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Adoption of Polymerase Chain Reaction Techniques for the Detection and Differentiation of Babesiosis, Anaplasmosis and Theileriosis in Clinically Infected and Slaughtered Cattle

MA Karim
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202.

Mahna Khan
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202.

MM Hossain
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202.

MA Habib
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202.

MS Islam
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202.

MZ Hossain
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202.

UK Rima
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202.

Abstract/ Executive Summary:

Economic losses attributed in cattle due to Babesiosis, Anaplasmosis and Theileriosis include morbidity, mortality, production loss with cost of treatment, diagnosis and tick control. Clinical examination and blood test have little value towards confirmatory rather a preliminary diagnosis of these diseases. In this study venous blood (N=14) and internal tissues (N=10) from suspected and slaughtered cattle were collected from different slaughter houses of Madhupur Upazila, Tangail District, Bangladesh and investigated for the presence of these diseases. Giemsa’s staining of blood smear and uniplex and multiplex polymerase chain reaction (PCR) were adopted with the extracted DNA of blood and body tissues. Giemsa’s staining of blood smear identified Babesia and Anaplasma organisms in three cases but unable to detect Theileria. Routine examination of blood did not reveal any significant change except anemia. Necropsy of a suspected dead bull was carried out to investigate the characteristics lesions associated with Anaplasmosis; grossly there were hemorrhages at the margin of the spleen and the gall bladder was distended. Histopathologic lesions were enlarged splenic trabeculae with hemosiderosis, testicular degeneration and necrosis, interstitial nephritis and hemorrhages in renal medulla, pyknosis in hepatocytes and focal accumulation of mononuclear cells in the hepatic tissues, bronchiolitis and interstitial pneumonia. The uniplex PCR (uPCR) with species specific primer gave nine positive bands at 166bp, two positive bands at 265bp and five positive bands at 312bp selective for B. bovis, A. marginale and T. annulata respectively. Results of multiplex PCR using primers of uPCR also amplified genomic fragments at 166bp (N=7), 265bp (N=02) and 312bp (N=03) levels specific for B. bovis, A. marginale and T. annulata respectively. Results of uniplex and multiplex PCR showed that five cattle were co-infected with Babesia and Theileria, one cattle with Babesia and Anaplasma and one cattle with Babesia, Anaplasma and Theileria. Results of this study suggested that the uniplex and multiplex PCR techniques were useful for the accurate detection and differentiation of Babesiosis, Anaplasmosis and Theileriasis in cattle. It needs further study to test large number of samples with wide range of primers selective for other species of Babesiosis, Anaplasmosis and Theileriasis in order to detect distribution of these diseases in animals and to design future preventive and control strategies.

Keywords: PCR, Babesiosis, Anaplasmosis, Theileriasis, Cattle

Location: Department of Pathology, Faculty of Veterinary Science, BAU, Mymensingh

Start Date:

End Date:

Program Area: Pest Management

Commodity: Cattle

Objectives:

To determine adoption of Polymerase Chain Reaction Techniques for the Detection and Differentiation of Babesiosis, Anaplasmosis and Theileriosis in Clinically Infected and Slaughtered Cattle

Methodology:

This study was carried out in the Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University (BAU), Mymensingh. Spleen and venous blood (N=10) of cattle were collected from the slaughter houses of Madhupur Upazilla, Tangail. A breeding bull used in artificial insemination center, BRAC, Shambhuganj, Mymensingh has been suffering from fever and suspected as a case of Anaplasmosis. The bull died later on, blood and tissues were collected, Giemsa staining, histopathology and PCR were carried out to confirm the etiology and pathology of clinical Anaplasmosis in Cattle. About 05ml Venous blood from the cattle were also collected in EDTA, shifted to the laboratory on ice for thin smear preparation and Giemsa’s staining, genomic DNA extraction and PCR amplification of gemonic DNA of TBDs. Cattle were selected randomly and both the indigenous and cross-bred animals were selected. The cattle were categorized into different age groups viz. calf (≤ 1year (n= 255)), young (1-3 years (n= 465)) and adult (≥ 3 years (n= 375)); sexes viz. male (n= 480) and female (n= 615); health conditions viz. normal (n= 375) and poor (n= 720) and types of floor of cattle houses viz. muddy floor (n= 795) and concrete floor (n= 300). Routine necropsy was carried out on to the breeding bull of Shambugonj bull station suspected to infect with Anaplasma sp. At necropsy, gross tissue changes observed were recorded. The spleen, liver, lungs, kidney and testis were collected in 10% neutral buffered formalin, embeded in paraffin, 5µ thick sections were stained with Hematoxylin & Eosin and examined changes in tissues under a microscope at low (10x) and high power (40x and 100x) microscopic objectives. The images were captured using an automated computer connected with the microscope and ZEN lite 2012 software. Genomic DNA from the spleen and blood (packed cell volume) were extracted using commercially available kit (Wizard Genomic DNA Purification kit, Promegra Corporation, U.S.A). The purity and concentration of genomic DNA was measured by using spectrophotometry (260°A/280°A) and gel (1.5%) electrophoresis. The concentration of genomic DNA was adjusted to 100ng/µl using nuclease free deionized distilled water. PCR was carried out in a final reaction volume of 25μl in the thin walled PCR tubes to amplify genomic DNA of Babesia, Anaplasma and Theileria species. The commercially available Promega mastermix kit was used to amplify fragments of genomic DNA in a programmable thermocycler. Electrophoresis grade agarose, the loading TAE buffer, 100bp DNA size marker and 10mg/ml ethidium bromide were used in 1.5% agarose gel to visualize fragments of amplicon in the dark chamber of an image documentation system. The images were captured by using computed softwares linked with the image documentation system and used in the detection and differentiation of TBDs in cattle.

Source of Information: Bangladesh Vet J (2012) 46(1-4):31-43

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

Results of this study indicated that TBDs are endemic in cattle of Madhupur Upazilla, Tangail, Bangladesh. The breeding bull died due to Anaplasmosis could be due to massive liver damage and or lack of sensitivity to Imidocarb. In order to accurately address future prevention and control strategies against TBDs, it needs further study to test large number of animals, identify vector ticks and confidently detect other species of Babesia, Anaplasma and Theileria involved.

Knowledge Management: Journal