Insights into the involvement of male Hyalomma anatolicum ticks in transmitting Anaplasma marginale, lumpy skin disease virus and Theileria annulata.

Ticks can transmit viruses, bacteria, and parasites to humans, livestock, and pet animals causing tick-borne diseases (TBDs) mechanically or biologically in the world. Lumpy skin disease virus, Anaplasma marginale, and Theileria annulata inflict severe infections in cattle, resulting in significant economic losses worldwide. The study investigated the potential transmissions of LSDV, A. marginale, and T. annulata through male Hyalomma anatolicum ticks in cattle calves. Two 6-month-old Holstein crossbred calves designated as A and B were used. On day 1, 15 uninfected female ticks (IIa) and infected batch of 40 male ticks (I) were attached on calf A for 11 days. Filial transmission of the infections was observed in female ticks (IIb) collected from calf A, where 8 female ticks had been co-fed with infected male ticks. The blood sample of calf B was found positive through PCR for the infections. The larvae and egg pools obtained from the infected ticks were also tested positive in PCR. The study confirmed the presence of these mixed pathogens and potential intra-stadial and transovarial transmissions of A. marginale, T. annulata, and LSDV in male and female ticks of H. anatolicum and experimental calves to establish the feasibility of infections through an in vivo approach.

CHANGES IN THE LEVELS OF THEILERIA ORIENTALIS IKEDA TYPE INFECTION IN HAEMAPHYSALIS LONGICORNIS NYMPHS OVER A SIX-MONTH PERIOD.

This study aimed to investigate whether the infection intensity of Theileria orientalis Ikeda type organisms within Haemaphysalis longicornis larvae and nymph stages fluctuated over 6 mo after feeding as larvae on infected calves in the field. Naïve larvae, hatched from eggs, were fed on infected calves for 5 days while contained within cotton socks glued over the calves' ears. Larvae were first sampled immediately post-feeding and then sampled every 3 wk for 23 wk in total, after molting to nymphs. All larvae and nymphs were tested for T. orientalis Ikeda organisms using quantitative PCR. The qPCR results showed that the infection intensity of Haemaphysalis longicornis larvae and nymphs was not constant over the sampling period, and after initially dropping after molting to nymphs, it then rose with fasting to a maximum at 17 and 23 wk post-feeding. The significant rise in T. orientalis Ikeda organisms observed at 23 wk postfeeding may explain why more severe clinical cases of bovine theileriosis in New Zealand are seen in the spring when nymphs are the predominant instar questing.

Detection and distribution of haematophagous flies and lice on cattle farms and potential role in the transmission of Theileria orientalis.

On the Northern Tablelands of New South Wales, Australia, endemic Theileria orientalis infection of cattle has been reported on farms where no ticks have been observed, raising the question of how Theileria is transmitted in these areas. To investigate the potential role of mechanical transmission by insects, the present study investigated the seasonal dynamics of biting fly species trapped between December 2017 and May 2019 on six cattle farms in the region where the presence of Theileria was confirmed. These flies, sucking lice collected from these farms and pools of Culicoides species affecting cattle also trapped in the region were subjected to qPCR detection of T. orientalis. Eleven species from eight genera of biting flies were collected using unbaited Nzi traps. Tabanid species were present in all farms, with Dasybasis oculata (43.6 %) and D. circumdata (27.6 %) being the most abundant and widespread species. The effect of season on the mean count per trapping event was statistically significant and highest in the summer months for Lilaea fuliginosa and D. oculata, and the autumn months for D. circumdata, with no seasonal effect on the abundance of the undescribed Dasybasis spp. No biting flies were trapped during the winter months. Sucking (Linognathus vituli and Haematopinus eurysternus) and biting (Bovicola bovis) cattle lice were also collected from all farms with the latter detected in only one farm. PCR screening for T. orientalis of trapped hematophagous insects resulted in parasite detection in the tabanid and Stomoxyini flies, biting midges and sucking lice with the highest proportion of positive samples for Haematopinus eurysternus (4/4 pools) and H. irritans exigua (6/15 individuals). The detection of the parasite in these potential vectors indicates a possible role in the mechanical transmission of T. orientalis and may partly explain the ubiquitous presence of Theileria in areas where ticks are absent.

First report of Theileria annulata in Nigeria: Findings from cattle ticks in Zamfara and Sokoto States.

BACKGROUND: Ticks and tick-borne pathogens (TBPs) represent a significant economic burden to cattle farming in sub-Saharan Africa including Nigeria. However, in the northern part of this country, where the largest livestock population resides, little is known about the contemporary diversity of ticks and TBPs. This area is particularly vulnerable to climate change, undergoing marked transformation of habitat and associated flora and fauna that is also likely to include ticks. This study aimed to document the occurrence of tick species and Apicomplexan TBPs in cattle from north-western Nigeria. METHODS: In 2017, ticks were collected from cattle in Zamfara and Sokoto States and identified morphologically. Additionally, a subset of ticks was screened molecularly for the detection of apicomplexan DNA. RESULTS: A total of 494 adult ticks were collected from 80 cattle in Zamfara and 65 cattle in Sokoto State. Nine tick species were encountered, among which the presence of one, Hyalomma turanicum, had not previously been recorded in Nigeria. Hyalomma rufipes was the most prevalent tick infesting cattle in Zamfara State (76%), while Hyalomma dromedarii was the most prevalent in Sokoto State (44%), confirming the widespread transfer of this species from camels onto livestock and its adaptation to cattle in the region. Of 159 ticks screened, 2 out of 54 (3.7%) from Zamfara State and 29 out of 105 (27.6%) from Sokoto State harboured DNA of Theileria annulata, the agent of tropical theileriosis. CONCLUSIONS: This study confirms the presence of a broad diversity of tick species in cattle from north-western Nigeria, providing the first locality records for Zamfara State. The occurrence of H. turanicum indicates a distribution of this tick beyond northern Africa. This study provides the first report for T. annulata in Nigerian ticks. Given its enormous burden on livestock farming in north Africa and across Asia, further investigations are needed to better understand its epidemiology, vector transmission and potential clinical significance in cattle from northern Nigeria and neighbouring Sahelian countries.

A U.S. isolate of Theileria orientalis, Ikeda genotype, is transmitted to cattle by the invasive Asian longhorned tick, Haemaphysalis longicornis.

BACKGROUND: Theileria orientalis is a tick-borne hemoparasite that causes anemia, ill thrift, and death in cattle globally. The Ikeda strain of T. orientalis is more virulent than other strains, leading to severe clinical signs and death of up to 5% of affected animals. Within the Asia-Pacific region, where it affects 25% of Australian cattle, T. orientalis Ikeda has a significant economic impact on the cattle industry. In 2017, T. orientalis Ikeda was detected in a cattle herd in Albermarle County, Virginia, United States. Months earlier, the U.S. was alerted to the invasion of the Asian longhorned tick, Haemaphysalis longicornis, throughout the eastern U.S. Abundant H. longicornis ticks were identified on cattle in the T. orientalis-affected herd in VA, and a subset of ticks from the environment were PCR-positive for T. orientalis Ikeda. A strain of T. orientalis from a previous U.S. outbreak was not transmissible by H. longicornis; however, H. longicornis is the primary tick vector of T. orientalis Ikeda in other regions of the world. Thus, the objective of this study was to determine whether invasive H. longicornis ticks in the U.S. are competent vectors of T. orientalis Ikeda. METHODS: Nymphal H. longicornis ticks were fed on a splenectomized calf infected with the VA-U.S.-T. orientalis Ikeda strain. After molting, a subset of adult ticks from this cohort were dissected, and salivary glands assayed for T. orientalis Ikeda via qPCR. The remaining adult ticks from the group were allowed to feed on three calves. Calves were subsequently monitored for T. orientalis Ikeda infection via blood smear cytology and PCR. RESULTS: After acquisition feeding on a VA-U.S.-T. orientalis Ikeda-infected calf as nymphs, a subset of molted adult tick salivary glands tested positive by qPCR for T. orientalis Ikeda. Adult ticks from the same cohort successfully transmitted T. orientalis Ikeda to 3/3 naïve calves, each of which developed parasitemia reaching 0.4-0.9%. CONCLUSIONS: Our findings demonstrate that U.S. H. longicornis ticks are competent vectors of the VA-U.S.-T. orientalis Ikeda strain. This data provides important information for the U.S. cattle industry regarding the potential spread of this parasite and the necessity of enhanced surveillance and control measures.

The role of sheep (Ovis aries) in maintaining Theileria orientalis Ikeda type infection.

Theileria orientalis is a tick‒borne intracellular parasite of red blood cells that causes severe and mild infections in various ruminants worldwide. To date there have been 11 types identified within this species, of which 4 types are presently found in New Zealand cattle. Since 2012, New Zealand has suffered a substantial epidemic of infectious bovine anaemia in both dairy and beef cattle associated with the Ikeda type. The speed at which the disease spread through the North Island suggested that other species could have been involved in transmission. The aim of a series of related experiments was to test the null hypothesis that sheep cannot maintain T. orientalis Ikeda type infection or infect ticks that feed on them. Several studies were conducted over 2 years to address this hypothesis which together showed that sheep can have detectable levels of T. orientalis Ikeda type infection in both the acute and chronic phase and that Haemaphysalis longicornis larvae can become infected when feeding on sheep. No anaemia, weight loss or clinical disease was recorded in the sheep in the acute phase of infection. The levels of infection recorded in the sheep were much lower than those found in cattle, consistent with the sheep being asymptomatic carriers of T. orientalis Ikeda type infection.

Prevalence of tick-transmitted pathogens in cattle reveals that Theileria parva, Babesia bigemina and Anaplasma marginale are endemic in Burundi.

BACKGROUND: Tick-borne diseases (TBDs) constitute a major constraint for livestock development in sub-Saharan Africa, with East Coast fever (ECF) being the most devastating TBD of cattle. However, in Burundi, detailed information is lacking on the current prevalence of TBDs and on the associated economic losses from mortality and morbidity in cattle as well as the costs associated with TBD control and treatment. The aim of this study was, therefore, to assess the prevalence and spatial distribution of tick-borne pathogens (TBPs) in cattle across the major agro-ecological zones (AEZs) in Burundi. METHODS: In a cross-sectional study conducted in ten communes spanning the five main AEZs in Burundi, blood samples were taken from 828 cattle from 305 farms between October and December 2017. Evidence of Theileria parva infection was assessed by antibody level, measured using a polymorphic immunodominant molecule (PIM) antigen-based enzyme-linked immunosorbent assay (ELISA) and by a T. parva-specific p104 gene-based nested PCR. Antibodies against Theileria mutans infection were detected using the 32-kDa antigen-based indirect ELISA, while the 200-kDa antigen and the major surface protein 5 (MSP5)-based indirect ELISA were used to detect antibodies against Babesia bigemina and Anaplasma marginale, respectively. RESULTS: The prevalence of T. parva across the ten communes sampled ranged from 77.5 to 93.1% and from 67.8 to 90.0% based on the ELISA and PCR analysis, respectively. A statistically significant difference in infection was observed between calves and adult cattle; however, T. parva infection levels were not significantly associated with sex and breed. The seroprevalence indicating exposure to T. mutans, B. bigemina and A. marginale ranged from 30 to 92.1%, 33.7 to 90% and 50 to 96.2%, respectively. Mixed infections of TBPs were detected in 82.91% of cattle sampled, with 11 different combinations of pathogen species detected . CONCLUSIONS: The findings indicate that T. parva, A. marginale and B. bigemina infections are endemic in Burundi. Knowledge of the spatial distribution of TBPs will facilitate the design of effective targeted strategies to control these diseases. There is a need for further investigations of the distribution of tick vectors and the population structure of TBPs in order to identify the key epidemiological factors contributing to TBD outbreaks in Burundi.

Utilizing attached hard ticks as pointers to the risk of infection by Babesia and Theileria species in sika deer (Cervus nippon yesoensis), in Japan.

Ticks are hematophagous ectoparasites that have a significant impact on their animal hosts. Along with mosquitoes, they are the main arthropod vectors of disease agents in domestic animals, wildlife and humans. To investigate the occurrence and prevalence of piroplasmids in ticks, DNA was extracted from 519 hard ticks collected from 116 hunted Hokkaido sika deer (Cervus nippon yesoensis). The success of the DNA extraction was confirmed by touchdown PCR targeting the mitochondrial 16S rDNA gene of ticks. Touchdown PCR and reverse line blot (RLB) hybridization targeting the 18S rRNA gene were used to detect 14 piroplasm species. All hard ticks parasitizing Hokkaido sika deer were identified as belonging to the genera Ixodes and Haemaphysalis. In total 163 samples (31.4%) were positive for Babesia and Theileria spp. among tick species according to RLB hybridization. Tick DNA hybridized to the oligonucleotide probes of Theileria sp. Thrivae (27.0% of ticks; 140/519), Theileria capreoli (10.6%; 55/519), Babesia divergens-like (1.7%; 9/519), Babesia sp. (Bab-SD) (0.6%; 3/519), Babesia microti U.S. (0.4%; 2/519), and B. microti Hobetsu (0.4%; 2/519). The partial sequencing and phylogenetic analyses of the 18S rRNA gene confirmed the RLB hybridization results. Further investigations are needed to reveal the epidemiology and respective vectors of these pathogens.

Variant analysis of the sporozoite surface antigen gene reveals that asymptomatic cattle from wildlife-livestock interface areas in northern Tanzania harbour buffalo-derived T. parva.

Buffalo-derived Theileria parva can 'break through' the immunity induced by the infection and treatment vaccination method (ITM) in cattle. However, no such 'breakthroughs' have been reported in northern Tanzania where there has been long and widespread ITM use in pastoralist cattle, and the Cape buffalo (Syncerus caffer) is also present. We studied the exposure of vaccinated and unvaccinated cattle in northern Tanzania to buffalo-derived T. parva using p67 gene polymorphisms and compared this to its distribution in vaccinated cattle exposed to buffalo-derived T. parva in central Kenya, where vaccine 'breakthroughs' have been reported. Additionally, we analysed the CD8+ T cell target antigen Tp2 for positive selection. Our results showed that 10% of the p67 sequences from Tanzanian cattle (n = 39) had a buffalo type p67 (allele 4), an allele that is rare among East African isolates studied so far. The percentage of buffalo-derived p67 alleles observed in Kenyan cattle comprised 19% of the parasites (n = 36), with two different p67 alleles (2 and 3) of presumptive buffalo origin. The Tp2 protein was generally conserved with only three Tp2 variants from Tanzania (n = 33) and five from Kenya (n = 40). Two Tanzanian Tp2 variants and two Kenyan Tp2 variants were identical to variants present in the trivalent Muguga vaccine. Tp2 evolutionary analysis did not show evidence for positive selection within previously mapped epitope coding sites. The p67 data indicates that some ITM-vaccinated cattle are protected against disease induced by a buffalo-derived T. parva challenge in northern Tanzania and suggests that the parasite genotype may represent one factor explaining this.

The Rhipicephalus appendiculatus tick vector of Theileria parva is absent from cape buffalo (Syncerus caffer) populations and associated ecosystems in northern Uganda.

Rhipicephalus appendiculatus is the major tick vector of Theileria parva, an apicomplexan protozoan parasite that causes the most economically important and lethal disease of cattle in East and central Africa. The African cape buffalo (Syncerus caffer) is the major wildlife host of T. parva from southern Uganda and Kenya to southern Africa. We show herein that R. appendiculatus appears to be absent from the two largest national parks in northern Uganda. Syncerus caffer is common in both of these national parks, specifically Murchison falls (MFNP) and Kidepo Valley (KVNP). We re-confirmed the previously reported absence of T. parva in buffalo sampled in the two northern parks based on RLB data using a nested PCR based on the T. parva p104 gene. By contrast, T. parva-infected R. appendiculatus ticks and parasite-infected buffalo were present in Lake Mburo (LMNP) in South central Uganda. This suggests that the distribution of R. appendiculatus, which is predicted to include the higher rainfall regions of northern Uganda, may be limited by additional, as yet unknown factors.

Detection of Theileria spp. in ticks, sheep keds (Melophagus ovinus), and livestock in the eastern Tibetan Plateau, China.

Theileria species, with a broad geographic distribution, infect a wide range of both domestic and wild animals and are transmitted by ixodid ticks. Currently, there is no comprehensive report regarding the distribution of Theileria spp. in the eastern Tibetan Plateau, especially in Ganze Tibetan autonomous prefecture (153,700 km2) and Ngawa Tibetan and Qiang autonomous prefecture (84,242 km2) of Sichuan province, China. In this study, we collected blood samples from yaks (n = 144) (Bos grunniens), Tibetan sheep (n = 92), and Tibet horses (n = 142) in Ganze and Ngawa.Theileria sinensis, T. luwenshuni, and T. equi were the dominant Theileria species detected in yaks, Tibetan sheep, and horses with the total infection rates of 25.7% (37/144), 75.0% (69/92), and 51.4% (73/142), respectively. For ectoparasites, T. luwenshuni was the only Theileria species detected in sheep keds (Melophagus ovinus) with an infection rate of 30.8% (8/26). The total infection rates of T. sinensis in Haemaphysalis qinghaiensis, Dermacentor everestianus, and Rhipicephalus microplus were 34.6% (36/104), 34.0% (17/50), and 51.3% (58/113), respectively. Theileria spp., belonging to T. sergenti/buffeli/orientalis group, were only detected in R. microplus collected in Danba county of Ganze with a total infection rate of 39.9% (19/48). Our results provide important data of the epidemiology of Theileria spp. in livestock and ectoparasites and will assist with the implementation of measures to control theileriosis transmission in eastern Tibetan Plateau, China.

Monitoring vaccinated cattle for induction and longevity of persistent tick-transmissible infection: Implications for wider deployment of live vaccination against East Coast fever in Tanzania.

The infection and treatment (ITM) procedure remains the only available method of immunization against Theileria parva infection. One constraint to deployment is the perception that the carrier state induced by ITM could result in enhanced disease problems. More than one million cattle have been ITM vaccinated in pastoralist systems in Tanzania over the last 2 decades. We present the results of a longitudinal study of six groups of cattle in Maasai villages in northern Tanzania exposed to natural tick challenge for between 2 weeks and 14 years post-vaccination. The p104 nested PCR revealed a higher frequency of T. parva carriers among vaccinates (30%) compared with controls (8%) (OR = 4.89, p = .000), with the highest frequency of carriers found in calves vaccinated 6 months previously, although carrier state was also detected in cattle vaccinated >10 years prior to the study. Variable number tandem repeat genotype analysis revealed 6 MS7 alleles with sizes ranging from 150 bp to 500 bp, but only two alleles were detected in cattle vaccinated >4 years earlier, relative to five alleles detected in recently vaccinated cattle and controls. In terms of heterozygosity, diversity was maximal in calves vaccinated within the last 2 weeks (h = 0.776) but lowest in cattle vaccinated 4 years earlier (h = 0.375). The analysis suggested close genetic relatedness of parasites in vaccinated and unvaccinated groups and up to 96% of variation was within rather than between the groups. These results confirm that ITM leads to a long-term T. parva carrier state in cattle and the detection of vaccine component VNTR in co-grazing unvaccinated cattle suggests potential vaccine transmission by ticks. However, vaccination stocks did not totally replace local genotypes, at least in cattle populations. These findings should mitigate concerns that ITM modifies T. parva field populations in a way that enhances disease in the medium term.

Multilocus genotyping of Theileria parva isolates associated with a live vaccination trial in Kenya provides evidence for transmission of immunizing parasites into local tick and cattle populations.

The live infection and treatment (ITM) vaccination procedure using the trivalent Muguga cocktail is increasingly being used to control East Coast fever, with potential implications for Theileria parva population genetic structure in the field. Transmission of the Kiambu V T. parva component to unvaccinated cattle has previously been described in Uganda. We monitored the T. parva carrier state in vaccinated and control animals on a farm in West Kenya where an ITM stabilate derived from the Kenyan T. parva Marikebuni stock was evaluated for field efficacy. A nested PCR-based Marikebuni-specific marker identified a carrier state in nine of ten vaccinated animals, detectable for a period of two years. We used 22 variable number tandem repeat (VNTR) markers to determine multilocus genotypes (MLGs) of 19 T. parva schizont-infected lymphocyte isolates derived from cattle and field ticks. Two isolates from unimmunized cattle were identical to the Marikebuni vaccination stock. Two cattle isolates were identical to a Muguga cocktail component Kiambu V. Seven isolates from ticks exhibited MLGs that were identical to the Serengeti/Muguga vaccine stocks. Six cattle and two tick-derived stocks exhibited unique MLGs. The data strongly suggest transmission of immunizing genotypes, from Marikebuni vaccine-induced carrier cattle to unimmunized cattle. It is possible that genotypes similar to those in the Muguga cocktail are present in the field in Western Kenya. An alternative hypothesis is that these parasites may have originated from vaccine trial sites in Eastern Uganda. If correct, this suggests that T. parva stocks used for immunization can potentially be disseminated 125 km beyond the immediate vaccination site. Regardless of their origin, the data provide evidence that genotypes similar to those in the Muguga cocktail are circulating in the field in East Africa, alleviating concerns about dissemination of 'alien' T. parva germplasm through live vaccination.

Potential tick vectors for Theileria equi in Israel.

Theileria equi Mehlhorn and Schein, 1998 (Piroplasmida: Babesiidae) is an important tick-borne pathogen of horses that is highly endemic in many parts of the world, including Israel. The present study evaluated the potential roles of five hard tick species [Hyalomma excavatum Koch, 1844; Hyalomma marginatum Koch, 1844; Rhipicephalus turanicus Pomerantsev 1936; Rhipicephalus annulatus Say, 1821; Haemaphysalis parva (Neumann, 1897) (all: Ixodida: Ixodidae)], previously found to infest horses in Israel, in acting as vectors for piroplasmosis. For this, DNA was extracted from whole ticks and, when possible, from the salivary glands in each species (n = 10-59). Polymerase chain reaction amplification and sequencing of the 18S rRNA gene were used to detect T. equi in 48 of the 127 ticks (37.8%) and in 21 of the 90 extracted salivary glands (23.3%) in all five species. All but two sequences were classified as T. equi genotype A; the remaining two were classified as genotype D. The findings of this study point to Ha. parva and R. annulatus as potential novel vectors of T. equi, and suggest that parasite genotype selection occurs within the tick vector.

Theileria parva: a parasite of African buffalo, which has adapted to infect and undergo transmission in cattle.

The tick-borne protozoan parasite Theileria parva causes an acute, often fatal disease in cattle throughout a large part of eastern and southern Africa. Infection of African buffalo (Syncerus caffer) is also widespread in this region but does not cause clinical disease in this species. This difference most likely reflects the evolutionary history of the parasites in these species, in that cattle were only introduced into Africa within the last 8000 years. In both hosts, T. parva establishes a carrier state, involving persistence of small numbers of parasites for many months following the acute phase of infection. This persistence is considered important for maintaining the parasite populations. Although cattle and buffalo parasites both produce severe disease when transmitted to cattle, the buffalo-derived parasites are usually not transmissible from infected cattle. Recent studies of the molecular and antigenic composition of T. parva, in addition to demonstrating heterogeneity in the populations in both host species, have revealed that infections in individual animals are genotypically mixed. The results of these studies have also shown that buffalo T. parva exhibit much greater genotypic diversity than the cattle population and indicate that cattle parasites represent a subpopulation of T. parva that has adapted to maintenance in cattle. The parasites in cattle and buffalo appear to be maintained largely as separate populations. This insight into the genotypic composition of T. parva populations has raised important questions on how host adaptation of the parasite has evolved and whether there is scope for further adaptation of buffalo-maintained populations to cattle.

Prevalence of Piroplasma in Ticks Collected from Dogs and Cattle in Guangxi, South China Determined by Reverse Line Blot Hybridization Assay.

Piroplasmosis is a disease of domestic and wild animals caused by tick-borne protozoa of the genera Theileria and Babesia. Piroplasmosis leads to substantial economic losses in the livestock industry. This disease has been frequently reported in subtropical and tropical regions worldwide. However, information regarding the prevalence of piroplasma in ticks collected from dogs and cattle is lacking in most areas. To assess the potential threat of piroplasmosis in South China, 671 ticks were collected in Guangxi Province. Deoxyribonucleic acid (DNA) was extracted from these ticks to evaluate the presence of piroplasma through a reverse line blot (RLB) hybridization assay using the hypervariable V4 region of the piroplasmic 18S ribosomal ribonucleic acid (r)RNA genes as probes to detect Theileria and Babesia species. The RLB results indicated that 144/671 (21.46%) ticks were infected with piroplasma species belonging to the genera Theileria or Babesia. Theileria buffeli, Babesia vogeli, Theileria annulata, and Theileria luwenshuni were present in the ticks at frequencies of 60/671 (8.94%), 21/671 (3.13%), 6/671 (0.89%), and 6/671 (0.89%), respectively. Mixed infections with 2 or more piroplasma species were present in 8/671 (1.19%) tick samples. The hypervariable V4 region of the piroplasmic 18S rRNA genes from 6 tick DNA samples with single infections was cloned, sequenced, and aligned to related sequences from GenBank. The Theileria and Babesia sequences were analyzed separately. 18S rRNA gene fragment sequences of T. annulata and T. buffeli were compared with previously reported homologous sequences. All 3 B. vogeli sequences examined in this study were grouped into the same cluster and belonged to the same genotype. The present study provides important epidemiological information regarding piroplasmosis occurrence in China. The existence of tick-borne piroplasma likely leads to high infection risks among the local animals in the studied areas.

A comprehensive review on past, present and future aspects of canine theileriosis.

Canine theileriosis is a notorious tick borne piroplasmid infection of wild and domestic canines. The causative agent has not yet been accurately classified. PCR studies revealed that causative agent resembles to Theileria genus and thus provisionally named as Theileria annae. The other Theileria species reported in canines is Theileria annulata, Theileria equi and unnamed Theileria specie. This emergent canine infection is considered to be endemic in most of the European countries. However in Asia this disease has not been reported till date. The vectors responsible for transmission of this disease have not been determined. It has been suggested that DNA of Theileria annae has been detected in hard tick Ixodes hexagonus in Northwestern Spain and several other tick species. Clinically canine theileriosis is characterized by severe weakness, fever, hemoglobinuria and anemia. Recently atovaquone or buparvaquone plus azithromycin therapy showed better clinical efficacy. This comprehensive review is intended to summarize the current knowledge on prevalence and epidemiology of canine theileriosis in different countries of the world and associated tick vectors.

Evaluation of the natural vertical transmission of Theileria orientalis.

Bovine theileriosis, caused by Theileria orientalis, is endemic from East Asia to Oceania. Even though the disease is mainly transmitted by Haemaphysalis ticks, the T. orientalis parasite can also be transmitted vertically. To develop proper control measures, the frequency of each transmission route must be elucidated. However, the frequency of vertical transmission, including transplacental transmission, of T. orientalis in naturally infected cattle is still controversial. This study aimed to clarify the frequency of the vertical transmission of T. orientalis in naturally infected cattle. Blood samples were collected from 204 T. orientalis-infected dams and their 211 newborn calves (including 7 sets of twins) within the first 24 h as well as 30 days after birth. Furthermore, 31 and 24 calves born to T. orientalis-infected and uninfected dams, respectively, were continuously surveyed for infection until 5 months of age. A total of 5 (2.4%) dams were diagnosed with mild anemia, whereas most of the dams were asymptomatic based on hematological examination and clinical signs. PCR analysis was performed on whole blood to determine the presence of T. orientalis in calves, and no calves were PCR positive 0 and 30 days after birth. However, 9.6% and 0% of the calves born to T. orientalis-infected and uninfected dams, respectively, tested positive at 3 and 5 months of age. The sampled calves were fed in-house, and the survey was conducted during the cold season; thus, horizontal transmission through blood-sucking insects rarely occurred. Therefore, the vertical transmission of T. orientalis took as long as 3 months to become detectable by PCR and occurred in approximately 10% of field cattle.

Molecular verification of transplacental transmission of Theileria lestoquardi in goat.

Ovine and caprine malignant theileriosis (OCMT), a critical condition in small ruminant production, causes lethal infections. In September 2016, a total number of 400 goats of Marghoz breed (the largest population of goat breed in Iran), in northwest of Iran, were examined for approximately 25 pre-partum abortions and 7 goats' mortality for a period of 3 days. A dead goat and her aborted fetus were brought into the Urmia Veterinary Hospital for further diagnostic investigations. The microbiological assessments including direct microscopical examination of the vaginal discharges and placentome with respect to the differential staining (Gram's staining method), conventional pure culturing, fetal abomasal contents, and the liver were negative. Microscopic examinations of blood smears of the goat and the fetus revealed characteristic of Theileria spp. piroplasms and impression smear samples from goat liver and fetal spleen were positive for Theileria Koch blue bodies. DNA analysis was performed using PCR technique and specific primers derived from the nucleotide sequences of 18S rRNA gene of T. lestoquardi; following extraction from blood samples, placentome, goat liver, and spleen of the fetus. The amplified DNA was sequenced afterwards and the corresponding sequence was registered under GenBank accession number MG208059. The sequence alignment showed that the products of PCR had a homology of 99% to known T. lestoquardi sequence registered under accession numbers of KY352037.1, KC778786.1, and JQ917458.1 in the GenBank. To our knowledge, this is a report demonstrating molecular verification of T. lestoquardi transplacental transmission in a neonatal kid of Marghoz breed of goats, its feasible role in induction of perinatal deaths and abortion in goat flocks.

Detection of Babesia spp., Theileria spp. and Anaplasma ovis in Border Regions, northwestern China.

Babesia spp., Theileria spp. and Anaplasma ovis are important intracellular agents that are transmitted by tick bites. However, Babesia spp., Theileria spp. and A. ovis in ticks have not been systematically reported along the border of northwestern China. In this study, a total of 1,084 adult ticks, including 134 Haemaphysalis punctata, 337 Hyalomma asiaticum, 233 Dermacentor nuttalli, 69 Rhipicephalus turanicus and 265 Dermacentor marginatus were collected from 11 counties or cities of Xinjiang Uygur Autonomous Region. The ticks were identified from morphological and molecular characteristics. Two fragments of 18S rRNA gene were used to determine the species level of Babesia and Theileria. Msp4 gene encoding major surface protein 4 was used to determine A. ovis. Of the 1,084 samples, five species of Babesia (B. occultans, B. caballi, B. motasi, B. major and Babesia sp. detected in this study), two kinds of Theileria (Theileria ovis and Theileria sp. detected in this study) and A. ovis with six phylogenic branches were detected in the border of northwestern China. Babesia occultans, first found in China, was first molecularly detected in D. nuttalli. Babesia caballi and Babesia sp. detected in this study were first molecularly detected in Hy. asiaticum. Genotype III of A. ovis was predominant in the border regions of northwestern China.