Investigation of Babesia spp. and Theileria spp. in ticks from Western China and identification of a novel genotype of Babesia caballi.

Babesia spp. and Theileria spp. are tick-borne protozoan parasites with veterinary importance. In China, epidemiological and genetic investigations on many Babesia and Theileria species were still absent in many areas and many tick species. From Aug 2021 to May 2023, 645 ticks were collected from the body surface of domestic animals (camels, goats, sheep, and cattle) using tweezers in seven counties in three provinces including Xinjiang (Qitai, Mulei, Hutubi, and Shihezi counties), Chongqing (Youyang and Yunyang counties), and Qinghai (Huangzhong county). Three tick species were morphologically and molecularly identified (334 Hyalomma asiaticum from Xinjiang, 245 Rhipicephalus microplus from Chongqing, and 66 Haemaphysalis qinghaiensis from Qinghai). A total of three Babesia species and two Theileria species were detected targeting the 18S gene. The COI and cytb sequences were also recovered from Babesia strains for further identification. In R. microplus from Chongqing, Babesia bigemina, the agent of bovine babesiosis, was detected. Notably, in H. asiaticum ticks from Xinjiang, a putative novel genotype of Babesia caballi was identified (0.90%, 3/334), whose COI and cytb genes have as low as 85.82% and 90.64-90.91% nucleotide identities to currently available sequences. It is noteworthy whether the sequence differences of its cytb contribute to the drug resistance of this variant due to the involvement of cytb in the drug resistance of Babesia. In addition, Theileria orientalis and Theileria annulata were detected in R. microplus from Chongqing (12.20%, 31/245) and H. asiaticum from Xinjiang (1.50%, 5/334), respectively. These results suggest that these protozoan parasites may be circulating in domestic animals in these areas. The pathogenicity of the novel genotype of B. caballi also warrants further investigation.

The first study of the prevalence and genetic diversity of Theileria equi and Babesia caballi in horses in Russia.

Equine piroplasmosis (EP) is a global worldwide infection, which can lead to the death of animals. Despite the causative agents of EP being well studied, there are no data on the distribution and genetic characteristics of EP agents in any region of Russia. In this study, blood samples from 750 horses from Novosibirsk province, Irkutsk province, and Altai region of Russian Siberia were examined for the presence of EP agents. Theileria equi and Babesia caballi were detected in all examined regions, with mean prevalence rates of 60.4% and 7.2%, respectively. The identified pathogens were genetically characterized by the 18S rRNA gene. The determined T. equi sequences were highly conserved and belonged to genotypes A and E, with genotype E being found in 88.6% of genotyped samples. In contrast to T. equi, B. caballi sequences were genetically diverse. Seven sequence variants of B. caballi were identified, and only two of them matched known sequences from the GenBank database. The determined B. caballi sequences belonged to four distinct branches within genotype A. Mixed infections with several variants of B. caballi or with T. equi and B. caballi were common. The conducted phylogenetic analysis based on all available B. caballi sequences of the 18S rRNA gene (> 900 bp) from GenBank and from this study first demonstrated the presence of five monophyletic clusters within genotype A and three clusters within genotype B. Thus, the genetic study of B. caballi from Siberia has significantly expanded the data on the genetic diversity of this pathogen.

Molecular detection of piroplasms in domestic donkeys in Xinjiang, China.

BACKGROUND: Piroplasmosis is a common and prevalent tick-borne disease that affects equids. OBJECTIVES: To determine the infection and molecular characteristics of the piroplasms in donkeys from Xinjiang, northwestern China, we undertook a cross sectional study by collecting representative samples across several counties within the region. METHODS: A total of 344 blood samples were collected from adult domestic donkeys from 13 counties in Xinjiang. PCR was conducted to test for T. equi and B. caballi in the blood samples based on the equine merozoite antigen-1 (Ema-1) gene and the 48 kDa rhoptry protein (BC48) gene, respectively. RESULTS: Sixteen blood samples tested positive for piroplasms and the overall infection rate was 4.7% (16/344). Seven of the 13 counties were positive for piroplasms. Among the 16 piroplasm-positive samples, 15 were singly infected with T. equi with an infection rate of 4.4% (15/344), and coinfection with T. equi and B. caballi was detected in one sample (0.3%, 1/344) from Wushi. Four T. equi sequence genotypes were identified and grouped into different branches of the evolutionary trees. CONCLUSION: These findings suggest that the infection rate of piroplasms is low in domestic donkeys in southern Xinjiang and that T. equi genotypes have a regional distribution.

Molecular genotyping of Babesia caballi.

Babesia caballi is an intra-erythrocytic parasite causing equine piroplasmosis. Three B. caballi genotypes (A, B, and C) have been identified based on the 18 S rRNA and rhoptry-associated protein (rap-1) gene sequences. These variant parasite genotypes compromise the diagnostic utility of the WOAH-recommended serological assays in declaring horses free of equine piroplasmosis. Although a gene encoding a spherical body protein 4 (sbp4) has recently been identified as a potential antigen for the serological detection of B. caballi, the ability of this antigen to detect the different geographical strains has not been determined. The molecular distinction between variant B. caballi genotypes is limited and therefore we developed molecular typing assays for the rapid detection and quantification of distinct parasite genotypes. Field samples were screened for the presence of B. caballi using an established multiplex equine piroplasmosis qPCR assay. In this study, B. caballi genotype A was not detected in any field samples screened. However, phylogenetic analysis of the amplified sbp4 and 18 S rRNA genes confirmed the phylogenetic groupings of the South African isolates into either B. caballi genotypes B or C. A multiple sequence alignment of the sbp4 gene sequences obtained in this study together with the published sbp4 sequences representing B. caballi genotype A, were used to identify conserved regions within the gene to design three primer pairs and three genotype-specific TaqMan minor-groove binder (MGB™) probes. The qPCR assays were shown to be specific and efficient in the detection and differentiation between B. caballi genotypes A, B, and C and could be used as a diagnostic assay to prevent the unintentional spread of variant B. caballi genotypes globally.

Detection of tick-borne pathogens in Rhipicephalus bursa ticks collected from the autochthonous Garrano breed of horses in Portugal.

The Garrano is a semi-feral horse breed native to several mountains in the northern Iberian Peninsula. Despite being endangered, this unique breed of pony has managed to survive in the wild and continues to be selectively bred, highlighting their remarkable resilience and adaptability to harsh environments. Wildlife plays a critical role in the survival of tick vectors in their natural habitats and the transfer of tick-borne pathogens, as they can serve as reservoir hosts for many agents and amplifiers for these vectors. The semi-feral lifestyle of the Garrano horses makes them particularly vulnerable to exposure to numerous tick species throughout the year. Therefore, the aim of this study was to investigate the occurrence of Anaplasma, Ehrlichia, Babesia, Theileria, and spotted fever rickettsiae in the Garrano horse ticks to obtain a knowledge of circulating agents in this host population. The collected ticks (n = 455) were identified as Rhipicephalus bursa. DNA specimens were organized in pools of 5 ticks, for molecular screening. Pools PCR results confirmed the presence of Candidatus Rickettsia barbariae (n = 12 for the ompB gene, n = 11 for the ompA gene and n = 6 for the gltA gene), Babesia bigemina (n = 1), Babesia caballi (n = 3), Theileria equi (n = 15) and Theileria haneyi (n = 1).These results confirm the circulation of an emerging rickettsial spotted fever group member, Candidatus R. barbariae, in R. bursa ticks. Our findings demonstrated that Candidatus R. barbariae co-circulates with B. bigemina and T. equi, which are vectored by R. bursa. We are reporting for the first time, the detection of T. haneyi among R. bursa ticks feeding in the Garrano horses in Portugal. Surveillance studies for tick-borne infections are essential to provide information that can facilitate the implementation of preventive and control strategies.

Molecular and Serological Detection of Vector-Borne Pathogens Responsible for Equine Piroplasmosis in Europe between 2008 and 2021.

Equine piroplasmosis (EP) is caused by Theileria (T.) equi and/or Babesia (B.) caballi. The aim was to assess the percentage of positive test results for EP in horses in Europe and to identify risk factors for pathogen contact/infection. This study included results from PCR and competitive enzyme-linked immunosorbent assay testing requested by European veterinarians between 2008 and 2021. Binary bivariate logistic regression was used to analyze risk factors. A total of 4060 horses were included. PCR testing was positive in 9.7% (154/1589), serology for T. equi in 15.2% (393/2591) and for B. caballi in 6.8% (175/2578). The odds of positive serology increased by 6.8% (B. caballi, p = 0.008) and 9.5% (T. equi, p < 0.001) each year. Regionality had a statistically significant impact on PCR (Eastern p = 0.047/OR = 1.605; Southern p = 0.029/OR = 1.451; Central p = 0.007/OR = 0.617) and serological testing for T. equi (Southern p < 0.001/OR = 2.521; Central p < 0.001/OR = 0.537; Northern p = 0.003/OR = 0.462), as well as breeds on seroprevalence of B. caballi (heavy horses: p = 0.016/OR = 2.239) and T. equi (ponies: p = 0.007/OR = 0.340; warmbloods: p = 0.025/OR = 1.602). In conclusion, there was a significant geographical impact on the results of PCR and serology, consistent with known vector habitats. The rising numbers of horses tested serologically positive highlights the importance of surveillance.

Diagnostic performance of a rapid immunochromatographic test for the simultaneous detection of antibodies to Theileria equi and Babesia caballi in horses and donkeys.

BACKGROUND: Equine piroplasmosis is caused by two tick-borne protozoan parasites, Theileria equi and Babesia caballi,, which are clinically relevant in susceptible horses, donkeys, and mules. Moreover, equine piroplasmosis significantly constrains international trading and equestrian events. Rapidly diagnosing both parasites in carrier animals is essential for implementing effective control measures. Here, a rapid immunochromatographic test for the simultaneous detection of antibodies to T. equi and B. caballi was evaluated using samples from horses and donkeys collected in Greece, Israel, and Italy. The results were compared with an improved competitive enzyme-linked immunosorbent assay (cELISA) for detecting antibodies to both parasites using the same panel of samples. METHODS: Blood samples were collected from 255 horses and donkeys. The panel consisted of 129 horses sampled at four locations in northern Greece, 105 donkeys sampled at four locations in Sicily, and 21 horses sampled at two locations in Israel. The rapid test and the cELISA were performed according to the manufacturer's instructions, and the results were subjected to a statistical analysis to determine the sensitivity and specificity of both tests and their association. RESULTS: The immunochromatographic test provided a result within 15 min and can be performed in the field, detecting both pathogens simultaneously. The overall coincidence rate between the rapid test and the cELISA for detecting antibodies against T. equi was 93% and 92.9% for B. caballi. The rapid test's sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for T. equi were above 91.5%. Sixteen samples were positive for both parasites in the rapid test and eight in the cELISA. Either test had no significant association between T. equi and B. caballi detection. The detection rates of both parasites were significantly higher in Italy than in Greece or Israel and in donkeys than in horses. The agreement for T. equi between the results of both tests was high in Greece (93.8%) and Italy (95.2%) and moderate in Israel (76.2%). For B. caballi, the specificity and NPV of the rapid test were high (94.2% and 98.3%, respectively), although the sensitivity and PPV were moderate (69.2% and 39.1%, respectively) due to the small sample size. However, for B. caballi, the sensitivity was higher with the rapid test. CONCLUSIONS: The rapid test detected T. equi and B. caballi simultaneously in the field, potentially replacing laborious cELISA testing and is recommended for import/export purposes. The test can also be helpful for the differential diagnosis of clinical cases, since seropositivity may rule out equine piroplasmosis since it does not indicate current or active infection.

Epidemiology and genetic diversity of Theileria equi and Babesia caballi in Mongolian horses.

Equine piroplasmosis is a tick-borne disease caused by Theileria equi and Babesia caballi in horses. Because of its impact on horse industry, control of this disease is crucial for endemic countries. The control of equine piroplasmosis may be influenced by the genotypic diversity of T. equi and B. caballi. Mongolia, a country with a thriving livestock industry, is endemic for T. equi and B. caballi. However, nationwide epidemiological surveys have not been conducted to determine the current status of infections and genetic diversity of these two parasite species. Therefore, the objective of this research was to investigate the infection rates and genotypes of T. equi and B. caballi in horses across Mongolia. Blood samples were collected from 1353 horses in 15 of Mongolia's 21 provinces, and their DNAs were analyzed with T. equi- and B. caballi-specific PCR assays. Additionally, blood smears were prepared from 251 horses, stained with Giemsa, and examined under a light microscope to identify T. equi and B. caballi. The microscopy revealed that 30 (11.9%) and 4 (1.6%) of the 251 horses were positive for T. equi and B. caballi, respectively. By contrast, PCR assays detected the T. equi and B. caballi in 1058 (78.2%) and 62 (4.6%) horses, respectively. Phylogenetic analysis of 18S rRNA sequences from 42 randomly selected T. equi-positive DNA samples detected the genotypes A and E. On the other hand, the rap-1 sequences from 19 randomly selected B. caballi-positive DNA samples occurred in clades representing the genotypes A and B1, as well as in a distinct clade closely related to the genotype A. Our findings confirm the widespread occurrence of T. equi and B. caballi infections in Mongolian horses, highlighting the need for a comprehensive control approach.

Equine Piroplasmosis in Asymptomatic Horses of Western Iran: Comparison of Microscopic Examination and Multiplex PCR.

PURPOSE: Piroplasmosis is responsible for anemia, fever, loss of physical activity and even death in equines. In epidemiological studies, accurate diagnostic tests are essential for detecting asymptomatic carriers. This study aimed to investigate the prevalence of infection in asymptomatic horses from Lorestan province, western Iran by developing a multiplex PCR. METHODS AND RESULTS: Blood samples were examined by microscopy and multiplex PCR targeting the SSU rRNA gene of Theileria equi and Babesia caballi. Out of the total of 165 horses, 19 (11.51%) and 31 (18.78%) cases were positive for piroplasms by microscopy and PCR, respectively. The detection rates of both genera were significantly higher in multiplex PCR compared to microscopy (p < 0.0001). Compared with multiplex PCR, the sensitivities of microscopy for the detection of Babesia were only 28.5%. The prevalence of T. equi infection was significantly higher in summer (p = 0.035). The prevalence of B. caballi was significantly higher in males (p = 0.038). CONCLUSION: Findings indicate that the multiplex PCR described here is a sensitive technique for the detection of piroplasm DNA in carriers. Furthermore, asymptomatic carriers must be considered as an important source of infection for equids living in this region.

Chromosome-level genome assembly of Babesia caballi reveals diversity of multigene families among Babesia species.

BACKGROUND: Babesia caballi is an intraerythrocytic parasite from the phylum Apicomplexa, capable of infecting equids and causing equine piroplasmosis. However, since there is limited genome information available on B. caballi, molecular mechanisms involved in host specificity and pathogenicity of this species have not been fully elucidated yet. RESULTS: Genomic DNA from a B. caballi subclone was purified and sequenced using both Illumina and Nanopore technologies. The resulting assembled sequence consisted of nine contigs with a size of 12.9 Mbp, rendering a total of 5,910 protein-coding genes. The phylogenetic tree of Apicomplexan species was reconstructed using 263 orthologous genes. We identified 481 ves1-like genes and named "ves1c". In contrast, expansion of the major facilitator superfamily (mfs) observed in closely related B. bigemina and B. ovata species was not found in B. caballi. A set of repetitive units containing an open reading frame with a size of 297 bp was also identified. CONCLUSIONS: We present a chromosome-level genome assembly of B. caballi. Our genomic data may contribute to estimating gene expansion events involving multigene families and exploring the evolution of species from this genus.

PCR detection of Theileria equi and Babesia caballi in apparently healthy horses in Paraguay.

Equine piroplasmosis (EP) is a tick-borne disease caused by Theileria equi and Babesia caballi in equids, including horses. EP has a global distribution and often leads to a significant socioeconomic impact on the equine industry. Infected animals remain as carriers and become a source of infection for tick vectors, thereby posing an immense challenge in the disease management. Therefore, detection of these carriers is crucial to assess the risk of transmission and to implement appropriate control measures in endemic countries. Paraguay is a tropical country where various tick-borne diseases are common among livestock; however, the status of EP remains unknown in this country. Because the tick vectors capable of transmitting T. equi and B. caballi are endemic in Paraguay, we hypothesised that Paraguayan horses are infected with these parasite species. To test our hypothesis, we prepared blood DNA samples from a total of 545 apparently healthy horses in 16 of the 17 departments of Paraguay and analysed them with specific PCR assays to detect T. equi and B. caballi. The PCR results showed that 178 (32.7%) and 8 (1.5%) of the horses were infected with T. equi and B. caballi, respectively. Among the infected horses, two (0.4%) were infected with both parasite species. Our analyses further indicated that the positive rates of T. equi infection did not differ between horse breeds, males and females, or age groups. We also found that haematological parameters were the same between the non-infected animals and animals with single infections. By contrast, the two horses co-infected with T. equi and B. caballi had haemoglobin and haematocrit values lower than the normal ranges. In conclusion, the present study demonstrated that Paraguayan horses are infected with T. equi and B. caballi and that the rate of T. equi infection is higher than that of B. caballi. Our findings highlight the need to add EP to the list of differential diagnoses when anaemic horses are presented to equine clinics in Paraguay.

A risk assessment of equine piroplasmosis entry, exposure and consequences in the UK.

BACKGROUND: Equine piroplasmosis (EP) is currently not endemic in the UK, despite a lack of formal surveillance and the presence of carrier horses in the equine population. Pathogen establishment would have significant welfare and economic impacts on the national equine industry, but the disease is often overlooked by UK practitioners. OBJECTIVES: To assess the risk of disease entry, exposure and consequences to the UK equine population. STUDY DESIGN: Qualitative risk assessment. METHODS: A qualitative risk assessment was constructed utilising the current World Organisation for Animal Health (OIE) published framework for importation risk assessment, assessing the key areas of disease entry, exposure and consequences to the UK equine population. RESULTS: The overall risk of EP entry to the UK via importation of infected equidae with acute disease is very low but considered medium with subclinical carrier animals. Entry via importation of ticks or the importation of blood is considered very low. The risk of EP exposure to susceptible equidae in the UK is considered low by the infection routes of tick-bites, contaminated needles and contaminated blood, but very high via transplacental transfer. However, the consequences of EP endemic establishment are considered of high significance to the UK equine industry. MAIN LIMITATIONS: A lack of available numerical data for events and variables in disease import risk meant a qualitative assessment was the most practical method for this scenario. CONCLUSIONS: This risk assessment highlights that EP positive animals are able to enter and are currently present in the UK, and that conditions do exist that could allow forward transmission of the disease. It has highlighted a gap in existing policy where the UK falls behind OIE guidelines and has suggested steps to correct this discrepancy and improve national biosecurity.

Prevalence and genetic characterization of Theileria equi and Babesia caballi in grazing horses in Xinjiang, northwestern China.

Piroplasmosis is a disease that negatively affects equine health worldwide. Hence, 324 blood samples were collected from grazing horses in ten sites in Xinjiang and testing them for the presence of Theileria equi and Babesia caballi by PCR of the EMA-1 gene and BC48 gene, respectively. Of the 324 blood samples, 161 (49.7%) were positive for equine piroplasms. The prevalence of T. equi was 38.9% (126/324), while that of B. caballi was 30.2% (98/324). The T. equi and B. caballi co-infection rate was 19.4% (63/324). From the 126 EMA-1 gene sequences and 98 BC48 gene sequences we obtained, 21 and 27 genotypes were identified, respectively. The EMA-1 sequences together with the GenBank reference sequences grouped into four clusters, with those from the present study forming two distinct clusters. In contrast, the BC48 sequences formed eight clusters with the GenBank reference sequences, while those obtained in the present study formed five distinct clusters. Our results highlight the widespread distribution and abundant gene polymorphism of T. equi and B. caballi in grazing horses from Xinjiang.

First molecular detection of Mycoplasma ovis in horses from Brazil.

This study aimed to determine the occurrence of hemoplasmas and tick-borne pathogens (TBP) (Theileria equi, Babesia caballi, and Ehrlichia sp.) in horses and ticks' salivary glands, and determine the factors associated with exposure/infection in a rural settlement in southern Brazil. Blood samples from 22 horses were screened for anti-T. equi and anti-Ehrlichia sp. antibodies by an indirect fluorescent antibody test (IFAT) assays. Samples were also tested by PCR assays for T. equi and B. caballi (18S rRNA and rap-1 genes, respectively), hemoplasmas (16S rRNA gene), and Ehrlichia sp. (dsb gene). Ticks were removed from the animals (inspection) and the environment (flannel trawling and dry ice traps), and morphologically identified. Additionally, salivary glands DNA was extracted from 28 adult ticks infesting the animals and four nymphs from the environment, and further screened for Ehrlichia sp. and hemotropic Mycoplasma sp. Anti-T. equi and anti-Ehrlichia sp. antibodies were detected in 40.91% (nine/22; 95% CI: 23.26-61.27) and 31.81% (seven/22; 95% CI: 16.36-52.68) horses, respectively. Theileria equi, B. caballi, and hemotropic Mycoplasma sp. DNA was detected in 59.09% (13/22), 4.55% (one/22), and 50% (11/22) horses, respectively. All horses tested negative in the PCR for Ehrlichia sp. All sequences showed ≥99% identity with multiple T. equi, B. caballi, and Mycoplasma ovis sequences deposited in GenBank database. Adult ticks were identified as Dermacentor nitens (44/47; 93.62%) and Rhipicephalus microplus (three/47; 6.38%). Ticks' salivary glands were negative for Ehrlichia sp., while 39.29% from adults (11/28) and 50% from nymphs (two/four) from the environment were positive for hemotropic Mycoplasma sp. This is the first report of M. ovis infection in horses from Brazil and the first detection of hemoplasma DNA in salivary glands of D. nitens and R. microplus ticks. Further studies are needed to elucidate the vector competence of ticks to transmit hemoplasmas.

Theileria equi in the horses of Iran: Molecular detection, genetic diversity, and hematological findings.

In all equids worldwide, Theileria equi and Babesia caballi are believed to be two important erythrocytic protozoa that cause equine piroplasmosis. In addition, it was recently discovered that Theileria haneyi is another potential equine piroplasmosis (EP) agent. Ixodid ticks are the major vectors of these parasites. Equine piroplasmosis is of international importance and affects enormously the equine industry. In this study, for the first time, molecular prevalence and genetic diversity of piroplasma parasites (T. equi and B. caballi) in horses from Fars province (south of Iran) were determined. Also, hematological alterations of naturally infected horses were analyzed. PCR positive horses showed anemia, thrombocytopenia, leukocytosis with a left shift of neutrophilia, and monocytosis. PCR results revealed that, from 133 blood samples of horses, 40 samples were positive (30.07%). The occurrence of T. equi in this area (30.07%) was more than the national average prevalence of T. equi (24.11%), but B. caballi prevalence in study area (0%) was less than the average of previous studies in Iran (5.47%). Our findings revealed that the T. equi was widespread in Fars province of Iran. PCR products of 18S rDNA and EMA-1 genes of T. equi strains were sequenced successfully. All 18S rDNA sequences collected in this experiment revealed 100% similarity together. According to the phylogenetic tree constructed using the 18S rDNA gene, Iranian T. equi is clustered with strains from Cuba (KY111762, KY111761) and USA (CP001669, JX177672). So, this could be concluded that T. equi studied in this research, and those strains are initiated from a common T. equi ancestor at an unknown time ago. Also, the phylogenetic tree based on EMA-1 gene demonstrated a genetically diverse population of Iranian T. equi strains (10 different genotypes). As EMA-1 is one of the most immunogenic antigens in this parasite, such variability could be a concern about the efficacy of T. equi vaccines. Finally, more studies on equine piroplasmosis in the provinces of the southern region of Iran are recommended to create a better vision of disease in this region.

Low seroprevalence of equine piroplasmosis in horses exported from the Netherlands between 2015 and 2021.

Equine piroplasmosis (EP) is a tick-borne disease affecting horses, donkeys, mules and zebras, caused by the intracellular apicomplexan protozoa Babesia caballi and Theileria equi. The geographical distribution of EP is closely related to the distribution of its vector tick species belonging to the genera of Dermacentor, Rhipicephalus and Hyalomma. Since the discovery of Dermacentor reticulatus ticks in 2007 and the first reported autochthonous cases in the South of the Netherlands in 2012, no data on the (sero)prevalence of EP in horses in the Netherlands have been reported and it remains unclear whether B. caballi and T. equi have been able to establish themselves in the Netherlands. This study aims to give an update on the current status of EP in horses in the Netherlands using data from serological tests performed in the context of export and screening of 12,881 horses from 2015 through 2020. Horses were categorized as "Dutch," "Foreign," or "Unknown" based on microchip number. The overall seroprevalence of EP in Dutch horses was found to be 0.5% (95% exact CI [0.4-0.7]), compared to 1.9% (95% exact CI [1.3-2.6]) in horses in the category "Foreign" and 1.7% (95% exact CI [1.2-2.3]) in horses in the category "Unknown." In addition, the seroprevalence per country in the category "Foreign" ranged from 0% (0.95% exact CI [0-2.8]) for Ireland to 6.0% (0.95% exact CI [3.5-9.3]) for Spain. In light of the reports on the seroprevalence during the outbreak of autochthonous EP reported in 2012 and on seroprevalences of EP in other countries in Northwestern Europe, the seroprevalence of EP in horses exported from the Netherlands is very low. However, the higher seroprevalence of EP in horses from abroad warrants the need for the monitoring of EP, as tick vectors are present in the Netherlands and the import of horses from endemic areas increases the chances of EP becoming more prevalent in the Netherlands.

Development of Nested PCR and Duplex Real-Time Fluorescence Quantitative PCR Assay for the Simultaneous Detection of Theileria equi and Babesia caballi.

Equine piroplasmosis (EP) is a type of blood protozoan disease caused by tick-borne parasites, Theileria equi (T. equi), Babesia caballi (B. caballi) and Theileria haneyi. While many studies have been conducted on EP diagnosis, diagnostic methods exhibiting high sensitivity and specificity remain lacking. Therefore, nested PCR (nPCR) and duplex real-time fluorescence quantitative PCR (qPCR) that can simultaneously detect both T. equi and B. caballi causing agents were established and compared. The two techniques were used to analyze 36 horse blood samples for EP. This set of samples was also detected by a multinested PCR (mnPCR) targeting the EMA-1 gene of T. equi and the RAP-1 gene of B. caballi. By nPCR, duplex real-time fluorescence qPCR and mnPCR, infections with B. caballi were detected in 16.67% (6/36), 2.78% (1/36), 19.44% (7/36) of the horses, respectively. The T. equi prevalence was 58.33% (21/36) by the nPCR, 33.33% (12/36) by the duplex real-time fluorescence qPCR and 2.78% (1/36) by the mnPCR. The overall prevalence of infection with mixed parasites by nPCR was 5.56% (2/36), by duplex real-time fluorescence qPCR was 2.78% (1/36) and by mnPCR 0% (0/36). Results suggest that nPCR can detect T. equi and B. caballi positive samples with good specificity and sensitivity, although distinguishing between the two parasites requires an electrophoresis with 4% agarose gels. The duplex real-time fluorescence qPCR can readily distinguish between T. equi and B. caballi infection, but with low sensitivity.

Detection of Tick-Borne Bacterial and Protozoan Pathogens in Ticks from the Zambia-Angola Border.

Tick-borne diseases (TBDs), including emerging and re-emerging zoonoses, are of public health importance worldwide; however, TBDs tend to be overlooked, especially in countries with fewer resources, such as Zambia and Angola. Here, we investigated Rickettsia, Anaplasmataceae, and Apicomplexan pathogens in 59 and 96 adult ticks collected from dogs and cattle, respectively, in Shangombo, a town at the Zambia-Angola border. We detected Richkettsia africae and Rickettsia aeschilimannii in 15.6% of Amblyomma variegatum and 41.7% of Hyalomma truncatum ticks, respectively. Ehrlichia minasensis was detected in 18.8% of H. truncatum, and Candidatus Midichloria mitochondrii was determined in Hyalomma marginatum. We also detected Babesia caballi and Theileria velifera in A. variegatum ticks with a 4.4% and 6.7% prevalence, respectively. In addition, Hepatozoon canis was detected in 6.5% of Rhipicephalus lunulatus and 4.3% of Rhipicephalus sanguineus. Coinfection of R. aeshilimannii and E. minasensis were observed in 4.2% of H. truncatum. This is the first report of Ca. M. mitochondrii and E. minasensis, and the second report of B. caballi, in the country. Rickettsia africae and R. aeschlimannii are pathogenic to humans, and E. minasensis, B. caballi, T. velifera, and H. canis are pathogenic to animals. Therefore, individuals, clinicians, veterinarians, and pet owners should be aware of the distribution of these pathogens in the area.

Molecular investigation of Babesia caballi in horses from the state of Rio de Janeiro, Brazil: Epidemiological aspects associated with the infection.

The epidemiological aspects of Babesia caballi infection were evaluated in 516 horse samples from Rio de Janeiro, Brazil. The presence and infestation level of ticks on horses, breed conditions, and animal management were evaluated on each farm through an epidemiological questionnaire. The gene that codes for rhoptry-associated protein-1 (RAP-1) of B. caballi was amplified by nested PCR (nPCR). Among the horses sampled, 17.2% (n = 89/516) presented B. caballi DNA. The characterized samples showed 99-100% similarity with other isolates of B. caballi based on the RAP-1 gene, available in GenBank. In the final logistic regression model, the variables associated with B. caballi infection in horses were as follows: age below two years (OR = 3.33; IC = 1.7-6.5), farms located in low altitudes (OR = 3.52; IC = 1.7-7.3) and Dermacentor nitens infestation (OR = 1.91; IC = 1.1-3.4). Furthermore, a high level of D. nitens infestation in horses was also a factor associated with positivity for B. caballi (OR = 2.11; IC = 1.25-3.54). In summary, young horses bred in low altitude regions characterized with high temperatures, and infested by D. nitens, mainly with a higher level of infestation, are more likely to be infected by B. caballi. This epidemiological study provides statical evidence that the D. nitens tick play a role as the biological vector of B. caballi in the studied region.

Comparison of direct and indirect methods to maximise the detection of Babesia caballi and Theileria equi infections in Central Southern Italy.

Equine piroplasmosis is a disease of equids, caused by tick-borne apicomplexan protozoan pathogens Babesia caballi and Theileria equi, which, according to the World Organisation for Animal Health (OIE), can be diagnosed by enzyme-linked immunosorbent assay (ELISA), immunofluorescent antibody test (IFAT) and polymerase chain reaction (PCR). The present study was conducted to evaluate and compare the assays available for the diagnosis of equine piroplasmosis. Data employed were obtained from 1300 blood samples collected between 2012-2014 from asymptomatic and symptomatic equines (horses and donkeys) of central-southern regions of Italy and analyzed by ELISA, IFAT, PCR (one commercial and one from literature) and blood smear microscopic examination. Statistical differences of the proportions of positivity for each parasite and group (asymptomatic and symptomatic) among the methods were verified by the z test to identify the most sensitive. The concordance between each pair of methods - for each parasite and within the groups - and trends in detection of suspect samples of four hypothetical diagnostic algorithms using serological and biomolecular assays were evaluated to identify the most suitable laboratory diagnostic workflow. The results of this study highlighted a lower capacity to detect suspect samples of commercial ELISA for B. caballi in all groups when compared to biomolecular methods and IFAT; and of the commercial PCRs in asymptomatic animals, identifying a PCR from literature and IFAT as the best choice for a combined diagnosis. For T. equi, IFAT detected more suspect samples than ELISA, even if the latter showed good performance and some samples were positive only by the ELISA and PCR, indicating that their simultaneous employment is still advantageous. Host-parasite interaction, amino-acid/genetic diversity and differences in detection limits among the assays could be among the reasons in explaining the present results. In view of further studies, ELISA should be used in combination with PCR, that should regularly be included in the laboratory diagnosis to maximise the detection of early infections and support the evaluation of pharmacological treatment.