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.

Survey of tick-borne pathogens in grazing horses in Kyrgyzstan: phylogenetic analysis, genetic diversity, and prevalence of Theileria equi.

Introduction: Tick-borne pathogens (TBP) are an important group of organisms that can affect animals and humans all over the world. Equine piroplasmosis (EP), caused by Theileria equi and Babesia caballi, is considered one of the most important tick-borne diseases and can cause significant clinical symptoms and mortality in horses. Moreover, EP plays a restrictive role in international horse traditions and transportation. Although these species can cause similar symptoms, there are different 18S rRNA genotypes of T. equi (five genotypes) and B. caballi (three genotypes). Besides piroplasma species, Anaplasma and hemotropic mycoplasmas (HM) are known as other important tick-borne pathogens reported in horses. Methods: In this study, we investigated the presence, prevalence, genetic diversity, and phylogenetic analyses of TBPs using PCRs and DNA sequencing in grazing horses in Kyrgyzstan. For these purposes, a total of 311 blood samples were collected from Chuy, Issyk-Kul, Naryn, Osh, Talas, and Jalal-Abad. Results: DNA amplification of TBP revealed that 23 (7.40%) out of 311 samples were found to be positive for T. equi. However, B. caballi, HM, A. phagocytophilum, and A. capra were not detected in this study. The infection rate of T. equi was higher in males (8.11%) than in females (6.35%) (p=0.2880) and in those older than 5 years (9.02%) than in the 1-4 age group (6.35%) (p=0.1950). Phylogenetic analysis of 18S rRNA revealed that A and E genotypes of T. equi have circulated in grazing horses in Kyrgyzstan. Discussion: Information about the genetic diversity of T. equi is important for understanding the population dynamics of the species and developing effective control strategies against this pathogen. This is the first molecular investigation of A. capra in horses in Kyrgyzstan. Although this pathogen has been detected in different hosts in Kyrgyzstan, it was not detected in this study. However, considering the wide host spectrum of A. capra, it is thought that more large-scale studies are needed to understand the effect of horses on the epidemiology of this pathogen.

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.

Expression of IL-10 and TGF-ß1 in horses experimentally infected with T. equi merozoites is associated with antibody production but not modulation of pro-inflammatory responses.

Theileria equi (T. equi) is an apicomplexan parasite that causes severe hemolytic anemia in equids. Presently, there is inadequate knowledge of the immune responses induced by T. equi in equid hosts impeding understanding of the host parasite relationship and development of potent vaccines for control of T. equi infections. The objective of this study was to evaluate the host-parasite dynamics between T. equi merozoites and infected horses by assessing cytokine expression during primary and secondary parasite exposure, and to determine whether the pattern of expression correlated with clinical indicators of disease. Our findings showed that the expression of pro-inflammatory cytokines was very low and inconsistent during both primary and secondary infection. There was also no correlation between the symptoms observed during primary infection and expression of the cytokines. This suggests that the symptoms might have occurred primarily due to hemolysis and likely not the undesirable effects of pro-inflammatory responses. However, IL-10 and TGF-ß1 were highly expressed in both phases of infection, and their expression was linked to antibody production but not moderation of pro-inflammatory cytokine responses.

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.

Molecular detection and characterization of prevailing Theileria equi genotype in equine from northern India.

Equine piroplasmosis caused by Theileria equi is a febrile, tick-borne disease of equids. However, there is limited literature about the genotyping of T. equi in India. Blood samples were collected from 202 horses and subjected to microscopy and PCR to detect T. equi. Initially, a universal screening primer pair targeting 18S ribosomal RNA genes common for Babesia caballi and T. equi was employed to amplify the DNA of both parasites. Thereafter additional primers were employed for species-specific detection resulting in amplification of approximately 435 bp specific for T. equi. T.equi was detected in 9.9% and 20.79% of horses screened by microscopy and PCR, respectively. The representative samples confirmed positive by PCR were sequenced, submitted to NCBI (OR651254, OR687254, OR685656, OR650830, OR650834), and used for genotype characterization and phylogenetic analysis. Employing Genetool and MEGA X software, the T. equi Indian isolates and across the globe were compared, and the results demonstrated 99.05-100% and 95.86-100% homologies, respectively. All the T. equi Indian isolates belonged to genotype A. Phylogeny based on the EMA-1 gene of five isolates (OR731831, OR731833, OR731829, OR731830, OR731832) were also characterized by sequencing and support the previous findings. Genotypes C and D, as well as genotypes B and E, exhibited lower levels of evolutionary divergence compared to other genotypes. The EMA-1 gene exhibited limited diversity and might not be the most suitable target for assessing variability within T. equi populations. The findings also reveal a significant association (p < 0.01) between T. equi infection and the presence of ticks.

Revisiting the genotypes of Theileria equi based on the V4 hypervariable region of the 18S rRNA gene.

Introduction: Equine theileriosis, an economically important disease that affects horses and other equids worldwide, is caused by a tick-borne intracellular apicomplexan protozoa Theileria equi. Genotyping of T. equi based on the 18S rRNA gene revealed the presence of two, three, four or five genotypes. In previous published reports, these genotypes have been labelled either alphabetically or numerically, and there is no uniformity in naming of these genotypes. The present study was aimed to revisit the phylogeny, genetic diversity and geographical distribution of T. equi based on the nucleotide sequences of the V4 hypervariable region of the 18S rRNA gene available in the nucleotide databases. Methods: Out of 14792 nucleotide sequences of T. equi available in the GenBank™, only 736 sequences of T. equi containing the complete V4 hypervariable region of the 18S rRNA gene (>207 bp) were used in multiple sequence alignment. Subsequently, a maximum likelihood phylogenetic tree was constructed based on the Kimura 2-parameter model (K2+I). Results: The phylogenetic tree placed all the sequences into four distinct clades with high bootstrap values which were designated as T. equi clades/ genotypes A, B, C and D. Our results indicated that the genotype B of Nagore et al. and genotype E of Qablan et al. together formed the clade B with a high bootstrap value (95%). Furthermore, all the genotypes probably originated from clade B, which was the most dominant genotype (52.85%) followed by clades A (27.58%), and C (9.78%) and D (9.78%). Genotype C manifested a comparatively higher genetic diversity (91.0-100% identity) followed by genotypes A (93.2-99.5%), and B and D (95.7-100%). The alignment report of the consensus nucleotide sequences of the V4 hypervariable region of the 18S rRNA gene of four T. equi genotypes (A-D) revealed significant variations in one region, between nucleotide positions 113-183, and 41 molecular signatures were recognized. As far as geographical distribution is concerned, genotypes A and C exhibited far-extending geographical distribution involving 31 and 13 countries of the Asian, African, European, North American and South American continents, respectively. On the contrary, the genotypes B and D exemplified limited distribution with confinement to 21 and 12 countries of Asian, African and European continents, respectively. Interestingly, genotypes A and C have been reported from only two continents, viz., North and South America. It was observed that genotypes A and C, and B and D exhibit similar geographical distribution. Discussion: The present study indicated the presence of only four previously described T. equi genotypes (A, B, C and D) after performing the molecular analyses of all available sequences of the complete V4 hypervariable region of the 18S rRNA gene of T. equi isolates in the GenBank™.

A cross-sectional study on performance evaluation in Italian standardbred horses' real-time PCR-positive for Theileria equi.

BACKGROUND: Inflammatory myopathy and perivasculitis have been recently described in horses with chronic equine piroplasmosis (EP). These alterations may be linked to poor performances. The aims of this study were to evaluate the prevalence for EP in clinically healthy Italian Standardbred (IS) racehorses and to compare laboratory parameters and performance metrics between positive and negative horses. Real-time PCR was applied for the detection of T. equi and B. caballi positivity. Haematology parameters, blood chemistry results, subjective muscle mass scores, and performance metrics were compared between PCR-positive and -negative horses. RESULTS: This cross-sectional study included 120 well-trained IS racehorses and was performed over a two-years period. The prevalence of T. equi was 36.3%, whereas all samples were negative for B. caballi. Red blood cells count, haemoglobin concentration, aspartate aminotransferase, alkaline phosphatase, and gamma-glutamyl transferase activities were significantly higher in PCR-positive horses, whereas blood urea nitrogen, globulin concentration and globulin-to-albumin ratio were significantly lower in PCR-positive horses compared to PCR-negative ones. Nonetheless, all values fell within the physiological range. The best racing time, which was selected as the most representative of the performance metrics at the principal component analysis, was not affected by PCR positivity, the muscle mass score or the training yard. The best racing time was significantly better in horses with a mild or no signs of muscular atrophy, within the PCR-positive group. The muscle mass score was associated with the training yard in PCR-negative horses. CONCLUSIONS: Prevalence of T. equi was high in IS racehorses in southern Italy. The absence of obvious changes in haematological and biochemical parameters, as well as performance metrics in positive horses, highlights the need for specific diagnostic tests to identify chronically infected horses.

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.

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.

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.

THEILERIA EQUI INFECTION IN WORKING HORSES OF PAKISTAN: EPIDEMIOLOGY, MOLECULAR CHARACTERIZATION, AND HEMATOBIOCHEMICAL ANALYSIS.

Theileria equi is 1 of the emerging and prevailing tick-borne hemoprotozoans adversely affecting the equids worldwide, including Pakistan. The current study aimed to investigate the prevalence and molecular characterization of T. equi in working horses (n = 194), the comparative efficacy of different diagnostic tests, associated risk factors, and hematobiochemical analysis. The blood samples of horses were subjected to microscopic examination, cELISA, and polymerase chain reaction (PCR) and the results revealed a prevalence of 9.79, 21.13, and 13.40%, respectively, for T. equi in working horses. The comparison of microscopy and cELISA results with PCR showed that cELISA had higher sensitivity (84.62%), but lower specificity (88.69%) and accuracy (88.14%) in comparison to microscopy (57.69, 97.62, and 92.27%). Molecular characterization of T. equi by phylogenetic analysis revealed a 61% resemblance of study isolates with each other OL662926, OL662925, and 82% similarity with isolate OL662924 while also showing homology with T. equi isolates of South Africa, South Korea, India, Pakistan, and Brazil. The risk factor analysis revealed a significant association (P < 0.05) of tick control status, previous tick history, tick infestation, house hygiene, deworming/vaccination, and the presence of other livestock species with T. equi infection in horses. The hematobiochemical profile revealed a significant (P < 0.05) decrease in red blood cells (RBCs), hemoglobin (Hb), packed cell volume (PCV), white blood cells (WBCs), platelet (PLT), phosphorus, and an increase in lymphocytes, granulocytes, aspartate aminotransferase (AST), glucose, bilirubin, blood urea nitrogen (BUN), and creatinine in T. equi-infected horses. The current study is the first comprehensive report for comparative evaluation of microscopy, cELISA, and PCR, assessment of epidemiological risk factors as well as hematobiochemical variations due to T. equi infection in Pakistan.

Development and evaluation of specific polymerase chain reaction assays for detecting Theileria equi genotypes.

BACKGROUND: Theileria equi causes equine piroplasmosis, an economically significant disease that affects horses and other equids worldwide. Based on 18S ribosomal RNA (18S rRNA sequences), T. equi can be classified into five genotypes: A, B, C, D, and E. These genotypes have implications for disease management and control. However, no conventional polymerase chain reaction (PCR) assays are available to differentiate the genotypes of T. equi. To overcome this limitation, we developed and evaluated PCR assays specific for the detection of each T. equi genotype. METHODS: A pair of forward and reverse primers, specifically targeting the 18S rRNA sequence of each genotype, was designed. The genotype-specific PCR assays were evaluated for their specificity using plasmids containing inserts of the 18S rRNA sequence of each genotype. Subsequently, the assays were tested on 270 T. equi-positive equine blood DNA samples (92 from donkeys in Sri Lanka and 178 from horses in Paraguay). 18S rRNA sequences derived from the PCR amplicons were analyzed phylogenetically. RESULTS: Each genotype-specific PCR assay accurately targeted the intended genotype, and did not produce any amplicons when 18S rRNA from other T. equi genotypes or genomic DNA of Babesia caballi or uninfected horse blood was used as the template. Previous studies employing PCR sequencing methods identified T. equi genotypes C and D in the Sri Lankan samples, and genotypes A and C in the Paraguayan samples. In contrast, our PCR assay demonstrated exceptional sensitivity by detecting four genotypes (A, C, D, and E) in the Sri Lankan samples and all five genotypes in the Paraguayan samples. All the Sri Lankan samples and 93.3% of the Paraguayan samples tested positive for at least one genotype, further emphasizing the sensitivity of our assays. The PCR assays also had the ability to detect co-infections, where multiple genotypes in various combinations were detected in 90.2% and 22.5% of the Sri Lankan and Paraguayan samples, respectively. Furthermore, the sequences obtained from PCR amplicons clustered in the respective phylogenetic clades for each genotype, validating the specificity of our genotype-specific PCR assays. CONCLUSIONS: The genotype-specific PCR assays developed in the present study are reliable tools for the differential detection of T. equi genotypes.

Antiparasitic activity of FLLL-32 against four Babesia species, B. bovis, B. bigemina, B. divergens and B. caballi, and one Theileria species, Theileria equi in vitro, and Babesia microti in mice.

Introduction: FLLL-32, a synthetic analog of curcumin, is a potent inhibitor of STAT3's constitutive activation in a variety of cancer cells, and its anticancer properties have been demonstrated both in vitro and in vivo. It is also suggested that it might have other pharmacological activities including activity against different parasites. Aim: This study therefore investigated the in vitro antiparasitic activity of FLLL-32 against four pathogenic Babesia species, B. bovis, B. bigemina, B. divergens, and B. caballi, and one Theileria species, Theileria equi. In vivo anti-Babesia microti activity of FLLL-32 was also evaluated in mice. Methods: The FLLL-32, in the growth inhibition assay with a concentration range (0.005-50 µM), was tested for it's activity against these pathogens. The reverse transcription PCR (RT-PCR) assay was used to evaluate the possible effects of FLLL-32 treatment on the mRNA transcription of the target B. bovis genes including S-adenosylhomocysteine hydrolase and histone deacetylase. Results: The in vitro growth of B. bovis, B. bigemina, B. divergens, B. caballi, and T. equi was significantly inhibited in a dose-dependent manner (in all cases, p < 0.05). FLLL-32 exhibits the highest inhibitory effects on B. bovis growth in vitro, and it's IC50 value against this species was 9.57 µM. The RT-PCR results showed that FLLL-32 inhibited the transcription of the B. bovis S-adenosylhomocysteine hydrolase gene. In vivo, the FLLL-32 showed significant inhibition (p < 0.05) of B. microti parasitemia in infected mice with results comparable to that of diminazene aceturate. Parasitemia level in B. microti-infected mice treated with FLLL-32 from day 12 post infection (pi) was reduced to reach zero level at day 16 pi when compared to the infected non-treated mice. Conclusion: The present study demonstrated the antibabesial properties of FLLL-32 and suggested it's usage in the treatment of babesiosis especially when utilized in combination therapy with other antibabesial drugs.

Prevalence and genetic diversity of Theileria equi from horses in Xinjiang Uygur Autonomous region, China.

Theileria equi is a tick-borne intracellular apicomplexan protozoan parasite that causes equine theileriosis (ET). ET is an economically important disease with a worldwide distribution that significantly impacts international horse movement. Horses are an essential part of the economy in Xinjiang which is home to ∼10% of all the horses in China. However, there is very limited information on the prevalence and genetic complexity of T. equi in this region. Blood samples from 302 horses were collected from May to September 2021 in Ili, Xinjiang, and subjected to PCR examination for the presence of T. equi. In addition, a Bayesian latent class model was employed to estimate the true prevalence of T. equi, and a phylogenetic analysis was carried out based on the 18S rRNA gene of T. equi isolates. Seventy-two horses (23.8%) were PCR positive. After accounting for the imperfect PCR test using a Bayesian latent class model, the estimated true prevalence differed considerably between age groups, being 10.8% (95%CrI: 5.8% - 17.9%) in ≤ 3-year-old horses and 35.7% (95%CrI: 28.1% - 44.5%) in horses that were > 3 year-old. All T. equi isolates had their 18S rRNA gene (430bp) sequenced and analyzed in order to identify whether there were multiple genotypes of T. equi in the Xinjiang horse population. All of the 18S rRNA genes clustered into one phylogenetic group, clade E, which is thus probably the dominant genotype of T. equi in Xinjiang, China. To summarize, we monitored the prevalence of T. equi in horses of Xinjiang, China, with a focus on the association between age and the occurrence of T. equi by Bayesian modelling, accompanied by the genotyping of T. equi isolates. Obtaining the information on genotypes and age structure is significant in monitoring the spread of T. equi and studying the factors responsible for the distribution.

Diminazene aceturate and imidocarb dipropionate-based combination therapy for babesiosis - A new paradigm.

In the present study, the effect of a combination therapy consisting of diminazene aceturate (DA) and imidocarb dipropionate (ID) on the in vitro growth of several parasitic piroplasmids, and on Babesia microti in BALB/c mice was evaluated using a fluorescence-based SYBR Green I test. We evaluated the structural similarities between the regularly used antibabesial medications, DA and ID, and the recently found antibabesial drugs, pyronaridine tetraphosphate, atovaquone, and clofazimine, using atom pair fingerprints (APfp). The Chou-Talalay approach was used to determine the interactions between the two drugs. A Celltac MEK-6450 computerized hematology analyzer was used to detect hemolytic anemia every 96 hours in mice infected with B. microti and in those treated with either mono- or combination therapy. According to the APfp results, DA and ID have the most structural similarities (MSS). DA and ID had synergistic and additive interactions against the in vitro growth of Babesia bigemina and Babesia bovis, respectively. Low dosages of DA (6.25 mg kg-1) and ID (8.5 mg kg-1) in conjunction with each other inhibited B. microti growth by 16.5 %, 32 %, and 4.5 % more than 25 mg kg-1 DA, 6.25 mg kg-1 DA, and 8.5 mg kg-1 ID monotherapies, respectively. In the blood, kidney, heart, and lung tissues of mice treated with DA/ID, the B. microti small subunit rRNA gene was not detected. The obtained findings suggest that DA/ID could be a promising combination therapy for treating bovine babesiosis. Also, such combination may overcome the potential problems of Babesia resistance and host toxicity induced by utilizing full doses of DA and ID.

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.

Tulathromycin and Diclazuril Lack Efficacy against Theileria haneyi, but Tulathromycin Is Not Associated with Adverse Clinical Effects in Six Treated Adult Horses.

Equine theileriosis, caused by Theileria haneyi and Theileria equi, leads to anemia, exercise intolerance, and occasionally, death. Theileriosis-free countries prohibit the importation of infected horses, resulting in significant costs for the equine industry. Imidocarb dipropionate is the only treatment for T. equi in the United States, but lacks efficacy against T. haneyi. The goal of this study was to assess the in vivo efficacy of tulathromycin and diclazuril against T. haneyi. Fourteen T. haneyi-infected horses were utilized. Six were treated with eight weekly 2.5 mg/kg doses of tulathromycin. Three were treated daily for eight weeks with 2.5 mg/kg diclazuril. Three were pre-treated with 0.5 mg/kg diclazuril daily for one month to determine whether low-dose diclazuril prevents infection. Following infection, the dose was increased to 2.5 mg/kg for eight weeks. Two infected horses remained untreated as controls. The horses were assessed via nested PCR, physical exams, complete blood counts, serum chemistry panels, and cytology. Tulathromycin and diclazuril failed to clear T. haneyi and the treated and control groups exhibited similar parasitemia and packed cell volume declines. To obtain additional safety data on tulathromycin use in adult horses, necropsy and histopathology were performed on tulathromycin-treated horses. No significant lesions were detected.

Diminazene aceturate and imidocarb dipropionate-based combination therapy for babesiosis - a new paradigm.

In the present study, the effect of a combination therapy consisting of diminazene aceturate (DA) and imidocarb dipropionate (ID) on the in vitro growth of several parasitic piroplasmids, and on Babesia microti in BALB/c mice was evaluated using a fluorescence-based SYBR Green I test. We evaluated the structural similarities between the regularly used antibabesial medications, DA and ID, and the recently found antibabesial drugs, pyronaridine tetraphosphate, atovaquone, and clofazimine, using atom pair fingerprints (APfp). The Chou-Talalay approach was used to determine the interactions between the two drugs. A Celltac MEK-6450 computerized hematology analyzer was used to detect hemolytic anemia every 96 h in mice infected with B. microti and in those treated with either mono- or combination therapy. According to the APfp results, DA and ID have the most structural similarities (MSS). DA and ID had synergistic and additive interactions against the in vitro growth of Babesia bigemina and Babesia bovis, respectively. Low dosages of DA (6.25 mg kg-1) and ID (8.5 mg kg-1) in conjunction with each other inhibited B. microti growth by 16.5, 32, and 4.5% more than 25 mg kg-1 DA, 6.25 mg kg-1 DA, and 8.5 mg kg-1 ID monotherapies, respectively. In the blood, kidney, heart, and lung tissues of mice treated with DA/ID, the B. microti small subunit rRNA gene was not detected. The obtained findings suggest that DA/ID could be a promising combination therapy for treating bovine babesiosis. Also, such combination may overcome the potential problems of Babesia resistance and host toxicity induced by utilizing full doses of DA and ID.