Evidence for unapparent Brucella canis infections among adults with occupational exposure to dogs.

Human serological assays designed to detect brucellosis will miss infections caused by Brucella canis, and low levels of periodic bacteremia limit diagnosis by blood culture. Recent B. canis outbreaks in dogs and concomitant illnesses in caretakers suggest that unapparent human infections may be occurring. With more than a quarter of a million persons in occupations involving dogs, and nearly 80 million dog owners in the United States, this pathogen is an under-recognized human health threat. To investigate occupational exposure to B. canis, we adapted a commercial canine serological assay and present the first controlled seroepidemiological study of human B. canis infections in recent years. 306 adults with occupational exposure to dogs and 101 non-matched, non-canine-exposed subjects were enrolled. Antibodies were detected using the canine D-Tec(®) CB rapid slide agglutination test (RSAT) kit with a secondary 2-mercaptoethanol (ME)-RSAT. Results were validated on a blinded subset of sera with an additional RSAT and indirect enzyme-linked immunoassay at the National Administration of Laboratories and Health Institutes (ANLIS) in Argentina. Seroprevalence ranged from 10.8% (RSAT) to 3.6% (ME-RSAT) among canine-exposed subjects. Kennel employees were more likely to test RSAT seropositive compared with other canine exposures (OR = 2.7; 95% CI, 1.3-5.8); however, low seroprevalence limited meaningful occupational risk factor analyses. Two seropositive participants reported experiencing symptoms consistent with brucellosis and having exposure to B. canis-infected dogs; however, temporality of symptom onset with reported exposure could not be determined. D-Tec(®) CB results had substantial agreement with ANLIS assays (Cohen's kappa = 0.60-0.68). These data add to a growing body of literature suggesting that people occupationally exposed to dogs may be at risk of unapparent B. canis infection. It seems prudent to consider B. canis as an occupational public health concern and encourage the development of serological assays to detect human B. canis infections.

Validation of a simple universal IELISA for the diagnosis of human brucellosis.

The definitive diagnosis of brucellosis requires isolation of the agent, although negative isolation does not rule out the infection. In contrast, serological testing is more sensitive and, therefore, preferred in clinical practice. The majority of reported cases around the world were caused by Brucella melitensis, B. abortus, B. suis and B. canis. The first three species contain O-polysaccharide (OPS) on the cell surface, but B. canis contains no measurable OPS on the rough lipopolysaccharide (R-LPS). A universal indirect enzyme immunoassay for the detection of serum antibody to smooth and rough Brucella spp. in both normal (u-IELISA®) and rapid forms (R-u-IELISA®) has been developed, and, therefore, the potential use of this method was assessed in comparison to cELISA, conventional tests, IELISA and RSAT on a total of 478 sera. The 77 sera from blood donors with no clinical or epidemiological evidence of brucellosis and negative serological tests showed a specificity of 100 % for both u-IELISA® and R-u-IELISA®, with a cut-off value of %P 24 and %P 18, respectively. Sera from 49 culture-positive cases (16 B. suis, 15 B. abortus, 12 B. melitensis and 6 B. canis) yielded a sensitivity of 98 % for u-IELISA® and 95.9 % for R-u-IELISA®. In general, u-IELISA® showed good correlation with cELISA and IELISA for the detection of antibodies to smooth and rough Brucella strains, as well as for monitoring patients during treatment, but R-u-IELISA® seems to need additional optimisation. u-IELISA® is simple to perform and could be a suitable test for field laboratories and hospitals lacking skilled personnel.

The feasibility of using antigens prepared with rough Brucella strains for diagnosis of canine brucellosis.

Clinical diagnosis of canine brucellosis is not sensitive enough and a negative blood culture cannot rule out the disease. Indirect methods of serological testing such as agar gel immunodiffusion (AGID), rapid slide agglutination test (RSAT) and indirect enzyme linked immunoassay (IELISA) are preferred for routine diagnosis. Since Brucella canis shares antigenic components with the Brucella ovis and Brucella abortus RB51 strain, it would seem that either strain could be used as antigen. We present data on AGID and IELISA tests using the B. ovis antigen, RSAT and IELISA using the B. canis antigen and IELISA using the B. abortus RB51 antigen. The cut-off values were adjusted by the ROC analysis; the IELISA-B. ovis cut-off value was 23 (%P) and the IELISA-B. abortus RB51, 24 (%P), with 100% sensitivity and 98.8% specificity. RSAT detected 100% of positive cases, while AGID was less sensitive. The sera from dogs treated with antibiotic showed that %P correlated well with the clinical course. Sera from dogs presumptively infected with B. suis were negative in all tests performed with the rough Brucella strains. RSAT is a very sensitive screening test and IELISA-B. canis, B. ovis and B. abortus RB51 could be used as confirmatory tests, since they show good specificity and sensitivity.

Human Brucella canis outbreak linked to infection in dogs.

The zoonotic risk of Brucella canis has been considered fairly high for persons who handle breeding dogs in kennels or are exposed to infected animals. Transmission to humans in other circumstances has been thought to be rare. We describe an uncommon outbreak of brucellosis caused by B. canis which, to the best of our knowledge, is the first reported in the literature. This outbreak involved six persons (three children and three adults), a bitch and three puppies which had close daily contact with the family. The clinical symptoms of the index case led to an erroneous diagnosis and the infection would have gone undiagnosed if culture had not been positive. This report aims to increase awareness of medical personnel of the need to order screening tests for children, immunodeficient persons or pregnant women presenting with fever of unknown origin, unexplained spleen or liver enlargement or other systemic signs. The emerging zoonotic potential of this disease in urban areas and the need to coordinate canine brucellosis surveillance systems should be evaluated.

A serological and bacteriological survey of dogs to detect Brucella infection in Lomas de Zamora, Buenos Aires province.

Canine brucellosis caused by Brucella canis is a disease of the reproductive tract that may cause miscarriage in females, infection of the sexual organs in males and infertility in both sexes. The prevalence of brucellosis in dogs is unknown and little has been done to control the disease, except in certain breeds and some commercial dog kennels. In the course of a free neuter program in Lomas de Zamora, Buenos Aires province, prevalence of antibodies to Brucella sp., bacteriological isolation and clinical observations were performed. Of 224 dogs studied, 33 (14.7%) were found positive for the rapid slide agglutination test (RSAT), 24 (10.7%) of which were confirmed by IELISA. Of the 33 RSAT positive, 17 (51.5%) blood cultures were done, and B. canis were isolated from 2 cases. Since infected dogs have been shown to remain bacteremic for prolonged periods, our results also suggest a risk of human infections in this area.

Brucella isolated in humans and animals in Latin America from 1968 to 2006.

We report a retrospective analysis of 1933 Brucella strains isolated from humans and animals in Latin American countries between 1968 and 1991 and in Argentina between 1994 and 2006. During the first period 50% of strains were from humans, mainly from Argentina, Mexico and Peru but, while B. suis was the main cause of infection in Argentina, B. melitensis was responsible for most infections in the other countries. In Argentina in the later years, B. melitensis and B. suis were observed more frequently than in the first period while isolation of B. abortus decreased. Of 145 B. melitensis human isolates, eight gave susceptibility patterns to dyes and penicillin and two were B. melitensis biovar 3, which has never been reported in animals. Forty-six percent of B. suis isolated were resistant to dyes which is an atypical feature in this species.

A new variant of Brucella melitensis.

Brucella melitensis is highly pathogenic and constitutes a serious risk to public health. In Argentina, biovar 1 has been isolated from infected animals, but the Rev.1 strain vaccine is not authorised for use. This report describes nine atypical B. melitensis isolates obtained from humans. These isolates grew slowly, produced small colonies and were susceptible to penicillin and dyes, similar to the B. melitensis Rev.1 vaccine strain, but were inhibited by streptomycin 2.5 mg/L. The isolation of such atypical B. melitensis variants has never been reported from animals in Argentina, and could indicate the emergence of a new mutant variant.

Detection of antibodies to Brucella ovis in sheep milk using B. ovis and B. canis antigen.

The diagnostic techniques most widely used for detecting brucellosis caused by Brucella ovis are serological tests such as complement fixation (CFT), agar gel immunodiffusion (AGID), and ELISAs. However, to our knowledge, milk tests, with the advantage that samples may be taken in a non invasive manner, have not been investigated as diagnostic tools. We studied 144 samples of milk and sera from lactating ewes, comparing bacteriological studies, serological and milk tests using Brucella canis and B. ovis antigens. A group of 75 ewes in an uninfected flock were serologically negative to rapid slide agglutination test (RSAT), indirect ELISA (IELISA)-B. canis, AGID and IELISA-B. ovis. The milk of these ewes had an IELISA-B. canis mean (%P) value of 16.18 (S.D. 5.63), while the IELISA-B. ovis had a mean (%P) value of 12.52 (S.D. 4.94). A cut-off value of (%P) 27.44 (+2 S.D.) or (%P) 33 (+3 S.D.) was determined by milk-ELISA-B. canis and (%P) 22.4 (+2 S.D.) and (%P) 27.34 (+3 S.D.) by milk-IELISA-B. ovis. These cut-off values were adjusted by receiver-operator characteristics (ROC) analysis using 23 positive samples from infected ewes, which indicated a milk-IELISA-B. canis cut-off value of (%P) 33 and milk-IELISA-B. ovis of (%P) 26 with 100% sensitivity and specificity. Based on our results, we propose that, following a study of a larger number of samples, the milk-IELISA-B. canis could be considered a suitable test for the diagnosis of B. ovis brucellosis in lactating ewes.

Use of Brucella canis antigen for detection of ovine serum antibodies against Brucella ovis.

Brucella ovis causes a genital disease of sheep manifested by epididymitis in rams and placentitis in ewes producing reduced fertility in the flock. Clinical diagnosis is not sensitive enough and bacteriological testing is not feasible for detection of the disease in large numbers of animals. Indirect methods of serological testing are preferred for routine diagnosis, of which agar gel immunodiffusion (AGID), complement fixation (CF) and ELISA tests are recommended as the most efficient. Since B. ovis shares antigenic components with Brucella canis, it would seem that either strain could be used as antigen with the same results; however, the advantage of the B. canis (M-) strain variant is that it can be used to develop a satisfactory antigen for agglutination tests. We present data on AGID and IELISA tests using B. ovis antigen and rapid screening agglutination test (RSAT), 2-mercapto-ethanol RSAT (2ME-RSAT) and IELISA using B. canis antigen. We tested 225 animals. The cut-off values were adjusted by ROC analysis using 51 negative and 32 positive sera; the IELISA-B. canis cut-off value was 39 (%P) and IELISA-B. ovis, 51 (%P), with 100% sensitivity and specificity. Of the 32 positive sera from the infected flock RSAT detected 32 (100%), 2ME-RSAT 29 (91%) and AGID 31 (97%). Of the 142 sera from suspicious flocks, 46 were negative and 56 positive in all the tests; 16 were positive by RSAT, IELISA-B. canis and IELISA-B. ovis, 20 positive only with RSAT and 2 positive only by both IELISAs. RSAT is a very sensitive screening test that, because of its simplicity and easy interpretation, following a study in larger sample, could replace AGID as a screening test for diagnosis of ovine brucellosis caused by B. ovis. The IELISA-B. canis or IELISA-B. ovis could be used as confirmatory tests, since they show equal specificity and sensitivity.

Sensitivity and specificity of an indirect enzyme-linked immunoassay for the diagnosis of Brucella canis infection in dogs.

The diagnosis of B. canis infection in dogs is based on bacteriological examination and serological methods including agglutination and gel diffusion tests. Bacteriological studies are the only methods that have been considered specific but, as intermittent periods of abacteraemia may occur, a negative blood culture cannot be used as a criterion for excluding canine brucellosis. Close contact between people and infected dogs increases the risk of transmission; however, its impact on public health is probably underestimated due to lack of reporting and inadequate diagnostic services. This paper describes an indirect enzyme-linked immunoassay (IELISA) procedure for the diagnosis of brucellosis caused by B. canis in a population of normal and infected dogs previously screened by the buffered plate antigen test (BPAT) and rapid slide agglutination test (RSAT). The serological survey was performed with 446 field sera. The 270 sera from the asymptomatic group found negative by BPA, RSAT and blood culture showed IELISA specificities of 96.7% and 100%, respectively, when cut-off values of OD 0.237 and 0.281 were selected. For 52 sera from culture-positive dogs, IELISA sensitivity was 100% with cut-off values of OD414 0.237 and 0.281. OD414 0.281 was selected because this value provided the highest accuracy with minimal false-negative and false-positive results. This cut-off value was used to study 124 blood culture-negative but RSAT positive sera. IELISA produced 107 positive results; the 17 sera that were negative by IELISA presented a wide range of reactivities by RSAT (2 were RSAT positive at 1 in 2 dilution and 15 were weakly positive with pure serum). These samples were probably from animals at an early stage of the infection or were false-positive results. The IELISA described here detects IgG and IgA antibodies that are useful for evaluating the clinical status of dogs. Although RSAT is a practical screening test, a supplementary technique such as IELISA should be used on all positive RSAT samples to ensure diagnostic specificity. Furthermore, people in contact with infected dogs could be investigated for possible transmission. The procedure described in this study was relatively simple and could have widespread applications.

Competitive enzyme immunoassay for diagnosis of human brucellosis.

The methods commonly used for human brucellosis serological testing are agglutination tests and the complement fixation test (CFT). Among the newer serological tests, primary binding assays were developed to improve sensitivity and specificity. The competitive enzyme immunoassay (CELISA) for the detection of serum antibody to Brucella is a multispecies assay which appears to be capable of differentiating vaccinal and cross-reacting antibodies from antibodies elicited by field infection in cattle. The competing monoclonal antibody used in this assay is specific for a common epitope of smooth lipopolysaccharide (S-LPS). In this study, we compared the CELISA to the classical tests for the diagnosis of human brucellosis. The CELISA cutoff value was determined to calculate its diagnostic specificity and sensitivity. A survey was performed with 911 sera. Of the sera, 341 were from an asymptomatic population that tested negative with conventional serological tests (screening and confirmatory). Based on these samples, the CELISA specificities were determined to be 99.7 and 100% with cutoff values of 28 and 30% inhibition (%I), respectively. In a further study with 393 additional sera from an asymptomatic population found negative by the conventional screening tests, the CELISA specificities were calculated to be 96.5 and 98.8% with cutoff values of 28 and 30%I. The CELISA sensitivities were determined to be 98.3 and 94.8% with cutoff values of 28 and 30%I, respectively, for sera from 116 individuals found positive by the classical tests. For the 51 culture-positive patients, CELISA was positive for 100%, the CFT was positive for 92%, and the standard tube agglutination test (TAT) was positive for 100%. The CELISA specificity was 100% for 31 sera from patients found negative by conventional serological tests but with brucellosis-like symptoms. The CELISA is fairly rapid to perform, somewhat faster than TAT, and cross-reacts less with other antigens (or antibodies) than the conventional tests. Further, the CELISA is simpler to perform that the CFT and may readily be standardized by the use of purified S-LPS antigen and monoclonal antibody for competition.