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.     

Serological, bacteriological and pathological changes in rams following different routes of exposure to Brucella ovis

Mature Merino rams were exposed to Brucella ovis by contact with infected semen, using either ewe transmission, intrapreputial, intranasal or intrarectal inoculation of infected semen or intrapreputial inoculation of B. ovis culture. Thirty-six of the 41 rams developed significant complement fixation (CF) test titres, but only 9 of these reactors showed clinical, bacteriological or pathological evidence of infection. Infection occurred in some of the rams from all groups. The results are discussed in relation to the transmission of the disease and the significance of CF titres in rams exposed to B. ovis.     

Evaluation of surface components of Brucella ovis as antigens for the detection of precipitin antibody in serums from artificially exposed rams

Surface components of Brucella ovis obtained by gentle physical shearing were tested as a potentially useful source of reagent for selective serological diagnosis. These antigens were used in a radial immunodiffusion (RID) test against serum from rams which had been inoculated with infective semen containing B. ovis by one of 4 routes namely mating rams with ewes previously inoculated intravaginally with infective semen, or by direct inoculation in the prepuce, rectum or nasal passage. Loosely attached surface antigens in the RID test formed precipitin bands with serums collected from rams 2 and 10 weeks after inoculation. In contrast, a detergent extracted membrane antigen B developed precipitin bands only with serum collected 10 weeks after inoculation from rams confirmed bacteriologically to be infected with B. ovis in the genital tract. The route by which the rams were artificially exposed did not affect the outcome of the RID test using the membrane B antigen. However, all experimentally exposed rams had demonstrable CF titres when a heat extracted antigen was used.     

Early sequential findings in the genitalia of rams experimentally infected with Actinobacillus seminis

AIM: To investigate and assess the sequential pathological changes in the epididymis and testis of young rams injected intra-epididymally with Actinobacillus seminis. METHODS: Twenty yearling Suffolk and Suffolk-cross rams were randomly divided into two groups comprising 16 test and four control animals. Each test ram received 2.3 x 109 cfu/ml of A. seminis injected intra-epididymally. Every 24 h post-inoculation (p.i.), two test rams were randomly selected, euthanised, and necropsied, until the end of the experiment at 192 h p.i. One control animal was euthanised at 24 h, 48 h, 96 h and 144 h p.i., respectively. The reproductive tract of each ram was carefully examined, lesions photographed, and tissues cultured. Thin sections of tissue samples were fixed and examined by light microscopy; additionally, epididymal tissues were examined by scanning electron microscopy (ScEM). RESULTS: Gross lesions were observed in the cauda epididymis of all test rams, and ranged from swelling at 24 h p.i. through enlargement and granuloma formation from 72 h p.i., to gradual enlargement and increasing firmness by 192 h p.i. Gross testicular atrophy was observed in three rams. Histologically, spermatic granulomas were evident in the epididymis and the tunica vaginalis of 10 and four rams, respectively. Cauda epididymitis was present in all rams, and caput and corpus epididymitis in eight and four rams, respectively. Interstitial orchitis was observed in seven, testicular degeneration in 14, and localised and diffuse tunica vaginalitis in 12 rams. Epididymal vasculitis and infiltration of eosinophils were observed as early as 24 h p.i. Moderate disruption of the epididymal duct from 72 h p.i., with subsequent release of spermatozoa into the interstitium, was revealed by ScEM. Actinobacillus seminis was cultured from the granuloma of six test rams from 72 h p.i. CONCLUSIONS: Actinobacillus seminis has the ability to persist in the genitalia of young rams following experimental infection. Suppurative epididymitis is observed as early as 24 h p.i., and spermatic granuloma within 72 h p.i. Infiltration of eosinophils appears to be an early host response to the bacterium, and may play a role in the pathogenesis of the epididymitis.     

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.     

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.     

Urogenital infection and seminal excretion after inoculation of bulls and rams with chlamydiae.

Five mature rams and 4 bulls were inoculated parenterally with bovine or ovine chlamydial strains of type 1 and 2. One to 3 days later, all animals developed a chlamydemia lasting 4 to 8 days. Chlamydial agents were isolated from the semen near the end of the chlamydemic phase. All rams and 3 of 4 inoculated bulls excreted chlamydiae in the semen for 22 to 29 days. From 8 to 39 days after inoculation, selected rams or bulls were killed to test for chlamydial infection in the urogenital tract and other organs. Chlamydiae were isolated in developing chicken embryos from testis, epididymis, and accessory sex glands. Bulls examined 29 and 39 days after inoculation did not harbor chlamydiae. Chlamydiae were also not isolated from 3 control bulls which were from the same herd as the principal bulls. All inoculated bulls and rams had a group-specific chlamydial antibody response within 7 days. The titers reached maximal levels of 128 to 512 at 14 days after inoculation. Subsequently, the antibody titers decreased gradually. Seminal plasma collected at different times after animals were inoculated did not fix complement in the presence of chlamydial group antigen. The number of polymorphonuclear leukocytes in the semen increased during the experiment. The semen was grossly purulent in 2 rams inoculated with the type 2 chlamydial strain of polyarthritis.     

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.     

Immune response of poults following intranasal inoculation with Artvax vaccine and a formalin-inactivated Bordetella avium bacterin

Poults 3 weeks and older developed temporary tracheal resistance to intranasal challenge following inoculation of either Artvax vaccine or formalin-inactivated Bordetella avium bacterin by the intranasal and eyedrop routes. Resistance usually persisted for 3-4 weeks after B. avium challenge. However, with constant exposure to infected controls, the vaccinated birds eventually developed tracheal infection. Day-old poults did not respond to either the Artvax or the bacterin and were completely susceptible to challenge. Two-week-old poults responded to some degree, but poults 3 weeks old and older responded best. Poults inoculated with bacterin by aerosol or by drinking water did not respond as well as those inoculated by the intranasal and eyedrop routes. When poults were given a single subcutaneous injection at 3 weeks of age and challenged 2 weeks later, three of five resisted infection for 18 days.     

A study of the ability of a TK-negative and gI/gE-negative pseudorabies virus (PRV) mutant inoculated by different routes to protect pigs against PRV infection

The capacity of a TK-negative (TK-) and gI/gE-negative (gI/gE-) pseudorabies virus (PRV) mutant to protect pigs against Aujeszky's disease carried out by experimental infection with a virulent PRV strain, was tested. There were three groups, each of four susceptible pigs which were inoculated twice by two different schedules. Group 1 received the modified virus by the intradermal (first inoculation)-intramuscular (second inoculation) routes; group 2 was treated by the intranasal (first inoculation)-intramuscular (second inoculation) routes. The third group was left untreated as the control. All of the pigs were challenged intranasally with a virulent PRV strain and they were subsequently injected with dexamethasone. Two pigs in each group were necropsied on days 5 and 15 after dexamethasone inoculation. The challenge exposure resulted in mild clinical signs, increase in growth and a shorter period of virus shedding in vaccinated pigs, whereas the control group showed severe signs of Aujeszky's disease. No difference in the titre of the virulent virus which was excreted by pigs of all three groups, was observed and all animals seroconverted. Both the mutant strain and the wild-type virus established a latent infection although only the latter was reactivated and shed. Slight lesions were observed in target tissues of the vaccinated animals and no significant differences were detected between the two inoculation schedules.     

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.     

Pathophysiologic correlates of acute porcine pleuropneumonia

OBJECTIVE: To develop and evaluate an in vivo model to study early events in the pathogenesis of acute porcine pleuropneumonia. ANIMALS: Thirty-six 6- to 8-week-old pigs. PROCEDURE: Pigs were inoculated intranasally or endotracheally with Actinobacillus pleuropneumoniae; inoculation routes were compared by evaluation of clinical signs, gross and microscopic lung lesions, hematologic changes, serum zinc, iron, and haptoglobin concentrations, and inflammatory cytokines. RESULTS: The 2 inoculation routes resulted in similar findings, although intranasal inoculation caused unilateral gross lung lesions, whereas endotracheal inoculation caused bilateral gross lesions. Clinical signs of disease were observed < 2 hours after endotracheal inoculation and 6 to 8 hours after intranasal inoculation. Total WBC counts did not differ significantly after inoculation by either inoculation route, although band neutrophils increased significantly. The earliest findings associated with A pleuropneumoniae inoculation, irrespective of route, were decreased serum zinc and iron concentrations. Serum haptoglobin concentrations were significantly increased after inoculation. Inoculation induced rapid influx of macrophages into the lung and local induction of proinflammatory cytokines. Northern blot analysis of total RNA from lung tissue indicated that inoculated pigs had increased concentrations of interleukin (IL)-1beta, IL-1alpha, and IL-8; tumor necrosis factor messenger RNA concentration was not increased. CONCLUSIONS: Endotracheal inoculation with A pleuropneumoniae rapidly and consistently induced diffuse bilateral pneumonia; thus, this method may be useful for the study of acute pathophysiologic changes associated with bacterial pneumonia and may provide an experimental model for testing modalities for prevention and treatment of this and other respiratory tract diseases of pigs.     

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.     

A Study of the Ability of a TK‐Negative and gI/gE‐Negative Pseudorabies Virus (PRV) Mutant Inoculated by Different Routes to Protect Pigs Against PRV Infection

The capacity of a TK‐negative (TK ^‐) and gI/gE‐negative (gI/gE ^‐) pseudorabies virus (PRV) mutant to protect pigs against Aujeszky's disease carried out by experimental infection with a virulent PRV strain, was tested. There were three groups, each of four susceptible pigs which were inoculated twice by two different schedules. Group 1 received the modified virus by the intradermal (first inoculation)‐intramuscular (second inoculation) routes; group 2 was treated by the intranasal (first inoculation)‐intramuscular (second inoculation) routes. The third group was left untreated as the control. All of the pigs were challenged intranasally with a virulent PRV strain and they were subsequently injected with dexamethasone. Two pigs in each group were necropsied on days 5 and 15 after dexamethasone inoculation. The challenge exposure resulted in mild clinical signs, increase in growth and a shorter period of virus shedding in vaccinated pigs, whereas the control group showed severe signs of Aujeszky's disease. No difference in the titre of the virulent virus which was excreted by pigs of all three groups, was observed and all animals seroconverted. Both the mutant strain and the wild‐type virus established a latent infection although only the latter was reactivated and shed. Slight lesions were observed in target tissues of the vaccinated animals and no significant differences were detected between the two inoculation schedules.     

NON-SURGICAL STERILISATION OF RAMS USING A SCLEROSING AGENT

Injection of 0.25 ml of a solution of 3.6% formaldehyde in 90% ethanol direct into the caudae epididymides caused sterility in the rams treated. No live sperm were seen in ejaculates collected 35, 57, 91 and 196 days post-injection. The rams retained their libido and were mated to ewes between 2 and 3 months post-injection with no pregnancy ensuing. The technique offers a simple, safe, quick and effective method of sterilising rams. Attempts to produce sterilisation by injection of the sclerosing agent into the vas deferens were not successful.     

Mycobacterium bovis lipids: virulence and vaccines

The objective of this study was to determine the amount and infectivity of porcine circovirus type 2 (PCV2) shed in nasal, oral and fecal secretions following experimental infection. Fecal, oral and nasal swabs and blood were collected at regular intervals until 69 days post-inoculation (DPI) from five PCV2-experimentally inoculated pigs (Trial 1). To assess the infectivity of the PCV2 present in excretions, secretions, and on a hypodermic needle, 26 PCV2-na?ve pigs (Trial 2) were inoculated with various samples obtained from Trial 1 pigs. In Trial 1, PCV2 DNA was detected in all sample types by 69 DPI. There were no differences in the amount of PCV2 DNA present in different sample types over time. In Trial 2, intraperitoneal inoculation with contaminated fecal, nasal and oral samples; intranasal inoculation of nasal secretions; and feces fed to na?ve animals resulted in viremia and seroconversion. Viremia and microscopic lesions were noted in one animal injected using a contaminated needle. In conclusion, experimental PCV2 exposure results in a long term infection. PCV2 is shed in similar amounts by nasal, oral and fecal routes and is infectious to na?ve pigs confirming that multiple routes of transmission are likely important in spread of PCV2 between pigs.     

Studies of ERA/BHK-21 rabies vaccine in skunks and mice.

ERA rabies vaccine virus grown in BHK-21 13S cells (ERA/BHK-21) and street rabies virus were titrated in mice by intracerebral, intranasal and intramuscular inoculation. Mice were also given undiluted ERA/BHK-21 in baits. Skunks were given undiluted ERA/BHK-21 in baits and by intramuscular, intranasal and intestinal inoculation. Virus neutralizing antibody titers against rabies virus were measured over a three month observation period. The surviving skunks were challenged by intramuscular inoculation with rabies street virus from a skunk salivary gland suspension. When titrated in mice, ERA/BHK-21 had titers of 10(7.0), 10(5.2) and 10(3.9) median lethal doses per mL by the intracerebral, intranasal and intramuscular routes, respectively. All skunks (8/8) inoculated intranasally developed paralytic rabies by 12 days after exposure to ERA/BHK-21 virus. None of the skunks that developed vaccine-induced rabies had infectious virus in the submandibular salivary glands. Vaccine-induced rabies also occurred in 1/8 skunks in the intramuscularly inoculated group and in 1/8 in the intestinally inoculated group. The survival rates of challenged skunks in the various groups were as follows: intramuscular, 7/7; intestinal, 2/7; bait, 0/8; and control, 0/8. These results indicate that ERA/BHK-21 virus has a significant residual pathogenicity in mice and in skunks by some routes of inoculation. Skunks given vaccine intramuscularly were protected against challenge, while those skunks given the vaccine in baits were not.     

Immune response to vaccinia virus and recombinant virus products in dogs

A study was undertaken to determine the safety and suitability of vaccinia virus as an eukaryotic expression vector in dogs. Clinical signs were not seen in inoculated dogs, with the exception of small nodules at the site of inoculation. Vaccinia virus did not spread from dogs that were inoculated by SC (n = 5), intradermal (ID; n = 13), or intranasal (n = 3) routes to noninoculated dogs maintained in close contact. Replication of vaccinia virus appeared to be restricted in dogs because greater than or equal to 10(5) plaque-forming units of virus were required to induce an immune response by ID inoculation. Results were better with ID inoculation than with SC or intranasal inoculations. Repeated inoculations enhanced serum antibody titers, and annual reinoculation resulted in boosting of antibody titers. Maternal antibody interfered with virus replication and antibody production. Recombinant virus products induced antibody formation in dogs to human influenza-A virus, herpes simplex virus, and human hepatitis-B virus antigens. It was concluded that vaccinia virus would be safe and suitable as an eukaryotic expression vector in dogs.     

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.     

Intranasal infection of Getah virus in experimental horses.

Aerosol transmission in equine Getah virus (GV) infection was examined by intranasal inoculation with 10(3.0) to 10(7.0) TCID50 of the MI-110 strain in 7 experimental horses. The establishment of intranasal infection of GV was confirmed in all these horses by detecting serum neutralizing antibody against the MI-110 strain. Horses inoculated with more than 10(4.0) TCID50 of the virus manifested mild pyrexia, eruptions, serous nasal discharge, lymphopenia or monocytosis. Viremia ranging from 10(1.0) to 10(3.5) TCID50/0.2 ml occurred in horses inoculated with 10(5.0) TCID50, or more. Virus recovery from the nasal cavity was observed only in horses inoculated with 10(7.0) TCID50, and the viral titers recorded were 10(3.0) TICD50/ml or less. From these results, it is assumed that GV disseminated from the nasal cavity of naturally infected horses, except for intranasal infection with a lot of the virus, is probably very low in titer. So it seems to be rare that GV in natural cycles is spread from horse to horse by aerosol transmission.