The role of elastase in the differentiation of Bacteroides nodosus infections in sheep and cattle

Eighty-seven Bacteroides nodosus isolates were examined for elastase production by clearing of elastin particles in TAS agar medium. These included 54 ovine virulent isolates, 28 ovine benign isolates and five bovine isolates. In addition 22 ovine virulent, 16 ovine benign and two bovine isolates were examined for decline in proteolytic activity over a 13-day period in the degrading proteinase test using hide power-azure as substrate. There was a remarkable correlation between elastase production, relative stability of proteolytic activity in the hide powder-azure test and virulence of B nodosus. Ovine virulent isolates invariably produced elastase whereas ovine benign isolates and bovine isolates were elastase negative. Bovine isolates produced only mild lesions in the feet of challenged sheep.     

A DEGRADING PROTEINASE TEST TO DISTINGUISH BENIGN AND VIRULENT OVINE ISOLATES OF BACTEROIDES NODOSUS

Bacteroides nodosus was recovered from naturally occurring cases of virulent ovine footrot (VFR) and benign footrot (BFR) using an artificial culture medium which incorporated trypticase, arginine, serine and 5% agar. A degrading proteinase (DP) test was developed which measured the proteinase activity of broth cultures of B. nodosus for a period of time after organism death to assess the stability of the enzyme. The spectrophotometric measurement of the release of dye from a hide powder--azure conjugate by the action of proteinase provided an objective analysis of enzyme activity. The DP test differentiated VFR and BFR isolates and promises to be a useful laboratory method for the diagnosis of benign and virulent footrot in sheep.     

Characterisation of virulent and benign strains of Bacteroides nodosus

The extracellular proteases of 395 isolates of B. nodosus from ovine, bovine and caprine foot lesions were classified as either thermostable or thermolabile. Stable protease was associated with one and unstable protease with four distinctive isoenzyme patterns, each pattern differentiated by the relative mobility of paired isoenzymes. Pathogenicity tests on 64 isolates showed a correlation between the production of stable protease and the production of virulent ovine footrot lesions. The mean values for total protease activity, twitching motility and colony diameter were significantly higher for virulent compared to benign isolates, but the range of values overlapped. SDS-PAGE whole-cell electrophoretic profiles of virulent isolates were similar to the profiles of some benign isolates.     

The pathogenicity and cultural characteristics of virulent, intermediate and benign strains of Bacteroides nodosus causing ovine foot-rot

The relationship between the cultural and biochemical characteristics of 22 strains of Bacteroides nodosus and their virulence for sheep was examined. Virulent, intermediate and benign strains were recognised. Although there was some relationship between virulence and colony morphology on hoof medium with 4% agar, colonies of one virulent and 4 intermediate strains resembled those of benign strains. However, on hoof medium with 2% agar and on blood Euonagar, colonies of this virulent and one intermediate strain differed from each other and the other 3 intermediate strains, which in turn differed from the benign. The degree of piliation, as assessed by electron microscopy, was not a reliable indicator of virulence in strains not possessing a beaded colony type. Together, the results of colony morphology and proteolytic tests such as zymogram, degrading proteinase and elastin-agar tests allowed better discrimination of virulent and benign strains. Intermediate strains generally possessed virulent protease activity. In strains with benign zymogram patterns, activity bands 2 and 3 were more labile than in strains with virulent patterns. The addition of CaCl2 to the culture medium resulted in greater stability of proteolytic activity, particularly with benign strains, and prevented the disappearance of protease activity in the band 5 position in virulent, intermediate and benign strains during prolonged incubation. There were slight differences in the sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) patterns of outer membrane proteins of some benign strains but those of intermediate category resembled virulent strains. There was some relationship between the apparent Mr of the pilin monomer on SDS-PAGE gels and serogroup specificity.     

Motility in relation to virulence of Bacteroides nodosus

Fourteen Bacteroides nodosus isolates from footrot lesions of sheep were examined microscopically and all were found to have twitching motility. The mean percentage of cells showing motility was 40% and 9% for virulent and benign strains, respectively. This corresponded with mean agar colony diameters of 17 mm and 7 mm, respectively, for these strains. Two strains of intermediate virulence had values of motility and colony diameter similar to the benign strains. However, the intermediate and the virulent strains produced relatively stable protease compared to the benign strains. All virulent, benign and intermediate strains produced abundant pili. Included for comparison in this study was an avirulent variant strain which was highly motile, formed large colonies and produced stable protease, but showed no pili on electron microscopy. It was concluded that the properties of motility and protease stability may be used to distinguish, in the laboratory, wild-type virulent, benign and intermediate strains of B. nodosus.     

ISOLATION AND CHARACTERISATION OF BACTEROIDES NODOSUS FROM FOOT LESIONS OF CATTLE IN WESTERN AUSTRALIA

SUMMARY Thirty one isolates of Bacteroides nodosus were obtained from foot lesions observed on cattle at 3 abattoirs. All isolates were similar to the B. nodosus of ovine benign footrot (BFR) in their response to the degrading proteinase test. At one abattoir, where the interdigital lesions were examined in detail, 9 of 10 isolates were obtained from hyperkeratotic lesions with deep fissures. Traceback to 8 of the farms of origin which carried both sheep and cattle, revealed BFR in sheep on 4 farms. The significance of B. nodosus in interdigital lesions in cattle, and its possible pathogenicity, are discussed.     

Genetic and antigenic characterization of the matrix protein of two genetically distinct ovine lentiviruses

Small Ruminant Lentiviruses (SRLV) are a group of non-oncogenic retroviruses including Maedi-Visna virus (MVV) and Caprine Arthritis-Encephalitis virus (CAEV), which cause a chronic, multisystemic disease in sheep and goats, respectively. Phylogenetic analyses of SRLV are based in most cases on partial pol sequences. Several reports indicate that the species specificity of these viruses is not as strict as previously thought; MVV-like viruses have been found in goat populations and vice versa. Recently, the sequencing of some Italian ovine isolates has shown the presence of a new cluster more similar to classical caprine isolates (CAEV-like). Few data are available on the variability of structural proteins involved in the antibody response of infected animals. In this study, the gag gene of two genetically distinct ovine isolates, namely the MVV-like It-561 and the CAEV-like It-Pi1, was sequenced and the epitopes of matrix protein (MA) were mapped. Recombinant MAs and their subunits from both ovine aforementioned strains were tested against a panel of sheep and goat sera. Reactive epitopes were found in all three subunits of MA, although the central subunit displayed a more consistent reactivity. Epitope mapping of this subunit demonstrated that the amino acid sequence of at least one immunodominant epitope was quite different in the two strains. This antigenic variability may affect the sensitivity of a single strain-based immunoassay and suggests that both SRLV genotypes should be used in the development of future diagnostic tests, to avoid viral strain selection during the eradication programmes.     

A recently introduced Dichelobacter nodosus strain caused an outbreak of footrot in Norway

Background

In 2008, an outbreak of ovine footrot occurred in Norway. Dichelobacter nodosus isolates collected between 2008 and 2011 have been characterised. Isolates defined as virulent by the gelatin gel test (GG-test) were only found in sheep in Rogaland County, where the severe cases of footrot were registered. The majority (96%) of the virulent isolates belonged to serogroup A. It is suspected that they represent a newly introduced strain, and the aim of the present study was to investigate whether they are genetically similar. Sixty-one virulent isolates from sheep and 116 benign isolates from sheep, cattle and goats were included. Four GG-test virulent isolates from Danish sheep were also included. All isolates were genotyped by pulsed-field gel electrophoresis (PFGE) and by PCR for pgr variant determination.

Results

The Norwegian virulent isolates were assigned to 8 pulsotypes (PTs), while the benign isolates were assigned to 66 PTs. Thirty-seven (68.5%) of the 54, virulent, serogroup A isolates belonged to the same PT, and included isolates from 2008 through 2011. Isolates belonging to this PT were defined as the outbreak strain. The remaining virulent serogroup A isolates belonged to 4 PTs differing by ≤3 bands from the outbreak strain. Two virulent, Danish, serogroup A isolates differed by 2 bands from the Norwegian outbreak strain. All but 3 (95%) of the virulent isolates had the pgrA variant while 85% of the benign isolates had the pgrB variant.

Conclusion

This study provides evidence that the footrot outbreak in Norway in 2008 most likely was caused by new introduction and local spread of one virulent D. nodosus strain.     

一例梅花鹿布氏杆菌和日本乙型脑炎混合感染的诊断

2018年3月17日,贵州省某梅花鹿场的梅花鹿(Cervus nippon)出现关节肿大、下颌肿大、睾丸单侧肿胀,还伴有严重的跛行等症状。采集4份该发病梅花鹿的血样送检。检测结果表明,通过虎红平板凝集试验2份血样检测出了布氏杆菌感染,4份血样经RT-PCR均扩增出了JEV NS1部分基因的特异性条带,PCR产物测序结果分析显示,4份梅花鹿血样的JEV NS1基因(GZ-015、GZ-019、GZ-022、GZ-039)间的核苷酸同源性分别为99. 9%、99. 3%、99. 4%,与强毒株SA-14、国内外的疫苗株及四川、吉林等地的JEV分离株之间核苷酸同源性为99%以上,GZ-015、GZ-019、GZ-022、GZ-039与强毒株SA-14、弱毒株、国内外的疫苗株及四川、吉林等地的JEV分离株处于同一个较大的遗传进化分支。     

Ovine Haemophilus somnus: experimental intracisternal infection and antigenic comparison with bovine Haemophilus somnus.

Experimental infection was produced by two of four isolates of ovine Haemophilus somnus given by intracisternal inoculation into two to three-month-old lambs. Isolate 2041 (originally obtained from a septicemic lamb in Alberta) caused lethal infection in eight of nine lambs, isolate 67p from the prepuce of a normal lamb produced less acute disease in four of nine lambs, and the other two isolates (93p and 1190) caused no detectable disease. Significant lesions were limited to the brain and spinal cord. Purulent meningitis was characteristic but vasculitis or septicemia were not detected, perhaps due to the route of inoculation. Since a difference in virulence was noted among strains, we analyzed surface proteins thought to be virulence factors of bovine H. somnus. Protein profiles of bovine and ovine H. somnus done by sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed similar patterns for virulent bovine isolates and ovine septicemic isolates. Preputial isolates showed a lower molecular mass major outer membrane protein than septicemic isolates. Antigenic analysis revealed that outer membrane proteins p270, p78, p76, p40, and p39 were detected in both ovine and bovine isolates except for 1190, which was probably not a true H. somnus isolate. Thus the preputial and septicemic isolates of ovine H. somnus were similar to bovine H. somnus in pathogenicity and in surface antigens.     

Epidemiology of ovine Campylobacter infection determined by numerical analysis of electrophoretic protein profiles

The relationship of 50 Campylobacter strains isolated from aborted ovine foetuses, and the faeces of sheep, cattle and chickens were determined by numerical analysis of electrophoretic (SDS-PAGE) protein profiles. Comparison of protein patterns by numerical methods revealed differences between C. fetus ssp. fetus, C. jejuni, and C. coli strains as well as heterogeneity among isolates from different outbreaks. Isolates from each farm produced a distinct cluster and flocks from different locations were found to be infected with relatively different strains. In most cases, protein patterns of ovine foetal isolates were very similar to those of ovine faecal isolates. Ovine isolates of C. fetus ssp. fetus, C. jejuni and C. coli gave similar protein patterns to the corresponding Campylobacter species isolated from cattle or chicken, on the same farm. Thus, it was concluded that certain protein types of ovine Campylobacter strains were more likely associated with local areas, and Campylobacter strains causing ovine abortions are distributed in the environment more widely than assumed to date.     

Comparison of virulence of ovine respiratory mycoplasmas in the mouse mammary gland

The virulence of isolates of Mycoplasma ovipneumoniae and M. arginini from pneumonic and unaffected ovine lungs was compared in a mouse mammary gland model. The isolates varied in their ability to induce a neutrophilic response in the mammary gland. A moderate to severe form of mastitis was induced by 3 M. ovipneumoniae isolates recovered from pneumonic lungs, while the remaining M. ovipneumoniae isolates from pneumonic lungs and those from unaffected lungs induced a very mild histopathological response. The severity of the mastitis could not be increased by the simultaneous inoculation of a mixture of 5 mycoplasma isolates. Mycoplasma arginini isolates induced only a very mild histopathological response despite having been isolated from pneumonic lungs. The finding that the 3 most virulent M. ovipneumoniae isolates were initially recovered from pneumonic ovine lungs suggested that these virulent isolates may contribute to ovine pneumonia. However, the isolation of M. ovipneumoniae from pneumonic ovine lungs does not necessarily imply that these organisms are the causal agents, since M. ovipneumoniae isolates may vary in virulence.     

Differences between strains of Bacteroides nodosus in their effects on the severity of foot-rot, bodyweight and wool growth in Merino sheep

The effects of 3 ovine and one bovine strains of Bacteroides nodosus on the severity of foot-rot, bodyweight and wool growth were compared in Merino sheep in a field experiment. Based on the severity of the induced foot lesions, one strain was classed as virulent (causing underrunning lesions in most feet), one was benign (causing lesions of the interdigital skin only), and 2, including the bovine strain, were of intermediate virulence (causing underrunning lesions in a small proportion of feet). Differences among strains in their effect on foot-rot severity and bodyweight were significant when compared over the whole experimental period, but were not significant at any single time of measurement, because of large differences between replicates. Bodyweight loss and severity of foot-rot caused by the virulent strain were significantly greater than that caused by the benign strain. The intermediate strains lay between these 2 extremes in terms of both bodyweight and foot-rot scores but were not significantly different from either in a statistical sense. Total greasy wool weight did not differ among groups over the whole experiment, but the rate of wool growth during the period when foot lesions were most prevalent and severe was reduced appreciably by the virulent strain and to a lesser extent by the intermediate strains.     

The thermostability of proteases from virulent and benign strains of Bacteroides nodosus

Protease enzymes, produced by Bacteroides nodosus strains isolated from animals with virulent and benign forms of ovine footrot, were partially purified by ultra-filtration, ion exchange chromatography and gel permeation chromatography. Each enzyme had a similar pH optimum, was inhibited by phenylmethylsulfonyl fluoride (PMSF), ethylene diamine tetraacetic acid (EDTA) and ethyleneglycot-bis-aminoethylether-N,N-tetraacetic acid (EGTA), but was not inhibited by 1,10-phenanthroline. The results suggest that these enzymes are serine proteases that require divalent cations for activity. The enzymes could be distinguished by their differential temperature stability and differing susceptibility to irreversible inactivation by EDTA. Both enzymes were stabilised by incubation in the presence of Ca2+, but the enzyme purified from the virulent isolate required less Ca2+ for maximum stability. These results suggest that the differential thermostability of the protease activity detected in virulence tests is an intrinsic property of the protease enzymes.     

Improved diagnosis of virulent ovine footrot using the intA gene

Footrot is a mixed bacterial infection of the hooves of sheep. The gram-negative anaerobic bacterium Dichelobacter nodosus is the principal causative agent, with different strains causing diseases of different severity, ranging from benign to virulent. In Australia, in the state of New South Wales (NSW), only virulent footrot is subject to regulatory action, including quarantine. However, it is often difficult to distinguish benign footrot from virulent footrot in the initial stages of infection, or under adverse climatic conditions. The gelatin gel test, which measures the thermostability of secreted bacterial proteases, is the laboratory test most widely used in Australia to aid in the differential diagnosis of footrot. The proteases of virulent strains are, in general, more thermostable than the proteases of benign strains. However, there are some false positives in the gelatin gel test, which may lead to unnecessary quarantine procedures. We used Southern blot analysis on 595 isolates of D. nodosus from 124 farms on which sheep had benign or virulent footrot to test for the presence of the intA gene. We found that for D. nodosus strains which are stable in the gelatin gel test, there is a high correlation between the presence of the intA gene and the ability of the strain to cause virulent footrot. We also developed a PCR-based assay for the rapid detection of intA, which can be used to test DNA extracted from colonies grown on plates, or DNA extracted from cotton swabs of culture plates.     

梅花鹿狂犬病的病原学研究

应用鼠脑传代,从具有神经症状和后躯麻痹的鹿尸脑组织分离获得5株弹状病毒。对其中一株——8202株,从形态学、生物学和理化学等方面进行了鉴定。应用弹状病毒料中狂犬病海和狂犬相关病毒等的抗血清或免疫腹水,在小鼠体内进行交叉中和试验,证明鹿毒8202株同狂犬病毒固定毒(北京株)有密切的抗原联系。经WHO狂犬病咨询和研究协作中心应用42个单克隆抗体,以间接荧光抗体试验检测鼠脑压印片,证明该株病毒的核衣壳抗原结构与大多数陆栖哺乳动物狂犬病毒相同,但有别于欧洲狐狸的狂犬病毒;用40个抗糖蛋白单克隆抗体,通过血清中和试验,证明8202株同欧洲狐狸毒株很相似。将该毒复旧鹿体,并接种犬及其他动物,发现其对鹿的毒力很强,而对犬及其他家畜毒力较弱,甚至没有毒力。结论认为,鹿毒8202株是狂犬病毒适应于鹿体的变异株。     

Spoligotype diversity of Mycobacterium bovis and Mycobacterium caprae animal isolates

The genetic diversity of 283 Mycobacterium bovis (M. bovis) and 10 Mycobacterium caprae (M. caprae) strains, isolated between 2002 and 2007 from cattle, goat, red deer and wild boar from six different geographical regions of Portugal was investigated by spoligotyping. The technique showed a good discriminatory power (Hunter-Gaston Index, h=0.9) for the strains, revealing 29 different patterns. One pattern (SB0121) was clearly predominant, accounting for 26.3% of the isolates; ten patterns, representing 20.7% of the isolates, had never been reported previously. Multiple spoligotypes were detected in thirteen cattle and one goat herd, most of which were found in beef cattle and extensive management regions, suggesting different infection sources. With the exception of two spoligotypes, those in wildlife species were also found in domestic species.     

弹性硬蛋白溶解试验鉴别反刍动物腐蹄病节瘤拟杆菌强毒株

根据腐蹄病节瘤拟杆菌毒力菌株具有溶解弹性硬蛋白的特性，利用弹性硬蛋白培养基建立了弹性硬蛋白溶解试验，以鉴别节瘤拟杆菌强毒株。试验表明，将不同毒力的菌株同时接种于弹性硬蛋白培养基，蛋白溶解带出现的时间和宽度都显著不同，强毒株大多在７ｄ内，少部分在７～１１ｄ内均出现一条明显的蛋白溶解带；而弱毒株在２１ｄ内基本上未见蛋白溶解带出现。     

Molecular genotyping of multinational ovine and caprine Corynebacterium pseudotuberculosis isolates using pulsed-field gel electrophoresis

Caseous lymphadenitis (CLA) is a chronic, suppurative disease, with a worldwide distribution, caused by Corynebacterium pseudotuberculosis. The clinical manifestation of CLA is known to vary between different countries, and has been postulated to be due to differences in the strains present in these countries. Forty-two sheep and goat isolates of C. pseudotuberculosis from Australia, Canada, Eire, The Netherlands and Northern Ireland were characterized by pulsed-field gel electrophoresis (PFGE), biotyping, antimicrobial susceptibility, and production of phospholipase D. The PFGE-determined genotypes of this multicentric collection were then compared with representative ovine and caprine isolates from a previously published panel of PFGE profiles of United Kingdom isolates. Digestion with SfiI generated 16-18 bands in the 48.5 and 290 kb range, and differentiated four distinct pulsotypes amongst the 36 ovine and 6 caprine strains which displayed remarkable homogeneity. Based on these results, it would appear that the genome of C. pseudotuberculosis is highly conserved, irrespective of the country of strain origin.