Microbiological, molecular, and histopathological findings in goats experimentally infected with Actinobacillus seminis.

The objective of this study was to experimentally evaluate the pathogenicity of an Actinobacillus seminis isolate named SAAS01 in goats. Animals were challenged with 2 mL of a suspension containing 1,5 × 108 CFU/mL of A. seminis (SAAS01 isolate) through the intrapreputial, epididymis tail, and conjunctival routes. Epididymis and testicular fragments were submitted to histopathological exam, and semen samples underwent microbiological and molecular diagnoses. Clinically, a unilateral increase in firm consistency was observed in the epididymis and testicles of two animals inoculated in epididymis tail and in one animal inoculated through conjunctival sac; this firmness continued until the day of euthanasia. Two goats inoculated through epididymis tail and conjunctival sac routes presented histopathological findings with macroscopically and microscopically significant changes. A. seminis was isolated from semen samples collected from goats inoculated through the epididymis tail and conjunctival sac routes. A. seminis DNA was amplified from six semen samples of three goats inoculated through the epididymis tail, two in conjunctival sac and one through intrapreputial route. The experimental infection model using goats confirmed the pathogenicity of the A. seminis isolate, demonstrating the predilection of the agent for the epididymis, with clinical signs, histopathological lesions, bacterial isolation, and a positive molecular diagnosis.

Achados microbiológicos, moleculares e histopatológicos em pequenos ruminantes experimentalmente infectados com Actinobacillus seminis / Microbiological, molecular and histopathological findings in small ruminants experimentally infected with Actinobacillus seminis

O objetivo deste trabalho foi avaliar a patogenicidade, em ovinos, de uma cepa de Actinobacillus seminis isolada de caprino no Brasil. Foram utilizadas amostras de sêmen, punção e fragmentos de epidídimo, ducto deferente, testículos e glândulas seminíferas de dois caprinos (animais 1 e 2) e dois ovinos (animais 3 e 4), e foram realizados exame histopatológico, cultivo microbiológico e diagnóstico molecular. O inóculo foi preparado com solução salina na diluição de 10-2 correspondendo ao padrão 1,0 da escala de McFarland, com colônias previamente cultivadas de A. seminis e administrado no volume de 2 mL pelas vias intra-prepucial (animais 1 e 3) e na cauda do epidídimo (animais 2 e 4). Na avaliação clínica observou-se aumento unilateral de consistência firme após 30 dias no epidídimo e testículo do animal 4 que continuou até o dia da eutanásia, bem como o animal 1 apresentou discreto aumento unilateral dos testículos. As lesões macroscópicas e microscópicas observadas nos animais 3 e 4 foram compatíveis com aquelas causadas pela infecção por A. seminis. A. seminis foi isolado de material de punção e sêmen de um ovino (animal 4). Conclui-se que o modelo de infecção experimental utilizando caprinos e ovinos comprovou a patogenicidade da amostra de A. seminis, isolada de um caprino no semiárido brasileiro e reproduzida em um ovino, comprovando a predileção do agente pelo epidídimo, com quadro clinico, achados histopatológicos, isolamento bacteriano e diagnóstico molecular positivo.(AU)

The aim of this study was to evaluate, in sheep, the pathogenicity of an Actinobacillus seminis strain isolated from a goat in Brazil. Samples of semen, puncture and fragments of epididymis, deferent duct, testicles and seminal vesicles from two goats (animals 1 and 2) and two sheep (animals 3 and 4) were used, and histopathological, microbiological culture and molecular diagnoses were performed. The inoculum was prepared with saline solution at 10-2 dilution corresponding to 1.0 McFarland standard, with A. seminis colonies previously cultured and administered on 2mL volume by intra-preputial (animals 1 and 3) and epididymis tail (animals 2 and 4) routes. At clinical evaluation it were found unilateral swelling of firm consistency after 30 days in epididymis and testicle from animal 4 that continued until the day of euthanasia, as well as animal 1 shown discrete unilateral swelling of testicles. Gross and microscopic lesions in animals 3 and 4 were compatibles with that caused by A. seminis infection. A. seminis was isolated from material of puncture and semen of one sheep (animal 4). It is concluded that the experimental infection model using goats and sheep has proved the pathogenicity of the A. seminis strain isolated from a goat in the Brazilian semiarid and reproduced in a sheep, which confirm the prediletion of the agent for epididymis, with clinical signs, histopathological findings, bacterial isolation and positive molecular diagnosis.(AU)

Distribution of lymphocytes, immunoglobulin-containing cells, macrophages, and dendritic cells in the accessory sex glands of rams experimentally infected with actinobacillus seminis / Distribuição de linfócitos, células carreadoras de imunoglobulinas, macrófagos e células dendríticas em glândulas sexuais acessórias de carneiros infectados experimentalmente com actinobacillus seminis

The distribution of cells involved in the immune response in accessory sex glands of rams experimentally infected with Actinobacillus seminis was studied. Twelve one-year old rams were experimentally infected by intraurethral (IU) (n=4) and intraepididymal (IE) (n=4) route, and four control (CON) animals were used. The animals were slaughtered 35 days post-inoculation, samples were taken from accessory sex glands, and bacteriology and histopathology tests were performed. The presence of CD4, CD8 and TCRγδ (WC1) lymphocytes, CD45RO cells, macrophages (CD14), dendritic cells (CD1b), IgA-, IgG- and IgM-containing cells (IgCC) was determined. Animals of the IE group developed clinical epididymitis. No lesions were seen in rams of the IU group; two of the intraepididymal inoculated CON developed small lesions in the epididymis. A. seminis isolates were achieved from 6:16 (37.5%) accessory sex glands in the IE group, but not in the IU and CON groups. In the CON group, IgA- and IgM- containing cells predominated in the bulbourethral glands and the disseminated prostate, and they were scarce or null in the vesicles and ampullae. A significant increase of IgA-, IgG- and IgM- containing cells was confirmed in the seminal vesicles, the ampullae and the bulbourethral glands in the IE group. In the IE and IU groups, an increase in CD4, CD8, WC1, CD45RO and CD14 was evidenced in the vesicles and ampullae. CD1b dendritic cells were present in the ampullae and vesicles with inflammatory processes. A. seminis triggered a local immune response in the IE and IU groups. These results indicate a different pattern of infiltrating immune cells in the accessory sex glands of infected A. seminis rams.

A distribuição das células envolvidas na resposta imune em glândulas sexuais acessórias de carneiros experimentalmente infectados com Actinobacillus seminis foi estudada. Doze carneiros de um ano de idade foram experimentalmente infectados via intrauretral (IU) (n=4) e via intraepididimal (IE) (n=4) e quatro animais controles (CON) foram utilizados. Os animais foram abatidos 35 dias após a inoculação, amostras foram retiradas das glândulas sexuais acessórias e testes bacteriológicos e histopatológicos foram realizados. A presença de linfócitos CD4, CD8 e TCRγδ (WC1), células CD45RO, macrófagos (CD14), células dendríticas (CD1b) e células contendo IgA, IgG and IgM (IgCC) foi determinada. Os animais do grupo IE desenvolveram epididimite clínica. Não foram visualizadas lesões nos carneiros do grupo IU, dois dos CON inoculados intraepididimalmente desenvolveram pequenas lesões no epidídimo. Isolados de A. seminis foram obtidos de 6:16 (37,5%) nas glândulas sexuais acessórias no grupo IE mas não nos grupos IU e CON. No grupo CON células contendo IgA and IgM predominaram nas glândulas bulbouretrais e na próstata e foram escassas ou ausentes nas vesículas e na ampola. Um incremento significativo de células contendo IgA, IgG and IgM foi confirmado nas vesículas seminais, na ampola e nas glândulas bulbouretrais no grupo IE. Nos grupos IE e IU foi evidenciado um aumento em CD4, CD8, WC1, CD45RO e CD14 nas vesículas e ampola. As células dendríticas CD1b estavam presentes na ampola e nas vesículas com processo inflamatório. A. seminis induziu uma resposta imune local nos grupos IE e IU. Estes resultados indicam um padrão diferente de células imunes infiltrantes nas glândulas sexuais acessórias de carneiros infectados por A. seminis.

Clinical and pathological changes in rams experimentally infected with Actinobacillus seminis and Histophilus somni.

Infectious epididymitis is considered a major cause of economic losses for the sheep industry worldwide. This study aimed to investigate clinical and pathological changes associated with experimental infections with A. seminis and H. somni in rams. Twenty rams of age 18 to 24 months were infected by intraepididymal inoculation of A. seminis (n = 10) and H. somni (n = 10). Rams were weekly examined and biological samples were collected during six weeks. All rams inoculated with A. seminis and 80% inoculated with H. somni became infected. The recovery of bacteria was possible in semen and urine samples and tissues in both experimental groups. Clinically, there were a decrease in testicular consistency and an increase in measures of the left epididymis tails in both experimental groups. The main gross changes were observed in the reproductive tract. Microscopically, the main lesions were inflammatory changes in the genitourinary tract and testicular degeneration. A. seminis and H. somni were able to colonize several organs of the genitourinary tract in rams, being indistinguishable by clinical exam, necropsy or histopathology. For differential diagnosis, it is important to use diagnostic techniques for direct confirmation of the etiologic agent.

Pathological changes in accessory sex organs of rams following experimental infection with Actinobacillus seminis.

AIM: To investigate and assess the susceptibility, lesions and route of dissemination in the accessory sex organs of young rams experimentally infected with Actinobacillus seminis. METHODS: The accessory sex organs were obtained from 64 young rams. The test rams (n=52) were infected by instillation, drenching or injection of 2.3 x 10(9) cells/ml A. seminis organisms via nine different routes: intra-epididymal (1 ml), I/V (3 ml), intra-urethral (3 ml), intra-preputial (3 ml), ductus deferens (1 ml), I/M (3 ml), oral (10 ml), intranasal (3 ml), and intra-conjunctival (3 drops). All test rams were necropsied 1-44 days post-inoculation (p.i.). Control rams (n=12) were necropsied at 1-46 days p.i. Accessory sex organs were cultured for A. seminis, and thin tissue sections were collected and examined for histopathological changes. RESULTS: Vesicular adenitis was the most frequent lesion (21/52; 40%), followed by ampullitis, deferentitis, urethritis and bulbo-urethral adenitis (20/52 (38%), 16/52 (31%), 12/52 (23%), and 8/52 (15%), respectively). The prostate and prepuce were the least affected, and lesions occurred equally at 4% (2/52). Actinobacillus seminis was isolated from the accessory sex organs of 10 (19%) rams: from the vesicular glands (n=6), ductus deferens (n=3), ampullae (n=1), and prepuce (n=3). No lesions were seen in rams inoculated via the nasal and conjunctival routes. The pattern of involvement of both ascending and descending A. seminis infection of the genital tract is discussed. CONCLUSIONS: Involvement of the accessory sex organ of rams following all but the intranasal and intra-conjunctival routes of the nine routes of experimental inoculation tested helped to clarify the epidemiology, pathogenesis, and pathology of ovine genital actinobacillosis.

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.

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.