Quantitative PCR detection of Theileria equi using laboratory workflows to detect asymptomatic persistently infected horses (original) (raw)
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Veterinary …, 2008
We developed a TaqMan real-time polymerase chain reaction (PCR) assay for the quantitative detection of Theileria equi from the in vitro-cultured parasite and field blood samples collected from horses living in Ghana and Brazil. The detection limit for the assay was determined to be 1.5 parasites/ml per sample, and the quantitative capacity was demonstrated using the in vitro-cultured parasite. For field applications, the real-time PCR assay was compared to a previously established nested PCR assay used as the gold standard for the real-time PCR assay. Of 65 field blood samples, 46 samples were T. equi-positive in the nested PCR assay, while the real-time PCR assay also detected the parasite in all 46 of the nested PCR-positive samples but did not detect T. equi in the remaining 19 negative blood samples. This quantitative real-time PCR assay provides a valuable tool for fast laboratory diagnostic assessment of T. equi infection in horses.
Brazilian Journal of Veterinary Research and Animal Science
This study was designed to detect equine piroplasmosis using the molecular technique in Al-Najaf province during the season that showed an increment in tick activities. Blood samples were collected from 110 horses with more than two signs of piroplasmosis. After DNA extraction, the product was examined by a polymerase chain reaction to amplify 18SrRNA. The results showed that the overall percentage of equine theileriosis was 38.18%. According to gender, the percentage of infection was 43.48% and 29.27% in females and males, respectively. Significant variations appeared between infected horses according to age, and the percentage of infection was 50% and 35.22% in less than 2 years and more than 2 years age, respectively. Moreover, the percentage of infection was 62.5% and 19.35% in animals with and without acariasis, respectively. Significant variations were also seen in equine theileriosis according to geographical areas, and the higher percentage was reported in Hera district (60....
The prevalence of Theileria equi infection was studied in 301 equine samples (133 donkeys and 168 horses) from Giza and Cairo governorate using microscopic examination (ME), nested (nPCR), competitive ELISA (cELISA) and indirect ELISA (iELISA). The used antigen in iELISA was prepared from blood of naturally infected splenectomized donkey at the peak of parasitemia In ME, the parasite was detected in 79 (26.2%) equine blood samples; 33 donkeys and 46 horses with a prevalence rate (24.8% and 27.4%), respectively. The prevalence rate in equine samples using iELISA was (33.5%) from which 71 donkeys and 30 horses were infected (53.4% and 17.9%), respectively. The T. equi antibodies were detected with cELISA in 60 (19.9%) equine serum samples, where 34 donkeys and 26 horses with a prevalence rate (25.6% and 15.5%), respectively. The nPCR based on the T. equi merozoite antigen gene (EMA-1) allowed the visualization of species-specific amplified product in 171 (56.8%) equine blood samples, 67 donkeys and 104 horses with a prevalence rate (50.4% and 61.9%), respectively. Approximately 229 bp of the ema-1 gene from 3 Eqyption samples were sequenced and BLASTN analysis confirmed all sequences to be merozoite surface protein genes, with an identity of 100% to previously published Babesia equi merozoite antigen-1 ema-1 gene reference sequence (our GenBank Accession number KX262963). Statistical analysis using Chi square indicated significant differences (P< 0.05) between ME and nPCR; microscopic examination and cELISA and between nPCR and cELISA on the detection of parasite carriers. In conclusion, the most sensitive technique in diagnosis of T. equi infection is nPCR, followed by cELIZA, iELISA and ME. The combination of ELISA and PCR was recommended for detection of acute and chronic stage.
Diagnosis of Theileria equi infections in horses in the Azores using cELISA and nested PCR
Ticks and Tick-borne Diseases, 2013
Equine piroplasmosis is a tick-borne disease of equids that is often caused by the parasite Theileria equi. We applied competitive ELISA (cELISA) and nested PCR diagnostic methods to detect this parasite in horses by screening 162 samples from mainland Portugal where the parasite is endemic, and 143 from the Azores representing both native and imported horse populations. We found that 2.8% of the Azorean samples tested positive exclusively by cELISA, 1.4% tested positive only by nested PCR, and 9.1% tested positive using both tests. Samples from the native Terceira Pony population were negative for both tests. The parasite was more prevalent in samples from mainland Portugal when both test methods were considered (9.3% positive exclusively by cELISA, 1.9% positive exclusively by nested PCR, and 16.7% positive for both tests). To our knowledge, this is the first time that molecular techniques have been used to detect T. equi in the Azores and the first report of this parasite in the archipelago. Based on this study, it is clear that the import of horses into the Azores and the movement of horses between the islands must be controlled to reduce the risk of new infections, contributing to the protection of native horse populations such as the Terceira Pony population.
Diagnosis and prevalence of Theileria equi horses in western Mexico by nested PCR
Parasitology International, 2017
Theileria equi infection prevalence was calculated from 1,000 blood samples obtained from apparently healthy horses in western Mexico. Samples were sent to the Animal Biotechnology Laboratory of the University of Guadalajara (Mexico) for T. equi diagnosis. Nested polymerase chain reaction (nPCR) was used as a diagnostic method to detect pathogen DNA. Using primers for the merozoite antigen-1 (EMA-1) gene, 19.70 ± 2.47% of the horses (95% CI, 17.23-22.17%) tested positive for T. equi. There was no significant association between gender and T. equi infection. However, prevalence was higher among stabled horses (25.81%) than that among grazing horses (15.02%). The positivity rate was also higher among Quarter Horse (24.70%), Lusitano (35.90%), and Costa Rican Saddle Horse (47.37%) breeds than that among the other seven breeds investigated in this study. The percentage of T. equi infection was higher among adult horses (≥ 4 years old, 25.05%) than that among colts and fillies (2-4 years old, 15.48%), yearlings (1-2 years old, 10.49%), and foals (< 1 year old, 10.34%). This is the first study of T. equi infection prevalence among horses in Mexico by nPCR. The results indicate that the equine piroplasmosis (EP) caused by T. equi is enzootic in western Mexico.
Serological and molecular detection of Theileria equi infection in horses in Hungary
Veterinary Parasitology, 2013
The prevalence of Theileria equi infection was studied in 324 healthy horses from 27 farms in Hungary with cELISA and IFAT and the blood samples of 101 horses selected randomly were also examined by PCR. The results indicate that there are many stud farms where one or more horses are infected with T. equi. Among 27 farms 17 (67.9%) were found to have seropositive horses. The seroprevalence of theileriosis among the tested stud farms ranged between 0 and 100%. No marked differences were found in seropositivity between geographical areas. The overall prevalence of positive samples was 32.0% with cELISA as well as with IFAT. The results obtained with cELISA and IFAT in this study had the strongest agreement, except for 9 samples in which the two serological tests gave different results. The prevalence of infection among 101 horses was 49% with PCR. All 14 sequenced samples were found by BLAST analysis to be closest to the T. equi 18S rRNA gene sequences in GenBank with a similarity of ≥99%.
Sequence heterogeneity in the 18S rRNA gene in Theileria equi from horses presented in Switzerland
Veterinary Parasitology, 2016
A reverse line blot (RLB) hybridization assay was adapted and applied for equine blood samples collected at the animal hospital of the University of Zurich to determine the presence of piroplasms in horses in Switzerland. A total of 100 equine blood samples were included in the study. The V4 hypervariable region of the 18S rRNA gene was amplified by polymerase chain reaction and analyzed using the RLB assay. Samples from seven horses hybridized to a Theileria/Babesia genus-specific and a Theileria genusspecific probe. Of these, two hybridized also to the Theileria equi-specific probe. The other five positive samples did not hybridize to any of the species-specific probes, suggesting the presence of unrecognized Theileria variants or genotypes. The 18S rRNA gene of the latter five samples were sequenced and found to be closely related to T. equi isolated from horses in Spain (AY534822) and China (KF559357) (≥98.4% identity). Four of the seven horses that tested positive had a documented travel history (France, Italy, and Spain) or lived abroad (Hungary). The present study adds new insight into the presence and sequence heterogeneity of T. equi in Switzerland. The results prompt that species-specific probes must be designed in regions of the gene unique to T. equi. Of note, none of the seven positive horses were suspected of having Theileria infection at the time of presentation to the clinic. Clinicians should be aware of the possibility of equine piroplasma infections outside of endemic areas and in horses without signs of piroplasmosis.
Iranian Journal of Parasitology, 2017
Background: Theileria equi is a tick borne protozoan parasite which causes piroplasmosis among equines worldwide. The present study was aimed to determine seroprevalence of T. equi in donkeys, horses, and mules from two equine populated districts (Peshawar and Charsadda) of Khyber Pakhtunkhwa (KPK), Pakistan. Methods: A total of 393 equine (195 horses, 194 donkeys and 4 mules) serum samples were collected from five and four randomly selected localities in Charsadda (n = 193) and Peshawar (n = 200), respectively. The presence of antibodies to T. equi was determined using a commercially available competitive enzyme-linked immunosorbent assay. Results: An overall seroprevalence of 38.2% (n=150) was observed among all the tested animals suggesting a higher seropositivity among equids belonging to Charsada (50.3%) as compared to Peshawar (27.5%). Binary logistic regression analysis revealed that being a donkey (OR 2.94), having tick infestation (OR 4.32), history of voiding red (i.e., bl...