M. Z. Hossain
Dept. of Pathology, BAU, Mymensingh
U. K. Rima
Dept. of Pathology, BAU, Mymensingh
M. Pervin
Dept. of Pathology, BAU, Mymensingh
G. A. Chowdhury
Dept. of Pathology, BAU, Mymensingh
M. A. Habib
Dept. of Pathology, BAU, Mymensingh
M. E. Haque
Dept. of Pathology, BAU, Mymensingh
E. H. Chowdhury
Dept. of Pathology, BAU, Mymensingh
M. A. H. N. A. Khan*
Dept. of Pathology, BAU, Mymensingh
Mycobacterium tuberculosis complex ((MTB), Tuberculin test, Lowenstein-Jensen (L-J) Media, Polymerase Chain Reaction (PCR)
Mymensingh, Sirajganj, Bogra, Sylhet, Chittagong, Dhaka, Tangail Districts.
Animal Health and Management
Comparative Intradermal Tuberculin test (CITT) were used as described in the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. Dairy cattle of total number 656 (six hundred and fifty six) were tested using single intra-dermal (Bovine PPD) and comparative intradermal (Bovine and avian PPD) Tuberculin tests at Mymensingh, Sirajganj, Bogra, Sylhet, Chittagong, Dhaka, Tangail Districts. A short needled syringe (McLintock® preset syringe and tuberculin testing equipment, Glasgow, G81 1NH) loaded with either avian type or bovine type tuberculin and 0.1ml of the content were injected. Bovituber PPD and Avituber PPD, Synbiotic, France were injected for tuberculin test. Tuberculin (+Ve) suspected cattle (n=2) were used for biopsy findings. The enlarged prescapular lymphnode were collected by biopsy procedure. Local anesthetic Xylazine 2% manufactured by Jayson, Bangladesh was used for biopsy with proper hygienic method without struggling the animal. Tuberculin (+Ve) small portion skin swelling also collected to observe delayed type of hypersensitivity skin reaction for histopathology. The tuberculin test positive cattle of (n=10) were euthanized with saturated MgSO4 and a thorough postmortem examination was carried out to investigate the gross lesions. In this study, postmortem samples were collected from suspected animals tuberculin positive cases. Following systemic necropsy lung, liver, kidney, spleen, bone marrow, prescapular lymphnode and mesenteric lymphnode were collected. For histopathologic examination collected samples preserved in 10% neutral buffered formalin. Lungs, lymphnodes and bone marrow were snap freeze and preserved at -20°C for PCR detection of TB. The formalin fixed tissues were processed, sectioned and stained with hematoxylin and eosin (H&E) stain and acid-fast stain. Most of the organisms localized in the macrophages, lymphocytes and neutrophils and these cells serve as the unique source of organisomal DNA. The Tuberculous organisms also localized in Lungs, liver, kidney, lymph nodes, uterus, intestine, conjunctiva and mammary gland. DNA was extracted from lung, lymphnode, bone marrow, L-J media culture product for cattle. The DNA purification from these tissues or cells will be crushed in liquid nitrogen, heated at 100ºC and extracted DNA using the phenol–chloroform–isoamyl (PCI) alcohol extraction and ethanol precipitation method. Wizard® Genomic DNA Purification Kit also used for DNA extraction. Lymphnode, lung, bone marrow of cattle were used for this purpose. The above showed primers were designed for amplifying the mature protein gene. The mature protein gene MPB83 was amplified by using the template of M. bovis chromosomal DNA and specific primers Primer-1 and Primer-2. PCR 2X Master Mix (GeNeiTM PCR Master Mix Kit) 12.5μl, Primer (forward) 0.5μl, Primer (reverse), RNAse free water 6.5μl, DNA template 5μl and total volume was 25μl. DNA amplification was carried out in a thermal cycler using the thermal profile a) 98°C for 5 min, one cycle; 95°C for 1 min, 56°C for 1 min, 72°C for 1 min, 30 cycles; 72°C for 10 min, one cycle and b) : initial denaturation at 94ºC for 5 min, followed by 30 three-step cycles, including denaturation at 94 ºC for 1 min, annealing at 52.3 ºC for 1.5 min, extension at 72ºC for 1 min and a final extension at 72ºC for 5 min. After completion of PCR reaction the tubes were held at 4°C.
Bangladesh J. Prog. Sci. & Tech. 10(1): 085-088, January, 2012 ISSN 2305-1809 (Online version)
Journal