Microscopic, Serologic and Molecular Identification of Mycobacterium avium Subsp. Para Tuberculosis in Clinically Suspected Small Ruminants

Abstract

This investigation was carried out in Al Qassim region during the period from February 2015 to January 2017 for identification of Mycobacterium avium subsp. paratuberculosis (MAP) the causative agent of Para tuberculosis (PTB) by using clinical examination, Ziehl- Neelsen (ZN) stained smears, ELISA test, gross and microscopic (semithin section) examination and real time polymerase chain reaction (RT-PCR) in small ruminants (sheep and goat). A total number of 1500 small ruminants were clinically examined and the animals showed signs of emaciation, diarrhea or softened feces, low-grade fever, lethargy and chronic history of weight loss were supposed to be infected with PTB. One hundred and thirty (130) PTB suspected animals were selected and subjected for microscopic screening using ZN stained smears test. Blood samples were randomly collected from the external jugular vein of 30 clinically suspected cases and serum obtained for ELISA test. Ten suspected cases showed acid fact bacilli were necropsied and gross lesion recorded and tissues samples were collected for histopathological and molecular examination. Fecal samples were collected in sterile containers from clinically suspected cases and preserved for molecular examination (RT-PCR) to detect MAP gene (IS900). The clinical screening indicate 130 suspected cases out of total 1500 examined animals (8.7%), Microscopic screening by Ziehl-Neelsen's stain of rectal scraping from suspected cases showed that 62 out of 130 (47.7 %), ELISA test revealed positive results of 11 out 30 (36.7%) samples and molecular examination ( RT-PCR) of 38 fecal samples revealed positive results of 25 (65.8%) samples. Gross pathology of suspected cases revealed thickening of the intestinal wall, corrugation and edematous of the mucosa and mesenteric lymph nodes hypertrophy and caseation. Histopathological examination of intestine tissues of suspected cases revealed cellular infiltration of epithelioid, lymphocyte, macrophage and giant cells and demonstration of free and phagocytosed acid fast bacilli. The molecular technique (RT-PCR) detected MAP (IS900) in all 10(100%) suspected tissues samples. The results of this study were detected the MAP organism in small ruminants in investigated area and evaluated the molecular technique (RT- PCR) were rapid, sensitive and specific test for detection of MAP in compare to ELISA and ZN stain smear tests. Histopathological examination showed the MAP bacilli and pathological changes of PTB in the section (semithin) and it was considered specific test when compared to molecular technique (RT-PCR) in tissues as standard.