PCR diagnosis of tick-borne pathogens in Maharashtra state, India indicates fitness cost associated with carrier infections is greater for crossbreed than native cattle breeds.

Tick-borne pathogens (TBP) are responsible for significant economic losses to cattle production, globally. This is particularly true in countries like India where TBP constrain rearing of high yielding Bos taurus, as they show susceptibility to acute tick borne disease (TBD), most notably tropical theileriosis caused by Theileria annulata. This has led to a programme of cross breeding Bos taurus (Holstein-Friesian or Jersey) with native Bos indicus (numerous) breeds to generate cattle that are more resistant to disease. However, the cost to fitness of subclinical carrier infection in crossbreeds relative to native breeds is unknown, but could represent a significant hidden economic cost. In this study, a total of 1052 bovine blood samples, together with associated data on host type, sex and body score, were collected from apparently healthy animals in four different agro-climatic zones of Maharashtra state. Samples were screened by PCR for detection of five major TBPs: T. annulata, T. orientalis, B. bigemina, B. bovis and Anaplasma spp.. The results demonstrated that single and co-infection with TBP are common, and although differences in pathogen spp. prevalence across the climatic zones were detected, simplistic regression models predicted that host type, sex and location are all likely to impact on prevalence of TBP. In order to remove issues with autocorrelation between variables, a subset of the dataset was modelled to assess any impact of TBP infection on body score of crossbreed versus native breed cattle (breed type). The model showed significant association between infection with TBP (particularly apicomplexan parasites) and poorer body condition for crossbreed animals. These findings indicate potential cost of TBP carrier infection on crossbreed productivity. Thus, there is a case for development of strategies for targeted breeding to combine productivity traits with disease resistance, or to prevent transmission of TBP in India for economic benefit.

Kolte SW ，Larcombe SD ，Jadhao SG ，Magar SP ，Warthi G ，Kurkure NV ，Glass EJ ，Shiels BR ... -  《PLoS One》

The impact of tick-borne pathogen infection in Indian bovines is determined by host type but not the genotype of Theileria annulata.

Tick-borne pathogens (TBP) are a major source of production loss and a welfare concern in livestock across the globe. Consequently, there is a trade-off between keeping animals that are tolerant to TBP infection, but are less productive than more susceptible breeds. Theileria annulata is a major TBP of bovines, with different host types (i.e. exotic and native cattle breeds, and buffalo) displaying demonstrable differences in clinical susceptibility to infection. However, the extent to which these differences are driven by genetic/physiological differences between hosts, or by different parasite populations/genotypes preferentially establishing infection in different host breeds and species is unclear. In this study, three different bovine host types in India were blood sampled to test for the presence of various TBP, including Theileria annulata, to determine whether native cattle (Bos indicus breeds), crossbreed cattle (Bos taurus x Bos indicus breeds) or water buffalo (Bubalus bubalis) differ in the physiological consequences of infection. Population genetic analyses of T. annulata isolated from the three different host types was also performed, using a panel of mini- and micro-satellite markers, to test for sub-structuring of the parasite population among host types. We discovered that compared to other host types, "carrier" crossbreed cattle showed a higher level of haematological pathology when infected with T. annulata. Despite this finding, we found no evidence for differences in the genotypes of T. annulata infecting different host types, although buffalo appeared to harbour fewer mixed parasite genotype infections, indicating they are not the major reservoir of parasite diversity. The apparent tolerance/resistance of native breed cattle and buffalo to the impacts of T. annulata infection is thus most likely to be driven by host genotype, rather than differences in the parasite population. Our results suggest that an improved understanding of the genetic factors that underpin disease resistance could help to ameliorate future economic loss due to TBP or tropical theileriosis.

Larcombe SD ，Kolte SW ，Ponnudurai G ，Kurkure N ，Magar S ，Velusamy R ，Rani N ，Rubinibala B ，Rekha B ，Alagesan A ，Weir W ，Shiels BR ... -  《-》

Simultaneous detection of Theileria annulata and Theileria orientalis infections using recombinase polymerase amplification.

Theileriosis is a disease of domesticated animals in tropical and subtropical countries and causes significant reductions in livestock productivity. The arid region of Punjab in Pakistan is notorious for the presence of the vector tick (Acari: Ixodidae) and tick-borne diseases, such as theileriosis and babesiosis. The distribution of Theileria annulata and T. orientalis in the Chakwal district of Punjab was determined by developing a multiplex recombinase polymerase amplification (RPA) assay as a scientific basis for formulating control strategies for bovine theileriosis. Specificity was evaluated using DNA from related piroplasm species, while analytical sensitivity was calculated using a long fragment of the enolase gene. A total of 188 blood samples were collected on FTA cards (Whatman) from tick-infested asymptomatic breeds of cattle (Bos indicus, Bos taurus, and Bos indicus × Bos taurus) in the study district. Finally, infections with of T. annulata and T. orientalis were detected using the multiplex RPA and compared with the conventional multiplex polymerase chain reaction (PCR). The multiplex RPA specifically amplified 282-bp and 229-bp fragments of the enolase gene from T. annulata and T. orientalis and had no cross-reaction with other piroplasm species. It was determined that 45 (23.9%) and 5 (2.6%) out of 188 blood samples were positive for T. annulata and T. orientalis, respectively, when examined using RPA. Multiplex PCR detection indicated that 32 (17.0%) and 3 (1.6%) blood samples were positive for T. annulata and T. orientalis, respectively. In the present study, a specific RPA method was developed for simultaneous differentiation and detection of T. annulata and T. orientalis infections and used for the first time for the detection of the two bovine Theileria infections.

Hassan MA ，Liu J ，Sajid MS ，Rashid M ，Mahmood A ，Abbas Q ，Guan G ，Yin H ，Luo J ... -  《-》

Epidemiology of tick-borne pathogens in the semi-arid and the arid agro-ecological zones of Punjab province, Pakistan.

Tick-borne diseases (TBDs) have a large impact on animal health and the livelihood of livestock owners, particularly in developing countries. Although climatic and ecological conditions in Pakistan may favour the transmission of tick-borne pathogens (TBPs), only a few systematic studies have been carried out on TBPs and the diseases that they cause in this country. To improve our understanding of the distribution of TBPs, 3,807 ticks were collected from ruminants (n = 369) on 108 livestock farms (semi-arid zone = 36, arid zone = 72) in Punjab Province. After morphological identification ticks were pooled into 405 pools (Hyalomma anatolicum = 300, Rhipicephalus microplus = 89, Hyalomma dromedarii = 9, Rhipicephalus turanicus = 7) based on their species, locality of collection, and the host. DNA from each pool was screened by a Reverse Line Blot (RLB) hybridization assay for the presence of Anaplasma, Ehrlichia, Rickettsia, Babesia, and Theileria species. DNA from at least one TBP was found in 142 (35.1%) pools. Among the positive pools, 91 (64.1%) had a mixed infection with two or more TBPs, whereas 51 (35.9%) pools were infected with a single TBP. The detected pathogens not only included species that were known to be endemic in Pakistan, such as Theileria annulata (6.7%), Theileria orientalis (3.5%), Anaplasma marginale (5.7%), Anaplasma centrale (2.7%), Anaplasma ovis (1.5%), Babesia bigemina (0.7%), and Babesia bovis (0.2%), but also several TBPs that had not been reported to occur in Pakistan before. This included Ehrlichia minasensis (3.2%), an Anaplasma platys-like organism (1.2%), Babesia occultans (0.2%), and Rickettsia massiliae (0.2%), as well as two previously uncharacterized species: Ehrlichia sp. Multan (18.0%) and Anaplasma sp. (BL099-6) (2.22%). The pathogenicity of these novel species remains to be examined. This study shows that a much broader spectrum of TBPs is present in Pakistan than previously thought, including several zoonotic pathogens.

Rehman A ，Conraths FJ ，Sauter-Louis C ，Krücken J ，Nijhof AM ... -  《-》

Development of multiplex PCR assay for concurrent detection of tick borne haemoparasitic infections in bovines.

This study describes development and evaluation of a multiplex PCR assay for simultaneous detection of Theileria annulata, Babesia bigemina and Anaplasma marginale infections in bovines. The assay was developed using parasites specific genomic DNA and three sets of PCR primers targeting the Tams1, 18S rRNA and 16S rRNA genes of T. annulata, B. bigemina and A. marginale, respectively. Blood samples collected from a total of 461 bovines, suspected for haemoparasitic infections, were examined microscopically to record the status of infection and simultaneously, genomic DNA extracted from these blood samples were utilized for the optimization and validation of multiplex PCR assay. Microscopic examination of blood samples revealed presence of single and multiple species of haemoparasites in 25.8% and 2.4% samples, respectively. Results of multiplex PCR revealed the presence of single haemoparasitic species infection in 159 cases (34.5%), whereas mixed infection was recorded in 82 (17.8%) samples. Occurrence of individual species infection detected by mPCR in the study was 26.03% (120/461) for T. annulata, 3.25% (15/461) for B. bigemina and 5.20% (24/461) for A. marginale. The detection limit of multiplex PCR assay was at the template dilutions of 10-6, 10-6 and 10-4, which corresponded to 0.1 pg, 0.1 pg and 10.0 pg of DNA for T. annulata, A. marginale, and B. bigemina, respectively. Based on the high diagnostic sensitivity and throughput, multiplex PCR assay developed in the present study could be exploited as a tool to conduct large-scale epidemiological survey for tick-borne haemoparasitic infection of bovines.

Kundave VR ，Ram H ，Banerjee PS ，Garg R ，Mahendran K ，Ravikumar GVPPS ，Tiwari AK ... -  《-》