A Multiplex Approach to Molecular Detection ofBrucella abortusand/orMycobacterium bovisInfection in Cattle

Abstract

A multiplex amplification and detection platform for the diagnosis of Mycobacterium bovis and Brucella abortusinfection simultaneously in bovine milk and nasal secretions was developed. This system (designated the bovine pathogen detection assay [BPDA]-PCR) consists of duplex amplification of species-specific targets (a region of the BCSP31K gene of B. abortus and a repeat-sequence region in the hsp65 gene of M. bovis, respectively). This is followed by a solid-phase probe capture hybridization of amplicons for detection. On the basis of spiking experiments with normal milk, the analytical sensitivity of the assay was 800 CFU equivalents/ml of milk for B. abortus and as low as 4 CFU equivalents per ml of milk for M. bovis. BPDA-PCR was validated with 45 liver samples from lemmings experimentally infected with B. abortus. The assay sensitivity, based on culture status as a “gold standard,” was 93.9%. In this experiment, BPDA-PCR also identified five culture-negative liver samples as positive (41.7%). Field studies for the evaluation of BPDA-PCR were performed with samples from dairy animals from geographically distinct regions (India, Mexico, and Argentina). A high prevalence of shedding of B. abortus(samples from India) and M. bovis (samples from Mexico) was identified by BPDA-PCR. In samples from India, B. abortusshedding was identified in 86% of milk ring test-positive animals (n = 15) and 80% of milk ring test-negative cows (n = 5). In samples from Mexico, M. bovis was identified by PCR in 32.6% of pools (n = 46) of milk that each contained milk from 10 animals and in 56.2% of nasal swabs (n = 121) from cattle from tuberculin test-positive herds. In contrast, the Argentine cattle (n = 70) had a modest prevalence of M. bovis shedding in nasal swabs (2.9%) and milk (1.4%) and ofB. abortus in milk (11.4%). On the basis of these analyses, we identify BPDA-PCR as an optimal tool for both screening of herds and testing of individual animals in a disease eradication program. A combination of the duplex assay, screening of milk samples in pools, and the proposed algorithm provides a highly sensitive, cost-effective, and economically viable alternative to serological testing.

Heterogeneous shedding ofBrucella abortusin milk and its effect on the control of animal brucellosis

“…More plausible is the alternative explanation that the low specificity of the serological tests observed in this study reflects the high number of false-positive reactions. The concordance between results from cultural and PCR tests reported here and the results from numerous studies (Blasco et al 1994;Dobson and Meagher 1996;Sreevatsan et al 2000) supports this contention. Notably, the rate of seropositive animals reported here, 2AE0 to 2AE5 times those positive to the cultural test (Table 4), is reasonably close (3AE0 times) to that reported studying brucellosis in the bison (Dobson and Meagher 1996).…”

“…Exposure does not necessarily imply that the animals are infected (Blasco et al 1994;Dobson and Meagher 1996;Sreevatsan et al 2000;Godfroid et al 2002). There is in fact evidence that animals can clear the infection (Harmon et al 1989;Price et al 1990;Sreevatsan et al 2000;Godfroid et al 2002). It could be argued that our data contain a large number of chronically infected animals, among which false culturenegative individuals occur frequently (Cheville et al 1998;Sreevatsan et al 2000).…”

“…There is in fact evidence that animals can clear the infection (Harmon et al 1989;Price et al 1990;Sreevatsan et al 2000;Godfroid et al 2002). It could be argued that our data contain a large number of chronically infected animals, among which false culturenegative individuals occur frequently (Cheville et al 1998;Sreevatsan et al 2000). This possibility, however, is not realistic because the herds included in the present study are monitored periodically by the veterinary service.…”

“…As is well known, B. abortus can be shed in the milk of infected animals intermittingly, in cattle (Sreevatsan et al 2000) and other species (Blasco et al 1994). This study supports this result and, in addition, shows that in the water buffalo, while the majority of the animals shed B. abortus at low levels (£10 3 CFU ml )1 ), a small number of individuals shed B. abortus persistently and at high levels ( ‡10 4 CFU ml )1 ) ( Table 1).…”

“…False positive reactions result from the presence in test sera of antibodies against organisms cross-reacting with B. abortus (Godfroid et al 2002;Gall and Nielsen 2004) or from exposure, but not infection of the animal to the pathogen. Exposure does not necessarily imply that the animals are infected (Blasco et al 1994;Dobson and Meagher 1996;Sreevatsan et al 2000;Godfroid et al 2002). There is in fact evidence that animals can clear the infection (Harmon et al 1989;Price et al 1990;Sreevatsan et al 2000;Godfroid et al 2002).…”