Investigating respiratory disease

Transcription

1 Vet Times The website for the veterinary profession Investigating respiratory disease Author : David Gibson Categories : Vets Date : August 3, 2009 David Gibson explores diagnostic methodologies and sampling techniques for a bovine disease that has dire consequences for livestock keepers BOVINE respiratory disease (BRD) in housed cattle is an annual problem that can prove devastating for livestock keepers and frustrating for veterinary surgeons. Despite the wide range of vaccines, antimicrobials and antiinflammatory drugs for treatment and prevention, respiratory disease continues to form a large part of the farm animal practice workload during the winter housing period. This article aims to provide a guide to the diagnostic tests available to the practitioner, how to collect samples of diagnostic quality to utilise these tests and how to apply this information in the field. While there are undoubtedly economic issues to be addressed within farm animal practice, money spent on sampling and testing early in an outbreak of BRD may prevent deaths at a later stage, thereby saving money in the long term. Common respiratory tract pathogens The major pathogens can be divided into two groups bacteria and viruses. The most commonly diagnosed bacterial causes of pneumonia are Mannheimia haemolytica, Histophilus somni (formerly Haemophilus somnus), Arcanobacterium pyogenes, Pasteurella multocida and Mycoplasma bovis. Viral pneumonias are caused by respiratory syncytial virus (RSV), parainfluenza virus (PI3) and bovine herpesvirus (BoHV-one), the latter the cause of infectious bovine rhinotracheitis (IBR) Figure 1. 1 / 16

2 It is also important to remember the negative impact that bovine viral diarrhoea (BVD) virus can have on the immunological response of calves to respiratory pathogens. In cases of unexpectedly high morbidity or poor response to treatment, screening for BVD virus should be considered. Laboratory testing methods Veterinary investigation laboratories are a valuable and often under-used resource for the practising veterinary surgeon. They offer a range of diagnostic tests that can identify respiratory tract pathogens, thereby allowing an informed and targeted approach to treatment, control and prevention. Bacteriology To achieve best results, samples for bacterial culture should be submitted to the laboratory within 24 hours of collection. Plates will be inoculated and then incubated for an initial 24 hours. If numerous colonies of a pathogenic bacterium are present, then identification may be possible that day. However, where mixed bacterial growths occur, or contaminants are present, it may be necessary to subculture specific colonies for a further 24 to 48 hours. Antibiotic sensitivity reports are usually available within 24 hours of identification of pathogenic bacteria. Most laboratories will endeavour to provide a result within 48 to 72 hours of receiving the sample, although pathogens such as M bovis may require typing at a reference laboratory (Figure 2). Fluorescent antibody technique This technique uses fluorescent conjugate staining to highlight specific antibody-antigen reactions. It is primarily used to look for the presence of RSV, PI3 or BoHV. It is possible to carry out fluorescent antibody technique (FAT) examinations of samples in a few hours, making same-day reporting quite common for this test. Virus isolation The isolation of viruses is a specialist technique in which samples collected from clinical cases are used to inoculate cell cultures, and the viruses that subsequently grow are then identified. Due to the complex nature of this testing, it can take a number of days to complete. However, it is a valuable technique that can, in some cases, be crucial in reaching a diagnosis. Serology Serum samples can be screened for evidence of significant levels of specific antibodies, usually by 2 / 16

3 using ELISA. Serological tests can identify antibodies raised against RSV, PI3, BoHV-one (IBR), M bovis and H somni. Serology is best carried out on paired samples, with one sample taken when the animal is acutely ill and another taken 21 days later once it has recovered. The samples are tested together to ensure the validity of the results, and specific titre elevations between acute and convalescent samples indicate seroconversion. It is important to remember that in younger calves (less than four months old), the presence of maternally derived antibodies is likely to make a significant contribution to the titres and may inhibit seroconversion. This does not preclude carrying out paired serology in calves of this age, but careful interpretation of results is essential (Figure 3). Histopathology This technique looks for specific changes at a cellular level within respiratory tract tissues. It can be a useful adjunct to the other tests listed above, and in some cases may be the only appropriate test of diagnostic value. Histopathology (Figure 4) can be particularly useful where other diagnostic techniques have been unable to detect specific pathogens. Necropsy A visual assessment of the respiratory tract can be of great value in diagnosing the cause of BRD. The presence of particular gross changes consistent with those caused by specific pathogens is a useful aid to diagnosis in many cases. Necropsy also allows targeted tissue selection for further testing (bacteriology, FAT, virus isolation and histopathology) and, in some cases, may identify concurrent diseases or alternative aetiologies. Diagnostic sampling A fundamental part of good veterinary practice is to understand exactly which pathogen or pathogens is causing disease in any given clinical situation. Whether you are dealing with a persistent otitis media in a dog, acute diarrhoea in a foal or, indeed, an outbreak of respiratory disease in housed cattle, knowledge of the aetiological agent(s) is essential if successful treatment is to be provided and relapses are to be avoided. Numerous diagnostic techniques are available to the large animal practitioner when presented with a case of BRD. Obviously, the most fundamental of these is the clinical examination. Visual assessment of stance and demeanour, ocular and nasal discharges, and respiratory rate can provide valuable information as to the nature and extent of disease. This can be augmented by thoracic auscultation (to assess heart rate and lung sounds) and rectal temperature. 3 / 16

4 The other diagnostic techniques available all involve the collection of samples from clinically affected animals and are outlined below. Ocular and/or nasopharyngeal swabs These swabs (Figure 5) can be submitted for general bacteriology, FAT examination for BoHV-one and virus isolation of BoHV-one. While the collection of ocular swabs is fairly straightforward, nasopharyngeal swabbing requires good animal restraint. Ideally, two swabs should be taken from each suitable animal one for bacteriology and a second to be placed in virus transport media (VTM) for subsequent virus FAT and/or isolation. If possible, four affected animals should be sampled. Swabs are only of value if collected from acute cases that are febrile and have a clear nasal discharge. Bronchoalveolar lavage Bronchoalveolar lavage (BAL), when carried out correctly and successfully, is of excellent diagnostic value. It allows the detection of both viruses and bacteria. BAL kits are usually available from veterinary investigation laboratories, which will also be able to provide guidance for inexperienced people on the technique of use. BALs tend to be underutilised by practitioners, most likely because the method is considered time consuming, requires good animal restraint and requires practice for its value to be realised. However, the author would encourage practitioners to try BALs as often as possible, as practice will make perfect. Blood samples Paired serology can be very useful in determining which pathogens were involved in an outbreak, particularly if cattle have been vaccinated and vaccine efficacy is in question. Results from paired serology can also help to inform vaccine protocols for subsequent years. It requires the collection of an acute sample and a convalescent sample, taken 14 to 21 days later from the same animal. The author recommends taking a blood sample (and recording the ear tag number) from all calves treated in an outbreak. These acute samples can be frozen in the practice or submitted to veterinary investigation laboratories for storage, pending the collection of convalescent samples. In outbreaks where cattle respond well to treatment and no further investigation is required, the 4 / 16

5 samples can be discarded. However, if an outbreak becomes a more complex ongoing problem, the sampled animals can be re-sampled, and a valuable diagnostic opportunity is not lost. Where cattle are more than four months of age, at least six animals should be sampled. If calves are less than four months of age, 12 animals should be sampled. Postmortem examination This represents the gold standard of diagnostic opportunities. Visual examination of the entire carcase and targeted sample collection maximises the likelihood of reaching a diagnosis. Ideally, carcases should be submitted to a veterinary investigation laboratory for examination as soon after death as possible. The advantages of a laboratory necropsy include aseptic sample collection, less risk of sample autolysis, more rapid processing of samples and faster results reporting. Where an on-farm necropsy cannot be avoided, trachea and lung tissue should be collected as aseptically as possible. Samples should be taken from pathological tissue and the margins of any lesions. A 2cm3 to 3cm3 section should be placed in a sealed container. This can be used for bacterial culture and FAT examination. A 0.5cm3 section should be placed in a transport medium (available from your local veterinary investigation laboratory). This can be used for FAT examination and virus isolation. Several 1cm3 sections from the edge of lesions should be placed in five to 10 times the volume of formal saline. Diagnostic protocols So, having outlined the tests that are available and the samples that can be collected to utilise these tests, how can this information be used in a practical way? It is helpful to adopt a structured approach to ensure that you are able to maximise the diagnostic potential of the samples you can take. Identify which aetiological pathogens are likely to be involved in the outbreak. Factors that will influence this differential list may include: clinical signs; the age of affected stock; the source of affected stock; the vaccination history of affected stock; and the BVD status of the herd. 5 / 16

6 Sampling Having (hopefully) narrowed down the list of likely aetiological agents, you need to decide which samples are most appropriate and likely to yield a positive diagnostic result. For example, where IBR is strongly suspected, ocular and nasopharyngeal swabs may be sufficient. However, where clinical signs are more ambiguous, BAL and paired serology may be of greater relevance. Determine the suitability of the cattle for the intended sampling. Animals may be too far through the disease process for ocular and nasopharyngeal swabbing, or the facilities on the farm may not be suitable to provide the restraint needed for bronchoalveolar lavage. If a bacterial pneumonia is suspected, then sampling or submission of animals that have been treated with antibiotics is rarely worthwhile. Decide how many cattle to sample. The number of animals that are clinically affected, the handling facilities that are available or the cost that sampling and testing will incur may govern this decision. The live animal sampling protocols below are intended as a guide, and consideration should always be given to the aforementioned factors before embarking on a sampling programme. Suspect primary bacterial pneumonia nasopharyngeal swabs for bacteriology; place second swabs in VTM; bronchoalveolar lavage for bacteriology; and blood samples for H somni and M bovis paired serology. Suspect IBR (ideally sample at least four animals) perform ocular swabs for IBR FAT; perform nasopharyngeal swabs for IBR FAT; place second swabs in VTM; and blood sample for IBR-paired serology. Suspect primary viral pneumonia perform BAL for IBR, RSV and PI3 FAT; and 6 / 16

7 blood sample for IBR, RSV and PI3-paired serology. Suspect mixed bacterial and viral pneumonia perform ocular swabs for IBR FAT; nasopharyngeal swabs for IBR FAT and bacteriology; place second swabs in VTM; bronchoalveolar lavage for IBR, RSV and PI3 FAT and bacteriology; and blood samples for IBR, RSV, PI3, H somni and M bovis paired serology. As a general rule, bronchoalveolar lavages, and ocular and nasopharyngeal swabs should be collected from at least four acutely infected animals to maximise the diagnostic potential of the samples. For the same reason, at least six animals should be blood sampled for paired serology if calves are more than four months of age, and 12 animals should be sampled if they are less than four months of age. 7 / 16

8 Figure 1. Incidence of viral and bacterial causes of pneumonia in cattle, Graph: SAC AND VLA VIDA DATA. 8 / 16

9 Figure 2 (above). A Mannheimia haemolytica growth on a blood agar plate. 9 / 16

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12 Figure 3 (right). A centrifuged clotted blood sample ready for serum removal. 12 / 16

13 Figure 4 (below). Histological section showing acute fibrinous pneumonia due to pasteurellosis. 13 / 16

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15 Figure 5. A guard with a long nasopharyngeal swab. 15 / 16

16 Figure 6. Lung tissue showing emphysema typical of RSV infection. 16 / 16 Powered by TCPDF (