J. Comp. Path. 2000, Vol. 122, 217–222 doi:10.1053/jcpa.1999.0359, available online at http://www.idealibrary.com on SHORT PAPER Isolation of Actinobacillus seminis from the Genital Tract of Rams in Spain V. A. de la Puente-Redondo*, N. Garcı ´a del Blanco*, C. Pe´rez-Martı ´nez , M. C. Gonza´ lez-Rodrı ´guez‡, E. F. Rodrı ´guez-Ferri * and C. B. Gutie´rrez-Martı ´n* *Unidad de Microbiologı ´a y Inmunologı ´a and Unidad de Histologı ´a y Anatomı ´a Patolo´gica, Facultad de Veterinaria, Universidad de Leo´n, Leo´n, and ‡Asociacio´n Nacional de Castellano Auto´ctono, Valladolid, Spain Summary Actinobacillus seminis isolates were cultured from the semen (two isolates) and the left testis (one isolate) of two one-year-old rams in Leo´n, Spain. One animal showed lesions of chronic unilateral orchitis and epididymitis while the other appeared to su er a subclinical infection and only a moderate number of pleomorphic rods and inflammatory cells were present in its semen. The isolates were biochemically similar to the A. seminis type strain NCTC 10851 and two other European A. seminis isolates, except that they produced acid from sorbitol; their identity was confirmed by arbitrarily primed polymerase chain reaction. The isolates were also tested against 30 antimicrobial agents, and only marbofloxacin was found active against all of them. As far as is known, this is the first report of A. seminis isolation from rams in southern Europe.  2000 Harcourt Publishers Ltd Several gram-negative, pleomorphic rods are petitive intergenic consensus (ERIC)-based PCR known to be associated with contagious epi- techniques have been described to distinguish A. didymitis and infertility in rams: Actinobacillus sem- seminis from other closely related organisms (Ap- inis, Brucella ovis, Corynebacterium pseudotuberculosis, puhamy et al. , 1998). The two latter methods are Haemophilus agni, Histophilus ovis, Pasteurella haemo- based on the use of repetitive elements (REP or lytica, Staphylococcus spp. and Streptococcus spp. ( Jan- ERIC) which generate DNA fingerprints that facil- sen, 1980; Cardenas and Maki, 1986). A. seminis itate discrimination between bacterial strains (Olive was first isolated in Australia from a ram with and Bean, 1999). A further method for DNA-based epididymitis (Baynes and Simmons, 1960). In Eur- typing of micro-organisms is the arbitrarily primed ope, reports of ovine genital infection with A. seminis polymerase chain reaction (AP-PCR) technique, are limited to Hungary (Hajto´s et al., 1987) and also referred to as random amplified polymorphic the UK (Heath et al. , 1991). There are relatively DNA (RAPD) assay. This technique, first described few distinguishing tests for this and phenotypically in 1990, is based on the use of short random similar organisms (Actinobacillus actinomycetem- sequence primers which hybridize so strongly with comitans, H. ovis, P. haemolytica), and there have been chromosomal DNA sequences at low annealing cases of misidentification (Walker et al., 1986). temperatures that they can be used to initiate Recently, polymerase chain reaction (PCR) ribo- amplification of regions of the bacterial genome typing, repetitive extragenic palindromic ele- (Olive and Bean, 1999). The AP form of PCR was ment (REP)-based PCR and enterobacterial re- chosen for the present study because the primer (Universal M13 primer) is less expensive than primers used in REP- or ERIC-PCR, and also Correspondence to: E. F. Rodrı ´guez-Ferri. 0021–9975/00/020217+06 $35.00  2000 Harcourt Publishers Ltd