Bacteriological characteristics of Histophilus ovis and its relationship to similar bacteria

Seventeen strains of Histophilus ovis were obtained from a variety of pathological conditions and geographical areas and were examined to determine the bacteriological characteristics of the organism. All were Gram-negative pleomorphic rods which only grew under micro-aerophilic conditions in media enriched with blood, serum or meat particles. All strains were catalase negative, indole positive and nitrate positive while most produced acid from glucose, fructose, galactose, mannose, xylose, mannitol and sorbitol. Eight strains were examined serologically and compared with two strains of Actinobacillus seminis. In agglutination and precipitation tests, all strains of H ovis appeared antigenically identical and showed antigenic relationships to A seminis. Results of complement fixation tests confirmed the close, if not identical, antigenic relationship of H ovis and A seminis. It is suggested that H ovis and A seminis are biochemical variants of the same organism.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Actinobacillus seminis infection in sheep in the Republic of South Africa. II. Incidence and geographical distribution

To obtain information on the incidence and distribution of Actinobacillus seminis infection in the Republic of South Africa, a clinical and serological survey was carried out on 409 farms situated in 29 districts. All rams submitted for certification to the Regional Laboratory from 1/1/69 to 31/1/74 were included in a separate investigation. These particular rams represented different breeds and originated from farms in over 48 districts. Examinations were also carried out on all rams on 11 stud farms in the Middelburg and adjacent districts with a high incidence of epididymitis, despite regular immunization with Elberg Rev. 1 vaccine. These investigations confirmed that genital infection of rams still presents a major problem in the main sheep breeds and the main sheep farming areas of South Africa. A high incidence of infection with A. seminis, an organism which appear to be the most important one associated with genital infection in this country, was also established. Genital infection due to A. seminis is geographically also very widespread.     

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.     

Pathology of acute experimental Actinobacillus seminis mastitis in ewes

Sequential pathological changes were studied for 9 days after inoculating Actinobacillus seminis in the mammary gland of sheep. The inoculated mammary glands became enlarged (2 to 4 times normal), turgid or consolidated and contained creamy or greenish-yellow viscid contents. A seminis was isolated from all inoculated udders at necropsy. Microscopically, the reaction in the udder to A. seminis may be divided into 4 overlapping phases; acute purulent, subacute purulent, necrotising, and proliferative. It was concluded that A. seminis was pathogenic for the ovine mammary gland, the clinical and pathological findings were nonspecific, and that A. seminis could survive within ovine mammary tissue for at least 9 days after inoculation.     

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.     

Genetic homogeneity of Actinobacillus seminis isolates

Isolates of Actinobacillus seminis from clinical cases and reference sources had markedly similar Bam H1 restriction endonuclease profiles but were readily distinguishable from the Bam H1 profiles of the Histophilus-Haemophilus group as well as from A lignieresii. For epidemiological purposes this lack of interstrain variation in Bam H1 profiles makes restriction endonuclease analysis of isolates of A seminis unsuitable.     

Detection of antibodies to Brucella ovis in sheep milk using B. ovis and B. canis antigen

The diagnostic techniques most widely used for detecting brucellosis caused by Brucella ovis are serological tests such as complement fixation (CFT), agar gel immunodiffusion (AGID), and ELISAs. However, to our knowledge, milk tests, with the advantage that samples may be taken in a non invasive manner, have not been investigated as diagnostic tools. We studied 144 samples of milk and sera from lactating ewes, comparing bacteriological studies, serological and milk tests using Brucella canis and B. ovis antigens. A group of 75 ewes in an uninfected flock were serologically negative to rapid slide agglutination test (RSAT), indirect ELISA (IELISA)-B. canis, AGID and IELISA-B. ovis. The milk of these ewes had an IELISA-B. canis mean (%P) value of 16.18 (S.D. 5.63), while the IELISA-B. ovis had a mean (%P) value of 12.52 (S.D. 4.94). A cut-off value of (%P) 27.44 (+2 S.D.) or (%P) 33 (+3 S.D.) was determined by milk-ELISA-B. canis and (%P) 22.4 (+2 S.D.) and (%P) 27.34 (+3 S.D.) by milk-IELISA-B. ovis. These cut-off values were adjusted by receiver-operator characteristics (ROC) analysis using 23 positive samples from infected ewes, which indicated a milk-IELISA-B. canis cut-off value of (%P) 33 and milk-IELISA-B. ovis of (%P) 26 with 100% sensitivity and specificity. Based on our results, we propose that, following a study of a larger number of samples, the milk-IELISA-B. canis could be considered a suitable test for the diagnosis of B. ovis brucellosis in lactating ewes.     

Experimental transmission of Actinobacillus seminis infection to rams

Nine groups of four 18- to 24-month-old rams were inoculated with Actinobacillus seminis by the following routes: intraconjunctival, intranasal, oral, intravenous, intramuscular, intraepididymal, vas deferens, intraurethral or intrapreputial. Eight similar rams were left uninoculated as controls. Systemic clinical signs were minimal and were confined primarily to the inoculation sites and the scrotal contents. Mild to severe epididymitis resulted from all the routes of inoculation except intraconjunctival and intranasal. Direct inoculation into the genital tract, especially into the cauda epididymis, was more effective. Intrapreputial and intraurethral inoculation led to ascending urethral infection, and inoculation into the vas deferens resulted primarily in descending infection of the accessory sex glands. A seminis was isolated from 11 of the 36 test rams (30.6 per cent); 26 of the 36 rams, some from each of the test groups except those inoculated intravenously, reacted serologically.     

Restriction endonuclease (EcoR1) analysis of Brucella ovis DNA

Seven Brucella ovis isolates from diverse sources (including two used in the manufacture of a Br. ovis vaccine, four field strains and a reference strain from the National Culture Type Collection in London) were subjected to bacterial restriction endonuclease DNA analysis using the endonuclease EcoR1. No genetic differences were detected. It was concluded that strain variation is unlikely to be a problem in Br. ovis control schemes, whether these are based on vaccination or on the eradication of the disease using serological tests.     

Epididymal and testicular lesions in rams following experimental infection with Actinobacillus seminis

AIM: To investigate and assess the epididymal and testicular lesions in rams up to 44 days after inoculation with Actinobacillus seminis via various routes. METHODS: Forty-four young (18-24 months old) rams were randomly divided into nine test and two control groups (n=4 per group). The test rams were infected by installation, drenching or injection of A. seminis organisms cultured for 24 h in a brain-heart infusion (BHI) broth containing 2.3 x 10(9) cells/ml, via the following nine routes: intra-epididymal (1 ml), intravenous (3 ml), intra-urethral (3 ml), intra-preputial (3 ml), vas deferens (1 ml), intramuscular (3 ml), oral (10 ml), intranasal (3 ml), and intra-conjunctival (3 drops). All test rams were necropsied 9-44 days post-inoculation (p.i.). Control rams were subdivided into in-contact and non-contact groups and necropsied at 45 and 46 days p.i., respectively. Thin tissue sections were examined for histopathology. RESULTS: Gross lesions were evident only in rams inoculated intra-epididymally. Epididymides on the inoculated side were two to three times larger than those on the un-inoculated side, and the testes attached to the inoculated epididymides were also enlarged. Fibrinopurulent periorchitis and tunica vaginalitis were seen in three rams and atrophy in one. Microscopically, epididymitis was present in 17 (47%) rams, the highest incidence being in the cauda, followed by the caput and the corpus epididymis. Seminiferous tubular degeneration with areas of lymphocytic infiltration were seen in four rams: three inoculated via the cauda epididymis and one via the urethra. No epididymal and/or testicular lesions were seen in rams inoculated via the nasal and conjunctival routes. CONCLUSIONS: Injection of A. seminis in young rams by all routes except intra-conjunctival and intranasal resulted in epididymitis, predominantly in the cauda epididymis. Development of lesions in the reproductive tract following non-genital routes of inoculation supports earlier suggestions that non-venereal transmission of genital actinobacillosis occurs. This study confirmed the predilection of A. seminis for the epididymis, especially the cauda.     

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.