the first ultrastructural and molecular confirmation of the development of S. neurona tissue cysts in the CNS of any animal.

Cytokine gene signatures in neural tissue of horses with equine protozoal myeloencephalitis or equine herpes type 1 myeloencephalopathy.

This study was designed to determine the relative levels of gene transcription of selected pathogens and cytokines in the brain and spinal cord of 12 horses with equine protozoal myeloencephalitis (EPM), 11 with equine herpesvirus type 1 (EHV-1) myeloencephalopathy, and 12 healthy control horses by applying a real time pcr to the formalin-fixed and paraffin-embedded tissues. Total rna was extracted from each tissue, transcribed to complementary dna (cDNA) and assayed for Sarcocystis neurona, Neospora hughesi, EHV-1, equine GAPDH (housekeeping gene), tumour necrosis factor (TNF)-alpha, interferon (IFN)-gamma, interleukin (IL)-1beta, IL-2, IL-4, IL-6, IL-8, IL-10 AND IL-12 p40. S neurona cdna was detected in the neural tissue from all 12 horses with EPM, and two of them also had amplifiable cDNA of N hughesi. The relative levels of transcription of protozoal cdna ranged from 1 to 461 times baseline (mean 123). All the horses with ehv-1 myeloencephalopathy had positive viral signals by PCR with relative levels of transcription ranging from 1 to 1618 times baseline (mean 275). All the control horses tested negative for S neurona, N hughesi and EHV-1 cdna. The cytokine profiles of each disease indicated a balance between pro- and anti-inflammatory markers. In the horses with epm the pro-inflammatory Th1 cytokines (IL-8, TNF-alpha and IFN-gamma) were commonly expressed but the anti-inflammatory Th2 cytokines (IL-4, IL-6 AND IL-10) were absent or rare. In the horses with ehv-1 the proinflammatory cytokine IL-8 was commonly expressed, but IL-10 and IFN-gamma were not, and TNF-alpha was rare. Tissue from the control horses expressed only the gene GAPDH.

Pulmonary besnoitiasis in captive maras (Dolichotis patagonum) associated with interstitial pneumonia.

Six captive maras (Dolichotis patagonum) were found to have cysts consistent with Besnoitia in the lungs by light microscopy and electron microscopy. Three were juveniles that died with no premonitory signs during a 17-month period. The most prominent finding consisted of severe, subacute, diffuse interstitial pneumonia with syncytia and Besnoitia cysts. The severity of pneumonia correlated with the number of cysts and the presence of lytic cysts, often surrounded by chronic granulomatous inflammation. Disseminated infection was observed in one of these maras. The three other maras died of unrelated conditions and had a few Besnoitia cysts in their lungs with mild or no inflammation associated. This is the first report of besnoitiasis in maras and of its association with fatal interstitial pneumonia in any species. Although other agents may have caused the interstitial pneumonia described here, it is possible that zoites released from lytic cysts were involved in the development of this lesion.

Meningoencephalitis associated with an unidentified apicomplexan protozoan in a Pacific harbor seal.

A Pacific harbor seal (Phoca vitulina richardsii) was found on the central California coast with neurologic signs and labored breathing, which were unresponsive to treatment. Necropsy revealed a nonsuppurative necrotizing meningoencephalitis, a multilocular thymic cyst, and nonsuppurative cystitis and renal pyelitis. Microscopic examination revealed protozoans in the brain, thymic cyst, and bladder mucosa. Ultrastructurally, the protozoal tachyzoites were different from those of Neospora caninum, Toxoplasma gondii, and Sarcocystis neurona; the rhoptries were small and had electron-dense contents, and the organism divided by endodyogeny. Specific antibodies were not detected in serum using agglutination (N. caninum, T. gondii) and immunoblot assays (S. neurona). Immunohistochemistry for these organisms was negative. Polymerase chain reaction on brain tissue using specific primers did not amplify T. gondii deoxyribonucleic acid. The meningoencephalitis in this seal thus appears to have been caused by a novel protozoan.

Characterization of monoclonal antibodies developed against Sarcocystis neurona.

Equine protozoal myeloencephalitis (EPM), caused by a protozoal parasite infection of the central nervous system, is the most commonly diagnosed neurologic disease of horses in North America. In specific regions of the United States approximately 50% of the horse population is seropositive to Sarcocystis neurona. However, not all seropositive horses develop clinical signs. Detailed clinical examination, along with cerebrospinal fluid antibody evaluation are often used to diagnose EPM. Postmortem evaluation of the brain stem and spinal cord for histopathologic lesions compatible with nonsuppurative meningoencephalomyelitis is used for reaching a diagnosis since organisms are difficult to detect by routine staining methods. Immunohistochemical staining aids detection of organisms; however, the polyclonal antibodies that react with S. neurona may react with merozoites of other closely related Sarcocystis species. In this study, two different monoclonal antibodies, mAb 2A7-18 and mAb 2G5-2, were developed against the merozoite stage of S. neurona UCD-SN1 strain. The antibodies were evaluated by immunoblot, immunofluorescence, immuno-electron microscopy and immunohistochemistry for their ability to react with S. neurona. MAb 2G5-2 reacted with antigenically distinct S. neurona isolates whereas mAb 2A7-18 appeared to be limited in its ability to recognize different isolates. These two monoclonal antibodies recognize protein epitopes of two different immunodominant proteins of S. neurona.

Evaluation of an indirect fluorescent antibody test (IFAT) for demonstration of antibodies to Toxoplasma gondii in the sea otter (Enhydra lutris).

An indirect fluorescent antibody test (IFAT) for detection of Toxoplasma gondii infection was validated using serum from 77 necropsied southern sea otters (Enhydra lutris nereis) whose T. gondii infection status was determined through immunohistochemistry and parasite isolation in cell culture. Twenty-eight otters (36%) were positive for T. gondii by immunohistochemistry or parasite isolation or both, whereas 49 (64%) were negative by both tests. At a cutoff of 1:320, combined values for IFAT sensitivity and specificity were maximized at 96.4 and 67.3%, respectively. The area under the receiver-operating characteristic curve for the IFAT was 0.84. A titer of 1:320 was used as cutoff when screening serum collected from live-sampled sea otters from California (n = 80), Washington (n = 21), and Alaska (n = 65) for T. gondii infection. Thirty-six percent (29 out of 80) of California sea otters (E. lutris nereis) and 38% (8 out of 21) of Washington sea otters (E. lutris kenyoni) were seropositive for T. gondii, compared with 0% (0 out of 65) of Alaskan sea otters (E. lutris kenyoni).

Redescription of Neospora caninum and its differentiation from related coccidia.

Neospora caninum is a protozoan parasite of animals, which before 1984 was misidentified as Toxoplasma gondii. Infection by this parasite is a major cause of abortion in cattle and causes paralysis in dogs. Since the original description of N. caninum in 1988, considerable progress has been made in the understanding of its life cycle, biology, genetics and diagnosis. In this article, the authors redescribe the parasite, distinguish it from related coccidia, and provide accession numbers to its type specimens deposited in museums.

Sarcocystis neurona reacting antibodies in Missouri feral domestic cats (Felis domesticus) and their role as an intermediate host.

Sarcocystis neurona is the parasite associated with equine protozoal myeloencephalitis (EPM). Recently, cats (Felis domesticus) have been implicated as a potential intermediate host in the life cycle of S. neurona. This study was initiated to determine whether cats have antibodies that react to S. neurona antigens similar to antibodies from horses with EPM, and to evaluate the role of cats as intermediate hosts in the parasite's life cycle. Nine feral cats were used for analysis. Only one had antibodies reacting to S. neurona antigens. Muscle tissue from this cat, with detectable sarcocysts in the tongue, was fed to an opossum (Didelphis virginiana). The opossum shed sporocysts, which were then fed to gamma-interferon receptor knockout mice. Histopathology, immunohistochemistry, parasite isolation and molecular analysis were used to examine the pathology and associated parasites in the mice. The study suggests that the domestic cat can serve as an intermediate host to S. neurona or a S. neurona-like organsim.

Immune responses in pregnant cattle and bovine fetuses following experimental infection with Neospora caninum.

Humoral and cell-mediated immune (CMI) responses [i.e. proliferative responses and gamma interferon (IFN-gamma) production], were elicited in five cows infected between 159 and 169 days of gestation by a combined intravenous-intramuscular inoculation of Neospora caninum tachyzoites. Analysis of antigen-specific immunoglobulin (IgG) subclasses revealed a predominant IgG2 response in two cows, a mixed IgG1-IgG2 response in two other cows and a predominant IgG1 response in one cow. No correlation was found between IgG2 titers and IFN-gamma levels. CD4-T cells were responsible for the CMI responses in peripheral blood mononuclear cells from three infected cows. All five fetuses removed from infected dams at week 9 post-infection (219-231 days of gestation) mounted strong Neospora-specific humoral responses and had a predominant IgG1 response, regardless of their ability to produce IFN-gamma. However, CMI responses were highly variable between fetuses. These data indicate the complexity of the immune mechanisms associated with Neospora infection in both the dams and their fetuses.

Isolation and characterization of two parasitic protozoa from a Pacific harbor seal (Phoca vitulina richardsi) with meningoencephalomyelitis.

Two species of protozoans were isolated from a harbor seal with fatal meninogoencephalitis. Serologic reactivity was detected to both Sarcocystis neurona and Toxoplasma gondii. Parasites associated with brain inflammation and necrosis reacted only with immunohistochemical stains utilizing polyclonal antisera raised against Sarcocystis neurona. However, 2 distinct parasites were observed in cell cultures derived from the seal's brain tissue. These parasites were separated by mouse passage and limiting dilution. Purified zoites from 1 isolate (HS1) reacted strongly with polyclonal antiserum to S. neurona and with the harbor seal's own serum (1:2,560 for each) on indirect immunofluorescent antibody tests (IFAT), but weakly to antisera to T. gondii and Neospora caninum (1:40). Zoites from the second isolate (HS2) reacted positively with T. gondii polyclonal antiserum (1:81,920) and with the harbor seal's own serum (1:640), but weakly to S. neurona and N. caninum antisera (1:80 or less). Amplification and sequence analysis of protozoal DNA encoding portions of the 18s ribosomal RNA (18s rDNA) and the adjacent first internal transcribed spacer (ITSI) were performed for both isolates, and resulting sequences were compared to those from similar protozoans. Based on molecular characterization, parasite morphology, serologic reactivity, histology, and immunohistochemistry, HS1 was indistinguishable from S. neurona, and HS2 was indistinguishable from T. gondii.

Acute Sarcocystis falcatula-like infection in a carmine bee-eater (Merops nubicus) and immunohistochemical cross reactivity between Sarcocystis falcatula and Sarcocystis neurona.

An unidentified Sarcocystis falcatula-like infection was diagnosed in a captive bee-eater (Merops nubicus) in a zoo in Florida. The bird died suddenly, probably due to protozoa-associated pneumonia. Protozoal schizonts were found in lungs and heart, and immature sarcocysts were seen in skeletal muscles. Ultrastructurally, schizonts were located in capillary endothelium and merozoites lacked rhoptries, consistent with the structure of Sarcocystis species. Sarcocysts were immature, microscopic, and contained only metrocytes. The sarcocyst wall had finger-like villar protrusions that were up to 0.7 microm long and up to 0.2 microm wide. The villar protrusions lacked microtubules, characteristically seen in sarcocysts of S. falcatula. Antigenically, parasites in lungs and muscles of the bee-eater reacted with a varying intensity with polyclonal rabbit antisera to S. falcatula and Sarcocystis neurona. Results indicated that sarcocysts in the bee-eater were morphologically different from the reported structure for sarcocysts of other S. falcatula infections.

Isolation and characterization of Sarcocystis from brain tissue of a free-living southern sea otter (Enhydra lutris nereis) with fatal meningoencephalitis.

A protozoan was isolated in cell culture from the brain of a free-ranging sea otter with fatal meningoencephalitis. The biological history of this otter, a study animal being monitored via an intraperitoneal radio transmitter, is summarized. Histologically, protozoal parasites were associated with areas of brain inflammation and necrosis in the cerebrum and cerebellum. Morphology and measurements of fixed, Giemsa-stained protozoal zoites growing on coverslips were consistent with Sarcocystis. These parasites reacted only with polyclonal antisera raised against S. neurona on immunohistochemistry. Cell culture-derived zoites reacted strongly with polyclonal antiserum to S. neurona on indirect fluorescent antibody tests. Amplification of portions of the 18S ribosomal DNA and the adjacent first internal transcribed spacer were performed. The resulting sequences were compared with published sequences from similar apicomplexan protozoa. This isolate (SO SN1), was indistinguishable from S. neurona, based on parasite morphology, antigenic reactivity and molecular characterization.

Acute pulmonary Sarcocystis falcatula-like infection in three Victoria crowned pigeons (Goura victoria) housed indoors.

Three free-roaming Victoria crowned pigeons (Goura victoria) housed in a completely enclosed tropical exhibit were found dead without antemortem signs of illness. The birds died within 9 days of each other. Gross necropsy revealed moderate pulmonary edema in all three birds. Histopathologic examination revealed pulmonary edema and pulmonary protozoal merozoites compatible with Sarcocystis spp., Toxoplasma gondii, or Neospora spp. infection. Immunohistochemical staining for T. gondii and Neospora spp. were negative. Immunohistochemical staining identified a Sarcocystis falcatula-like parasite in all three birds. It is suspected that new exhibit soil contaminated with feces from the Virginia opossum (Didelphis virginiana) was the source of the infective sporocysts.

A POLYGEN-adjuvanted killed Neospora caninum tachyzoite preparation failed to prevent foetal infection in pregnant cattle following i.v./i.m. experimental tachyzoite challenge.

Cattle immunised with a POLYGEN-adjuvanted killed Neospora caninum tachyzoite preparation were previously shown to produce interferon (IFN)-gamma at levels similar to those of tachyzoite-infected cattle. In view of the critical role of IFN-gamma in resistance of mice to N. caninum infection, these results prompted us to test the POLYGEN-adjuvanted preparation in pregnant cattle to determine whether it will be able to prevent foetal infection following an experimental tachyzoite challenge. Seven heifers were immunised at 35 and 63 days of gestation with the POLYGEN-adjuvanted preparation, while five heifers were inoculated with POLYGEN alone at the same days of gestation. Four weeks later, all heifers were challenged with a combined i.v./i.m. inoculation of tachyzoites. The same challenge was given to seven unimmunized heifers at the same stage of gestation. An additional unimmunized heifer was inoculated with uninfected monolayer cell culture material. All challenged heifers, immunized and unimmunized, had infected foetuses. Immunized heifers developed both parasite-specific humoral and cellular immune responses, characterised by increased IFAT titres, a predominant IgG1 response, elevated lymphoproliferative response and IFN-gamma production. Following tachyzoite challenge, they developed an anamnestic humoral response and produced similar amounts of IgG1 and IgG2 antibodies, but did not have an anamnestic cellular immune response. The lack of anamnestic cellular immune response and/or the large i.v/i.m tachyzoite inoculum may have contributed to the failure of the preparation.