SEROLOGICAL COMPARISON BETWEEN HISTOPHILUS OVIS, ACTINOBACILLUS SEMINIS AND BRUCELLA OVIS

SUMMARY: The serological relationships between 4 strains of Histophilus ovis , the neotype strain of Actinobacillus seminis and Brucella ovis were examined using a cross-absorption complement-fixation technique.
It was found that the 4 strains of H. ovis were serologically homologous and that an incomplete relationship existed between these organisms and A. seminis. Anteriserums prepared against one strain of H. ovis and the strain of A. seminis gave a weak, apparently nonspecific cross-reaction with Br. ovis antigen.
The practical significance of these results is discussed.     

Trials with an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of subclinical genital infections in rams caused by Histophilus ovis and Actinobacillus seminis

The performance of an enzyme-linked immunosorbent assay (ELISA) was evaluated in the serological diagnosis of subclinical genital infection in 6 naturally infected ram flocks and 2 experimentally infected ram hoggets. The test employs lipopolysaccharide (LPS) antigen prepared by autoclaving from Actinobacillus seminis and Histophilus ovis. A total of 193 sheep (118 unmated virgin rams and 75 mature breeding rams) were examined clinically, serologically (by ELISA) and bacteriologically (semen bacteriology) at the same time. Serum samples from all animals were also tested by an ELISA employing LPS antigen prepared from Brucella ovis in the same way. Shedding of A. seminis and H. ovis did not show close correlation with serological positivity (Table 1), as only 9 (15.0%) out of the 60 A. seminis shedders were ELISA seropositive at the same time. As regards H. ovis only 10 (19.2%) out of the 52 H. ovis shedders were ELISA seropositive at the same time. The results indicate that, when used alone, the ELISA employing LPS antigen is unsuitable for diagnosing subclinical genital infection caused by H. ovis and A. seminis in rams. The authors discussed shortly the employing fields of this ELISA test in the diagnostic work.     

Rapid identification of Haemophilus somnus, Histophilus ovis and Actinobacillus seminis using the API ZYM system

The API ZYM system, a commercially-available technique that measures bacterial enzyme activity was used to test 43 isolates identified as H. somnus, H. ovis or A. seminis and 19 from related genera. The enzyme patterns resulting from the API ZYM differentiated H. somnus and H. ovis from A. seminis and related genera but not from each other. An identification scheme based on 9 of the enzymes in the API ZYM and a few simple biochemical tests is proposed for the rapid and reliable identification of these bacteria in a diagnostic bacteriology laboratory.     

An evaluation of the API ZYM system as a means of identifying Haemophilus somnus and related taxa.

The commercially available API ZYM microbiological identification system was evaluated for the rapid identification of Haemophilus somnus. Eighty-seven isolates of the organism had API ZYM profiles which were characteristic. The API ZYM profiles demonstrate clear differences between H. somnus and other genera but suggest a close association to three related organisms. Enzyme activity of H. somnus isolates were similar to organisms identified as Histophilus ovis, Haemophilus agni and strains UQV of Actinobacillus actinoides and Actinobacillus seminis but was clearly different from isolates of Pasteurella haemolytica, Pasteurella multocida, Bordetella bronchiseptica and group EF4. The API ZYM system allowed more rapid identification of H. somnus than conventional biochemical tests and may be a useful adjunct to conventional methods used for identification of H. somnus isolates. The test did not reveal obvious differences between isolates from various anatomic locations.     

Multiplex PCR for the detection of Brucella ovis, Actinobacillus seminis and Histophilus somni in ram semen

OBJECTIVE: To develop a multiplex polymerase chain reaction (PCR) assay for the rapid detection of Brucella ovis, Actinobacillus seminis, Histophilus somni in fresh ram semen samples. DESIGN: The multiplex assay was based on the single PCR assays published for the detection of A seminis and B ovis, and the forward primer published for the detection of H somni; an alternative reverse primer for H somni was designed in this study. PROCEDURE: Culture and PCR of 295 fresh semen samples were carried out. RESULTS: The multiplex PCR was far more successful in the detection of H somni (45/295) than culture (23/295). A seminis was also detected in more semen samples by multiplex PCR (29/295) than culture (13/295) and B ovis was detected in three samples using both PCR and culture. No amplifications were detected with DNA from a range of bacterial isolates including species associated with epididymitis in rams. CONCLUSION: This PCR could be used as a complementary test, or alternative to culture of ram semen and other biological samples for the detection B ovis, H somni and A seminis.     

Prevalence of Histophilus ovis and Actinobacillus seminis in the genital tract of sheep

Histophilus ovis and Actinobacillus seminis were isolated from the preputial cavity of 6-month-old rams and the vagina of 6-month-old ewes at a substantially higher rate than that in mature (greater than 2 years old) rams and ewes. These organisms appeared to be a transitory component of the ovine genital flora, the prevalence of which was associated with age regardless of gender. Additional evaluation of the recoverability of H ovis and A seminis from the preputial cavity of rams from birth to 1 year of age indicated that the isolation rate from rams and predominance of the organisms in the preputial cavity differed greatly over this age period. These organisms were not recoverable until ram lambs were 12 weeks of age and were most prevalent at 20 weeks of age, after which recoverability of H ovis and A seminis from the preputial cavity steadily decreased, continuing through the time of the last evaluation at 1 year of age. The time period with which these organisms can be isolated from the preputial cavity is closely correlated with the time period when epididymitis associated with these organisms develops, and may be an important factor in the pathogenesis of epididymitis.     

A study for the differentiation of Actinobacillus seminis, A. actinomycetem-comitans, Histophilus ovis and Pasteurella haemolytica

By using well-defined techniques under optimum conditions it is possible adequately to define the biochemical characteristics of typical A. seminis strains. A. seminis can be distinguished from Histophilus ovis on the latter's distinctive colony morphology, but it cannot be distinguished from Actinobacillus actinomycetem-comitans. These organisms, however, can be differentiated from Pasteurella haemolytica on serological grounds and the latter's greater pathogenicity for mice. It is appreciated, however, that intermediate forms occur which cannot as yet be satisfactorily allocated to any of the above-mentioned genera.     

Characterisation of Histophilus ovis and related organisms by restriction endonuclease analysis

The banding profiles generated by Bam H1 restriction endonuclease cleavage of bacterial DNA from clinical and reference isolates of Histophilus ovis, Haemophilus somnus and related bacteria were compared. H. ovis, H. somnus and Haemophilus agni isolates were found to have distinct similarities in banding profiles characterised by 10 common bands between 2.0 and 9.6 kilobases (kb). The close taxonomic relationship of these isolates was reinforced by these findings. The reference isolates examined in this study--Actinobacillus lignieresii, Actinobacillus seminis, H. agni, H. somnus, H. ovis, Haemophilus influenzae, Haemophilus parainfluenzae, Haemophilus parahaemolyticus--could be distinguished from each other on the basis of their characteristic banding profiles. Actinobacillus sp were observed to have more bands between 2 and 23 kb compared with the H. ovis and Haemophilus sp isolates studied. Analysis of isolates from an experimental infection trial illustrated the potential of restriction endonuclease analysis in molecular epidemiological applications. It was possible to demonstrate by this means that the post-challenge isolates had identical banding profiles to the challenge (or infecting) isolate which had a distinctly different banding profile from that of pre-challenge H. ovis isolates. Furthermore, restriction endonuclease analysis of H. ovis isolates obtained from follow-up investigations of a recurrent problem of epididymitis in unmated rams, indicated that the H. ovis isolates implicated in epididymitis, were present as a single strain in a number of sheep over a period of time. This suggested that the mechanism of transmission was by perinatal perputial contamination.     

The usefulness of the API 20 E classification system in the identification of Actinobacillus actinomycetem comitans, Actinobacillus seminis and Pasteurella haemolytica

The prepuces of lambs aged 6--8 months and semen of 2 adult rams were found to be infected with gram negative, non-motile, non-haemolytic, pleomorphic bacilli. These organisms were compared with those of known strains of actinobacillus actinomycetem comitans. Actinobacillus seminis and Pasteurella haemolytica, using the API 20 E classification system. Applying the principles of numerical taxonomy, the majority of suspected strains of A. seminis could be classified as A. actinomycetem comitans and 3 examples as Histophilus ovis. Although some of the suspected strains of A. seminis could be classified as P. haemolytica, obvious differences between the genera Actinobacillus and Pasteurella were evident.     

Identification of an immunogenic protein of Actinobacillus seminis that is present in microvesicles

Actinobacillus seminis is a gram-negative bacterium of the Pasteurellaceae family that is involved in ovine epididymitis. Looking for a protein specific to this species, we determined the protein profile of subcellular fractions of A. seminis (American Type Culture Collection number 15768): proteins from the outer membrane (OMPs), inner membrane (IMPs), and cytoplasm (CPs). These profiles provide the first data, to our knowledge, regarding subcellular fractions of A. seminis. In the OMP fraction, we identified a protein with a molecular mass of 75 kDa that proved to be immunogenic and apparently specific for A. seminis. This conclusion was based on the reaction of hyperimmune serum of rabbits inoculated with whole cells of A. seminis that was tested against sonicated complete cells of reference strains and field isolates of Brucella ovis, Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni. No protein of these bacteria cross-reacted with the 75-kDa protein of A. seminis. Furthermore, when each type of hyperimmune serum was tested against the sonicated cells and each of the subcellular fractions of A. seminis, it did not recognize the A. seminis 75-kDa protein. We also isolated and identified this protein in microvesicles released to the culture supernatant. The results suggest that the 75-kDa protein could be used to establish a diagnostic test specific for ovine epididymitis caused by A. seminis.     

Isolation of Actinobacillus seminis from rams in the United Kingdom.

Actinobacillus seminis was isolated from the semen of five rams on four farms. Four of the rams had abnormal semen and three were also infertile. The isolates of A seminis showed similar phenotypic profiles and electrophoretic protein patterns to the type strain of A seminis but were distinct from Histophilus ovis previously isolated from rams with epididymitis in Scotland. The infection appeared to be subclinical but two of the five rams had palpable abnormalities of their testes. Three rams were treated with antibiotics but the infection persisted. No gross lesions were found in the genitalia of two of three rams examined post mortem but one had necrotic abscesses in the testes and epididymis. A seminis was isolated from the seminal vesicles and epididymis of one ram without gross lesions but not from the genitalia of the other two. On one farm the infection in a recently purchased ram led to the detection of another case as a result of the bacteriological screening of 11 stock rams not in contact with the initial case. These five subclinical cases, which included a supposedly healthy stock ram, suggest that A seminis infection may be widespread and should be considered in cases of infertility.     

Comparison of three serological tests for Brucella ovis infection of rams using different antigenic extracts

The sensitivity and specificity of the complement fixation, gel diffusion and ELISA tests for the diagnosis of Brucella ovis infection of rams have been compared using three different antigenic preparations. The antigens obtained by petroleum ether - chloroform - phenol, or cold saline extractions gave poorer diagnostic results than those obtained by hot saline extraction in all the tests. The best sensitivity was obtained with the ELISA (97.6 per cent) followed by the gel diffusion (96.4 per cent) and complement fixation tests (92.7 per cent). The gel diffusion test detected as positive the two rams negative in the ELISA, while the complement fixation test did not improve the sensitivity of the other tests. Under these conditions all the tests were 100 per cent specific when testing sera from rams free of B ovis.     

Serology of Haemophilus (Actinobacillus) pleuropneumoniae serotype 5 strains: establishment of subtypes a and b

The serological characteristicis of 14 strains assigned to serotype 5 were examined by the slide agglutination test, the indirect haemagglutination (IHA) test and by gel diffusion.All the strains possessed identical capsular antigenic determinants of polysaccharide (PS) nature and lipopolysaccharide (LPS) nature. However, based upon a capsular antigenic determinant of PS nature the strains could be divided in two subtypes. It is therefore proposed to refer to the H. pleuropneumoniae strains of serotype 5 to two subtypes: subtype a with strain K17 as the subtype strain and subtype b with strain L20 as the subtype strain.     

Actinobacillus seminis infection in sheep in the Republic of South Africa. I. Identification of the problem

A clinical palpation and semen smear examination of 647 rams submitted to the Regional Veterinary Laboratory during 1967 revealed that 42 (6,5%) of these animals had clinical epididymitis or orchitis, 6 (0,9%) showed other types of genital lesions and 98 (15,1%) suffered from subclinical genital infection. A. seminis and A. seminis-like organisms were isolated from semen specimens of 18 out of 35 rams with clinical epididymitis or orchitis, 25 out of 33 rams with subclinical infection and none out of 13 rams which showed no neutrophils in their semen. On 4 stud farms where Elberg Rev. 1 vaccine was meticulously applied and the complete absence of Brucella ovis infection was established, of a total of 327 rams examined, 10 (3,6%) were found to be clinically and 72 (22,0%) subclinically affected. A. seminis was isolated from 5 out of 6 of these rams with clinical lesions and 10 out of 15 of those which showed evidence of subclinical infection.     

Use of Brucella canis antigen for detection of ovine serum antibodies against Brucella ovis

Brucella ovis causes a genital disease of sheep manifested by epididymitis in rams and placentitis in ewes producing reduced fertility in the flock. Clinical diagnosis is not sensitive enough and bacteriological testing is not feasible for detection of the disease in large numbers of animals. Indirect methods of serological testing are preferred for routine diagnosis, of which agar gel immunodiffusion (AGID), complement fixation (CF) and ELISA tests are recommended as the most efficient. Since B. ovis shares antigenic components with Brucella canis, it would seem that either strain could be used as antigen with the same results; however, the advantage of the B. canis (M-) strain variant is that it can be used to develop a satisfactory antigen for agglutination tests. We present data on AGID and IELISA tests using B. ovis antigen and rapid screening agglutination test (RSAT), 2-mercapto-ethanol RSAT (2ME-RSAT) and IELISA using B. canis antigen. We tested 225 animals. The cut-off values were adjusted by ROC analysis using 51 negative and 32 positive sera; the IELISA-B. canis cut-off value was 39 (%P) and IELISA-B. ovis, 51 (%P), with 100% sensitivity and specificity. Of the 32 positive sera from the infected flock RSAT detected 32 (100%), 2ME-RSAT 29 (91%) and AGID 31 (97%). Of the 142 sera from suspicious flocks, 46 were negative and 56 positive in all the tests; 16 were positive by RSAT, IELISA-B. canis and IELISA-B. ovis, 20 positive only with RSAT and 2 positive only by both IELISAs. RSAT is a very sensitive screening test that, because of its simplicity and easy interpretation, following a study in larger sample, could replace AGID as a screening test for diagnosis of ovine brucellosis caused by B. ovis. The IELISA-B. canis or IELISA-B. ovis could be used as confirmatory tests, since they show equal specificity and sensitivity.     

Serology and semen culture for the diagnosis of Brucella ovis infection in chronically infected rams

The serological response to Brucella ovis and the shedding of the organism in semen was followed for a period of 13-14 months in 42 naturally infected rams. Most rams remained chronically infected and excreted the organism in their semen throughout the investigation. B. ovis was isolated from 87.9% of the semen samples from the infected rams. The most common sites from which B. ovis could be isolated at necropsy were the epididymides and accessory sexual glands. In one ram the organism was isolated from lung, spleen, kidney and iliac lymph nodes. Three rams ceased to shed B. ovis in their semen during the course of the investigation. Seventy-five (11%) of 686 sera from infected rams were negative in the complement fixation test (CFT) although 76% and 77% of CFT-negative sera were positive in the gel diffusion precipitin test (GDT) and enzyme labelled immunosorbent assay (ELISA) respectively. The high incidence of CFT-negative infected rams was due to the selection for the investigation of many rams with histories of negative or vacillating CFT titres. Sera from five rams which never shed B. ovis in their semen reacted erratically in the three serological tests. The five rams were from heavily infected flocks and were kept in contact with infected rams throughout the investigation.     

Cultural and biochemical characterization of Actinobacillus and Actinobacillus-like species from ram lambs with epididymitis

Cellular, colonial, cultural, and biochemical characteristics of 25 field strains of gram-negative pleomorphic bacilli from rams with epididymitis were compared with Actinobacillus actinomycetemcomitans American Type Culture Collection (ATCC) strain 29522 and Actinobacillus seminis ATCC strain 15768. Three field strains were identified as A. actinomycetemcomitans, 15 as A. seminis, and 2 as Haemophilus agni; however, 5 strains (3 in group A and 2 in group B) were not identified as species in the genera Actinobacillus, Haemophilus, or Pasteurella based on the taxonomic criteria in Bergey's manual of systematic bacteriology. The 5 Actinobacillus-like organisms in groups A and B were predominantly gram-negative coccobacilli and exhibited less pleomorphism than the 2 Actinobacillus species. The colonial morphologies of groups A and B were similar to the 2 Actinobacillus species but were smaller in diameter and had a pale yellow color. Groups A and B, like the actinobacilli, were facultative anaerobic and capnophilic, did not grow on MacConkey agar, and were catalase-positive and oxidase-positive. Group A reduced nitrate but group B did not. The A. seminis strains utilized ornithine, and group A utilized arginine; but group B did not utilize either ornithine or arginine. All strains failed to utilize lysine or tryptophane. All strains produced acid but no gas from glucose, and the utilization of other carbohydrates varied markedly both between and within the 5 groups of bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)     

The complement fixation test for the diagnosis of Brucella ovis infection in rams--an Animal Health Division Report

Complement fixation tests using three B. ovis antigen preparations in warm fixation tests (WCFT) and cold fixation (CCFT) tests were done on 541 ram sera. Semen samples from the same rams were examined culturally to identify B. ovis excretors. The CCFT, using an antigen prepared by heat extraction of B. ovis cells, had a sensitivity of 97% in 124 rams which were shedding B.ovis. The specificity was 99% in 144 rams from non-infected flocks. Seventy-seven per cent of 156 rams which reacted to this test were shedding B. ovis in their semen. Tests with other antigens were inferior in sensitivity and/or specificity. The WCFT gave lower titres than CCFT. Vaccination caused large numbers of false positive reactions in 4 flocks.