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.     

Validation of an ELISA method for the serological diagnosis of canine brucellosis due to Brucella canis

In the present study, the validation of an enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of canine brucellosis is described. Two different antigenic extracts, obtained by heat or ultrasonic homogenization of microbial antigens from a wild isolate of Brucella canis bacteria, were compared by ELISA and Western blot (WB). A total of 145 canine sera were used to define sensitivity, specificity and accuracy of the ELISA as follows: (1) sera from 34 animals with natural B. canis infection, confirmed by blood culture and PCR, as well as 51 sera samples from healthy dogs with negative results by the agar–gel immunodiffusion (AGID) test for canine brucellosis, were used as the control panel for B. canis infection; and (2) to scrutinize the possibility of cross reactions with other common dog infections in the same geographical area in Brazil, 60 sera samples from dogs harboring known infections by Leptospira sp., Ehrlichia canis, canine distemper virus (CDV), Neospora caninum, Babesia canis and Leishmania chagasi (10 in each group) were included in the study. The ELISA using heat soluble bacterial extract (HE-antigen) as antigen showed the best values of sensitivity (91.18%), specificity (100%) and accuracy (96.47%). In the WB analyses, the HE-antigen showed no cross-reactivity with sera from dogs with different infections, while the B. canis sonicate had various protein bands identified by those sera. The performance of the ELISA standardized with the heat soluble B. canis antigen indicates that this assay can be used as a reliable and practical method to confirm infection by this microorganism, as well as a tool for seroepidemiological studies.     

Use of Brucella abortus vaccine strain RB51 in pregnant cows after calfhood vaccination with strain 19 in Argentina

One hundred and seven pregnant cows, which had been calfhood vaccinated with Brucella abortus strain 19 (S-19) were revaccinated with either S-19 or strain RB51 (S-RB51). All S-19-revaccinated animals seroconverted, while none of the RB51-revaccinated animals seroconverted. Two out of 25 (8%) S-19-revaccinated animals aborted, while none of the 57 RB51-revaccinated group aborted. Four of the S-19-revaccinated animals shed S-19 in the milk for at least 7 days, while only 1 cow shed S-RB51 for at least 3 days (but <7 days) post-parturition. Revaccination of strain 19 calfhood-vaccinated, pregnant cattle with S-RB51 appears to be a safe procedure with no diagnostically negative consequences.     

Discrimination between sheep antibodies to Brucella melitensis and to Brucella ovis

When preparations containing smooth Brucella abortus lipopolysaccharide (LPS) were used as antigens in an ELISA, strong positive reactions were obtained with sera from sheep infected with Brucella melitensis or with Brucella ovis. Oxidation of the LPS with sodium metaperiodate greatly reduced the extent of the cross-reactions with antisera to B. ovis, with little effect on the reactions with antisera to smooth B. melitensis. Periodate oxidation of hot saline extract (HSX) antigen of B. ovis markedly reduced its reactivity in ELISA with anti-B. ovis sera and eliminated cross-reactivity with anti-B. melitensis sera. The reactivity of HSX was maintained after treatment with proteinase K.A simple ELISA system, in which replicate samples from a single serum dilution were tested in parallel against both B. ovis HSX antigen and periodate-oxidised smooth phase B. abortus LPS, was evaluated. It was found to discriminate well between antibodies induced by vaccination or virulent infection with B. melitensis strains and those induced by infection with B. ovis.     

The genome of Brucella melitensis

The genome of Brucella melitensis strain 16M was sequenced and contained 3,294,931 bp distributed over two circular chromosomes. Chromosome I was composed of 2,117,144 bp and chromosome II has 1,177,787 bp. A total of 3198 ORFs were predicted. The origins of replication of the chromosomes are similar to each other and to those of other -proteobacteria. Housekeeping genes such as those that encode for DNA replication, protein synthesis, core metabolism, and cell-wall biosynthesis were found on both chromosomes. Genes encoding adhesins, invasins, and hemolysins were also identified.     

Seeking a niche: putative contributions of the hfq and bacA gene products to the successful adaptation of the brucellae to their intracellular home

Long-term residence of the brucellae in the phagosomal compartment of host macrophages is essential to their ability to produce disease in both natural and experimental hosts. Correspondingly, the Brucella spp. appear to be well adapted to resist the multiple environmental stresses they encounter in their intracellular home. This brief review will focus on the contributions of the hfq and bacA gene products to this adaptation. Studies with Brucella hfq mutants suggest that stationary phase physiology is critical for successful long-term residence in host macrophages. Analysis of Brucella bacA mutants, on the other hand, reveal very striking parallels between the strategies employed by the rhizobia to establish and maintain protracted intracellular residence in their plant host and those used by the brucellae during their long-term survival in the phagosomal compartment of host macrophages.     

The intramacrophagic environment of Brucella suis and bacterial response

Phagocytes have developed various antimicrobial defense mechanisms to eliminate pathogens. They comprise the oxidative burst, acidification of phagosomes, or fusion of phagosomes with lysosomes. Facultative intracellular bacteria, in return, have developed strategies counteracting the host cell defense, resulting in intramacrophagic survival.Until lately, only very little was known about the phagosomal compartment containing Brucella spp., the environmental conditions the bacteria encounter, and the pathogen’s stress response. Recently, we have determined that the phagosomes acidify rapidly to a pH of 4.0–4.5 following infection, but this early acidification is crucial for intracellular replication as neutralization results in bacterial elimination. A vacuolar proton-ATPase is responsible for this phenomenon that is not linked to phagosome–lysosome fusion. On the contrary, in vitro reconstitution assays revealed association only between phagosomes containing killed B. suis and lysosomes, describing the absence of phagolysosome fusion due to specific recognition inhibition for live bacteria. Further evidence for the necessity of an intact, acidic phagosome as a predominant niche of brucellae in macrophages was obtained with a strain of B. suis secreting listeriolysin. It partially disrupts the phagosomal membranes and fails to multiply intracellularly.How does B. suis adapt to this environment? We have identified and studied a series of genes that are involved in this process of adaptation. The bacterial heat shock protein and chaperone DnaK is induced in phagocytes and it is essential for intracellular multiplication. A low-level, constitutive expression of dnaK following promoter exchange does not restore intramacrophagic survival. Another chaperone and heat shock protein, ClpB, belonging to the family of ClpATPases, is important for the resistance of B. suis to several in vitro stresses, but does not contribute to intramacrophagic survival of the pathogen. Additional bacterial genes specifically induced within the phagocyte were identified by an intramacrophagic screen of random promoter fusions to the reporter gene gfp. A large majority of these genes are encoding proteins involved in transport of nutrients (sugars, amino acids), or cofactors, such as nickel. Analysis of the intracellular gene activation reveals that low oxygen tension is encountered by B. suis.Altogether, these results suggest three major stress conditions encountered by brucellae in the phagosome: acid stress, starvation and low oxygen tension.     

Evaluation of a rough mutant of Brucella melitensis in pregnant goats

Brucella melitensis strain VTRM1, a rough derivative of B melitensis strain 16M, is able to colonise the lymph nodes of goats, does not induce abortion in pregnant goats when used at doses leading to abortions with virulent strain 16M, and does not induce anti-O chain antibodies. However, strain VTRM1 as a single dose vaccine induces only partial protection against both infection and abortion following challenge.     

羊布鲁氏菌甲酰基转移酶的生物信息学研究

本研究旨在分析和预测羊布鲁氏菌甲酰基转移酶基因及其编码蛋白的结构与功能,用于指导其生物学功能的实验室研究。综合利用生物信息学软件(DNAMAN和Vector NTI)、数据库(prosite和PDB)和网络服务器对甲酰基转移酶进行基本性质分析、三维结构预测和功能预测,展示了甲酰基转移酶的氨基酸分布情况,二级结构中形成螺旋、折叠、卷曲的氨基酸区段及结构域;预测并综合阐释了B细胞表位的柔韧性、表面可及性和亲水性参数。生物信息学预测和分析结果可为研究在布鲁氏菌致病及机体免疫应答中甲酰基转移酶的结构与功能的关系提供丰富资料。     

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.     

In vitro antagonistic activities of Lactobacillus spp. against Brachyspira hyodysenteriae and Brachyspira pilosicoli

The sensitivity of Brachyspira hyodysenteriae and Brachyspira pilosicoli, respectively the causative agents of Swine Dysentery and Porcine Intestinal Spirochaetosis to two probiotic Lactobacillus strains, L. rhamnosus CNCM-I-3698 and L. farciminis CNCM-I-3699 was studied through viability, motility and coaggregation assays. The cell-free supernatant of these lactobacilli contains lactic acid, that is stressful for Brachyspira (leading to the formation of spherical bodies), and lethal. It was demonstrated for the first time the in vitro coaggregation properties of two probiotic Lactobacillus strains (active or heat-treated) with two pathogenic strains of Brachyspira, leading to (1) trapping of spirochaetal cells in a physical network as demonstrated by SEM; (2) inhibition of the motility of Brachyspira. Such in vitro studies should encourage in vivo studies in animal model to evaluate the potential of the use of probiotic lactobacilli through a feeding strategy for the prevention of B. hyodysenteriae and B. pilosicoli.     

家养观赏地图鱼肺炎克雷伯氏菌、维氏气单胞菌与黏质沙雷氏菌的分离鉴定及耐药性分析

本试验旨在通过细菌分离鉴定,明确家养观赏地图鱼的死因,筛选敏感药物。采用常规方法分离纯化细菌后,进行细菌形态学观察,并通过小白鼠致病性试验、细菌主要生化鉴定、16SrDNA序列测定分析、药敏试验及耐药基因检测等方法对分离的细菌进行鉴定及耐药分析。分离出3株革兰氏阴性短杆菌,根据细菌形态特征及理化特性,结合16SrDNA序列测定与系统发育分析结果,判定其分别为肺炎克雷伯氏菌(Klebsiella pneumoniae)、维氏气单胞菌(Aeromonas veronii)和黏质沙雷氏菌(Serratia marcescens),其中肺炎克雷伯氏菌具有较强致病性。3株细菌均对洛美沙星、氧氟沙星、阿米卡星、卡那霉素敏感,对阿莫西林、氟苯尼考、克林霉素、甲硝唑等具有较强耐药性。结果表明,肺炎克雷伯氏菌、维氏气单胞菌、黏质沙雷氏菌的混合感染是家养观赏地图鱼的死亡原因。     

Phylogentic analysis of Anaplasma ovis strains isolated from sheep and goats using groEL and mps4 genes

Evidence of Anaplasma spp. in goats and sheep in Cyprus has been demonstrated by previous research. Herein, further research was performed for the identification of the exact Anaplasma spp. resulting in the identification of Anaplasma ovis strains in all samples examined. We used a bioinformatics as well as a molecular approach (study of groEl and mps4 genes) in order to verify the validity of the results. All samples depicted the presence of A. ovis regardless of the host (goat or sheep).     

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.     

Seroprevalence of antibodies to Encephalitozoon cuniculi and Encephalitozoon intestinalis in humans and animals

The presence of antibodies against Encephalitozoon cuniculi (E. cuniculi) and Encephalitozoon intestinalis (E. intestinalis) was examined in 215 samples from humans and in 488 samples from five different species of domestic and companion animals in Slovakia. The 215 human samples and samples from 90 swine, 123 non-infected cattle (cattle), 24 cattle infected with bovine leukosis virus (BLV-positive cattle), 140 sheep and 111 dogs were examined by the enzyme-linked immunosorbent assay (ELISA). Samples with serum titres 1:200 or higher were considered as positive. Specific anti-E. cuniculi antibodies were found in humans (0.9%), swine (52%), cattle (2%), sheep (9%) and dogs (15%) except for the BLV-positive cattle at the titre of 1:200. The titre of 1:400 was detected only in humans (0.5%). The presence of specific anti-E. intestinalis antibodies at the titre of 1:200 was confirmed in humans (6%), swine (51%), cattle (11%), BLV-positive cattle (13%) and dogs (6%) but not in sheep. The anti-E. intestinalis antibodies reached the 1:400 in humans (1%), swine (4%) and BLV-positive cattle (17%). The presence of specific anti-E. intestinalis antibodies at the titre of 1:600 was observed only in one swine (1%). Significant differences were observed in animals at titres 1:200 and 1:400 (chi-squared test: p < 0.0001) for both pathogens and in humans only for E. cuniculi at the titre of 1:400 (chi-squared test: p < 0.0075).     

Anaplasma marginale infection in a Japanese Black cow 13 years after eradication of Rhipicephalus (Boophilus) microplus in Okinawa, Japan

In October 2007, a 15-year-old Japanese Black cow on Ishigaki Island, Okinawa, Japan, was diagnosed with Anaplasma marginale infection based on clinical symptoms, blood examination, smear observation, 16S rRNA and groEL gene sequence analysis, and the result of a CF test. The cow was introduced into the farm from mainland Japan as a calf in 1993, one year before the eradication of Rhipicephalus (Boophilus) microplus, the main vector of A. marginale in Okinawa Prefecture. It is possible that the cow was first infected with A. marginale as a calf in Ishigaki Island and had been persistently infected since then. This is the first reported clinical case of A. marginale infection of cattle since the eradication of R. microplus in Okinawa Prefecture. Additional analysis of major surface protein 1α amino acid sequences revealed that the A. marginale Okinawa strain presented four new repeat forms which were not seen in other strains. This indicates that the Okinawa strain may be a unique geographical variant of A. marginale.     

Destruction of Anoplocephala perfoliata Eggs by the Nematophagous Fungus Pochonia chlamydosporia

The in vitro effect of an isolate of the nematophagous fungus Pochonia chlamydosporia (VC1) on the eggs of Anoplocephala perfoliata was evaluated. The eggs were morphologically analyzed for their integrity using light microscopy (10× objectives), plated on 9.0-cm diameter petri dishes containing 2% WA culture medium with and without fungal isolate (control), grown for 10 days, and 10 replicates were prepared per group. In all, 1000 eggs of A perfoliata were plated on petri dishes containing 2% water agar culture medium with (VC1) and without the fungal isolate (control). After 3, 5, 7, and 10 days, approximately 100 eggs were removed from each plate and classified on the basis of the following parameters: without alteration; type 1, lytic effect without morphological damage to eggshell; type 2, lytic effect with morphological alteration of embryo and eggshell; and type 3, lytic effect with morphological alteration of embryo and eggshell, in addition to hyphal penetration and internal egg colonization and destruction. The P chlamydosporia fungus demonstrated ovicidal activity (P < .05) on the eggs of A perfoliata in the studied intervals presenting type 3 effects of 35%, 42.5%, 53.83%, and 71.17% for the intervals 3, 5, 7, and 10 days, respectively. P chlamydosporia is a potential biological control agent for the eggs of A perfoliata.