Santiago S. Diab

Divergent Astrovirus Associated with Neurologic Disease in Cattle

Using viral metagenomics of brain tissue from a young adult crossbreed steer with acute onset of neurologic disease, we sequenced the complete genome of a novel astrovirus (BoAstV-NeuroS1) that was phylogenetically related to an ovine astrovirus. In a retrospective analysis of 32 cases of bovine encephalitides of unknown etiology, 3 other infected animals were detected by using PCR and in situ hybridization for viral RNA. Viral RNA was restricted to the nervous system and detected in the cytoplasm of affected neurons within the spinal cord, brainstem, and cerebellum. Microscopically, the lesions were of widespread neuronal necrosis, microgliosis, and perivascular cuffing preferentially distributed in gray matter and most severe in the cerebellum and brainstem, with increasing intensity caudally down the spinal cord. These results suggest that infection with BoAstV-NeuroS1 is a potential cause of neurologic disease in cattle.

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 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, β-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.

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.

Necrotizing Enteritis and Hyperammonemic Encephalopathy Associated With Equine Coronavirus Infection in Equids

Equine coronavirus (ECoV) is a Betacoronavirus recently associated clinically and epidemiologically with emerging outbreaks of pyrogenic, enteric, and/or neurologic disease in horses in the United States, Japan, and Europe. We describe the pathologic, immunohistochemical, ultrastructural, and molecular findings in 2 horses and 1 donkey that succumbed to natural infection with ECoV. One horse and the donkey (case Nos. 1, 3) had severe diffuse necrotizing enteritis with marked villous attenuation, epithelial cell necrosis at the tips of the villi, neutrophilic and fibrinous extravasation into the small intestinal lumen (pseudomembrane formation), as well as crypt necrosis, microthrombosis, and hemorrhage. The other horse (case No. 2) had hyperammonemic encephalopathy with Alzheimer type II astrocytosis throughout the cerebral cortex. ECoV was detected by quantitative polymerase chain reaction in small intestinal tissue, contents, and/or feces, and coronavirus antigen was detected by immunohistochemistry in the small intestine in all cases. Coronavirus-like particles characterized by spherical, moderately electron lucent, enveloped virions with distinct peplomer-like structures projecting from the surface were detected by negatively stained transmission electron microscopy in small intestine in case No. 1, and transmission electron microscopy of fixed small intestinal tissue from the same case revealed similar 85- to 100-nm intracytoplasmic particles located in vacuoles and free in the cytoplasm of unidentified (presumably epithelial) cells. Sequence comparison showed 97.9% to 99.0% sequence identity with the ECoV-NC99 and Tokachi09 strains. All together, these results indicate that ECoV is associated with necrotizing enteritis and hyperammonemic encephalopathy in equids.

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.

Infection with a Hepatozoon sp. closely related to Hepatozoon felis in a wild Pampas gray fox (Lycalopex - Pseudalopex - gymnocercus) co-infected with canine distemper virus

A species of Hepatozoon closely related to Hepatozoon felis found in the skeletal and cardiac muscle of a wild Pampas gray fox (Lycalopex gymnocercus) is described. The fox was euthanized after showing severe incoordination. On necropsy and histopathology there was bilateral, diffuse, severe, sub-acute, necrotizing bronchointerstitial pneumonia, with intracytoplasmic and intranuclear eosinophilic inclusion bodies. Canine distemper virus was detected by immunohistochemistry in the bronchiolar epithelium, syncytial cells, alveolar macrophages and pneumocytes. The skeletal muscle and myocardium contained multiple round to oval protozoan cysts ranging from 64 μm × 75 μm to 98 μm × 122 μm, with a central eosinophilic meront-like core surrounded by concentric rings of mucinous material resembling Hepatozoon americanum cysts but smaller in size. Macrophages within rare pyogranulomas and monocytes/macrophages in adjacent sinusoidal blood vessels in the skeletal muscle contained intracytoplasmic round protozoa consistent with merozoites or developing gamonts of Hepatozoon. Hepatozoon sp. infection was confirmed by PCR of skeletal muscle and the sequenced 18S rRNA PCR product was found to be 99% identical to H. felis by BLAST analysis and deposited in GenBank as accession number HQ020489. It clustered together in the phylogenetic analysis with published H. felis sequences and separately from H. canis, H. americanum and other Hepatozoon species. However, the close relatedness of the fox Hepatozoon to H. felis does not rule out infection with a different and possibly unknown Hepatozoon species.

Equine leukoencephalomalacia (ELEM) due to fumonisins B1 and B2 in Argentina

In August 2007 an outbreak of neurological disease and sudden death in Arabian horses occurred in a farm located in Coronel Rosales County, Buenos Aires Province, Argentina. The animals were on a pasture of native grasses and supplemented ad libitum with corn kernels and wheat bran. Three horses were observed having acute neurologic signs including blindness, four leg ataxia, hyperexcitability, aimless walking and circling, followed by death in two of them. Four other horses were found dead overnight without a history of neurologic signs. The morbidity, mortality and lethality rates were 11.6%, 10% and 85.7%, respectively. Grossly, the brain showed focal areas of hemorrhage, brown-yellow discoloration and softening of the sub-cortical white matter. The microscopic brain lesions consisted of extensive areas of malacia within the white matter of the cerebral hemispheres, brainstem and cerebellum, characterized by rarefaction of the white matter with cavitations filled with proteinaceous edema, multifocal hemorrhages and mild infiltration by neutrophils, and rare eosinophils. Swollen glial cells with abundant eosinophilic cytoplasm, distinct cell borders, intracytoplasmic deeply eosinophilic globules and eccentric, hyperchromatic, occasionally pyknotic nucleus were present throughout the areas of rarefaction hemorrhage, edema and necrosis. The feed supplements contained 12,490µg/kg of fumonisin B1 and 5,251µg/ kg of fumonisin B2. This is the first reported outbreak of ELEM associated with consumption of feed supplements containing high concentrations of fumonisins in Argentina.

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.

Retrospective study of fatal pneumonia in racehorses

Respiratory diseases have a major impact on racehorses in training and are often cited as the second most common reason of horses failing to perform. Cases were submitted by the California Horse Racing Board to the California Animal Health and Food Safety laboratory for postmortem examination between January 1, 2005 and December 31, 2014. We determined the demographics of racehorses with fatal pneumonia, characterized the pathologic findings in animals with a postmortem diagnosis of respiratory infection, and determined the most significant pathogens associated with lower respiratory tract disease. We analyzed autopsy reports from 83 horses with a diagnosis of pneumonia, bronchopneumonia, and/or pleuropneumonia. The most common presentation was pleuropneumonia (71% of cases), with extensive areas of lytic necrosis and abscesses of the pulmonary parenchyma. Streptococcus equi ssp. zooepidemicus, a normal mucosal commensal of the upper respiratory tract of healthy horses, was the most commonly isolated organism (72% of cases), either in pure culture or accompanied by other aerobic or anaerobic bacteria. Its presence in the pulmonary parenchyma is associated with severe and extensive damage to the lung. Furthermore, this agent has zoonotic potential, which stresses the importance of early detection and proper management of cases of pneumonia in racehorses.