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Apply Traditional and Molecular Protocols for the Detection of Carrier State of Visceral Leishmaniasis in Black Bengal Goat

Sharmin Shahid Labony
Department of Parasitology, Bangladesh Agricultural University, Bangladesh

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

Mohammad Ahsan Habib
Department of Pathology, Bangladesh Agricultural University, Bangladesh

Mohammad Zakir Hossain
Department of Pathology, Bangladesh Agricultural University, Bangladesh

Umme Kulsum Rima
Department of Pathology, Bangladesh Agricultural University, Bangladesh

Md. Golam Azam Chowdhury
Department of Pathology, Bangladesh Agricultural University, Bangladesh

Nurjahan Begum
Department of Parasitology, Bangladesh Agricultural University, Bangladesh

Abstract/ Executive Summary:

Visceral leishmaniasis (VL) is the second largest parasitic killer of human in the world after malaria which is responsible for an estimated 500,000 new cases of VL in each year with 8-10% mortality. The parasite is transmissible to humans and animals by the bite of phlebotomine sand fly. The clinical manifestations are highly diverse, humans and dogs are naturally infected, and the diseases are associated with several risk factors, yet to understand. The aims of this study were to apply traditional and molecular detection tools and more emphasis was given to identify goat as a carrier of visceral leishmaniasis. To demonstrate the promastigote and amastigote phases of Leishmania in tissues, traditional impression smear staining technique was used. For the confirmation of the species of Leishmania involved specific technique like polymerase chain reaction (PCR) was applied. A total of twenty goats were investigated and samples were tested using impression smear staining, histopathology and PCR. Blood smear and impression smears were prepared from spleen, liver, bone marrow and stained with Giemsa’s stain. Using Giemsa’s staining out of twenty goats investigated, six (6/20) were found to contain promastigote and amastigote stages of Leishmania in their visceral organs. Histopathological examination from the liver section of suspected goats showed degeneration, necrosis and non specific fibrous connective tissue proliferation compared to non-reactive goats. There was accumulation of macrophages in lymphoid follicles of spleen in five suspected goats. A highly sensitive and specific primers were used in PCR amplification with the extracted DNA from liver and spleen of suspected six goats. Results of PCR showed that two of them were generated 145bp amplicon selective for L. donovani in their liver and spleen. Leishmaniasis has a great public health significance and the protozoa found in goats of Fulbaria Upazilla may possess threat for transmission in human and other animals, require further investigation.

Keywords: Black Bengal Goats, Visceral leishmaniasis, Giemsa’s staining, Polymerase Chain Reaction (PCR)

Location: Fulbaria Upazilla, Mymensingh

Start Date: 00-05-2013

End Date: 00-05-2013

Program Area: Animal Health and Management

Commodity: Goat

Objectives:

To standardize traditional and molecular tools for the detection of leishmanial protozoa in black bengal goat and identify goat as a carrier of VL in Fulbaria Upazilla, Mymensingh, Bangladesh.

Methodology:

A total of twenty goats of ill health and both sexes were randomly selected and systemic dissection and investigation were carried out and liver, spleen, blood and bone marrow were collected. Thin smears of blood and impression smears from liver, spleen and bone marrow were made on to clean grease free glass slides, dried in air and fixed in ice cold absolute methanol (acetone free) for 15 minutes. The slides were dried in air and placed in Coplin jar containing working Giemsa’s solution and allowed to stain for 50 minutes. Slides were washed in running tape water for 30sec, dried in air and examined at low (10x) and high power (40x and 100x) microscopic field. Extensive investigation was carried out for the presence of promastigote and amastigote stages of leishmanial protozoa in blood, in tissue spaces and in macrophages of various organs were employed and protozoal morphology was investigated and images were captured. During necropsy, portion of liver and spleen were collected and fixed in 10% buffered neutral formalin for histopathological studies. Formalin fixed tissue samples were trimmed, processed, sectioned and stained with H&E. A low (10x) and high power (40x, 100x) microscopic investigation were carried out to observe the changes in the internal organs specific to VL. For DNA extraction from the liver and spleen of goats Wizard1 Genomic DNA Purification Kit (Promega) was used according to the manufacturer’s protocol. Briefly 100μl of 20% liver samples suspension was mixed with 600 μl of nuclei lysis solution, incubated for 15 min at 65⁰C temperature and 3μl of RNase Solution was added to the nuclear lysate and mixed by inversion. The mixture was incubated for 30minutes at 37°C. 200μl of Protein Precipitation solution was added and vortexed vigorously for 20 s and centrifuged at 13000 rpm for 4 min and the precipitated protein was formed a tight white pellet. The supernatant was transferred to a new tube containing 600μl of room temperature isopropanol and centrifuged at 13000 rpm for 1 min. The supernatant was discharged and 600μl of 70% ethanol was added. After the centrifugation at 13000 rpm for 1 min, the ethanol was aspirated and the pellet air dried. The pellet was suspended in 100μl of the DNA rehydration solution. Then incubating at 65°C for 1 hour and finally stored the DNA at 4°C. The DNA samples were evaluated quantitatively and qualitatively using spectrophotometry (A260 and A280) and agarose gel electrophoresis. One set of primers were used to identify the species of leishmanial protozoa. PCR reactions were performed onto each DNA sample in 25μl reaction volume containing 7 μl of Nuclease free water, 2x PCR master mix (Promega Corporation, USA), .25μl forward and .25μl reverse primers and 5 μl of DNA template. A total of 45 cycles of DNA amplification reaction for L. donovani was carried out. The thermal profile used for L. donovani comprised an initial denaturation for two minutes at 94°C followed by 45 cycles of DNA amplification reaction in a Master Cycler (Master Cycler Gradient, Eppendorf, Germany). The condition of PCR amplifications were denaturation for 60sec at 94°C, primer annealing for 90sec at 62°C and extension for 30sec at 70°C followed by a final extension for 10min at 70°C. The PCR reactions were finally terminated by adding 3μl 50mM EDTA and PCR products were analyzed by electrophoresed in 2% agarose gel, stained with ethidium bromide and examined under UV light using an image documentation system (Cell Biosciences, Alphalmager HP, USA).

Source of Information: IOSR Journal of Agriculture and Veterinary Science (IOSR-JAVS); e-ISSN: 2319-2380, p-ISSN: 2319-2372.Volume 7, Issue 2 Ver. III (Mar-Apr. 2014), pp.13-18

Article Link (Online Journal): www.iosrjournals.org

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

This study concludes that probably this is the first study in Bangladesh detecting Leishmania sp. in goats by using PCR. However further studies are needed by increases sample size & to identify genomic DNA in sand flies by using PCR which transmitting VL to other animals and human beings.

Knowledge Management: Journal