Development and evaluation of a diagnostic cytokine-release assay for Mycobacterium suricattae infection in meerkats (Suricata suricatta)

We have developed a novel diagnostic assay for M. suricattae infection in meerkats which measures antigen-specific IP-10 release in whole blood incubated with Bovigam® PC-HP peptides. All meerkats with no known exposure to M. suricattae (n?=?10) tested negative with the IPRA, while 35 out of 101 (34.6%) animals with known exposure to this pathogen tested positive. Although M. suricattae infection in these individuals was not confirmed, the odds of a meerkat testing positive were significantly greater for animals with a high risk of infection when compared to those with a low risk of infection, supporting the validity of the IPRA.

IP-10 has been shown to be a useful diagnostic biomarker for detection of M. tuberculosis infection in humans [9] and M. bovis infection in African buffaloes [11]. In both these species, IP-10 is produced in greater amounts than IFN-? in antigen-stimulated whole blood and has been shown to be a more sensitive marker of mycobacterial infection [8, 9, 13]. In the present study, despite the use of ELISA antibodies produced against distantly related species, measurement of IP-10 proved to be a useful marker of immune activation. While this indicates the sensitivity of the ELISA for meerkat IP-10, it may, in part, reflect the abundance of this molecule in antigen-stimulated samples. Moreover, the similarity between the amino acid sequences of cattle, horses, cats and humans is significantly greater for IP-10 than for IFN-? (data not shown) and this may explain the increased potential for cross-species reactivity of the anti-IP-10 antibodies. For these reasons, IP-10 may also be a useful diagnostic target in other species.

In order to define a diagnostic cut off value for the IPRA, we tested 10 captive meerkats with no known exposure to M. suricattae. These animals showed no significant difference in their IP-10 responses to PBS and PC-HP peptides indicating that their selection as uninfected controls was appropriate. Moreover, a threshold value of OD^HP-Nil??0.038 classified all control animals as test-negative. Such a low threshold will increase the sensitivity of the assay, thereby reducing the number of false negative test results; however, this could be at the expense of specificity [14]. In part, this low cut off value resulted from plasma samples being diluted 1:4 in the present study and a lower dilution factor might improve test accuracy. However, this was not tested in the present study because of limited sample volumes.

In contrast to the control animals, 35 of 101?M. suricattae-exposed meerkats displayed significant IP-10 responses to the PC-HP peptides, confirming the antigenicity of this peptide pool for these animals. However, it is currently unclear which components of the PC-HP antigens might be responsible for this immune sensitization. The genetic region of difference 1 (RD1), a variant of which is deleted from the M. suricattae genome [15], encodes both ESAT-6 and CFP-10 and is also required for the secretion of Esx-1 substrate protein C (espC) which is encoded by the gene Rv3615c [16]. While Rv3615c is present in the M. suricattae genome (pers. comm., Anzaan Dippenaar), it is possible that this component of the PC-HP peptide pool, in addition to ESAT-6/CFP-10, would have a limited diagnostic contribution to detection of M. suricattae-infected animals. Nonetheless, the use of the PC-HP reagent is supported by the fact that the odds of an animal testing IPRA-positive were significantly greater for meerkats with the greatest risk of M. suricattae infection. Moreover, our results suggest that the PC-HP peptides might be useful for diagnostic testing of species infected with related RD1-deleted strains, i.e. M. microti, M. mungi and the dassie bacillus [5].

In both the captive and free-living populations, the median OD^PWM was significantly higher than the values for either OD^Nil or OD^HP, indicating that PWM is an appropriate mitogen in this species. Seven animals were excluded from our analysis based on negative OD^PWM-Nil values; however, in 5 of these cases, the exclusion criterion was met as a result of unusually high OD^Nil values, not failure to respond to PWM. Similar spontaneous release of IP-10 has previously been seen in cattle [17], although the mechanism for this phenomenon is not currently understood. In the present study, although the positive control was intended to confirm the viability of cytokine production in blood samples, it additionally served to identify samples where high OD^Nil values would have affected the test interpretation. In such cases, repeat sampling of the animal would be recommended.