Development of a polymerase chain reaction method for diagnosis of Babesia ovis infection in sheep and goats

Aktaş, Münir; Altay, Kürşat; Dumanlı, Nazir

doi:10.1016/j.vetpar.2005.05.057

Cited by 96 publications

(70 citation statements)

References 16 publications

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“…Extraction of DNA was performed according to the procedure described by Aktas et al (2005) with some modifications. Briefly, 125 µl of the blood sample was added to 250 µl of lysis buffer (0.32 M sucrose, 0.01 M Tris, 0.005 M MgCl 2 , 1% Triton X-100, pH 7.5) and the mixture was centrifuge at 11600 × g for 1 min.…”

Section: Pcr Amplificationmentioning

confidence: 99%

“…The primer's specificity and sensitivity and also the PCR condition have been described previously by Aktas et al (2005). PCR was carried out in 50 µl total reaction volume containing 5 µl of 10 × PCR buffer, 2 mM MgCl 2 , 250 µM of each of the four deoxynucleotide triphosphate, 1.25 U Taq DNA polymerase (Fermentas, Germany), 50 pmol of each primer and 5 µl (≈ 25 ng) of extracted DNA.…”

Section: Pcr Amplificationmentioning

confidence: 99%

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Status of lipid peroxidation and antioxidant enzymes in goats naturally infected with Babesia ovis

Esmaeilnejad

Tavassoli

Asri‐Rezaei

et al. 2012

Acta Parasitologica

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This study aimed to assess lipid peroxidation and antioxidant enzymes in goats naturally infected with Babesia ovis. Red blood cell count (RBC), hemoglobin (Hb) concentration, packed cell volume (PCV), malondialdehyde (MDA) concentration, erythrocyte superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) activities and total antioxidant capacity (TAC) were determined in 15 goats naturally infected with B. ovis as well as same number of healthy goats. The parasitological diagnosis was confirmed using polymerase chain reaction (PCR) analysis by amplifying a partial 18S rRNA gene sequence of B. ovis. Percentage of parasitemia varied from 0.01 to 1%. The activities of erythrocyte GSH-Px, SOD, CAT and TAC were significantly lower (p<0.05) in the infected goats than in healthy ones. MDA concentration in erythrocytes of infected goats was significantly higher in infected goats than in healthy ones (p<0.05). Severity of parasitemia showed a positive correlation with the MDA and negative correlation with PCV, SOD, CAT, GSH-Px and TAC. Also, MDA was negatively correlated with PCV, SOD, CAT, GSH-Px and TAC. The results of this study suggested that oxidative damage to RBCs may contribute to the pathogenesis of anemia in caprine babesiosis.

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Section: Pcr Amplificationmentioning

confidence: 99%

Section: Pcr Amplificationmentioning

confidence: 99%

Status of lipid peroxidation and antioxidant enzymes in goats naturally infected with Babesia ovis

Esmaeilnejad

Tavassoli

Asri‐Rezaei

et al. 2012

Acta Parasitologica

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“…Extraction of DNA was performed according to the method previously reported by Aktas et al (2005). Briefly, 125 μl of blood were added to 250 μl of lysis mixture.…”

Section: Dna Isolation and Pcr Reactionsmentioning

confidence: 99%

Determination of prevalence and risk factors for infection with Babesia ovis in small ruminants from Turkey by polymerase chain reaction

2006

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In this study, PCR and thin blood smear-based diagnostic methods were used to assess the frequency of Babesia infection in small ruminants. A total of 300 sheep and 100 goats from 37 randomly selected herds located in eight locations of eastern Turkey were examined for the presence of Babesia infection and any tick species on the body of the animals. Of 400 blood samples examined, 6 (1.5%) were positive for Babesia spp. piroplasms upon microscopic examination, whereas 33 (8.25%) were positive for the presence of B. ovis by PCR. The prevalence of babesiosis in small ruminants detected by PCR was significantly higher than obtained in microscopic examination of thin blood smears (P< 0.05). Thirty-three animals produced the DNA fragment specific for Babesia ovis of which 32 (10.66%) were sheep and 1 (1%) was goat. The difference between the prevalence of Babesia infection in sheep and goats were statistically significant (P < 0.05). The prevalence of Babesia infection in different age groups of sheep were statistically non-significant (P > 0.05). The frequency of B. ovis infection was higher in herds with tick burden than no tick burden (P < 0.05). Seven amplicons (six from sheep and one from goat) were sequenced. The resulting sequences were identical to the recently reported nucleotide sequence of B. ovis. A total of 510 ticks belonging to the Rhipicephalus spp. were collected from sheep. Ticks were identified to be R. bursa and R. turanicus on the basis of morphological features.

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“…For construction of the cDNA expression library, a field strain of B. ovis was obtained from a natural case (50% parasitemia levels) located in an area in the central Anatolian region of Turkey in which ovine babesiosis is enzootic. The parasites were monitored by microscopic examination of Giemsa-stained thin blood smears, and results were verified by PCR analysis, as described previously (22). The serum samples used for the ELISA were available in our laboratories from previous studies (8,14,23) and were as follows: 15 negative samples, confirmed by microscopy and the IFAT, from experimentally infected lambs prior to infection (negative-control samples); 120 serum samples from the same experimentally infected lambs, at 6,7,8,11,21,30,45, and 75 days postinfection; and 76 serum samples from 38 naturally infected sheep, at the time of clinical infection and 20 to 30 days after treatment, in which the presence of the parasite was confirmed by microscopy.…”

Section: Methodsmentioning

confidence: 99%

Identification and Expression of Babesia ovis Secreted Antigen 1 and Evaluation of Its Diagnostic Potential in an Enzyme-Linked Immunosorbent Assay

Sevinç

Cao

et al. 2015

J Clin Microbiol

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bIn order to identify immunoreactive proteins that are usable for the immunological diagnosis of Babesia ovis infections, a phage lambda cDNA expression library was constructed and screened using parasite-specific immune serum. Immunoscreening resulted in the identification of a full-length cDNA clone encoding a secreted protein designated Babesia ovis secreted antigen 1 (BoSA1). The full-length BoSA1 cDNA contained a 1,137-bp open reading frame that encoded a protein of 378 amino acids, with a signal peptide and 2 internal repeat domains. The theoretical molecular mass of the mature protein was 42.5 kDa. Recombinant BoSA1 (rBoSA1) protein was expressed in Escherichia coli strain DH5␣ cells as a glutathione S-transferase (GST) fusion protein and was purified by affinity chromatography. Purified rBoSA1 was tested for reactivity with sera from animals experimentally or naturally infected with B. ovis, in an indirect enzyme-linked immunosorbent assay (ELISA). The results showed that specific antibodies against rBoSA1 were detectable on days 7 and 8 of the experimental infection and were maintained during the sampling period. Additionally, 38 field sera taken from sheep naturally infected with B. ovis gave strong positive reactions in the ELISA between day 20 and day 30 of treatment. As a result, the identified recombinant BoSA1 protein seems to be a promising diagnostic antigen that is usable for the development of serological assays for the diagnosis of ovine babesiosis. This is the first report on the molecular cloning, expression, and potential use of a recombinant antigen for the diagnosis of ovine babesiosis. O vine babesiosis, caused by the tick-borne intraerythrocytic apicomplexan parasite Babesia ovis, is an acute disease characterized by clinical signs such as fever, hemolytic anemia, hemoglobinuria, and icterus in small ruminants. The disease is endemic in European, African, Asian, and Far Eastern countries (1-8). Babesia ovis is highly pathogenic and, in serious infections, causes pancytopenia. Untreated cases usually result in the death of sick animals, and even some treated animals may die as a result of heavy infection. Many recurrences also occur after the treatment period. Compensation for the abnormalities in the hematological profiles of acutely infected animals takes a long time after the treatment period (8). Currently, control of ovine babesiosis is based only on chemotherapy and limited tick control measures. In areas in which the status of B. ovis endemicity is unstable, an immunization program is needed (9). However, immunoprophylaxis is lacking for Babesia species in sheep.Currently, the diagnosis of ovine babesiosis includes microscopic examination of Giemsa-stained thin blood smears. Although intraerythrocytic parasites are apparent in clinical cases, it is very difficult to detect the organisms by microscopy in chronic infections, because of the low levels of parasitemia (10-12). The analysis of blood smears usually depends on the experience of the observer. The morphological changes in ...

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Development of a polymerase chain reaction method for diagnosis of Babesia ovis infection in sheep and goats

Cited by 96 publications

References 16 publications

Status of lipid peroxidation and antioxidant enzymes in goats naturally infected with Babesia ovis

Status of lipid peroxidation and antioxidant enzymes in goats naturally infected with Babesia ovis

Determination of prevalence and risk factors for infection with Babesia ovis in small ruminants from Turkey by polymerase chain reaction

Identification and Expression of Babesia ovis Secreted Antigen 1 and Evaluation of Its Diagnostic Potential in an Enzyme-Linked Immunosorbent Assay

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