Maria M. Wanke

Diagnosis of canine brucellosis by detection of serum antibodies against an 18 kDa cytoplasmic protein of Brucella spp.

An antigenic capture ELISA was developed to measure serum antibodies against an 18 kDa cytoplasmic protein of Brucella. This assay was used to detect anti-18 kDa reactivity in sera from 30 dogs having confirmed or suspected brucellosis. Antibodies against the 18 kDa protein were found in 26 of them, which were also positive by the slide agglutination test (2ME-RSAT). The overall correlation (positive and negative results) between the ELISA and 2ME-RSAT tests was 93.3%. Additionally, these sera were assayed by an indirect ELISA using a whole extract of cytoplasmic proteins of B. abortus (LPS-free CYT). The results of both ELISAs were coincident in 28 of 30 (93.3%) dogs having confirmed or suspected brucellosis. When a serological follow-up was performed on some dogs having confirmed brucellosis, antibody titers measured by both ELISAs showed a parallel progression. On the other hand, the capture ELISA showed good specificity, since a positive result was obtained only in 2 of 103 sera from healthy dogs. These preliminary results show that the ELISA for detecting serum antibodies against the 18 kDa cytoplasmic protein of Brucella could be useful for the diagnosis of canine brucellosis. This study also shows that the results obtained with this single protein of Brucella are equivalent to those obtained with the whole extract of cytoplasmic proteins.

Brucella abortus cytoplasmic proteins used as antigens in an ELISA potentially useful for the diagnosis of canine brucellosis

A preparation of Brucella abortus cytoplasmic proteins was depleted of lipopolysaccharide (LPS) by immunoadsorption with a monoclonal antibody (BC68) specific for the O antigen of B. abortus smooth LPS. This extract was used as antigen in an indirect ELISA for measuring antiprotein humoral immune response in dogs suffering from brucellosis and in healthy controls. All of the affected dogs studied showed IgG antiprotein response, while 2% (2 of 103) of the healthy dogs used as controls gave a positive result. All sera found to be positive by ELISA were also positive by the rapid slide agglutination test. These preliminary results show that the ELISA using B. abortus cytoplasmic proteins could be useful for the specific diagnosis of canine brucellosis.

Comparative performance of tests using cytosolic or outer membrane antigens of Brucella for the serodiagnosis of canine brucellosis.

Although some ELISA tests using cytoplasmic or outer membrane antigens of Brucella have been developed to improve the diagnosis of canine brucellosis, the performance of these assays has not been compared. In the present study three ELISA tests using lipopolysaccharide (LPS)-free cytoplasmic proteins (CPs) of Brucella abortus, the lumazine synthase (LS) of Brucella spp. or a hot-saline (HS) extract of Brucella canis containing outer membrane antigens were used to test sera from dogs with suspected or confirmed brucellosis (n=36) and from dogs with pathological conditions other than brucellosis (n=212). In the first group the proportion of positive results was 92, 92 and 81% for the ELISAs with HS, CP and LS, respectively, and 94% of the samples were positive by at least one ELISA test. Three dogs that were negative by agglutination (2ME-RSAT) had a positive result by at least one ELISA, and this discrepancy was attributed to the lower analytical sensitivity of agglutination tests. This hypothesis was confirmed by a serological follow-up of seven dogs recently infected with B. canis in three of which the illness was diagnosed earlier by one or more ELISA tests than by 2ME-RSAT. Among dogs having pathological conditions other than brucellosis, specificities were 94.3, 96.7 and 96.7% for the ELISAs with HS, CP and LS, respectively. This study shows that HS-ELISA and CP-ELISA are highly specific and sensitive for the diagnosis of canine brucellosis and can detect the infection by B. canis shortly after the exposure to the pathogen.

Preliminary Study of an Immunochromatography Test for Serological Diagnosis of Canine Brucellosis

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.

Immunization with Brucella VirB Proteins Reduces Organ Colonization in Mice through a Th1-Type Immune Response and Elicits a Similar Immune Response in Dogs

ABSTRACT VirB proteins from Brucella spp. constitute the type IV secretion system, a key virulence factor mediating the intracellular survival of these bacteria. Here, we assessed whether a Th1-type immune response against VirB proteins may protect mice from Brucella infection and whether this response can be induced in the dog, a natural host for Brucella. Splenocytes from mice immunized with VirB7 or VirB9 responded to their respective antigens with significant and specific production of gamma interferon (IFN-γ), whereas interleukin-4 (IL-4) was not detected. Thirty days after an intraperitoneal challenge with live Brucella abortus, the spleen load of bacteria was almost 1 log lower in mice immunized with VirB proteins than in unvaccinated animals. As colonization reduction seemed to correlate with a Th1-type immune response against VirB proteins, we decided to assess whether such a response could be elicited in the dog. Peripheral blood mononuclear cells (PBMCs) from dogs immunized with VirB proteins (three subcutaneous doses in QuilA adjuvant) produced significantly higher levels of IFN-γ than cells from control animals upon in vitro stimulation with VirB proteins. A skin test to assess specific delayed-type hypersensitivity was positive in 4 out of 5 dogs immunized with either VirB7 or VirB9. As both proteins are predicted to locate in the outer membrane of Brucella organisms, the ability of anti-VirB antibodies to mediate complement-dependent bacteriolysis of B. canis was assessed in vitro. Sera from dogs immunized with either VirB7 or VirB9, but not from those receiving phosphate-buffered saline (PBS), produced significant bacteriolysis. These results suggest that VirB-specific responses that reduce organ colonization by Brucella in mice can be also elicited in dogs.

Hypo-osmotic test in cat spermatozoa.

The hypo‐osmotic (HOS) test has been used in other species as an indicator of the fertilising capacity of spermatozoa. The aims of this study were to assess the response of domestic cat spermatozoa to the hypo‐osmotic test, to determine the type of solution, concentration and time of incubation needed to obtain a maximum percentage of swelling, to correlate the selected combination with the percentages of progressive motility and to evaluate whether dilution of the ejaculate alters the results. Incubation for 30 and 45 min in solutions of fructose and of citrate of 50 and 100 mOsmol kg−1 was evaluated. The highest percentage of swelling was obtained using the 50 mOsmol kg−1 solution, and no significant differences were observed between the times of exposure to the solutions. A positive correlation was observed between the percentage of individual progressive motility and the percentage of sperm swelling in a 50 mOsmol kg−1 fructose solution, with no significant differences being observed between raw and diluted semen samples. The results of this study suggest that the HOS test could be useful for evaluating membrane function in domestic cat spermatozoa, both in raw semen and in samples diluted in the EZ Mixin® commercial extender, and thus could be incorporated into routine semen evaluation protocols.