RUMINANTS

Verotoxigenic E.coli associated with diarrhea and mortality in young calves

Andrew Brooks, Durda Slavic, Margaret Stalker, Murray Hazlett, Maria Spinato, Beverly McEwen

VTEC are a heterogeneous group of E.coli that express one or more Shiga toxins (stx). VTEC have important public health significance because some strains cause hemorrhagic colitis and hemolytic uremia syndrome in people. VTEC are carried subclinically in the intestine of healthy cattle, which are an important reservoir for human infection, and sporadically cause diarrhea and dysentery in young calves.

Verotoxigenic E.coli (VTEC) was determined to be the cause of diarrhea or death in 9 calves submitted to the AHL for postmortem or histological examination from January 2013 to December 2014. The major clinical signs were diarrhea and sudden death in calves 4-30 days of age (Table 1). The most common gross lesion was enterocolitis that was often mild; in some cases fibrinonecrotic or fibrinohemorrhagic intestinal exudates were present. The common histologic lesion was attachment of bacilli to the apical border of small intestinal or colonic enterocytes (Fig. 1). E. coli isolates from the intestine (1 isolate was from feces) were genotyped at the AHL with a PCR assay that detects the intimin (eaeA), hemolysin (hlyA), and Shiga toxin 1 and 2 (stx1/2) virulence genes. The majority of isolates from these cases were positive for eaeA, hlyA, and stx1. Co-infections with other pathogens associated with calf diarrhea were common.   

Figure 1.  VTEC attached to surface enterocytes of the small intestine (from case 4).

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Table 1. Case summaries of diarrhea and mortality in calves associated with VTEC infection

Case	Breed	Age (days)	Major clinical problems	Major postmortem lesions	E. coli virulence factor genotype				Co-infections
					eaeA      hylA        stx1      stx2
1	Holstein	4	Bloody feces Found dead	Fibrinonecrotic enteritis	+	+	+	-	Cryptosporidium
2*	not given	10	Diarrhea	Erosive enterocolitis	+	+	+	-	Cryptosporidium
3	Holstein	10	Diarrhea, pneumonia	Enterocolitis, mycotic rumenitis	+/- 1	+/-	+/-	-	Enterotoxigenic E. coli Cryptosporidium Coronavirus
4	Mixed-beef	9	Diarrhea, sudden death	Enterocolitis Septicemia	+	+	+	-	Rotavirus
5	Charolais	14	Diarrhea	Enterocolitis Mycotic rumenitis/reticulitis	+	+	+	-	Rotavirus
6	Holstein	30	Found dead	Enteritis, rumenitis, meningitis, pyelonephritis	+	+	+	-	-
7	Jersey	15	Diarrhea	Enterocolitis, abomasitis, rumenitis	+	+	+	-	Cryptosporidium Rotavirus
8	Holstein	3	Found dead	Fibrino-hemorrhagic enteritis, abomasitis	-	+	+	+	-
9	Holstein	10	Acute death	Enterocolitis, abomasitis	+/-	+	+	-	Coronavirus Salmonella Muenster Cryptosporidium

* Field postmortem; 1.  +/- denotes suspicious PCR result.

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An outbreak of Campylobacter jejuni abortions in a small dairy herd

Maria Spinato, Andrew Vince, Durda Slavic, Geert Jongert

The producer of a closed, well-managed herd of 85 Holstein cattle reported 9 abortions during a 2-week period. Cattle are vaccinated with a modified live, 5-way vaccine (IBRV, BVDV types 1 and 2, BRSV, PI3V) at freshening. Most of the aborted fetuses were between 200-260 days gestation. Three fetuses and placentas were submitted to the AHL for postmortem examination and ancillary testing. Placentas contained variably-sized, relatively uniform tan cotyledons; slight marginal cupping and congestion were noted in several cotyledons in the placenta of fetus 3. All 3 fetuses were moderately autolysed. The only remarkable observation in fetal tissues was the finding of an edematous sheet of fibrin overlying the lung of fetus 3. Fibrinous pleuritis is most often observed in abortions caused by bacterial infections; therefore, Campylobacter culture and Leptospira MAT were performed, in addition to routine aerobic bacterial culture. Campylobacter jejuni subsp. jejuni was isolated in one or more samples of lung, abomasal fluid and placenta cultured from all 3 fetuses. Leptospira MATs and PCR tests for BVDV, BoHV-1 (IBRV), and Coxiella burnetii were negative. Neospora caninum ELISAs performed on maternal sera of fetuses 1 and 2 were also negative.  Histologic lesions included multifocal neutrophilic necrotizing placentitis, colonization of chorionic stroma and occasional trophoblastic epithelial cells by abundant Gram negative bacteria, and hypercellular alveolar septa in lung sections due to circulating neutrophils. The allantois of fetus 1 also had a few arterioles characterized by hyaline walls and fibrin thrombi.

Upon further investigation, it was discovered that a cow had aborted on this farm approximately 2.5-3 weeks prior to the outbreak. This cow was housed in a hospital pen that is scraped out twice weekly using the same tractor and bucket used for making the total mixed ration (TMR). Although the bucket was rinsed with water after scraping the hospital pen, it was not thoroughly cleaned or disinfected. It is suspected that this abortion was the index case (not submitted for testing), and that the placenta contaminated the TMR which was the source of infection for the other aborting cows via ingestion.  Subsequent to this outbreak, a premature live calf and a first trimester abortion at 60 days gestation were reported on this farm; however, no additional testing was performed and their relation to the outbreak could not be confirmed.

Campylobacter jejuni is a normal commensal organism found in the gastrointestinal tract of ruminants and other food-producing animals. Although sporadic abortions have been reported in cattle, abortion outbreaks due to C. jejuni are extremely rare.  Conversely, outbreaks of C. jejuni abortion in small ruminants are a more common occurrence.  The emergence of tetracycline-resistant strains in Canada and the USA is of significant concern to the small ruminant industries, as tetracycline is used both therapeutically and prophylactically for infectious causes of abortion (1). Minimum inhibitory concentration (MIC) analysis of the C. jejuni isolate in this case confirmed that it was susceptible to tetracycline.

This case highlights the importance of expanding on-farm biosecurity protocols to include appropriate disposal and disinfection procedures for aborted fetuses and placentas. Many abortifacient bacterial species in ruminants also pose a significant zoonotic risk for producers, their families and veterinarians. Campylobacter jejuni is recognized world-wide as a significant cause of gastroenteritis in humans, and contamination of food and water supplies by bovine feces is a known risk factor.  

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An alternative method for brain removal

Josepha DeLay, Andrew Brooks

An alternative approach and, for large animals especially, often easier method to expose brain is by using a lateral approach, cutting through a coronal plane.

                                                                        

Figure 1. Landmarks for brain removal by lateral approach.                           Figure 6. Brain removal in rostral and caudal sections. These may be sagittally sectioned, with half of each section fixed in

                                                                                                               formalin for histopathology, and the remaining halves stored fresh or frozen for  microbiologic tests.

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Field and clinic postmortems. I: Simplified protocol and image list

Josepha DeLay

Digital images captured during postmortem (PM) examinations provide a permanent record of lesions. The images are a very useful communication tool when consulting with pathologists and other specialists. PM images provide valuable supplemental information for pathologists evaluating tissue samples submitted to a diagnostic laboratory for histologic examination. Images may be emailed to AHL pathologists at ahlpath@uoguelph.ca

The image list below provides both a step-wise guide to the postmortem procedure and a suggested set of images that are applicable to all species of companion and food-producing animals. Establishing and following a standard routine for PM procedures is important. This allows the practitioner to spend more time identifying and interpreting lesions, rather than concentrating on the logistics of the exam. Developing a PM routine is similar to having a routine protocol for physical examination in a live patient.

Remove ear tag or create ID label, and include with all photos.

Image 1. External views: full body, head, thorax / abdomen, perineum

- for unexpected deaths, take image in situ, in location and position where body was discovered.

- include views that depict body condition, hydration (eyes), evidence of predation or trauma, etc.

Open abdominal and thoracic cavities.

Image 2. Opened thorax (with organs in situ).

Image 3. Heart in situ, with pericardial sac opened (check for fluid, exudate, etc.).

Remove pluck.

Image 4. Pluck, with focus on lungs (dorsoventral view, with right and left lung visible).

Image 5. Cross-section of right and left lung.

Image 6. Cross-section of heart through both ventricles.

Image 7. Larynx (including thyroid glands) and trachea: opened and mucosal surface exposed.

Image 8. Opened abdomen (with organs in situ).

In ruminants, remove omentum. In all species, fan out intestines and locate cecum and ileum.

Image 9. Opened abdomen with intestines fanned out.

Image 10. Open cecum, ileum, and jejunum to expose mucosal surface.

Image 11. Open colon to expose mucosal surface.

Image 12. Open duodenum to expose mucosal surface.

Image 13. Liver – capsular surface. For ruminants, include opened caudal vena cava.

Image 14. Liver – cross section.

Image 15. Abomasum / stomach – serosal surface.

Image 16. Abomasum / stomach – mucosal surface.

Image 17. Ruminants: rumen – serosal surface.

Image 18. Ruminants: rumen – mucosal surface and content.

Image 19. Solid organs:

- kidneys: sagittal sections, with cut surfaces exposed

- spleen: cross section

- adrenal glands

Unexpected death / neurologic cases: Remove brain. Also remove spinal cord if required, based on clinical signs.

Image 20. Brain.  The next installment in this series will focus on ancillary test selection and sample collection during PM exams.   

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Theileriosis in a dairy cow from eastern Ontario

Kris Ruotsalo, Jan Shapiro

A 3-year-old Holstein cow from eastern Ontario had a 2-month clinical history of pyrexia, diarrhea, lameness, and weight loss. Significant hematological changes included marked, mildly responsive anemia; hematocrit 0.15 L/L (reference interval 0.21-0.30 L/L), hemoglobin 48 g/L (reference interval 84-120 g/L), and mild lymphopenia. Significant biochemistry changes included hypoproteinemia (total protein 50 g/L) and hyperbilirubinemia (total bilirubin 20 µmol/L).

Peripheral blood smear examination revealed numerous intraerythrocytic organisms (Fig. 1). These organisms were pleomorphic, exhibiting round, rod, comma, and signet ring forms. A tentative diagnosis of Theileria spp. infection was made. No commercially available test to confirm theileriosis is available, and the species of Theileria cannot be morphologically distinguished on the basis of a blood smear. Confirmatory testing by 18S RNA amplification and gene sequence analysis was performed at the Animal Health Diagnostic Laboratory, Cornell University, Ithaca, NY. Analysis revealed that the Theileria organisms within this blood sample were part of the Theileria buffeli complex.

The affected cow continued to deteriorate clinically, and euthanasia was elected. Repeated CBC analysis just prior to euthanasia revealed the ongoing presence of Theileria organisms within erythrocytes, although the anemia had marginally improved (hematocrit 0.18 L/L and hemoglobin 60 g/L). The cow was submitted to AHL-Kemptville for postmortem examination. Significant gross postmortem lesions consisted of marked edema and diffuse enlargement of the pelvic, hepatic, and peri-ruminal lymph nodes, mildly watery pale blood, and mild icterus of internal fat. Lameness was attributed to localized fibrinopurulent myositis of left thigh muscles, and periarthritis and arthritis of the left femorotibial joint. Histology of the enlarged lymph nodes showed cortical lymphoid hyperplasia, with germinal centers. In one node, medullary cords were populated predominantly by small lymphocytes, with scattered foci of extramedullary hematopoiesis. Lymphocytic schizonts were not seen in the lymph nodes, and are reported as uncommon in cows infected with T. buffeli. Bone marrow taken at postmortem was autolysed, and only adipose tissue with mild intercellular hemorrhage and scattered foci of hematopoiesis were identified.

This is the first documented identification of this parasite in cattle in Canada. Neither the affected cow nor her clinically unaffected herd mates had ever travelled outside of Ontario. Theileria are tick transmitted, protozoal erythroparasites of ruminants. The tick species associated with T. buffeli transmission has not been identified. The role of wildlife such as white-tailed deer as a possible parasite reservoir is also unclear. Although this cow had been on pasture, it is not known how she became infected.

Theileria buffeli has been reported previously in individual cows in the United States (Kansas 1950, Texas 1975, Missouri 2000, Michigan 2002 and 2014). T. buffeli has been considered non-pathogenic or less pathogenic than other Theileria spp. such as T. parva and T. annulata which are the agents of the rapidly fatal East Coast fever in Africa, and tropical theileriosis in the Mediterranean and Asia, respectively. Unlike the intralymphocytic schizonts which are considered the major pathogenic stage for T. parva and which also play a role in T. annulata infections, the intraerythrocytic piroplasms are the major pathogenic state for T. buffeli. T. buffeli has been identified both in asymptomatic cattle, as well as those with clinical evidence of anemia. The pathogenesis of anemia has not been clearly established and may be multifaceted, involving both immune-mediated mechanisms as well as erythrocyte fragmentation and damage by proteases and oxygen radicals. Lymphoid hyperplasia and lymphoma have been previously identified with bovine theileriosis. It is known that intra-lymphocytic theilerial parasites can transform cells, and lead to the clonal proliferation of lymphocytes, although it is unclear if this occurs with T. buffeli infections.

Currently, Theileria can only be detected by examination of peripheral blood smears. Therefore evaluation of a well-prepared blood smear is strongly recommended for all anemic ruminants.

Theileriosis is on the federal and provincial lists of immediately notifiable diseases, and therefore this case was reported to OMAFRA and the CFIA.   

Figure 1. Intraeryrthrocytic piroplasms of Theileria buffeli (arrows).