Author's address (for correspondence): María M. Wanke, Facultad de Ciencias Veterinarias, Cátedra de Teriogenología Chorroarín 280 (1427) CABA, Argentina. E-mail: email@example.com
The most widely used screening test for the diagnosis of brucellosis in the dog is the rapid slide agglutination test in the presence of 2-mercaptoethanol (2ME-RSAT). The diagnosis is partially confirmed by the agar gel immunodiffusion test (AGID) and definitively confirmed by bacteriological isolation. Some chronic cases not detected by these tests may be detected by ELISA tests. The use of 2ME-RSAT in routine clinical practice requires a microscope and an experienced operator. An immunochromatographic diagnostic test for canine brucellosis (FASTest®Brucella c., Megacor, Hörbranz, Austria) has been recently released. In this study, we compared the diagnostic performance of the FASTest with those of 2ME-RSAT, AGID and ELISAs. Sera from 17 healthy dogs used as negative controls yielded negative results by FASTest, indicating a 100% specificity in this sample. Among 27 sera of dogs with acute or subacute brucellosis confirmed by B. canis isolation, all of which were positive by RSAT and ELISAs, the FASTest was positive in 24 cases and AGID in 23. In acute and subacute cases, the sensitivity of FASTest was 89%. Sera from six dogs with bacteriologically confirmed chronic brucellosis, which were positive by ELISAs but negative by 2ME-RSAT, were also tested; 1 was positive by FASTest and 4 were positive by AGID. These preliminary results indicate a good specificity of the FASTest (100% in this sample) but an unacceptable sensitivity as a screening test. In cases with chronic brucellosis, the sensitivity of the FASTest was lower than that of ELISAs but this assay could make a good intermediate test to be run after a positive RSAT and before running an AGID.
In countries where canine brucellosis is endemic, it constitutes a serious problem for dog breeders and pet owners. The causative agent, Brucella canis, is transmitted through the placenta, by sexual contact and the oro-nasal route. Canine brucellosis is usually diagnosed by means of serological tests and culture; however, isolating the organism is a lengthy process and its sensitivity may be compromised by intermittent abacteraemia (Zoha and Carmichael 1982). For these reasons, detecting anti-Brucella antibodies has a central role in the diagnosis of canine brucellosis.
The most widely used serological test is the rapid slide agglutination test (RSAT), with and without 2-mercaptoethanol (2ME-RSAT) that uses the M(–) strain of B. canis (Carmichael and Joubert 1987). This test detects antibodies directed against surface antigens of the bacteria, mainly to the rough lipopolysaccharide (R-LPS). The use of 2ME-RSAT in the routine clinical practice is complicated by the need of a microscope to read the reaction and an experienced operator to interpret the results. While visual reading with the naked eye was used in the original description of the technique (Carmichael and Joubert 1987), the use of a light microscope for reading the reaction improves the accuracy of the test (Dr. Carmichael, personal communication) and is regularly used in all the canine brucellosis reference centres in Argentina (ANLIS, SENASA, School of Veterinary Medicine). Taking into account the false-negative results obtained with naked eye reading, we suggest to use microscope reading, especially for detecting weak reactors. The agar gel immunodiffusion test (AGID) has been reported to be a more specific, but less sensitive method to confirm a serological diagnosis (Zoha and Carmichael 1982). An ELISA using a hot saline (HS) extract of B. canis M(–) is also presumed to detect antibodies to surface antigens, and an ELISA for measuring serum antibodies directed at cytoplasmic proteins (CPs) has been tested. Both have improved sensitivity and specificity as compared with agglutination, resulting in the detection of acute infections not detected by 2ME-RSAT (Wanke et al. 2002).
An immunochromatographic diagnostic test for canine brucellosis (FASTest® Brucella c., Megacor, Hörbranz, Austria) has been recently released. This test is very simple to perform and could be potentially used in the routine clinical practice (Wanke et al. 2011. This study was performed to compare the diagnostic performance of the FASTest with the 2ME-RSAT, AGID and ELISA tests.
Materials and Methods
A total of 50 sera were used, which included 17 sera from healthy dogs, 27 sera from dogs with acute (<3 months of infection) or subacute (3–12 months of infection) brucellosis confirmed by B. canis isolation and six sera from dogs with bacteriologically confirmed chronic (>12 months of infection) brucellosis. Each sample was tested by RSAT, 2ME-RAST, AGID, HS-ELISA, CP-ELISA and FASTest®.
The RSAT (Carmichael and Joubert 1987) used a suspension of B. canis (M-) (Brucellosis service, ANLIS-Malbrán, Buenos Aires). The strength of agglutination was scored from + to ++++. Samples positive by RSAT (any +) were assayed by 2ME-RSAT. For the AGID, a hot saline extract of B. ovis (SENASA, Argentina) was used as the antigen and the test was performed according to the Manual of Standards for Diagnostic Tests and Vaccines (OIE Manual of Standards for Diagnostic Tests and Vaccines 2004). The positive control serum was also provided by SENASA.
Two ELISAs were evaluated. One test used the HS from B. canis (M-) as an antigen (Wanke et al. 2002), and the other test used the CP fraction of B. abortus depleted of LPS (Wanke et al. 2002). A commercial test employing immunochromatography (FASTest®Brucella c., Megacor, Hörbranz, Austria) was used according to the manufacturer's instructions. A drop of the serum sample or positive control serum was dispensed onto the cassette using the provided pipette, followed by four drops of diluent. The reaction was read 20 min later. If a pink-purple line appeared in the control zone (C) and no pink-purple line was visible in the test zone (T), the result was negative. However, if a pink-purple line also appeared in the test zone (T), the result was positive. It made no difference when each band appeared to indicate a positive result.
The results obtained with the FASTest in the different groups of canine sera are summarized in Fig. 1. Sera from 17 healthy dogs used as negative controls yielded negative results by the FASTest, 2ME-RSAT and ELISA, indicating a 100% specificity for these tests. One of these control negative samples was positive by AGID, indicating a 94% specificity. Of the 27 sera from dogs with acute or subacute brucellosis confirmed by B. canis isolation, all were positive by 2ME-RSAT and both ELISAs, 23 were positive by AGID and 24 by FASTest, indicating sensitivities of 100%, 100%, 85% and 89%, respectively. Two of the acute/subacute infection group sera (one from a dog recently infected and the other from a dog with late infection) were positive by AGID but not by FASTest, and three positive sera from dogs with recent infections were positive by FASTest and negative by AGID. One of the positive serum samples from a dog with a recent infection was negative on both the AGID and the FASTest. An additional group of sera from six dogs with bacteriologically confirmed chronic brucellosis was tested. All sera were positive by the ELISAs and negative by 2ME-RSAT, 1 was positive on FASTest, and four were positive on AGID.
These preliminary results indicate a good specificity of the FASTest (100% in this sample). In acute and subacute cases, the sensitivity of the test was 89%. Two of the false-negative results of FASTest corresponded to samples taken during early stages of the infection (one was also negative by AGID) and may have been caused by a low concentration of circulating anti-Brucella antibodies. Because the antigen used in the FASTest is a hot extract of B. canis, this test most likely detects antibodies to surface antigens, in a similar fashion to 2ME-RSAT, AGID and ELISA with HS. As these last tests were all positive for the two sera having false-negative FASTest, the lack of detection by FASTest is probably due to a lower analytical sensitivity of this test rather than to antigenic differences with other tests.
In the six cases of chronic brucellosis (all of which were negative by 2ME-RSAT), the sensitivity of the FASTest [1 of 6 (17%)] was lower than that of ELISAs (100%) and AGID (67%) tests, which yielded positive results in all cases and four cases, respectively.
While this preliminary evaluation of the FASTest yielded promising results, it is necessary to test a higher number of samples at least in duplicate to draw sound conclusions on the reliability of this test as a screening tool for canine brucellosis. The test appears to be a poorer screening test than the RSAT or ELISA tests but does have high specificity. It could be a good intermediate test run after a positive RSAT, but before running an agar gel immunodiffusion test (AGID).
Conflicts of interest
None of the authors have any conflicts of interest to declare.
Dr. Wanke conceived the experiment, choose and provided the sera from the serum bank of the Small Animal Theriogenology area, ran the ELISA tests and wrote the draft of the manuscript. Dr. F Cairó performed the chromatographic tests. Dr. M Rossano performed the chromatographic tests. Dr. M Laiño performed the AGID tests. Dr. PC Baldi prepared and provided the antigens for both ELISA tests, and contributed to manuscript writing and translation to English. Dr. NE Monachesi contributed to blood sampling and RSAT and 2ME-RSAT testing during the 5 years follow-up of the dogs. Dr. EA Comercio contributed to blood sampling and RSAT and 2ME-RSAT testing during the 5 years follow-up of the dogs. Dr. MM Vivot conceived the experiment. All the authors contributed to the discussion and conclusions of the paper.