A comparison of two different techniques for the detection of blood parasite, Theileria annulata , in cattle from two districts in Khyber Pukhtoon Khwa Province (Pakistan) (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(...
Scientific Reports
Theileria annulata is an intracellular parasite that causes active and latent forms of bovine theileriosis. Diagnosis of the disease is primarily based on traditional methods such as microscopy, however, PCR based methods have proven to be superior in the absence of clear disease symptoms. However, diagnosis is difficult in cases of lower parasitaemia by conventional PCR. Hence, a rapid and sensitive method which can detect early infection and low parasite load is required. Therefore, we have developed an absolute quantification based real-time PCR (qPCR) assay. Reference standard curve using recombinant plasmids of a host (hprt) and a parasite gene (tasp) was constructed, and the assay was initially standardised using in vitro T. annulata cell lines. Further, 414 blood samples from suspected theileriosis cases were also evaluated using qPCR. The assay can estimate host to parasite ratios, calculate parasitaemia and treatment effectiveness in the clinical cases of theileriosis. In comparison with the conventional PCR results, 44 additional positive cases were found. Therefore, the assay holds importance in a clinical setting due to its ability to quantify the parasite load in clinical samples. It may be further used in distinguishing active and latent theileriosis infections and detection of drug resistance in the field. Bovine theileriosis is caused by an apicomplexan parasite Theileria spp. which is an important tick-borne disease of livestock 1. Theileria annulata and Theileria parva are the two economically important species responsible for livestock morbidity and mortality worldwide 2-4. In India, T. annulata is the primary causative agent which hampers animal health and productivity 5,6. The economic loss to the tune of $800 million has been reported due to infection caused by T. annulata in India 7. It mainly infects crossbreed cattle, however, native breed cattle like water buffalo, and small ruminants are also known to be affected 5. The prevalence of T. annulata from different parts of India has been reported from 3 to 41% with the help of microscopy and molecular tests 8,9. The existing diagnostic tools include microscopy, PCR, and serological assays. The need of the hour is a sensitive, and reliable diagnostic tool which can perform timely detection along with an estimation of host-parasite ratio of clinical samples. The life cycle of T. annulata is complex, the tick vector while feeding on cattle releases the sporozoites in the bloodstream, which later enters into the host leukocytes (monocytes or B-lymphocytes) 10,11. Following host leukocyte invasion, T. annulata hijacks the host cell machinery and transforms the cells with a cancer-like phenotype 11. T. annulata parasites multiply in synchrony along with the host cells and form schizonts which is the symptomatic stage of the disease. The transformed T. annulata infected bovine leukocyte cells can be cultured in vitro for an infinite time in the parasite-specific culture medium. Currently, an attenuated T. annulata schizont stage vaccine is available in India for controlling the disease 12,13. The diagnosis of theileriosis heavily relies on the microscopy, where Giemsa stain is used to check for Theileria infected multinucleated host cells (Koch's bodies) and the piroplasm stage in the blood smear 14. Microscopy has certain drawbacks of being tedious, labour
Advances in Animal and Veterinary Sciences, 2021
T ropical theileriosis is one of the most prevalent serious problem with greatest economic and mortality impact of cattle in Iraq (Aktas et al., 2004). This disease is one of tick-borne protozoal disease caused by Theileria of the suborder Piroplasmorina which its transmitted by ticks from genus Hyalomma spp (Salih et al., 2007; Aziz and AL-Barwary, 2020). This protozoan is obligatory intracellular hemoparasites, and two species invaded to cattle T. parva (causes East Coast fever) and T. annulata (causes tropical theileriosis) (Sandhu et al., 1998; Singh et al., 2001). The infection occurs due to the presence and multiplication of parasite inside WBCs and then RBCs and resulting in progressive and severe macrocytic hemolytic anemia (Radostits et al., 2000). The main clinical signs are enlargement of prescapular lymph nodes, fever, diarrhea, pale of mucous membrane, decrease of milk production, abortion in preg
Iranian journal of veterinary research, 2020
Background Theileria annulata is a tick-borne apicomplexan parasite that affects bovine and causes severe economic losses. Aims: Our study aimed to determine the molecular prevalence of T. annulata infection in asymptomatic carrier cattle in Odisha, India, to study the association of potential risk factors with theileriosis, and to investigate the effect of the parasite infection on hematological parameters in naturally affected animals. Methods A total of 226 cattle blood samples were collected from seven districts of Odisha, India. Molecular diagnoses of tropical theileriosis by polymerase chain reaction (PCR), cloning, sequencing, and phylogenetic analysis of isolated parasites were performed. Potential risk factors were investigated by univariable and multivariable logistic regression statistical analysis. Hematological parameters were compared between positive and negative animals. Results All animals included in our study were clinically normal, however, 54.86% (124/226) of ex...
This is the first report on tropical theileriosis in Cholistani cattle, with the aim of 1) assessing the reliability of PCR as a tool for diagnosis of the early/carrier state; 2) determining the prevalence of theileriosis; and 3) comparing haematological profiles of parasite-positive and parasite-negative cattle. A total of 264 cattle (142 female and 122 male; 127 adult and 137 young) were examined for tropical theileriosis through clinical examination, stained smear screening, and polymerase chain recation. No cattle showed clinical signs of the disease. Of the diagnostic tests, PCR was more sensitive for detection of the early/carrier state of theileriosis (19.3%) compared to stained thin blood smear examination (1.9%). Female (24.6%) and young animals (23.4%) showed higher prevalence than males and adults, but not significant (P>0.05). Prevalence of the disease (51.6%) was significantly higher (P<0.05) in summer. Haematological indices were not significantly different in parasite-positive compared to parasite-negative cattle, except for total protein and creatinine which were significantly higher in infected animals. The study revealed a substantial prevalence of tropical theileriosis in Cholistani cattle. Nevertheless, their adaptation to the climate and their potential for tick and disease resistance may reflect in the absence of clinical signs and in normal haematological indices. Pakistan'ın Cholistan Çölünde Başıboş Dolaşan Cholistan Sığırlarında Tropikal Theileriosisin Prevalansı ve Kan Değerleri Özet Cholistan sığırlarında tropical theileriosis ile ilgili ilk rapor olan bu çalışma ile; 1 teşhis yöntemi olarak taşıyıcı hayvanlarda PCR metodunun güvenirliliğinin belirlenmesi; theileriosisin prevalansının tespit edilmesi; parazit pozitif ve negative bireylerde kan parametrelerinin karşılaştırılması hedeflenmiştir. Toplam 264 sığır klinik, mikroskopik ve PCR ile tropical theileriosis yönünden muayene edilmiştir. Sığırların hiç birinde klinik bulgu gözlenmemiştir. PCR (%19.3), subklinik enfeksiyonların belirlenmesinde mikroskopik bakıya (%1.9) göre oldukça duyarlı bulunmuştur. Hastalğın yaygınlığı dişi (24.6%) ve genç hayvanlarda (%23.4) erkek ve erişkinlere göre daha yüksek bulunmuş, ancak bu farklılığın istatistiksel olarak anlamlı olmadığı (P>0.05) görülmüştür. Hastalığın, yaz aylarında (%51.6) daha yüksek olduğu tespit edilmiştir. T. annulata yönünden pozitif ve negative hayvanların kan değerlerinde total protein ve creatinine hariç (bu değerler enfekte hanvanlarda yüksek bulunmuştur) bir farklılık gözlenmemiştir. Bu çalışma Cholistan sığırlarında hastalığın prevalansının yüksek olduğunu ortaya koymuştur. Muayene edilen hayvanların hiç birinde klinik bulguların gözlenmemesi, kan değerlerinde değişikliğin olmaması, bu sığırların kenelere ve hastalığa karşı dirençlerini yansıtmaktadır.
Animal Biotechnology, 2020
The objective of the present study was to investigate the epidemiological and haematological parameters with simultaneous molecular detection of Theileria orientalis infection of crossbred jersey (CBJ) cattle. Haematological values like mean hemoglobin (Hb) (7.31 ± 2.3 g/dl), packed cell volume (PCV) (21.69 ± 6.11%), red blood cells count (RBCs) (4.40 ± 1.6 M/ll), white blood cells count (WBCs) (6.93 ± 3.06 103/ml) and mean corpuscular hemoglobin concentration (MCHC) (33.56 ± 3.51 g/dl) were decreased significantly (p < 0.05), whereas mean corpuscular volume (MCV) (51.06 ± 6.14fl) and eosinophil count (0.39 ± 0.44 103/ml) were significantly (p < 0.05) increased in cattle infected with T. orientalis. Analysis of major piroplasm surface protein (MPSP) of 110 blood samples randomly collected from cattle from seven districts by PCR indicated that an average of 70% of cattle was positive for T. orientalis infection. In particular, Puri and Khorda districts were identified as relatively highrisk areas for T. orientalis infection, with infection rates of 76.66% and 72.4%, respectively. The phylogenetic analysis of isolated T. orientalis MPSP gene (MN334767) classified it into type 5. Earlier Indian isolates were classified into three types viz.type 1, type 3 and type 7 and this is the first time to detect type 5 in Odisha, India.
Detection of tropical bovine theileriosis by polymerase chain reaction in cattle
Journal of Parasitic Diseases, 2013
Tropical bovine theileriosis, a tick borne disease, caused by, Theileria annulata with marked clinical signs of pyrexia (102-105°F), enlargement of lymphnodes etc., causes heavy economic losses in terms of high mortality and morbidity rates. Diagnosis of theileriosis is mainly based on clinical symptoms and microscopic examination of stained blood smears and lymph node biopsy smears but limitations of these methods against Theileria sp. limits the specificity. Hence, to overcome the limitations, the present study reports the detection of T. annulata in blood samples of cattle by polymerase chain reaction. The study was conducted on 155 cattle having typical clinical symptoms and blood smear after staining with Giemsa stain was examined for the presence of T. annulata in RBC. The Primer sequences were used as per d'Oliveira et al. The assay employs primers specific for the gene encoding the 30-kDa major merozoite surface antigen of T. annulata and the amplification of 721 bp was done. Out of the total 155 animals, 34 were positive for T. annulata by blood smear method whereas 134 samples were positive by PCR. So diagnosis of blood samples by PCR is found to be the most sensitive and specific methodology as compared to cytological blood smear examination. The sensitivity was 23.88 % and specificity was 90.47 % of blood smear method considering PCR as gold standard and it was found that PCR is more sensitive than the conventional method of examination.
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.