Experimental Infection of Captive Red Foxes (Vulpes vulpes) with Mycobacterium bovis

Abstract

In Europe, animal tuberculosis (TB) due to Mycobacterium bovis involves multi-host communities that include cattle and wildlife species, such as wild boar (Sus scrofa), badgers (Meles meles) and red deer (Cervus elaphus). Red fox (Vulpes vulpes) infections have also been recently reported in some TB endemic regions in the Iberian Peninsula and France, with some of the infected animals shedding M. bovis in urine and feces. In order to understand the pathogenesis of M. bovis infection in foxes and the associated risk of transmission, 12 captive foxes (6 females and 6 males) were inoculated orally with 2 × 10⁷ colony-forming units of a French field isolate of M. bovis. Clinical samples (urine, feces and oropharyngeal swabs) were collected every four weeks and tested for molecular diagnosis and bacteriology. Serological responses were measured by IDEXX M. bovis Ab Test and Multi Antigen Print Immunoassay (MAPIA). At a post-mortem examination performed 12 weeks post infection (wpi), tissues were tested for the presence of M. bovis and associated gross and microscopic TB-like lesions. M. bovis was detected by PCR in bladder swabs of 3 animals at 12 wpi. It was also detected pre-mortem at different time points of the experiment in the oropharyngeal mucus of three individuals and in the feces of nine foxes, with two of them confirmed by bacteriology. All 12 foxes had at least 4 PCR positive samples (out of the 23 tested), and all but 1 fox had at least 1 culture positive sample. The culture negative fox was PCR positive in both retropharyngeal and mesenteric lymph nodes, in line with the results of the other animals. Seroconversion was observed in all foxes except one during the experiment, and in nine at the final time point. No gross visible lesions were found in any animal at the post-mortem examination. The histology showed small granulomas within the lymph nodes, tonsils, liver and lungs from eight animals, with the presence of few acid-fast bacilli. These results confirmed that all orally-infected foxes developed mild TB lesions but they were able to shed mycobacteria in about 75% of cases, 1 month post-infection (9 out 12 foxes). These results show that it is possible to induce typical TB infection experimentally in captive foxes, with measurable M. bovis excretion; such an experimental system could be useful for future evaluations of diagnostics and vaccines in this species.

Keywords: Vulpes vulpes; excretion; pathogenesis; red fox; serology; tuberculosis.

Figure 1

PCR and bacteriology (in green), and PCR only (in yellow) results in all tissues. LN: lymph node.

Figure 2

Number of infected tissues by bacteriology (blue) and PCR (yellow) for each of the 12 foxes (A to L) experimentally infected with Mycobacterium bovis.

Figure 3

Bacteriology (colony forming units (CFU/1.8 mL) (in blue) and PCR IS6110 (Cycle threshold (CT)) (yellow points) levels of infection in the left (a) and right (b) retropharyngeal lymph nodes (LN), tonsils (c) and mesenteric LN (d).

Figure 4

Number of foxes positive by PCR and bacteriology simultaneously (in green), and by PCR only (in yellow) in shedding material.

Figure 5

Examples of lesions observed by histopathology in foxes. (A) Multiple coalescing type I granulomas (*) within the liver (fox D); bar = 100 µm. (B) Necrotic type III granuloma (*) in the periphery of the mandibular lymph node (fox B); bar = 200 µm. (C) Necrotic type III granuloma (*) in the retropharyngeal lymph node; bar = 200 µm. (D) Large type IV granuloma with caseotic necrosis (*) in the mesenteric lymph node (fox G); bar = 200 µm; insert = acid-fast bacilli (arrows) within the necrotic area stained by the Ziehl–Neelsen technique. (E) Multiple type IV granulomas (arrows) within the lung parenchyma (fox I); bar = 500 µm. (F) Solid type I granuloma (*) in the tonsil (fox D).