Professional Documents
Culture Documents
9th edition
Veterinary Laboratory Service
Edition Year Under the Title
1st edition 1945 Instructions for the Collection and Despatch of Material for Pathological and
Bacteriological Examination.
2nd edition 1947 Instructions for the Collection and Despatch of Material for Pathological and
Bacteriological Examination.
3rd edition 1950 Instructions for the Collection and Despatch of Material for Laboratory
Examination
4th edition 1961 Collection and Despatch of Material for Laboratory Examination
5th edition 1974 Collection and Despatch of Material for Laboratory Examination
The general format of the previous edition, edited by Graeme Eamens, has been retained.
Many laboratory staff have contributed to this revised Manual. Also, field veterinarians have provided
suggestions, which have been incorporated.
The Manual includes information about specimen collection and submission in general, as well as for
specific diseases.
For further information about tests for diseases of commercial livestock, please contact your nearest
Regional Veterinary Laboratory or Customer Service at 1800 675 623.
Specimens are generally only accepted on Laboratory charges are subsidised for
referral from a veterinarian. Exceptions testing in the following circumstances*:
include WormTest kits, which can be • Notifiable disease
submitted by primary producers. The • Emergency (exotic) disease
Genetics section, at EMAI also accepts • National TSE Surveillance Program
bovine hair samples collected by cattle • Mortality investigations
owners for genetic disease testing where
certification is not required. *See ‘Disease Surveillance’ below for more
details.
CASES LIKELY TO INVOLVE CRIMINAL
INVESTIGATIONS Because it is often difficult for submitters to
Examination is not undertaken on specify the precise tests that will be
specimens likely to involve criminal required, many find it useful to indicate a
prosecutions (e.g. in cases of suspected maximum laboratory fee on the Specimen
malicious poisoning). If a case is likely to Advice Form. The Pathologist will consult
involve police matters or matters controlled with the submitter before exceeding this
by other regulatory authorities, those bodies amount.
should be contacted by phone and
consulted before any action is taken. Often SPECIMEN SUBMISSION FORM
they have special procedures to be followed
with respect to sampling/examination, and Field veterinarians should supply all
with respect to sealing, custody, care and pertinent details of the history, clinical signs
transport of exhibits. They may also have and necropsy findings. This information will
analyses performed by the Government allow the laboratory veterinarian interpret
Analytical Laboratories. the laboratory results and suggest
additional testing as appropriate. All
Laboratory Reports on samples submitted information supplied will be included in the
by field veterinarians as routine diagnostic Laboratory Report.
Please print clearly. The same name must For breed, give breed or predominant breed
be used in all specimen submissions from a in crosses.
property to allow previous submissions from
the property to be traced. For sex, select from the following: male,
female, castrate, spayed, mixed group,
RURAL LANDS PROTECTION BOARD unknown.
and RLPB Property ID
The affected property's RLPB (Rural Lands NUMBER OF ANIMALS
Protection Board) district and its RLPB We use the numbers provided to calculate
property ID is required for statistical and the epidemiological statistics, 'period
reporting purposes. The Property ID is the prevalence' and 'cumulative mortality'. For
Property Identification Code (PIC), tail tag the statistics to have meaning, the following
number, or Assessment Number. conventions should be followed:
Clearly state the breed of the subject and STANDARD CONTAINERS AND
DNA test required. EQUIPMENT
Blood Specimens for gross pathology should be
Fill evacuated blood tubes, to end of draw, submitted in glass or plastic screw-top
from the coccygeal vessels or the jugular containers or strong plastic bags. When live
vein. Immediately after drawing the sample or dead animals are submitted, the
ensure mixing of anticoagulant and blood containers used should prevent
by repeated but gentle inversion of the contamination of the environment with
tubes. Label each tube with the identity of possible pathogens.
the subject.
COLLECTION OF SPECIMENS FOR
Semen GROSS PATHOLOGY
A semen straw or 0.5mL of raw semen in a
serum vial. When submitting whole animals, ensure
that they are typical of the syndrome being
STORAGE OF SPECIMENS AND investigated.
DESPATCH OF SPECIMENS When the necropsy is done in the field and
Samples of tissues other than hairs must be a laboratory opinion is sought on gross
chilled until despatch. changes detected, then a large portion of
the tissue or organ should be submitted
Do not freeze blood samples. chilled but not frozen, containing the lesion
and adjoining normal tissue for comparison.
Clean, dry hairs are stable at ambient
temperature for an extended period of time. If material is submitted for macroscopic
examination, small portions in buffered
Specimens other than hairs should be formalin should also be taken and
forwarded in an insulated container with an submitted for histopathological examination.
icebrick. Use sufficient icebricks to ensure
samples arrive at least chilled. Refer to STORAGE OF SPECIMENS PRIOR
Guidelines for Packaging Specimens for TO DESPATCH
more detail.
Tissue samples should be kept chilled from
Always seal the specimen advice form in a the time of collecting until they are received
separate plastic bag. at the laboratory.
Key
AGID Agar gel immunodiffusion test;
CFT Complement fixation test
ELISA Enzyme linked immunosorbent assay
IFAT Indirect fluorescent antibody test;
LAT Latex agglutination test
MAT Microscopic agglutination test
RPT Rapid plate test
RBT Rose Bengal test
SAT Serum agglutination test;
* Commercial test only
* Liver arsenic may be in range 2 to 8 mg/kg if several days elapsed since toxic exposure.
** Typical liver copper for cattle is > 20 and sheep > 40.
† In sheep, liver copper may increase up to 200 mg/kg (wet wt) before poisoning occurs
NB
i. Interpret concentrations between normal and toxic according to clinical and pathological
findings.
ii. Concentrations based on dry wt are approximately 5 times the above (wet wt) values.
iii. Conversion from mg/kg to SI units is as follows:
• As mg/kg x13.3 = As μmol/kg
• Pb mg/kg x 4.8 = Pb μmol/kg
• Cu mg/kg x 0.0157 = Cu mmol/kg
VIROLOGY
Presence of antibody can be a result of
clinical disease, unapparent infection,
Virological examinations involve demonstration passive immunity or vaccination.
of a pathogenic virus or detection of antibody
to virus. Findings must be interpreted in the Tests variously demonstrate group specific
light of history, clinical findings, lesions, etc. antibody, type specific antibody or a cross
reaction.
It is not possible to screen for a wide range of
viruses. Submitters should forward specimens Viral serology can be applied with the
to be tested for specific viruses. If there is any following limitations:
doubt about the availability of a test, the
laboratory should be contacted for advice.
Single Serum
• Useful only to eliminate a diagnostic
DIAGNOSIS OF VIRAL DISEASE possibility. Presence of antibody in the
The diagnosis of a viral disease can be acute phase may eliminate the
based on Histopathology, Virus isolation, diagnostic possibility; absence of
Virus Serology, or Virus or Virus Antigen antibody in the convalescent phase
Detection. eliminates the diagnostic possibility.
• Accurate interpretation often difficult
HISTOPATHOLOGY because time of collection may be
Cytological changes can indicate a viral critical. A negative test on serum < 3
aetiology. weeks after a suspected viral condition
leaves the possibility that the animal
VIRUS ISOLATION was infected but had not yet produced
Cultivation and identification of virus grown detectable antibody.
in tissue culture or eggs inoculated with
specimen. Paired Sera
• A change from negative to positive
NB Failure to cultivate the virus does not antibody status is known as
rule out a viral aetiology. seroconversion and indicates infection.
• A four-fold rise in antibody titre between
Cultivation of a virus does not necessarily acute and convalescent samples can
mean it caused the disease process. also indicate infection by the specific
pathogen.
VIRAL SEROLOGY
Detection and quantitation of virus specific However both the above may also be due
antibody in the serum of an infected animal. to cross reaction or booster immunisation.
Plasma is also an acceptable sample for They may also indicate stress induced
most serological tests. reactivation of latent infection.
Each test sample for viral antibody is tested Enzyme linked immunosorbent assay
against a known viral antigen to examine (ELISA)
the relationship of any precipitin line formed ELISA results can express the detection of
with an adjacent standard reference line antibody in a qualitative (+ or -) or a
formed between known positive serum and quantitative way. The three common means
virus antigen. This reference line is of expressing a quantitative result are
optimised to give a strong visible precipitin absorbance (optical density), ELISA ratio
line centrally located between the antigen and ELISA value (unit).
and positive serum wells (termed a 3
reaction - see below). Absorbance (Optical Density)
The amount of antibody in a sample is
For viral serology, a specific precipitin line proportional to the absorbance (optical
formed by the tested serum sample is density, OD) given by it. The OD may
recorded as 1, 2, 3, or >3 to describe its be standardised against one or more
relative position to the serum well and the serum controls on the ELISA plate.
antigen well: ELISA ratio
The ELISA ratio indicates the strength
Description / Position of Precipitin Line of the sample OD compared to that of a
Result
Turn on end of reference line negative 1 serum control on the same
Line, closer to serum well ELISA plate.
2 (E/R = OD test/OD neg
Line, midway between serum well and antigen well control) 3
Line, closer to antigen well ELISA value
>3 or ELISA units
The sample OD is fitted to a curve
A 1, 2, 3 or >3 antibody reaction is a determined by the performance of
positive test for antibody, and indicates that control positive and negative samples,
an animal has been infected with the and the result given as an ELISA value
specific virus (or a related virus). (eg 0 100). This is often a
measurement of how the sample OD
Strength of viral antibody levels in the test compares with that of a high positive
generally does not reflect the severity and control (taken as a value of 100).
stage of infection with any certainty. Percent inhibition
The results of blocking or competitive
Virus Neutralisation Test (VNT) ELISAs are expressed as the reduction
in OD (as a %) relative to the OD given
Crustacea White spot syndrome (WSS) White spot syndrome virus PCR
Key
AGID Agar gel immunodiffusion test
ELISA Enzyme linked immunosorbent assay
EM Electron microscopy
HA Haemagglutination test
HI Haemagglutination inhibition test
LA Latex agglutination test
VI Virus isolation
VNT Virus neutralisation test
* available by arrangement
Comment: Interpret these results with caution due to low egg counts in the controls. However it
appears that:
i. BZ resistance(*) is present.
ii. LEV resistance (*) is present.
iii. BZ + LEV resistance (*) is present.
Interpretation depends on the interval between exposure and sampling. Maximum concentrations
of arsenic in tissues occur about eight hours after ingestion, and animals that survive for 2 to 3
days may have liver levels as low as 2 mg/kg. Conversely, clinically normal stock, chronically
exposed to arsenic (eg via arsenic contaminated feed over a long period or, in the past stock
regularly treated in arsenical dips) may have liver levels up to 8mg/kg and milk levels to 1.5 mg/kg.
Diagnosis
ARTHROGRYPOSIS AND
Based on clinical examination supported by
HYDRANENCEPHALY history and laboratory examination to define
Causes include infections of the dam with the specific cause.
Akabane virus, Aino virus, Palyam virus
and Pestivirus (Mucosal disease and Specimens required
Border disease), as well as inherited i. Brain, spinal cord and peripheral
defects. nerves in buffered formalin for
histopathology.
Diagnosis ii. Sections of skeletal and heart
Based on clinical signs and gross muscle in buffered formalin for
pathology. Diagnosis of the cause depends histopathology.
on history and samples from the dam and iii. Sections of lung and other organs
affected animals. with lesions, in buffered formalin for
histopathology.
Specimens required iv. At least 3 ml of serum, free of cells
i. Sample of foetal pericardial or and haemolysis, submitted chilled
pleural fluid submitted chilled for for serum enzymology and
virus isolation and serology, or, a biochemistry.
blood sample from the neonatal
animal if it has not sucked.
Diagnosis
CITRULLINAEMIA
History of ill thrift, in young animals
(Holstein/Friesian calves) particularly after elimination of other likely
causes, e.g. parasitism, deficiency of other
Affected calves are clinically normal at birth, minerals, malnutrition. Response to cobalt
become depressed by 24 hours, develop supplements or injections of vitamin B12,
signs of neurological dysfunction by 48 preferably in young, weaned animals.
hours. Typical clinical signs are seemingly Evidence of a mild to moderate macrocytic
aimless wandering, tongue protrusion, anaemia associated with weepy eyes.
chewing on fixed objects, head pressing
and ultimately collapse and death between Specimens required
the third and fifth day of life. ii. 10 ml of blood, clotted in vacuum
The mutation responsible for this defect tubes, from each of 3 to 5 animals in
was imported into Australia by a US-born the affected group, submitted chilled
bull whose descendants were selected for for biochemistry.
high EBVs for butter fat content of milk. The iii. Specimens should be submitted to
mutation is now relatively rare in the eliminate other causes of ill thrift.
Australian Holstein herd due to selection
against heterozygotes by AI centres.
CYSTICERCOSIS DIARRHOEA
Diagnosis See Scouring
Diagnosis Diagnosis
A history of severe haemorrhages in Clinical signs, bacteriological and
grandsons of a sire should raise a suspicion macroscopic examination of affected wool.
of haemophilia A.
Specimens required
FACTOR XI DEFICIENCY i. Sample of affected wool, submitted
for gross examination
(Holstein cattle) ii. Skin biopsy from affected area
submitted in buffered formalin for
An autosomal recessive defect reported in histopathology.
Holsteins in North America and Europe.
The mutation responsible for the defect has
been defined. The mutation has been
Submitters are requested to specify breed Registered animals of most cattle breeds
of the subject. Submitters are advised to have been derived from a limited gene pool.
determine (via the Australian Brahman Within modern breeds, herd books are
Breeders’ Association) if a Brahman subject generally closed. Consequently, most
is a descendant of the founder for the rare recessive defects are usually “breed-
E13 mutation. specific” and are caused by founder effects
relating to a single mutation. There are
Diagnosis exceptions where multiple mutations cause
Clinical signs and history, histopathology, the same clinical disease within and across
confirm by DNA analysis. breeds. An affected individual may be
heterozygous for two distinct mutations.
Specimens required The term ‘compound heterozygote’ is used
See to describe these animals. An example
http://www.dpi.nsw.gov.au/agriculture/vetm would be a Brahman calf affected with
anual/specimens-by-disease- generalised glycogenosis if it inherited the
exon7 mutation from one parent and the
* Consign samples direct to Dr. Van Haeringen, Laboratorium B.V. PO Box 408, 6700 AK,
Wageningen, The Netherlands.
** Consign samples direct to Reprogen, Faculty of Veterinary Science, University of Sydney,
PMB 3, Camden, New South Wales 2570
See Specimens (by disease) for details of specimen collection for diagnosis of a specific disease.
Diagnosis
C strain S strain
Method Conventional Bactec Bactec
Decontamination 5-7 d 5-7 d 5-7 d
Incubation 20 wks 8 wks* 12 wks
Confirmation by PCR/REA 1-4 wks (if reqd) 1-4 wks 1-4 wks
Confirmation by subculture
Up to 10 wks Up to 10 wks Up to 10 wks
(mycobactin dependency test)
Total time if no growth detected 21 wks 9 wks 13 wks
Total time if growth detected and M.
12-26 wks 10-21 wks 10-23 wks
paratuberculosis confirmed
Total time if growth detected and not
12-24 wks 12-21 wks 12-23 wks
M. paratuberculosis
* If growth is detected late (e.g. at week 8), incubation to week 10 may be required to reach a
satisfactory level for confirmation.
Kidney lead concentrations below 4 mg/kg are considered non-significant. Higher concentrations
should be interpreted on the basis of clinical findings and histopathology.
Faecal lead concentrations below 10 mg/kg are considered non-significant. Higher levels could be
significant, depending on the source of lead.
LEPTOSPIROSIS
Serovar Maintenance Host
Leptospirosis is transmissible to man and L. hardjo Cattle
represents an occupational hazard to L. pomona, L. Pigs
veterinarians, abattoir workers, dairy and tarassovi
pig farmers. L. copenhageni Rats
The most commonly encountered serovars
associated with clinical disease are: In the maintenance host, infections by
• Cattle L hardjo, L pomona these specific serovars are likely to be
• Pigs L pomona, L tarassovi endemic, whereas serovar infections in non
• Horses L pomona maintenance hosts are usually associated
• Dogs L copenhageni with sporadic outbreaks of disease. The
most common manifestations of
Other serovars do affect domestic animals leptospirosis in animals are:
in Australia, particularly Northern Australia, • Abortion (all species)
but such infections are rare in NSW. • Milk drop syndrome/ mastitis
(cattle)
Zoonotic infections are usually acquired • Pyrexia/Septicaemia (all species)
from the maintenance host for each • Icterus and/or haemoglobinuria
serovar: (mainly dogs and calves)
• Kidney lesions found at routine
slaughter (pigs, cattle)
NB. It is more productive to inspect a larger ELISA serology is available at EMAI for the
number of sheep rather than search a detection of infection with both immature
smaller number of sheep more thoroughly. and adult fluke. Using the Institut Pourquier
(France) antibody ELISA recently adopted
A thorough examination of the dip operation (2004) by NSW DPI laboratories, titres
and thoroughness of wetting is appear 2-4 weeks (cattle) after infection
recommended, particularly in the case of and remain high for at least 20 weeks after
shower dipping failures with insecticides. tretment. The false negative rate is low in
cattle (<2%). The test has not been
Specimens required validated for sheep under Australian
conditions.
Testing for OP resistance is not routinely
available; it requires controlled temperature Faecal egg counts are only of value in
and humidity and is a laboratory based test chronic fascioliasis where eggs are being
at the Elizabeth Macarthur Agricultural excreted (patent infection); they are of no
Institute (contact Dr Garry Levot). value in acute and subacute infections.
Submission of live affected sheep would be ELISA serology is more sensitive than
required. faecal egg counts for the detection of
chronic fascioliasis in both cattle (30% more
infected animals detected) and sheep (15-
LISTERIOSIS 20% more infected animals detected).
Listeriosis can be transmitted to man. ELISA serology is the only antemortem
diagnostic method for the detection of acute
Diagnosis and subacute fascioliasis (before fluke eggs
are excreted), which typically occurs in
GSHPx U/g Hb
Interpretation Cattle Sheep
Disease < 10 <10
Production response < 25 <25
Normal > 100 >50
Note that current blood concentration of be toxic to mycoplasmas if left in contact for
GSHPx reflect the selenium intake of the extended periods.
animal at the time the present circulating
erythrocytes were formed. An ‘average’ Diagnosis
time would be the half life of the erythrocyte
for the species and age of animals to be Pigs
examined (e.g. adult cattle approx. 80 days, Culture of some pathogenic mycoplasma
adult sheep approx. 60 days, calves and species can be undertaken using media
lambs approx. 50 days). specific for pig mycoplasmas.
Identification of M. hyopneumoniae in lung
The interpretation of selenium status and tissues or nasal swabs by PCR.
prognosis is further complicated by factors Identification of M. hyopneumoniae herd
which include: infection by ELISA serology.
• Season (winter and summer peaks,
autumn and spring troughs of Se NB: M. hyorhinis is a common inhabitant of
availability). the URT and ears (Eustachian tube), and is
• Soil type (acid soils < pH 5.5 have a common co-inhabitant of pneumonic
50% reduction in Se availability to lungs. It has no pneumonic potential and
plants). can rapidly overgrow M. hyopneumoniae
• Pasture type (legumes and other leafy cultures. It can cause a serofibrinous
plants lower in Se than grasses). polyserositis, or fibrinous arthritis in suckers
• Fertilizer history (competitive or weaners less than 10 weeks old. It
interaction from sulphur results in produces a milder, more sporadic disease
reduced selenium in pastures on than Glasser's disease, and can
superphosphate fertilized soils). occasionally affect young adults. (DD:
• Age of animals preruminant animals Glasser's disease, Streptococcus suis,
more prone to deficiency than Erysipelas).
ruminants.
M. hyosynoviae causes synovitis and
For diagnosis of selenium deficiency in arthritis
sheep, it is recommended that weaners be
sampled after access to good feed (e.g. (DD: Erysipelas; M. hyorhinis polyserositis
clover). Older sheep normally have lower of young pigs; Glasser's disease
levels. Diagnosis should be considered on polyserositis or respiratory disease;
a property basis. Pasteurella spp synovitis; Streptococcus
suis purulent arthritis or polyserositis; other
Streptococci causing purulent arthritis with
MYCOPLASMOSIS
percutaneous infections/abrasions; leg
See also Porcine enzootic pneumonia weakness/osteochondrosis)
Specimens required
The egg laying capacity of Ostertagia spp considered. This is of particular significance
and Nematodirus spp is poor and severe with Nematodirus, Ostertagia, Chabertia,
clinical signs may be seen before Fasciola hepatica and paramphistomes.
appreciable numbers of eggs are present in
the faeces. Dictyocaulus filaria is often associated with
mixed gastrointestinal infections on the
Low and medium egg counts will be more tablelands and slopes.
significant where the stocking rate is high,
when weather conditions are conducive to Cattle
epidemics (warmth, rain, humidity) and The clinical history and knowledge of the
where the biotic potential is high, e.g. seasonal pattern of worm parasites in
Haemonchus contortus. different areas of the State will assist in
interpretation of faecal egg counts.
Infections with one parasite only are rarely
seen and the additive effects of mixed In cattle greater than 18 months of age, the
infections will require assessment. The egg count gives little indication of the level
pathogenicity of immature stages not of the parasite burden.
indicated by egg count should always be
Note that the stability of these enzymes is variable, as is their half life in the blood:
ml
BOUIN'S FIXATIVE
Saturated aqueous picric acid 75
Formalin (Commercial solution; 38% formaldehyde w/v) 25
Acetic acid 5
DAVIDSON'S SOLUTION
Glycerine 10
Formalin (38% formaldehyde) 20
95% Alcohol 30
3.5% NaCl solution (or Filtered seawater) 30
Glacial acetic acid 10