Bovine theileriosis is a disease of increasing significance in Australia, particularly along the east coast. It is caused by the haemoprotozoan Theileria orientalis that infects erythrocytes in the host animal. Infection with T. orientalis results in clinical signs of lethargy, fever, anaemia, jaundice, abortions and mortalities in naive cattle. T. orientalis has three common subtypes based on differences in their major piroplasm surface protein (MPSP). (Eamens and Jenkins, 2013) Of the three subtypes only the Ikeda subtype is considered to cause clinical disease in Australia. The Ikeda subtype was first detected in Australia in 2007. The Buffeli subtype has occurred in Australia prior to 2007 and is considered benign. The Chitose subtype while being occasionally related to infection (Eamens et al., 2013) is not considered to contribute greatly to disease in Australian herds.
A previous study (Swilks et al., 2014) investigated the early stages of infection in calves in the Gloucester area of NSW. The area has a large number of Theileria cases reported each year. In this study six properties were tested with a total number of 55 calves and their dams tested four times over a 16 week period. Prevalence was found to be 95% in both calves and their dams across the six properties, demonstrating the high levels of infection in this area. A number of calves in this study showed evidence of infection with T. orientalis based on blood smears as early as four days of age. Previous research has indicated that it takes approximately 20 days post infection for piroplasms to appear in erythrocytes of the infected animal (Eamens and Jenkins, 2013). This lead to the proposal that infection can be passed to the calf in-utero which is a potential source of continued transmission of disease within the herd.
This group has recently developed and validated a highly sensitive multiplex quantitative real time PCR (qPCR) which can differentiate between the different subtypes of T. orientalis and provide a good measure of the disease load on the animal (Bogema et al.., in press). The following study aims to use this qPCR along with other clinical parameters to determine if transplacental transmission is occurring and to examine the role that transplacental transmission plays in continuation of infection in these herds.
Two properties were selected in the Gloucester region to be sampled for the study. On both of these properties dams (Property 1 n=31, Property 2 n=28) were selected at random for initial screening to determine their infection status. All calves from these dams were sampled as soon as practicable after birth (within 48hrs). A subset of 10 calves from each property was then sampled over a time course to examine disease progression. Eight samples were taken over a 56 day period from each calf from birth. Samples collected from each animal were whole blood (EDTA) for qPCR, blood smear analysis and PCV along with sera for antibody testing. Clinical signs of disease were recorded for all calves. Additionally, calves from property 1 were weighed at first sampling.
One dairy property from Victoria was also sampled as per the Gloucester properties regime with the exception that no time course study was carried out. Additional samples were also collected from pregnant cows and their foetuses at Wingham abattoir. These came from a wide area of north-eastern NSW as part of the normal intake of cattle through the abattoir. In addition to the samples collected as per the on-farm component of the study, spleen and lung samples were also collected from abattoir foetuses.
Laboratory and clinic testing
PCV and blood smears were carried out at Gloucester vet clinic within 4 hours of collection. Smears were submitted to EMAI for staining and analysis. EDTA blood and sera were frozen before submission to EMAI for qPCR and ELISA respectively. DNA for qPCR was extracted from blood samples using a simplified in-house extraction procedure (manuscript in preparation). We have found this method to have comparable sensitivity to commercially available extraction kits currently available. DNA for qPCR was extracted from tissue samples using DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany)
DNA testing was carried out using the qPCR method as described by Bogema et al. (in press). This multiplex qPCR includes a universal target which detects all three subtypes of T. orientalis along with typing targets for the Ikeda and Chitose subtypes. A qPCR specific for the Buffeli subtype was also carried out for all samples (manuscript in preparation)
Blood smears and sera for ELISA are awaiting testing, PCVs have not been fully analysed.
On property 1, all dams (n=31) tested positive by qPCR at initial sampling within two weeks of the start of calving. Of these one was Ikeda negative but positive for Chitose/Buffeli. The levels of organisms detected by qPCR for all dams were consistent with subclinical levels of infection. Three of the calves tested positive by qPCR at day zero (n=29). Of these one gave a weak positive result below the standard limit of detection (LOD) for Ikeda subtype only, one was positive for Buffeli only but below the LOD and the third was moderately positive for all three subtypes.
Time course results for property 1 (figure 1) show that for the ten calves sampled the first positive results were detected around 10-11 days post birth. All calves detected as positive by the universal qPCR over subsequent samplings.
On property 2, all dams (n=28) tested positive by qPCR at initial sampling within two weeks of the start of calving. Of these, one was positive for Chitose only, one for Buffeli only and one for Ikeda and Chitose. The levels of organisms detected by qPCR for all dams were consistent with subclinical levels of infection. No calves tested positive for any of the subtypes at day zero (n=25).
Time course results for property 2 (figure 2) show for the ten calves sampled the first positive results were detected at day 9 post birth with the first positives over the LOD at 12 days post birth.
Of the Victorian samples, 19 dams (n=30) tested positive with different mixes of subtypes detected. Of these dams, 16 were found to be positive for the Ikeda subtype. Like the Gloucester properties, these levels were consistent with subclinical infection. All calves (n=30) were negative for all subtypes.
In the abattoir sampling, 16 of the dams (n=20, not all dams able to be sampled) were found to be positive by qPCR. The Ikeda subtype was detected in all but one of the positive dams. One calf (n=24) was found to be Ikeda positive, however below LOD by qPCR from whole blood. From foetal lung samples collected (n=24), one sample tested positive by qPCR for Chitose subtype, below the LOD. For spleen samples (n=24), three samples were positive below the LOD. All three samples were Ikeda subtype. The positive Chitose subtype foetal lung was also positive for Ikeda subtype in the spleen sample. The whole blood sample for this same foetus was negative for all subtypes.
The low rates of detection of T. orientalis in new born calves indicates that transplacental transmission has not likely a major source of persistence of disease on these properties over the study period. While there is some evidence of infection in the calves sampled, only one calf approached levels of moderate infection when sampled just after birth.
For the Gloucester properties, forming the main part of this trial, there are a number of factors that could be contributing to these low levels of transplacental transmission detected. Firstly, the dams on these properties have long been exposed to T. orientalis and while all were found to be positive, the levels of infection were sufficiently low, suggesting disease would not be observed in these animals. Testing of newly infected dams may yield quite different results for both them and their offspring.
In the period leading up to and during calving the Gloucester region experienced below average rainfall. This in turn, appeared to reduce the number of ticks present, hence reducing infection loads on dams. Discussion with the owners of both properties confirmed abnormally low levels of ticks found on any cattle during this period.
With the exception of one calf, all positive results obtained to date have been below what would normally be used as the standard limit of detection (LOD) for a positive result in a diagnostic test. This level is set to eliminate any chance of including false positive results from samples which become positive late in the qPCR reaction. Possible sources of false positives are sample to sample cross contamination and possible issues of "cross-talk" between filter channels in the qPCR machine. For the samples tested it is believed that these are real results and merely demonstrate the low levels of infection. It is also observed, through the foetal positive of both lung and spleen, that results can be variable at low levels of infection. In the case of this calf it was positive for Ikeda in one sample and Chitose in the other. This most likely indicates presence of both subtypes but at a level that it is difficult to detect consistently by qPCR.
On the basis of available results, a change in sampling approach may be required in order to detect transplacental transmission as it appears it is low in persistently infected herds. Preliminary findings from abattoir samplings suggest this may be an avenue to revisit. However, a more targeted approach to on farm sampling may prove worthwhile using clinically infected dams and/or calves or samples from naive herds with new infections.
This project is internally funded by NSW DPI. Cheryl Jenkins provided invaluable guidance over the course of this project. Daniel Bogema assisted with some of the early sample processing and was always on hand for advice when required. Arthur Poynting and the staff at Gloucester Veterinary Clinic were very accommodating with facilities to process samples and advice on sampling and processing of samples. Assistance from the staff at Wingham abattoir with sample collections was greatly appreciated. Jim Kerr also contributed greatly with sample collection and advice. Kate Sawford and Damian Collins provided valuable input into project design.