Unexpected high responses to tuberculin skin-test in farmed red deer: implications for tuberculosis control.

Tuberculosis (TB) in deer is a serious zoonotic disease of worldwide distribution. Detection of infected animals is usually performed using single or comparative skin-testing (SST/CST), although false responses due to sensitization to other mycobacteria may occur, hampering diagnostic specificity. We describe the evolution of the responses to the SST, CST and to an in-house serological assay in a red deer farm subjected to regular TB testing in southern Spain in an attempt to understand the dynamics of possible non-specific reactions occurring under field conditions. We performed 2288 skin-tests and ELISAs in nine sampling periods between May 2009 and January 2011. In May 2010, a strong increase in skin fold thickness in response to avian purified protein derivative (PPD) (mean=4.0mm, 95% CI=3.5-4.5) and bovine PPD (mean=1.8mm, 95% CI=1.6-2.0) was observed in yearling deer hinds (n=150), compared to values recorded for the same individuals in November 2009 (avian PPD: mean=0.7 mm, 95% CI=0.6-0.8 and bovine PPD: mean=0.7 mm, 95% CI=0.6-0.7) and in January 2011 (avian PPD: mean=2.2mm, 95% CI=1.9-2.4 and bovine PPD: mean=1.1mm, 95% CI=1.0-1.2). Using SST, 54 animals (36%) of the yearlings tested in May 2010 would have been classified as positive reactors, while none of them was positive in the CST. The five animals with highest skin fold increases to mycobacterial antigens were culled and subjected to post-mortem analysis, which confirmed the absence of Mycobacterium tuberculosis complex (MTBC) infection but demonstrated the presence of environmental mycobacteria and closely related bacteria in four out of the five analyzed animals. Our results demonstrated how non-specific responses to mycobacterial antigens can adversely affect the specificity of TB diagnosis based on the SST. Thus, once TB infection has been ruled out using confirmatory techniques, application of comparative diagnostic tests is highly advisable to maximize test specificity and avoid the slaughter of false positive reactors.

No evidence that wild red deer (Cervus elaphus) on the Iberian Peninsula are a reservoir of Mycobacterium avium subspecies paratuberculosis infection.

The potential role of red deer (Cervus elaphus) as a reservoir of Mycobacterium avium subspecies paratuberculosis (MAP) infection is largely unknown. A total of 332 wild red deer were investigated using post-mortem examination, bacteriology and serology. Only three animals (1.12%) were found to have lesions on histopathological examination and no MAP bacteria were recovered on culture. The results suggest it is unlikely that wild red deer make a significant contribution to the maintenance of MAP infection in the region. The cross-reactivity of the ELISAs used indicates this diagnostic modality is ineffective in the detection of MAP infection in this species. The implications of these results for the control of this important pathogen in both livestock and wildlife are discussed.

The testing season affects red deer skinfold increase in response to phytohaemagglutinin.

Red deer (Cervus elaphus) have a pronounced seasonality in their physiology. The aim of this study was to assess the effect of the season on red deer responsiveness to skin testing with the phytohaemagglutinin (PHA) mitogen. Study subjects included 270 farmed adult red deer (19 stags and 251 hinds). The skin testing was carried out between January 2009 and August 2010. The animals were injected intradermally with a 0.1 ml volume containing 250 µg of PHA diluted in phosphate buffered saline. The skinfold thickness was measured immediately prior to injection and 72 h after administration, always by the same person and with three repeats per measurement. Single effects of sex and time on skin test responsiveness were significant (p < 0.001) as well as their interaction (p < 0.001). In winter (January), and considering the average of two years, the skinfold increase in response to the intradermal injection of 250 µg PHA was 2.1 times larger in stags and 1.4 times in hinds than in summer (August). While stags had 1.3 times larger responses than hinds in winter, the inverse occurred in summer, with 1.1 times larger responses in hinds. We also evidenced a limited inter-annual variation of skinfold increase in response to PHA in red deer. These findings have important consequences regarding the interpretation of skin test results in the ante-mortem diagnosis of tuberculosis and paratuberculosis, at least in deer.

Ante-mortem testing wild fallow deer for bovine tuberculosis.

This study aimed to maximize the sensitivity of bovine tuberculosis detection in living wild fallow deer (Dama dama) under field conditions. We evaluated the rapid test (RT; CervidTB STAT-PAK Assay, Chembio Diagnostic Systems, Inc., USA) in comparison with the comparative cervical skin test (CCT). A total of 134 fallow deer were captured between January and March 2008. At time 0, 0.1 ml of avian purified protein derivative (avian PPD; Cooper-Zeltia, Spain), 0.1 ml bovine PPD (Cooper-Zeltia, Spain), 0.1 ml negative control PBS and 0.1 ml of a positive control (the mitogen phytohaemagglutinin, PHA; containing 250 mg PHA, diluted in PBS) were injected intradermally at four shaved sites in the neck. The skin fold thickness at each injection site was measured at time 0 and 72 h (3 repeats each time). Animals with a skin test response of 2mm or more at the bovine PPD injection site and animals with any visible reactivity in the RT were necropsied and tissues submitted for culture and for histopathology. A total of 36 fallow deer were considered reactors to bovine PPD or to the RT (apparent prevalence 27%). Regarding both bovine PPD reactivity and the skin fold increase at the PHA injection site, we found significant effects of age and sex by age interaction. Adult males had the largest responses. Mycobacterium bovis was isolated from lymphoid tissues of 21 fallow deer. Skin test sensitivity, as compared to M. bovis culture confirmed deer, was 80.1% (17/21). But, the CCT alone would have missed 4 of 21 culture confirmed animals. RT sensitivity, based on culture confirmed deer, was also 80.1% (17/21). Similarly, the RT alone would have missed another 4 of 21 culture confirmed deer. However, combining the CCT and the RT allowed for detecting all 21 culture positive fallow deer. We conclude that the combined application of the RT and the skin testing can maximize the sensitivity of bTB detection in living fallow deer, thus facilitating control programs for wildlife disease surveillance.

Distribution of lesions in red and fallow deer naturally infected with Mycobacterium bovis.

Wild deer have an important role in the epidemiology of bovine tuberculosis (bTB). The aims of this study were (1) to compare the pattern of lesions present in wild red (Cervus elaphus) and fallow (Dama dama) deer that were naturally infected with Mycobacterium bovis, and (2) to use this information to develop a sampling strategy for the isolation of M. bovis from the lymphoid tissues of the head of these animals. Culture of head lymphoid tissues demonstrated that 28 of 95 red deer and 22 of 100 fallow deer sampled were infected with M. bovis. Approximately 30% of each deer population had no gross lesions. Fallow deer were significantly more likely to have thoracic lesions than red deer. Lesions were observed in the retropharyngeal lymph nodes of 64% of the culture-positive red deer and 43% of the culture positive fallow deer. One third of the red deer, but none of the fallow deer, had well-encapsulated abscess lesions. There were no microscopical differences in the lesions in the lymph nodes of the red and fallow deer. Bacteriological culture from both the tonsil and retropharyngeal lymph nodes increased the rate of isolation of M. bovis by 22% over culture of the retropharyngeal lymph nodes alone in both species. These findings indicate that investigation of wild deer for bTB-compatible lesions should include examination of the medial retropharyngeal, left tracheobronchial, mediastinal, mesenteric and ileocaecal lymph nodes. Sampling for bacteriological culture from head lymphoid tissues should be from the tonsil and the medial retropharyngeal lymph nodes. These protocols may prove useful in bTB surveillance and control in regions where wild deer contribute to the circulation of M. bovis.

First data on Eurasian wild boar response to oral immunization with BCG and challenge with a Mycobacterium bovis field strain.

The Eurasian wild boar (Sus scrofa) is considered a reservoir for bovine tuberculosis (bTB) caused by Mycobacterium bovis and closely related members of the Mycobacterium tuberculosis complex in south-central Spain. The vaccination of wildlife with BCG offers an alternative to culling and to movement restriction for the control of bTB among wildlife reservoirs. In this study, we hypothesized that oral BCG immunization of wild boar would affect the expression of immunoregulatory genes and confer protection against M. bovis. Three groups were used to describe the infection, pathological findings and gene expression profiles in wild boar: BCG-vaccinated and M. bovis-challenged (vaccinated challenged group; N=6), non-vaccinated and M. bovis-challenged (non-vaccinated challenged group; N=4), and non-vaccinated and mock-infected (control group; N=2) animals. M. bovis was isolated from 50% (3/6) and 75% (3/4) of vaccinated challenged and non-vaccinated challenged animals, respectively. All four wild boar from the non-vaccinated challenged group developed bTB-compatible lesions 114 days after challenge. In contrast, only 50% of vaccinated challenged wild boar developed lesions. The PBMC mRNA levels of IL4, RANTES, C3, IFN-gamma and methylmalonyl-CoA mutase (MUT) were analyzed at several days post-vaccination (dpi). When vaccinated challenged animals were compared to controls, all five genes were significantly upregulated at the time of M. bovis infection at 186dpi but IFN-gamma levels were also upregulated at 11 and 46dpi. The C3 and MUT mRNA levels were higher at 46dpi, and 11 and 186dpi, respectively, in vaccinated protected wild boar when compared to non-vaccinated challenged animals. At the end of the experiment (300dpi), the mRNA levels of selected genes were lower in non-vaccinated challenged animals when compared to control wild boar. Exposing wild boar to a dose of 10(4)cfu of M. bovis by the oropharyngeal route is an adequate protocol to produce an infection model in this species. Our results suggested that oral BCG immunization of wild boar results in the upregulation of immunoregulatory genes that may be associated with protective response to M. bovis infection in this species. More studies on vaccine efficacy, delivery, and safety will be needed to confirm if oral vaccination with BCG could be used in bTB control programs for reducing M. bovis infection and clinical disease in wild boar.

Massive presence of Echinococcus granulosus (Cestoda, Taeniidae) cysts in a wild boar (Sus scrofa) from Spain.

We report a heavy infestation of a free-living wild boar sow from Spain with Echinococcus granulosus cysts and state its molecular characterization. We found >65 hydatid cysts in the thoracic and abdominal cavities of the sow. Parasites were routinely processed for their identification and histopathology and DNA molecular characterization of the E. granulosus cysts were carried out. The polymerase chain reaction results confirmed the E. granulosus identity of the cysts and restriction fragment length polymorphism and sequencing revealed its G1 genotype. Our results suggest that wild boar could be involved in the epidemiology of E. granulosus, particularly considering that large amounts of carcass remains are available to dogs and wolves during the hunting season. The recent population increase of the wild boar in Spain and the DNA confirmation that the wild boar isolate shared identical sequences to the sheep strain emphasize the importance of the reported finding in public health.