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.     

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.     

Association of age of ram with distribution of epididymal lesions and etiologic agent

A 2-year study of the frequency of isolation of various organisms from mature and yearling rams with epididymitis was conducted at the US Sheep Experiment Station at Dubois, Idaho. Investigation into the distribution of lesions in the epididymis in relation to age of the ram also was studied. Serologic or bacteriologic evidence of Brucella ovis infection was demonstrated in 79.5% of the mature rams with epididymal lesions. Actinobacillus seminis and Histophilus ovis were the 2 most frequently isolated organisms from yearling rams with lesions. The tail(s) of the epididymis was the most frequent site of lesion development in the mature rams (86.4%). Yearling rams developed lesions twice as frequently in the tail(s) of the epididymis as in the head(s) of the epididymis. When lesions were localized in the head(s) of the epididymis, an etiologic agent usually was not demonstrated.     

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.     

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.     

Detection of antibody in rams with contagious epididymitis, using the enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay, using whole-cell and sonicated antigens prepared from Brucella ovis, Actinobacillus seminis, and Actinobacillus seminis-like cultures isolated from rams in Wyoming, was able to detect antibody to these antigens in rams with epididymitis. The whole-cell antigens used in this procedure gave lower background values, compared with those of the sonicated antigens. The procedure was able to detect antibody in rams before clinical signs of epididymitis became apparent.     

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.     

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.     

SPERMATIC GRANULOMA IN THE RAM: DIAGNOSIS BY ULTRASONOGRAPHY AND SEMEN CHARACTERISTICS

Ultrasonographic appearance of the testis and epididymis, and seminal characteristics, with regard to localization of spermatic granuloma were studied. In rams with spermatic granuloma (n = 9), diagnosed by clinical or ultrasonographic examinations before histopathological confirmation, localization of each lesion was recorded. Epididymal granulomas, caput and cauda, were seen as anechoic or hyperechoic areas with a distinct margin with or without a hyperechoic capsule. Granulomas in the testis were microscopic and, therefore, could not be detected by ultrasonography. Enlargement in the mediastinum testis was detected in all rams when there were granulomas in the caput. Heterogeneous testis parenchyma invaded by numerous hyperechoic foci, representing testicular degeneration, was associated with granulomas both in the epididymis and testis. Ultrasonographic appearance of the lesions did not differ with regard to season. Seminal characteristics varied between rams. It was concluded that ultrasonographic evaluation may give valuable information in the diagnosis of sperm granuloma in the ram.     

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.     

Ovine contagious epididymitis: A review

Ovine contagious epididymitis is predominantly associated with Brucella ovis, Actinobacillus seminis and a variety of organisms including Histophilus ovis. Transmission occurs venereally or by homosexual activity and, in the case of Actino bacillus seminis, ewe to lamb transmission is probably important. A chronic infection, mainly in the cauda epididymis may result in formation of spermatic granulomata and a reduction in ram fertility. Eradication of ovine brucellosis can be achieved by serological testing using a complement-fixation test in conjunction with palpation of lesions and removal of reactors. Vaccination to control ovine brucellosis is advocated in some countries.The nomenclature of the group of Gram-negative pleomorphic organisms to which Actinobacillus seminis and Histophilus ovis belong is urgently in need of review. This paper reviews the organisms capable of producing ovine contagious epididymitis, the pathogenesis of the condition as well as methods of diagnosis and control.     

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.     

Immunization with Brucella melitensis Rev 1 against Brucella ovis infection of rams

The efficacy of Brucella Melitensis Rev 1 vaccine (Rev 1) for the prophylaxis of Brucella ovis ram epididymitis was evaluated. Twenty-nine 3-month-old rams were vaccinated with 2 X 10(9) Rev 1 and 14 were revaccinated with 5 X 10(8) at 14 months of age. Six rams remained unvaccinated as a control group. All rams were challenged with 5 X 10(8) B. ovis at 21 months of age. Before being slaughtered 8 weeks later, only one vaccinated ram developed epididymitis while four of the six control rams developed testicular alterations. Genital and selected extragenital organs and lymph nodes were removed at slaughter and inoculated on selective media. B. ovis was isolated from 26.6% of the vaccinated rams, 21.4% of the revaccinated rams and 100% of control rams. Portions of epididymis, testes and vesicular glands were also used for pathological studies. More severe lesions were observed in control rams than in vaccinated ones. In conclusion, these results show that vaccination of young lambs, followed or not by revaccination, is a suitable method for the prophylaxis of B. ovis infection of rams.     

Association of sexual experience with isolation of various bacteria in cases of ovine epididymitis

Testicles from rams in flocks experiencing ram epididymitis in Idaho and eastern Oregon were cultured. Twenty-six breeding rams from 6 flocks were cultured and only Brucella ovis was isolated. Virgin rams (65) harbored numerous species of small fastidious gram-negative rods, including Actinobacillus actinomycetemcomitans, Haemophilus spp, Moraxella spp, and Pasteurella spp. Thus, there appeared to be 2 separate disease entities, dependent on sexual experience of the animal.     

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)     

Serodiagnosis of Histophilus ovis-associated epididymitis in rams

An ELISA for the detection of antibodies to Histophilus ovis was used to evaluate the association of epididymal lesions in rams with serologic response to His ovis. Comparison of ELISA results for His ovis in groups of rams with epididymal lesions with ELISA results of clinically normal rams (control group) revealed a significant difference (P less than 0.01) between the control group and those rams from which His ovis was isolated. A significant difference (P less than 0.01) was noticed between the control group and rams with lesions from which an organism other than His ovis or Brucella ovis was isolated. Additionally, a significant difference (P less than 0.01) was noticed in ELISA results between the control group and affected rams from which no organism was recovered and in which the epididymal lesion was not limited to the head of the epididymis. A difference was not detected in the His ovis ELISA results between control rams and rams with lesions associated with a B ovis infection or rams from which no organism was recovered and in which the epididymal lesion was limited to the head of the epididymis. The serologic findings in our study suggest that His ovis is more important in the development of epididymitis in rams than culture results alone would indicate.     

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.     

Comparison and partial characterization of the protein profiles and outer membrane antigens of Actinobacillus species isolated from ram lambs with epididymitis

Three monoclonal antibodies (LG17, LG30, LG33) were used in the indirect fluorescent antibody test, the ELISA, and the immunoelectrotransfer blot technique to identify group-specific and strain-specific epitopes on the outer membranes of Actinobacillus seminis, A actinomycetemcomitans, and 17 field isolates of Actinobacillus spp. The field isolates had been obtained by bacteriologic culture of specimens from ram lambs with epididymitis. Only antibody LG33 consistently had specificity for an outer membrane epitope shared by most of the bacterial isolates tested. Staining of polyacrylamide gels with periodic acid-Schiff reagent, Sudan black B, and Coomassie brilliant blue R250 indicated that target antigens for antibodies LG17 and LG33 contained carbohydrate and lipoprotein components, respectively. The chemical composition of the LG30 target antigen was not determined because of its instability after exposure to sodium dodecyl sulfate. Discontinuous-gradient polyacrylamide gel electrophoresis in sodium dodecyl sulfate and spectrophotometric scans of the gels were used to analyze n-octyl-beta-D-glucopyranoside protein extracts from A seminis, A actinomycetemcomitans, and 13 representative field isolates of Actinobacillus spp. Bacterial isolates could be grouped according to their protein profiles. The first group consisted of A seminis, A actinomycetemcomitans, and 7 field isolates of Actinobacillus spp, all of which shared common protein bands with molecular masses of approximately 94 kilodaltons (kD), 64 kD, 60 kD, 52 kD, 44 kD, and 26 kD. The second group was composed of 6 field isolates, each with unique protein profiles; isolates had relatively few protein bands in common. These data suggested that members of the genus Actinobacillus cultured from ram lambs with epididymitis probably include a number of various strains.     

Pathogenicity of different strains of Histophilus somni in the experimental induction of ovine epididymitis

The purpose of this study was to determine any differences in pathogenicity when sheep are experimentally infected with different Histophilus somni isolates: a) 2336 bovine origin strain; b) an isolate from ram orchitis and epididymitis; c) an isolate from the brain of a sheep with neurological signs; d) an isolate from the vagina of a clinically healthy ewe. A total of 20 rams divided in groups of 5 animals each were inoculated in the epididymis with 1 × 10⁷ CFU/mL of H. somni; a negative control group of 5 rams was used. All groups inoculated with H. somni showed some epididymitis, but the most pathology was caused by the epididymitis isolate, followed by the vaginal isolate. It was demonstrated that there is a difference in experimental infection capacity among isolates from different origins, as epididymitis occurred and the bacteria was recovered only from groups inoculated with isolates originating from epididymitis and vaginal exudate.     

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.