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Necropsy equipment, containers for collecting samples, and paper or electronic templates for recording observations were all provided on board each ship. The AAVs accompanying
the voyages were encouraged to collect a defined set of data and samples from each animal that died, but this was not always possible due to the demands of other tasks and duties that
they are routinely expected to perform during voyages. For 1 out of the 20 voyages, data and sample collection was performed by a member of the research team SJ Moore.
Standardized data recording forms were used to collect animal and epidemiological data for each animal that died. Data included the animal’s location on the ship, animal characteristics
including visual ear tag, electronic identification tag, breed, and weight, clinical signs displayed before death, risk factors or events that may have contributed to death, gross
necropsy findings if available, and a preliminary cause of death.
Necropsy samples included fresh tissue samples collected into 10 buffered formalin at a maximum ratio of 1 part tissue to 10 parts formalin, and tissue samples approximately 5
mm² andor swabs collected into a 5 ml plastic screw top container filled with 2 ml of viral transport media VTM; Hanks balanced salt solution, penicillin G [1,000 unitsml],
streptomycin [25 mgml], and amphotericin B [0.1 mgml]; Department of Agriculture and Food, Western Australia DAFWA. Samples stored in VTM were stored frozen on-board
the vessel until they were collected when unloaded at an Australian port.
The number and type of samples collected at necropsy depended on the animal’s clinical signs prior to death and gross necropsy findings. These protocols were all defined in the
Veterinary Export Handbook W.LIV.0252. Core samples collected from all animals were: lung grossly normal and abnormal, trachea, heart, ileocecal junction, kidney, liver, and
rumen into 10 buffered formalin, and nasal and lung swabs. When the animal showed clinical signs prior to death which were suggestive of a specific disease and which was
confirmed at necropsy, then a range of additional samples were collected according to the suspected disease. When there was no obvious cause of morbiditymortality and the cause
of death could not be determined from gross necropsy findings then the core samples plus fixed skeletal muscle, reticulum, abomasum, small intestine, large intestine, pancreas,
mesenteric lymph node, gall bladder, spleen, adrenal gland, and the brainstem and cervical spinal cord were collected into 10 buffered formalin.
Samples and data collection forms remained on the ship until the next time it berthed in Fremantle Western Australia. At that time a member of the project team boarded the ship
and collected all forms and samples, processed them through importation inspection and transported them directly to the quarantine approved premises at the Department of
Agriculture and Food, Western Australia, for processing. Care was taken during this process to ensure that VTM samples remained frozen during transit and were held frozen at DAFWA
facilities until they were processed and analysed.
3.4 Collection of samples from assembly depots
For four voyages voyage 5, 8, 17, 18 nasal swabs and serum samples were collected from cattle during the assembly period.
The animal was restrained in a head bail and a 20cm cotton swab was inserted approximately 10 cm into the nasal cavity. The swab was rotated across the nasal mucosa
to collect a sample of the nasal secretions. During both insertion and removal care was
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taken to prevent the swab being contaminated by dirt and other debris on the nostrils. The swab was immediately placed into a 5mL plastic tube filled with 1-2 mL VTM and kept chilled
until transported back to the laboratory.
To investigate whether the prevalence of viral and bacterial shedding changed with time, 2 cohorts of animals were sampled 7 days and 9 days apart. It was not possible to select the
same individuals for sampling at the first and second sampling sessions, but animals that were sampled at the second session were selected from pens containing animals that had
been sampled at the first session.
Nasal swab samples were processed and nucleic acids from the organisms of interest –
bovine coronavirus BCoV, bovine herpes virus 1 BoHV-1, bovine viral diarrhoea virus BVDV, bovine respiratory syncytial virus BRSV, bovine parainfluenza virus 3 BPIV-3,
Histophilus somni, Mycoplasma bovis, Mannheimia haemolytica, Pasteurella multocida –
were detected using quantitative polymerase chain reaction qPCR assays as described in the following section.
Serological samples were collected from a subset of 334 animals that were also sampled by nasal swabbing by harvesting a small amount of serum from samples that had been
collected as part of health certification requirements for export animals.
Whole blood was collected by tail-vein bleeding by accredited third party veterinarians as part of routine pre-export health checks and serum from these samples then submitted to the
Department of Agriculture and Food, Western Australia, for commercial Bluetongue and Bovine leukaemia virus antibody testing. An aliquot of serum was separated from a subset of
animals and used for separate disease testing as part of this project.
Collection of serum aliquots for testing as part of this project did not interfere with routine pre-export health tests and the project team had no knowledge of the results of any routine
certification testing procedures at the time sampling was completed for this project.
Aliquots of 200 μl of serum were tested for the presence of circulating antibodies to the viruses of interest using commercially available enzyme-linked immunosorbant assay
ELISA kits according to manufacturer instructions for the following viruses: BoHV-1 Infectious Bovine Rhinotracheitis IBR gB X2 Ab Test, IDEXX, Montpellier, France, BRSV
Bovine Respiratory Syncytial Virus BRSV IgG Antibody Test Kit, IDEXX, Montpellier, France, BVDV Bovine Viral Diarrhoea Virus BVDV Antibody Test Kit, IDEXX, Montpellier,
France, BPIV-3 Parainfluenza-3 Virus PI3 Antibody Test Kit, IDEXX, Montpellier, France.
It was not possible to test for antibodies to BCoV since a validated commercial test was not available.
Results were classified as positive, negative or inconclusive using thresholds defined by the manufacturer for each test kit. Inconclusive samples were not retested, and animals with
inconclusive test results were not included in the analyses.
Where possible, the ear tag, RFID, sex, breed and weight of each sampled animal was recorded. Not all data was available for all animals.
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3.5 Processing and analysis of samples
