Control of small ruminant brucellosis by use of Brucella melitensis Rev.1 vaccine: laboratory aspects and field observations

Brucellosis vaccines are essential elements in control programs. Since first developed in the mid-1950s, the Brucella melitensis vaccine strain Rev.1 has been used worldwide and its significant value in protecting sheep and goats in endemic areas recognized. This review provides historical background on the development of the vaccine, its use and field complications arising in Israel following changes in the strain’s pathogenicity. The urgent need for resolving cases of vaccine strain excretion in the milk, horizontal transfer and a unique case of human infection has led to identification of an atypical B. melitensis biovar 1 strain that resembles strain Rev.1 in susceptibility to penicillin and dyes. An omp2 based PCR method has been developed that traced the lineage of Israeli B. melitensis biovar 1 strains. This locus serves as an epidemiological tag for the Rev.1 vaccine strain. Despite the rapid development of new approaches in the field of vaccination, it is anticipated that in the near future the Rev.1 vaccine would remain the only accepted vaccine in national control programs.     

Seroprevalences and lack of abortions after vaccination of Churra sheep with reduced doses of Rev. 1 Brucella melitensis vaccine by subcutaneous or conjunctival routes

A field study to evaluate the serological response and the safety of different doses and administration routes of the Rev. 1 vaccine was carried out on two Churra breed flocks. Reduced doses of 2.3 × 10⁶ and 3 × 10⁷ live organisms were administered by the subcutaneous or the conjunctival route, respectively. In those animals which were seropositive before vaccination, the percentage of positive sera declined progressively in a similar way in all groups over the 36 weeks that the study lasted; the antibody titers also dropped continuously in the group vaccinated by the conjunctival route with the lower dose, while in the remaining three groups there was a transitory increase in the 4th week after vaccination. In those animals which were serologically negative prior to vaccination, the percentage of positive sera and the antibody titers generally reached their peak in the 4th week after vaccination, followed by a progressive decline in succeeding weeks. Similarly, titers were higher in animals vaccinated subcutaneously than in those vaccinated by the conjunctival route. The differences between the frequencies of positive sera and the levels of antibodies were important when routes were compared. Animals receiving a dose of 2.3 × 10⁶ CFU subcutaneously had a satisfactory serological response, with a more rapid decline in their level of antibodies than in the animals which were vaccinated with 3 × 10⁷ CFU by the same route. No cases of abortion were reported in the 461 vaccinated ewes.     

动物布鲁氏菌Rev.1疫苗研究进展

布鲁氏菌病（布病）是由布鲁氏菌引起的一种重要的人畜共患病,对公共卫生有着巨大的威胁。布鲁氏菌Rev.1疫苗研制于上世纪50年代中期,并在欧洲、中东和蒙古等地区被广泛应用于小反刍动物布病的防控,一度被认为是防控小反刍动物布病最为有效的疫苗。但是,由于Rev.1疫苗存在毒力偏强及毒力不稳定等现象,其安全性仍值得关注。本文主要从Rev.1疫苗背景及其菌株的生物学特性、疫苗使用情况以及存在的不足等几个方面进行阐述,以期为Rev.1疫苗的应用和布鲁氏菌相关疫苗的开发提供借鉴。     

Brucella proteomes--a review

The proteomes of selected Brucella spp. have been extensively analyzed by utilizing current proteomic technology involving 2-DE and MALDI-MS. In Brucella melitensis, more than 500 proteins were identified. The rapid and large-scale identification of proteins in this organism was accomplished by using the annotated B. melitensis genome which is now available in the GenBank. Coupled with new and powerful tools for data analysis, differentially expressed proteins were identified and categorized into several classes. A global overview of protein expression patterns emerged, thereby facilitating the simultaneous analysis of different metabolic pathways in B. melitensis. Such a global characterization would not have been possible by using time consuming and traditional biochemical approaches. The era of post-genomic technology offers new and exciting opportunities to understand the complete biology of different Brucella species.     

布鲁菌疫苗株Rev.1全基因序列的测定与分析

为促进布鲁菌病Rev.1疫苗及相关疫苗的研发, 该研究提取Rev.1疫苗株核酸, 应用PacBio平台进行全基因序列测定与分析。结果表明, Rev.1疫苗株基因组大小约3 299 187 bp, G+C含量为57.2%, 组装为染色体1、染色体2两条环状基因组, 大小分别为2 121 370、1 177 817 bp, G+C含量分别为57.2%、57.3%。将其Ery、BLS和VirB10基因序列与9株GenBank上发表的布鲁菌参考菌株的Ery、BLS和VirB10基因序列进行比较分析, 存在不同程度的差异, 同源性为97.4%~100%。     

Identification of Brucella abortus, B. canis, B. melitensis, and B. suis by carbon substrate assimilation tests

By using the results of seven carbon substrate assimilation tests from the Biotype 100 system (bioMérieux, Marcy-l’Etoile, France), we correctly identified 79 (85.9%) of 92 Brucella strains tested. The specificity of the method varied from 97.4 to 100% depending on the species. Although a biological safety cabinet must be used, this method represents an easy and fast alternative for the identification of Brucella species.     

Protection against Brucellosis in Goats,Five Years after Vaccination with Reduced-Dose Brucella melitensis Rev 1 Vaccine

The protection conferred by the reduced-dose Rev 1 Brucella melitensis vaccine in goats that had been immunized 5 years previously was evaluated. Sixteen goats vaccinated 5 years before with Rev 1 (1 x 10(5) cfu) and 5 non-vaccinated goats were challenged with B. melitensis 16M (4 x 10(5) cfu) using the conjunctival route. After giving birth or aborting, the goats were sacrificed and tissue samples were taken for bacteriological study. The challenge strain was recovered in 12%, of the animals from the vaccinated group, and in (80% of the control group. It is concluded, therefore that the use of reduced-dose Rev 1 protects goats vaccinated in endemic areas for at least 5 years after immunization.     

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.     

Risk factors for brucellosis seroprevalence of sheep and goat flocks in Spain

Risk factors for ovine and caprine brucellosis in the Ávila region (center of Spain) were evaluated using data from a cross-sectional study of the most important diseases of small ruminants in this Spanish region between 1996 and 1997. Questionnaire data from 56 herds (35 ovine and 21 caprine) were used. Sixteen (29%) flocks (3 caprine and 13 ovine) were brucellosis-seropositive. Overall, 0.7% of sheep and 0.1% of goats were seropositive. Eleven risk factors were studied at the group level by logistic regression using flock brucellosis-status as outcome, and by linear regression using percentage of brucellosis-seropositivity as outcome. Both final models contained the same variables: contact with sheep and grazing in communal pastures as risk factors, and frequency of disinfecting practices as a protective factor.     

The immune response of goats vaccinated with low and high doses of Brucella melitensis Rev 1

The serological response, lymphocyte reactivity, and dermal hypersensitivity reactions of goats vaccinated with high and low doses of $\text{Brucella melitensis}$ Rev 1 organisms to Brucella antigens were studied. Antibodies to soluble antigen A2 were detectable by immunoelectrophoresis (IE), appeared later than agglutinating antibodies but disappeared faster in most vaccinated animals. Anti-A2 antibodies appeared earlier in goats which received the high dose. Antibodies to polysaccharide B antigen were not detected. Lymphocytes reactive to Brucella antigens in lymphocyte transformation assays appeared at variable times after vaccination. In contrast to the humoral response to antigen A2, the appearance of circulating, reactive lymphocytes was not dependent on vaccine doses. All vaccinated animals demonstrated dermal hypersensitivity reactions to one of the antigenic extracts. Skin reactions peaked at 24 hrs post inoculation. The reactions were elicited when all but one goat had undetectable anti-A2 antibodies and no circulating, reactive lymphocytes as measured in whole blood lymphocyte transformation assay.     

Toxoplasma gondii surface antigen 1 (SAG1) as a potential candidate to develop vaccine against toxoplasmosis: A systematic review

Toxoplasma gondii is an intracellular parasite that infects a broad range of animal species and humans. As the main surface antigen of the tachyzoite, SAG1 is involved in the process of recognition, adhesion and invasion of host cells. The aim of the current systematic review study is to clarify the latest status of studies in the literature regarding SAG1-associated recombinant proteins or SAG1-associated recombinant DNAs as potential vaccines against toxoplasmosis. Data were systematically collected from six databases including PubMed, Science Direct, Web of Science, Google Scholar, EBSCO and Scopus, up to 1st of January 2019. A total of 87 articles were eligible for inclusion criteria in the current systematic review. The most common antigens used for experimental cocktail vaccines together with SAG1 were ROP2 and SAG2. In addition, the most parasite strains used were RH and ME49. Freund’s adjuvant and cholera toxin have been predominantly utilized. Furthermore, regarding the animal models, route and dose of vaccination, challenge methods, measurement of immune responses and cyst burden have been discussed in the text. Most of these experimental vaccines induce immune responses and have a high degree of protection against parasite infections, increase survival rates and duration and reduce cyst burdens. The data demonstrated that SAG1 antigen has a high potential for use as a vaccine and provided a promising approach for protecting humans and animals against toxoplasmosis.     

A single vaccine for the control of capripox infection in sheep and goats

A single vaccine against capripox was developed to protect sheep and goats throughout the capripox enzootic area. The vaccine was stable and safe to use, and provided substantial protection against capripox for at least a year. Preliminary field trials in the Middle East indicated that the vaccine was effective in controlling capripox in indigenous breeds of sheep and goats.     

Auxotrophic, plasmid-cured Salmonella enterica serovar typhimurium for use as a live vaccine in calves

Calves were vaccinated orally, subcutaneously or intraperitoneally with a smooth, plasmid-cured strain of Salmonella enterica serovar typhimurium, strain 81. Oral vaccination was not effective, as only 1/5 calves survived challenge with virulent S. typhimurium. Strain 81 was attenuated for calves, as only a slight rise in rectal temperatures was detected after vaccination. The organism was excreted by some calves in the faeces, but no signs of diarrhoea were observed after vaccination. After parenteral vaccination, strain 81 was able to reach the intestines, gastric associated lymphoid tissues and other internal lymphoid tissues and remained viable for up to 14 days in the bovine host. After oral challenge with a virulent strain, 9/10 vaccinated calves survived challenge as opposed to 4/10 control calves (p<0.5). Diarrhoea was present in all calves of the control groups, but in only 4/10 of the vaccinated calves. The clinical reactions of the vaccinated calves were milder than in the control calves, as the rises in rectal temperatures were lower, diarrhoea was less severe, and the challenge strain was present in fewer organs from vaccinated calves than control calves. This study showed that parenterally administered Salmonella vaccines can induce both mucosal and systemic immunity, and it is postulated that this capability of strain 81 is related to its colonisation of lymphoid tissues and other systemic and intestinal tissues. This study confirmed that plasmid-cured strains were attenuated in the bovine host and conferred significant protection after parenteral vaccination, but not oral vaccination.     

布鲁菌Rev.1株eryA基因的原核表达及间接ELISA方法的建立

通过对布鲁菌优势抗原eryA基因进行原核表达,在获得目的蛋白的基础上建立以该蛋白为包被抗原的间接ELISA方法。构建原核表达载体pET-30a-eryA,并将其转入大肠杆菌BL21（DE3）,IPTG诱导表达,镍柱亲和纯化eryA重组蛋白,SDS-PAGE 鉴定纯化蛋白,Western-Blotting检测反应原性后建立间接ELISA方法。反应条件优化后,抗原包被浓度为0.312 5 μg/mL,37 ℃包被2 h;封闭液为5%猪源明胶,37 ℃作用2 h;血清稀释度为1∶100,37 ℃作用1 h;酶标抗体稀释度为1∶8 000,37 ℃作用45 min。血清稀释度为1∶1 600时,仍然检测为阳性,证明建立的间接ELISA方法灵敏性良好;其他菌种及病毒经过特异性检测,均为阴性,证明建立的间接ELISA方法特异性良好;通过重复性检测,批间及批内变异系数均小于10%,证明建立的间接ELISA方法重复性良好。检测临床血清样品,建立的方法与试管凝集试验总符合率为95.7%,证明该方法可初步应用于临床诊断。     

Etude preliminaire du vaccin antibrucellique P.B. Rev. 1 destine aux especes ovine et caprinePreliminary study of vaccines against brucellosis P. B. Rev. 1 destined for sheep and goats

A new inactivated anti-brucellosis vaccine, prepared according to a process previously described under the name of P.B. has been carried out from the strain Brucella melitensis Rev. 1 Elberg. The absence of agglutinogen content was verified through rabbits and mice. Its immunogenic content, tested on DBA₂ mice by means of an infection test, produced excellent results. Tests were planned for sheep. If the results on this species were satisfactory, this type of vaccine could then better replace the live vaccine Rev 1 which, in addition to the post-vaccine agglutinines which it takes after, is not without danger to man.     

Control of brucellosis in Kuwait by vaccination of cattle,sheep and goats withBrucella abortus strain 19 orBrucella melitensis strain Rev. 1

Summary In Kuwait, approximately 12,000 diary cows were vaccinated with a reduced dose of 3×10⁹ Brucella abortus strain 19 and approximately 350,000 sexually mature sheep and goats with a reduced dose of 10⁷B.melitensis strain Rev. 1. Using the criteria of prevaccinal and postvaccinal incidences of antibodies, abortions, and human cases of brucellosis, the programme was very successful. Widespread vaccination of adult animals is the most effective method of controlling brucellosis among cattle, sheep and goats in many countries.

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Resumen En Kuwait, se vacunaron aproximadament 12,000 vacas lecheras con una dosis reducida de 3×10⁹ organismos deBrucella abortus cepa 19 y approximadament 350,000 ovejas y cabras sexualmente maduras fueron vacunadas con una dosis reducida de 10⁷ organismos deB. melitensis opa Rev. 1. Utilizando los criterios prevacunales de incidencia de anticuerpos, abortos, y casos humanos de brucelosis, el programa fuvo gran exito. Ef método mas efectivo de control de la brucelosis bovina, ovina y caprina an numerosas paises es la amplia utilización de vacuna en animales adultos.

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Résumé Au Kuwait, environ 12,000 vaches laitiěres ont été vaccinées avec une dose réduite de souche 19Brucella abortus (3×10⁹) et environ 350,000 moutons et chevres qui étaient matures sexuellement, one été vaccinés avec une dose réduite de soucheB. melitensis Rev. 1 (10⁷). Basé sur la présence d'anticorps, sur le nombre d'avortements et de cas de brucellose chez les humains avant et aprés vaccination, le programme a été un succěs. La méthode la plus effective pour contr?ler la brucellose chez les bovins, les ovins et les caprins dans beaucoup de pays et de vacciner sans exception tous les animaux adultes.

     

Validation of a competitive ELISA for diagnosis of Brucella melitensis infection in sheep and goats

A competitive enzyme-linked immunosorbent assay (cELISA) was validated for the serodiagnosis of Brucella melitensis infection in small ruminants using 2108 positive and 2154 negative reference sera from sheep and goats. The optimum cut-off values, offering the highest diagnostic sensitivity (DSn) and diagnostic specificity (DSp), determined by receiver operating characteristic analysis, were at 23.6%, 21.8% and 25.0% inhibition of the conjugate control for sheep, goats and both species, respectively. The DSns of the cELISA for sheep, goats and both species at these cut-off values were 89.2% (95% confidence interval 87.1-91.1%), 74.0% (95% CI 71.4-76.5%) and 77.9% (95% CI 76.1-79.7%), whereas DSps were 96.4% (95% CI 95.2-97.4%), 92.9% (95% CI 91.1-94.3%) and 97.2% (95% CI 96.4-97.8%), respectively. Compared to cELISA, indirect ELISA and fluorescence polarisation assay have higher DSns and DSps. However, the results obtained with the cELISA were in good agreement with those of the complement fixation test (CFT) under field conditions using 5735 sheep and goat sera. The cELISA can be used as an alternative to the CFT for diagnosing B. melitensis infection in small ruminants.     

Mic1-3 Knockout Toxoplasma gondii is a good candidate for a vaccine against T. gondii-induced abortion in sheep

This study assessed the effectiveness of a mutant strain of Toxoplasma gondii (RH strain) lacking the mic1 and mic3 genes (Mic1-3KO) against Toxoplasma abortion in sheep. Ewes were inoculated subcutaneously with 10⁵ Mic1-3KO tachyzoïtes in three independent experiments. Following vaccination, Mic1-3KO induced a mild febrile response and serum IgG antibodies, which persisted throughout the experiments. Tissue cysts formed in the sheep, but were not, under our experimental conditions, infectious when given orally. Ewes were mated two months after vaccination and were orally challenged with the PRU strain of T. gondii at mid-gestation (400 oocysts in Experiments 1 and 2; 100 oocysts in Experiment 3). Challenge of vaccinated pregnant ewes resulted in a slight febrile response, whereas unvaccinated ewes developed a more severe, characteristic febrile response of longer duration. After challenge, all unvaccinated ewes aborted whereas 62%, 91% and 64% (Experiments 1, 2 and 3 respectively) of the lambs from vaccinated ewes were viable, with no clinical signs of infection. Mic1-3KO was as effective as S48, the strain used as a live vaccine for sheep (Toxovax^®). A dose of 10⁵ Mic1-3KO tachyzoites was sufficient to induce protection (versus a dose of 2 × 10⁶). Both subcutaneous and intraperitoneal injections were effective. Moreover, preliminary results showed the potential of Mic1-3KO to reduce the development of tissue cysts in lambs born to vaccinated ewes. This study demonstrates that Mic1-3KO is a potent vaccine candidate.