Homologies of Deoxyribonucleic Acids from Brucella ovis, Canine Abortion Organisms, and other Brucella Species

The bacterium that causes canine abortion has polynucleotide sequences similar, in deoxyribonucleic acid (DNA)-DNA homology studies, to those of Brucella suis and, by inference from previous data, those of B. abortus and B. melitensis as well as B. neotomae. Therefore, the organism causing canine abortion appears to be a member of the genus Brucella. DNA preparations from Serratia marcescens, Alcaligenes faecalis, and Bordetella bronchiseptica, 58, 62, and 66 mole% guanine plus cytosine, respectively, do not have detectable polynucleotide sequence homologies with B. suis DNA which is 56 mole% guanine plus cytosine. B. ovis DNA lacks some of the polynucleotide sequences present in B. suis DNA and appears to be a deletion mutant. However, a large proportion of B. ovis polynucleotides are similar to those of other Brucella species, which supports the inclusion of B. ovis in the genus.     

A phylogeny of members of the family Taeniidae based on the mitochondrial cox1 and nad1 gene data

The cestode family Taeniidae consists of 2 genera, Taenia and Echinococcus, which both have been the focus of intensive taxonomic and epidemiological studies because of their zoonotic importance. However, a comprehensive molecular phylogeny of this family has yet to be reconstructed. In this study, 54 isolates representing 9 Taenia species were characterized using DNA sequences in the mitochondrial cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 1 (nad1) genes. Phylogenetic relationships within the family Taeniidae were inferred by combining cox1 and nad1 sequence data of the present and previous studies. In the phylogenetic analysis, the genus Echinococcus was shown to be monophyletic, but Taenia proved to be paraphyletic due to the position of T. mustelae as a probable sister taxon of Echinococcus. This indicates that T. mustelae should form a genus of its own. Taenia ovis krabbei was placed distant from T. ovis ovis, as a sister taxon of T. multiceps, supporting its recognition as a distinct species, T. krabbei. High intraspecific sequence variation within both T. polyacantha and T. taeniaeformis suggests the existence of cryptic sister species.     

Antigenic affinity of cestodes in the genus Taenia

Antigens of four species of the genus Taenia (T. ovis ovis, T. ovis krabbei, T. hydatigena and T. parenchimatosa) were studied by means of immunodiffusion reaction in agar gel with the use of hyperimmune sera. It has been established that extracts of the studied cestodes contain a great number of antigens, which during parenteral administration cause a synthesis of antibodies in rabbits. In homologous systems the number of recorded antigen-antibody complexes varied from 5 to 10. The most close antigenic affinity was found between T. ovis ovis and T. ovis krabbei, T. ovis ovis and T. hydatigena, as far as the main mass of precipitation bands in the immunodiffusion reaction fused together that suggests the identity of corresponding antigenic components. In all cases when analysing antiserum to T. parenchimatosa extract no differences of species-specific character in heterologous systems were traced.     

Cattle and small ruminant piroplasmosis in Macedonia, Greece

Macedonia is an endemic region of cattle as well as of sheep and goat piroplasmosis In cattle, Theileria orientalis (=T. buffeli?), the agent of Eurasian benign theileriosis, is the most common and widespread piroplasm species. T. annulata, the agent of tropical theileriosis, seems to be rare and limited to few foci, but causes very severe clinical cases, especially in imported pure-bred or cross-bred animals. Babesia bovis and B. bigemina are present in several localities, and often coexist. Cattle babesiosis cases are due to both piroplasms, but B. bovis is considered responsible for the severe and acute clinical cases. Ovine and caprine babesiosis is due to B. ovis and mainly affected imported animals as well as indigenous animals which have been transported from localities where the infection was absent. B. ovis is extremely widespread in both sheep and goats. During the last decades, no clinical cases of small ruminant theileriosis have been registered in this region. However, T. ovis, a non-pathogenic theileria, is common in sheep, but not in goats. Anaplasma ovis, a protobacterium of small ruminants, is also present in the region.     

Polymorphism in Brucella spp. due to highly repeated DNA.

The species of Brucella are very closely related, but Brucella ovis does not express detectable amounts of a protein, designated BCSP31, that is common to the other species. We studied the lack of expression of BCSP31 by Southern analysis. DNAs from the B. ovis culture collection strains and field isolates were probed with a 1.3-kb HindIII fragment encoding BCSP31 of Brucella abortus. The probe hybridized to a 1.6-kb HindIII fragment of all B. ovis strains tested, showing that the gene is present in B. ovis but occurs on a larger restriction fragment. DNA linkage studies and restriction mapping of the cloned polymorphic region of B. ovis showed that the polymorphism was due to a DNA insertion of approximately 0.9 kb at a site downstream of the BCSP31-coding region. When the 1.6-kb polymorphic B. ovis fragment was used to probe a HindIII Southern blot of cellular DNA of strains of B. ovis and of B. abortus, at least 24 fragments of B. ovis and 6 fragments of B. abortus hybridized to the inserted DNA. Specimens of B. ovis collected over a 30-year period on two continents had similar hybridization patterns. The large difference between B. ovis and B. abortus in the number of copies of the repeated DNA is interesting in the context of the closeness of the Brucella species.     

DNA polymorphism in the omp25/omp31 family of Brucella spp.: identification of a 1.7-kb inversion in Brucella cetaceae and of a 15.1-kb genomic island, absent from Brucella ovis, related to the synthesis of smooth lipopolysaccharide

Five genes homologous to the well-known omp25 and omp31 genes, that code for two major Brucella spp. outer membrane proteins (OMPs), have been detected in the genome of Brucella melitensis 16M and Brucella suis 1330. In this work we have determined the nucleotide sequence of these five genes, named omp31b, omp25b, omp25c, omp25d and omp22, in the six classical Brucella species reference strains and in representative strains of the recently proposed species Brucella cetaceae and Brucella pinnipediae that classify the Brucella strains isolated in the last years from marine mammals. Although these genes are quite conserved in the genus Brucella, several important differences have been found between species (i) omp31b contains a premature stop codon in B. canis and B. ovis truncating the encoded protein; (ii) the 5' end of omp31b is deleted in the three biovars of B. melitensis which probably prevents synthesis of Omp31b in this species; (iii) only B. melitensis, B. suis and B. neotomae would be able to synthesize the Omp25b protein with the characteristics shared by the Omp25/Omp31 group of proteins (characteristic signal sequence and C-terminal phenylalanine); (iv) a DNA inversion of 1747 bp including omp25b was detected in B. cetaceae strains; (v) a DNA deletion of about 15 kb was detected in all the six B. ovis strains tested. This deletion in B. ovis includes, among other genes, omp25b and wboA, a gene that has been shown to be required for the synthesis of the O-polysaccharide chain of the Brucella spp. smooth lipopolysaccharide. Several features of the DNA region absent from B. ovis suggest that this DNA fragment is a genomic island acquired by the Brucella ancestor by horizontal transfer and later deleted from B. ovis. The DNA polymorphism we have found in this work within the genus Brucella might be involved in the differences in pathogenicity and host preference displayed by the Brucella species.     

DNA polymorphism in strains of the genus Brucella

Preparations of DNA from 23 Brucella strains including 19 reference strains were compared by restriction endonuclease analysis. Pulsed-field gel electrophoresis resulted in optimal resolution of fragments generated by digestion with low-cleavage-frequency restriction enzymes such as XbaI. By this technique, five electrophoretypes were distinguished in five reference strains of the different species, i.e., B. abortus, B. melitensis, B. suis, B. canis, and B. ovis. Minor profile differences allowed us to discriminate between most biovars within a species. However, the differences in the DNA patterns of different field strains of biovar 2 of B. melitensis were not sufficient to serve as markers for epidemiological studies. From the XbaI fragments, we were able to estimate the size of the genomes of B. abortus 544T and B. melitensis 16 MT. This method revealed a relationship between DNA fingerprints, species, and pathovars which could shed light on problems concerning the classification and evolution of members of the genus Brucella.     

Experimental Brucella ovis infection in mouflon (Ovis musimon)

Brucella ovis was isolated for the first time in Italy in 1994 from the genital organs of two domestic rams. In subsequent years bacteriologic and serologic investigations demonstrated an increasing distribution of this disease in domestic sheep. Mouflon (Ovis musimon) occur in several hilly and mountainous areas of Italy where they can potentially contact domestic sheep. To determine if this species may have a role in the epidemiology of B. ovis, four male and four female mouflon, serologically negative for B. ovis and other Brucella spp., were infected intra-conjunctivally with B. ovis strain BG1/94. Physical examinations, including collection of blood samples for serology and bacteriology, were performed weekly. The animals were euthanized 8 mo postinoculation (p.i.). Samples of retropharyngeal, parotid, and iliac lymph nodes; bone marrow; kidneys; spleen; epididymis; testicle; bulbourethral glands; seminal vesicles; uterus; and oviducts were collected from each animal as appropriate for histopathology and bacteriology. At the time of euthanasia none of the animals exhibited obvious clinical signs of brucellosis. The animals seroconverted 2 wk p.i. and became seronegative 24 wk p.i. Bacterial cultures, including hemocultures, were negative. No lesions due to B. ovis infection were revealed by histologic examinations. Brucella ovis probably did not infect mouflon and this wild sheep is not likely to play a role in the epidemiology of contagious epididymitis caused by B. ovis.     

Naturally occurring egg parasitoids of the genus Trichogramma (Hymenoptera: Trichogrammatidae) in a pomegranate orchard in Tunisia

Four Trichogramma species were found in a pomegranate orchard in Gabès, an arid region of Tunisia, from parasitized eggs of Ectomyelois ceratoniae Zeller (Lepidoptera: Pyralidae), an economically important insect pest. Identification based on assessment of male genitalia and internal transcribed spacer 2 ( ITS 2) sequences showed that they were T. bourarachae Pintureau and Babault, 1988, T. oleae Voegelé and Pointel, 1979, T. cacoeciae Marchal, 1927 and T. evanescens Westwood, 1833. Trichogramma evanescens is reported for the first time in Tunisia. Trichogramma cacoeciae was the largely dominant species in the analyzed samples, whereas T. bourarachae was present in a minor portion of 1.38%. The implications of these results for attempts at controlling E. ceratoniae are discussed.     

Burkholderia, a genus rich in plant-associated nitrogen fixers with wide environmental and geographic distribution

The genus Burkholderia comprises 19 species, including Burkholderia vietnamiensis which is the only known N(2)-fixing species of this bacterial genus. The first isolates of B. vietnamiensis were recovered from the rhizosphere of rice plants grown in a phytotron, but its existence in natural environments and its geographic distribution were not reported. In the present study, most N(2)-fixing isolates recovered from the environment of field-grown maize and coffee plants cultivated in widely separated regions of Mexico were phenotypically identified as B. cepacia using the API 20NE system. Nevertheless, a number of these isolates recovered from inside of maize roots, as well as from the rhizosphere and rhizoplane of maize and coffee plants, showed similar or identical features to those of B. vietnamiensis TVV75(T). These features include nitrogenase activity with 10 different carbon sources, identical or very similar nifHDK hybridization patterns, very similar protein electrophoregrams, identical amplified 16S rDNA restriction (ARDRA) profiles, and levels of DNA-DNA reassociation higher than 70% with total DNA from strain TVV75(T). Although the ability to fix N(2) is not reported to be a common feature among the known species of the genus Burkholderia, the results obtained show that many diazotrophic Burkholderia isolates analyzed showed phenotypic and genotypic features different from those of the known N(2)-fixing species B. vietnamiensis as well as from those of B. kururiensis, a bacterium identified in the present study as a diazotrophic species. DNA-DNA reassociation assays confirmed the existence of N(2)-fixing Burkholderia species different from B. vietnamiensis. In addition, this study shows the wide geographic distribution and substantial capability of N(2)-fixing Burkholderia spp. for colonizing diverse host plants in distantly separated environments.     

细菌学方法和HOOF-Prints技术在绵羊种布鲁氏菌019株鉴定中的比较研究

应用细菌学常规方法和分子生物学检测方法对绵羊种布鲁氏菌非典型株019进行分类研究。利用高变8聚核苷酸DNA指纹技术(HOOF-Prints)对绵羊种布鲁氏菌019株可变数目重复片段(VNTR)的8个位点进行PCR扩增和序列测定,将测定结果与GenBank数据库比较分析,应用DNAMAN进行同源性分析,并构建系统进化树。结果表明,绵羊种布鲁氏菌019株和绵羊种布鲁氏菌参考株63/290的亲缘关系高于绵羊种布鲁氏菌019株与其他参考株的亲缘关系,该结论与细菌学常规鉴定结果一致。应用HOOF-Prints技术可以对绵羊种布鲁氏菌非典型株019进行鉴定,该技术有望弥补传统分类方法的不足。     

Genetic variation at the omp2 porin locus of the brucellae: species-specific markers

The omp2 locus of Brucella abortus is composed of two closely related genes (omp2a and omp2b) that encode, and potentially both express, homologous porin proteins. Genetic variation at this locus is revealed in the form of restriction-fragment-length polymorphisms which can be used to distinguish the type strains of all six Brucella species. Five of the six species contain single copies of omp2a and omp2b, whereas Brucella ovis appears to have two copies of the omp2a gene. The implications of these results with regard to the physiological functions of the omp2a and the omp2b gene products, phylogeny of the genus, and species-specific adaptation are discussed.     

Studies of Antigens for Complement Fixation and Gel Diffusion Tests in the Diagnosis of Infections Caused by Brucella ovis and Other Brucella

Sonically treated and saline-extracted antigens of Brucella ovis, B. canis, B. abortus, and B. melitensis were compared in gel diffusion, complement fixation, and serum absorption tests. All the sonically extracted antigens showed cross-reactions with sera from animals infected or immunized with these species, whereas the saline-extracted antigens were specific for the surface of the rough or smooth colonial phase of the species or strain. The saline-extracted antigens of B. ovis and B. melitensis were both eluted as a single peak in the void volume by Sephadex G-200 column chromatography, in gel diffusion had staining characteristics of lipoproteins, but in immunoelectrophoresis showed distinct mobility patterns. Serological activity for both gel diffusion and complement fixation tests was demonstrated in the immunoglobulin G-containing fraction of sera taken from sheep 12 to 412 days after infection with B. ovis. The gel diffusion test with saline extract of B. ovis is as sensitive as the complement fixation test for the diagnosis of ram epididymitis and is more practical.     

Ecological and genetic differentiation of Barbus callensis populations in Tunisia

Enzyme electrophoresis on horizontal starch gel was carried out on 356 barbel individuals. The sampling comprised 278 individuals of the species Barbus callensis from 10 rivers in Tunisia. The other individuals belonged to reference species (outgroups) from France and Morocco. An ecological study was also carried out on Tunisian rivers. The results show a clear differentiation of the two samples from northwestern Tunisia, which was only partly correlated with ecological characteristics of the rivers they inhabit. There is no genetic cline, but rather a discontinuity between populations in the northwestern-most watershed and all the other Tunisian populations. This differentiation probably has a paleohistoric origin not only related to adaptation to ecological conditions but also to difficulties in colonizing the watersheds. The results do not indicate clearly a colonization direction for the genus Barbus in North Africa. Analysis of the Algerian populations would appear to be indispensable. Lastly, in contrast with the usual taxonomy, Morocco and Tunisia are populated by two closely related species, but B. callensis should remain the name of the Tunisian species, which was the first to be described in the small El Kebir basin, a river that flows from Tunisia to Algeria.     

Gastrointestinal parasites of cougars (Felis concolor) in Washington and the first report of Ollulanus tricuspis in a sylvatic felid from North America.

Gastrointestinal helminths including two species of cestodes (Taenia omissa and T. ovis krabbei) and three species of nematodes (Toxocara cati, Cylicospirura subequalis and Ollulanus tricuspis) are reported from two free-ranging cougars (Felis concolor) in Washington (USA). Ollulanus tricuspis is reported for the first time from cougars and represents the first occurrence of this parasite in a sylvatic felid from North America.     

Antibody responses of experimentally infected lambs to antigens collected during in vitro maintenance of the adult, metacestode or oncosphere stages of Taenia hydatigena and Taenia ovis with further observations on anti-oncospheral antibodies

The antigenicity and specificity of crude antigens collected during the in vitro maintenance of Taenia hydatigena and T. ovis, excretory/secretory (ES) antigens, were assessed in a peroxidase microenzyme-linked immunosorbent assay (ELISA), using sera from lambs given experimental monospecific infections with T. hydatigena, T. ovis, Echinococcus granulosus or Fasciola hepatica. ES antigens of larval cysts of T. ovis and T. hydatigena were less reactive than those of adult or oncosphere stages. Strong interspecific cros-reactions occurred between all antigen preparations, and these antigens offered no better specificity than crude somatic extracts. IgG1 was the major immunoglobulin detected in sera from lambs experimentally infected with T. ovis or T. hydatigena using antigens prepared from sonicated oncospheres. Discrete peaks of anti-oncospheral antibodies were detected following initial and challenge infections with eggs (whether the homologous or heterologous species), when sera were assayed with a PBS sonicate or an ES antigen from oncospheres. However, when oncospheres solubilised with sodium deoxycholate were used, the antibody response was prolonged and resembled that reported previously when somatic extracts of adult and metacestode stages were used as antigen. The results showed that oncospheres share antigens in common with other life-cycle stages, but also support the notion that they may possess some unique stage-specific antigenic determinants.     

Genomic island 2 is an unstable genetic element contributing to Brucella lipopolysaccharide spontaneous smooth-to-rough dissociation

Brucella is a Gram-negative bacterium that causes a worldwide-distributed zoonosis. The genus includes smooth (S) and rough (R) species that differ in the presence or absence, respectively, of the O-polysaccharide of lipopolysaccharide. In S brucellae, the O-polysaccharide is a critical diagnostic antigen and a virulence determinant. However, S brucellae spontaneously dissociate into R forms, a problem in antigen and S vaccine production. Spontaneous R mutants of Brucella abortus, Brucella melitensis, and Brucella suis carried the chromosomal scar corresponding to genomic island 2 (GI-2) excision, an event causing the loss of the wboA and wboB O-polysaccharide genes, and the predicted excised circular intermediate was identified in B. abortus, B. melitensis, and B. suis cultures. Moreover, disruption of a putative phage integrase gene in B. abortus GI-2 caused a reduction in O-polysaccharide loss rates under conditions promoting S-R dissociation. However, spontaneous R mutants not carrying the GI-2 scar were also detected. These results demonstrate that the phage integrase-related GI-2 excision is a cause of S-R brucella dissociation and that other undescribed mechanisms must also be involved. In the R Brucella species, previous works have shown that Brucella ovis but not Brucella canis lacks GI-2, and a chromosomal scar identical to those in R mutants was observed. These results suggest that the phage integrase-promoted GI-2 excision played a role in B. ovis speciation and are consistent with other evidence, suggesting that this species and B. canis have emerged as two independent lineages.