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T heileria orientalis is a vector-borne hemoprotozoan that infects cattle and buffalo and is generally spread by ticks of the Haemaphysalis genus (1-3). Historically, this organism has been referred to as Theileria sergenti, Theileria buffeli, or the T. orientalis/T. sergenti/T. buffeli complex; however, the name T. sergenti is now considered invalid (4), and T. orientalis is commonly used to refer to all (5). From a clinical perspective, T. orientalis can cause anemia, lethargy, jaundice, fever, abortion, and mortality in cattle (6). Clinical infection can also result in decreased milk production in dairy cattle (7). T. orientalis is a conditional pathogen, and while pathogenic forms are largely limited to Eastern Asia and Australasia (5, 6, 8-10), it is frequently detected in asymptomatic animals; these benign forms are globally spread (5,(11)(12)(13)(14). Analysis of the most common genotyping locus (p32), encoding the variable major piroplasm surface protein (MPSP), currently identifies 11 distinct T. orientalis genotypes (13,15,16). Of these genotypes, type 2 (Ikeda) and to a lesser extent type 1 (Chitose) are typically found in association with clinical disease (6,9,10,(17)(18)(19)(20)(21)(22). The presence of pathogenic and benign forms of T. orientalis greatly complicates its clinical diagnosis, with standard blood film analysis unable to identify the pathogenic genotypes.Multiple conventional PCR (cPCR) assays have been published for the identification of T. orientalis in blood samples (9,21,23,24). The most commonly cited assays detect and genotype T. orientalis by amplifying unique regions of the MPSP gene (21). These assays use two universal primers to detect T. orientalis infection and specific forward primers to identify the Ikeda, Chitose, and Buffeli genotypes. While this method is highly sensitive and has been validated in prior studies (19,21), it is not multiplexed and is highly susceptible to PCR inhibitors (6), which are often found in nucleotide extractions obtained from blood (25,26). To overcome inhibition, undiluted and diluted nucleotide extracts can be examined in parallel to prevent false-negative results (6,19,27). However, if multiple reactions per sample are required to determine the presence of infection, the procedure becomes both expensive and time-consuming. Furthermore, cPCR does not give an accurate representation of parasite load and therefore cannot provide an indication of the severity of T. orientalis infection. A recently developed multiplexed tandem PCR (28) is able to discriminate between four genotypes of T. orientalis, but it is only semiquantitative and therefore cannot be used to define a clinical threshold.Hydrolysis probe quantitative PCR (qPCR) assays employ sequence-specific, fluorescently labeled probes attached to duplex-

Ovine Johne's disease, or paratuberculosis, occurs in many countries. In Australia, surveillance using serology is used as part of a control program, but the testing regime is costly relative to its sensitivity. For this reason, culturing of Mycobacterium avium subsp. paratuberculosis in fecal samples pooled from a number of sheep was evaluated. Initially, the effect of pooling on the sensitivity of fecal culture was evaluated using samples from 20 sheep with multibacillary paratuberculosis and 20 sheep with paucibacillary paratuberculosis, each confirmed histologically. All multibacillary cases and 50% of paucibacillary cases were detected by culturing of feces at a pooling rate of 1 infected plus 49 uninfected sheep. In a pilot-scale study in 1997,M. avium subsp. paratuberculosis was detected by pooled fecal culture on 93% of 27 infected farms which were identified originally based on history, clinical signs, and one or more rounds of testing using serologic and histopathologic examinations. Pooled fecal culture was compared with serologic examination for submissions from 335 farms where both tests had been conducted on the same sheep and was significantly more sensitive (P < 0.001). Computer simulation of random sampling indicated that the testing of 6 pools of 50 sheep would provide 95% confidence in detecting ≥2% prevalence of infection. The estimated laboratory cost of pooled fecal culture when applied as a flock test is approximately 30% that of serologic examination, and sample collection costs are lower. It is recommended that pooled fecal culture replace serologic examination for detection of M. avium subsp.paratuberculosis infection at the flock level.

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BackgroundBovine theileriosis, caused by the haemoprotozoan Theileria orientalis, is an emerging disease in East Asia and Australasia. Previous studies have demonstrated transplacental transmission of various Theileria spp. but molecular confirmation of transplacental transmission of T. orientalis has never been confirmed in the field. In this study, cow-calf (< 48 h old) pairs were sampled across 3 herds; opportunistic samples from aborted foetuses or stillborn calves were also examined. Molecular (multiplex qPCR) and serological (ELISA) methods were used to determine infection prevalence and the presence of anti-Theileria antibodies in each herd. In addition, pregnant heifers and foetal calves were sampled at abattoir and tested for the presence of T. orientalis by qPCR.ResultsThe qPCR results indicated that, even though there was a high prevalence of T. orientalis infection in cows, the rate of transplacental transmission to their calves was low, with only one newborn calf from one herd and one foetus from the abattoir testing positive for T. orientalis DNA. Five aborted foetuses and stillborn calves, 3 of which were derived from a herd experiencing a high number of clinical theileriosis cases at the time of sampling, all tested negative for T. orientalis by qPCR. This suggests that in utero infection of calves with T. orientalis may not be a major driver of abortions during theileriosis outbreaks. Temporal monitoring of 20 calves born to T. orientalis-positive mothers indicated that T. orientalis was detectable in most calves between 10 and 27 days post-partum, consistent with prior field studies on adult cattle introduced to Theileria-affected herds. There was a positive correlation between the ELISA ratio of newborn calves and their mothers within 48 h of calving; however, maternal antibodies were only detectable in some calves and only for 4–4.5 weeks post-partum. All calves displayed high parasite loads peaking at 4–8 weeks post-partum, with only some calves subsequently mounting a detectable adaptive antibody response.ConclusionsThese findings indicate transplacental transmission of T. orientalis appears to play only a minor role in persistence of T. orientalis infection in the field; however calves are highly susceptible to developing high level T. orientalis infections at 4–8 weeks of age regardless of whether maternal antibodies are present post-partum.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-017-2166-9) contains supplementary material, which is available to authorized users.

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