High prevalence of extrapulmonary tuberculosis in dairy farms: Evidence for possible gastrointestinal transmission

Abstract

Bovine tuberculosis (bTB) caused by Mycobacterium bovis (M. bovis) represents one of major zoonotic diseases among cattle, it also affects the health of human, other domestic animals and wild life populations. Inhalation of infected aerosol droplets is considered as the most frequent route of the infection. This study aims to investigate the current forms of tuberculosis in cattle and identify the possible transmission modes in dairy farms of China. 13,345 cows from eight dairy farms in three provinces were comprehensively diagnosed by a multitude of assays, including SIT, CIT, IFN-γ assay and ELISA. It has been indicated that advanced infection of bTB was found in 752 (5.64%) cattle, suggesting a high prevalence of tuberculosis in these dairy farms. In the necropsy examination of 151 positive cattle, typical bTB lesions were observed in 131 cattle (86.75%), of which, notably, 90.84% lesions appeared in liver, spleen, mesenteric lymph nodes, mammary lymph nodes and other organs, taking up a large proportion among cattle with advanced bTB infection. 71.26% extrapulmonary tuberculosis (EPTB) was related to gastrointestinal system. M. bovis nucleic acid was further found in milk and feces samples and M. bovis was even isolated from milk samples. Phylogenetic analysis based on whole genome sequencing unraveled that six isolates were closely related to M. bovis AF2122/97 originated from UK, whereas four isolates shared close relation to M. bovis 30 from China, respectively. Our data demonstrate that the increase of EPTB transmitted by digestive tract is implicated in the current high prevalence rate of bTB in China, which also provides leads for bTB control in other countries with high prevalence of bTB in the future.

Fig 1. Lesion distribution.

(A) Proportion of extrapulmonary tuberculosis and pulmonary tuberculosis in slaughtered cattle. (B) Proportion of lesion sites in extrapulmonary tuberculosis. (C) Proportion of lesions associated with gastrointestinal system and other system.

Fig 2. Lesions observed by necropsy.

(A) Extensive cavitary lesions in liver. (B) Granulomas with caseous necrosis in mammary lymph node. (C) Granulomas with caseous necrosis in gastric lymph node. (D) Granulomas with caseous necrosis and mineralization in mesenteric lymph node. (E) Tuberculous granuloma in spleen. (F) Granulomas with caseous necrosis and mineralization in intestinal lymph node. (G) Granulomas with caseous necrosis and mineralization in hilar lymph node. (H) Big granulomas with caseous necrosis and mineralization in lung. (I) Miliary generalised tuberculosis on the thoracic cavity pleura (tuberculous “pearls”).

Fig 3. H&E staining of sections of hilar lymph nodes.

(A) Typical tuberculous granulomas consisted of three parts, as shown at the box: the center of the granuloma displays evident necrosis and mineralization (*); numerous epithelioid cells and Langhans’ giant cells are present in the intermediate layer of the granuloma (†); A large number of lymphocytes are present in the external layer of the granuloma (‡). Magnification ×200. Box (*), Box (†), and Box (‡) indicate areas enlarged in panel B, C, and D, respectively. (B) Amorphous pink caseous material composed of the necrotic elements of the granuloma as well as the infectious organisms. Magnification ×400. (C) Epithelioid cells aggregation and Langhans’ giant cells could be seen subsequently. Magnification ×400. (D) Redundant lymphocytes infiltrated. Magnification ×400.

Fig 4. Phylogenetic trees of ten isolates.

The optimal tree with the sum of branch length = 1.71277593 was shown in Fig 4. The evolutionary distances were calculated using the Maximum Composite Likelihood method and were in the units of the number of base substitutions per site. This analysis involved 17 nucleotide sequences. All ambiguous positions from each sequence pair were removed (pairwise deletion option). There were a total of 3,914 positions in the final dataset. Evolutionary analyses were conducted in MEGA 5.