CASE NOTES


THE FUTURE OF VETERINARY DIAGNOSTICS: MORE THAN THE LABORATORY

Adrian W. Philbey, NSW Department of Primary Industries, State Veterinary Diagnostic Laboratory, Elizabeth Macarthur Agricultural Institute

Posted Flock & Herd March 2012

Molecular diagnostics

We are moving into the age of molecular diagnostics, with potential for high throughput, rapid turnaround and highly specific molecular detection of pathogens in veterinary species. Real-time quantitative polymerase chain reactions (PCRs), multiplex PCRs, microarrays and rapid sequencing are now in routine use in state of the art veterinary diagnostic laboratories at relatively favourable costs. The increased application of molecular diagnostic techniques will bring rewards, but will not be without challenges. It will bring solutions, but will not solve all problems. As always, strong communication between laboratory and field staff will continue to be essential in veterinary disease investigations as we enter this new era and explore the range of advanced technologies that are becoming available to us.

Advances in molecular bacteriology

The State Veterinary Diagnostic Laboratory (SVDL) currently has polymerase chain reaction (PCRs) for a wide range of bacteria. Some of these assays can be used directly on tissue samples, while others are used to confirm the identity of bacteria isolated in culture:

Actinobacillus pleuropneumoniae: Porcine pleuropneumonia

Bordetella sp.: Respiratory disease in pigs

Haemophilus parasuis: Glasser's disease

Pasteurella multocida: Pneumonia in pigs, cattle and sheep

Erysipelothrix rhusiopathiae: Swine erysipelas

Streptococcus suis: Porcine meningitis, septicaemia and arthritis

Brachyspira hyodysenteriae: Swine dysentery

Brachyspira pilosicoli: Swine dysentery

Lawsonia intracellularis: Porcine proliferative enteropathy

Histophilus somni: Ovine epididymitis; Bovine pneumonia; Thromboembolic meningoencephalitis; Mastitis; Abortions; Otitis; Conjunctivitis

Actinobacillus seminis: Ovine epididymitis

Brucella ovis: Ovine epididymitis

Campylobacter fetus subspecies venerealis and fetus: Ruminant abortion and infertility

Tritrichomonas fetus: Trichomoniasis

Actinobacillus equuli: Foal pneumonia

Streptococcus equi subspecies equi: Strangles

Taylorella equigenitalis: Contagious equine metritis

Chlamydia sp./Chlamydophila sp.: Chlamydiosis/chlamydophilosis; Ovine polyarthritis

Bacillus anthracis: Anthrax

Listeria ivanovii and Listeria monocytogenes: Listeriosis

Yersinia pseudotuberculosis: Yersiniosis

Escherichia coli virulence factors: Haemolysin gene (hlyA, ehxA), intimin gene, genes for Shiga toxins 1 and 2

Mycobacterium avium subspecies paratuberculosis: Johne's disease

Leptospira spp.: Leptospirosis

Avibacterium (Haemophilus) paragallinarum: Infectious coryza of chickens

Streptococcus iniae: Septicaemia in fish

Mycoplasma bovis: Pneumonia, polyarthritis and mastitis in cattle

Mycoplasma hyopneumoniae: Enzootic pneumonia of pigs

Mycoplasma mycoides cluster: Contagious bovine pleuropneumonia; Contagious caprine pleuropneumonia

Mycoplasma (Eperythrozoon) ovis: Anaemia in sheep

Melissococcus pluton: European foulbrood of bees

Nosema apis: Nosemosis of bees

Paenibacillus larvae subspecies larvae: American foulbrood of bees

Multiplex assays:

Brucella ovis, Actinobacillus seminis and Histophilus somni

Pasteurella multocida and Histophilus somni

Streptococcus suis and Pasteurella multocida

Advances in anthrax diagnosis

The standard method for confirmation of anthrax requires submission of smears of peripheral blood or tissues to be stained with polychrome methylene blue (PMB) for demonstration of Bacillus anthracis. Blood smears should be relatively thick and should be sent from more than one animal if there are multiple deaths. Smears should be air dried and, if possible, should be fixed with methanol for 20 minutes prior to submission to improve adherence and reduce infectivity. Samples should be packaged appropriately and despatched as standard diagnostic submissions. A clear warning note indicating a suspicion of anthrax should be placed on top of the packaging inside the box or esky. The warning note should not be placed on the outside of the consignment, since this may result in a delay in transportation. At the State Veterinary Diagnostic Laboratory (SVDL), containers with anthrax submissions are opened in a class I microbiological safety cabinet and decontaminated with a disinfectant (Virkon) before being handled.

An immunochromatographic test (ICT) is now available as a rapid field test for anthrax. ICT kits for detection of B. anthracis supplied by the Victorian Department of Primary Industries are available from the NSW SVDL to be used in the field. They provide a rapid on-farm test that is reliable in relatively fresh carcasses but may give false negative results in decomposed carcasses. A positive field test result may preclude the need for a more detailed postmortem examination, thereby avoiding unnecessary occupational risks and environmental contamination.

However, the assay still requires laboratory confirmation since the ICT is not formally approved by the Sub-Committee on Animal Health Laboratory Standards (SCAHLS). Thus, it is still necessary to submit smears to the SVDL for PMB staining and, if necessary, polymerase chain reaction (PCR) for confirmation. This is also relevant with respect to consignment of samples to the laboratory, since samples not yet confirmed as 'anthrax positive' can be submitted as routine diagnostic submissions. Please let us know on the specimen advice form if you have used the ICT in the field, since this will help us to monitor the use and efficacy of the kits.

Sometimes PMB staining of blood and tissue smears will give equivocal results, particularly if there is postmortem decomposition with contaminating clostridia and pink-staining debris that resembles B. anthracis capsular material. We are able to perform the PCR for B. anthracis in these equivocal cases by scraping material from blood or tissue smears. PCR results are usually available on the same day or the next day after receival at the SVDL.

1. Please contact the State Veterinary Diagnostic Laboratory for supplies of anthrax immunochromatographic test (ICT) kits.

2. Please indicate on your specimen advice form whether the anthrax ICT kit has been used as part of your field investigation and whether the result was positive, negative or inconclusive.

3. Please indicate the state of decomposition of the carcasses on which the ICT kit was used and from which blood or tissue smears have been collected.

4. Anthrax submissions should be packaged with care and a warning note should be placed on top of other packaging inside the box or esky (not on the outside of the box or esky).

Advances in molecular parasitology

At present, faecal egg counts (FECs) for monitoring nematode infestations in sheep are performed using the modified McMaster method, usually on 10 individual faecal samples from the flock. In submissions with moderate or high FECs, the faecal samples are pooled for larval culture to assess the proportion of each species of nematode contributing to the parasite burden. Larval culture takes 10 days, which represents a substantial delay when it is important to differentiate Haemonchus contortus burdens from infestations with Teladorsagia spp. or Trichostrongylus spp.

Several studies have been published in which PCR has been used to differentiate nematode larvae in DNA extracted from sheep faecal samples. Multiplex PCR for larval differentiation is likely to be more expensive than 10 day larval culture, but could deliver differential results within 1 or 2 days of receiving the faecal samples. It is envisioned that PCR eventually will be offered as a diagnostic assay for larval differentiation, giving submitters the choice of cost versus turn-around time for delivery of results.

Diagnosis of anthelminthic resistance in sheep nematodes is still largely reliant on the faecal egg count reduction test (FECRT). In vitro tests for anthelminthic resistance are available, but are not widely used as routine diagnostic tests. Interpretation of these assays may be limited by cost, the presence of multiple species of nematodes in any one sample and the range of anthelminthics that might need to be tested for. In the future, it is likely that multiplex PCRs will be available for direct detection of anthelminthic resistance genes in DNA extracted from nematode eggs in faecal samples.

1. Introduction of a multiplex polymerase chain reaction for larval differentiation of nematodes in sheep would result in more rapid reporting of results but would cost more than the standard 10 day larval culture.

2. There is potential to replace the faecal egg count reduction test with a multiplex polymerase chain reaction for direct detection of known anthelminthic resistance genes in DNA extracted from nematode eggs in faecal samples.

Advances in diagnosis of bovine theileriosis

With the emergence of theileriosis in cattle in New South Wales, there is a need to develop improved diagnostic assays for routine laboratory testing. Currently, the standard diagnostic approach is to submit blood in ethylene diamine tetraacetic acid (EDTA) and blood smears for Diff-Quik or Giemsa staining. The packed cell volume (PCV) is determined from the EDTA-treated blood. The smears are examined for evidence of intraerythrocytic organisms consistent in morphology with Theileria spp. and the percentage of infected erythrocytes is estimated. There is still some debate as to whether the percentage of infected erythrocytes is correlated with clinical signs, since there will be fluctuations in parasitaemia related to immunity and stage of disease.

A PCR for detection of Theileria spp. is available at the Elizabeth Macarthur Agricultural Institute (EMAI) and has been used as a research tool. Real-time and multiplex PCRs for detection, quantification and differentiation of Theileria orientalis sub-types Ikeda, Chitose and Buffeli are under development in the State Veterinary Diagnostic Laboratory (SVDL). This will allow strains of different pathogenicity to be distinguished and also will allow mixed infections to be identified.

There is also a need to be aware of the potential for cattle to be infected with Babesia spp., Anaplasma spp. and Mycoplasma (Eperythrozoon) wenyonii.

1. The current standard diagnostic method for bovine theileriosis is examination of stained blood smears by microscopy.

2. Blood in EDTA should also be submitted for determination of the packed cell volume.

3. The polymerase chain reaction for detection of Theileria spp. and differentiation of types currently is a research tool but should be available as a diagnostic test during 2012.

Submission of samples for bacteriology

Due to the cost of laboratory testing, a staged approach often will be taken to working up each diagnostic case. We are often asked to delay processing fresh tissues for bacterial culture until histopathology or other testing has been completed. However, please note that it may take several days for initial results to be reported. Therefore, usually it is best for fresh tissues to be processed for bacteriology immediately on receipt at the diagnostic laboratory.

When collecting samples of fresh lung, liver or other solid visceral tissues for bacteriology, collect a block of tissue with dimensions of several centimetres and submit it in a clean plastic screw topped container (e.g. 50 mL yellow topped container). For routine culture, the laboratory technicians will sear the surface with a heated spatula to remove surface contamination and then will use a Pasteur pipette to remove an uncontaminated sample from the centre of the sample for inoculation onto agar.

Segments of intestine with contents can be placed directly into clean plastic screw topped containers. Some submitters prefer to tie off each end of the segment of intestine with string, but this is not usually necessary. Stomach or intestinal contents or samples of faeces can be poured or placed into clean or sterile plastic screw topped containers for bacteriology.

Samples of fluid for bacteriology from body cavities or hollow organs can be collected using a syringe with or without a needle. However, please transfer the fluid from the syringe into a sterile screw topped plastic container to be submitted to the laboratory. Please do not submit material to the laboratory in syringes and please ensure that no needles are included.

Swabs are useful for collection of small volumes of material for bacteriology from surfaces such as the conjunctiva. However, generally swabs are less suitable for sampling from sites such as the intestines, lungs or liver, or other solid visceral tissues, when larger amounts of sample are available. Usually it is better to collect samples of faeces from the rectum rather than swabbing material from the rectal mucosa. Note that Ames charcoal transport medium is suitable for bacteriology, but samples collected into this medium will not be suitable for virology.

When submitting swabs for culture of Dichelobacter nodosus from sheep with footrot for virulence testing, please ensure that the footrot transport medium is within its specified use by date, otherwise the results may not be valid.

When performing postmortem examinations on cases with suspected arthritis, you could consider submitting the entire unopened joint for bacteriology to minimise contamination during sampling.

Samples for bacteriology should be submitted chilled with a cold block but not frozen. Sample containers can be placed near to a frozen cold block but should not have direct contact to reduce the risk of freezing. Please ensure that samples are contained within appropriate sealed containers and plastic bags. Absorbent material should be placed around the samples in case of leakage of fluid.

Please specify if selective cultures for specific pathogens, such as Salmonella spp., Yersinia spp., Campylobacter spp., Haemophilus spp., Mycobacterium avium subspecies paratuberculosis, Mycoplasma spp. or anaerobes such as Clostridium spp., are required. Note that samples from cases of infectious keratoconjunctivitis ('pinkeye') need to be plated directly onto agar plates in the field for isolation of Moraxella bovis.

1. Usually it is best to avoid any delay in processing fresh tissues for bacteriology.

2. Blocks of tissue from the lung, liver or other solid visceral tissues are often the preferred samples for bacteriology.

3. Segments of intestine with contents can be placed directly into containers.

4. Stomach or intestinal contents or samples of faeces can be poured or placed into clean or sterile plastic screw topped containers for bacteriology.

5. Samples of fluid for bacteriology from body cavities or hollow organs should be transferred into sterile screw topped plastic containers. Please do not submit material to the laboratory in syringes.

6. Swabs are useful for collection of small volumes of material for bacteriology from surfaces such as the conjunctiva.

7. Samples for bacteriology should be submitted chilled with a cold block but not frozen.

8. Please specify if selective culture for specific pathogens is required.

Submission of samples for histopathology

Histopathological examination of formalin fixed tissues will continue to play an important role in veterinary diagnostic investigations as a routine complement to gross pathological examinations.

Collection of tissues

We are commonly experiencing problems with submission of fixed tissues for histopathology. Frequently, portions of tissue are too thick to allow formalin fixative to penetrate adequately. Often the volume of formalin is not sufficient. We also have problems with unsuitable containers and leakage of formalin.

Ideally, portions of tissue from the lung, heart, liver, kidney, spleen and muscle should be cut to about 1 cm thickness to allow adequate penetration of formalin fixative. If the portions of tissue are thicker than this, the deeper tissue will not fix adequately. If sections are too thin, the tissue may be damaged during cutting and sometimes will become distorted during fixation, making it difficult for the veterinary pathologist to cut representative portions of tissue into cassettes for histological processing.

Portions of intestine should be cut open to allow formalin fixative to penetrate to the mucosal surface. Although the wall of the intestine is usually thin, it is relatively resistant to penetration of fixative. This is compounded further by the rapid rate of autolysis of the mucosa. In most cases, the intestine can be opened longitudinally with scissors. If the samples are from a freshly killed animal, contraction of the intestinal musculature may result in distortion. This can sometimes be prevented by allowing the intestine to adhere to cardboard for around 30 seconds before being opened up and placed in fixative. Some pathologists recommend submitting intestine as a tube with nicks cut at each end with scissors, but this does not always allow adequate penetration of fixative.

Brains should be fixed whole. Despite the large size of the brain, penetration of formalin fixative into central nervous tissue is generally adequate for most diagnostic purposes, whereas attempting to slice fresh brain into thin portions introduces substantial handling artefacts. Brains may be perfused with formalin for improved fixation, but special apparatus is required and the process of perfusion may introduce artefacts if excessive pressure is applied.

Please note that usually it is not necessary to place the tissues collected from one animal into separate labelled containers. Generally, the veterinary pathologist is able to differentiate tissue types when cutting the fixed tissues into cassettes and subsequently when examining the histological section. Separate containers are usually only necessary when (1) it is necessary to clearly identify and differentiate similar tissues from different sites, e.g. muscles from different locations; left and right lung lobes; (2) it is desirable to prevent contamination of tissues from one site with loose material from another, e.g. contamination of other tissues with contents from the gastrointestinal tract; or (3) if each container is not large enough to fit in the appropriate amount of tissues with an appropriate amount of fixative.

Tissues being collected for histopathology should be handled carefully to avoid handling or crush artefacts. If handling tissues with forceps, it is often good practice to cut off the portion used to hold the tissue as you drop the untouched portion of tissue into fixative.

Containers should be selected carefully so that they are the appropriate size and shape for collection of samples. It is best to avoid placing large portions of tissue (especially brain) into containers with small necks, since it can be difficult to remove the tissue when it hardens with fixation. It is best to add tissues to fixative rather than fixative to tissues to ensure that all surfaces of the tissue are exposed to formalin. Plastic containers with screw topped or press sealed lids are preferred. The main criterion is that the sample must not leak in transit. Avoid overfilling containers, since changes in pressure due to handling and temperature fluctuations can increase the risk of leakage. There is little value in placing tape around the lids. Generally, it does not reduce the risk of leakage and is often difficult to remove. Please avoid glass containers, since they are more prone to breakage.

Fixation of tissues

The appropriate fixative for histopathology is 10% neutral buffered formalin. Special fixatives are rarely necessary. Bouin's solution frequently has been used for fixation of eyes, since it penetrates tissue more rapidly than formalin, but adequate fixation of eyes can usually be achieved with routine formalin fixation. Also, samples should be transferred from Bouin's solution to 10% neutral buffered formalin the next day to prevent degradation of tissues. A small quantity of formalin can be injected into large eyes to aid fixation. Glutaraldehyde fixative is often used for electron microscopy, although for many investigations it is acceptable to fix tissues first in formalin and then to post-fix in glutaraldehyde for subsequent electron microscopy.

Please use an adequate volume of fixative relative to tissues. A generous rule is ten volumes of fixative to one volume of tissue. It is acceptable to fix tissues in formalin overnight and then to decant the tissue into a smaller volume of formalin for transportation to reduce the risk of leakage. Some submitters wrap pre-fixed tissues in formalin-soaked tissue paper with good results.

Histological processing

The usual minimal time scale for histological processing is 2 days. Tissues are fixed overnight in 10% neutral buffered formalin, allowing them to be cut into cassettes the next day and then processed overnight (this involves being dehydrated in graded ethanol solutions and infused with molten paraffin wax). The processed tissues are embedded in paraffin wax, sectioned, allowed to adhere to glass slides, stained with haematoxylin and eosin, and then finally sealed with a glass cover slip.

1. The standard fixative is 10% neutral buffered formalin.

2. Solid portions of tissue should be cut to no more than 1 cm thickness to allow adequate fixation.

3. Portions of intestine should be cut open to allow formalin fixative to penetrate to the mucosal surface.

4. Brains should be fixed whole.

5. Usually it is not necessary to place tissues collected from one animal into separate labelled containers.

6. Tissues being collected for histopathology should be handled carefully to avoid handling or crush artefacts.

7. Containers should be selected carefully so that they are the appropriate size and shape for collection of samples. The lid must have an adequate seal to prevent leakage in transit. There is little value in placing tape around the lids.

8. Use an adequate volume of fixative relative to tissues. A generous rule is ten volumes of fixative to one volume of tissue.

9. It is acceptable to fix tissues in formalin overnight and then to decant the tissue into a smaller volume of formalin for transportation to reduce the risk of leakage.

Immunohistochemistry

Immunohistochemistry (IHC) is widely used in veterinary diagnostic laboratories for detection of infectious agents and tumour markers. IHC for Newcastle disease virus, West Nile virus, Kunjin virus, porcine circovirus type 2, Bungowannah virus and Lawsonia intracellularis has been applied in the research laboratories at the Elizabeth Macarthur Agricultural Institute (EMAI). In the State Veterinary Diagnostic Laboratory (SVDL), we intend to develop IHC as a routine diagnostic tool in our histology laboratory.

For most diagnostic purposes, IHC can be performed on formalin-fixed paraffin-embedded tissues. This will allow us to continue to use our present processing and embedding system, along with the current microtomes. For detection of some markers, it is necessary to use antigen retrieval systems such as microwaving and/or pressure cooking to unmask antigens modified by formalin fixation. For detection of markers that cannot be unmasked by these techniques, it will be necessary to prepare cryostat sections cut from specially prepared unfixed frozen tissues, but we do not have this capacity at present.

Systems of immunostaining currently available include the avidin-biotin complex (ABC), polymer-based detection systems (EnVision, ImmPress or MultiVision), labelled streptavidin-biotin (LASB), peroxidase-antiperoxidase (PAP) and immunofluorescent antibody (IFA) techniques.

Our initial aim is to work up commercial monoclonal antibodies for detection of

Neospora caninum and Toxoplasma gondii. The N. caninum immunostaining in particular would have application in brains and hearts from aborted foetuses. Currently, we send samples to the Tasmanian Department of Primary Industries and Water (DPIW) for these IHC assays.

Transmissible spongiform encephalopathies

(Acknowledgements: Rod Reece and Dermot McNerney)

Australia is free from bovine spongiform encephalopathy (BSE) and scrapie, and has been designated as having a 'negligible risk' status (the lowest risk category) by the World Organisation for Animal Health (OIE). To verify our ongoing freedom from TSEs, active surveillance commenced in Australia in 1990 and the National TSE Surveillance Program (NTSESP) was established in 1998. Specified diagnostic samples collected from a designated number of cattle and sheep are examined each year for TSE exclusion and the results are reported to the OIE. The animals must be of an appropriate age and must exhibit neurological signs similar to those observed in cattle with BSE or sheep with scrapie. 'Fallen' and casualty slaughter cattle and sheep are also examined as part of the programme.

The NTSESP encourages submission of samples by (1) providing incentives to farmers to have affected animals examined; (2) reimbursing veterinarians for collecting samples, providing case details and completing the paperwork; and (3) covering the cost of laboratory testing to exclude TSE and to provide an alternative diagnosis. The latter is often of direct benefit to the farmer.

Postmortem examinations are performed on cattle and sheep exhibiting appropriate clinical signs. The whole brain is removed from each animal and submitted in 10% neutral buffered formalin to the State Veterinary Diagnostic Laboratory at the Elizabeth Macarthur Agricultural Institute, Menangle, along with a 2 cm long segment of the cervical spinal cord submitted unfixed and chilled; these samples are essential. Other samples relevant to the diagnostic investigation may be submitted, e.g. serum for clinical biochemistry, non-nervous tissues fixed for histopathology, fresh liver and kidney for lead analysis.

TSEs affect specific regions of the brain and these target areas must be included in the fixed brain for the case to be accepted into the NTSESP and for payments to be forthcoming to both the farmer and veterinarian. These specific regions are the caudal mid-brain at the level of the superior colliculus, the brain stem at the level of the cerebellar peduncles and the caudal brain stem at the obex. Unless all of these target areas are present and intact, the case cannot be accepted into the programme. The fixed brain is processed for histopathology and routinely stained sections are examined by the veterinary pathologists for vacuoles within the cytoplasm of neurones and in the grey matter. Samples from any suspicious cases are submitted to AAHL for further testing for specific accumulation of abnormally folded prion proteins by immunohistochemistry.

Cattle are only eligible for inclusion in the programme if more than 30 months and less than 9 years of age. Sheep must be more than 18 months of age. A maximum of two animals per clinical episode per property will be accepted.

The property identification code (PIC) must be included in the accompanying documentation. See: http://www.dpi.nsw.gov.au/agriculture/livestock/nlis/pic

Further information is available on the website:

http://www.animalhealthaustralia.com.au/aahc/programs/adsp/tsefap/tsefap_home.cfm

DVDs are available from the NSW Department of Primary Industries depicting clinical signs of endemic diseases in sheep and cattle for which the differential diagnosis could be TSE. A DVD is also available showing how to remove the brain for submission for TSE exclusion.

The NSW Department of Primary Industries coordinator for the NTSESP is Dermot McNerney, NSW Department of Primary Industries, Dareton NSW 2717: Telephone: 03 5019 8411; Mobile: 0428 223 978; Email: dermot.mcnerney@dpi.nsw.gov.au

1. If the fixed brain does not include the specified target areas, the case cannot be accepted into the National Transmissible Spongiform Encephalopathy Surveillance Program

2. Cattle are only eligible if more than 30 months and less than 9 years of age.

3. Sheep must be more than 18 months of age.

4. A maximum of two animals per clinical episode per property will be accepted.

5. The property identification code (PIC) must be included in the accompanying documentation.

6. Animals must exhibit nervous signs and behavioural changes such as might be observed in TSEs, such as ataxia, weakness, head-pressing, tremors, depression and/or hyperexcitability.

7. All of the above criteria must be satisfied, otherwise no payment will be forthcoming to the veterinarian or farmer.

 


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