Abortus Biovar Research Articles

Emergence of a Clonal Lineage of Brucella abortus Biovar 3 in Clinical Cases in Spain

Brucella abortus biovar 3 is an uncommon cause of human brucellosis in Spain, where Brucella melitensis has accounted for 97.5% of all cases. B. abortus biovar 3 was detected in specimens from four patients in Caceres (west-central Spain) with a febrile syndrome associated with arthralgia and

Characterization of some Brucella species from Zimbabwe by biochemical profiling and AMOS-PCR

BackgroundBovine 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.FindingsFourteen 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.ConclusionWe 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.

Characterisation of Brucella abortus biovar 3 isolates from Turkey as biovar 3b

Between 2002 and 2007, 75 strains of Brucella abortus were isolated from aborted bovine fetuses collected from several regions of Turkey. The isolates were all identified as B abortus biovar...

Brucellosis in Camels (Camelus dromedarius) in Darfur, Western Sudan

In a field outbreak of brucellosis in 21 camels mixed with cattle, sheep and goats, five camels, three of which showed clinical signs, were serologically positive. In a subsequent abattoir survey of apparently healthy camels, six animals were seropositive, albeit with titres that tended to be lower than those found in the field outbreak. Of the six seropositive slaughtered camels, five were shown to have lymph nodes (prescapular and supramammary) infected with brucellae (Brucella melitensis biovar 3, two camels; Brucella abortus biovar 6, three camels). Infection of camels with B. abortus biovar 6 had not previously been reported. Infection of the supramammary lymph nodes presents a potential hazard to those who consume raw camels' milk, a common practice in nomadic camel owners.

Genotipos de aislados de campo de Brucella abortus de distintas regiones geográficas de Chile

SuMMAry Bovine brucellosis is an endemic, zoonotic disease of high economic impact. The genetic identification of the prevalent Brucella abortus strains, the pathogen, is key to pursue further epidemiological strategies for disease control. The insertion sequence IS711 has been used as genetic marker to differentiate among Brucella species, members of the same specie and within the same biovar. We have analyzed the IS711- RFLP pattern for 46 B. abortus isolates, collected during the period of 1997-2005 from 16 different geographical areas of Chile. All isolates were previously identified by conventional techniques as B. abortus biovar 1. Of these, 87% shared the same IS711 DNA profile, while an 8.7 % corresponded to the pattern of RB51 vaccine strain. We report the finding of two new strains, not differentiated by AMOS PCR, which showed unreported patterns of IS711-RFLP.

Repuesta serológica y bacteriológica a la exposición al biovar 1 de Brucella abortus en búfalos de agua (Bubalus bubalis) vacunados con la cepa RB51 de Brucellus abortus

Serological and bacteriological responses to Brucella abortus biovar 1 following vaccination with B. abortus strain RB51 (RB51) were evaluated in thirty domestic water buffalo (Bubalus bubalis) randomly divided into five treatment groups. Groups I to V received, respectively, the recommended dose (RD) of RB51 vaccine once, RD twice 4 weeks apart, double RD once, double RD twice 4 weeks apart, and saline once (control). Vaccination did not result in a serological response. Experimental animals released 27 weeks post initial inoculation (27 PIIW) into a brucellosis-positive herd failed to seroconvert after 29 weeks. Experimental challenge commenced at 57 PIIW. All animals received B. abortus biovar 1 intraconjunctivally at 0, 5 and 9 weeks post experimental exposure (PEEW). Serum samples collected at 4, 8 and 13 PEEW were negative. At 16 PEEW all animals received B. abortus biovar 1 subcutaneously (SC), and all seroconverted by 20 PEEW. Five of twenty-six animals were positive for Brucella infection on bacterial culture. Brucella abortus biovar 1 was isolated from three animals; B. abortus RB51 was isolated from two. Treatment group, age and sex had no effect on the isolation of Brucellae (P>0.05).

BRUCELLOSIS IN ELK OF EASTERN IDAHO

Brucellosis occurs in free-ranging elk (Cervus elaphus) and bison (Bison bison) in the Greater Yellowstone Area, which includes portions of Idaho, Wyoming, and Montana. Brucella abortus was first detected in elk in Idaho in 1998, and from 1998 to 2002, serologic surveillance of hunter-killed elk was conducted in northeastern and southeastern Idaho. Prevalence of antibodies in these elk varied annually, but averaged between 2% and 3%. Elk were also trapped in northeastern Idaho from 1998-2002 and tested for brucellosis using serology and tissue culture. In areas where artificial feeding of elk was done, antibody prevalence ranged from 12% to 80% depending on site, age, and sex. At one feeding site (Rainey Creek), a decline in the prevalence of antibodies (from 56.8% in 1999 to 13.5% in 2002) was detected after the removal of seropositive elk over 4 yr. Seropositive elk removed from two artificial winter feeding sites (Rainey Creek and Conant Creek) were euthanized and sampled or held in captivity and allowed to calve prior to euthanasia and necropsy. At necropsy, B. abortus biovar 1 and B. abortus biovar 4 were isolated from both cows and calves; however, biovar 4 was predominant. A dual infection with both biovars was found in one calf born to a seropositive cow from which biovar 4 was isolated. Abortions (16%), stillbirths (8%), and weak calves (4%) were observed in these elk. These findings confirm the presence of brucellosis in elk in eastern Idaho and provide information on disease management options.

Multiplex PCR Assay for the Identification and Differentiation of all Brucella Species and the Vaccine Strains Brucella abortus S19 and RB51 and Brucella melitensis Rev1

The genus Brucella consists of 6 recognized bacterial species (1) and 2 proposed new species recently isolated from marine mammals (2). Some species have several biovars or biotypes, distinguishable by time-consuming analysis of phenotypic characteristics (3). Such analyses are subject to variable interpretations and must be performed by skilled technicians who are at high risk because most clinical Brucella strains are highly pathogenic. Accurate typing procedures are critical for eradication and control of disease-causing organisms, but DNA-based techniques are challenging because the various Brucella species and strains have a high degree of genetic homology, as demonstrated by whole-genome comparative analyses of the 3 Brucella genomes sequenced, Brucella melitensis (4), Brucella suis (5), and Brucella abortus (6). Unique fragments occur among the different Brucella genomes, however, which allowed us to develop a rapid, specific single-test-tube assay for molecular identification of all known Brucella species. We used the following species- or strain-specific genetic differences to design PCR primers: ( a ) a 25-kb DNA deletion leading to the loss of omp31 gene in the reference strains of all B. abortus biovars (6)(7); ( b ) a 15-kb deletion comprising …

Abortos provocados por Brucella abortus en ovejas de Nigeria

This paper reports on a sporadic, naturally acquired infection of sheep with Brucella abortus on a privately owned farm in Toro near Bauchi, Nigeria. The abortions, which occurred in a flock of 28 Yankassa sheep, involved five ewes at the third month of gestation. Serum and milk samples from the flock were examined for Brucella antibodies by the Rose Bengal plate test, serum agglutination test (SAT) and milk ring test (MRT). The proportion shown as positive by SAT was 14.3%. All the five milk samples examined by MRT were positive. A total of seven isolates of Brucella were obtained from three milk samples and four vaginal swabs collected from aborting ewes. All isolates were identified and biotyped as B. abortus biovar 1. This biovar was also isolated from cattle maintained on the farm in association with the sheep. The infection was attributed to the animal husbandry practices employed on the farm.

Development of a new PCR assay to identify Brucella abortus biovars 5, 6 and 9 and the new subgroup 3b of biovar 3

One hundred twenty-nine Brucella field strains isolated from cattle in Cantabria, Spain, from March 1999 to February 2003, were analysed by using the AMOS-ERY PCR assay and by Southern blot hybridisation with a probe from insertion sequence IS711. Most of the field isolates produced only the ery band in the AMOS-ERY assay and showed a hybridisation pattern identical to that exhibited by reference strains of biovars 5, 6 and 9 of Brucella abortus, but different from strain Tulya, belonging to biovar 3 of B. abortus. However, typing of these strains by standard methods demonstrated that they belonged to biovar 3 of B. abortus. These results indicated that B. abortus biovar 3 was not genetically homogeneous and at least could be divided in two. In one class, that we called biovar 3a, would be the Tulya strain, while the local field strains would belong to biovar 3b. Cloning and nucleotide sequencing of a DNA fragment containing an IS711 copy exclusive of the B. abortus field strains from biovar 3b and reference strains from biovars 5, 6 and 9, revealed the existence of a 5.4 kb deletion close to an IS711 copy. Based on these data, we designed a new primer, which together with the IS711 AMOS primer produced a PCR fragment of 1.7 kb only from the isolates of biovars 3b, 5, 6 and 9 of B. abortus. No amplification products were produced with these primers from strains of the rest of species and biovars of Brucella and from bacteria phylogenetically close to Brucella analysed in this work. Addition of this primer to the AMOS-ERY PCR primer cocktail allows the positive distinction of B. abortus biovars 3b, 5, 6 and 9 from the rest of Brucella species and biovars.

Restriction site polymorphisms in the genes encoding new members of group 3 outer membrane protein family of Brucella spp.

Thirty-seven Brucella reference and field strains representing all the species and their biovars were analysed by PCR–RFLP to determine the degree of variation in the genes encoding the new members of group 3 outer membrane protein (Omp) family. Analysis of the omp22 and omp25c/ omp25d genes indicated that the restriction patterns were identical for all species and biovars with all restriction enzymes tested, except for Brucella ovis that showed a short 30 bp deletion close to omp22 gene, and for B. abortus biovar 6 and B. ovis that lacked a DdeI site and a HinfI site, respectively, in the omp25c/ omp25d genes. Analysis of PCR products of the omp31b gene digested with 20 restriction enzymes revealed that this gene has a greater level of DNA polymorphism than the other genes encoding the new members of group 3 Omp family. A deletion of 232 bp was detected in fourteen B melitensis strains from different hosts and from different geographic origins, confirming that this feature is indeed a hallmark of B. melitensis. PCR–RFLP analysis of omp31b with DdeI allowed us to identify species-specific markers for B. abortus, B. melitensis, and B. ovis. Finally, by PCR analysis, Southern blot hybridization and DNA sequencing we showed that a large deletion of 15 kb, comprising the entire omp25b gene and 21 more genes, is present in all B. ovis strains, thus confirming previous observations from other authors.

Phenotypic characterization of Brucella strains isolated from livestock in Nigeria

Isolation of brucellae from aborted fetuses, hygroma fluids, milk and vaginal swabs obtained from aborting cattle, sheep, goats, pigs, and horses in Nigeria was carried out. A total of 25 isolates, obtained mainly from cattle, sheep and horses, were biotyped. All strains belonged to one species, Brucella abortus biovar 1. The epidemiological significance of this finding is discussed. Some preliminary observations on the zoonotic and public health implications of Brucella infection in Nigerian livestock are presented. A control programme involving improved management, animal movement restrictions, public health education and mass vaccination of animals is suggested.

Brucellosis in captive Rocky Mountain bighorn sheep (Ovis canadensis) caused by Brucella abortus biovar 4.

Nine (four female, five male) captive adult Rocky Mountain bighorn sheep (Ovis canadensis) contracted brucellosis caused by Brucella abortus biovar 4 as a result of natural exposure to an aborted elk (Cervus elaphus) fetus. Clinical signs of infection were orchitis and epididymitis in males and lymphadenitis and placentitis with abortion in females. Gross pathologic findings included enlargement of the testes or epididymides, or both, and yellow caseous abscesses and pyogranulomas of the same. Brucella abortus biovar 4 was cultured in all bighorn sheep from a variety of tissues, including testes/epididymides, mammary gland, and lymph nodes. All bighorn sheep tested were positive on a variety of standard Brucella serologic tests. This is the first report of brucellosis caused by B. abortus in Rocky Mountain bighorn sheep. It also provides evidence that bighorn sheep develop many of the manifestations ascribed to this disease and that infection can occur from natural exposure to an aborted fetus from another species. Wildlife managers responsible for bighorn sheep populations sympatric with Brucella-infected elk or bison (Bison bison) should be cognizant of the possibility of this disease in bighorn sheep.

Genetic diversity at alkB locus in Brucella abortus.

DNA polymorphism of the alkB gene, a DNA repair gene, was assessed by PCR on Brucella abortus biovars 1 (strains 99, S19, 45/20, RB51 and 2308), 3 (Tulya strain), 5 (B3196 strain) and 6 (870 strain). A DNA repetitive element, named IS711, was detected in all studied biovars 1 and its complete nucleotide sequence was determined. We found that the element in alkB gene, bounded by 14 bp imperfect inverted repeats (IRs), is 840 bp long and appears to duplicate a consensus target site, CTAG. Analysing its nucleotide sequence of both forward and reverse strands, more than 10 open reading frames (ORFs) were found. Two potential transposase coding regions were chosen comparing all possible ORFs with the database. Comparing IS711 elements isolated from Brucella species, including both those characterized in our work and the published ones, differences in length and in nucleotide composition were observed among Brucella species, members of the same species and within the same strain. Our results confirm the heterogeneity of IS711 elements in Brucella genus and suggest the possibility to use this element to assess gene and genome diversity and to identify new molecular markers for Brucella species.

Safety of revaccination of pregnant bison with Brucella abortus strain RB51.

From December 1998 through February 1999, a study was conducted in a Brucella-infected bison herd to evaluate the safety of booster vaccination of adult bison (Bison bison) with 6 x 10(9) colony forming units (CFU) of Brucella abortus strain RB51 (SRB51) that had previously been vaccinated as yearlings with 1 x 10(10) CFU of SRB51. Abortions or other adverse effects were not observed after SRB51 booster vaccination. At 10 wk after adult vaccination, pregnant and nonpregnant bison (n = 65) were randomly selected for bacteriologic sampling of targeted maternal tissues during abattoir processing. Fetal tissues were also sampled in pregnant bison. The SR351 recovered from tissue samples of eight of 48 pregnant bison and none of 17 nonpregnant bison. In three of the eight culture-positive bison, SRB51 was recovered from fetal tissues. In three additional bison, one pregnant and two nonpregnant, B. abortus biovar 1 field strain was recovered from internal iliac or supramammary lymphatic tissues. Results of this study suggest the possibility that the SRB51 vaccine can be safely used to booster vaccinate pregnant bison in a Brucella-infected bison herd. Our data also reaffirms the potential for B. abortus field strains to persist in bison until attainment of reproductive age, despite extensive use of vaccination and serologic testing.

Evaluation of brucellosis RB51 vaccine for domestic water buffalo ( Bubalus bubalis) in Trinidad

Thirty-two young domestic water buffalo ( Bubalus bubalis) were obtained from a brucellosis-free farm to determine effectiveness of RB51 vaccination for prevention of Brucella infection under natural-exposure conditions in Trinidad. Study animals (20 males and 12 females 5–20 months old) were assigned to vaccination or control groups, using a block randomization design ensuring equal sex distributions between groups. The vaccination group received commercially available RB51 at the recommended calfhood dose of (1.0–3.4)×10 10 colony-forming units (CFU) and controls received 2 ml sterile saline. Vaccination did not result in positive serologic results as measured by four traditional agglutination tests: standard tube agglutination test (STAT), standard plate agglutination test (SPAT), buffered plate agglutination test (BPAT), and card agglutination. Study animals were maintained in a brucellosis-positive herd in southern Trinidad with an estimated 56% prevalence to allow for natural exposure to B. abortus, which was evaluated using STAT, SPAT, BPAT, and card tests. Animals were sampled seven times over 2 years and were classified as positive if they had persistent agglutination titers or had Brucella isolated from specimens collected at completion of the study. Five of the original 32 study animals were lost to follow-up during the field trial. Six of the 14 (43%) vaccinated animals completing the study were classified as positive for Brucella infection—as were two of the 13 (15%) control animals ( P=0.21). Isolates from four vaccinates and one control were confirmed as B. abortus biovar 1.

Brucella 'HOOF-Prints': strain typing by multi-locus analysis of variable number tandem repeats (VNTRs)

BackgroundCurrently, there are very few tools available for subtyping Brucella isolates for epidemiological trace-back. Subtyping is difficult because of the genetic homogeneity within the genus. Sequencing of the genomes from three Brucella species has facilitated the search for DNA sequence variability. Recently, hypervariability among short tandem repeat sequences has been exploited for strain-typing of several bacterial pathogens.ResultsAn eight-base pair tandem repeat sequence was discovered in nine genomic loci of the B. abortus genome. Eight loci were hypervariable among the three Brucella species. A PCR-based method was developed to identify the number of repeat units (alleles) at each locus, generating strain-specific fingerprints. None of the loci exhibited species- or biovar-specific alleles. Sometimes, a species or biovar contained a specific allele at one or more loci, but the allele also occurred in other species or biovars. The technique successfully differentiated the type strains for all Brucella species and biovars, among unrelated B. abortus biovar 1 field isolates in cattle, and among B. abortus strains isolated from bison and elk. Isolates from the same herd or from short-term in vitro passage exhibited little or no variability in fingerprint pattern. Sometimes, isolates from an animal would have multiple alleles at a locus, possibly from mixed infections in enzootic areas, residual disease from incomplete depopulation of an infected herd or molecular evolution within the strain. Therefore, a mixed population or a pool of colonies from each animal and/or tissue was tested.ConclusionThis paper describes a new method for fingerprinting Brucella isolates based on multi-locus characterization of a variable number, eight-base pair, tandem repeat. We have named this technique "HOOF-Prints" for Hypervariable Octameric Oligonucleotide Finger-Prints. The technique is highly discriminatory among Brucella species, among previously characterized Brucella strains, and among unrelated field isolates that could not be differentiated by classical methods. The method is rapid and the results are reproducible. HOOF-Printing will be most useful as a follow-up test after identification by established methods since we did not find species-specific or biovar-specific alleles. Nonetheless, this technology provides a significant advancement in brucellosis epidemiology, and consequently, will help to eliminate this disease worldwide.

Incidence and control of brucellosis in the Near East region

In countries of the Near East region, brucellosis was reported in almost all domestic animals, particularly cattle, sheep and goats. Brucellosis in camels has been reported in Saudi Arabia, Kuwait, Oman, Iraq, Iran, Sudan, Egypt, Libya and Somalia. It has been reported even in racing camels in the United Arab Emirates. In Egypt, brucellosis has been reported also in buffaloes, equines and swine. Brucella melitensis biovar 3 is the most commonly isolated species from animals in Egypt, Jordan, Israel, Tunisia and Turkey. B. melitensis biovar 2 was reported in Turkey and Saudi Arabia, and B. melitensis biovar 1 in Libya, Oman and Israel. B. abortus biovar 1 was reported in Egypt, biovar 2 in Iran, biovar 3 in Iran and Turkey, and biovar 6 in Sudan. The countries with the highest incidence of human brucellosis are Saudi Arabia, Iran, Palestinian Authority, Syria, Jordan and Oman. Bahrain is reported to have zero incidence. Most human cases are caused by B. melitensis, particularly biovar 3. However, B. abortus has been responsible for an increasing number of cases in recent years, e.g. in Yemen, where B. abortus was identified in 45 cases and B. melitensis in 7 cases out of 330 cultures performed in 1995. Concerning control of brucellosis in animals, there is a controversy on the choice of policy. In some countries, the test and slaughter policy together with the vaccination of young females is adopted, in others, particularly with regard to sheep and goats; mass vaccination has been recently started. The most commonly used vaccines are B. abortus S19 and B. melitensis Rev.1 vaccines. B. abortus RB51 vaccine is used in some countries on small scale. Vaccination is limited to cattle and small ruminants.

How to substantiate eradication of bovine brucellosis when aspecific serological reactions occur in the course of brucellosis testing

Collaborative work was financed by the EU to develop and assess new diagnostic tools that can differentiate between bovine brucellosis and bovine infections due to Yersinia enterocolitica O:9 either in conjunction with, or as an alternative to, the classical serological, bacteriological or allergic skin tests. Sixteen heifers were experimentally infected with Brucella abortus biovar 1 (five heifers), Brucella suis biovar 2 (two heifers), Y. enterocolitica O:9 (six heifers) and Y. enterocolitica O:3 (three heifers). Four heifers, naturally infected with Y. enterocolitica O:9 that presented aspecific brucellosis serological reactions were also included in the experiment. A self-limited infection was induced in cattle by B. suis biovar 2. All the brucellosis serological tests used, i.e. the slow agglutination test (SAW), the Rose Bengal test (RB), the complement fixation test (CFT), indirect and competitive ELISA’s, lacked specificity when used to analyze sera from Y. enterocolitica O:9 infected animals. A Yersinia outer membrane proteins (YOPs)-ELISA was also used and although the test is able to detect a Yersinia group infection, it provided no evidence of whether or not there is a possible brucellosis infection when dual infections are present. The brucellergen IFN-γ test showed a lack of specificity also. The only test that was proven to be specific is the brucellergen skin test. All brucellosis serological tests, except the indirect ELISA, were limited in their ability to detect B. abortus persistently infected animals. Based on these experimental studies, a strategy was implemented as part of the year 2001 Belgian Brucellosis Eradication Program to substantiate the eradication of bovine brucellosis. Epidemiological inquiries have identified risk factors associated with aspecific serological reactions, possible transmission and infection of cattle by B. suis biovar 2 from infected wild boars; and both legal and administrative measures taken by the veterinary services. No cases of bovine brucellosis have been confirmed in Belgium since March 2000.

Field investigations of brucellosis in cattle and small ruminants in Syria, 1990-1996

The authors present the epidemiological status of brucellosis in cattle and small ruminants in Syria from 1990 to 1996, based on laboratory findings at the Brucellosis Centre, Damascus. Initial investigations using the Rose Bengal plate test, the complement fixation test and a miniaturised variant of the slow agglutination test were conducted throughout the country in 1990 and 1991, revealing an overall herd seroprevalence rate of 3.14% in cattle herds and 2.94% in small ruminant flocks. Although partially biased by previous vaccination of young female cattle with S19 vaccine, these figures indicate that brucellosis in cattle is widespread, particularly in the urban governorates (provinces) of Damascus, Aleppo and Suwaydah. Brucellosis seroprevalence in sheep and goats was relatively high in the governorates of Damascus, Aleppo and Dara'a. The results of a second series of investigations, performed between 1992 and 1996, show that herd seroprevalence in cattle decreased steadily from 17.48% in 1992, to 2.59% in 1996, in the Government-owned farms, while seroprevalence increased in the private sector during the same period. The difference may be explained by the restriction of brucellosis vaccination to public farms (although this was far from systematic), combined with partial application of a 'test-and-slaughter' policy. In sheep and goats, brucellosis seroprevalence fluctuated in the two sectors, but was higher in the private sector where husbandry is principally extensive. Bacteriological investigations led to the isolation of Brucella melitensis biovars 2 and 3 in sheep and B. abortus biovar 9 in cattle. Although no specific methodology was employed, particularly with regard to sampling, this study is significant as the first international report of the distribution of brucellosis in Syria. Further, well-structured studies are required, the results of which could be used to plan an appropriate national control programme for brucellosis.