Toxoplasmosis in ring-tailed lemurs (Lemur catta) and a peahen (Pavo cristatus) in a zoological collection caused by the common toxoplasma genotype in wild animals in the US.

Toxoplasmosis is caused by the ubiquitous Apicomplexan protozoan Toxoplasma gondii. This pathogen affects domestic and wildlife species, but prosimians including ring-tailed lemurs (Lemur catta) are highly susceptible to infection with high mortality rates. Avian species are considered resistant to infection and are often used in surveillance efforts to determine genotypes of T. gondii present in geographical areas. This study describes the gross and histologic lesions of an outbreak of toxoplasmosis in a university-run zoological collection involving three ring-tailed lemurs and a peahen (Pavo cristatus). DNA was extracted from the liver of the lemurs and peahen to determine the genotype of T. gondii by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP), which revealed that all samples were ToxoDB PCR-RFLP genotype #5 (haplogroup 12) that is common in wildlife in North America.

Spatial and seasonal analysis and geovisualization of Fasciola hepatica-free bovine bacillary hemoglobinuria outbreaks in eastern Uruguay, 1999-2019.

Bovine bacillary hemoglobinuria (BBH) produced by Clostridium novyi type D, is an endemic, highly fatal disease of cattle in the temperate grassland region of eastern Uruguay. A previous study showed that in this region, BBH is not associated with Fasciola hepatica or any other known focal-ischemic liver injury, so the reasons for its high incidence remains undetermined. The objective of this study was to analyze data from 45 Fasciola hepatica-free BBH outbreaks (1999-2019) in order to find common animal, seasonal and/or geographical risk factors, which may explain the occurrence of the epizootics. Fisher's goodness-of-fit testing showed a significantly higher case proportion of adult cows (N = 368, 80.5%) and lower of calves (N =8, 1.8%), as compared to the expected proportions of the censused population in the study area and historical submissions computed from the laboratory database (Chi-Sq = 346.2 and 174.8, df = 7, P < 0.00). Time series decomposition showed a bi-seasonal pattern, with a larger peak in spring and early summer (October to January) and a smaller increase in autumn (March-May). The lowest seasonal indices were on mid-summer (February) and winter (June-September). A combination of spatial statistics was used to assess the different spatial features of the disease and consistency of the findings. Global spatial autocorrelation showed BBH was significantly clustered (Moran's I = 0.407, P < 0.001). Both smoothed Anselin's Local Indicator of Spatial Autocorrelation and Kulldorff's spatial scan Poisson and Bernoulli models, detected roughly the same high-risk areas in the southeastern part of the Merin Lagoon basin, with the most likely cluster centered in the large wetland biosphere reserve "Eastern Wetlands and Coastal Strip" (RR = 9.12, P < 0.001). Outbreaks were georeferenced (latitude, longitude) and thematic dot-mapping geovisualization in Google Earth™ showed that the results were robust and truly geographic in nature. Most outbreaks (40/45, 88.8%) occurred on wetlands areas and large river valleys, characterized by poorly drained and frequently flooded soils, indicating that moisture-laden soils are the natural habitat of C. novyi type D. Grasslands in these endemic areas support rapid fattening of cattle during spring-summer, and somewhat less in autumn, in almost exact correspondence with BBH peaks, suggesting a close causal association in high-risk areas. Risk is significantly higher in adult cows probably because the spore content in the liver is highest in this category. The altered lipid metabolism and lipotoxicity in the liver may be the precipitating factor for spore germination and epizootic occurrence.

Death of a neonatal lamb due to Clostridium perfringens type B in New Zealand.

Case history and clinical findings: A flock of 20 sheep was kept within three paddocks on a single property. None of the animals in the flock had been vaccinated against any disease for at least three years. Abdominal bloating and haemorrhagic diarrhoea were observed in Lamb 1 at 24 hours-of-age. The lamb subsequently died within an hour of the onset of clinical signs. Lamb 2 was 3-days-old when observed to be recumbent with opisthotonus. The lamb was treated with dextrose, vitamins B1 and B12, and penicillin G, but died 4 hours later.Pathological findings: Examination of Lamb 1 revealed markedly increased gas within the peritoneum and within dilated loops of intestine. The intestines were dark red and contained large quantities of haemorrhagic fluid. Histology of the intestines revealed peracute mucosal necrosis with minimal accompanying inflammation. The intestinal lumen contained cell debris, haemorrhage, and myriad large Gram-positive bacilli. The intestines of Lamb 2 did not appear bloated or reddened. However, multiple fibrin clots were visible within the pericardial sac. Histopathological examination revealed small foci of necrosis within the mucosa of the distal intestine. The necrotic foci were often associated with large numbers of large Gram-positive bacilli.Immunohistochemsitry and molecular biology: Intestinal samples from Lamb 1 were processed for Clostridium perfringens immunohistochemistry, which revealed large numbers of intralesional, positively immunostained rods. Fragments corresponding to the expected sizes for genes encoding alpha, beta, and epsilon C. perfringens typing toxins were amplified by PCR from DNA extracted from formalin-fixed sections of intestine.Diagnosis: Lamb dysentery due to C. perfringens type B.Clinical relevance: C. perfringens bacteria have a worldwide distribution, but disease due to C. perfringens type B has only been diagnosed in a small number of countries and has never been reported in New Zealand or Australia. C. perfringens type B produce both beta toxin and epsilon toxins, therefore both haemorrhagic enteritis and systemic vascular damage can develop. As many animals are exposed to C. perfringens without developing disease, there must be additional unknown factors that resulted in disease in these particular sheep. Vaccines that specifically protect against C. perfringens type B are available and may be recommended for use in smaller non-commercial flocks, as in the present case.

Pathology of Naturally Occurring Bacillary Hemoglobinuria in Cattle.

Clostridium haemolyticum causes bacillary hemoglobinuria (BH), an infectious and usually fatal disease that occurs mostly in cattle, which is clinically characterized by jaundice, hemoglobinuria, and anemia. The trematode Fasciola hepatica has been commonly reported as the main predisposing factor that triggers this condition. The authors evaluated 20 naturally occurring cases of bovine BH to characterize the pathology and pathogenesis of the disease. Grossly, the most consistent finding was a large, frequently single focus of necrosis surrounded by a red to purple halo, observed most frequently on the parietal surface of the right and left hepatic lobes. Other findings were jaundice, dark-brown discoloration of kidneys, and red urine in the urinary bladder. Microscopically, characteristic lesions were locally extensive, necrotizing hepatitis with thrombosis and numerous intralesional Gram-positive rod-shaped bacteria, and acute renal tubular necrosis. By immunohistochemistry, many hepatocytes outside the necrotic focus in the liver were positive for activated caspase 3, suggesting that those cells were undergoing apoptosis. Ultrastructural evaluation revealed hepatocyte necrosis, hemolysis, and clumps of vegetative and sporulating bacilli within the liver. Polymerase chain reaction for the C. haemolyticum beta toxin gene was positive in randomly selected liver samples. No gross or microscopic lesions indicative of fascioliasis were detected in the liver of any animal, suggesting that other yet undetermined predisposing factors were associated with these cases of BH.

Non-Clostridium perfringens infectious agents producing necrotic enteritis-like lesions in poultry.

Necrotic enteritis (NE) produced by Clostridium perfringens is amongst the most prevalent enteric diseases of chickens and turkeys. However, several other bacterial, parasitic and viral agents can cause clinical signs, gross and microscopic lesions in poultry very similar to those of NE and the diseases produced by those agents need to be differentiated from NE. The main differential diagnoses for C. perfringens NE include bacterial (Clostridium colinum, Clostridium sordellii, Clostridium difficile, Pasteurella multocida, Brachyspira spp.), parasitic (Eimeria spp., Histomonas meleagridis) and viral (Duck Herpesvirus type 1, Avian Paramyxovirus type 1) diseases. Confirmation of the diagnosis of these diseases requires identification of the aetiological agents by morphological, cultural and/or molecular methods.

Caudal lumbar vertebral fractures in California Quarter Horse and Thoroughbred racehorses.

REASONS FOR PERFORMING STUDY: To gain insight into the pathophysiology of equine lumbar vertebral fractures in racehorses. OBJECTIVES: To characterise equine lumbar vertebral fractures in California racehorses. STUDY DESIGN: Retrospective case series and prospective case-control study. METHODS: Racehorse post mortem reports and jockey injury reports were retrospectively reviewed. Vertebral specimens from 6 racehorses affected with lumbar vertebral fractures and 4 control racehorses subjected to euthanasia for nonspinal fracture were assessed using visual, radiographic, computed tomography and histological examinations. RESULTS: Lumbar vertebral fractures occurred in 38 Quarter Horse and 29 Thoroughbred racehorses over a 22 year period, primarily involving the 5th and/or 6th lumbar vertebrae (L5-L6; 87% of Quarter Horses and 48% of Thoroughbreds). Lumbar vertebral fractures were the third most common musculoskeletal cause of death in Quarter Horses and frequently involved a jockey injury. Lumbar vertebral specimens contained anatomical variations in the number of vertebrae, dorsal spinous processes and intertransverse articulations. Lumbar vertebral fractures examined in 6 racehorse specimens (5 Quarter Horses and one Thoroughbred) coursed obliquely in a cranioventral to caudodorsal direction across the adjacent L5-L6 vertebral endplates and intervertebral disc, although one case involved only one endplate. All cases had evidence of abnormalities on the ventral aspect of the vertebral bodies consistent with pre-existing, maladaptive pathology. CONCLUSIONS: Lumbar vertebral fractures occur in racehorses with pre-existing pathology at the L5-L6 vertebral junction that is likely predisposes horses to catastrophic fracture. Knowledge of these findings should encourage assessment of the lumbar vertebrae, therefore increasing detection of mild vertebral injuries and preventing catastrophic racehorse and associated jockey injuries.

Comparative neuropathology of ovine enterotoxemia produced by Clostridium perfringens type D wild-type strain CN1020 and its genetically modified derivatives.

Clostridium perfringens type D causes enterotoxemia in sheep and goats. The disease is mediated by epsilon toxin (ETX), which affects the cerebrovascular endothelium, increasing vascular permeability and leading to cerebral edema. In the present study, we compared the distribution and severity of the cerebrovascular changes induced in lambs by C. perfringens type D strain CN1020, its isogenic etx null mutant, and the ETX-producing complemented mutant. We also applied histochemical and immunohistochemical markers to further characterize the brain lesions induced by ETX. Both ETX-producing strains induced extensive cerebrovascular damage that did not differ significantly between each other in nature, neuroanatomic distribution, or severity. By contrast, lambs inoculated with the etx mutant or sterile, nontoxic culture medium did not develop detectable brain lesions, confirming that the neuropathologic effects observed in these infections are dependent on ETX production. Lambs treated with the wild-type and complemented strains showed perivascular and mural vascular edema, as well as serum albumin extravasation, particularly severe in the cerebral white matter, midbrain, medulla oblongata, and cerebellum. Brains of animals inoculated with the ETX-producing strains showed decreased expression of glial fibrillary acidic protein and increased expression of aquaporin-4 in the end-feet processes of the astrocytes around blood vessels. Early axonal injury was demonstrated with anti-amyloid precursor protein immunohistochemistry. Perivascular accumulation of macrophages/microglia with intracytoplasmic albumin globules was also observed in these animals. This study demonstrates that ETX is responsible for the major cerebrovascular changes in C. perfringens type D-induced disease.

Prevention and treatment of Clostridium perfringens epsilon toxin intoxication in mice with a neutralizing monoclonal antibody (c4D7) produced in Nicotiana benthamiana.

Epsilon toxin (ETX), produced by Clostridium perfringens types B and D, is among the most lethal toxins known. ETX is a potential bioterrorism threat that was listed as a Category B agent by the U.S. Centers for Disease Control until 2012 and it still remains a toxin of interest for several government agencies. We produced a monoclonal antibody (MAb) against ETX (ETX MAb c4D7) in Nicotiana benthamiana and characterized its preventive and therapeutic efficacy in mice. The ETX preparation used was highly lethal for mice (LD50 = 1.6 µg/kg) and resulted in a mean time from inoculation to death of 18 and 180 min when administered intravenously or intraperitoneally, respectively. High lethal challenge resulted in dramatic increases of a variety of pro-inflammatory cytokines in serum, while lower, but still lethal doses, did not elicit such responses. ETX MAb c4D7 was highly effective prophylactically (ED50 = 0.3 mg/kg; ED100 = 0.8 mg/kg) and also provided protection when delivered 15-30 min post-ETX intoxication. These data suggest that ETX MAb c4D7 may have use as a pre- and post-exposure treatment for ETX intoxication.

Diagnostic exercise: hemolysis and sudden death in lambs.

Within a 24-hour period, 7 out of 200 three- to four-week-old pastured Katahdin lambs died after showing clinical signs of hemoglobinuria, red-tinged feces, weakness, and recumbency. One of the lambs that was examined clinically before natural death also had abdominal pain, trembling, tachycardia, and severe anemia with a packed cell volume of 4%. Pathologic findings included icterus, hemoglobinuric nephrosis, dark red urine, pulmonary edema, hydrothorax, splenomegaly, and acute centrilobular to midzonal hepatocellular degeneration and necrosis with cholestasis. The differential diagnoses and diagnostic workup to achieve the diagnosis are briefly discussed.

Clostridium difficile infection in horses: a review.

Clostridium difficile is considered one of the most important causes of diarrhea and enterocolitis in horses. Foals and adult horses are equally susceptible to the infection. The highly resistant spore of C. difficile is the infectious unit of transmission, which occurs primarily via the fecal-oral route, with sources of infection including equine feces, contaminated soil, animal hospitals, and feces of other animals. Two major risk factors for the development of C. difficile associated disease (CDAD) in adult horses are hospitalization and antimicrobial treatment, although sporadically, cases of CDAD can occur in horses that have not received antimicrobials or been hospitalized. The most common antibiotics associated with CDAD in horses are erythromycin, trimethoprim/sulfonamides, ß-lactam antimicrobials, clindamycin, rifampicin, and gentamicin. Clinical signs and intestinal lesions of CDAD infection are not specific and they cannot be used to distinguish infections by C. difficile from infections by other agents, such as Clostridium perfringens or Salmonella sp. The distribution of lesions throughout the intestinal tract seems to be age-dependent. Small intestine is invariably affected, and colon and cecum may or may not have lesions in foals<1-month old. Naturally acquired disease in older foals and adult horses has a more aboral distribution, affecting colon and sometimes cecum, but rarely the small intestine. Detection of toxin A, toxin B or both in intestinal contents or feces is considered the most reliable diagnostic criterion for CDAD in horses. Isolation of toxigenic strains of C. difficile from horses with intestinal disease is highly suggestive of CDAD. A better understanding of pathogenesis, reservoirs of infection, and vaccines and other methods of control is needed. Also further studies are recommended to investigate other possible predisposing factors and/or etiological agents of enteric diseases of horses.

Pathology and diagnostic criteria of Clostridium difficile enteric infection in horses.

Clostridium difficile is commonly associated with diarrhea and colitis in humans and other mammals, including horses. To this date, the epidemiologic, microbiologic, clinical, and diagnostic aspects of C. difficile-associated disease (CDAD) in horses have been thoroughly described. However, reports describing the enteric pathology of this disease in horses are limited. This study presents a comprehensive description of the pathologic characteristics of CDAD in 21 horses and discusses the criteria for the diagnosis of the disease. Case selection was based on C. difficile A/B toxins detection (enzyme-linked immunosorbent assay) in intestinal content samples accompanied by compatible gross and microscopic enteric lesions. Grossly, multifocal, segmental, or diffuse hemorrhage; congestion; and/or marked gelatinous edema of the intestinal wall with abundant bloody or green watery contents were observed. Histologically, the most common lesion was severe necrotizing or necrohemorrhagic enteritis, colitis, or typhlocolitis, with mucosal and/or submucosal thrombosis and marked submucosal edema. The pathology of CDAD in horses is similar to that caused by other equine enteric pathogens; therefore, a definitive diagnosis requires detection of C. difficile A/B toxins in the intestinal contents.

Epsilon toxin is essential for the virulence of Clostridium perfringens type D infection in sheep, goats, and mice.

Clostridium perfringens type D causes disease in sheep, goats, and other ruminants. Type D isolates produce, at minimum, alpha and epsilon (ETX) toxins, but some express up to five different toxins, raising questions about which toxins are necessary for the virulence of these bacteria. We evaluated the contribution of ETX to C. perfringens type D pathogenicity in an intraduodenal challenge model in sheep, goats, and mice using a virulent C. perfringens type D wild-type strain (WT), an isogenic ETX null mutant (etx mutant), and a strain where the etx mutation has been reversed (etx complemented). All sheep and goats, and most mice, challenged with the WT isolate developed acute clinical disease followed by death in most cases. Sheep developed various gross and/or histological changes that included edema of brain, lungs, and heart as well as hydropericardium. Goats developed various effects, including necrotizing colitis, pulmonary edema, and hydropericardium. No significant gross or histological abnormalities were observed in any mice infected with the WT strain. All sheep, goats, and mice challenged with the isogenic etx mutant remained clinically healthy for ≥24 h, and no gross or histological abnormalities were observed in those animals. Complementation of etx knockout restored virulence; most goats, sheep, and mice receiving this complemented mutant developed clinical and pathological changes similar to those observed in WT-infected animals. These results indicate that ETX is necessary for type D isolates to induce disease, supporting a key role for this toxin in type D disease pathogenesis.

Brain lesions associated with clostridium perfringens type D epsilon toxin in a Holstein heifer calf.

A 6-month-old dairy heifer calf with no premonitory signs was acutely down after the morning feeding and could not rise. On presentation, the heifer was in right lateral recumbency and moribund with opisthotonus and left hind limb paddling. Following euthanasia, gross examination of the brain revealed multifocal loss of gray-white matter distinction and extensive petechiae throughout the brainstem. On histopathological examination, there was striking white matter edema and marked perivascular proteinaceous edema surrounding many arterioles and venules (microangiopathy), mainly in the white matter of the internal capsule, thalamus, midbrain, cerebellum, and cerebellar peduncles. The perivascular neuropil was strongly positive for Alzheimer precursor protein A4. Clostridium perfringens epsilon toxin was detected in the intestinal contents. This is the first report of microangiopathy in postneonatal cattle associated with the detection of epsilon toxin in the intestinal contents.

Encephalomyelitis by Toxoplasma gondii in a captive fossa (Cryptoprocta ferox).

Encephalomyelitis due to Toxoplasma gondii was diagnosed in a fossa (Cryptoprocta ferox). The animal had ataxia, atrophy of hind limb muscles and progressive wasting before dying 12 months after the onset of clinical signs. Toxoplasmosis was suspected antemortem based on clinical signs and the detection of T. gondii DNA by PCR on EDTA-blood from live animal. Necropsy revealed necrotizing gastritis and severe emaciation. The main histological lesions included non-suppurative encephalomyelitis, with dilation of myelin sheaths and swollen axons in the spinal cord, and multifocal gliosis in the brain with intralesional protozoan cysts that stained positive for T. gondii immunohistochemistry. To the authors' knowledge, this is the first report of toxoplasmosis in a fossa, and a new host record.

Clostridium novyi type B as a causative agent of bovine meat spoilage.

A series of bovine meat spoilage cases in which meat from clinically healthy Belgian Blue cattle showed green discoloration are described. Histology of skeletal muscle revealed numerous spore-forming rods in the discolored areas of the meat. These organisms stained positively for Clostridium novyi by immunohistochemistry. A combination of 16S rDNA and fliC gene sequencing of bacterial DNA, isolated from the spoiled meat samples, revealed the unique presence of C. novyi type B. Although this bacterium has been implicated in clinical necrotic hepatitis in cattle, the cases described here are the first implicating C. novyi type B as a cause of bovine meat spoilage.

Acute respiratory distress in an alpaca.

An alpaca was presented with a history of respiratory difficulty and death. Histology of the phrenic nerves and diaphragm revealed degenerative changes consistent with denervation atrophy, and a diagnosis of diaphragmatic paralysis was established. No gross or histological abnormalities were observed in the spinal cord or other organs. The etiology of the phrenic nerve neuropathy could not be determined. The need to examine phrenic nerves and diaphragm in camelids with respiratory distress is emphasized, as failure to examine these samples will preclude a diagnosis of diaphragmatic paralysis.

The effect of Clostridium perfringens type C strain CN3685 and its isogenic beta toxin null mutant in goats.

Clostridium perfringens type C is an important cause of enteritis and/or enterocolitis in several animal species, including pigs, sheep, goats, horses and humans. The disease is a classic enterotoxemia and the enteric lesions and associated systemic effects are thought to be caused primarily by beta toxin (CPB), one of two typing toxins produced by C. perfringens type C. This has been demonstrated recently by fulfilling molecular Koch's postulates in rabbits and mice. We present here an experimental study to fulfill these postulates in goats, a natural host of C. perfringens type C disease. Nine healthy male or female Anglo Nubian goat kids were inoculated with the virulent C. perfringens type C wild-type strain CN3685, an isogenic CPB null mutant or a strain where the cpb null mutation had been reversed. Three goats inoculated with the wild-type strain presented abdominal pain, hemorrhagic diarrhea, necrotizing enterocolitis, pulmonary edema, hydropericardium and death within 24h of inoculation. Two goats inoculated with the CPB null mutant and two goats inoculated with sterile culture media (negative controls) remained clinically healthy during 24h after inoculation and no gross or histological abnormalities were observed in the tissues of any of them. Reversal of the null mutation to partially restore CPB production also increased virulence; 2 goats inoculated with this reversed mutant presented clinical and pathological changes similar to those observed in goats inoculated with the wild-type strain, except that spontaneous death was not observed. These results indicate that CPB is required for C. perfringens type C to induce disease in goats, supporting a key role for this toxin in natural C. perfringens type C disease pathogenesis.

Pathology of Clostridium perfringens type C enterotoxemia in horses.

Clostridium perfringens type C is an important cause of enteritis and enterocolitis in foals and occasionally in adult horses. The disease is a classic enterotoxemia, and the enteric lesions and systemic effects are caused primarily by beta toxin, 1 of 2 major toxins produced by C. perfringens type C. Until now, only sporadic cases of C. perfringens type C equine enterotoxemia have been reported. We present a comprehensive description of the lesions in 8 confirmed cases of type C enterotoxemia in foals and adult horses. Grossly, multifocal to segmental hemorrhage and thickening of the intestinal wall were most common in the small intestine, although the colon and cecum were also frequently affected. All horses had variable amounts of fluid, often hemorrhagic intestinal contents. The most characteristic microscopic lesion was necrotizing or necrohemorrhagic enteritis, with mucosal and/or submucosal thrombosis. Numerous gram-positive rods were occasionally seen in affected mucosa. A definitive diagnosis of C. perfringens type C enterotoxemia in all 8 cases was based on the clinical history, gross and histologic lesions, and detection of the beta toxin in intestinal contents.

Clostridium perfringens type C and Clostridium difficile co-infection in foals.

Clostridium perfringens type C is one of the most important agents of enteric disease in newborn foals. Clostridium difficile is now recognized as an important cause of enterocolitis in horses of all ages. While infections by C. perfringens type C or C. difficile are frequently seen, we are not aware of any report describing combined infection by these two microorganisms in foals. We present here five cases of foal enterocolitis associated with C. difficile and C. perfringens type C infection. Five foals between one and seven days of age were submitted for necropsy examination to the California Animal Health and Food Safety Laboratory. The five animals had a clinical history of acute hemorrhagic diarrhea followed by death and none had received antimicrobials or been hospitalized. Postmortem examination revealed hemorrhagic and necrotizing entero-typhlo-colitis. Histologically, the mucosa of the small intestine and colon presented diffuse necrosis and hemorrhage and it was often covered by a pseudomembrane. Thrombosis was observed in submucosal and/or mucosal vessels. Immunohistochemistry of intestinal sections of all foals showed that many large bacilli in the sections were C. perfringens. C. perfringens beta toxin was detected by ELISA in intestinal content of all animals and C. difficile toxin A/B was detected in intestinal content of three animals. C. perfringens (identified as type C by PCR) was isolated from the intestinal content of three foals. C. difficile (typed as A(+)/B(+) by PCR) was isolated from the intestinal content in 3 out of the 5 cases. This report suggests a possible synergism of C. perfringens type C and C. difficile in foal enterocolitis. Because none of the foals had received antibiotic therapy, the predisposing factor, if any, for the C. difficile infection remains undetermined; it is possible that the C. perfringens infection acted as a predisposing factor for C. difficile and/or vice versa. This report also stresses the need to perform a complete diagnostic workup in all cases of foal digestive disease.