Detection of tropical bovine theileriosis by polymerase chain reaction in cattle (original) (raw)
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Tropical biomedicine, 2014
Bovine tropical theileriosis caused by Theileria annulata is a tick-borne disease associated with high morbidity and mortality in the livestock. The conventional method of diagnosis is by the demonstration of the parasite stages by microscopic examination. This method suffers from low sensitivity, making it even more difficult to detect piroplasms in the carriers. PCR based assays are known to be more sensitive. The present study was undertaken to detect and quantify T. annulata in the blood of clinically infected and carrier animals using a quantitative PCR protocol targeting the gene encoding the major merozoite piroplasm surface antigen Tams 1. A total of 116 samples were collected from infected as well as apparently healthy cattle and buffaloes. Of these, 74 samples (63.79%) were positive for T. annulata by real-time PCR, including the 15 samples that were positive by Giemsa staining. The parasite load ranged from 1.39 x 10(6) to 3.35 x 10(9) and 0.35 x 10(6) to 2.83 x 10(7) ml(...
Journal of Parasitic Diseases, 2013
Theileria annulata, a protozoan parasite of cattle is causes tropical theileriosis. Polymerase chain reaction (PCR) was used to assess the presence and the frequency of T. annulata infection in blood samples obtained from carrier cattle in Kerman, Southeast of Iran. Blood samples were collected in citrate solution from 150 native cattle with mean age of 1 year which selected randomly. Primarily, a thin layer smear was prepared from their ear sublime vein blood and was fixed with methanol and stained with Giemsa dye. Blood smears were examined for the presence of parasites, and blood samples were analyzed by PCR. Piroplasmic forms of T. annulata were seen in 16 of 150 (10.66 %) by examination the blood smears with light microscope, whereas 68 of 150 (45.33 %) cattle were positive by PCR method. All animals that were positive by blood smears were also positive by PCR. Difference between these methods was significant (P \ 0.05). Our results demonstrate that this PCR assay in diagnosing T. annulata parasites in carrier cattle is more sensitive than method of smear preparation and can be used in epidemiological studies.
Journal of Parasitic Diseases, 2020
Theileria annulata (T. annulata) is a tick-borne apicomplexan parasite that affects bovine. It is endemic in many tropical and subtropics areas, including Odisha, India. The objective of this study is to identify T. annulata infection in the peripheral blood of cattle as a biological sample by conventional PCR (cPCR) and quantitative PCR (qPCR). The phylogenetic analysis was done using the T. annulata merozoite surface antigen (Tams 1) gene. Out of 552 samples of examined blood smears by microscopy, 454 (82.24%) animals were positive for Theileria species. Out of 454 samples, 96 samples were further examined by both cPCR and qPCR, 52 samples (54.16%) were found positive for T. annulata in both PCR methodologies. Phylogenetic analysis revealed that T. annulata Odisha isolate was closely related to T. annulata Uttarakhand, India isolate (KM061799) and Hyderabad, India isolate (MK034702) with Nucleotide sequence identity 95.36%, 95.25%, respectively. This is the first study to detect T. annulata by qPCR in Odisha and supported that both PCR techniques were equally effective for the detection of Tams 1 gene of T. annulata in cattle's blood.
Purpose Bovine theileriosis caused by Theileria annulata is a tick-borne disease of livestock animals in tropical and subtropical regions of the world. The diagnosis of theileriosis is usually carried out by blood smear staining technique, which is not sufficiently sensitive to detect the piroplasms in the carrier animals. The objective of this research was comparison of efficacy of routine microscopic and PCR methods for diagnosing of theileriosis. Methods In this study a total number of 281 blood samples were obtained from healthy cattle in different range of ages and in native and crossbreed cattle in northwest of Iran in summer 2018. Samples were detected by Giemsa staining and microscopic observation and PCR method based on using the specific primers from the major merozoite-piroplasm surface antigen sequence of T. annulata (Tams-1) gene. Results In microscopic method (8.89%) samples were positive. However, the PCR detected 108 samples (38.43 %) positive for T. annulata. In positive samples of cattle, highest prevalence was recorded in cattle 2-5 years' old (22.4%). This differences in age results was significant. Out of 108 samples that were positive by PCR, 45(41.66%) were native cattle and 63(58.33%) were crossbreed cattle whereas, this difference was not significant. Conclusions Our results suggest that the PCR screening tests are sensitive and accurate enough to be used as diagnostic tool for detection of theileriosis in cattle. Moreover, results propose that high percentage of carriers in cattle of Iran-northwest indicates a high potential risk for infestation of healthy animals and vectors of the disease.
Indian Journal of Animal Research, Volume 58 Issue 3: 490-494 (March), 2024
Background: Theileria annulata is predominant and of utmost economical importance tick borne pathogen of bovines in the region which is routinely diagnosed based on the microscopic examination of Romanowsky stained thin blood smears. Present study was intended to evaluate and analyze the detection efficacy of a commercial polymerase chain reaction kit based assay in comparison to conventional PCR assay and classical microscopy for detection of T. annulata from blood samples of bovines from Punjab state. Methods: In this comparative study 360 bovine blood samples from various districts of agro-climatic zones in Punjab were first screened for T. annulata by Giemsa-stained thin blood smear (GSTBS) examination. The same panel of blood samples was tested for T. annulata by a commercial PCR kit Bovi-TheiDX Theileria annulata (Genext Genomics) (PCR1) and established conventional PCR assay targeting merozoite piroplasm surface antigen (Tams1) gene of T. annulata (PCR2). Result: Out of 360 samples screened, positivity of T. annulata by GSTBS was found to be 12.5% (45/360) with a sensitivity of 37.20% and specificity of 96.00% when compared with commercial kit (PCR1), the difference was statistically significant (p<.0001). The detection prevalence by PCR1 29.70% (107/360) and PCR2 assay 33.90% (122/360) showed significant (p<.0001) difference. The conventional PCR targeting the Tams1 gene (PCR2) was found to be more sensitive (84.6%) (p<0.001) than PCR1 (69.7%).
Journal of Parasitic Diseases, 2015
Theileria annulata is common in tropical and subtropical regions especially in Iran and causes great economic losses in cattle industry. In Iran the epidemiological aspects of bovine theileriosis in different breeds of cattle is poorly understood. The aim of present study is comparison of the number of T. annulata carriers in the two major cattle breeds (Holstein-Friesian and Sistani) in Sistan of Iran by giemsa and polymerase chain reaction (PCR) methods. During winter 2013, 160 native cattle, from the two major breeds in Sistan, with the mean age of more than one year and without typical clinical symptoms of theileriosis were selected. At first, a thin layer smear was held from their ear sublime vein blood for Giemsa staining method. In order to do PCR assay, jugular vein blood sample of each cow was taken. The PCR employs primers specific for the 721-bp gene fragment encoding the 30-kDa major merozoite surface antigen of T. annulata. By PCR method, 38 (47.5 %) Holstein blood samples and 22 (27.5 %) Sistani blood samples had DNA of T. annulata and considered positive (The correlation was significant at values of P \ 0.05). By checking 160 blood smears with light microscope and lens 9 100, only 10 samples (6.25 %) were positive for T. annulata. Statistical comparison between PCR and smear method showed that the PCR method is more sensitive and accurate in comparison to Giemsa staining method to diagnose the asymptomatic carriers of T. annulata.
Parasite, 2012
The present study was carried out to determine the prevalence of Theileria annulata in large ruminants from two districts, Peshawar and Kohat, in Khyber Pukhtoon Khwa (Pakistan). Blood samples were collected from 95 cattle. Data on the characteristics of animals and herds were collected through questionnaires. No significant risk factors were found associated with the spread of tropical theileriosis in the study area. Two different parasite detection techniques, PCR amplification and screening of Giemsa stained slides, were compared and it was found that PCR amplification is a more sensitive tool (33.7 % parasite detection), as compared to smear scanning (5.2 % parasite detection) for the detection of Theileria annulata. 32 out of 95 animals, from both districts, produced the 721-bp fragment specific for Theileria annulata. Résumé : COMPARAISON DE DEUX TECHNIQUES DE DÉTECTION DE THEILERIA ANNULATA CHEZ DES BOVINS DE DEUX DISTRICTS DE LA PROVINCE DE KHYBER PUKHTOON KHWA (PAKISTAN) Une étude a été menée afin de déterminer la prévalence de Theileria annulata chez 95 bovidés de deux districts (Peshawar et Kohat) de la province de Khyber Pukhtoon Khwa au Pakistan. L'âge des bovins, la présence de tiques chez ceux-ci, ainsi que la présence de tiques chez les chiens du troupeau ne sont pas des facteurs de risque impliqués dans la diffusion de la theilériose dans la zone étudiée. La comparaison de deux techniques de détection du parasite (PCR et frottis sanguin coloré au Giemsa) a montré que la PCR était plus sensible (33,7 %) que le frottis (5,2 %).
Detection of Theileria annulata in cattle and vector ticks by PCR using the Tams1 gene sequences
Parasitology, 2000
A Polymerase Chain Reaction (PCR) and Southern blot hybridization for the detection of Theileria annulata are described. The PCR used primers amplifying a 785 base-pair fragment of the T. annulata gene which encodes the 30 kDa major merozoite surface antigen, Tams1. The sensitivity of the PCR in bovine blood was 1 piroplasm in 1 μl of blood. T. buffeli, T. parva, Babesia bigemina, B. bovis and B. divergens were not detected. The PCR detected down to 1 infected acinus/tick in resting and partially fed adult Hyalomma anatolicum anatolicum ticks and was negative for T. lestoquardi and T. equi, which are transmitted by this tick but are not infective to cattle. The specificity of the PCR was checked using 30 stocks of T. annulata, all of which were detected. Three stocks of T. lestoquardi, 4 of T. equi and 1 each of T. buffeli, T. parva, B. bigemina, B. bovis and B. divergens were used to ascertain there were no cross-reactions. A nested PCR using separate primers for the first reaction...
Journal of Parasitic Diseases, 2014
Tropical theileriosis is a progressive bovine lymphoproliferative disease caused by the intracellular protozoan parasite Theileria annulata. In this study 138 blood samples and 289 ticks were collected and examined from cattle that belonged to 10 randomly selected flocks. The Tbs-S/Tbs-A primer set was used for PCR amplification of Theileria spp. and the Ta-S/Tbs-A specific primer set was used in semi-nested PCR technique for detection of T. annulata. Blood smears of each case were examined by Giemsa staining method. The semi-nested PCR accurately revealed 22 (15.94 %) positive samples; whereas Giemsa staining method could detect 15 (10.86 %) out of 138 blood samples. The examination of 289 ticks by semi-nested PCR revealed that, 32.86 % of Hyalomma anatulicum anatulicum, 26.47 % of Hyalomma anatulicum excavatum and 22.42 % of Hyalomma asiaticum asiaticum, were infected with T. annulata. The results suggest that H. anatulicum anatolicum may play a major role in transmission of T. annulata infection in Iran. The results indicated that the Giemsa staining method, having low sensitivity, while the semi-nested PCR technique can be used as a gold standard method for this purpose.