Electron Microscopic Examination of Corynebacterium ovis

Corynebacterium ovis (C. pseudotuberculosis) was examined by electron microscopy after being subjected to various methods of fixation. The organism exhibited a fine structure similar to other corynebacterial species in the appearance of its cell wall, plasma membrane, nuclear apparatus, cytoplasmic matrix, wealth and complexity of intracytoplasmic membrane systems, and polyphosphate granules. An outstanding structural feature was the existence of an electron-dense, floccular layer external to the cell wall which both ligroin and acetone-methanol extractions demonstrated to be the previously postulated surface lipid of this organism. The only variations in structure evident between virulent and attenuated strains was a quantitative difference in the thickness and appearance of the surface lipid. The observation of this layer provided a basis for explaining the surface properties of C. ovis, with particular respect to its clumping capacity in suspension, the waxiness of its growth on solid media, and its ability to grow as a pellicle on suitable liquid media. The variation in the visible amount of surface lipid between the virulent and avirulent strains adequately explained the divergence of these three surface properties between the strains.     

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。     

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.     

A note on hydrolysis of tributyrin by Branhamella and Neisseria

Sixty-three strains of Branhamella and Neisseria were tested by two methods for their ability to hydrolyse glycerol tributyrate. After the conventional plate test, gas liquid chromatographical (GLC) analysis of the agar medium was carried out to detect the hydrolysis product, butyric acid, and other volatile fatty acids. All strains of Branhamella catarrhalis, Neisseria caviae, N. cuniculi and N. ovis but no other Neisseria spp. gave positive results with the conventional test. With GLC, however, most strains of Branhamella and Neisseria were shown to liberate butyric acid. In addition, some strains liberated acetic and isovaleric acids. Greater amounts of butyric acid were produced by clinical strains, in particular B. catarrhalis, compared with reference strains. It was concluded that the conventional plate test for tributyrin hydrolysis differentiates B. catarrhalis, N. caviae, N. cuniculi and N. ovis from other Neisseria.     

A note on hydrolysis of tributyrin by Branhamella and Neisseria

Sixty-three strains of Branhamella and Neisseria were tested by two methods for their ability to hydrolyse glycerol tributyrate. After the conventional plate test, gas liquid chromatographical (GLC) analysis of the agar medium was carried out to detect the hydrolysis product, butyric acid, and other volatile fatty acids. All strains of Branhamella catarrhalis, Neisseria caviae, N. cuniculi and N. ovis but no other Neisseria spp. gave positive results with the conventional test. With GLC, however, most strains of Branhamella and Neisseria were shown to liberate butyric acid. In addition, some strains liberated acetic and isovaleric acids. Greater amounts of butyric acid were produced by clinical strains, in particular B. catarrhalis , compared with reference strains. It was concluded that the conventional plate test for tributyrin hydrolysis differentiates B. catarrhalis, N. caviae, N. cuniculi and N. ovis from other Neisseria.     

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 232bp 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.     

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.     

Do sheep regulate the size of their mallophagan louse populations?

Alternatives to chemicals for controlling parasites are required to minimise problems from resistance, residues in animal products and occupational exposure. Utilisation of host response to parasites through selection of resistant types or vaccination is an appealing option. To date most studies have been with haematophagous or invasive parasites which directly contact elements of the host immune system. Sheep lice (Bovicola ovis) feed superficially on the skin of sheep ingesting lipid, scurf, bacteria and loose stratum corneum squames. Evidence is presented that despite their surface feeding habit Bovicola ovis stimulate an immune response in sheep and that this response may play a part in regulating the size of louse populations.     

Transferrin-dependent expression of TbpA by Histophilus ovis involves a poly G tract within tbpA

A poly G tract in tbpA of Histophilus ovis strain 3384Y was suspected of being responsible for the transferrin (Tf)-dependent expression of TbpA. The region encompassing the poly G tract was amplified using DNA from H. ovis strains 9L and 3384Y grown under iron-replete conditions and under iron-restricted conditions in the presence of bovine Tf. Sequence analysis of the amplification products revealed that regardless of the growth conditions, the poly G tract in strain 9L contained eight Gs, a situation that maintains the correct reading frame of the gene. Similarly, the poly G tract in strain 3384Y contained eight Gs when the organisms were grown under iron-restricted conditions in the presence of bovine Tf but when grown under iron-replete conditions, the poly G tract contained nine Gs resulting in a frame shift and the introduction of a premature stop codon. It is concluded that the Tf-dependent expression of TbpA in H. ovis strain 3384Y is due to a form of phase variation.     

Brucella melitensis B115-based complement fixation test to detect antibodies induced by Brucella rough strains

Aims:  To assess the efficiency of a Brucella melitensis B115 rough strain, naturally devoid of anticomplementary activity, used as antigen in a complement fixation test (CFT) to detect antibodies induced by Brucella strains with rough phenotype, such as Brucella abortus RB51, Brucella ovis and Brucella canis .
Methods and Results:  Complement fixation testing was performed on sera from RB51-vaccinated cattle and buffaloes, B. ovis -infected sheep and B. canis -infected dogs using B115, RB51 and the hot saline extract (HSE) as antigens. The B115-based CFT proved highly sensitive and specific in detecting rough antibodies and its efficiency was comparable with that of RB51 and HSE-based CFT.
Conclusions:  Brucella melitensis B115 can be successfully used as an antigen in CFT to detect antibodies induced by Brucella rough strains.
Significance and Impact of the Study:  Brucella melitensis B115 antigen may represent an improvement over Brucella rough strains for Brucella antibody detection by CFT, thus enhancing the efficiency of brucellosis surveillance systems. Owing to the absence of anticomplementary activity, it does not require particular growth conditions or modifications and can be accurately standardized. The B115-based CFT may constitute a suitable supplementary test for the diagnosis of human infections owing to rough Brucellae .     

Ecology of Staphylococcus aureus: comparative characterization of strains isolated from man, cattle and sheep in Bulgaria and in the GDR

Staphylococcus aureus strains from man, cattle and sheep have been differentiated by biochemical tests and by phage typing. Strains from pathological material of human origin mostly belong to the host-specific variety hominis, strains from mastitis in cattle mostly belong to the host-specific variety bovis. However, there are also strains from cattle which cannot be allotted to one of the known host-specific varieties and also strains which belong to the host-specific variety hominis. Strains from mastitis in sheep clearly differ from strains of human and bovine origin; these strains are designated as variety ovis. The frequency distribution of the host-specific varieties in man, cattle and sheep is the same in Bulgaria and in the GDR.     

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.     

Phospholipids of bacteria of Brucella genus

The phospholipid composition of 6 Brucella species (B. melitensis, B. abortus, B. suis, B. ovis. B. canis, B. neotomae) and Australian mouse-derived strains of Brucella N 4, 11, 12 were studied. Comparison of phospholipid composition of Brucella cells with that of serologically related microorganisms revealed that all Brucella biotypes contain phosphatidyl-(N-methyl)ethanolamine and phosphatidylcholine while Y. enterocolitica, Sh. disenteriae, E. coli cells do not contain these two substances. It is concluded that the specific phospholipid pattern of Brucella biotypes may be useful in typing of new Brucella strains.     

Pyrethroid resistance in Australian field populations of the sheep body louse, Bovicola (Damalinia) ovis

Abstract. Synthetic pyrethroid (SP) resistance has developed in Australian field populations of the sheep body louse, Bovicola ( Damalinia ) ovis. Laboratory bioassays were used to measure the susceptibility of lice to cypermethrin and the other registered SPs. Results of these bioassays indicated resistance to cypermethrin, deltamethrin, cyhalothrin and alphacypermethrin. So far, high-level resistance has been diagnosed in only a few strains. The toxicological responses of these strains were clearly separated from those of the majority of louse strains tested. Furthermore, these strains had survived immersion in commercial SP dips. The level of resistance described in some strains was sufficient to cause pour-on products to fail despite the fact that the LC50s of these strains fell within the normal range of field responses.     

Fatty acid makeup of various Brucella species and its relationship to the culture medium]

Gas chromatographic method was applied to the study of the fatty acid composition (in Br. melitensis, Br. abortus, Br. suis, and Br. ovis strains. Fatty acid composition was similar in the mentioned brucellae species, except Br. suis No. 1330 significantly differing by this sign. Methyleneoctadecanoic acid content was considerably elevated, and that of octadecenoic -- reduced in brucellae grown on liver agar with the addition of serum and on meat-peptone agar in comparison with brucellae grown on liver agar; apparently this represents one of the mechanisms of the microorganism adaptation to the less favourable conditions of the nutrient medium. Passage of Br. ovis strain through the guinea pig organism led to the appearance of brucellae forming two types of colonies when grown on liver agar with the addition of serum. The fatty acid composition of brucellae forming small transparent colonies was the same as that of the initial culture with the prevalence of methyleneoctadecanoic acid; as to brucellae with larger colonies with irregular margin and nontransparent centre of the colony--octadecenoic acid prevailed in their fatty acid composition, i.e. their composition was similar to such in brucellae of the melitensis and abortus species grown on liver agar.     

Cloning and characterization of the Brucella ovis heat shock protein DnaK functionally expressed in Escherichia coli.

The Brucella ovis dnaK gene, homolog to the eukaryotic hsp70 genes, was cloned by using a Drosophila melanogaster probe. Comparison of B. ovis and Escherichia coli sequences revealed a similar organization for the dnaK and dnaJ genes and putative regulatory signals. In E. coli transfected with the cloned fragment, B. ovis hsp70 was expressed at 30 and 50 degrees C apparently under the control of its own promoter. The recombinant protein and a B. ovis native protein displaying the same molecular weight were both recognized by anti-E. coli DnaK serum. Native B. ovis protein was also recognized by sera of sheep either infected or vaccinated with an attenuated Brucella strain, suggesting that Brucella hsp70 could be up-regulated during host colonization. A thermosensitive E. coli dnaK mutant transfected with the cloned fragment recovered colony-forming ability at 42 degrees C, showing that the B. ovis DnaK protein could behave as a functional heat shock protein in E. coli.     

The recombinant Omp31 from Brucella melitensis alone or associated with rough lipopolysaccharide induces protection against Brucella ovis infection in BALB/c mice

Immunogenicity and protective activity against Brucella ovis of detergent-extracted recombinant Omp31 (rOmp31 extract) from Brucella melitensis produced in Escherichia coli, purified rough lipopolysaccharide from B. ovis (R-LPS) and a mixture of rOmp31 extract and R-LPS (rOmp31 extract + R-LPS) were assessed in BALB/c mice. The experimental vaccines were compared with a hot saline extract (HS extract) from B. ovis mainly composed of outer membrane proteins (OMPs) and R-LPS, and known to be protective in mice against a B. ovis infection. Serum antibodies to Omp31 and R-LPS were detected in the corresponding mice using Western blotting with B. ovis whole-cell lysates and ELISA with purified antigens. Protection was evaluated by comparing the levels of infection in the spleens of vaccinated mice challenged with B. ovis. A significantly lower number of B. ovis colony-forming units in spleens relative to unimmunized (saline injected) controls were considered as protection. Mice immunized with rOmp31 extract or rOmp31 extract mixed with R-LPS developed antibodies that bound to the B. ovis surface with similar titers. Vaccination with rOmp31 extract plus R-LPS provided the best protection level, which was comparable with that given by HS extract. Similar protection was also obtained with rOmp31 extract alone and, to a lesser degree, with R-LPS. Comparisons between groups showed that an extract from E. coli-pUC19 (devoid of Omp31) provided no protection relative to either HS extract, rOmp31 extract or rOmp31 extract mixed with R-LPS. In conclusion, the recombinant Omp31 associated or not with B. ovis R-LPS, could be an interesting candidate for a subcellular vaccine against B. ovis infection.     

Experimental infections of Anaplasma ovis in pronghorn antelope

Anaplasma ovis was experimentally transmitted from sheep to pronghorn antelope (Antilocapra americana) and back to sheep. Anaplasma ovis was recovered in splenectomized sheep, from two of three spleen-intact pronghorns following their inoculation with blood from known A. ovis carrier sheep. These two pronghorns exhibited a 0.5% or higher A. ovis parasitemia within 48 days after exposure, and an anaplasmosis-positive serological response 91 days after exposure. Clinical signs of illness were not observed. Blood from the infected pronghorns produced disease in four splenectomized sheep.     

Serum and skin surface antibodies and their associations with sheep biting lice, Bovicola ovis, on experimentally infested sheep

The sheep biting louse ( Bovicola ovis ) feeds superficially on the skin of sheep but appears to stimulate an immune response. In this study we examined the association between louse infestation and serum and skin surface antibodies. Louse numbers were monitored on experimentally infested Polypay and Columbia ewes for two years and on their lambs in the second year. Serum and skin wash samples were tested for antibodies to soluble extracts of B. ovis , Stomoxys calcitrans and Musca autumnali s by enzyme linked immunosorbent assay (ELISA). In addition, the effects of skin wash extracts on B. ovis were examined in vitro .
The titre of anti- B. ovis antibodies in the serum did not differ significantly between infested and naive ewes. However, there was an increase in serum antibody titre which coincided with periods of high louse density in ewes with high louse counts. Infested lambs had higher serum antibody levels than naive lambs. Substantial cross reactivity was evident among extracts of the different insects.
Densities of lice on the ewes during population decline were negatively related to the titre of skin surface antibodies. Skin washings collected from sheep during B. ovis population decline reduced the number of louse progeny when incorporated into louse diet. These results indicate that B. ovis stimulates an immune response in sheep and suggest that compounds on the skin surface may play a role in the regulation of louse populations.