Serological investigation and genotyping of Mycobacterium avium subsp. paratuberculosis in sheep and goats in Inner Mongolia, China

Abstract

Paratuberculosis a contagious and chronic disease in domestic and wild ruminants, is caused by Mycobacterium avium subspecies paratuberculosis (MAP). Typical clinical signs include intractable diarrhea, progressive emaciation, proliferative enteropathy, and mesenteric lymphadenitis. Paratuberculosis is endemic to many parts of the world and responsible for considerable economic losses. In this study, different types of paratuberculosis and MAP in sheep and goats were investigated in Inner Mongolia, a northern province in China contiguous with two countries and eight other provinces. A total of 4434 serum samples were collected from six cities in the western, central, and eastern regions of Inner Mongolia and analyzed using the ELISA test. In addition, tissue samples were collected from seven animals that were suspected to be infected with MAP. Finally, these tissues samples were analyzed by histopathological examination followed by polymerase chain reaction (PCR), IS1311 PCR-restriction enzyme analysis (PCR-REA), and a sequence analysis of five genes. Among all 4434 ruminant serum samples collected from the six cities in the western, central, and eastern regions of Inner Mongolia, 7.60% (337/4434) measured positive for the MAP antibody. The proportions of positive MAP antibody results for serum samples collected in the western, central, and eastern regions were 5.10% (105/2058), 6.63% (85/1282), and 13.44% (147/1094), respectively. For the seven suspected infected animals selected from the herd with the highest rate of positivity, the gross pathology and histopathology of the necropsied animals were found to be consistent with the pathological features of paratuberculosis. The PCR analysis further confirmed the diagnosis of paratuberculosis. The rest of the results demonstrated that herds of sheep and goats in Inner Mongolia were infected with both MAP type II and type III. To the best of our knowledge, this is the first study of the two subtypes of MAP strains in sheep and goats in Inner Mongolia.

Fig 1. Bowel wall thickening and mucosal folding in the cecum.

Fig 2. Inflammation of mesenteric lymph nodes; grayish-white lesions of different sizes were observed on the surface and the cut section.

Fig 3. Proliferation of epithelioid cells and lymphocytes in ileum propria (hematoxylin and eosin, ×200).

Fig 4. Epithelial hyperplasias in the cortex of the lymph node (hematoxylin and eosin, ×100).

Fig 5. A significant number of acid-fast bacilli were observed in the cytoplasm of epithelioid cells in cecum propria (Ziehl–Neelsen, ×400).

Fig 6. PCR results of seven pathological tissue samples obtained from the amplification of the DMC gene (M: Maker; NC: Negative control; 1 to 4: Case-1 to Case-4; 5 to 6: Case-6 and Case-7; 7: Case-5).

Fig 7. PCR-REA results of seven pathological tissue samples (M: Maker; 1 to 4: Case-1 to Case-4; 5 to 6: Case-6 and Case-7; 7: Case-5).

Fig 8. Phylogenetic tree of IS900 gene sequences from MAP.

The evolutionary history was inferred using the neighbor-joining method base on the Kimura 2-parameter model. The analysis involved 26 nucleotide sequences. There were a total of 400 positions in the final dataset. Evolutionary analyses were conducted in MEGA5. Sequences of this study were marked with a black triangle (▲).