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.

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.

Risk factors for equine trypanosomosis and hematological analysis of horses in Paraguay.

Animal trypanosomosis, caused by Trypanozoon trypanosomes (Trypanosoma evansi and T. equiperdum), and Trypanosoma vivax, is endemic to South American countries and has a negative impact on the livestock industry. However, the risk factors for trypanosomosis in Paraguay remain unknown. This study aimed to determine the risk factors for equine trypanosomosis in Paraguay based on a PCR-based molecular survey and individual horse sampling data. In this study, 739 blood samples were collected from horses in 16 departments of Paraguay between August 2019 and November 2020. To elucidate the risk factors for trypanosome infection, the relationship between trypanosome infection status detected by PCR and the location, sex, age, breed of horses, and season of sample collection was analyzed. There were no significant differences in trypanosome prevalence in horses between the eastern and western regions, ages, or breeds of horses in Paraguay. Sex and season were identified as risk factors for trypanosome infection in horses in Paraguay in the current study. These results suggest that the rainy-summer season, when vectors increase in number and their blood-sucking activity, could be the most important risk factor for trypanosome infection in Paraguay horses. Preventive measures and treatments should be developed to address these factors.

First molecular survey of animal trypanosomes in Paraguayan horses.

Despite the epidemic situation of animal trypanosomosis caused by Trypanosoma evansi, Trypanosoma equiperdum and Trypanosoma vivax in South American countries, there are no reports for the prevalence of animal trypanosomes in Paraguay. In this study, 408 blood samples were obtained from apparently healthy horses from sixteen departments of Paraguay, for routine medical check-up from August to September 2019, and a polymerase chain reaction (PCR)-based cross-sectional study was carried out to identify trypanosome prevalence. The prevalence of Trypanozoon (T. evansi and T. equiperdum) and T. vivax was 7.11% (29/408) and 26.23% (107/408), respectively. Mixed infections were detected in 4.90% (20/408) of the samples. Some of the selected trypanosome positive samples were confirmed as T. vivax and T. evansi Type A by sequence analysis of the internal transcribe spacer region and RoTat1.2 variant surface glycoprotein gene, respectively. In conclusion, we found higher prevalence of T. vivax than Trypanozoon in Paraguayan horses. However, the genotypic variation should be verified in further studies.