Comparison of serology, culture, and PCR for detection of brucellosis in slaughtered camels in Iran

Brucellosis is a zoonotic disease which is characterized by abortion and reduced fertility in many species. Camel brucellosis is caused by Brucella abortus and Brucella melitensis. To investigate sensitive methods in the detection of camel brucellosis, PCR was used to overcome the limitations of serology and culture methods. Three hundred ten camels were examined for brucellosis infection using serological tests (RBPT, mRB, Wright, and 2-ME). In addition, 100 serological tested cases (39 mRB positive and 61 mRB negative) were analyzed with both bacteriological (lymph node culture on Brucella agar supplemented with antibiotics) and PCR (nested-PCR on sera and blood samples) methods. The nested-PCR was genus-specific and amplified the 16S rDNA locus. Six out of 310 (1.94 %) of the examined camels were positive using the serological tests, whereas, no bacteria was isolated from lymph tissues. Nested-PCR was positive in six and nine individuals in sera and blood samples, respectively. The genus-specific nested-PCR assay on blood samples detected a higher number of camel brucellosis compared with serological and classical culture methods. These results have identified a sensitive PCR method which could be used as a complement test for the detection of brucellosis in live camels with the lowest risk of infection to laboratory workers.     

Improved Serodiagnosis of Bovine Brucellosis by Novel Synthetic Oligosaccharide Antigens Representing the Capping M Epitope Elements of Brucella O-Polysaccharide

Members of the genus Brucella have cell wall characteristics of Gram-negative bacteria, which in the most significant species includes O-polysaccharide (OPS). Serology is the most cost-effective means of detecting brucellosis, as infection with smooth strains of Brucella leads to the induction of high antibody titers against the OPS, an unbranched homopolymer of 4,6-dideoxy-4-formamido-d-mannopyranosyl residues (d-Rha4NFo) that are variably α(1→2)- and α(1→3)-linked. Six d-Rha4NFo homo-oligosaccharides were synthesized, each containing a single α(1→3) link but with a varied number of α(1→2) links. After conjugation to bovine serum albumin (BSA), glycoconjugates 1 to 6 were used to develop individual indirect enzyme-linked immunosorbent assays (iELISAs). The diagnostic capabilities of these antigens were applied to panels of cattle serum samples, including those falsely positive in conventional assays, and the results were compared with those of the complement fixation test (CFT), serum agglutination test (SAT), fluorescent polarization assay (FPA), smooth lipopolysaccharide (sLPS) iELISA, and competitive enzyme-linked immunosorbent assay (cELISA) methods. Results from field serum samples demonstrated that all of the synthetic antigens had excellent diagnostic capabilities. Assays developed with the α(1→3)-linked disaccharide conjugate 1 were the best at resolving false-positive serological results. This was supported by the results from serum samples derived from experimentally infected cattle. Data from synthetic trisaccharide antigens 2 and 3 and tetrasaccharide antigen 4 identified an OPS epitope equally common to all Brucella abortus and Brucella melitensis strains but unique to Brucella. Synthetic oligosaccharide conjugates function as effective surrogates for naturally derived antigens. The creation of discrete OPS epitope antigens reveals not only the previously untapped diagnostic potential within this key diagnostic structure but also holds significance for the design of brucellosis vaccines and diagnostics that enable the differentiation of infected from vaccinated animals.     

Efficacy of Several Serological Tests and Antigens for Diagnosis of Bovine Brucellosis in the Presence of False-Positive Serological Results Due to Yersinia enterocolitica O:9

Yersinia enterocolitica O:9 bears a smooth lipopolysaccharide (S-LPS) of Brucella sp. O-chain A + C/Y epitopic structure and is a cause of false-positive serological reactions (FPSR) in standard tests for cattle brucellosis. Brucella S-LPS, cross-reacting S-LPSs representing several O-chain epitope combinations, Brucella core lipid A epitopes (rough LPS), Brucella abortus S-LPS-derived polysaccharide, native hapten polysaccharide, rough LPS group 3 outer membrane protein complexes, recombinant BP26, and cytosolic proteins were tested in enzyme-linked immunosorbent assays (ELISA) and precipitation tests to detect cattle brucellosis (sensitivity) and to differentiate it from FPSR (specificity). No single serological test and antigen combination showed 100% sensitivity and specificity simultaneously. Immunoprecipitation tests with native hapten polysaccharide, counterimmunoelectrophoresis with cytosolic proteins, and a chaotropic ELISA with Brucella S-LPS were 100% specific but less sensitive than the Rose Bengal test, complement fixation, and indirect ELISA with Brucella S-LPSs and native hapten or S-LPS-derived polysaccharides. A competitive ELISA with Brucella S-LPS and M84 C/Y-specific monoclonal antibody was not 100% specific and was less sensitive than other tests. ELISA with Brucella suis bv. 2 S-LPS (deficient in C epitopes), Escherichia hermannii S-LPSs [lacking the contiguous α-(1-2)-linked perosamine residues characteristic of Y. enterocolitica S-LPS], BP26 recombinant protein, and Brucella cytosolic fractions did not provide adequate sensitivity/specificity ratios. Although no serological test and antigen combination fully resolved the diagnosis of bovine brucellosis in the presence of FPSR, some are simple and practical alternatives to the brucellin skin test currently recommended for differential diagnosis.     

Evaluation of Lipopolysaccharides and Polysaccharides of Different Epitopic Structures in the Indirect Enzyme-Linked Immunosorbent Assay for Diagnosis of Brucellosis in Small Ruminants and Cattle

Brucella abortus and Brucella melitensis have surface lipopolysaccharides and polysaccharides carrying B. melitensis-type (M) and B. abortus-type (A) epitopes as well as common (C) epitopes present in all smooth Brucella biotypes. Crude lipopolysaccharides, hydrolytic O polysaccharides, and native hapten polysaccharides of MC or AC specificity were evaluated in indirect enzyme-linked immunosorbent assays with polyclonal, monoclonal, or protein G conjugates by using sera from cattle, sheep, and goats infected with AC, MC, or AMC Brucella biotypes. Regardless of the antigen, the levels of antibodies were lower in goats than in sheep and highest in cattle. The diagnostic performance of the assay was not affected by the absence of lipid A-core epitopes, the presence of contaminating outer membrane proteins, the AC or MC epitopic structure of the absorbed antigen, or the conjugate used. Moreover, with sera from cattle vaccinated with B. abortus S19 (AC) or from sheep and goats vaccinated with B. melitensis Rev 1 (MC), AC and MC antigens showed similar levels of reactivity. The results show that antibodies to the C epitopes largely dominate in infection, and this is consistent with the existence of multiple overlapping C epitopes (V. Weynants, D. Gilson, A. Cloeckaert, A. Tibor, P. A. Denoel, F. Godfroid, J. N. Limet, and J.-J. Letesson, Infect. Immun. 65:1939–1943, 1997) rather than with one or two C epitopes. It is concluded that, by adaptation to the corresponding antibody levels, brucellosis in cattle, sheep, and goats can be diagnosed by immunosorbent assay with a single combination of conjugate and antigen.     

Antibody Reactivity to Omp31 from Brucella melitensis in Human and Animal Infections by Smooth and Rough Brucellae

Group 3 of outer membrane proteins (OMPs) of Brucella includes Omp25 and Omp31, which share 34% identity. Omp25 is highly conserved in Brucella species, and Omp31 is present in all Brucella species, except Brucella abortus. Antibodies to Brucella melitensis Omp31 have been sought only in infected sheep, and Western blotting of sera from infected sheep did not reveal anti-Omp31 reactivity. We obtained recombinant purified Omp31 (B. melitensis) and tested its recognition by sera from humans and animals suffering from brucellosis by an indirect enzyme-linked immunosorbent assay (ELISA). Serum samples from 74 patients, 57 sheep, and 47 dogs were analyzed; brucellosis was confirmed by bacteriological isolation in all ovine and canine cases and 31 human cases of brucellosis. Thirty-five patients (47%) were positive for antibodies to Omp31, including seven cases of Brucella suis infection, two cases of B. abortus infection, and three cases of B. melitensis infection. Of 39 sheep naturally infected with B. melitensis (biovars 1 and 3), 23 (59%) were positive for antibodies to Omp31. Anti-Omp31 antibodies were also detected in 12 of 18 rams (67%) in which Brucella ovis was isolated from semen. Antibodies to Omp31 were also found in 41 (87%) of the 47 dogs, including 13 with recent infection. These results suggest that an indirect ELISA using recombinant purified Omp31 from B. melitensis would be of limited value for the diagnosis of human and animal brucellosis. Nevertheless, the potential usefulness of this antigen in combination with other recombinant proteins from Brucella should not be dismissed.     

Evaluation of North American Antibody Detection Tests for Diagnosis of Brucellosis in Goats

The sensitivities and specificities of 17 antibody detection tests for brucellosis in goats were estimated. Tests evaluated included the U.S. Department of Agriculture (USDA) card test with 8% cell concentration (8%Card), USDA rapid automated presumptive test (RAP), Mexican rose bengal plate tests with 8 and 3% cell concentrations (8%RB and 3%RB), French rose bengal plate test with 4.5% cell concentration (4.5%RB), USDA standard plate test (SPT), USDA buffered acidified plate agglutination test (BAPA), USDA and Mexican rivanol tests (URIV and MRIV), USDA standard tube tests with Brucella abortus and Brucella melitensis antigens (SATA and SATM), serum enzyme-linked immunosorbent assay (ELISA), USDA cold-fixation complement fixation tests with B. abortus and B. melitensis antigens (CFA and CFM), USDA and Mexican milk ring tests (UBRT and MBRT), and a milk ELISA. Test sensitivity was evaluated by using two groups of 10 goats experimentally infected with B. melitensis or B. abortus and monitored for 24 weeks. Specificity was evaluated by using 200 brucellosis-free nonvaccinated goats from 10 California herds. The 3%RB was considered a good screening test because of high sensitivity at week 24 postinfection (90%), ease of performance, and low cost. The cold-fixation CFA and CFM had 100% specificity in the field study and were considered appropriate confirmatory tests. The milk ELISA was significantly more sensitive (P < 0.05) than the UBRT and significantly more specific (P < 0.05) than the MBRT. The milk ELISA also had the advantage of objectivity and ease of interpretation.     

Enzyme-Linked Immunosorbent Assay To Differentiate the Antibody Responses of Animals Infected with Brucella Species from Those of Animals Infected with Yersinia enterocolitica O9

Enzyme-linked immunosorbent assays using antigens extracted from Brucella abortus with n-lauroylsarcosine differentiated natural Brucella-infected animals from Brucella-vaccinated or Yersinia enterocolitica O9-infected animals. A field trial in Mongolia showed cattle, sheep, goat, reindeer, camel, and human sera without infection could be distinguished from Brucella-infected animals by conventional serological tests.     

Bacteriological and molecular investigation of B. melitensis in dairy cows in Iran

It is extremely important to investigate the presence of Brucella melitensis as a nonspecific and heterogeneous agent in dairy cows in Iran due to mixed populations of sheep, goats, and cattle. B. melitensis from infected sheep or goat herds may be introduced into the cattle population in this area. Hence, it is essential to obtain epidemiological data on the probable existence of B. melitensis as a different source of the infection or new exotic Brucella phenotypes development in dairy cow herds in Iran. The purpose of this study was to investigate the presence of B. melitensis infection in cows, using both traditional bacteriological tests and polymerase chain reaction (PCR)-based assay. Traditional biotyping and PCR results of 42 Brucella spp. isolates from Tehran and Fars (northern and southern) provinces of Iran identified five (11.9%) and 37 (88.1%) as B. melitensis (four biovar 1 and one biovar 2) and 37 (88.1%) Brucella abortus biovar 3. This study demonstrated that B. melitensis infection in dairy cows of Iran is still present at a low level. However, the Iranian Veterinary Organization should be careful to have dairy cow herds free of B. melitensis infection as a nonspecific agent of brucellosis, since this type of brucellosis may be extended and act as a potential source of severe infection in humans.     

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

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.     

Serological,Bacteriological, and Molecular Diagnosis of Brucellosis in Domestic Animals in Croatia

Aim

To present the surveillance data on Brucella melitensis, B. suis, and B. ovis infection in cattle, sheep, goats, and swine in Croatia obtained in 2008 by serological, bacteriological, and molecular methods for diagnostics of brucellosis in domestic animals.

Methods

We serologically tested 42 785 cattle serums, 22 686 sheep and goat serums, and 28 520 swine serums using the Rose Bengal test, complement fixation test, and various immunosorbent assays. We also tested 10 173 ram blood samples for B. ovis infection using the complement fixation test. Bacteriological examination was conducted on 214 samples collected from 34 serologically positive animals. Different molecular methods were employed in the identification and typing of 20 isolates from the samples.

Results

B. melitensis biovar (bv.) 3 was confirmed with different identification methods in 2 flocks in 2 Croatian counties and B. suis bv. 2 in 3 herds in 3 counties. B. melitensis in cows was confirmed for the first time in Croatia. Infection with B. ovis was serologically confirmed in 202 rams in 12 counties.

Conclusions

In 2008, the size of the brucellosis-affected area in Croatia and the efficiency of detection and prevention of brucellosis in sheep, goats, and swine were satisfactory. Infection with B. melitensis in cattle was confirmed for the first time and possible links for infection in humans were detected. More efficient measures for suppression and control of ovine epididymitis are required and a new strategy may be necessary for complete eradication of this disease.Brucellosis is a chronic infectious disease caused by bacteria of the genus Brucella that affects animals and humans. Each species of Brucella has their preferred host: B. abortus infects cattle, B. metitensis sheep and goats, B. suis swine, B. canis dogs, and B. ovis sheep, although they can also infect other animals (1). Brucellosis in sheep and goats is endemic in the Mediterranean region but is spread throughout Asia, Africa, and Central and South America (2,3). Along with tuberculosis and rabies, brucellosis is the most important bacterial zoonosis and remains an important public health and economic concern.With the exception of B. ovis and B. neotomae, all Brucella species can cause infections in humans. New Brucella species pathogenic for humans – B. ceti and B. pinnipedialis – have recently been discovered in marine mammals (4). Infection is transmitted to humans though direct contact with the infected animals or by consuming infected milk or fresh cheese (1).In Croatia, brucellosis in domestic animals is controlled in accordance with the annual order issued by the Ministry of Agriculture. Serological blood examination of all male breeding animals is mandatory twice per year, and all cases of abortion must be reported and tested for brucellosis. On large cattle and pig farms, 20% of breeding animals must be tested annually. Castration of seropositive rams without the obligation of bacteriological testing is required as an eradication measure for B. ovis infection.Bovine brucellosis (B. abortus) was eradicated in Croatia in 1964, while brucellosis in sheep and goats has occurred sporadically in the recent years, limited to 1-2 sheep flocks per year. All of the occurrences have resulted from epizooty originating in the neighboring country of Bosnia and Herzegovina (BH) (5,6). Swine brucellosis has been detected in swine and wild boars during regular controls (7,8) and B. suis isolates were determined as biovars (bv.) 1, 2, or 3 (7-11).B. ovis in rams and sheep causes either clinical or subclinical disease and is not pathogenic for humans (12). According to simulation models, B. ovis infection causes significant economic losses in flocks with no control measures, but there is no exact confirmation of the extent of such losses (13,14). Eradication is possible, but requires considerable resources.The aim of this study was to determine the effectiveness of the existing programs for diagnosis and control of brucellosis in domestic animals in order to prevent transmission of disease to humans and to reduce economic losses in animal production. This article describes the spread of brucellosis caused by B. melitensis, B. suis, and B. ovis in cattle, sheep, goats, and swine in the Republic of Croatia in 2008, as determined using different diagnostic methods.     

Interaction of Brucella suis and Brucella abortus rough strains with human dendritic cells

Brucella is a facultative intracellular pathogen of various mammals and the etiological agent of brucellosis. We recently demonstrated that dendritic cells (DCs), which are critical components of adaptive immunity, are highly susceptible to Brucella infection. Furthermore, Brucella prevented the infected DCs from engaging in maturation processes and impaired their capacity to present antigen to naive T cells and to secrete interleukin-12 (IL-12). The lipopolysaccharide (LPS) phenotype is largely associated with the virulence of Brucella. Depending on whether they express the O-side chain of LPS or not, the bacteria display a smooth or rough phenotype. Rough Brucella mutants are attenuated and induce a potent protective T-cell-dependent immune response. Due to the essential role of DCs in the initiation of T-cell-dependent adaptive immune responses, it seemed pertinent to study the interaction between rough Brucella strains and human DCs. In the present paper, we report that, in contrast to smooth bacteria, infection of DCs with rough mutants of Brucella suis or Brucella abortus leads to both phenotypic and functional maturation of infected cells. Rough mutant-infected DCs then acquire the capacity to produce IL-12 and to stimulate naive CD4⁺ T lymphocytes. Experiments with rough and smooth purified LPS of Brucella supported the hypothesis of an indirect involvement of the O-side chain. These results provide new data concerning the role of LPS in Brucella virulence strategy and illuminate phenomena contributing to immune protection conferred by rough vaccine strains.     

Intracellularly Induced Cyclophilins Play an Important Role in Stress Adaptation and Virulence of Brucella abortus

Brucella is an intracellular bacterial pathogen that causes the worldwide zoonotic disease brucellosis. Brucella virulence relies on its ability to transition to an intracellular lifestyle within host cells. Thus, this pathogen must sense its intracellular localization and then reprogram gene expression for survival within the host cell. A comparative proteomic investigation was performed to identify differentially expressed proteins potentially relevant for Brucella intracellular adaptation. Two proteins identified as cyclophilins (CypA and CypB) were overexpressed in the intracellular environment of the host cell in comparison to laboratory-grown Brucella. To define the potential role of cyclophilins in Brucella virulence, a double-deletion mutant was constructed and its resulting phenotype was characterized. The Brucella abortus ΔcypAB mutant displayed increased sensitivity to environmental stressors, such as oxidative stress, pH, and detergents. In addition, the B. abortus ΔcypAB mutant strain had a reduced growth rate at lower temperature, a phenotype associated with defective expression of cyclophilins in other microorganisms. The B. abortus ΔcypAB mutant also displays reduced virulence in BALB/c mice and defective intracellular survival in HeLa cells. These findings suggest that cyclophilins are important for Brucella virulence and survival in the host cells.     

Improved immunogenicity and protective efficacy of a divalent DNA vaccine encoding Brucella L7/L12-truncated Omp31 fusion protein by a DNA priming and protein boosting regimen

Brucellosis is one of the most common zoonotic diseases caused by species of Brucella. At present, there is no commercially available vaccine for the human brucellosis. Brucella melitensis and Brucella abortus are the main causes of human brucellosis, worldwide. The outer membrane protein 31 (Omp31) and L7/L12 are immunodominant and protective antigens conserved among human Brucella pathogens. The purpose of the current study was to evaluate and compare the immunogenicity and protective efficacy of the L7/L12-TOmp31 construct administered as DNA/DNA and DNA/Pro vaccine regimens. Vaccination of BALB/c mice with the DNA/Pro regimen provided more protection levels against B. melitenisis and B. abortus challenge than did the DNA/DNA regimen. IgG1 and IgG2a titers were higher in the sera from DNA/Pro-immunized mice than in those from mice immunized with DNA alone. Moreover, splenocytes from DNA/Pro-immunized mice produced significantly higher levels of IFN-γ than did those from mice given DNA alone. The pcDNA-L7/L12-TOmp31 priming followed by rL7/L12-TOmp31 boosting led to improved protection against B. abortus or B. melitensis infection.     

Field Validation of the Use of RB51 as Antigen in a Complement Fixation Test To Identify Calves Vaccinated with Brucella abortus RB51

In order to confirm the efficiency of an experimental RB51-based complement fixation (CF) test in identifying cattle vaccinated with Brucella abortus strain RB51, 831 sera from 110 vaccinated and 48 unvaccinated Hereford heifers of Iowa, collected for studies conducted in different years, were sent to Italy without coding to be tested in a CF test using RB51 as antigen. Most of the calves, aged from 3 to 10 months, were vaccinated subcutaneously with the recommended dosage of 10¹⁰ CFU of RB51 commercial vaccine, while only six calves received 10⁹ CFU of the same vaccine. Serum samples for serologic testing, collected until 16 postinoculation weeks (PIW), were also tested by routine surveillance tests for brucellosis such as rose bengal plate and CF tests performed with B. abortus smooth strain 99 as control antigen. RB51 CF test results obtained by testing sera from cattle vaccinated in 1999 indicate that the sensitivity of the reaction is 97% at 2 to 3 PIW and 90% until 8 PIW and decreases to 65% at 12 PIW, the specificity remaining at 100%. Collectively, the results of this study confirm that serologic standard tests fail to detect antibodies to RB51 while the RB51-based CF test is able to monitor antibody responses to RB51 until 15 to 16 PIW with a specificity of 100%. In addition, unlike the RB51-based dot blot assay, which is the only test currently used to monitor antibody responses to RB51, the CF test also detected specific responses following vaccination with 10⁹ CFU of RB51, although seroconversion was only 50% at 8 PIW. In conclusion, because of high specificity and sensitivity, the CF test described here can be used to efficaciously monitor serologic responses following RB51 vaccination in cattle and could also be employed to detect RB51 infection in humans exposed to this strain.     

Brucellosis and its associated risk factors to humans and domestic ruminants in Kagera Ecosystem,Tanzania

BackgroundBrucellosis is an important disease for both veterinary and public health. A study was conducted to understand the seroprevalence of brucellosis and its associated risk factors in pastoral areas of Kagera, Tanzania.MethodsSera from 156 patients with malaria-like symptoms were analyzed using the commercial rapid agglutination test (specific for B.abortus and B.melitensis detection) and Fluorescence Polarization Assay (FPA). Sera from 426 cattle, 206 goats and 197 sheep were analyzed using Rose Bengal Plate (RBPT) and Competitive ELISA (c-ELISA) tests.ResultsIn humans, overall brucellosis, B. abortus, and B. melitensis sero-prevalences were 7.7% (95%CI: 3.8–12.2%), 1.9% (95% CI: 0.4–4.5%), and 5.8 % (95%CI: 2.6–10.6%), respectively. At animal level, seropositivity was 5.9% (95%CI: 4.0–8.6%), 2.5% (95%CI: 0.8–5.7%) and 0.5% (95%CI: 0.01–2.8%) in cattle, goats and sheep, respectively. At herd level, seropositivity was 18.2% (95%CI: 12.0–25.8%) in cattle and 6.9% (95%CI: 2.2–15.3%) in small ruminants. Brucellosis was associated with assisting in parturition without wearing protective gears (OR= 5.6; p= 0.02) in humans, herds of 50–200 animals (OR= 4.2, p= 0.01) and cattle (OR=3.5; p=0.01). The knowledge of brucellosis among pastoralists (OR=0.1; p<0.01) was a protective factor.ConclusionBrucella infections could be occurring in pastoralists and domestic ruminants in Kagera. Community health education is necessary for the control of brucellosis in Tanzania.     

Enzyme Immunoassays for Serological Diagnosis of Bovine Brucellosis: A Trial in Latin America

The results of a field trial conducted in Latin America with two indirect enzyme-linked immunosorbent assays (ELISAs) and two competitive ELISAs (CELISAs) for the detection of bovine antibody to Brucella abortus are reported. One of the CELISA formats performed most accurately. The percentage of positive reactions in the CELISA relative to the selected positive rose bengal agglutination test (RBT) and complement fixation test (CFT) results was 97.47%, the percentage of negatives relative to the selected negative RBT and CFT results for unexposed cattle was 98.32%, and the percentage of negatives in cattle vaccinated with B. abortus 19 was 96.51%. The same assay format under Canadian conditions had an actual sensitivity of 100%, a specificity of 99.90% in nonvaccinates, and a specificity of 97.7% in a strain 19-vaccinated population. Overall, the CELISA performed as expected and the results were not dissimilar from the results obtained in the Canadian study. This provided further evidence that this CELISA can in many instances differentiate infected cattle from those that are vaccinated or infected with a cross-reacting organism while still giving very few false-positive or false-negative results.     

Chronic Brucellosis and Persistence of Brucella melitensis DNA

After acute brucellosis infection, symptoms persist in a minority of patients for more than 1 year. Such patients are defined as having chronic brucellosis. Since no objective laboratory methods exist to confirm the presence of chronic disease, these patients suffer delays in both diagnosis and treatment. The aim of the current study was to evaluate the usefulness of quantitative real-time PCR (Q-PCR) in the diagnosis and follow-up of these patients. Thirty-five subjects with a well-documented history of brucellosis that had been diagnosed between 2 and 33 years previously were screened by Q-PCR for the presence of Brucella melitensis DNA and by serological tests and blood culture. Subjects were divided into three groups: 8 (23%) focal-disease subjects, 9 (26%) nonfocal-disease subjects with subjective complaints, such as fatigue, malaise, arthralgia, and/or myalgia, and 18 (51%) asymptomatic subjects. All (100%) focal-disease patients and symptomatic nonfocal-disease patients had at least one positive Q-PCR sample. Only six (33%) of the asymptomatic subjects had Q-PCR-positive samples (P < 0.05). Eleven patients (five focal-disease patients and six nonfocal-disease patients with subjective complaints) received therapy during the study. For those patients who completed treatment, six (60%) still had Q-PCR-positive samples at the posttreatment follow-up. The proportion of individuals with B. melitensis DNA was significantly higher for symptomatic nonfocal-disease patients than for asymptomatic subjects. Therefore, Q-PCR appears to be a useful method for identifying chronic brucellosis patients.Human brucellosis is a multisystem disease that may present with a broad spectrum of clinical manifestations. The causative organisms, Brucella spp., are facultatively intracellular bacteria that are capable of evading a number of host defense mechanisms and can survive within phagocytic cells for long periods. These properties may account for focal complications, relapses, and chronic disease (21).The chronic course of the disease was initially explored during the 1930s by Evans (6a) and was further explored in the 1950s by Davies (5). In 1951, Spink and associates stated, with respect to the duration of the illness, that the majority of patients with brucellosis recover within a year after the administration of an antibiotic, while a small but significant number of patients continue to have clinical manifestations despite such therapy (22). These patients can be divided into two groups: those with a focal disease, such as spondylitis, and those without a focal disease who complain, nevertheless, of poor health and have symptoms such as chronic fatigue syndrome (CFS), musculoskeletal pain, depression, or anxiety.The diagnosis of chronic brucellosis is often based on clinical complaints together with the presence of high immunoglobulin G titers (2). However, the specificity of current serological assays is considered to be low, since titers may remain positive for years after the successful resolution of symptoms. In 1980, Buchanan and Faber described a serological test for evaluation of the effectiveness of treatment and for excluding a diagnosis of chronic brucellosis (3). The authors observed that, in assays with 2-mercapthoethanol, results remained positive for 9% of patients 1 year after the initiation of treatment. Among these patients, 50% still had signs and symptoms of brucellosis and required further treatment. Recently, PCR has been used to detect Brucella spp. in the diagnosis of primary infections, relapse, and focal complications of the disease (12, 13, 20). Initially, the persistence of Brucella sp. DNA after therapy was linked to relapse (9, 12, 17). However, our group and others have demonstrated the persistence of Brucella sp. DNA for long periods of time after the conclusion of therapy in asymptomatic patients (10, 15, 24). The practical role of quantitative real-time PCR (Q-PCR) in the laboratory diagnosis of chronic brucellosis and the assessment of clinical manifestations remains to be demonstrated. In the present study, we examined 35 subjects with a well-documented history of brucellosis and followed them to evaluate the usefulness of Q-PCR and the relationship between a positive result by the Q-PCR assay and clinical course.     

Development and Characterization of Mouse Models of Infection with Aerosolized Brucella melitensis and Brucella suis

There is a need to identify vaccines that can protect against Brucella, a potential bioterrorism agent. We have developed mouse models of infection with aerosolized Brucella melitensis and Brucella suis and demonstrated their utility for the evaluation of vaccines using the model live B. melitensis vaccine strain Rev.1.Brucellosis is a zoonotic disease that is caused by Brucella species. Four species, Brucella melitensis, Brucella abortus, Brucella suis, and Brucella canis, are currently known to be pathogenic to humans (1, 5, 14). In animals, brucellosis can have a huge economic impact, since infection can lead to abortions, stillbirths, and the loss of fertility in livestock. In comparison, brucellosis in humans is a debilitating disease characterized by fever, sweats, and aches. In approximately 5% of cases it can be fatal when complications, usually endocarditis, arise (4). The illness can last a number of weeks, and even with antibiotic treatment, relapses can occur. Brucellosis is common in developing countries and areas without effective animal disease control policies. In these countries, the microorganisms are usually transmitted through ingestion, inhalation, or direct skin contact. Unpasteurized milk is a common source of infection, as is inhalation from carcasses among abattoir workers (5, 11). There are several live attenuated vaccines licensed for use in animals. Of these, the most widely used are B. melitensis Rev.1 and B. abortus S19 or RB51 (reviewed in reference 17). These vaccines are unsuitable for use in humans since they are insufficiently attenuated and still cause disease (1, 17).Brucellosis is one of the most-common laboratory-acquired infections; it is readily aerosolized and highly infectious. Brucella species have been considered potential biological warfare agents and are classed as category B threat agents (15). B. suis was the first agent weaponized by the United States, in 1952 (6). Furthermore, there are also claims that Brucella bacteria were used by the Japanese Manchuria Unit and were developed by the former Soviet Union Biopreparat offensive biological weapon program (15). Since it is possible that a bioterrorist attack with Brucella bacteria would result in aerosolized bacteria causing inhalational infection, there is a requirement to develop and utilize appropriate animal models of aerosolized Brucella infection in order to evaluate the efficacy of vaccines or therapies for human brucellosis.A small-rodent model of brucellosis offers advantages over the use of larger animals for preliminary studies, including the relative ease of use, ethical acceptability, and cost. Here we describe the development and characterization of mouse models of infection with aerosolized B. melitensis and B. suis bacteria and demonstrate the utility of these models in evaluating vaccines and therapeutics for inhalational brucellosis using the model live attenuated B. melitensis Rev.1 vaccine. A mouse model of intranasal (i.n.) infection with B. melitensis has previously been described (12), and since we started this work, two other laboratories have described studies of infection of mice with aerosolized Brucella bacteria. Kahl-McDonagh et al. described aerosol infection of BALB/c mice with B. abortus 2308 and B. melitensis 16 M and the use of these models for the evaluation of protective efficacy of deletion mutants (10). Olsen et al. similarly described the infection of BALB/c mice with aerosolized strain 16 M or 2308 and demonstrated that vaccination with the live attenuated animal vaccine B. abortus RB51 provided protection against intraperitoneal but not aerosolized B. abortus challenge (13). Our findings both support and extend the data generated in these studies.     

Evaluation of Indirect Enzyme-Linked Immunosorbent Assays and IgG Avidity Assays Using a Protein A-Peroxidase Conjugate for Serological Distinction between Brucella abortus S19-Vaccinated and -Infected Cows

This study aimed to evaluate the use of protein A-peroxidase (horseradish peroxidase [HRPO]) in indirect enzyme-linked immunosorbent assays (iELISAs) and IgG avidity assays for serological distinction between Brucella abortus S19-vaccinated and -infected cows. Four groups were analyzed: GI, 41 nonvaccinated seropositive cows; GII, 79 S19-vaccinated heifers analyzed at 3 months postvaccination; GIII, 105 S19-vaccinated cows analyzed after 24 months of age; and GIV, 278 nonvaccinated seronegative cows. IgG levels and avidity to B. abortus smooth lipopolysaccharide (S-LPS) were determined using anti-bovine IgG-HRPO or protein A-HRPO conjugates. Similar levels of IgG anti-S-LPS were found with GI using both conjugates. Lower IgG levels were detected with GII, GIII, and GIV using protein A-HRPO. Both conjugates showed high performance in discriminating GI from GIII, with high sensitivity (Se; 97.6%) and specificity (Sp; 97.1%). Protein A-HRPO was better in distinguishing GI from GIV (Se, 97.6%; Sp, 94.6%) and GI from GII (Se, 80.5%; Sp, 94.9%). Protein A-HRPO excluded a higher number of positive samples with GII and GIV. IgG avidity showed that protein A-HRPO, but not anti-IgG-HRPO, was able to distinguish nonvaccinated from vaccinated cattle, showing a higher avidity index (AI) with GI than with GII, with 78% of serum samples in GII showing an AI of <50%. Therefore, the iELISA using B. abortus S-LPS antigen and protein A-HRPO conjugate for preferential detection of the IgG2 subclass was shown to be suitable for serological distinction between S19-vaccinated and -infected cows. Also, antibodies generated after vaccination showed lower avidity, suggesting a role for the IgG2 subclass as an antibody of higher-affinity maturation after infection, constituting an additional tool for differentiating vaccinated from infected cattle.Brucellosis is a major zoonosis and has been considered an emerging or reemerging disease worldwide (11). Pathogens from the genus Brucella can infect a wide variety of mammals, causing abortion and infertility in domestic herds as well as debilitating illness in humans that may persist intermittently for years (13, 21).Diagnosis of brucellosis in cattle caused by Brucella abortus is difficult to establish because of the variable time of incubation and the absence of clinical signs other than abortion (24). Bacteriological isolation of the microorganism confirms the diagnosis, and this result is taken as the gold standard against which other tests have to be compared (31). However, as the rate of isolation of B. abortus from blood or tissue cultures generally presents low sensitivity and the results are not available immediately because this procedure may be time consuming and processing a large number of samples is cumbersome, the diagnosis is based mainly on serological methods (5, 9). Accordingly, most conventional serological assays, such as the rose bengal test (RBT), serum agglutination test (SAT), and complement fixation test (CFT), use whole-cell preparations or lipopolysaccharide (LPS)-enriched fractions, which are often obtained from smooth (S) B. abortus strains, being rich in S-LPS, which induces a strong antibody response (5, 27). The classical serological techniques rely mainly on the detection of antibodies to this antigen fraction (4).Enzyme-linked immunosorbent assays (ELISAs) have been evaluated for their diagnostic performance to detect serum antibodies to B. abortus in cattle, and such techniques offer several advantages over other tests, including that sera do not need to be heat inactivated as for CFT or pretreated with 2-mercaptoethanol (2ME) as for SAT/2ME; the reactivity is measured objectively, reducing subjective errors; and they are particularly advantageous in mass testing programs (24, 33, 34).In many countries, control programs are based on different protocols of B. abortus S19 vaccination to reduce prevalence, followed by the diagnosis and elimination of reactors (1). S19 vaccine is antigenically similar to the virulent strain, and therefore, serodiagnosis by conventional tests does not permit precise differentiation of vaccinated from infected animals (10, 27). Consequently, other tests have been developed, including the competitive ELISA (cELISA) and fluorescence polarization assay (FPA), which have eliminated most reactions due to residual antibodies produced in response to S19 vaccination (27, 31). However, the use of highly specific reagents as monoclonal antibodies in cELISA or costly equipment in FPA has made these assays unfeasible for various laboratories throughout the world (30).Immunoglobulin-binding proteins, such as protein A, protein G, or recombinant protein A/G labeled with horseradish peroxidase (HRPO), have been used as valuable tools in the ELISA for detection of anti-Brucella antibodies in various animal species (29, 30). While protein G reacts with both bovine IgG subclasses (IgG1 and IgG2), protein A reacts more specifically with the IgG2 subclass, whose levels increase with the intensity of antigen exposure in Brucella infection (22, 32). On the other hand, the avidity of specific IgG antibodies has been used often to discriminate acute from chronic infections for several diseases, such as toxoplasmosis (19) and neosporosis (3), as well as for human brucellosis (12, 15, 20), showing that antibodies with high avidity would be useful in excluding recent infection. However, there is currently no information on IgG avidity assays for bovine brucellosis, particularly with an emphasis on differentiating vaccinated from infected cattle.The purpose of the present study was to evaluate the use of protein A-peroxidase as conjugate in indirect ELISAs and IgG avidity assays in order to establish a serological distinction between B. abortus S19-vaccinated and -infected cows.     

Evaluation of Competitive ELISA for Detection of Antibodies to Brucella Infection in Domestic Animals

Aim

To evaluate competitive enzyme-linked immunosorbent assay (cELISA) for its suitability as an additional serological test for the diagnosis of animal brucellosis.

Methods

cELISA, which was developed at the Veterinary Laboratories Agency, has been evaluated for its accuracy and suitability as an additional serological test for the diagnosis of animal brucellosis. Samples from naturally and experimentally infected animals and those from Brucella-free flocks and herds were tested.

Results

Data obtained since 1991 were analyzed from routine surveillance, animals experimentally infected with Brucella, and stored sera to validate cELISA for the detection of antibodies to Brucella in cows, small ruminants, and pigs. The sensitivity of the test ranged from 92.31% to 100%, in comparison with 77.14% to 100% for the complement fixation test (CFT). Specificities for cELISA, indirect enzyme-linked immunosorbent assay, and CFT were greater than 90%.

Conclusion

cELISA can be used on a variety of animal species, and an added advantage is its suitability for use on poor-quality samples such as those affected by hemolysis.In accordance with EC Directive 91/68/EEC, flocks of sheep and herds of goats in the United Kingdom (UK)are monitored serologically to prove that they are free from Brucella melitensis. In 2006, competitive enzyme-linked immunosorbent assay (cELISA) was introduced to screen these animals as part of a surveillance program in Great Britain (GB), the territory including all of the UK except for Northern Ireland. It replaced the complement fixation test (CFT) because of its much higher specificity and ease of automation. Currently, in excess of 35 000 animals are tested annually.In 2001, a revision to the pig semen directive was introduced by EC Directive 99/608 so that CFT was replaced with the Rose Bengal test (RBT) as the test used for brucellosis on all pigs whose semen is used for artificial insemination. RBT and CFT were run in parallel in addition to cELISA prior to this date in order to assess the effects of changing the testing regime and, at the same time, to validate the use of cELISA for pigs. During 2001, all routine samples that were tested for artificial insemination purposes and were positive by RBT were also tested by cELISA and the results analyzed using different diagnostic thresholds. The aim was to set an appropriate threshold that would provide optimal specificity and sensitivity for cELISA.CFT, RBT, and indirect enzyme linked immunosorbent assay (iELISA) are the conventionally used tests for diagnosis of bovine brucellosis. These tests are described in the Manual for Diagnostic Tests and Vaccines for Terrestrial Animals produced by the World Organisation for Animal Health, previously the Office International des Epizooties (OIE) (1), and this manual gives details of all the diagnostic methods. It also describes the strain of Brucella required for antigen preparation and the procedure for standardization for each test.The cELISA for the detection of antibodies against Brucella spp. was adapted at the Veterinary Laboratories Agency (VLA) from the method described by MacMillan et al in 1990 (2). It was initially developed for the diagnosis of brucellosis in small ruminants and was tested extensively on British sheep and on sheep and goats from France. It has also since been tested on large numbers of cattle and pigs.The aim of this study was to bring together and compare all brucellosis testing results carried out using cELISA, RBT, and iELISA at the VLA since 1991. The samples had been collected and analyzed within the framework of various surveillance screening programs and experimental studies. The present study demonstrates the effectiveness of cELISA compared with other assays currently used as diagnostic tests of brucellosis in domestic animals.