Phenotypic characterization of Brucella strains isolated from livestock in Nigeria

Isolation of brucellae from aborted fetuses, hygroma fluids, milk and vaginal swabs obtained from aborting cattle, sheep, goats, pigs, and horses in Nigeria was carried out. A total of 25 isolates, obtained mainly from cattle, sheep and horses, were biotyped. All strains belonged to one species, Brucella abortus biovar 1. The epidemiological significance of this finding is discussed. Some preliminary observations on the zoonotic and public health implications of Brucella infection in Nigerian livestock are presented. A control programme involving improved management, animal movement restrictions, public health education and mass vaccination of animals is suggested.     

Serogroups of Campylobacter fetus and Campylobacter jejuni isolated in cases of ovine abortion

Of 38 aborted ovine fetuses from 23 sheep flocks 29 C. fetus subsp. fetus and 22 C. jejuni were isolated and examined biochemically and serologically for heat-stable antigens. Serologic examinations were carried out by passive haemagglutination test. In case of C. fetus subsp. fetus strains alkaline antigen extraction was used. Antisera to two serogroups of C. fetus and to Penner serotype reference strains 1 to 60 were produced in rabbits. Abortion was caused in 18 (78.3%) flocks by C. fetus subsp. fetus and in 5 (21.7%) flocks by C. jejuni. Six C. fetus subsp. fetus strains grew well at both 43 and 25 degrees C. With one exception all C. fetus subsp. fetus were resistant, whereas all 29 C. fetus subsp. venerealis strains were sensitive to 30 micrograms/ml cefoxitin and cefamandole. These two cephalosporins can be used to differentiate the two subspecies of C. fetus. Passive haemagglutination test using alkaline antigen extraction is a proper method for the examination of heat-stable antigens of both C. fetus subspecies. Out of 24 C. fetus subsp. fetus strains 13 belonged to serogroup A(01), and 11 to serogroup B(02). C. jejuni strains examined belonged to Penner serogroup 1 (6 strains), to serogroup 5 (4 strains) and to serogroup 8 (4 strains).     

Serology of Campylobacter fetus fetus strains from four outbreaks of ovine abortion

As a result of a Ministry of Agriculture & Fisheries survey on ovine abortions, 76 isolates of Campylobacterfetus fetus were obtained. These isolates were from four farms in the southern Hawkes Bay, with an abortion incidence of 2.8% to 9.1%. Antisera to eight different strains of C. fetus fetus were made in rabbits. Strains were then examined using whole cell tube agglutination tests and sensitised Staphylococcal Protein A slide agglutination tests. Heat labile antigens were examined by absorbing antisera with heat-treated bacteria. Two broad serogroups were found, but within-group variation was demonstrated by cross-absorbing antisera. The isolates from one farm were all of a single broad serogroup. Both serogroups were found on the remaining three farms. Evidence for the presence of two major serogroups was also obtained by immobilisation tests and antigen analysis by gel diffusion.     

牛源胎儿弯曲菌PCR检测方法的建立

根据胎儿弯曲菌的16S rRNA基因序列设计引物,以微需氧培养的胎儿弯曲菌标准株菌体裂解物为模板,进行PCR扩增目的片段。同法检测了胎儿弯曲菌的10个参考菌株,结果均为阳性。采用异硫氰酸胍快速提取流产牛阴道分泌物中胎儿弯曲菌的DNA,然后用PCR方法检测,5份样品阳性,该试验可在24 h内完成,比病原分离法快5~7 d;而检测空肠弯曲菌、大肠杆菌、布氏杆菌、葡萄球菌、链球菌等相关病原时,均无特异性扩增产物,表明该检测方法具有快速、特异、实用性强的特点。     

Diagnosis by ELISA of bovine abortion due to Campylobacter fetus

An enzyme-linked immunosorbent assay (ELISA) was developed to detect antigen-specific secretory IgA antibody in bovine vaginal mucus after abortion due to Campylobacter fetus subsp venerealis. Abortions were diagnosed by isolating the organism from 8 foetuses and/or foetal membranes and by histopathology. Vaginal mucus was collected from 7 cows shortly after abortion. All showed a high level of IgA antibody in their vaginal mucus when they were compared with an uninfected control group. The new ELISA is simple and practical and provides a useful tool for diagnosis of bovine venereal campylobacteriosis.     

Immunohistochemical identification of Campylobacter fetus in natural cases of bovine and ovine abortions

An immunohistochemistry (IHC) procedure for the detection of Campylobacter fetus antigens using an avidin-biotin complex technique was performed on formalin fixed bovine and ovine fetal tissues from 26 natural cases of Campylobacter spp. abortion (four ovine and 22 bovine). The species of Campylobacter isolated included C. fetus ssp. venerealis from 13 bovine fetuses, C. fetus ssp. fetus from two ovine and one bovine fetus, Campylobacter jejuni from seven bovine fetuses, Campylobacter lari from two ovine fetuses and an unspeciated Campylobacter species in one bovine fetus. Histologic lesions identified in the aborted fetuses included placentitis, serositis, pneumonia, gastroenteritis, hepatitis and encephalitis. Campylobacter fetus antigens were identified by IHC in 13 of 13 bovine fetuses from which C. fetus ssp. venerealis was isolated and in two of two ovine fetuses from which C. fetus ssp. fetus was isolated. The IHC stains were negative in tissues from seven bovine fetuses from which C. jejuni was isolated, one bovine fetus infected with C. fetus ssp. fetus, one bovine fetus infected with the unspeciated Campylobacter and two ovine fetuses infected with C. lari. In positive cases, the IHC stain most frequently identified bacteria in the lung and gastrointestinal tract. The C. fetus IHC procedure performed on formalin fixed tissues is a practical tool for the diagnosis of natural cases of ovine and bovine abortion caused by C. fetus.     

Immunological and molecular characterization of Leptospira interrogans isolated from a bovine foetus

Cattle are commonly infected with pathogenic leptospires, and similarly to rodents, they excrete the bacteria in their urine and can transmit the pathogen from animal to animal or animal to human. Thus, surveillance and monitoring systems for detection of new Leptospira serovars are important for the control of leptospirosis. Here, we report the isolation of a spirochete from a stillborn bovine foetus and its characterization by immunological and molecular techniques. A variable number tandem repeat profile using seven discriminatory primers identified the spirochete as belonging to species Leptospira interrogans serogroup Australis serovar Muenchen. A phenotypic analysis using monoclonal antibodies (mAbs) against leptospiral membrane-associated proteins confirmed the expression of important virulence and pathogenicity factors (LipL32 and LigBrep). Out of 120 reference sera tested, 22 positive (36.66%) and 9 negative (15%) also reacted with the new isolate. Furthermore, the serovar Muenchen isolate was virulent in hamster model. The animal inoculated developed acute lethal infection characterized by hepatic, pulmonary and renal lesions. Local isolates exhibited unique characteristics that differed from those of reference strains; therefore, isolation of leptospires is useful in the surveillance of local pathogenic serovars. In conclusion, the data obtained from this study can contribute to the epidemiological understanding and control of leptospirosis in southern Brazil.     

Phenotypic and molecular characterization of Brachyspira spp. isolated from laying hens in different housing systems

Several species of intestinal spirochaetes, Brachyspira (B.) alvinipulli, B. intermedia and B. pilosicoli, may cause reduced egg production and faecal staining of eggshells in chickens. The aim of this study was to characterize potentially pathogenic and presumably non-pathogenic Brachyspira spp. from commercial laying hens. Selective culture, phenotyping, PCR and 16S rRNA gene sequencing were used and clinical data were collected. Phenotypic profiles were obtained for 489 isolates and 351 isolates obtained after subculture, and 30 isolates were selected for molecular characterization. Seven isolates were positive by a B. intermedia-specific PCR based on the nox gene, and two were positive in a B. hyodysenteriae-specific 23S rRNA gene based PCR. By comparative phylogenetic analysis in combination with PCR and phenotyping, seven isolates were identified as B. intermedia, eight isolates as B. innocens, five as B. murdochii, and three isolates each as B. alvinipulli and "B. pulli". The remaining four isolates could not be assigned to any presently recognized species. Co-infection with several species or genetic variants of Brachyspira spp. were detected in some flocks and samples, suggesting a high level of diversity. Organic flocks with access to outdoor areas were at higher risk (RR=2.3; 95% CI 1.5-3.6) for being colonized than chickens in other housing systems. No significant differences between colonized and non-colonized flocks were found regarding clinical parameters, i.e. mortality, egg production, faecally contaminated eggshells, and wet litter. Our results show that a combination of traditional laboratory diagnostics, molecular tests and phylogeny is needed for identification of Brachyspira sp. from chickens.     

Enzyme-linked immunosorbent assay for antibodies to Campylobacter fetus in bovine vaginal mucus

An enzyme-linked immunosorbent assay (ELISA) was used to detect antibodies to Campylobacter fetus subspecies venerealis in bovine vaginal mucus. The results of testing 168 samples from experimentally infected, field cases and control cows showed that the ELISA was more sensitive than the vaginal mucus agglutination test and also detected antibodies in earlier stages of infection.     

Campylobacter fetus fetus abortions in vaccinated ewes

AIMS: To investigate the cause of an outbreak of ovine abortion in 1996 in a flock of 300 two-tooth (rising 2-year-old) ewes vaccinated against Campylobacter fetus fetus infection and to subsequently characterise the strain of C. fetus fetus isolated from aborted foetuses. METHODS: Standard bacteriological methods were used to identify C. fetus fetus isolates which were then antigenically typed and subjected to pulsed-field gel electrophoresis (PFGE) and compared to the vaccine strain. RESULTS: C. fetus fetus was identified as the causal agent of the abortions despite the ewes having been vaccinated before ram introduction and at the time of ram removal. Four isolates cultured from aborted material were indistinguishable when compared using antigenic typing and PFGE, but all differed from the vaccine strain. CONCLUSIONS: Within the limitations of the available typing systems, it is proposed that PFGE may be a useful tool to establish the distribution and strain variation of C. fetus fetus. CLINICAL RELEVANCE: This field case indicates the need for further study of non-vaccine C. fetus fetus strains which cause abortion in vaccinated ewes, and of the importance of these strains to the New Zealand sheep industry.     

A comparative study of the growth of Campylobacter fetus strains in liquid media

The growth of C. fetus fetus, C. fetus venerealis and C. fetus venerealis biotype intermedius was examined in 10 liquid media. From the data obtained, a 10% inoculum size and an oxygen level of 6% seemed imperative for consistent growth, especially for the C. fetus venerealis strain. A lowered redox potential obtained by the addition of 0, 1% cysteine-HC1 to the media was stimulatory. The medium which yielded the best growth was the one described by Dennis & Jones (1959). The fastidious C. fetus venerealis strain yielded maximum values of 0,5% packed cell volumes after 48 h cultivation in a microaerophilic atmosphere on this medium. The other strains yielded higher values.     

Pulsed-field gel electrophoresis typing of Campylobacter fetus subsp. fetus isolated from sheep abortions in New Zealand

AIMS: To genotype Campylobacter fetus subsp. fetus isolates cultured from sheep abortions submitted to diagnostic laboratories in New Zealand during the year 2000 breeding season. To compare the types found nationally with those found in the Hawke's Bay region in 1999, and strains held in the New Zealand Reference Culture Collection, Medical Section (NZRM) from a study published in 1987. METHODS: Campylobacter fetus subsp. fetus isolates cultured by veterinary diagnostic laboratories in the year 2000 breeding season, from sheep abortions from throughout New Zealand, were typed using pulsed-field gel electrophoresis (PFGE). In addition, seven freeze-dried C. fetus subsp. fetus isolates (strain numbers 2939-2945) from the NZRM, representing restriction types a-g found amongst sheep abortion isolates in a study published in 1987, were typed using PFGE. RESULTS: In total, 293 C. fetus subsp. fetus isolates from 200 farms were obtained from veterinary diagnostic laboratories. Twenty-two distinct PFGE profiles were identified amongst the isolates. PFGE type B1 was predominant in each region of New Zealand and was identified from 66% of farms overall. Of the C. fetus subsp. fetus restriction types a-g lodged with the NZRM, 3/7 had PFGE profiles indistinguishable from profiles found in the current study. The other four restriction types had PFGE profiles that were unique but similar to those found in the current study. CONCLUSIONS: PFGE type B1 was predominant amongst the C. fetus subsp. fetus isolates cultured from sheep abortions in each region of New Zealand in the year 2000, as was found in Hawke's Bay in 1999. The similarity between PFGE profiles of C. fetus subsp. fetus sheep abortion isolates from 1987 and 2000, and the relative prevalence of the PFGE groups, suggests that there has been no major genotypic shift in the population of C. fetus subsp. fetus implicated in sheep abortion in New Zealand during this time.     

Pulsed-field gel electrophoresis typing of Campylobacter fetus subsp. fetus isolated from sheep abortions in New Zealand

AIMS: To genotype Campylobacter fetus subsp. fetus isolates cultured from sheep abortions submitted to diagnostic laboratories in New Zealand during the year 2000 breeding season. To compare the types found nationally with those found in the Hawke' Bay region in 1999, and strains held in the New Zealand Reference Culture Collection, Medical Section (NZRM) from a study published in 1987.

METHODS: Campylobacter fetus subsp. fetus isolates cultured by veterinary diagnostic laboratories in the year 2000 breeding season, from sheep abortions from throughout New Zealand, were typed using pulsed-field gel electrophoresis (PFGE). In addition, seven freeze-dried C. fetus subsp. fetus isolates (strain numbers 2939–2945) from the NZRM, representing restriction types a–g found amongst sheep abortion isolates in a study published in 1987, were typed using PFGE.

RESULTS: In total, 293 C. fetus subsp. fetus isolates from 200 farms were obtained from veterinary diagnostic laboratories. Twenty-two distinct PFGE profiles were identified amongst the isolates. PFGE type B1 was predominant in each region of New Zealand and was identified from 66% of farms overall. Of the C. fetus subsp. fetus restriction types a–g lodged with the NZRM, 3/7 had PFGE profiles indistinguishable from profiles found in the current study. The other four restriction types had PFGE profiles that were unique but similar to those found in the current study.

CONCLUSIONS: PFGE type B1 was predominant amongst the C. fetus subsp. fetus isolates cultured from sheep abortions in each region of New Zealand in the year 2000, as was found in Hawke' Bay in 1999. The similarity between PFGE profiles of C. fetus subsp. fetus sheep abortion isolates from 1987 and 2000, and the relative prevalence of the PFGE groups, suggests that there has been no major genotypic shift in the population of C. fetus subsp. fetus implicated in sheep abortion in New Zealand during this time.     

Phenotypic and genotypic characterization of Mannheimia (Pasteurella) haemolytica-like strains isolated from diseased animals in Denmark.

Trehalose-negative strains of the Pasteurella haemolytica complex have recently been transferred to a new genus, Mannheimia. This genus presently consists of five named species: M. haemolytica, M. glucosida, M. granulomatis, M. ruminalis and M. varigena. The purpose of this study was to investigate the occurrence of these species and lesions associated with these isolates in Denmark. In all 106 M. haemolytica-like strains isolated from pathological material from cattle, sheep, pigs and hares submitted to the Danish Veterinary Laboratory between 1994 and 1998 were investigated. Phenotypic characterization and ribotyping were used for identification in addition to sequencing of the 16S rRNA genes for selected strains. The species allocation was determined by comparison to results from a previous polyphasic taxonomic study. Seventy-one percent of the strains belonged to M. haemolytica, 18% to M. varigena and 8% to unnamed groups within the genus Mannheimia. Single isolates identified as M. glucosida and P. trehalosi, respectively, were detected. Two isolates belonged to M. granulomatis. Forty-three percent of the strains belonged to serotype 1, 41% were untypeable, while the rest belonged to serotypes 2, 7, 9, and 16. The present investigation also showed that a simplified phenotypic characterization using Diatabs Diagnostic Tablets (Rosco, Denmark) represents a useful method for obtaining a quick and reliable species identification. Finally, the investigation confirmed that serotyping does not represent a reliable method for species identification. The heterogeneity of species associated with bovine "pasteurellosis" should be considered in future studies to improve our understanding of the pathogenesis of pneumonic disease.