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Comparison of Primer Sets for Amplification of 30 kDa Merozoite Surface Antigen of Bovine Theileriasis
Abstract
The efficiency of two primer sets that used for diagnosis of Theileria annulata infection in cattle were evaluated in 28 cows that showed clinical signs of theileriosis. Both conventional method and Polymerase Chain Reaction (PCR) were used to confirm theileria infection. For PCR, two primer sets that amplify the 30 kDa major merozoite surface antigen gene were used. Only 12 cows (42.85%) were positive for theileria infection with blood film. Higher number of cows were diagnosed positive by using PCR, the number of positive cows using primer set one (N516/N517) was 21 cows (75%) and by using primer set two (TarnslF/TspmlR) was 27 (96.42%). It could be concluded that primer set TamslF/TspmlR is the first choice when diagnosis of Theileria annulata is decided.
... Significant difference was observed in the count of RBC in all the three groups; however, it was more severe in case of parasitologically positive cases resulting into severe anaemia due to T. annulata infection (Fig. 4). Omer et al. (2002) and Ellah and AL-Hosary (2011) also observed similar changes in animals naturally infected with T. annulata infection. The marked decrease in the RBC count causing anaemia, might be due to intra-erythrocytic piroplasms (Preston et al. 1992) and/or an autoimmune reaction (Hooshmand-Rad 1976), although it may also be attributed to an erythro-phagocytosis rather than a parasite-induced lysis (Boulter and Hall 2000). ...
Bovine tropical theileriosis, caused by Theileria annulata,is one of the economically important fatal tick borne haemoprotozoandiseases of dairy animals. The aim of present investigation was to map the distribution of T. annulatain bovines of Punjab stateof India in relation to various risk factors including age, sex of animals, location and management of farms. In a cross sectionalstudy, a total of 1278 blood samples were randomly collected from twenty districts falling in five major agro-climatic zones ofPunjab. All the samples were screened by blood smear examination followed by polymerase chain reaction targeting SSU rRNAgene for Theileriaspp. PCR positive samples (n = 386) for Theileriaspp. were then analyzed for T. annulataby amplificationof Tams1gene.Overall prevalence of T. annulatawas found to be 29.26% in Punjab, with highest in western Zone (40.49%, 95% CI = 35.57–45.41) and lowest in submountain zone (18.90%, 95% CI = 13.73–24.06). The propensity of incidence of T. annulata was found to be highest in cross bred cattle (32.40%, 95% CI = 29.87–34.94), followed by indigenous cattle (19.64%,95% CI = 10.67–28.61) and buffaloes (19.2%, 95% CI = 14.99–23.41). Between the two sexes, incidence of T. annulatawashigher in female animals. Calves less than 6 months of age were found to be more prone to theileriosis.
... Detection of T. annulata was carried out using primers specific for Tams1 gene and the PCR assay found to be sensitive and specific (Haibibi et al. 2007). Mahmoud et al. (2011) studied, the efficiency of two primer sets, primer set one (N516/N517) and primer set two (Tams1F/ Tspm1R) to amplify the 30 kDa major merozoite surface antigen gene for the diagnosis of T. annulata infection in cattle, and concluded that primer set Tams1F/Tspm1R is the first choice when diagnosis of T. annulata is concerned. This justifies the reason for using primers specific for the Tams1 gene in our study, and also shows the efficacy of PCR technique for confirmational diagnosis of theileriosis. ...
Bovine tropical theileriosis caused by Theileria annulata is a tick-borne disease of great economic importance in tropical and subtropical regions of the world. The present study was undertaken to detect theilerosis in cattle and buffaloes by polymerase chain reaction (PCR). 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. In this study, a total of 116 samples were collected from infected as well as apparently healthy cattle and buffaloes. Screening of blood smears by Giemsa staining detected 15 samples (12.93 %) positive for Theileria piroplasms out of 116 samples. However, the PCR based screening using the specific primers from the major merozoite-piroplasm surface antigen sequence of T. annulata (Tams1) gene detected 74 samples (63.79 %) positive for T. annulata which included 59 samples found negative by Giemsa staining. Our study suggests that the PCR based screening is more sensitive and accurate method for diagnosis of tropical theileriosis in cattle and buffaloes.
Bovine theileriosis, caused by Theileria annulata, presents a significant economic challenge to India’s livestock industry. This study investigates the molecular epidemiology of T. annulata in bovine populations from Haryana, India, focusing on the genetic diversity of the Tams1 gene (785 bp).
The prevalence of T. annulata was evaluated in 800 blood samples using microscopy and PCR techniques, specifically targeting SSU rRNA and Tams1 genes. Genetic diversity among T. annulata isolates was analyzed through DNA sequencing and phylogenetic studies. Additionally, the impact of various risk factors on T. annulata infection was assessed.
Microscopy revealed that 29.25% (117/400) of cattle and 2.75% (11/400) of buffalo were detected positive for Theileria spp. in Giemsa-stained blood smears. The Theileria genus-specific primers amplified a 1098 bp fragment of the SSU rRNA gene in 39.5% (158/400) cattle and 6% (24/400) buffalo. The T. annulata-specific PCR targeting the partial Tams1 gene revealed a prevalence rate of 37% (148/400) in cattle and 3.5% (14/400) in buffalo. A genetic analysis of the Tams1 gene in 389 sequences, including 14 T. annulata isolates from Haryana (8 from cattle, 6 from buffaloes), revealed significant variation within Indian T. annulata parasites. Analysis of Tams1 gene sequences (389 from 16 countries) revealed 35 haplotypes globally, with four identified from the 14 isolates sequenced in this study. Nucleotide homology among 14 northern Indian isolates ranged from 89.46–100%, compared to a broader range of 78.42–100% when global sequences were included. Among 35 haplotypes, Hap_1 is the most dominant and shows geographic clustering. Globally, low genetic distance (Fst < 0.15) and high gene flow (Nm > 1) were observed among the five populations (South Asia, East Asia, West Asia, Europe, and Africa), suggesting minimal genetic differentiation among T. annulata populations. Negative values in Tajima’ s D (-1.21941) and Li’s F (-2.97801) tests suggest recent population expansion. Risk factors such as age, sex, and host species are significantly associated with T. annulata infection.
This study offers comprehensive insights into T. annulata genetic diversity, population structure, and haplotype networks using the Tams1 gene.
Bovine theileriosis caused by Theileria annulata ( T. annulata ) that result in high mortality and financial losses for the livestock industry in Egypt. For this study, fifty cattle were utilized. Whole blood samples were collected for laboratory analysis. Giemsa-stained blood smears were employed to detect Theileria infection. Polymerase chain reaction (PCR) was used to evaluate various target genes like 30-kDa and Cyto B of T. annulata . Nine (18%) samples tested positive for piroplasm of Theileria by microscopic examination of blood smear. Twenty one (42%) of the analyzed samples tested molecularly positive depended on 30-kDa gene, while 10 (20%) samples were positive based on Cyto B gene. In our study, we carried out DNA sequencing and phylogenetic analysis of T. annulata using the Cyto B gene. The PCR products' phylogenetic analysis of the Egyptian strain of T. annulata (Assiut) showed nucleotide identity ranging from 96.16–98.92% with T. annulata strains of various governorates (Sharkia and Qulyubia) of Egypt, Sudan, Tunisia, Turkey, and India. The isolates obtained were found to be closely clustered with an isolate from Sudan (accession number LC431533). We identified thirty point mutations at the amino acids sequences. There was substantial variance (P < 0.05 and P < 0.01) between age and sex of tested cattle, respectively and percentages of T. annulata infection. The data obtained from our study in characterization of Cyto B gene of T. annulata in Assiut Governorate suggest that the Cyto B gene may be used as a genetic marker to identify resistant isolates of T. annulata .
Bovine theileriosis is considered as a major barrier to improving output from India's vast bovine population. A one-year study was conducted to determine the hematobiochemical alterations and Clinical signs in Theileria infected cattle and buffaloes in Indore district from July 2022 to June 2023. In a group of 480 dairy animals, 72 tested positive, resulting in an incidence rate of 15%. The incidence of theileriosis was found to be significantly different (p<0.01) between cattle (21.25%) and buffaloes (8.75%). Theileria-infected cattle showed significant declines (p<0.01) in Hb, PCV, TEC, lymphocytes, and MCHC, while total leukocyte count increased (p<0.01). Basophils and MCV did not change significantly. Infected buffaloes had increased TLC, basophils and decreased mean Hb, PCV and lymphocytes. No changes were found in eosinophils. Significant increases in ALT, AST, creatinine, bilirubin, and BUN were found (p<0.01) in dairy animals. Predominant clinical signs observed in animals suffering from theileriosis were reduced milk yield
To determine the presence and distribution of bovine theileriosis in the North Central region of Algeria, 358 DNA samples and 359 blood smears were analyzed from nine provinces. Theileria DNA extracted from cattle blood was amplified by fluorescence resonance energy transfer polymerase chain reaction (FRET-PCR). Blood smears were examined for Theileria piroplasms by microscopical examination (ME) of Giemsa-stained slides. While microscopical identification revealed only 42 animals being infected with Theileria piroplasms, PCR-positive amplification using Theileria genus-specific primers was obtained from 132 Theileria spp. (P < 0.0001). Among the 132 positives, 108 animals (81.8 %) were found positive of Theileria annulata, while 24 (18.2 %) were found positive for Theileria sp. (P < 0.0001). However, melting curve analysis of these latter samples revealed the presence of two different peaks, 51.5 ± 0.5 °C corresponding to Theileria sp1 and 52.5 ± 0.5 °C for Theileria sp2. Cloning and sequencing of Theileria sp1 and Theileria sp2 using the Cox primers indicated that these species are very closely related to Theileria buffeli. There is a highly significant difference in the distribution of theileriosis between different provinces (P < 0.0001). This disparity between provinces is probably due to differences in tick contact, influenced by the subhumid bioclimatic gradient and differences in agricultural land use.
During March and April 2007, 140 native cows with the mean age of more than one year were selected randomly and their age and sex were registered in the related papers. Primarily, a thin layer smear was prepared from their ear sublime vein blood and was fixed with methanol and then it stained with Gimsa dye. Also, 9 mL blood from the jagular vein of the same cow was taken and collected in autoclaved tubes (containing 1 mL of 3.2% buffered citrate solution) conversely for extracting their DNA for PCR. Two primers were used: N516/N517 belonged to a huge gene (30 kDa) which is responsible for coding the surface antigen of Theileria annulata merozoite. By detection of 140 prepared blood smear with light microscope and lens 100, piroplasmic forms of Theileria annulata were seen in 12 smear (8.57%). By PCR method, 56 DNA of Theileria annulata was separated from 140 samples (40%). Statistical comparison of PCR and smear methods in diagnosing of Theileria annulata carriers can explain a significant difference between these two methods (p<0.05). The results of this study shows that sensitivity and accuracy of PCR method in diagnosing of Theileria annulata carriers is more than common method of smear preparation and can be used in epidemiological studies for the sake of controlling, prevention and determining of immunological condition of cows in region.
The isolation of Theileria annulata-infected lymphocytes using blood from an animal suffering from Mediterranean theileriosis as a source of parasites is described. The present work reports the first isolation and establishment in in vitro culture of a T. annulata-infected cell line from southwestern Europe, where Mediterranean theileriosis causes important economic losses, especially in southern Spain. The parasite was identified by staining of cells from culture with Giemsa, by immunofluorescent antibody techniques (IFAT), and by isoenzyme characterization. The possibility of using this T. annulata-infected lymphoblastoid cell line to obtain an antigen for diagnosis of Mediterranean theileriosis by IFAT and to develop a tissue-culture vaccine against this disease in our geographic area shows the significance of this isolation and culture.
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 microl 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 and the same primers for the second reaction detected T. annulata to the same sensitivity and specificity in saponin-extracted DNA samples stored for long periods at -20 degrees C.
The recombinant surface protein of Theileria annulata (TaSP) was used in the standardization and validation of an enzyme linked immunosorbent assay (ELISA) for the detection of circulating antibodies against tropical theileriosis. ELISA data were expressed as the percentage positivity (PP) of the reactivity of an internal positive control. A total of 50 sera samples from a disease-free area were used for the calculation of the cut-off value which served as a threshold between the positive and the negative sera samples. This was determined as the mean PP plus two standard deviations or the twice the mean PP of the results obtained with these negative samples. The obtained thresholds were 17.8% and 18.3%, respectively. Accordingly, the reactivity of 140 field sera samples collected at random from an area known to be endemic for tropical theileriosis in Sudan was determined as PP values which were then compared to the results obtained using the indirect fluorescence antibody test (IFAT) from the same samples. Both tests showed a high degree of correlation. The TaSP-ELISA had a sensitivity of 99.1% and specificity of 90.47% when taking the IFAT as a reference test. Our test has proved its suitability for the diagnosis of tropical theileriosis and could be used in serological surveys to map out the prevalence of the disease or to monitor vaccination efficiencies in disease-free populations.
A total of 2388 cattle and 442 shelters, from two provinces (Elazig and Malatya) endemic for tropical theileriosis in the east of Turkey, were studied for Hyalomma tick populations from July 1993 to July 1995 in Elazig and from May 1998 to January 1999 in Malatya. Four thousand five hundred and eighty one of 7455 Hyalomma ticks were collected from cattle, the other ticks (2874) were collected from shelters. All of the ticks collected from shelters were Hyalomma anatolicum anatolicum. Two thousand eight hundred and ninety five (63.1%) of 4581 Hyalomma ticks collected from cattle were H.a. anatolicum. 23.8% (1047/4581), 11.7% (536/4581) and 0.6% (3/4581) of Hyalomma ticks were Hyalomma anatolicum excavatum, Hyalomma detritum and Hyalomma marginatum marginatum, respectively. A total of 5909 Hyalomma adult ticks collected from cattle (3362/5909) and shelters (2447/5909) were dissected and salivary glands were stained with Methylgreen/Pyronin method. Thousand one hundred and fifty (46.9%) of 2447 H.a. anatolicum collected from shelters and 412 (19.1%) of 2147 H.a. anatolicum collected from cattle were positive for Theileria infection. Twenty (2.4%) of 820 H.a. excavatum and 23 (4.6%) of 495 H. detritum collected from cattle were positive. The mean number of infected acini per infected male and female ticks collected from cattle were 11.3 and 22.4 in H.a. anatolicum, 4 and 6.8 in H.a. excavatum, 17.9 and 18.3 in H. detritum, respectively. In H.a. anatolicum collected from shelters, the above rates were 11.8 and 17.6 in male and female ticks, respectively. The prevalence and intensity of Theileria infection was greater in female ticks than in males.
A herd-based study was carried out in Central Equatoria State, Southern Sudan, to study epidemiological aspects of tick-borne diseases. Six herds of cattle situated in three different locations were selected and investigated every 3 months during the year 2005. Blood smears for Giemsa staining and blood spots on filter paper for deoxyribonucleic acid extraction were collected from 600 apparently healthy indigenous cattle. A total of 69 (11.5%) samples showed the presence of piroplasms in Giemsa-stained blood smears, and polymerase chain reaction increased the detection limit to 297 (49.5%). Using reverse line blot, it was possible to detect and differentiate eight different piroplasms namely, Theileria parva (71.2%), Theileria mutans (73%), Theileria velifera (45.3%), Theileria taurotragi (2.7%), Theileria buffeli (0.5%), Theileria annulata (0.2%), Babesia bovis (1.7%), and Babesia bigemina (0.3%). Mixed infections were detected in 406 samples (67.7%) accounting for 17 different combinations. High infection of Theileria parva was reported among young calves compared to older cattle. The highest prevalence of Theileria parva was reported in the rainy season (October). The implications of these results on the epidemiology of tick-borne diseases are discussed with emphasis on East Coast fever.
In the present study, we aimed to provide information on the serum content of sialic acids (TSA, LBSA, and PBSA), total antioxidant capacity (TAC), and adenosine deaminase (ADA) activity in cattle affected with naturally acquired theileriosis and anaplasmosis. A total of 55 Holstein cattle, comprising of 15 clinically healthy control animals, 20 cattle with theileriosis, and 20 with anaplasmosis, were used. Diagnosis was based on clinical signs, Giemsa stained blood or lymph node aspirate, and PCR assay. For the PCR assay, Tarns 1 primers were used. The obtained results suggested that the concentration of sialic acids and ADA activity were significantly higher; and TAC were significantly lower in the theileriosis and anaplasmosis groups in contrast to the control group. In conclusion, the increased level of sialic acids and ADA in theileriosis and anaplasmosis may be attributable to the stimulation of the host immune response. In contrast, the reduced level of TAC may reflect a decrease in the antioxidant capacity.
Adult Hyalomma ticks were examined for the presence of Theileria annulata infection using the Polymerase Chain Reaction (PCR). A 372 bp DNA fragment derived from the small ribosomal RNA gene of T. annulata was amplified from 45 out of 50 (90%) H. dromedarii ticks and from 36 out of 50 (72%) H. marginatum marginatum ticks. No product was amplified from non-infected control ticks. Restriction enzyme digestion with Sac II confirmed that the product was derived from the targeted T. annulata gene. As a further confirmation it was shown that both species of Hyalomma ticks were able to transmit T. annulata to experimental calves. PCR detection of Theileria parasites in ticks was compared with conventional staining of dissected salivary glands using methyl green pyronin and its comparative advantages are discussed.
A survey of Theileria parasites in cattle in eastern Turkey was carried out using specific polymerase chain reaction. A total of 252 blood samples were collected from clinically healthy cattle between June and July 2004. Of 252 blood samples examined, 41 (16%) were positive for piroplasms by microscopy, whereas 114 (45%) were positive for the presence of at least one species of Theileria by PCR. The percentages of positive animals for Theileria annulata and benign Theileria species (Theileria sergenti/buffeli/orientalis) were 39% (99/252) and 7% (18/252), respectively. By allele-specific PCR examination of 18 field isolates which were positive for benign Theileria parasites, 8 samples were only amplified by B-type specific primers and 10 samples were amplified by both of the B and C-type specific primers, indicating a mixed infection with B and C-type of the parasite. None of the field isolates was amplified by I-type specific primers. Three samples were co-infected with T. annulata and benign Theileria parasites. Two of them which were infected with B-type parasite were also infected with T. annulata, the other sample which was infected both of B and C-type parasites was also infected with T. annulata. A total of 724 ixodid ticks were collected from the cattle. Hyalomma anatolicum anatolicum was the dominant species with 32% (230/724) in the region. H. a. excavatum, Boophylus annulatus and Rhipicephalus bursa represented 25% (183/724), 19% (140/724) and 15% (112/724) of the total number of ticks, respectively. R. sanguineus was the minor species and represented 8% (59/724) of the tick population.
Tams1 is a merozoite surface antigen of Theileria annulata. Genetical variations of Tams1 make studies of vaccine and diagnostic tests like ELISA difficult. In this study, Tams1 genes of 89 T. annulata isolates obtained from natural infected cattle in Elazig and Bingöl regions were tested with PCR-RFLP. Six different restriction profiles (a, b, c, d, e, f) were detected. The number of restriction profiles of 89 samples was found to be as follows: 78(a), 2(b), 2(c), 5(d), 1(e), and 1(f).