INTRODUCTION

Bovine tuberculosis (BTB) has affected wildlife in South Africa (SA) for almost a century (Paine and Martinaglia 1929; Jolles et al. 2005) and the disease is now enzootic in the Greater Kruger National Park Complex and Hluhluwe-iMfolozi Park (Michel et al. 2009; Hlokwe et al. 2011). The disease status of animals in private game parks, reserves, and farms in the country is largely unknown, but sporadic disease reports indicate a number of BTB cases involving different wildlife species (Hlokwe et al. 2014).

Wildlife maintenance hosts of tuberculosis pose major difficulties to controlling infection in cattle and other wildlife species with which they interact (Michel et al. 2006). African buffaloes (Syncerus caffer) are maintenance hosts of BTB (Michel and Bengis 2012), and there is growing evidence that greater kudu (Tragelaphus strepsiceros) have maintenance host potential (Keet et al. 2001; Michel et al. 2009). Movement of infected livestock and wildlife species is one of the most important risk factors in the transmission of BTB (Skuce et al. 2012). In SA, movement control measures in the wildlife industry apply only to African buffaloes. Outside the foot-and-mouth disease control zones, there are no restrictions for other wildlife species that may play a role in the spread of several high-impact livestock diseases such as BTB (Woodford and Rossiter 1993; De Vos et al. 2001). The wildlife industry is the fastest growing agricultural sector in SA and provides sources of income not only through ecotourism but also game sales, hunting, and consumption (van der Merwe and Saayman 2003).

Our aim was to unravel the descriptive and molecular epidemiology underlying the BTB scenario that led to the infection of African buffaloes in the Madikwe Game Reserve (GR) with Mycobacterium bovis, detected during routine testing of buffaloes for auction.

MATERIALS AND METHODS

RESULTS

Intradermal tuberculin testing

Single intradermal tuberculin testing: Two buffaloes reacted to bovine PPD with increases in skinfold thickness ≥6 mm (including PH713; Table 1). Nine buffaloes had an inconclusive skin reaction (increase in skinfold thickness ≥4 mm).

Table 1.

Culture, skin test, and interferon gamma results of 2012–13 testing for bovine tuberculosis (TB; Mycobacterium bovis) of euthanized African buffaloes (Syncerus caffer) captured in Madikwe Game Reserve, South Africa.

a 

IDT = intradermal tuberculin testing.

b 

ND = not done.

Comparative intradermal tuberculin testing (3 mo after the single IDT): Three of 51 buffaloes were BTB-positive based on the reaction to bovine PPD of >4 mm of the reaction to avian PPD. Five buffaloes were regarded as suspect based on an increase in bovine skinfold thickness of 1–4 mm greater than the avian reaction (including 39-221392 and 4-256762; Table 1).

Comparative intradermal tuberculin testing (twelve months after the single IDT): Eight buffaloes (including 33-257215, 38-240440, 32-252380, 39-221392, and 4-256762; Table 1) were BTB positive. These included two positive animals detected in the initial comparative IDT. Results for seven animals were inconclusive.

Genotyping

Spoligotyping of the isolates from different tissue samples from all three buffaloes yielded a pattern corresponding to SB0140 in the international M. bovis database ( www.mbovis.org). We found two VNTR profiles with 90% relatedness differing only at one VNTR locus (M tub 12). We detected one VNTR profile (Fig. 1; TB 8146) in animal PH713 and another in isolates from animals 4-256762 and 38-240440 (Fig. 1; TB 8277C and TB 8278A).

Figure 1.

Dendrogram based on variable number of tandem repeat (VNTR) typing data illustrating the genetic relationships of Mycobacterium bovis isolates from African buffaloes (Syncerus caffer) in South Africa. KZN=KwaZulu-Natal Province; NW=North West Province; MGR=Madikwe Game Reserve; GP=Gauteng Province; MP=Mpumalanga Province; LP=Limpopo Province; EC=Eastern Cape Province; Baboon=Papio ursinus; Buffalo=Syncerus caffer; Cattle=Bos taurus; Nyala=Tragelaphus angasii; greater Kudu=Tragelaphus strepsiceros; Black rhinoceros=Diceros bicornis.

DISCUSSION

We have used traditional outbreak investigations and molecular typing to confirm and characterize M. bovis isolated from African buffaloes in a reserve previously free of M. bovis infection in the North West Province of SA. The source and time of introduction of this M. bovis strain into the game reserve are unknown. We investigated several possible sources. The Madikwe GR is bordered by three cattle farms on the southern border and by communal villages in the west. The M. bovis strain we detected could not be linked to any of the cattle strains isolated in the North West Province (Fig. 1); the prevalence of BTB in the province is very low. It is unlikely that buffaloes in this particular game reserve could have contracted the disease from cattle on the neighboring farms. In addition, the game reserve had a well maintained electric fence bordering the communal grazing land to prevent wild animals from escaping and causing conflict with cattle owners. This measure is considered acceptably effective in preventing cattle from straying into the reserve. Alternatively, the disease could have been introduced with the founder buffalo between 1992 and 1996. This is, however, unlikely because of their negative skin test status and the negative tuberculosis history of the herds of origin. The introduction of the plains game species in large numbers and from different sources during the 8 yr prior to the confirmation of the index case is, in retrospect, considered a high-risk management intervention which most-likely facilitated the establishment of BTB in the resident buffalo population.

Spoligotype SB0140 was detected in the Madikwe GR buffaloes and also in cattle and wildlife species in other parts of the country (Hlokwe et al. 2014). Strains bearing the same spoligotype pattern are assumed to be a set of individuals derived relatively recently by clonal replication from a single ancestral cell (Smith et al. 2006). On the other hand, VNTR analysis allows for higher resolution of genetic patterns within the SB0140 clone and therefore leads to improved discrimination between isolates associated with individual, epidemiologically unrelated outbreaks. With further tracing, we found that the VNTR genotype of the M. bovis strain from Madikwe GR was identical to the genotype isolated from a moribund chacma baboon associated with the Mkuze GR in KZN Province. As shown in Figure 1, all isolates with more than 85% genetic similarity with the M. bovis strain in the Madikwe GR stemmed from KZN, with or without intermediate translocation. These molecular findings are strongly suggestive of an epidemiologic link between BTB detected in KZN and the Madikwe GR. The VNTR genotypes from the buffaloes in Madikwe GR, the chacma baboon, greater kudu, and nyala are suggested to have evolved from the most-recent common ancestor, which is a genotype isolated from cattle (Fig. 1). These findings further support our knowledge that cattle historically served as source of BTB for wildlife species (Thorburn and Thomas 1940).

According to official premovement testing records, no M. bovis reactors were detected in any of the groups of buffalo sold in auctions during the previous 2 decades. However, this testing was sporadic and selective for prime animals. It is therefore plausible that, in the absence of a sound surveillance program for the entire African buffalo population, the presence of the BTB remained undetected until it had reached a prevalence that made it more likely for infected buffalo in prime body condition to be selected for preauction testing. This is supported by the observation that none of the three culture-positive buffaloes showed an indication of advanced or generalized tuberculosis. We speculated that the introduction of BTB in the game reserve could have been a recent event. The diagnosis of BTB in this game reserve suggests that control measures previously applied were inadequate due to the insidious nature of the disease. In SA, the mandatory test for BTB in African buffalo is the IDT test, and IFNγ is only recommended as ancillary. Overall, our findings emphasize that caution should be taken not to rely solely on ad hoc testing for the diagnosis of BTB in wild animals because the time of infection is unknown in most cases.

Knowledge of the fact that BTB-infected African buffaloes may have been sold to both national and international buyers further complicates the situation, as BTB may have been translocated with auctioned buffaloes to several properties free of M. bovis infection. Madikwe GR is on the national border with Botswana. There is also a concern of the disease spreading into Botswana, whose national cattle population is officially free of BTB (Jori et al. 2013). Detection of M. bovis in the buffalo population in Madikwe GR has already had negative financial implications for the reserve's income generation because the land remains under quarantine and no further buffalo sales have been allowed. Our study highlights the significance of M. bovis as a silent, growing, disease risk factor in game translocation in the absence of herd or property-based surveillance.

We highlight the importance of suitable long-term surveillance programs for early detection of BTB at the herd level. In SA, movement control measures in wildlife apply only to African buffaloes. The current rate of BTB spread among wildlife properties, however, suggests expansion of testing requirements to at least those species that previously have been diagnosed with BTB (Hlokwe et al. 2014) and those in high demand for sale at game auctions. In view of the limitations of available tests, we need to develop and validate diagnostic tests for wildlife species other than buffalo.