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3.5 Processing and analysis of samples
Details of the methods for handling, processing and examining biological samples have been described.
4
A brief summary is provided here. Representative samples were taken from each fixed tissue and processed routinely for
embedding in paraffin wax. Histological sections were cut at 5 μm, stained with hematoxylin
and eosin, and examined for pathological changes using light microscopy. Samples in VTM were processed using conventional methods for viral RNA extraction using
commercial nucleic acid extraction kits. Quantitative polymerase chain reaction qPCR was used to detect nucleic acids from organisms commonly associated with BRD. The qPCR
was performed using a commercial kit QuantiTect virus +ROX vial kit, Qiagen Inc., Valencia, CA according to th
e manufacturer’s instructions. Primer and probe sequences were sourced from available sequences for each of the
pathogens of interest: BCoV, BoHV-1, BPIV-3, BRSV, BVDV, Mannheimia haemolytica, Pasteurella multocida, Mycoplasma bovis and Histophilus somni.
To reduce the number of tests, required reactions were multiplexed as follows: BCoV and BPIV-3, BRSV and BVDV, M. haemolytica and P. multocida, H. somni and M. bovis. Bovine
herpesvirus 1 was run as a single assay.
Each run contained duplicate samples of a positive control either a virus isolate or a clinical extract that had previously been characterized, a negative control an extract of cell culture
– grade fetal bovine serum, and a blank PCR-grade water. Runs were only considered valid
if the positive control was amplified at the expected threshold cycle, and the negative and blank controls showed no amplification.
Positive samples were those with a characteristic sigmoidal curve similar to the positive control, crossing the threshold before 40 cycles. Negative samples were those with no
characteristic sigmoidal curve. Samples crossing the threshold after 40 cycles were regarded as suspicious for containing the nucleic acid of interest and were retested before
classifying as negative or positive.
3.6 Determining the cause of death
More details can be found in Moore.
5
The final diagnosis for each animal was based on the combination of all available data and information relating to that animal:
description of pre-mortem clinical signs and gross necropsy findings and any other relevant information recorded on the dead cattlenecropsy form;
histology results from tissue samples collected from the animal and stored in formalin; qPCR results on samples collected in VTM;
digital photographs of pre-mortem or gross necropsy findings.
4
Moore et al. 2014; Moore 2014
5
Moore 2014
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Not all data was available for all animals. Respiratory disease was considered the cause of death if there was cranioventral
consolidation of more than 30 of the lung
6
andor the AAV had assigned a preliminary diagnosis of pneumoniaBRD and there were histological findings of moderate to severe
pneumonia in lung tissue. Heat stroke was considered the cause of death if the animal was noted to be panting heavily
prior to death or was found dead with no previous clinical signs, the core body temperature CBT was ≥ 43°C measured using a deep temperature probe, and necropsy findings were
consistent with those described previously for heat stroke in live export cattle : eyes sunken, muscles glowing pink and dry rather than red-brown and moist, heart tightly contracted with
epicardial ecchymoses, severe acute diffuse pulmonary congestion, and edema causing the lungs and mucosa of the trachea and bronchi to be dark red. Ambient environmental
temperatures were not recorded.
Other causes of death were based on review of the findings from all available data on dead animals and assignment of the most plausible cause or causes of death.
3.7 Retrospective data
Retrospective data for sea voyages between January 1995 and December 2012 were obtained from the Shipboard Mortality Database SMDB which is funded by Meat
Livestock Australia and administered by the Department of Agriculture and Food, Western Australia. The SMDB provides a repository of information, including livestock mortalities, on
every voyage on which sheep, cattle, andor goats are transported live by sea from Australia.
7
For the purposes of this report only voyages carrying cattle, either as the only species or as a mixed sheepcattle shipment, were included in the analysis.
Load regions were coded as described in Norris et al.
7
Northern ports were those north of 20° latitude south Port Hedland, Broome, Wyndham, Darwin, Karumba, Weipa, Mourilyan,
Townsville; southern ports are those south of 31° latitude south Fremantle, Bunbury, Esperance, Thevenard, Adelaide, Portland, Geelong, Devonport, Port Kembla, Sydney,
Newcastle; other ports are those located between 20° and 31° latitude south Geraldton, Denham, Carnarvon, Dampier, Mackay, Gladstone, Brisbane.
Destination regions were coded based on destination port and country location using a similar approach to that described in earlier annual reports. Regions included Middle East
and North Africa MENA: Bahrain, Egypt, Israel, Jordan, Kuwait, Libya, Oman, Pakistan, Qatar, Saudi Arabia, Sudan, United Arab Emirates, South East Asia SE Asia: Brunei,
Indonesia, Malaysia, Philippines, Singapore, Vietnam, North East Asia NE Asia: China, Japan, Russian ports on the Pacific facing coast of Russia, South Korea, South East
Europe SE Europe: Turkey and Russian ports on the Black Sea, Miscellaneous East Timor, Mauritius, Mexico, New Caledonia, Russian ports on the west coast near Finland,
Samoa, Solomon islands, Sri Lanka.
The primary outcome variable voyage mortality rate was based on a numerator measuring the count of deaths that occurred during loading, voyage and discharge for each voyage,
6
Gagea et al. 2006
7
Norris and Norman 2003
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and a denominator representing animal time at risk. Animal time at risk was calculated by multiplying the total cattle loaded by the sum of voyage days plus discharge days.
More recent years of data from the SMDB contained detailed records of mortalities per voyage day for export voyages. These records were used to create a dataset with one row
per animal that recorded survival time for each animal. Each row included a variable measuring time at risk for that animal. For animals that survived, time at risk was equal to
the length of the voyage in days and for animals that died it was equal to the voyage day when death occurred. These data were used for survival analysis.
3.8 Cattle movement data
Kategori