RUMINANTS

Tissue sampling and test selection for field PMs

Josepha DeLay, Jan Shapiro, Maria Spinato, Andrew Brooks

The type and quality of samples submitted to the diagnostic laboratory has a tremendous impact on the diagnostic information that you (and your client) will glean from testing. Your clinical and postmortem differential diagnoses will determine test selection for each case. Plan ahead for those tests that will likely be necessary, and know which specific samples are required for each test. Remember that tissues fixed in formalin are suitable only for histopathology and immunohistochemistry. Microbiologic tests require fresh tissue; frozen tissue may be substituted, if available. The general guidelines listed below for sampling are based on common, specific clinical conditions.

Diarrhea

Intestinal samples will obviously be important, and careful handling is required to prevent contamination of other tissues. For microbiologic testing, fresh intestinal segments 5-10 cm long should be placed into labelled whirl-pak bags. Feces may be collected in sterile screw-top containers.

General sample requirements:

· 1 sample of small intestine (jejunum or ileum) and colon for bacterial culture

· 1 sample of small intestine (jejunum) and colon for various viral/bacterial PCRs (dependent on species/age group)

· 1 fecal sample for parasitology

· multiple (6-10) samples from intestine for histopathology, including all levels of the intestinal tract and stomach (in formalin).

Optimal fixation will occur if the intestinal loop is opened longitudinally before placing in formalin (rather than fixing cross-sectional ‘doughnuts’ of intestine). For neonatal piglets, pups, and kittens, this is nearly impossible, and the thin-walled intestinal segments may be fixed intact (no opening necessary).

Example of sampling and testing for bovine neonatal diarrhea:

· Bacterial culture (SI / ileum and colon)

· Bovine coronavirus and ruminant rotavirus PCR (SI / jejunum and colon)

· Sucrose wet mount for Cryptosporidium (feces)

· Histopathology (multiple intestinal samples as above; also include mesenteric lymph node, forestomachs, abomasum, lung, thymus, heart, liver, spleen, kidney, adrenal gland, and skeletal muscle – to evaluate for other neonatal conditions including septicemia and nutritional myopathy)

Pneumonia

Lung is a large organ, with regional variation in susceptibility to various infectious agents. Samples should be taken using a clean scalpel blade, and placed in separate, labelled whirl-pak bags.

General sample requirements:

· 3 approximately 5 X 5 cm samples from the margin of affected and unaffected lung for bacterial culture, mycoplasma PCR, and viral PCR

· bronchial lymph node, trachea, heart, and 3 samples from each of cranial, middle, and caudal lobes of lung, and including the margin of affected and unaffected lung, for histopathology.

Example of sampling and testing for bovine pneumonia:

· Bacterial culture (lung, at margin of lesion and normal tissue)

· Mycoplasma bovis PCR (lung, at margin of lesion and normal tissue)

· BRSV, IBRV, PI-3, +/- BVDV PCR (lung, at margin of lesion and normal tissue)

· Histopathology (multiple samples as above)

Unexpected death with no gross lesions on PM

Sampling should include a wide range of tissues for histopathologic examination. A similar range of tissues should be frozen for potential microbiologic and / or toxicologic testing, based on histopathology results. Brain and heart are especially important. Head may be submitted to the diagnostic lab intact for brain removal, if desired. Heart may also be submitted intact for gross examination.

Histopathology: BRAIN (1 half of sagittally sectioned brain), HEART (left papillary muscle, interventricular septum, right ventricular free wall), lung x3 (cranial, middle, and caudal lobes), thyroid gland, thymus, kidney (include cortex and medulla), adrenal gland, pancreas, liver, spleen, mesenteric lymph node, skeletal muscle, gastrointestinal tract (1 section from each of forestomachs, stomach / abomasum, duodenum, jejunum, ileum, colon, rectum).

Freeze the following tissues, each in separate labelled whirl-pak bags: brain (half of sagittally sectioned brain), heart (left ventricle), lung (cranial lobe), kidney, liver, spleen, ileum, colon. Approximately 5 cm diameter samples from solid organs are appropriate.

**Also include STOMACH CONTENT and URINE among the samples to freeze. Each may be collected in a sterile screw-top container.

Additional information on PM methods and procedures, tissue sampling, and sample submission to the AHL can be found at the following links:

Tips for Practitioners for Field Postmortems https://www.uoguelph.ca/ahl/ahl-labnote-2-tips-practitioners-field-postmortems

Field and Clinic Postmortems: Simplified Protocol and Image List https://www.uoguelph.ca/ahl/ahl-labnote-42-field-and-clinic-postmortems-simplified-protocol-and-image-list

Brain Removal in Field Postmortems https://www.uoguelph.ca/ahl/ahl-labnote-33-brain-removal-field-postmortems

Submission Instructions https://www.uoguelph.ca/ahl/submissions/ahl-labnote-27-submission-instructions

Additional information on sampling and test selection information for specific disease conditions in various species can be found in the AHL Users’ Guide – Disease Investigation section: http://www.guelphlabservices.com/AHL/UsersGuide.aspx

Salmonella Dublin update

Durda Slavic, Andy Vince, Jim Fairles

Salmonella enterica ssp. enterica serovar Dublin is considered a host adapted Salmonella of cattle. It can affect cattle of all ages but it is usually more severe in younger, immunologically ‘naive’ animals. Calves can be infected at calving but do not necessarily show any clinical signs until they are 2-3 weeks old. In general, S. Dublin disease is most frequently reported in cattle over 3 months of age.

Lesions caused by S. Dublin usually differ from lesions caused by other Salmonella serotypes. S. Dublin more consistently results in septicemia in young calves, and less consistently in necrotizing enteritis.

Frequently, lesions include:

· multifocal necrotizing hepatitis and splenitis (but without producing the inflammatory “paratyphoid nodules” seen with other strains);

· arthritis (often involving the growth plates of long bones);

· bronchopneumonia;

· dry gangrene of the distal limbs, tail, and ears;

· abortion is common in endemic areas.

Although S. Dublin has been causing problems in cattle in the northeastern United States and in the Prairies for years, the first case detected in Ontario by our laboratory was in December of 2012. Since then, the bacteriology laboratory has been screening all bovine lungs submitted for culture and susceptibility for the presence of this pathogen through an enrichment method. No S. Dublin was detected in 2013. There was one veal operation with the clinical presentation of diarrhea and pneumonia in 6-8 week-old Holstein calves diagnosed with S. Dublin in 2014. To date in 2015, S. Dublin has been detected in 3 veal operations (2 new sites, and 1 from 2014). Multiple cases were received from these operations and the clinical history indicates that these animals predominantly suffer from pneumonia that is not responsive to treatment. In addition, these animals usually come from multiple sources including other Canadian provinces where cases of S. Dublin are on the rise.

Given that S. Dublin has been identified in Ontario, we recommend adding S. Dublin to a differential diagnosis for cases of septicemia and respiratory problems in young calves, particularly in veal operations.

The AHL bacteriology laboratory continues to screen all bovine lungs for the presence of S. Dublin and will start reporting results for routine submissions. If Salmonella sp. is isolated from lungs or from any other internal organs on primary culture, then serogrouping for group D Salmonella will be also reported. S. Dublin belongs to group D Salmonella but it is not the only member of group D. Therefore, isolation of group D Salmonella does not necessary means that it is S. Dublin, but in combination with clinical history it may be highly suggestive. All Salmonella isolates from the AHL are sent to the Public Health Agency of Canada for confirmation of serotype.

Bovine coronavirus (BCV) and rotavirus (BRV) – on the increase?

Murray Hazlett, Davor Ojkic, Josepha DeLay, Jim Fairles

A subjective increase has been noted in the number of bovine coronavirus (BCV) cases submitted to the AHL. This may in part be the result of better testing - we no longer rely on the bovine coronavirus ELISA, or the rotavirus A latex agglutination test (RLA) - these have been replaced by polymerase chain reaction (PCR) and immunohistochemistry (IHC) testing.

Figure 1 illustrates the percentage of positive bovine submissions by quarter for both coronavirus (blue) and rotavirus (red). It shows a dramatic increase in both the number of submissions that had at least one positive test (of any sort) for either bovine coronavirus or bovine rotavirus. For bovine rotavirus (BRV), this started in the first quarter of 2013, with a marked increase starting in 2014 Q1. For bovine coronavirus, this started in 2014 Q1 and continues to escalate. Our PCR tests for bovine coronavirus and bovine rotavirus were both instituted in late 2012 and our IHC for bovine coronavirus in 2007, so the dramatic increases do not seem to correlate with improved testing.

During this period, if a positive coronavirus submission was also tested for rotavirus, it had a 55% chance of being positive for both. Most of the rotavirus identified by PCR was type A (224 cases) with only 4 rotavirus B cases.

The data suggest an increase in both bovine coronavirus and bovine rotavirus cases among AHL submissions. Figure 2 shows the percent positive of cases tested for BRV by year and the percent positive of cases tested for bovine coronavirus by year. It shows an increase for both BRV, with a spike in 2013, and BCV, with a spike in the first two quarters of 2015. The rotavirus spike shown in Fig 1, 14-3 and 14-4, is likely the result of increased testing.

In the winter of 2014, we identified 2 cases that were strongly IHC positive for coronavirus (Fig. 3) but were PCR negative, and the primer was modified to identify the strain in these samples and to improve test sensitivity. This occurrence may have represented either a new strain of coronavirus, or simply that we were better at IHC testing, however an increase in positive BCV PCR tests was happening before this event.

While it does appear that the AHL is seeing more cases of BCV this year, this may not reflect the “real world” situation regarding BCV and BRV, as there may be other factors influencing lab submissions.


Figure 1. Percent positive bovine submissions by quarter, 2009 4^th quarter to 2015 2^nd quarter. There were totals of 208 BCV positive submissions and 233 BRV positive submissions.	Figure 2. Percent of tests that were positive by year.	Figure 3. Strong positive IHC staining for bovine coronavirus antigen in colon from a calf that was BCV PCR negative, showing the value of using more than one test for diagnostic evaluation.