Approaches to the Diagnosis of Emerging and Major Endemic Viral Diseases of Cattle
Peter D. Kirkland
Virology Laboratory, Elizabeth Macarthur Agriculture Institute,
Woodbridge Rd, Menangle NSW Australia
peter.kirkland@dpi.nsw.gov.au
1. Introduction
There are some diseases for which a diagnosis can be made based on either clinical signs
and/or gross pathology observations. However, a high proportion of infectious diseases
require laboratory testing to be undertaken to achieve an aetiological diagnosis. Further,
laboratory investigations are essential to unravel the potential involvement of several
pathogens and to establish their relative significance in a multi-factorial disease problem.
Identifying the primary pathogen is also essential for effective disease control to be
undertaken. The role of the animal that is persistently infected with BVDV in respiratory
disease in intensively managed cattle provides an excellent example of these points.
During the investigation of a disease event in production animals, both the time to achieve a
test result (‘turn around time’) and the cost are usually important considerations for the
clinician and are likely to influence the extent to which samples are submitted for laboratory
testing. In the last 15 years there have been a number of changes in the technologies and range
of tests available to the diagnostic virology laboratory. These have made a significant impact
on the capacity to rapidly confirm infectious diseases and at a realistic cost. However, to be
able to take best advantage of these advances, it is important for clinicians and ultimately
veterinary pathologists who are submitting samples to the virology laboratory to be aware of
the most appropriate specimens, the limitations of the technologies and the interpretation of
results. These issues will be discussed in detail in the context of major endemic viral diseases
and also some diseases that are thought to be uncommon, perhaps mainly due to difficulties in
achieving an aetiological confirmation.
2. Evolution of diagnostic tools
In the past, diagnostic virology laboratories relied on methods such as virus isolation,
immunofluorescence or immunoperoxidase staining and agglutination based methods for
detection of a virus or its components. In some laboratories agarose gel based PCR was used
with increasing frequency but was prone to issues with contamination and is relatively labour
intensive. Enzyme linked immunosorbent assays (ELISA) have been used more frequently for
some diseases. Antibody detection relied on cell culture based virus neutralisation assays,
haemagglutination inhibition, agar gel immunodiffusion assays and more recently ELISAs.
Even though some of these assays (eg ELISAs) can be completed relatively quickly and at
low cost, most are dependent on the availability of high quality antigens or antibodies. This
has sometimes limited more widespread application of ELISA technology to diseases for
which there is a high demand and hence potential for develop of commercial kits.
Nevertheless, some ELISAs have had a major impact on the diagnosis of important diseases
of cattle, with antigen detection ELISAs for BVDV being an exceptional example while
assays for the detection of antibodies for viruses such as bluetongue, bovine herpesvirus 1
(IBR), RSV, PI3 and enzootic bovine leucosis commonly in use for testing individual
animals.
While there has been a gradual evolution of testing technologies, the greatest step forward has
come through the widespread adoption of real time PCR (qPCR, or for RNA detection, qRTPCR). Real time PCR offers numerous benefits for application to diagnostic investigations.
These include turn around time, high analytical and diagnostic sensitivity, a capacity to test
samples of inferior quality and usually over a longer time frame during the course of a disease
compared to virus isolation. There are many other opportunities to capitalise on the power of
this technology and is limited mostly by the creativity of laboratory scientists. There is
considerable merit in re-investigating the biology of a disease of interest as this may provide a
foundation for selection of alternative sample types (eg hair, skin, milk or body fluids) and
provide data that is needed to establish the practicality of sample pooling. qPCR is also highly
prescriptive and new assays can be readily adopted/transferred to other laboratories.
The capacity for multiplexing of real time PCR assays (ie testing for multiple agents on the
same sample and in the sample test) provides an efficient method for the investigation of
disease syndromes. There is also a variation on multiplexing – referred to as multitasking
whereby several different assays are run under uniform conditions at the same time and on the
same test plate. Both multiplexing and multitasking provide considerable economies of scale,
on one hand reducing the cost of assays for individual samples and significantly reducing
‘turn around’ time – allowing most tests to be completed on a ‘same day’ basis.
These new technologies have greatly enhanced the capacity of diagnostic laboratories,
allowing provision of a test capacity for an uncommon disease. This has allowed more
frequent laboratory confirmation of apparently uncommon diseases (eg BMC) or rapid
confirmation of acute diseases (eg BEF) for which there were either no agent-specific
confirmatory assays, or virus isolation was difficult or depended on serological confirmation
using paired sera. Being able to provide rapid laboratory confirmation of a viral disease has
had a marked impact on the submission patterns of clinicians, usually with more frequent use
of the laboratory and in turn increasing the frequency of confirmation of a disease.
While ELISA assays may not be seen as a new technology, assays for some diseases have
undergone significant refinement with an increase in analytical sensitivity (eg BVDV) or they
have been utilised in a more creative manner. Applications include the use of ELISAs for the
detection of antibodies in individual or pooled samples. The high analytical sensivity of some
ELISAs has led to the development or protocols to support cost effective testing of pools of
individual samples or establishment of herd profiles (eg testing of tank milk samples).
There are several other technologies that are progressively being introduced into diagnostic
laboratories. These include nucleic acid sequencing for the identification of pathogen strains,
subtypes or topotypes to guide decisions for disease control (eg selection of vaccines) or assay
platforms that allow multiplexing for the detection of either nucleic acids or antibodies (eg
Luminex/Magpix). While whole genome sequencing is being utilised widely, it is unlikely to
become a tool for routine diagnostic use but rather be used for the identification of new or
widely variant organisms.
3. Specimen collection
In order to maximise the benefits of new rapid diagnostic procedures, it is important to have
appropriate samples submitted to the laboratory. The veterinary pathologist is a key person in
the diagnostic chain as he/she is frequently the front line person or interface between the
client and the specialist virology laboratory. The pathologist may be involved in the selection
or collection of specimens or perhaps to advise the clinician on specimen collection.
However, there are also occasions when suitable specimens have been collected and
submitted to a laboratory but may not have been referred for virology testing. Further,
collection or selection of appropriate sample types can be critical. For example, the type of
anticoagulant used and its concentration in a sample can be critical for optimal detection of
some viruses. Testing for an RNA virus on a half filled tube containing heparin will
invariably produce a very different result to a tube containing EDTA as the anticoagulant. In
contrast, adverse effects are likely to be less pronounced or minimal for a DNA virus, while
similar results will be obtained for both DNA and RNA viruses on serum from clotted blood.
The availability of assays with extremely high analytical sensitivity presents a range of
special requirements for sample collection. During collection of samples at a post mortem
examination, steps must be taken to minimise cross contamination between samples from an
individual animal, or from other animals or the working environment. Similarly, situations
have been documented where handling of vaccine by a clinician can result in contamination
of swabs that are collected later during the day and result in misleading positive results. While
these results may be considered to be false positives in the context of an investigation, they
are not false from the perspective of assay specificity. The assay has correctly detected
residual nucleic acid or antigen, although it did not originate from an infected animal.
Sampling methods that may appear to be convenient in the field can at times lead to ‘downstream’ false positives. For example, collection of milk samples can be very convenient for
the detection of animals persistently infected with BVDV. If samples are collected using
automated ‘in line’ sampling devices, residual RNA can be carried over for many successive
animals passing through that sampling point. While these risks are greater with PCR assays,
problems can also be encountered with ELISAs. Inappropriate collection of tank milk samples
for testing for antibodies to enzootic bovine leucosis virus has resulted in false positive results
on several farms that have followed sampling of an infected farm.
Storage conditions for samples are also obvious considerations and may be different for PCR
and virus isolation. On occasions it may be more appropriate to hold samples chilled rather
than frozen. The careful selection of a stabilising agent or preservative may be useful to
overcome the demands of long distance transport of specimens. Even the type of sample
container can affect the capacity of a laboratory to operate efficiently and can potentially
compromise sample quality.
The widespread introduction of real time PCR has brought many advantages, the greatest of
which is probably ‘turn around’ time. In a laboratory that is focussed towards high throughout
and ‘rapid turn’ around, there is a strong preference for testing of samples in a liquid format to
minimise the need for sample processing prior to testing. This may encompass blood, body
fluids, semen, milk or swabs collected from mucosal surfaces (eg nasal, tracheal, cloacal). Reinvestigation of the biology of a disease can be rewarding and identify sample types that are
both easy to collect and also to test. While fresh tissues have traditionally been collected at
post mortem for the investigation of viral infections, these require considerable processing
prior to testing. Comparable and often superior results are obtained by testing of swabs
collected from the freshly cut surface of organs. These are very easy to collect, transport and
store but should be collected into a suitable viral transport medium. Phosphate buffered
gelatine saline (PBGS) is a recommended universal transport medium.
4. Investigation of specific disease syndromes
The preceding sections have provided general information for consideration during the
investigation of a disease with a possible viral aetiology. The section that follows offers
comments on approaches to maximise the rapid detection of an infectious agent, with specific
reference to what are considered to be some of the most important viral infections in cattle
populations in North America (and also many other countries). These will be considered on a
syndromic rather than individual agent basis because the emphasis should be directed towards
collection of appropriate samples that will allow testing for a number of different viruses.
a) Reproductive disease
This should be considered in a broad context and extends, at the least, to infections that
impact on conception rates to the viability of animals at least into the neonatal period. For
some agents, such as persistent infections with BVDV, even disease later in life could be
considered to be a form of delayed reproductive loss because infection occurred in utero.
Where specific agents are mentioned, it is considered that these are probably the more
common but not the sole agents to be encountered.
(i) Conception failure and early embryonic loss – common viral causes include BVDV and
BHV1 (IBR virus). There can be effects on either the conceptus or the female reproductive
tract and the hormonal environment. In many instances it may not be possible to detect the
aetiological agent due to a delay between the time of infection and the clinical expression of
disease. However, there can be benefit in collection of swabs from the reproductive tract.
Where suitable serological assays (or serially collected samples) are available, providing
evidence of ‘recent’ infections (in the absence of vaccination), while not diagnostic, can guide
future investigations to incriminate an agent.
(ii)Abortion, still birth and perinatal mortality – BVDV is one of the most common causes,
although some strains of BHV1 are known to be abortigenic. If there is a history of
undiagnosed abortion, examination of stillborn calves or perinatal deaths can be productive.
In regions where there is scope for arbovirus transmission, when clusters of abortion, still
birth and perinatal deaths, sometimes with congenital defects are observed on multiple
properties, an arbovirus aetiology should be considered. These may be the first signs of an
incursion of an emerging or exotic virus. The possibility of a vector borne (or other) virus can
often be overlooked because of an apparent disconnection between the occurrence of the
reproductive loss and exposure to an agent. Under these circumstances, infection may have
occurred 3-6 months prior to the observed reproductive loss.
A thorough post-mortem examination should be undertaken. The thoracic cavity and later,
pericardial sac, should be carefully opened, with the objective of aseptically collecting pleural
and pericardial fluids, preferably avoid gross contamination with blood. A range of solid
organs (especially lung, liver, spleen and kidney) should be collected (fresh and formalin
fixed). When a solid organ is first incised, the freshly cut surface prior to collection of tissue.
Examination of the brain is essential as gross abnormalities of the cerebral hemispheres
and/or cerebellum are often observed. A segment of spinal cord should be collected. If the
placenta is available, a sample for virus detection can be taken by extensively swabbing clean
portions. During the investigation of perinatal deaths, it is important to establish whether the
calf has been fed colostrum as this can confound testing and interpretation of results.
(iii)Infection of the male reproductive tract – when conception failure, early embryonic loss
or vulvovaginitis are observed, either the bull or semen should be considered as a possible
source of virus. Both BHV1 and BVDV have been involved as a result of either
contamination of semen used for artificial breeding or directly from an infected bull. Blood
samples and swabs of the penis and prepuce should be collected.
b) Respiratory disease
Respiratory disease is most frequently encountered in intensively managed cattle. Commonly
encountered agents include BHV1, BVDV, parainfluenza virus 3 (PI3), bovine respiratory
syncitial virus (BRSV) and bovine coronavirus (BCoV). While each of these viruses may play
a role in respiratory disease, the relative importance of PI3 and BRSV may be more variable
than the major agents that affect cattle of all ages. BCoV may be more common than has
previously been recognised. In live animals, deep nasal and conjunctival swabs should be
collected while at post-mortem examination, as well as a range of formalin fixed tissues,
samples include swabs of trachea and cut surface of lung as well as fresh lung and tracheal
epithelium. In complex cases, prospectively collected paired serum samples can be useful to
establish viral transmission patterns that may be more evident before overt disease is
observed.
c) Enteric/mucosal disease
While enteric disease is most frequently encountered in neonates and pre-weaning age
animals, occasionally outbreaks occur in older animals. Agents commonly encountered in
outbreaks are BCoV and rotavirus but acute BVDV infections may be important and can be
difficult to detect due to their transient nature. Enteric disease and illthrift are common
manifestations of persistent infections with BVDV which, though more common in weaner
and yearling age animals, may become clinically apparent at any age. BCoV has been
associated with disease outbreaks in older animals. Faecal samples are commonly collected
but blood and nasal swabs may also be of value for the detection of BVDV and BCoV.
Rarely, BHV1 may also be associated with enteric disease. At post mortem examination,
samples should be collected from the gastrointestinal and respiratory tracts.
d) Systemic/febrile/production/agalactia
A number of different viral infections will cause a febrile illness in cattle of any age, but there
are usually other clinical signs (eg respiratory, enteric) that will guide an investigation.
However, at times systemic disease characterised by fever, inappetance and drop in milk
production is observed. There may also be locomotor signs and recumbency. These signs are
usually more readily observed in dairy cattle. When observed in a number of animals, perhaps
on several farms in a district, an emerging or exotic disease should be considered. While
bovine ephemeral fever virus (BEFV) is one of the viruses most commonly recognised in
Australia, Asia and the Middle East, strains of epizootic haemorrhagic disease virus (EHDV)
may also cause similar signs. Although considered an uncommon presentation for an
orthobunyavirus, fever and drop in milk production in numerous animals were the first signs
observed when European cattle were infected with the recently emergent Schmallenberg
virus. In the absence of other clinical signs, blood samples (serum, EDTA and heparin treated
whole blood) should be collected from animals as early in the course of disease as possible.
5. Testing of specimens.
The selection of specific laboratory assays may be heavily influenced by the likelihood of a
particular agent being present, cost considerations, ‘turn around’ time, availability of
syndromic test panels and local knowledge. The full range of assay types has a role in the
investigation of viral infections. The following comments may help to make a decision to
request particular examinations. Not all test types are relevant to the investigation of each
syndrome.
a) Serology
Before undertaking serology, care should be taken to ensure that animals have been affected
for sufficient time to mount an immune response. In some instances (eg reproductive disease),
there are usually long intervals between infection and a clinical event. Where suitable assays
are available, maternal serology can suggest ‘recent’ infection in a population and direct
further investigations. In other situations a negative result will usually exclude a specific virus
with a high degree of confidence. Collection and testing of foetal (especially pericardial)
fluids can be very useful. Initial testing for total IgG levels can guide subsequent testing. If
IgG levels are elevated, agent-specific testing should be undertaken. Positive results are
usually considered to be significant. In contrast, if IgG levels are not elevated, an infectious
aetiology cannot be excluded but testing should be directed towards specific pathogen
detection.
b) Antigen ELISA
Due to the relatively low cost and rapid turn around, antigen capture ELISA assays are
extensively used for the detection of BVDV. A wide range of sample types (whole blood,
serum, extracts of hair, skin, fresh spleen, lung, lymph node) can be tested. ELISAs and
agglutination (eg dip stick) assays are also used for detection of corona and rotavirus
infections in young calves but a negative result does not exclude an agent.
c) PCR
With the widespread availability of many real time PCR assays, sometimes with either
multiplexing or multitasking capabilities, real time PCR is now a preferred choice for direct
detection of viral pathogens. Selection of samples in a liquid format aids rapid processing and
reduces turn around time. Swabs taken from cut surfaces of tissue usually give comparable or
even superior results to homogenates of tissue, save considerable time and minimise the
likelihood of cross contamination. qPCR is also a valuable diagnostic tool because residual
nucleic acid may be detected even when infectious virus may no longer be present. This can
be particularly beneficial where there has been degradation of tissues or a long delay between
infection and testing (eg testing of swabs of tissue or placenta from aborted foetuses and still
born calves). qPCR will also readily detect a pathogen in the presence of antibody, which is
likely to prevent detection by other methods (both antigen ELISA and culture). For example,
where foetal fluids indicate an immune response (elevated IgG), it may still be possible to
detect residual nucleic acid in tissues or to detect an acute viral infection in the presence of
colostral antibody.
d) Histopathology
Although histopathology can guide virus detection or in some cases provide a presumptive
diagnosis (eg bovine malignant catarrh, herpesvirus abortion) the absence of specific lesions
should not exclude the possibility of a viral infection. Few if any pathological changes are
seen in many cases of BVDV infection that results in abortion or stillbirth. When lesions are
detected, specific antigen/nucleic acid detection assays based on immunohistochemistry (IPX,
IFA) or in situ hybridisation/PCR may be employed but these are usually not practical for
routine diagnostic investigations and are reserved for special situations and research.
e) Virus isolation
Although virus isolation has been widely practised, rapid diagnostic methods (eg antigen
ELISA, qPCR) are often used as ‘front line- tools. The higher sensitivity and greater
flexibility of qPCR is also an advantage. However, virus isolation is still important and is
required for difficult cases where an infectious aetiology is suspected (eg based on
histopathology) or for suspected new or emerging diseases. Collecting virus isolates from
routine diagnostic submissions is valuable to support agent characterisation (eg subtyping, or
monitoring for antigenic changes) and may be guided by qPCR (eg selection of samples with
high virus loads).
f) Other genome detection methods
The use of microarray technology and whole genome sequencing are becoming increasingly
important but usually as ‘back up’ tools for agent typing or characterisation or for the
detection of novel agents.
6. Interpretation of results
While sample selection, collection and test methods are all important, care must also be
exercised during the interpretation of results, especially with assays of high analytical
sensitivity. There are many occasions where any detection of nucleic acid from a pathogen
could be considered to be significant or warrant further investigation. However, the possibility
of the detection being the result of residual nucleic acid or antigen from a past infection or
vaccination should be considered. Many rapid diagnostic assays (especially real time PCR
and ELISAs) provide a semi-quantitative result which may provide insight into whether or not
an infection is recent or longstanding. Interpretation may also be guided by the nature of an
agent (eg persistence of RNA in orbivirus infections such as BTV or EHDV at low levels for
many moths after infection), the likelihood of its occurrence (eg rare/sporadic vs endemic) or
possibly by the strength of reactivity (eg acute compared to persistent pestivirus infections).
With assays of high sensitivity, while weak positive results may sometimes be difficult to
interpret, a negative result forms a strong basis for the exclusion of an agent.