The protoxin Cry1Ac of Bacillus thuringiensis improves the protection conferred by intranasal immunization with Brucella abortus RB51 in a mouse model

Brucellosis is a zoonotic disease affecting many people and animals worldwide. Preventing this infection requires improving vaccination strategies. The protoxin Cry1Ac of Bacillus thuringiensis is an adjuvant that, in addition to increasing the immunogenicity of different antigens, has shown to be protective in different models of parasitic infections. The objective of the present study was to test whether the intranasal co-administration of pCry1Ac with the RB51 vaccine strain of Brucella abortus confers protection against an intranasal challenge with the virulent strain B. abortus 2308 in BALB/c mice. The results showed that co-administration of pCry1Ac and RB51, increased the immunoprotection conferred by the vaccine as evidenced by the following: (1) decrease of the splenic bacterial load when challenged intranasally with the virulent strain; (2) greater in vivo cytotoxic activity in response to the transference of previously infected cells; (3) further proliferation of cytotoxic TCD8+ cells in response to stimulation with heat-inactivated bacteria; (4) increased production of TNF-α and IFN-γ; and (5) significant IgG2a response. These results indicate that the use of the Cry1Ac protein as a mucosal adjuvant via the intranasal route can be a promising alternative for improving current RB51 vaccine against brucellosis.     

Protective effects of recombinant Brucella abortus Omp28 against infection with a virulent strain of Brucella abortus 544 in mice

The outer membrane proteins (OMPs) of Brucella (B.) abortus have been extensively studied, but their immunogenicity and protective ability against B. abortus infection are still unclear. In the present study, B. abortus Omp28, a group 3 antigen, was amplified by PCR and cloned into a maltose fusion protein expression system. Recombinant Omp28 (rOmp28) was expressed in Escherichia coli and was then purified. Immunogenicity of rOmp28 was confirmed by Western blot analysis with Brucella-positive mouse serum. Furthermore, humoral- or cell-mediated immune responses measured by the production of IgG1 or IgG2a in rOmp28-immunized mice and the ability of rOmp28 immunization to protect against B. abortus infection were evaluated in a mouse model. In the immunogenicity analysis, the mean titers of IgG1 and IgG2a produced by rOmp28-immunized mice were 20-fold higher than those of PBS-treated mice throughout the entire experimental period. Furthermore, spleen proliferation and bacterial burden in the spleen of rOmp28-immunized mice were approximately 1.5-fold lower than those of PBS-treated mice when challenged with virulent B. abortus. These findings suggest that rOmp28 from B. abortus is a good candidate for manufacturing an effective subunit vaccine against B. abortus infection in animals.     

Brucellosis in domestic water buffalo (<Emphasis Type="Italic">Bubalus bubalis</Emphasis>) of Trinidad and Tobago with comparative epidemiology to cattle

The water buffalo is an important domestic animal worldwide, and the local Buffalypso variety was developed in Trinidad to have improved beef qualities. Brucellosis was diagnosed in Trinidad and Tobago during 1998 in both cattle and domestic water buffalo (Bubalus bubalis) populations. Brucellosis in the latter species is caused by infection with Brucella abortus, similar to bovine brucellosis. Control of brucellosis is of paramount importance to preservation of the genetic diversity of these animals in Trinidad, and this has been complicated by differences in the epidemiology of water buffalo and bovine brucellosis. Some diagnostic tests do not have comparable accuracy between the two species, and the RB51 vaccine does not adequately protect against infection in water buffalo. The water buffalo in Trinidad may also be more resistant to infection than cattle. Development of effective vaccination protocols is key to brucellosis control in Buffalypso in Trinidad, and prohibitions on import of virulent B. abortus strains for vaccine efficacy studies has impeded progress in this area. These Trinidadian strains are of variable virulence; some might be effective for challenge in vaccine efficacy studies, while other, of lower virulence, may be vaccine candidates for use in water buffalo.     

Seroprevalence of brucellosis in yaks (Poephagus grunniens) in India and evaluation of protective immunity to S19 vaccine

The present study was carried out to explore the seroprevalence of brucellosis in yaks of North-Eastern hilly yak tracts of Arunachal Pradesh, India. Of 374 animals tested, 23.79, 21.11 and 18.98% were found positive for brucellosis using avidin-biotin ELISA (AB-ELISA), Rose-Bengal plate test (RBPT) and standard tube-agglutination test (STAT), respectively. The relative sensitivity and specificity for STAT were 79.77 and 100%, respectively and the same for RBPT were 88.76 and 100%, respectively in comparison to AB-ELISA. The alarming prevalence as recorded was highest among the yak cows (31.42%) followed by heifers (23.85%) and bulls (8.88%). The immune response in yaks following standard dose of calfhood vaccination with Brucella abortus strain 19 vaccine showed that protective antibody level persisted up to 210 days. This is the first report from India on prevalence of brucellosis and immunization with B abortus strain 19 vaccine in yaks. The present investigation would be a valuable guideline for future control measure and eradication programme of brucellosis in yaks.     

Virulence of <Emphasis Type="Italic">Brucella abortus</Emphasis> isolated from cattle and water buffalo

Brucellosis has been documented in domestic water buffalo (Bubalus bubalis) but published literature is limited despite the importance of this species in tropical agricultural systems. The objective of this study was to compare the virulence of Brucella abortus isolates recovered from cattle and water buffalo. Nineteen strains of B. abortus from cattle and domestic water buffalo in Trinidad were intraperitoneally inoculated into BALB/c mice. Spleens were cultured for B. abortus and histopathological severity scores were calculated based on lymphoid depletion, lymphoid necrosis, splenitis, and macrophage accumulation. A general linear model approach was used to estimate the effect of isolate source (cattle versus water buffalo) on virulence. Isolates of water buffalo origin were significantly less virulent in the mouse model based on recovered B. abortus from splenic tissues, spleen/weight ratio, and lymphoid necrosis but not overall histopathological severity scores. Further investigation of isolates recovered from water buffalo might provide the key to the development of procedures for brucellosis control in tropical environments.     

Immunogenicity of adenovirus and DNA vaccines co-expressing P39 and lumazine synthase proteins of <Emphasis Type="Italic">Brucella abortus</Emphasis> in BALB/c mice

Brucella poses a great threat to animal and human health. Vaccination is the most promising strategy in the effort to control Brucella abortus (B. abortus) infection, but the currently used live vaccines interfere with diagnostic tests and could potentially result in disease outbreak. Therefore, new subunit vaccines and combined immunization strategies are currently under investigation. In this study, immunogenicity and protection ability of a recombinant adenovirus and plasmid DNA vaccine co-expressing P39 and lumazine synthase proteins of B. abortus were evaluated based on the construction of the two molecular vaccines. Four immunization strategies (single adenovirus, single DNA, adenovirus/DNA, DNA/adenovirus) were investigated. The results showed that the immunization strategy of DNA priming followed by adenovirus boosting induced robust humoral and cellular immune responses, and it significantly reduced the numbers of B. abortus in a mouse model. These results suggest that it could be a potential antigen candidate for development of a new subunit vaccine against B. abortus infection.     

A comparison of the potency of several Brucella allergens used to detect brucellosis in cattle

The potency of Brucella allergens prepared from a smooth Brucella abortus strain S-99, mucoid strain Leewarden, rough strain 45/20, and rough Brucella melitensis strain B-115 was assessed. The potency of these allergens was compared with that of a standard allergen prepared from smooth Brucella abortus S-99 that efficiently detected bovine brucellosis in other studies. Eight cattle experimentally inoculated with Brucella abortus 544 were tested with the allergens 4 and 10 weeks after infection, and again 8 months after infection. All the allergens effectively detected infection but there was a clear distinction in the mean skin reactions 48 and 72 h after injection of the allergens. The skin reactions provoked by the allergens prepared from smooth or mucoid strains of Brucella were most pronounced 48 h after injection. Skin reactions provoked by allergens prepared from rough strains of Brucella were strongest 72 h after injection. Allergens prepared from smooth or mucoid Brucella strains were more potent in detecting brucellosis than those prepared from rough strains of Brucella.Abbreviations Bruc/OCB Brucellergen OCB - cfu colony-forming units - CFT complement fixation test - ID-DLO Institute voor Dierhouderij en Diergezondheid-Dienst Landbouwkundig Onderzoek - ICFTU international complement fixation units - IU international units - LPS lipopolysaccharide - SAT serum agglutination test - SDTH skin delayed-type hypersensitivity     

Frequent Exposure of Mice to Crude Brucella abortus Proteins Down‐regulates Immune Response

Mice repeatedly immunized via the intraperitoneal route with a Brucella abortus antigen lost their ability to develop a strong in vitro lymphoproliferative response. This result correlates with a decreased tendency of the lymphoid population to produce interferon‐γ when stimulated in culture with the immunizing antigen. With respect to the humoral response, as the number of immunizations increased, the animals produced more specific immunoglobulin M and immunoglobulin G1 antibodies. It is postulated that the long‐term exposure of an animal to Brucella antigen changes the nature of the immune response from a T‐cell‐mediated response to a humoral response favouring the establishment of the disease.     

Molecular typing of Brucella species isolates from livestock and human

Although host specificity has been observed in different species of Brucella, crossing the animal host boundary is likely to occur at any time. In this study, Bruce ladder PCR and abortus–melitensis–ovis–suis (AMOS) PCR assays were used to characterize 47 Brucella isolates from Indian origin in order to know exact species for understanding epidemiology of brucellosis. Out of them, 28, 14, and 5 isolates were found to be Brucella abortus, Brucella melitensis, and Brucella suis, respectively. Further analysis by AMOS PCR has identified that all the B. abortus isolates belong to any one of the biovar 1, 2, or 4; of the five B. suis isolates, three belong to biovar 1 and two belong to any one of the biovar 2, 3, 4, or 5. Although this multiplex Bruce ladder PCR is useful in differentiating all Brucella species, elaborate study is required to further characterize the isolates at exact biovar level.     

Ultracentrifugal Studies of Sera from Cattle Vaccinated or Naturally Infected with Brucella Abortus

In conformity with the findings of previous investigators, it was shown by density gradient ultracentrifugation that the antibodies in sera collected from calves shortly after vaccination with Brucella abortus, strain 19, were entirely or mainly rapidly-sedimenting. These macroglobulin (19S or IgM) antibodies showed complement-fixing as well as agglutinative activity with Br. abortus antigen. In later bleedings from the same vaccinated calves, antibodies with an intermediate sedimentation rate, (IgG), were present, as well as IgM. Sera from 15 of 22 non-vaccinated, relatively recent field cases of brucellosis appeared to contain only the IgG class of antibodies. In one herd, however, two cows with IgM only and five with both IgM and IgA were found; all seven of these cattle had been serologically negative before their introduction into this known infected herd a few months earlier. The agglutinative activity of sera from four cases of brucellosis of long standing and from eight cows, 4 to 13 years of age, that had been vaccinated as calves, was confined to the IgG fraction.     

Infection of cattle in Kenya with Brucella abortus biovar 3 and Brucella melitensis biovar 1 genotypes

Brucella melitensis biovar 1 was isolated from bovine milk samples from a herd in central Kenya, and Brucella abortus biovar 3 was isolated from aborted fetus materials and vaginal discharge fluids from cattle in central and eastern provinces of Kenya. All infections including those with B. melitensis were in cattle with reproductive problems kept in mixed herds indicating that cross infection occurs from small ruminants. Multiple-locus variable-number tandem repeat analysis genotyping revealed a close molecular homology of the B. melitensis isolates with an isolate from Israel and a close homology of the B. abortus isolates with an isolate from Uganda indicating that these genotypes have a wide geographic distribution. Infection of cattle with B. melitensis may complicate the control of brucellosis in this country.     

Brucella abortus infection in indigenous Korean dogs

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.     

IMMUNOGLOBULIN CLASSES IN SERUM ANTIBODY REACTIONS IN CATTLE FOLLOWING VACCINATION WITH BRUCELLA ABORTUS STRAIN 19 AND KILLED 45/20 VACCINES

SUMMARY A group of 4 cows was vaccinated with Brucella abortus strain 19, followed 8 weeks later by a single dose of B. abortus 45/20 vaccine. A similar group received 2 doses of B. abortus 45/20 vaccine 8 weeks apart. The antibody responses of the groups were compared by testing whole serums and separated IgM and IgG fractions by the Rose Bengal Plate (RBP) agglutination and the complement fixation tests (CFT) using rough and smooth B. abortus antigens. Animals that had received B. abortus strain 19 responded to the 45/20 vaccine with increased titres to the smooth antigen. These relevant antibodies were predominantly of the IgG class. Standard CFT and RBP test antibodies could be detected in IgM and IgG fractions after the primary inoculation with B. abortus strain 19 vaccine.     

Characterization of culture supernatant proteins from Brucella abortus and its protection effects against murine brucellosis

In this study, we characterized the secreted proteins of Brucella abortus into the enriched media under the bacterial laboratory growth condition and investigated the pathogenic importance of culture supernatant (CS) proteins to B. abortus infection. CS proteins from stationary phase were concentrated and analyzed using 2D electrophoresis. In MALDI TOF/TOF analysis, more than 27 proteins including CuZn SOD, Dps, Tat, OMPs, Adh, LivF, Tuf, SucC, GroEL and DnaK were identified. Cytotoxic effects of CS proteins were found to increase in a dose-dependent manner in RAW 264.7 cells. Upon B. abortus challenge into phagocytes, however, CS proteins pre-treated cells exhibited lower bacterial uptake and intracellular replication compared to untreated cells. Immunization with CS proteins induced a strong humoral and cell mediated immune responses and exhibited significant higher degree of protection against virulence of B. abortus infection compared to mice immunized with Brucella broth protein (BBP). Taken together, these results indicate that B. abortus secreted a number of soluble immunogenic proteins under laboratory culture condition, which can promote antibody production resulted in enhancing host defense against to subsequently bacterial infection. Moreover, further analysis of CS proteins may help to understand the pathogenic mechanism of B. abortus infection and host–pathogen interaction.     

Letters to the Editor

Extract

Sir.—There has been some correspondence in the Velerinary Record recently concerning brucella abortion breakdowns in heifers vaccinated with Strain 19 Brucella abortus. This year we have been alarmed by the sudden occurrence of abortion storms in vaccinated heifers. Brucella abortus has been seen in large numbers in direct smears taken from cotyledons and foetal stomach contents stained by the modified Z-N technique, both in our own practice laboratory and at the Department of Agriculture laboratory. To remove any possible doubt as to the identity of the organism, it has been cultured. Blood samples have been taken from the heifers concerned and sent to the Department of Agriculture laboratory where agglutination titres to Br. abortus were observed at dilutions of up to 1 in 1,280. Our practice here serves 550 dairy farms in one of the most closely stocked areas in New, Zealand. In 18 herds more than 25% of the heifers have aborted owing to brucellosis.     

Non-specific seroreactions against Brucella abortus in ruminants in New Zealand and the presence of Yersinia enterocolitica 0:9

The level of non-specific reactions found in the brucellosis serology of ruminants in New Zealand was very low until July 1992. This changed when, in an export consignment of 1071 deer, 35% reacted in the Brucella abortus tube agglutination test with titres varying from 50 to 200 IU. The reactors were also positive in the Rose-Bengal agglutination test and most of them reacted in the complement fixation test with titres varying from 10 to 80 IU. Yersinia enterocolitica 0:9 was later isolated from one deer of this consignment. It was the first isolate of this serotype recovered in New Zealand from an animal. Shortly after, false reactors occurred more frequently than before in sera from Brucella abortus accredited free cattle herds. As the involvement of Yersinia enterocolitica 0:9 was suspected in these cases, faecal samples from reactors and in-contact animals were cultured for this organism. Yersinia enterocolitica 0:9 was isolated from nine of 19 herds showing one or more false Brucella abortus seroreactions.

Prior to 1990, Yersinia enterocolitica serotype 0:9 had not been isolated in New Zealand, despite the recovery of a number of other bio— or serotypes of the organism from humans and animals. From 1990 onward, serotype 0:9 began to be isolated from human faecal samples with increasing frequency. Since the first isolations from deer and cattle in 1992, it has now also been recovered from a cat and an alpaca and from cattle without any association with false positive Brucella abortus reactions. All serotype 0:9 isolates were of biotype 2.     

Immunoadjuvant effects of bacterial genomic DNA and CpG oligodeoxynucleotides on avian influenza virus subtype H5N1 inactivated oil emulsion vaccine in chicken

This study investigated the immunoadjuvant effects of three types of bacterial genomic DNA and CpG oligonucleotides (CpG ODN) on the avian influenza virus (AIV) subtype H5N1 inactivated oil emulsion vaccine under two immunization strategies. The genomic DNA extracted from Escherichia coli O₂, Staphylococcus aureus, Streptococcus faecalis FQ68, and synthetic CpG ODN were used as adjuvants, and their effects on the AIV oil emulsion vaccine were examined in chickens. The results indicated that when administered separately from the vaccine, adjuvants induced lower haemagglutination inhibition (HI) titres and serum IgG titres but resulted in higher concentrations of IFN-γ and IL-10. In contrast, when combined with the oil emulsion vaccine prior to inoculation, CpG ODN induced higher HI, IgG titres and IFN-γ concentration but resulted in lower IL-10 concentration. These data suggest that, depending on the immunization approaches, adjuvants may exert distinct immune effects in chickens receiving AIV H5N1 oil emulsion vaccine: the prior incorporation of CpG ODN into the vaccine may augment both the humoral and Th1 type immune responses, while separate inoculation of adjuvants has not shown better adjuvanticity.     

Outbreak of Brucellosis in Domestic Elk in Korea

Seven of 18 elk on a deer farm were found by the official Rose‐Bengal agglutination test (RBT) and tube agglutination test to be brucellosis reactors/suspects. Evaluation with the competitive ELISA (C‐ELISA) and the fluorescence polarization assay (FPA) tests revealed that six and five sera were positive respectively. The seven reactors/ suspects were slaughtered and their blood and tissues were collected. Brucella species could be isolated from three of the slaughtered animals, with nine isolates being obtained from the popliteal, supramammary and submandibular lymph nodes, vaginal discharge, mammary tissue and spleen. Brucella genus‐specific PCR based on 16S rRNA and AMOS‐PCR, which is specific for differential Brucella species, revealed that all nine isolates were Brucella abortus. These nine were further confirmed to be B. abortus biovar 1 by classical biotyping scheme assays. This is the first report of an outbreak of brucellosis in domestic elk in Korea. Our observations suggest that deer should be included in the routine Brucella surveillance programme for the effective control and prevention of brucellosis in Korea.     

Comparison of the Antibody Response in Adult Cattle Against Different Epitopes of Brucella abortus Lipopolysaccharide

The comparison of serological responses in a sample of adult, vaccinated and field‐infected bovines with Brucella abortus is reported. Indirect enzyme immunoassay (EIA) titration curves and Western blotting tests for smooth‐type lipopolysaccharide (S‐LPS), rough‐type LPS (R‐LPS) and lipid A were performed. In the initial screening of sera, an overall prevalence of 20.5 % was found, which corresponds to a country with a high incidence of brucellosis. End‐point EIA titres against LPS antigens from vaccinated and field‐infected cows were not significantly different. However, the absorbance values in the titration curves were significantly higher for S‐LPS as compared with the other antigens. A high correlation coefficient (r=0.933) was obtained when the titres to R‐LPS versus lipid A were compared. Western blotting reactions of vaccinated and field‐infected animals were indistinguishable. S‐LPS, R‐LPS and lipid A epitopes were recognized in a heterogeneous manner. In general, the number of bovines that reacted against LPS was higher in the field‐infected group, with a stronger binding to S‐LPS. Based on our observations, the vaccinated and field‐infected bovines are capable of producing similar antibody responses to the Brucella main outer surface antigen, LPS. It should be emphasized that the humoral response of cattle to Brucella LPS contains significant amounts of antibodies to other antigenic moieties of this important surface molecule, which may contribute to the immunity to brucellosis.     

Leptospirosis as the most frequent infectious disease impairing productivity in small ruminants in Rio de Janeiro,Brazil

Despite the importance of small ruminants breeding in developing countries, milk/meat productivity remains unsatisfactory. Infectious diseases, such as leptospirosis, brucellosis, and small ruminant lentiviruses (SRLVs), contribute to this scenario. The objective of the present study was to determine the role of each of these diseases in the productivity of small ruminants breeding in Rio de Janeiro, Brazil. In goats, 343 samples were tested for leptospirosis, 560 for Brucella abortus, and 506 for caprine arthritis-encephalitis (CAE), whereas in sheep, 308 samples were tested for leptospirosis, 319 for B. abortus, 374 for Brucella ovis, and 278 for Maedi-Visna (MV). Regarding leptospirosis, 25.9% of goats and 47.4% sheep were seroreactive, with serovar Hardjo the most prevalent in both species. Anti-B. abortus agglutinins were found in 0.7% of all samples, exclusively in goats. In relation to SRLVs, 8.6% of goats and 3.2% of sheep samples were positive for CAE and MV, respectively. Leptospirosis was the major infectious problem in the small ruminants sampled and may contribute to impaired productivity of these animals.