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Pasteurellosis as a cause of genital lesions in rams. A descriptive study
Small Ruminant Research 87 (2009) 111–115
Contents lists available at ScienceDirect
Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres
Short communication
Pasteurellosis as a cause of genital lesions in rams. A descriptive study L. García-Pastor a , J.M. Blasco b , M. Barberán a,∗ a b
Dpto. de Patología Animal, Facultad de Veterinaria, Universidad de Zaragoza, Miguel Servet 177, 50013 Zaragoza, Spain Unidad de Sanidad Animal, CITA-Gobierno de Aragón, Av. Monta˜ nana 930, 50059 Zaragoza, Spain
a r t i c l e
i n f o
Article history: Received 25 June 2009 Received in revised form 9 October 2009 Accepted 9 October 2009 Available online 14 November 2009 Keywords: Pasteurella Genital infections Genital lesions Epididymitis Ram
a b s t r a c t Pasteurellosis is one of the most prevalent diseases of sheep, but the involvement of Pasteurellae in genital pathology of rams has been described rarely. One hundred and eighty-four rams showing palpable lesions in testes, epididymides or scrotum were submitted to bacteriological studies, and seven mature rams found infected with bacterial species belonging to the Pasteurella cluster (i.e., Mannheimia, Pasteurella and Bibersteinia (M/P/B)). The M/P/B cultures obtained were pure and/or heavy, and were confirmed after necropsy in the five M/P/B infected rams that could be slaughtered for further pathological examinations. Pasteurella multocida infected rams exhibited fibrinous exudate and generalized adhesions between the vaginal and the external scrotal layers. Testicular atrophy and epididymal sperm granulomas were also evident in these rams. Microscopically, epithelial hyperplasia with intraepithelial cysts, fibrosis and spermatic granulomas were present in the epididymis, while testis showed sperm stasis foci, microcalcifications and fibrosis. Mannheimia haemolytica infected rams showed severe unilateral epididymitis and testicular atrophy, being microscopically similar to the lesions found in P. multocida infected rams. The ram found infected with B. threalosi had severe unilateral lesions in testis, epididymis and scrotum. Microscopically, abscesses in epididymis and testis, and severe fibrosis and interstitial round cells infiltrates in testis were observed. Further studies should be conducted to determine properly the role played by the Pasteurella cluster in the pathogenesis of genital lesions in rams. Crown Copyright © 2009 Published by Elsevier B.V. All rights reserved.
Bacterial infections inducing genital lesions in rams are widespread in most sheep raising countries, and the cause of reduced fertility and important economical losses. The best known primary pathogens associated to this problem are Brucella ovis (B. ovis) (Blasco, 1990) and Actinobacillus seminis (A. seminis) (Heath et al., 1991), but other microorganisms have been reported as a cause of genital lesions in rams: Corynebacterium ovis (Ekdahl et al., 1968; Krishna et al., 1977), Yersinia pseudotuberculosis (DeLong et al., 1979; Williamson and Nairn, 1980; Bulgin and Anderson, 1983), Histophilus ovis (Rahaley, 1978; Webb, 1983; Walker and LeaMaster, 1986), Haemophilus somnus (Palomares et
∗ Corresponding author. Tel.: +34 976 761600x4186. E-mail address: [email protected] (M. Barberán).
al., 2005), Arcanobacterium pyogenes (Ekdahl et al., 1968; DeLong et al., 1979; Bulgin and Anderson, 1983; Walker et al., 1986; Gouletsou et al., 2004), Brucella melitensis (Chand et al., 2002), Burkholderia pseudomallei (Bulgin and Anderson, 1983), Stenotrophomonas maltophilia (DeLong et al., 1979), Escherichia coli (Constable and Webber, 1987), Streptococcus sp. (Ekdahl et al., 1968), Staphylococcus sp. (Bulgin and Anderson, 1983; Walker et al., 1986; Palomares et al., 2005), Chlamydophila abortus (Rodolakis and Bernard, 1977), and Pasteurella sp. (Ekdahl et al., 1968; Jansen, 1980, 1983; Bulgin and Anderson, 1983; Walker et al., 1986; Fodor et al., 1987). Descriptive studies of lesions induced have been mainly focused to B. ovis and A. seminis infections (Blasco, 1990; Heath et al., 1991), but very few information is available on this respect for the other microorganisms, and particularly in the case of Pasteurel-
0921-4488/$ – see front matter. Crown Copyright © 2009 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2009.10.010
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L. García-Pastor et al. / Small Ruminant Research 87 (2009) 111–115
lae. Mannheimia haemolytica (M. haemolytica), Pasteurella multocida (P. multocida) and Bibersteinia trehalosi (B. trehalosi), are the primary agents of pneumonic or systemic Pasteurellosis, one of the most prevalent diseases of sheep. Moreover, M. haemolytica has been reported as a cause of mastitis in ewes (el-Masannat et al., 1991) and isolated also in rams suffering genital lesions (Ekdahl et al., 1968; Jansen, 1980, 1983; Bulgin and Anderson, 1983; Lozano, 1986; Walker et al., 1986; Fodor et al., 1987), but the involvement of M. haemolytica in genital pathology is seldom reported. The objective of this work was to describe the main clinical and pathological features in scrotal organs of rams found infected with bacteria belonging to the formerly Pasteurella cluster (i.e., Mannheimia, Pasteurella and Bibersteinia (M/P/B)).
and eosin and submitted to microscopic examination. Microscopic lesions were graded subjectively as severe (S), mild (M) or recorded as (0) when absence of tissue damage. Samples for bacteriology were taken again directly from the lesions or from semen and cultured as above.
2. Results When obtaining either a pure or heavy culture containing two or less different bacterial species, the culture result was considered significant and submitted to the identification procedures described above. Having in consideration the limitations of the above bacteriological methods, it is possible that some slow or fastidious growing microorganisms were not isolated. However, a significant isolate was obtained in 93 (50.54%) of the 184 rams bacteriologically studied, and several bacterial species considered as a primary cause of genital lesions in rams were identified (Table 1). Seven of these isolates corresponded to the Pasteurella cluster, being isolated from seven rams belonging to seven different flocks. Four of these seven isolates were identified as P. multocida, two as M. haemolytica, and one as B. threalosi. Two out of the four P. multocida strains were isolated from samples taken from lesions in the epididymides, other P. multocida strain from lesions in testes and the remaining P. multocida strain was exclusively isolated from a scrotal abscess. The two M. haemolytica strains were isolated from lesions in the epididymides. Interestingly, one of these M. haemolytica strains was also excreted in semen. Finally, the unique B. threalosi strain identified was isolated only from semen samples of the corresponding clinically affected ram. These bacteriological results were confirmed again in the five animals that could be necropsied after owner’s agreement. The main clinical lesions in P. multocida infected rams were scrotal adhesions and unilateral or bilateral epididymitis (Table 2). A scrotal abscess was also evidenced in one of P. multocida infected rams. The gross lesions observed at necropsy in these P. multocida infected rams were generalized adhesions between the vaginal and the external scrotal layers, fibrinous exudate around tail epididymis, spermatic granulomas in epididymides, testicular atrophy and small size testicular calcifications (Table 2 and Fig. 1). Microscopically, the affected epididymides were showing spermatic granulomas, fibrosis and round cell infiltrates. Epididymal ducts
1. Materials and methods The rams belonging to 234 semi-extensive sheep flocks (mainly composed of the local Aragonesa breed) in the North-East of Spain were clinically examined once a year during a seven-year period to detect scrotal abnormalities. A systematic palpation of the scrotal contents was carried out in around 2500 rams per year, checking for the presence of any alteration in testes, epididymides and scrotum. A total of 184 rams having palpable lesions were identified along the seven years of study, and further submitted to bacteriological studies. Samples for bacteriology were obtained directly from open lesions (through sterile swabs) or by direct aspiration using a sterile needle and syringe. When possible, semen samples were additionally obtained by electro-ejaculation (Ruakura Ram Probe; New Zealand) and collected in sterile bags. The same day of sampling all samples were smeared onto at least two plates of both Blood Agar Base (Biolife, Italy) containing 5% sheep blood and MacConkey No. 2 (Difco, USA) media, and incubated for 1–5 days at 37 ◦ C in 10% CO2 . Bacterial yields were preliminary identified by growth characteristics, colonial morphology, Gram-staining, oxidase, catalase, urease and indol tests by standard procedures and/or using the adequate API strips (Biomerieux, France). Representative isolates were submitted to further sugar fermentation assays and DNA sequencing when required. For this, colonial DNA was extracted and 5 fragment of 16S-RNA was amplified using bacterial universal primers. PCR yields were sequenced (around 450 nucleotides) and sequences compared with those described in Gen Bank for the corresponding bacterial species (the assistance in molecular tests from Dr. G. Aduriz; Neiker, Bilbao, Spain, is acknowledged). For definitive Pasteurella/Mannheimia/Bibersteinia (P/M/B) identification at least glucose, lactose, maltose, salicin, sucrose, trehalose, arabinose and xylose tests were performed. No additional typing procedures were used to determine the different serotypes isolated. When farmers agree, Pasteurella positive animals were necropsied for further pathological examinations. Samples from affected tissues were then fixed in 10% buffered formalin and embedded in paraffin. Five micrometer sections were stained with haematoxylin
Table 1 Bacteriological results obtained in the 93 out of 184 Aragonesa rams affected by genital lesions in which a significant isolate was obtained. Number of rams
24 15 14 12 7 7 4 3 3 2 1 1 a
Bacterial isolate
Streptococcus sp. Staphylococcus sp. Arcanobacterium pyogenes. Actinobacillus seminis Pasteurella (P/M/B) cluster Corynebacterium ovis Gram +ve catalase −ve cocci Brucella ovis Escherichia coli Stenotrophomonas maltophilia Haemophilus somnus Histophilus ovis
Sample positive Scrotum
Semena
Testes and/or epididymides
7 3 10 1 1 2
5 1a 2a 2 2a 3 3 1a 1
12 12 3 9 5 2 1 3 2 2 1 1
When indicated, at least one of strains was isolated simultaneously from both semen and other samples.
Age (years)
2 5 7 7 4 5 3
Number
1 2 3 4 5 6 7
Clinical signs: 0: absence, U: unilateral, B: bilateral; gross lesions: 0: absence, +: presence, ND: not determined; epididymitis location: H: head, B: body, T: tail; microscopic lesions: 0: absence, M: mild, S: severe, ND: not determined.
Scrotum Testes
ND M ND S ND S S
Epididymides
ND + + 0 ND 0 + ND + + + ND + 0
Testicular calcifications
ND S ND S ND S S
Testicular Atrophy
ND + + + ND + +
Epididymitis
ND H/B/T 0 H ND H/B/T H/B
Scrotum
B 0 B 0 0 0 0 0 0 0 U 0 0 U
Testes Epididymides
P. multocida P. multocida P. multocida P. multocida M. haemolytica M. haemolytica B. threalosi
0 U 0 U U U U
Adhesions
Microscopic lesions Gross lesions Clinical signs Strain isolated Rams
Table 2 Clinical signs and gross and microscopic lesions identified in the rams in which pasteurellae were isolated.
ND M ND 0 ND M S
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were showing epithelial hyperplasia with intraepithelial cysts with neutrophils, and tubular lumens contained cellular debris but few spermatozoa. The seminiferous germinal epithelium was completely sloughed or only Sertoli cells and some spermatogonia remained. There were also marked fibrosis, foci of sperm stasis and intratubular sperm granulomas with giant cells in the testis. In the unique animal infected by M. haemolytica that could be necropsied, the only clinical lesion evidenced was unilateral epididymitis. Right epididymis was enlarged and showing several spermatic granulomas and fibrosis, and the right testis was atrophic and showing some calcifications (Fig. 2). Diffuse infiltrates of round cells, spermatic granulomas, epithelial hyperplasia and empty intraepithelial cysts were evidenced microscopically in the epididymis of this ram. In testis of this ram desquamation, stasis and mineralization of seminiferous tubules, and presence of intratubular sperm granulomas with giant cells were evidenced microscopically. A three-year-old ram showing unilateral epididymitis and testicular atrophy (Table 2) excreted B. trehalosi in semen. Right epididymis in this ram was enlarged and showing wide adhesions with the external layer of the tunica vaginalis and several well-encapsulated spermatic granulomas (Fig. 3). The corresponding right testis was slightly atrophic and carried a large abscess. Microscopically, the epididymal sperm granulomas seen macroscopically were actually abscesses devoid of spermatozoa. Epididymal ducts were showing severe hyperplasia, intraepithelial cysts with neutrophils, perivascular loose accumulations of round cells and moderate sclerosis. Severe fibrosis, interstitial round cells foci and an abscess, were detected microscopically in the atrophic testis of this ram. 3. Discussion In agreement with similar reports (Ekdahl et al., 1968; Jansen, 1980; Bulgin and Anderson, 1983), the isolation frequencies of Pasteurellae in relation to other pathogens causing genital lesions in rams was also low in our study (Table 1). However, the seven Pasteurella isolated were always obtained from mature rams (Table 2), whereas genital pasteurellosis has been associated generally to young lambs (Fodor et al., 1987) or ram hoggets (Ekdahl et al., 1968; Bulgin and Anderson, 1983; Walker et al., 1986). Pasteurellosis in sheep is generally associated to a systemic or respiratory disease. Pasteurellae can be asymptomatically carried in the mouth, tonsils and nasopharynx of apparently healthy sheep and goats (Al-Tarazi and Dagnall, 1997; Poulsen et al., 2006), and transition to clinical disease appears to be facilitated by various stressors. Apart from sporadic cases of mastitis in ewes, genital lesions due to these bacteria are considered as a very rare event, and the role of Pasteurellae as a cause of genital lesions in rams could be questioned. Due to the ubiquity of Pasteurellae (Burriel, 1997), semen or tissue samples from clinically affected rams due to other pathologies could be potentially contaminated by commensal Pasteurellae, leading to a misdiagnosis. However, this was improbable in our study since in all cases, the M/P/B cultures obtained were always pure and/or heavy, and confirmed
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Fig. 1. Gross lesions found in P. multocida infected rams: generalized adhesions between the vaginal and the external scrotal layers (left), and fibrinous exudates around tail epididymis (right).
Fig. 2. Gross lesions in testis and epididymidis in a M. haemolytica infected ram: chronic epididymitis in tail epididymis (left), and cut section showing big spermatic granulomas (arrowheads) and fibrosis (asterisk) in tail epididymis and calcification (arrow) in testis (right).
after necropsy in all cases, thus suggesting that a causeeffect relationship exists among M/P/B isolation and the induction of genital lesions in rams. In fact, it has been described that a heat labile toxin from some Pasteurellae is capable to induce testicular lesions in rats (Ackermann et al., 1992). Moreover, M. haemolytica can colonise the vaginal mucous membranes of sheep, and it has been isolated
from the stomach content of aborted or still-born lambs and from the epididymis of a sucking lamb (Fodor et al., 1987). Altogether, the most reasonable hypothesis is that the M/P/B isolated in our study could be the responsible of the pathological lesions observed in rams. Pasteurellae could reach epididymides as a consequence of bacteraemia or by ascending infection. It has been reported that M.
Fig. 3. Gross and histopathological findings in the ram infected with B. threalosi: chronic epidiyimitis and severe vaginal adhesions in the head and body of epididymis (left), and histological section of the epididymal abscess. H–E (right).
L. García-Pastor et al. / Small Ruminant Research 87 (2009) 111–115
haemolytica is able to migrate from the contaminated prepuce foreskin to the testicle (Jansen, 1980, 1983), although if this M. haemolytica contamination of the testicle leads to the development of lesions remains to be determined experimentally. The initial preputial infection can be originated by skin contact with contaminated manure and food residues on the floor. Moreover, sexual contacts with ewes excreting Pasteurellae in vaginal exudates (Fodor et al., 1987) may also be a source of genital infection for rams. Most of rams necropsied were showing lesions in either testes or epididymides (Table 2). However, one animal (ram no. 1, Table 2) in which P. multocida was isolated, did not show lesions in testes and epididymides and was showing exclusively a subscrotal abscess. The entry route in this animal was unknown but could have been through a wound in the scrotal skin. In any case, the precise pathogenic mechanisms that M/P/B use for inducing testicular lesions in rams remain undetermined and out of the scope of this work. A heat labile toxin from some P. multocida strains has been reported to induce severe degenerative testicular lesions in rats but unable to produce orchitis or epididymitis (Ackermann et al., 1992). M. haemolytica and B. trehalosi synthesise a lipopolysaccharide (LPS) similar to that produced by other Gram-negative bacteria (Keiss et al., 1964). These bacterial LPSs are usually endotoxic and considered a major virulence factor in some Gramnegative bacteria. These antigens are known to activate complement leading to the production of anaphylatoxins, and stimulate the secretion of several cytokines such as IL-1, TNF-␣ and IL-2. These proinflammatory mediators amplify PMN recruitment at the sites of inflammation, hence contributing to the production and perpetuation of tissue injury (Morrison and Ryan, 1987). With the exception of the orchitis and the abscesses evidenced in testis and epididymis in the ram in which B. trehalosi was isolated, the severe lesions observed in all M/P/B infected rams were closely related to those induced by B. ovis and other bacterial pathogens causing genital lesions in rams and, therefore, are not pathognomonic. Further studies should be conducted to determine properly the role played by the Pasteurella cluster in the pathogenesis of genital lesions in rams. Acknowledgements This work has been financed in part by CICYT (AGL20047088-C03-03/GAN and AGL2008-04514-C03-03) research projects, and the Gobierno de Aragón (Grupo de Investigación Consolidado, code A14, BOA 55, May 9, 2008, p. 6986). The authors are very grateful to Dr. G. Aduriz from the Instituto Vasco de Investigación y Desarrollo Agrario (NEIKER), Bilbao, Spain, for his kind help with the laboratory confirmation and identification of the different strains isolated; S. Becerra, necropsy technician at the Veterinary Faculty, is also acknowledged for his excellent technical assistance.
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Contents lists available at ScienceDirect
Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres
Short communication
Pasteurellosis as a cause of genital lesions in rams. A descriptive study L. García-Pastor a , J.M. Blasco b , M. Barberán a,∗ a b
Dpto. de Patología Animal, Facultad de Veterinaria, Universidad de Zaragoza, Miguel Servet 177, 50013 Zaragoza, Spain Unidad de Sanidad Animal, CITA-Gobierno de Aragón, Av. Monta˜ nana 930, 50059 Zaragoza, Spain
a r t i c l e
i n f o
Article history: Received 25 June 2009 Received in revised form 9 October 2009 Accepted 9 October 2009 Available online 14 November 2009 Keywords: Pasteurella Genital infections Genital lesions Epididymitis Ram
a b s t r a c t Pasteurellosis is one of the most prevalent diseases of sheep, but the involvement of Pasteurellae in genital pathology of rams has been described rarely. One hundred and eighty-four rams showing palpable lesions in testes, epididymides or scrotum were submitted to bacteriological studies, and seven mature rams found infected with bacterial species belonging to the Pasteurella cluster (i.e., Mannheimia, Pasteurella and Bibersteinia (M/P/B)). The M/P/B cultures obtained were pure and/or heavy, and were confirmed after necropsy in the five M/P/B infected rams that could be slaughtered for further pathological examinations. Pasteurella multocida infected rams exhibited fibrinous exudate and generalized adhesions between the vaginal and the external scrotal layers. Testicular atrophy and epididymal sperm granulomas were also evident in these rams. Microscopically, epithelial hyperplasia with intraepithelial cysts, fibrosis and spermatic granulomas were present in the epididymis, while testis showed sperm stasis foci, microcalcifications and fibrosis. Mannheimia haemolytica infected rams showed severe unilateral epididymitis and testicular atrophy, being microscopically similar to the lesions found in P. multocida infected rams. The ram found infected with B. threalosi had severe unilateral lesions in testis, epididymis and scrotum. Microscopically, abscesses in epididymis and testis, and severe fibrosis and interstitial round cells infiltrates in testis were observed. Further studies should be conducted to determine properly the role played by the Pasteurella cluster in the pathogenesis of genital lesions in rams. Crown Copyright © 2009 Published by Elsevier B.V. All rights reserved.
Bacterial infections inducing genital lesions in rams are widespread in most sheep raising countries, and the cause of reduced fertility and important economical losses. The best known primary pathogens associated to this problem are Brucella ovis (B. ovis) (Blasco, 1990) and Actinobacillus seminis (A. seminis) (Heath et al., 1991), but other microorganisms have been reported as a cause of genital lesions in rams: Corynebacterium ovis (Ekdahl et al., 1968; Krishna et al., 1977), Yersinia pseudotuberculosis (DeLong et al., 1979; Williamson and Nairn, 1980; Bulgin and Anderson, 1983), Histophilus ovis (Rahaley, 1978; Webb, 1983; Walker and LeaMaster, 1986), Haemophilus somnus (Palomares et
∗ Corresponding author. Tel.: +34 976 761600x4186. E-mail address: [email protected] (M. Barberán).
al., 2005), Arcanobacterium pyogenes (Ekdahl et al., 1968; DeLong et al., 1979; Bulgin and Anderson, 1983; Walker et al., 1986; Gouletsou et al., 2004), Brucella melitensis (Chand et al., 2002), Burkholderia pseudomallei (Bulgin and Anderson, 1983), Stenotrophomonas maltophilia (DeLong et al., 1979), Escherichia coli (Constable and Webber, 1987), Streptococcus sp. (Ekdahl et al., 1968), Staphylococcus sp. (Bulgin and Anderson, 1983; Walker et al., 1986; Palomares et al., 2005), Chlamydophila abortus (Rodolakis and Bernard, 1977), and Pasteurella sp. (Ekdahl et al., 1968; Jansen, 1980, 1983; Bulgin and Anderson, 1983; Walker et al., 1986; Fodor et al., 1987). Descriptive studies of lesions induced have been mainly focused to B. ovis and A. seminis infections (Blasco, 1990; Heath et al., 1991), but very few information is available on this respect for the other microorganisms, and particularly in the case of Pasteurel-
0921-4488/$ – see front matter. Crown Copyright © 2009 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2009.10.010
112
L. García-Pastor et al. / Small Ruminant Research 87 (2009) 111–115
lae. Mannheimia haemolytica (M. haemolytica), Pasteurella multocida (P. multocida) and Bibersteinia trehalosi (B. trehalosi), are the primary agents of pneumonic or systemic Pasteurellosis, one of the most prevalent diseases of sheep. Moreover, M. haemolytica has been reported as a cause of mastitis in ewes (el-Masannat et al., 1991) and isolated also in rams suffering genital lesions (Ekdahl et al., 1968; Jansen, 1980, 1983; Bulgin and Anderson, 1983; Lozano, 1986; Walker et al., 1986; Fodor et al., 1987), but the involvement of M. haemolytica in genital pathology is seldom reported. The objective of this work was to describe the main clinical and pathological features in scrotal organs of rams found infected with bacteria belonging to the formerly Pasteurella cluster (i.e., Mannheimia, Pasteurella and Bibersteinia (M/P/B)).
and eosin and submitted to microscopic examination. Microscopic lesions were graded subjectively as severe (S), mild (M) or recorded as (0) when absence of tissue damage. Samples for bacteriology were taken again directly from the lesions or from semen and cultured as above.
2. Results When obtaining either a pure or heavy culture containing two or less different bacterial species, the culture result was considered significant and submitted to the identification procedures described above. Having in consideration the limitations of the above bacteriological methods, it is possible that some slow or fastidious growing microorganisms were not isolated. However, a significant isolate was obtained in 93 (50.54%) of the 184 rams bacteriologically studied, and several bacterial species considered as a primary cause of genital lesions in rams were identified (Table 1). Seven of these isolates corresponded to the Pasteurella cluster, being isolated from seven rams belonging to seven different flocks. Four of these seven isolates were identified as P. multocida, two as M. haemolytica, and one as B. threalosi. Two out of the four P. multocida strains were isolated from samples taken from lesions in the epididymides, other P. multocida strain from lesions in testes and the remaining P. multocida strain was exclusively isolated from a scrotal abscess. The two M. haemolytica strains were isolated from lesions in the epididymides. Interestingly, one of these M. haemolytica strains was also excreted in semen. Finally, the unique B. threalosi strain identified was isolated only from semen samples of the corresponding clinically affected ram. These bacteriological results were confirmed again in the five animals that could be necropsied after owner’s agreement. The main clinical lesions in P. multocida infected rams were scrotal adhesions and unilateral or bilateral epididymitis (Table 2). A scrotal abscess was also evidenced in one of P. multocida infected rams. The gross lesions observed at necropsy in these P. multocida infected rams were generalized adhesions between the vaginal and the external scrotal layers, fibrinous exudate around tail epididymis, spermatic granulomas in epididymides, testicular atrophy and small size testicular calcifications (Table 2 and Fig. 1). Microscopically, the affected epididymides were showing spermatic granulomas, fibrosis and round cell infiltrates. Epididymal ducts
1. Materials and methods The rams belonging to 234 semi-extensive sheep flocks (mainly composed of the local Aragonesa breed) in the North-East of Spain were clinically examined once a year during a seven-year period to detect scrotal abnormalities. A systematic palpation of the scrotal contents was carried out in around 2500 rams per year, checking for the presence of any alteration in testes, epididymides and scrotum. A total of 184 rams having palpable lesions were identified along the seven years of study, and further submitted to bacteriological studies. Samples for bacteriology were obtained directly from open lesions (through sterile swabs) or by direct aspiration using a sterile needle and syringe. When possible, semen samples were additionally obtained by electro-ejaculation (Ruakura Ram Probe; New Zealand) and collected in sterile bags. The same day of sampling all samples were smeared onto at least two plates of both Blood Agar Base (Biolife, Italy) containing 5% sheep blood and MacConkey No. 2 (Difco, USA) media, and incubated for 1–5 days at 37 ◦ C in 10% CO2 . Bacterial yields were preliminary identified by growth characteristics, colonial morphology, Gram-staining, oxidase, catalase, urease and indol tests by standard procedures and/or using the adequate API strips (Biomerieux, France). Representative isolates were submitted to further sugar fermentation assays and DNA sequencing when required. For this, colonial DNA was extracted and 5 fragment of 16S-RNA was amplified using bacterial universal primers. PCR yields were sequenced (around 450 nucleotides) and sequences compared with those described in Gen Bank for the corresponding bacterial species (the assistance in molecular tests from Dr. G. Aduriz; Neiker, Bilbao, Spain, is acknowledged). For definitive Pasteurella/Mannheimia/Bibersteinia (P/M/B) identification at least glucose, lactose, maltose, salicin, sucrose, trehalose, arabinose and xylose tests were performed. No additional typing procedures were used to determine the different serotypes isolated. When farmers agree, Pasteurella positive animals were necropsied for further pathological examinations. Samples from affected tissues were then fixed in 10% buffered formalin and embedded in paraffin. Five micrometer sections were stained with haematoxylin
Table 1 Bacteriological results obtained in the 93 out of 184 Aragonesa rams affected by genital lesions in which a significant isolate was obtained. Number of rams
24 15 14 12 7 7 4 3 3 2 1 1 a
Bacterial isolate
Streptococcus sp. Staphylococcus sp. Arcanobacterium pyogenes. Actinobacillus seminis Pasteurella (P/M/B) cluster Corynebacterium ovis Gram +ve catalase −ve cocci Brucella ovis Escherichia coli Stenotrophomonas maltophilia Haemophilus somnus Histophilus ovis
Sample positive Scrotum
Semena
Testes and/or epididymides
7 3 10 1 1 2
5 1a 2a 2 2a 3 3 1a 1
12 12 3 9 5 2 1 3 2 2 1 1
When indicated, at least one of strains was isolated simultaneously from both semen and other samples.
Age (years)
2 5 7 7 4 5 3
Number
1 2 3 4 5 6 7
Clinical signs: 0: absence, U: unilateral, B: bilateral; gross lesions: 0: absence, +: presence, ND: not determined; epididymitis location: H: head, B: body, T: tail; microscopic lesions: 0: absence, M: mild, S: severe, ND: not determined.
Scrotum Testes
ND M ND S ND S S
Epididymides
ND + + 0 ND 0 + ND + + + ND + 0
Testicular calcifications
ND S ND S ND S S
Testicular Atrophy
ND + + + ND + +
Epididymitis
ND H/B/T 0 H ND H/B/T H/B
Scrotum
B 0 B 0 0 0 0 0 0 0 U 0 0 U
Testes Epididymides
P. multocida P. multocida P. multocida P. multocida M. haemolytica M. haemolytica B. threalosi
0 U 0 U U U U
Adhesions
Microscopic lesions Gross lesions Clinical signs Strain isolated Rams
Table 2 Clinical signs and gross and microscopic lesions identified in the rams in which pasteurellae were isolated.
ND M ND 0 ND M S
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were showing epithelial hyperplasia with intraepithelial cysts with neutrophils, and tubular lumens contained cellular debris but few spermatozoa. The seminiferous germinal epithelium was completely sloughed or only Sertoli cells and some spermatogonia remained. There were also marked fibrosis, foci of sperm stasis and intratubular sperm granulomas with giant cells in the testis. In the unique animal infected by M. haemolytica that could be necropsied, the only clinical lesion evidenced was unilateral epididymitis. Right epididymis was enlarged and showing several spermatic granulomas and fibrosis, and the right testis was atrophic and showing some calcifications (Fig. 2). Diffuse infiltrates of round cells, spermatic granulomas, epithelial hyperplasia and empty intraepithelial cysts were evidenced microscopically in the epididymis of this ram. In testis of this ram desquamation, stasis and mineralization of seminiferous tubules, and presence of intratubular sperm granulomas with giant cells were evidenced microscopically. A three-year-old ram showing unilateral epididymitis and testicular atrophy (Table 2) excreted B. trehalosi in semen. Right epididymis in this ram was enlarged and showing wide adhesions with the external layer of the tunica vaginalis and several well-encapsulated spermatic granulomas (Fig. 3). The corresponding right testis was slightly atrophic and carried a large abscess. Microscopically, the epididymal sperm granulomas seen macroscopically were actually abscesses devoid of spermatozoa. Epididymal ducts were showing severe hyperplasia, intraepithelial cysts with neutrophils, perivascular loose accumulations of round cells and moderate sclerosis. Severe fibrosis, interstitial round cells foci and an abscess, were detected microscopically in the atrophic testis of this ram. 3. Discussion In agreement with similar reports (Ekdahl et al., 1968; Jansen, 1980; Bulgin and Anderson, 1983), the isolation frequencies of Pasteurellae in relation to other pathogens causing genital lesions in rams was also low in our study (Table 1). However, the seven Pasteurella isolated were always obtained from mature rams (Table 2), whereas genital pasteurellosis has been associated generally to young lambs (Fodor et al., 1987) or ram hoggets (Ekdahl et al., 1968; Bulgin and Anderson, 1983; Walker et al., 1986). Pasteurellosis in sheep is generally associated to a systemic or respiratory disease. Pasteurellae can be asymptomatically carried in the mouth, tonsils and nasopharynx of apparently healthy sheep and goats (Al-Tarazi and Dagnall, 1997; Poulsen et al., 2006), and transition to clinical disease appears to be facilitated by various stressors. Apart from sporadic cases of mastitis in ewes, genital lesions due to these bacteria are considered as a very rare event, and the role of Pasteurellae as a cause of genital lesions in rams could be questioned. Due to the ubiquity of Pasteurellae (Burriel, 1997), semen or tissue samples from clinically affected rams due to other pathologies could be potentially contaminated by commensal Pasteurellae, leading to a misdiagnosis. However, this was improbable in our study since in all cases, the M/P/B cultures obtained were always pure and/or heavy, and confirmed
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Fig. 1. Gross lesions found in P. multocida infected rams: generalized adhesions between the vaginal and the external scrotal layers (left), and fibrinous exudates around tail epididymis (right).
Fig. 2. Gross lesions in testis and epididymidis in a M. haemolytica infected ram: chronic epididymitis in tail epididymis (left), and cut section showing big spermatic granulomas (arrowheads) and fibrosis (asterisk) in tail epididymis and calcification (arrow) in testis (right).
after necropsy in all cases, thus suggesting that a causeeffect relationship exists among M/P/B isolation and the induction of genital lesions in rams. In fact, it has been described that a heat labile toxin from some Pasteurellae is capable to induce testicular lesions in rats (Ackermann et al., 1992). Moreover, M. haemolytica can colonise the vaginal mucous membranes of sheep, and it has been isolated
from the stomach content of aborted or still-born lambs and from the epididymis of a sucking lamb (Fodor et al., 1987). Altogether, the most reasonable hypothesis is that the M/P/B isolated in our study could be the responsible of the pathological lesions observed in rams. Pasteurellae could reach epididymides as a consequence of bacteraemia or by ascending infection. It has been reported that M.
Fig. 3. Gross and histopathological findings in the ram infected with B. threalosi: chronic epidiyimitis and severe vaginal adhesions in the head and body of epididymis (left), and histological section of the epididymal abscess. H–E (right).
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haemolytica is able to migrate from the contaminated prepuce foreskin to the testicle (Jansen, 1980, 1983), although if this M. haemolytica contamination of the testicle leads to the development of lesions remains to be determined experimentally. The initial preputial infection can be originated by skin contact with contaminated manure and food residues on the floor. Moreover, sexual contacts with ewes excreting Pasteurellae in vaginal exudates (Fodor et al., 1987) may also be a source of genital infection for rams. Most of rams necropsied were showing lesions in either testes or epididymides (Table 2). However, one animal (ram no. 1, Table 2) in which P. multocida was isolated, did not show lesions in testes and epididymides and was showing exclusively a subscrotal abscess. The entry route in this animal was unknown but could have been through a wound in the scrotal skin. In any case, the precise pathogenic mechanisms that M/P/B use for inducing testicular lesions in rams remain undetermined and out of the scope of this work. A heat labile toxin from some P. multocida strains has been reported to induce severe degenerative testicular lesions in rats but unable to produce orchitis or epididymitis (Ackermann et al., 1992). M. haemolytica and B. trehalosi synthesise a lipopolysaccharide (LPS) similar to that produced by other Gram-negative bacteria (Keiss et al., 1964). These bacterial LPSs are usually endotoxic and considered a major virulence factor in some Gramnegative bacteria. These antigens are known to activate complement leading to the production of anaphylatoxins, and stimulate the secretion of several cytokines such as IL-1, TNF-␣ and IL-2. These proinflammatory mediators amplify PMN recruitment at the sites of inflammation, hence contributing to the production and perpetuation of tissue injury (Morrison and Ryan, 1987). With the exception of the orchitis and the abscesses evidenced in testis and epididymis in the ram in which B. trehalosi was isolated, the severe lesions observed in all M/P/B infected rams were closely related to those induced by B. ovis and other bacterial pathogens causing genital lesions in rams and, therefore, are not pathognomonic. Further studies should be conducted to determine properly the role played by the Pasteurella cluster in the pathogenesis of genital lesions in rams. Acknowledgements This work has been financed in part by CICYT (AGL20047088-C03-03/GAN and AGL2008-04514-C03-03) research projects, and the Gobierno de Aragón (Grupo de Investigación Consolidado, code A14, BOA 55, May 9, 2008, p. 6986). The authors are very grateful to Dr. G. Aduriz from the Instituto Vasco de Investigación y Desarrollo Agrario (NEIKER), Bilbao, Spain, for his kind help with the laboratory confirmation and identification of the different strains isolated; S. Becerra, necropsy technician at the Veterinary Faculty, is also acknowledged for his excellent technical assistance.
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