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Background

Brucellosis is an important cause of morbidity and mortality in patients living in areas that are endemic for the infection.

Case Presentation

A 20 years old Saudi male was diagnosed to have severe aplastic anemia at King Faisal Specialist Hospital and Research Centre in Riyadh in April 2006. One hundred and twelve days following his successful allogeneic hematopoietic stem cell transplant, he presented with pyrexia in addition to neutropenia and mild thrombocytopenia. Brucella serology was strongly positive and blood cultures grew Brucella melitensis. The bacteremic episode of brucellosis was successfully treated with streptomycin, doxycyclin and ciprofloxacin at the outpatient clinic. To our knowledge, this is the first case of a naturally occurring Brucella infection complicated by Brucella bacteremia in a recipient of hematopoietic stem cell transplant.

Conclusion

Brucellosis may cause systemic infections, complicated bacteremias and serious morbidity in immunocompromised patients living in countries that are endemic for the infection. It should be considered as a possible cause of fever and pancytopenia in hematopoietic stem cell transplant recipients living in these geographical locations. Nevertheless, the infection is curable provided the diagnosis is made early and an appropriate antimicrobial therapy is promptly initiated.

A serological and bacteriological study was performed with sera taken from 2,228 swine from six states in Venezuela. None of the animals were vaccinated against brucellosis, and the prevalence of the disease varied from 5 to 89% on farms located in these states. Our studies indicated that the animals could be categorized into four groups depending on the degree of reactivity in serological tests. Brucella suis biovar 1 was isolated from the lymph nodes, spleens, and semen samples of seropositive animals and identified by oxidative metabolic techniques. B. suis could not be isolated from tissues of seronegative swine even from farms with cases of the disease (detected by serology). Results suggest that, although the immunodiffusion assay using Brucella melitensis B115 polysaccharide B or B. abortus 1119-3 O-polysaccharide could be useful in the detection of active infections, it is perhaps not as sensitive as some of the other standard serological tests used in this study for the detection of swine brucellosis.

An enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of brucella-specific IgG, IgM and IgA in 173 patients with acute brucellosis, 22 patients with chronic brucellosis and in 281 controls consisting of 98 patients with other infectious etiologies, 20 patients with non-infectious diseases and 163 normal healthy adults. The ELISA results were compared with culture findings, the results of slide agglutination tests with Brucella melitensis (M), B. abortus (A) and Ross Bengal (RB) antigens, and of tube and microagglutination tests. Brucella cultures were positive in 53 and 5% of patients with acute and chronic brucellosis respectively. The slide agglutination tests with A, M, A plus M and RB antigens were positive in 42, 44, 51 and 98% of patients with acute brucellosis and in 23, 27, 27 and 64% of patients with chronic brucellosis. There was no significant difference in the results between the tube and microagglutination tests regardless of the type of antigen used. At a titre of greater than or equal to 80 or greater than or equal to 160 these tests were positive in 98% and 92% of patients with acute brucellosis and 60 and 40% of patients with chronic brucellosis. The brucella culture and agglutination tests were negative for all the controls. Brucella ELISA immunoglobulins (Ig) were detected in some individuals in the control groups but the majority of these had titres of less than or equal to 100 for IgG, IgM, and IgA. However, patients with brucellosis had significantly higher ELISA titres in all classes of Ig than controls but the sensitivity and specificity within each Ig class varied with the titre considered. At a titre of greater than or equal to 1600 the brucella IgG had a sensitivity and specificity of 98% for patients with acute or chronic brucellosis; this decreased with lower reciprocal titres. The brucella IgM titre of greater than or equal to 400 had a sensitivity of 98% and a specificity of 98% for patients with acute brucellosis. However, in patients with chronic brucellosis the brucella IgM was very low. The brucella IgA titre of greater than or equal to 200 showed a sensitivity of 98% and a specificity of 99% for patients with either acute or chronic brucellosis. This study indicates that brucella ELISA is a rapid, sensitive and specific assay, provides a profile of Ig classes in the diagnosis of acute and chronic brucellosis, is useful for mass screening and could be considered the method of choice for the serological diagnosis of brucellosis.

In the search to find discriminative serological markers to differentiate between patients with acute brucellosis and those with chronic brucellosis, an enzyme-linked immunosorbent assay (ELISA) was used to determine and compare the brucella-specific IgE response in 80 sera from patients with acute brucellosis, 37 sera from patients with chronic brucellosis, 26 sera from patients with positive blood cultures for bacteria other than brucella and 51 sera from healthy controls. The IgE findings were compared to brucella-specific IgG, IgM, IgA and IgG1-4 demonstrated by ELISA, and to microagglutination test (MAT) results. Elevated (positive) antibrucella IgE titres were detected in 89 and 81% of sera from patients with acute and chronic brucellosis respectively. The predominant antibodies found in patients with acute brucellosis were of the IgG, IgM, IgA, IgE, IgG1 and IgG3 types while in chronic brucellosis IgG, IgA, IgE and IgG4 were found. Although IgE can be detected in patients with brucellosis, it does not discriminate between the acute and chronic stages of the disease.

The reactivity of monoclonal antibody (MAb) 12G12 was analyzed in regard to the main biovars of Brucella species and some members of the families Enterobacteriaceae and Vibrionaceae which present serological cross-reactions with the smooth lipopolysaccharide (S-LPS) of Brucella species. This MAb was strictly directed against the common specific epitope of the Brucella S-LPS. It recognized all of the smooth Brucella strains and biovars except B. suis biovar 2. In order to improve the specificity of the serological diagnosis of brucellosis, a competitive enzyme-linked immunosorbent assay (cELISA) was developed with the horseradish peroxidase-conjugated MAbs 12G12 and S-LPS of B. melitensis Rev1. The specificity of the cELISA was analyzed with 936 serum samples from healthy cattle. The assay was evaluated with sera from heifers (n = 18) experimentally infected with B. abortus 544. After infection, the performance of the cELISA was in agreement with those of the complement fixation test and the rose Bengal plate test. Finally, the specificity of the assay was also evaluated in regard to false-positive serological reactions by using sera from heifers experimentally infected with Yersinia enterocolitica 0:9 (n = 4) and with field sera presenting false-positive reactions (n = 74). The specificity of the cELISA was greater than the specificities of the complement fixation test and the rose Bengal plate test. Indeed, the new assay detected only 31 of the 101 false-positive serum samples detected by at least one serological test.

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.

 

Five serological assays were evaluated for the diagnosis of brucellosis in goats: the rose bengal test (RBT), complement fixation test (CFT), radial immunodiffusion (RID) with Brucella and Yersinia enterocolitica O:9 polysaccharides, counterimmunoelectrophoresis (CIEP) with cytosol, and enzyme-linked immunosorbent assay (ELISA) with polyclonal and protein G conjugates and smooth lipopolysaccharide (S-LPS), native hapten polysaccharide (NH), or cytosol antigens. For optimal sensitivity, RBT had to be used with sera-antigen at a 3:1 dilution. In the RID test, Brucella melitensis biotype 1 NH could not be replaced by Brucella abortus biotype 1 or Y. enterocolitica 0:9 polysaccharides. In the ELISA, S-LPS and NH gave similar results and the protein G conjugate increased the specificity. With the sera from 55 B. melitensis culture-positive goats, the sensitivity was 100% for RBT, CFT (titer > or = 4), and ELISA with S-LPS or NH; 94% for RID; and 93% for CIEP. All tests were negative (100% specific) when testing the sera from 127 brucella-free goats. Larger discrepancies among the results of the serological tests were obtained with sera from goats of areas where brucellosis is endemic. When the sera of 20 young goats vaccinated subcutaneously (10(9) CFU of B. melitensis Rev 1) and bled 6 months later were examined, the specificities were as follows: NH ELISA, 60%; CFT and S-LPS ELISA, 75%; RBT, 80%; CIEP, 90%; and RID, 94%. With the sera from 10 young goats vaccinated conjunctivally (10(9) CFU of B. melitensis Rev 1) all tests were 100% specific 4 months after vaccination. The proportion of goats giving a positive reaction after vaccination decreased faster in RID than in other tests.

In order to improve the specificity of the diagnosis of bovine brucellosis, we developed a test which can be regarded as an in vitro correlate of the delayed-type hypersensitivity test (DTH). A mixture of cytoplasmic proteins from Brucella melitensis B115 was used as a specific antigenic stimulus in bovine whole blood culture. Supernatants harvested at 18 to 24 h after the in vitro antigenic stimulus were assayed for their gamma interferon (IFN-gamma) content by using a commercial sandwich enzyme-linked immunosorbent assay kit. The IFN-gamma assay was evaluated with 10 heifers during the course (80 days) of an experimental infection and with 14 cows from an ongoing brucellosis outbreak. All of these animals were slaughtered, and pertinent organs were subjected to classical bacteriological analyses. In addition, we analyzed 23 field cases in which false-positive serological reactions occurred. The IFN-gamma results were compared with those of the standard DTH and a battery of serological assays, and they were correlated with bacteriological data. Both for the experimental infection and for the field brucellosis outbreak, the IFN-gamma assay detected infection in more animals than any combination of the serological tests, and it detected infection earlier than these tests. Finally, none of the samples from cows showing false-positive serological reactions was classified as positive by the IFN-gamma assay, attesting to its specificity and to its usefulness in interpreting ambiguous serological results. A rapid and convenient alternative to the DTH, the IFN-gamma assay appears to be an ideal method that is complementary to the serological diagnosis protocols.

Three dogs reared on a dairy farm with a high incidence for Brucella abortus were serologically positive for B. abortus and no other Brucella spp. The identity of the organism was confirmed to be B. abortus by AMOS (abortus melitensis ovis suis)-polymerase chain reaction with specific primers for B. canis. One hundred percent homology of the canine isolate and the bovine pathogen isolated from the farm was demonstrated. The only possible source of infection was infected cattle on the same farm. It is suggested that dogs be routinely included in brucellosis surveillance and eradication programs.

Brucella melitensis is the cause of brucellosis in sheep and goats, which often results in abortion. Few cases of B. melitensis infection in goats have occurred in the United States over the last 25 years. However, vigilance must be maintained, as it is for the bovine milk industry, to ensure that brucellosis is not introduced into the U.S. goat population. The objective of this study was to develop a sensitive and specific indirect enzyme-linked immunosorbent assay (iELISA) for the detection of B. melitensis-specific antibodies in goat milk. Brucella salt-extractable protein extract was employed as an antigen, and a horseradish peroxidase-labeled polyclonal anti-goat antibody was used as an anti-species conjugate. Thirteen of 13 (100%) individual infected goat milk samples tested positive and 134 of 134 (100%) uninfected bulk milk samples tested negative by the developed iELISA. Three positive milk samples with high, medium, and low absorbance values were used to simulate one positive animal in an otherwise negative herd. By this estimation, one high-titer animal could be detected in a herd of >1,600 animals. Detection estimates for medium- and low-titer animals were one positive animal per herd of <200 and 50 animals, respectively. Based on this estimation, it is recommended that herds be sampled in groups of 50 animals or less for bulk milk testing. The iELISA developed for this study was found to be sensitive and specific and shows potential for use as a bulk milk test for the detection of B. melitensis-specific antibodies in goat milk.

The facultative intracellular bacterial pathogen Brucella infects a wide range of warm-blooded land and marine vertebrates and causes brucellosis. Currently, there are nine recognized Brucella species based on host preferences and phenotypic differences. The availability of 10 different genomes consisting of two chromosomes and representing six of the species allowed for a detailed comparison among themselves and relatives in the order Rhizobiales. Phylogenomic analysis of ortholog families shows limited divergence but distinct radiations, producing four clades as follows: Brucella abortus-Brucella melitensis, Brucella suis-Brucella canis, Brucella ovis, and Brucella ceti. In addition, Brucella phylogeny does not appear to reflect the phylogeny of Brucella species' preferred hosts. About 4.6% of protein-coding genes seem to be pseudogenes, which is a relatively large fraction. Only B. suis 1330 appears to have an intact β-ketoadipate pathway, responsible for utilization of plant-derived compounds. In contrast, this pathway in the other species is highly pseudogenized and consistent with the “domino theory” of gene death. There are distinct shared anomalous regions (SARs) found in both chromosomes as the result of horizontal gene transfer unique to Brucella and not shared with its closest relative Ochrobactrum, a soil bacterium, suggesting their acquisition occurred in spite of a predominantly intracellular lifestyle. In particular, SAR 2-5 appears to have been acquired by Brucella after it became intracellular. The SARs contain many genes, including those involved in O-polysaccharide synthesis and type IV secretion, which if mutated or absent significantly affect the ability of Brucella to survive intracellularly in the infected host.

A single-step PCR assay with genus-specific primers for the amplification of a 223-bp region of the sequence encoding a 31-kDa immunogenetic Brucella abortus protein (BCSP31) was used for the rapid diagnosis of human brucellosis. We examined peripheral blood from 47 patients, with a total of 50 cases of brucellosis, and a group of 60 control subjects, composed of patients with febrile syndromes of several etiologies other than brucellosis, asymptomatic subjects seropositive for Brucella antibodies, and healthy subjects. Diagnosis of brucellosis was established in 35 cases (70%) by isolation of Brucella in blood culture and in the other 15 cases (30%) by clinical and serological means. The sensitivity of our PCR assay was 100%, since it correctly identified all 50 cases of brucellosis, regardless of the duration of the disease, the positivity of the blood culture, or the presence of focal forms. The specificity of the test was 98.3%, and the only false-positive result was for a patient who had had brucellosis 2 months before and possibly had a self-limited relapse. In those patients who relapsed, the results of our PCR assay were positive for both the initial infection and the relapse, becoming negative once the relapse treatment was completed and remaining negative in the follow-up tests at 2, 4, and 6 months. In conclusion, these results suggest that the PCR assay is rapid and easy to perform and highly sensitive and specific, and it may therefore be considered a useful tool for diagnosis of human brucellosis.

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.

Background and Objectives

Brucellosis is one of the most common zoonotic diseases in Iran and human brucellosis is endemic in all parts of the country. Because of the difficulty in the diagnosis of brucellosis, particularly in endemic areas, the use of new and feasible diagnostic tests seem to be of great importance for resolving the diagnostic obstacles. We evaluated the usefulness of a new serological test based on an immunocapture-agglutination technique in comparison with ELISA test for serological diagnosis of brucellosis.

Materials and Methods

A total of 11 patients with brucellosis, who had positive blood cultures for Brucella species, and 47 suspected patients were included in this study. Serum samples collected from these patients were tested by brucellacapt and ELISA and the results were, consequently, compared.

Results

In patients with positive blood culture, all the samples gave positive results with brucellacapt test while IgM ELISA, IgG ELISA and (IgG + IgM) ELISA tests were positive in 8, 9 and 11 patients, respectively. Out of the 46 suspected patients, (IgG + IgM) ELISA, Brucellacapt, IgG ELISA and IgM ELISA were positive in 37, 15, 34 and 37 patients, respectively.The best cut-off point of ELISA-IgG was 10.78 IU/ml which produced the maximal sensitivity and specificity for the diagnosis of human brucellosis.

Conclusion

Both the (IgG + IgM) ELISA and Brucellacapt tests demonstrate a high specificity in this study. According to the results of the current study, it is found that both tests are valuable tools for diagnosis of brucellosis in Iran as an endemic area of brucellosis. It is strongly suggested that a combination of both tests to be used for the diagnosis of brucellosis.

Brucellosis is a zoonotic infection transmitted from animals to humans by the ingestion of infected food products, direct contact with an infected animal or inhalation of aerosols. The last method is remarkably efficient given the relatively low concentration of organisms (10 – 100 bacteria) needed to establish infection in humans, and has brought renewed attention to this old disease. Brucella is a facultative intracellular pathogen that has the ability to survive and multiply in the phagocytes and cause abortion in cattle and undulant fever in humans. Brucella spp particularly B. melitensis, B. abortus, and B. suis represent a significant public health concern. At present, B. melitensis is the principle cause of human brucellosis in India. Molecular studies have demonstrated the phylogenetic affiliation of Brucella to Agrobacterium, Ochrobactrum, and Rhizobium. Human brucellosis still presents scientists and clinicians with several challenges, with regard to the understanding of its pathogenic mechanism, severity, progression, and development of improved treatment regimens. Molecular studies have now highlighted the pathogenesis of Brucella, for the development of newer diagnostic tools that will be useful in developing countries where brucellosis is a common, but often a neglected disease. This review compiles all these issues in general and the pathogenicity and newer diagnostic tools in particular.

Brucella ceti causes disease in Odontoceti. The absence of control serum collections and the diversity of cetaceans have hampered the standardization of serological tests for the diagnosis of cetacean brucellosis. Without a “gold” standard for sensitivity and specificity determination, an alternative approach was followed. We designed an indirect enzyme-linked immunosorbent assay (iELISA) that recognizes immunoglobulins G (IgGs) from 17 odontocete species as a single group. For the standardization, we used Brucella melitensis and Brucella abortus lipopolysaccharides, serum samples from seven resident odontocetes with no history of infectious disease displaying negative rose bengal test (RBT) reactions, and serum samples from seven dolphins infected with B. ceti. We compared the performance of the iELISA with those of the protein G ELISA (gELISA), the competitive ELISA (cELISA), and the immunofluorescence (IF) and dot blot (DB) tests, using 179 odontocete serum samples and RBT as the reference. The diagnostic potential based on sensitivity and specificity of the iELISA was superior to that of gELISA and cELISA. The correlation and agreement between the iELISA and the gELISA were relatively good (Ri/g2 = 0.65 and κi/g = 0.66, respectively), while the correlation and agreement of these two ELISAs with cELISA were low (Ri/c2 = 0.46, Rg/c2 = 0.37 and κi/c = 0.62, κg/c = 0.42). In spite of using the same anti-odontocete IgG antibody, the iELISA was more specific than were the IF and DB tests. An association between high antibody titers and the presence of neurological symptoms in dolphins was observed. The prediction is that iELISA based on broadly cross-reacting anti-dolphin IgG antibody would be a reliable test for the diagnosis of brucellosis in odontocetes, including families not covered in this study.

Background

Bovine brucellosis caused by Brucella abortus is endemic in most large commercial and smallholder cattle farms of Zimbabwe, while brucellosis in other domestic animals is rare. The diagnosis of brucellosis is mainly accomplished using serological tests. However, some Brucella spp. have been isolated from clinical cases in the field and kept in culture collection but their biochemical profiles were not documented. We report biochemical profiling and AMOS-PCR characterization of some of these field isolates of Brucella originating from both commercial and smallholder cattle farming sectors of Zimbabwe.

Findings

Fourteen isolates of Brucella from culture collection were typed using biochemical profiles, agglutination by monospecific antisera, susceptibility to Brucella-specific bacteriophages and by AMOS-PCR that amplifies species- specific IS711. The results of the biochemical profiles for B. abortus biovar 1 (11 isolates) and biovar 2 (2 isolates) were consistent with those of reference strains. A single isolate from a goat originating from a smallholder mixed animal farm was identified as B. melitensis biovar 1. The AMOS-PCR produced DNA products of sizes 498 bp and 731 bp for B. abortus (biovar 1 and 2) and B. melitensis biovar 1, respectively.

Conclusion

We concluded that the biochemical profiles and AMOS-PCR characterization were consistent with their respective species and biovars. B. abortus biovar 1 is likely to be the predominant cause of brucellosis in both commercial and smallholder cattle farms in Zimbabwe.

Aim

To describe and discuss the merits of various direct and indirect methods applied in vitro (mainly on blood or milk) or in vivo (allergic test) for the diagnosis of brucellosis in animals.

Methods

The recent literature on brucellosis diagnostic tests was reviewed. These diagnostic tests are applied with different goals, such as national screening, confirmatory diagnosis, certification, and international trade. The validation of such diagnostic tests is still an issue, particularly in wildlife. The choice of the testing strategy depends on the prevailing brucellosis epidemiological situation and the goal of testing.

Results

Measuring the kinetics of antibody production after Brucella spp. infection is essential for analyzing serological results correctly and may help to predict abortion. Indirect ELISAs help to discriminate 1) between false positive serological reactions and true brucellosis and 2) between vaccination and infection. Biotyping of Brucella spp. provides valuable epidemiological information that allows tracing an infection back to the sources in instances where several biotypes of a given Brucella species are circulating. Polymerase chain reaction and new molecular methods are likely to be used as routine typing and fingerprinting methods in the coming years.

Conclusion

The diagnosis of brucellosis in livestock and wildlife is complex and serological results need to be carefully analyzed. The B. abortus S19 and B. melitensis Rev. 1 vaccines are the cornerstones of control programs in cattle and small ruminants, respectively. There is no vaccine available for pigs or for wildlife. In the absence of a human brucellosis vaccine, prevention of human brucellosis depends on the control of the disease in animals.

Brucella suis, Brucella abortus, and Brucella melitensis were shown by microscopic and cultural procedures to multiply extensively within normal rat, mouse, and guinea pig monocytes maintained in vitro in cell cultures for 3 days. Intracellular growth of brucellae had no observable toxic effects on most monocytes, although many of the cells became completely engorged with brucellae within 3 days. Non-smooth brucellae and strain 19 multiplied slowly within normal monocytes. In contrast, "immune" monocytes) i.e. those derived from animals previously infected with smooth brucellae, greatly restricted the intracellular growth of smooth and non-smooth brucellae and strain 19. Growth of smooth Brucella, within either normal or "immune" monocytes, was not influenced by addition of Brucella antiserum to the culture medium. Desensitization of immunized guinea pigs did not diminish the refractory state of their monocytes. Cellular resistance did not develop when animals were vaccinated with heat-killed brucellae, though these animals did produce agglutinating antibody. Similarly, vaccination of animals with living, rough B. suis failed to induce a refractory state in their monocytes, even though the vaccinated animals developed delayed hypersensitivity to smooth Brucella antigen. In vivo studies of Brucella survival in the spleens of normal and vaccinated mice (treated with streptomycin to prevent extracellular survival) gave strong support to the in vitro demonstrations of acquired "cellular immunity." Some implications of these results are discussed.

Abstract

Vertical transmission of Brucella abortus in Sprague-Dawley (SD) rats was verified with microbiologic, serologic, and polymerase chain reaction (PCR) methods. The 38 initially Brucella-free SD rats, weighing 200 to 250 g, were injected subcutaneously with 50 μL of a suspension containing 1 × 109 colony-forming units (cfu) of B. abortus biotype 1 Korean isolate. The rats were allowed to mate with uninfected SD rats. The isolate was detected by culture and by AMOS (abortus, melitensis, ovis, suis) PCR in testis tissue of infected male rats and splenic tissue of infected female rats. By 7 d after inoculation, the results of both the rose bengal test (RBT) and the plate agglutination test (PAT) were positive for antibody against B. abortus; the reciprocal antibody titre ranged from 200 to 400 in the 1-mo-old offspring and 800 in their dams. The infected rats directly transmitted Brucella to their breeding partners and offspring. Fetuses of infected dams were found to be infected at 20 d of gestation. These data are discussed in relation to a model for epizootic and zoonotic cases possibly involving wild animals. Additional rigorous experiments are warranted to explore the value of this model in developing measures to prevent congenital brucellosis.

A study was performed to evaluate the previously described PCR (C. Romero, C. Gamazo, M. Pardo, and I. López-Goñi, J. Clin. Microbiol. 33:615-617, 1995) for the diagnosis of brucellosis in dairy cattle. Milk samples from 56 Brucella milk culture-positive cattle and from 37 cattle from Brucella-free herds were examined for Brucella DNA by PCR and for specific antibodies by an indirect enzyme-linked immunosorbent assay (ELISA). The specificities of both tests were 100% when testing the milk samples from Brucella-free cattle. The milk samples from 49 infected cattle were positive by PCR (87.5% sensitivity), and 55 were positive by ELISA (98.2% sensitivity). A PCR-positive sample was negative by ELISA, and 7 ELISA-positive samples were PCR negative, yielding an observed proportion of agreement of 0.91 for the two tests. Although the results suggest that ELISA is a better screening test than PCR, the combined sensitivity of the two assays was 100%, and their simultaneous application could be more useful than one test alone for a rapid screening of brucellosis in dairy cattle.

The diagnostic usefulness of an enzyme-linked immunosorbent assay (ELISA) using a purified recombinant ribosome recycling factor from Brucella melitensis (CP24 antigen) was tested in human and canine infections caused by smooth and rough Brucella species, respectively. Anti-CP24 antibodies were detected in 9 (43%) of 21 consecutive cases of canine brucellosis and in 8 (53%) of 15 dogs followed for 60 days after the diagnosis of acute brucellosis. Among eight patients with acute brucellosis, anti-CP24 antibodies were detected in four in the 10 weeks following diagnosis, but the remaining four were negative during the whole follow-up (22 weeks). The frequency of anti-CP24 antibodies was also low among 24 patients with subacute brucellosis and 23 patients with chronic illness (29 and 26%, respectively). While all patients positive for anti-CP24 antibodies were also positive for antibodies to total cytoplasmic proteins of Brucella (CP), five were negative for antibodies to another cytoplasmic protein, the Brucella lumazine synthase (BLS). When a larger sample of 35 human sera negative for anti-BLS antibodies was assayed, 85.7% were positive for anti-CP24 antibodies, suggesting that the combined measurement of both reactivities could yield a higher sensitivity than any test alone. To test this hypothesis, an ELISA combining both antigens was designed. The percentage of positive results among chronic cases was higher for this assay than for the individual measurement of anti-CP24 or anti-BLS antibodies (83 versus 26 and 65%, respectively) and was closer to the value obtained for anti-CP antibodies (91%). The frequency of anti-CP24 antibodies is low in both canine and human brucellosis. In the latter case, however, an ELISA combining CP24 and BLS is more sensitive than assays measuring anti-CP24 or anti-BLS antibodies separately and almost as sensitive as the ELISA using CP.

We describe a retrospective analysis of Brucella enzyme immunoassay (EIA) IgM and IgG results compared to those of the standard tube agglutination test (SAT). Among 1,091 samples tested, 104 (9.5%) and 24 (2.2%) sera were positive by IgM and IgG EIA, respectively. Supplemental testing by SAT showed that 82.7% (86/104) of IgM EIA-reactive samples and 54.2% (13/24) of IgG EIA-reactive samples were negative by SAT. Testing all EIA screen-reactive samples by SAT is required when evaluating patients for potential brucellosis. Due to the limitations of serology, culture remains the gold standard for detecting Brucella infection.

A case of brucellosis due to Brucella abortus, biotype 5, occurred in a bulk milk-tanker driver who collected milk from refrigerated tanks on 23 farms.

The organism was isolated from blood cultures by using a serum dextrose broth containing antibiotics. Serological investigations indicate that both abortus and melitensis antigen suspensions should be used in the investigation of cases of suspected brucellosis.

As Brucella abortus is an intracellular parasite heavy, prolonged, antibiotic therapy is necessary in the treatment of the disease. The danger of inadequate treatment of the acute disease is that it may become chronic, and response to antibiotic therapy in chronic brucellosis is not good.

We report a case of metastatic abscesses caused by a chronic form of brucellosis in a shepherd. When she was admitted the patient was cachectic with haematological signs of phlogosis. An abdominal computed tomography scan revealed the presence of multiple hepatic and renal abscesses with a fluid mass in the abdominal wall. The blood cultures, tuberculin skin test, and Wright reaction all gave negative results, but the brucellosis Coombs test for Brucella species was highly positive. Diagnosis was confirmed by a high titre of anti-Brucella IgM antibodies. The patient started antibiotic treatment with a progressive clinical improvement, but after discharge she was lost to follow-up and died 7 months later.