A systematic review of current immunological tests for the diagnosis of cattle brucellosis
Introduction
Bacteria of the genus Brucella cause brucellosis, a zoonosis of worldwide impact inducing abortions and infertility in a variety of wild life forms and domestic livestock, the latter being the most common source of human brucellosis, a grave and debilitating disease (Zinsstag et al., 2011). Cattle and small ruminants are respectively the preferential hosts of B. abortus and B. melitensis, but the latter species also infects cattle in mixed breeding systems (Corbel, 1997; Verger, 1985) and, although rarely, cattle can also be infected by some B. suis biovars (Corbel, 1997; Ewalt et al., 1997; Musser et al., 2013; Szulowski et al., 2013; Tae et al., 2012). Diagnosis and vaccination are essential for control and eventual elimination and surveillance of this disease.
The diagnosis of brucellosis in cattle is based on bacteriological and immunological tests, which include DTH3 and serological tests. In infected animals, DTH is elicited by protein antigens, and antibodies recognize the different sections of the S-LPS, mostly its O-polysaccharide or OPS, the cognate NH polysaccharide or proteins (Ducrotoy et al., 2016). Anti-S-LPS antibodies in serum include IgM, IgG and IgA. IgM dominates in the early phase but is rapidly replaced by IgG and to a lesser extent by IgA. During the early stages of infection, all these antibodies display agglutinating ability at neutral pH; as the infection evolves, non-agglutinating IgG and IgA antibodies progressively replace agglutinating antibodies. Non-agglutinating antibodies, however, become agglutinating at acid pH. Accordingly, S-LPS tests vary in their ability to detect those immunoglobulin classes, depending on pH (neutral in SAT and acid in the buffered plate agglutination tests [i.e. RBT, CT and BPAT), specificity of immuno-conjugate (immunosorbent assays) and the effect detected (agglutination, precipitation, complement consumption or primary binding; see Table 1). Vaccination, however, also elicits both DTH and antibody responses (Ducrotoy et al., 2016). The vaccines recommended by OIE are B. abortus S19 and B. abortus RB51 (OIE, 2016). Vaccine S19 elicit antibodies to the S-LPS whose intensity depends on age, dose and route of vaccination, and RB51 (a rough vaccine) triggers antibodies to the LPS lipid A-core but not to the OPS (Ducrotoy et al., 2016). Moreover, bacteria carrying OPS structurally close to the OPS of S brucellae can cause FPSR in S-LPS or OPS tests but not in protein tests (Ducrotoy et al., 2016).
There is a long list of brucellosis diagnostic tests and candidates (Ducrotoy et al., 2016; McGiven, 2013). Those that have come into use have only been the topic of partial reviews (Bale, 2002; Godfroid et al., 2010; Kaltungo et al., 2014; Nielsen, 2002; Nielsen and Yu, 2010; Poester et al., 2010; Smirnova et al., 2013) that with two exceptions (Anon., 2006; Greiner et al., 2009) have not critically examined the methodology of the studies reviewed. To fill this gap, in the present work the determination of the DSe and DSp4 is discussed first, and then strict criteria applied to a systematic review of the literature. Similarly, no work has so far discussed the application of the tests in the different contexts of brucellosis (infection and no vaccination, mass vaccination; elimination [i.e. local eradication]) based on vaccination and test-and-slaughter, and surveillance with presence of FPSR). Thus, the systematic review is complemented by a discussion of the application of tests.
Section snippets
Diagnostic sensitivity and specificity in the context of brucellosis
The capacity of a test to accurately predict the infection status of the host depends essentially on its DSe and DSp, parameters that are determined using samples of sufficient numbers5
Studies reviewed
A systematic review was conducted following Prisma guidelines (Moher et al., 2009) (Fig. 2 and Supplemental Table S0). The eligibility criteria (Table 2) overlap with those of the scientific panel of experts commissioned by the European Food Safety Agency to evaluate brucellosis tests for ruminants (Anon., 2006). For eligibility, full texts were thoroughly examined by three of the authors (M.D., J.M.B. and I.M.) and studies classified into three groups: Bayesian, eligible gold standard and
Blood serum tests in non-vaccinated cattle and absence of FPSR
SAT, RBT, CT, BPAT and CFT are usually referred to as “classical” or “conventional” tests. All but SAT are OIE prescribed or alternative tests for trade (OIE, 2016). SAT shows low DSe (Anon., 2006; Table 3 and Table S1; for discrepant results see below) and its use is particularly troublesome in chronically infected herds (Davies, 1971) because of the dominance of non-agglutinating IgG after the acute phase (Ducrotoy et al., 2016). On the other hand, and in keeping with historical and more
Blood serum tests in Brucella-free cattle in FPSR contexts
Only two studies meet the criteria in Table 2. One study (Muñoz et al., 2005) tested sera positive in S-LPS tests (RBT and CFT) confirmed as true positives by bacteriological isolation of B. abortus or B. melitensis (Table 2, inclusion criterion 7), and adjusted the DSp of the immunosorbent assays to 100% in Brucella-free cattle not exposed to Y. enterocolitica O:9. This study (Table 4) shows that, whereas iELISAs with S-LPS or OPS-core display 100% DSp in the Brucella-free not exposed to Y.
Blood serum tests in vaccinated cattle
Although there are numerous reports of the DSp of tests after vaccination with S19, few contain precise information on the time lapse after vaccination, age of cattle at vaccination and vaccine route and dose, variables relevant to the antibody response (Ducrotoy et al., 2016). Table 5 summarizes those studies that provide this information for calf subcutaneous vaccination. Only few of these studies included a parallel DSe/DSp assessment in infected and Brucella-free cattle. However, as all
Milk antibody tests
The diagnostic aspects of the MRT have been reviewed elsewhere (Pietz, 1977). The MRT can yield false positive reactions in S19 cattle vaccinated less than 4 months before testing, in samples containing abnormal milk (such as colostrum) or in cases of mastitis. It is not sensitive enough in individual animals, and its main interest is to determine the infection status of dairy herds in bulk milk, where it has been reported to identify 88% of the herds in which infected animal(s) were in
Cellular immunity tests
The γ-interferon assay has been only the topic of work aimed to set up the method and to assess its ability to discriminate FPSR using experimentally infected animals (Kittelberger et al., 1997; Weynants et al., 1995, Weynants et al., 1998). For the brucellin skin test, it is essential to employ protein extracts free of LPS, OPS, NH or polysaccharide B to avoid dermal toxicity and/or Arthus-type hypersensitivity reactions, which requires the use of rough strains (Ducrotoy et al., 2016).
Validation studies using bayesian models
Table 6 lists DSe and DSp Bayesian estimates of relevant tests taken from key studies of diagnostic interest (for the origin of priors and other information, see Table S3). As expected, most of these studies have been performed in resource-limited areas where data on the prevalence of disease are scanty.
The studies that analyzed iELISAs coincide in reporting high DSe/DSp, which is in line with the studies using gold standard sera (compare Table 3, Table 6). However, of the three studies that
Conclusions: diagnostic tests in the main contexts of cattle brucellosis
Immunological tests are required at each step of intervention against cattle brucellosis: the evaluation of the prevalence, the assessment of the efficacy of control and elimination measures, and the confirmation of freedom of disease through surveillance.
Effective progress for each step depends on a set of requisites: (i) a proficient veterinary infrastructure and individual animal tagging, (ii) repeated access to the animals, (iii) the control of animal movements, and (iv) the involvement of
Author contributions
M.D., J.M.B. and I.M reviewed and discussed all the literature cited in the paper and supplemental materials. R.C.-A. and P.M. specifically contributed to Sections 4–6. MD and IM wrote the paper.
Competing interests
The authors declare not to have competing interests.
Acknowledgements
We are very thankful to Professor Mathias Greiner for a critical reading of the manuscript.
This work was supported by the “Ministerio de Economía y Competitividad” of Spain (project AGL2014-58795-C4), INIA (project Bru-Epidia 291815-FP7/ERANET/ANIHWA) and consolidated group from Aragón Government A14.
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- 1
Present address: Ceva Santé Animale, 10 Avenue de la Ballastière, 33500-LIBOURNE, France.
- 2
These authors contributed equally to this work.