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.

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.

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.

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.

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.

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.

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.

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.

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 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.

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.

Sudden death in racing Thoroughbred horses: an international multicentre study of post mortem findings.

REASONS FOR PERFORMING STUDY: To improve the understanding of exercise related sudden death in Thoroughbred racehorses. OBJECTIVES: To describe the post mortem findings in cases of sudden death associated with exercise in 268 Thoroughbred racehorses. METHODS: Gross and histological post mortem findings of 268 cases of sudden death were collated and reviewed. Cases originated from 6 racing jurisdictions around the world. Sudden death was defined as acute collapse and death in a closely observed and previously apparently healthy Thoroughbred racehorse, during, or within one hour after, exercise. Cause of death as determined by the attending pathologist was categorised as definitive, presumptive or unexplained and compared between the different populations. Cardiopulmonary lesions recorded at post mortem examination were compared between different populations. RESULTS: Pathologists recorded a definitive cause of death in 53% (143/268) of cases. Major definitive causes of sudden death included cardiac failure, apparent pulmonary failure, pulmonary haemorrhage, haemorrhage associated with pelvic fractures or with idiopathic blood vessel rupture, and spinal cord injury. A presumptive cause of death was made in 25% (67/268) of cases and death remained unexplained in 22% (58/268) of cases. There were several statistically significant inter-population differences in the cause of death and in reporting of cardiopulmonary lesions. CONCLUSIONS: Sudden death can be attributed to a variety of causes. Causes of sudden death and the lesions found in cases of exercise-related sudden death are similar in different racing jurisdictions. However, the lesions are often not specific for the cause of death and determination of the cause of death is therefore affected by interpretation by the individual pathologist.

Zygomycotic lymphadenitis in slaughtered feedlot cattle.

During the 12 months of 2006, zygomycotic lymphadenitis was diagnosed in 194 of 198 feedlot steers (0.04% of cattle slaughtered during that period) in a California slaughterhouse as part of bovine tuberculosis surveillance. Mesenteric lymph nodes were involved in 190 cases. Affected lymph nodes were enlarged (2 to 42 cm in greatest dimension), firm, and mottled gray-white to yellow with multiple granular or caseocalcareous foci. Histologically, nodal architecture was effaced by necrosis, granulomatous inflammation, and fibrosis. In approximately 20% of the cases, granulomas were mainly restricted to subcapsular sinuses and afferent lymphatic vessels, causing granulomatous lymphangitis. Nonseptate, irregularly branching hyphae with nonparallel walls and bulbous enlargements were common in necrotic areas and within the cytoplasm of multinucleated giant cells. Fungal cultures were performed on 124 affected lymph nodes using 7 media, but no zygomycetes were cultured. Fungal DNA was amplified from 20 lymph nodes. Amplicons from 16 nodes had nearly 100% homology with sequences for Rhizomucor pusillus; 4 amplicons had (> 98%) homology with Absidia corymbifera sequences. Zygomycosis should be considered in the differential diagnosis for granulomatous lymphadenitis in feedlot steers.

Clinicopathologic features of experimental Clostridium perfringens type D enterotoxemia in cattle.

This study was designed to experimentally reproduce enterotoxemia by Clostridium perfringens type D in cattle and to characterize the clinicopathologic findings of this disease. Fourteen 9-month-old calves were inoculated intraduodenally according to the following schedule: group 1 (n = 4), C. perfringens type D whole culture; group 2 (n = 3), C. perfringens type D washed cells; group 3 (n = 5), C. perfringens type D filtered and concentrated supernatant; group 4 (n = 2), sterile, nontoxic culture medium. In addition, all animals received a 20% starch solution in the abomasum. Ten animals from groups 1 (4/4), 2 (3/3), and 3 (3/5) showed severe respiratory and neurologic signs. Gross findings were observed in these 10 animals and consisted of acute pulmonary edema, excessive protein-rich pericardial fluid, watery contents in the small intestine, and multifocal petechial hemorrhages on the jejunal mucosa. The brain of one animal of group 2 that survived for 8 days showed multifocal, bilateral, and symmetric encephalomalacia in the corpus striatum. The most striking histologic changes consisted of perivascular high protein edema in the brain, and alveolar and interstitial proteinaceous pulmonary edema. The animal that survived for 8 days and that had gross lesions in the corpus striatum showed histologically severe, focal necrosis of this area, cerebellar peduncles, and thalamus. Koch's postulates have been met and these results show that experimental enterotoxemia by C. perfringens type D in cattle has similar clinical and pathologic characteristics to the natural and experimental disease in sheep.

Clostridium perfringens epsilon toxin is absorbed from different intestinal segments of mice.

Clostridium perfringens epsilon toxin is a potent toxin responsible for a rapidly fatal enterotoxaemia in several animal species. The pathogenesis of epsilon toxin includes toxicity to endothelial cells and neurons. Although epsilon toxin is absorbed from the gastrointestinal tract, the intestinal regions where the toxin is absorbed and the conditions favoring epsilon toxin absorption are unknown. The aim of this paper was to determine the toxicity of epsilon toxin absorbed from different gastrointestinal segments of mice and to evaluate the influence of the intestinal environment in the absorption of this toxin. Epsilon toxin diluted in one of several different saline solutions was surgically introduced into ligated stomach or intestinal segments of mice. Comparison of the toxicity of epsilon toxin injected in different sections of the gastrointestinal tract showed that this toxin can be absorbed from the small and the large intestine but not from the stomach of mice. The lethality of epsilon toxin was higher when this toxin was injected in the colon than in the small intestine. Low pH, and Na(+) and glucose added to the saline solution increased the toxicity of epsilon toxin injected into the small intestine. This study shows that absorption of epsilon toxin can occur in any intestinal segment of mice and that the physicochemical characteristics of the intestinal content can affect the absorption of this toxin.

Cluster of cases of malignant schwannoma in cattle.

Between 1998 and 2001, several cases of ataxia and paresis followed by recumbency and death were reported in cows from different farms in a restricted area of the Argentinian Patagonia. Five cases of this cluster were studied and a diagnosis of malignant schwannoma was established. Electron microscopy (em) of tumour samples from three of the animals revealed intracytoplasmic or interstitial structures resembling retroviral particles. Attempts to isolate a viral agent from the tumours were unsuccessful but the epidemiological data and the em findings suggest a viral aetiology.

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.

Detection of Clostridium perfringens type D epsilon antitoxin in serum of goats by competitive and indirect ELISA.

Indirect and competitive ELISA techniques were developed and their ability to detect antibodies to Clostridium perfringens epsilon toxin in goat serum was compared. Different dilutions of a hyperimmune goat serum, in serum from a colostrum-deprived kid, were used as positive controls, while sera from eleven colostrum-deprived kids were used as negative controls. The epsilon toxin antibodies in the hyperimmune serum were also measured by mouse neutralisation test (MNT). The correlation coefficient between both the indirect ELISA technique and MNT was 0.99, while the same coefficient for the competitive ELISA was 0.98. Both the indirect and competitive ELISAs proved to be rapid, simple, sensitive and specific for detecting antibodies to C. perfringens epsilon toxin in serum of goats.