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Identification of Vector Borne Blood Protozoa in Cattle and Sheep in Bangladesh

Md Zakir Hassan*
Animal Health Research Division, Bangladesh Livestock Research Institute, Savar, Dhaka-1341, Bangladesh

Md Giasuddin
Animal Health Research Division, Bangladesh Livestock Research Institute, Savar, Dhaka-1341, Bangladesh

Md Mamunur Rahman
Conservation and Improvement of Native Sheep through Community & Commercial Farming Project, Bangladesh Livestock Research Institute, Savar, Dhaka-1341, Bangladesh

Md Ershaduzzaman
System Research Division, Bangladesh Livestock Research Institute, Savar, Dhaka-1341, Bangladesh

Mahmudul Hasan
Animal Health Research Division, Bangladesh Livestock Research Institute, Savar, Dhaka-1341, Bangladesh

Md Abu Hadi Noor Ali Khan
Department of Pathology, Bangladesh Agricultural University, Mymensingh, Bangladesh

Abstract/ Executive Summary:

Babesiosis, Anaplasmosis and Theileriais are the most common vector (Tick) borne blood protozoan diseases (TBDs) in Bangladesh. This study was conducted in cattle and sheep in a different area of Bangladesh. A total number of 1150 blood samples were randomly collected from Dhaka, Sirajganj and Nikhangsori for blood smear microscopy. However, co-infections, temperature, humidity, season, farming and prophylaxis were also under consideration. From the clinically positive sample PCR was done followed by gel electrophoresis. Prevalence of blood protozoa were 100% (55), 80% (n=320), 30% (n=120), 22% (n=44), 31% (n=22), 65% (n=16) in exotic sheep, intensive farming, milk-vita area, local cattle, hill tracts and native sheep respectively. The overall prevalence was 50.17% (n=577). Among the protozoa, Anaplasma spp. was 43%, Babesia spp. 19%, Anaplasma spp. with Babesia spp. 33%, Theileria spp 4% and Anaplasma spp. with Babesia and Theileria spp was 1%. The prevalence of blood protozoa in local breed ≥50%, up to 75% and above 75% cross or pure breed were 17.58% (n= 103), 31.91% (n=187) and 50.51% (n=296) respectively. Prevalence of blood protozoa during October to March was 16.041% (n= 94) and April to September was 83.959% (n=492). In PCR Anaplasma marginale showed positive band as 265 bp, Babesia bovis in 166 bp, and Theileria annulata in 312 bp, Babesia ovis in 422bp and Babesia motasi in 518bp respectively. Therefore, the tick is act as vector and high humidity and temperature is the main risk factor for vector borne diseases. In conclusion, blood protozoa are the silent emerging disease in livestock and need to improve the control strategy.

Keywords: Anaplasma spp; Bangladesh; PCR; Prevalence; Vector

Location: Bangladesh Livestock Research Institute (BLRI), Savar, Dhaka

Start Date: 00-07-2016

End Date: 00-06-2017

Program Area: Animal Health and Management

Commodity: Cattle, Sheep

Objectives:

Therefore, considering the importance of TBDs the research work was done for the identification and molecular detection of vector borne blood protozoan infection notably Babesia spp, Anaplasma spp and Theileria spp in cattle and sheep in Bangladesh with seasonal variation.

Methodology:

Study area: An epidemiological study was carried out in Parasitology Laboratory under Animal Health Research Division in Bangladesh Livestock Research Institute (BLRI), Savar, Dhaka from July 2016 to June 2017. Sample collection About 2.5 ml of peripheral blood samples were collected from different cattle and sheep farm from several of Savar, Sirajganj Sadar, Shajadpur Upazila and Nikhangsori, Chottrogram within Ethylene Diamine Tetra Acetate (EDTA) tube with ice-cool storage and shifted to the Parasitology laboratory in BLRI. A total number of 1150 blood samples were collected randomly on questionnaire basis, among them 55 from Australian sheep (BLRI), 400 from a high yielding dairy farm of Savar, 400 from high yielding cattle from milk vita Bathan area (Baghabari), 200 from native cattle (Sirajganj), 70 from native hilly cattle of Nikhangsori, 25 from native sheep. Laboratory identification of blood protozoa through Giemsa’s stain Method Samples were examined by Giemsa’s stained blood smear (GMS) microscopy (FAO, 2016) and confirmatory diagnosis through Polymerase Chain reaction (PCR). The effect of topography, season, age and sex was remaining in consideration in this study. In GMS protocol thick and thin blood smear was done. After air dry absolute methanol fixation was done and stained with 10% Giemsa’s stain. After washing, air dry and emulsification, magnification under 100x objectives. The haemoprotozoa were microscopically identified based on the characteristic morphology illustrated by Soulsby. Molecular Identification and confirmation of tick borne protozoa (Blood Protozoa) DNA extraction of blood protozoa Blood protozoan DNA was extracted using a commercially available kit (Invitrogen Purelink Genomic DNA mini kit, Cat. no. K1820- 01) from blood sample through chloroform method. At a glance, 200 μl whole blood was mixed with 200 μl of lysis buffer containing 20μg/ml proteinase K and incubation was done at 55 0C for 10 minutes. Thereafter, washing and centrifugation was done at 13,000x g for 3 minutes. Finally, the spin column was discarded and collecting the eppendorf tube containing extracted DNA and stored in -20 0C in the refrigerator. The purity of genomic was visualized by using spectrophotometry (260°A/280°A) with 1.5% gel electrophoresis (Sigma Aldrich, USA). The compaction of the DNA genome was adjusted to 100ng/µl nuclease free water. Multiplex Polymerase Chain Reaction (PCR) 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 master mix kit (Thermo Scientific) was used to amplify fragments of genomic DNA in a programmable thermocycler (Eppendorf, Germany). Furthermore, after an initial enzyme activation step at 95°C for 5 min, the reaction mixture was subjected to 35 cycles each containing a denaturation step at 95°C for 30 sec, an annealing step at 68°C for 30 sec, and an extension step at 72°C for 1.5 min. After a final elongation step at 72°C for 5 min, PCR products were resolved by agarose gel electrophoresis, stained with ethidium bromide, and then observed under UV light. Oligonucleotide primers were used in the PCR amplification cycle (First BASE Laboratories sdnbhd, Malaysia). The PCR images were captured though computer software (Carl Zeiss, GmbH, Germany) and the positive samples were detected by specific band size of the PCR product.

Source of Information: J Virol Antiviral 2019, 2: 004

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

Tick borne blood protozoan disease (Babesiosis, Anaplasmosis, and Theileriosis) are now a days a crucial factor for livestock production in Bangladesh. Local animal act a as carrier but it indicating future havoc in livestock industry especially high yielding exotic animal (70 % to 100 % pure breed). Moreover, they are more susceptible to TBDs and it is very difficult to control because high temperature and humidity provoke the tick multiplication. To introduce high yielding animal in a farm strict biosecurity is essential for farming.

Knowledge Management: Journal