Karlsson et al. Acta Veterinaria Scandinavica 2010, 52:2 http://www.actavetscand.com/content/52/1/2 route. Additionally, artificial insemination (AI) is per- formed yearly on more than 95% of the approximately 400,000 Swedish dairy cows [25]. As there is a possibi- lity of transmission of Chlamydophila spp. via this route, it is important to determine whether breeding bulls are infected through screening of semen before AI in order to minimize this risk. The aim of this study was to investigate the presence of chlamydial agent in semen and in tissue of cauda epididymidis and to esti- mate the seroprevalence of Cp. abortus in Swedish bulls. Methods Animals and samples Beef bulls This study comprises samples from a subset of 166 beef bulls from 124 herds from different parts of Sweden that were taken to the only performance testing station in Sweden in September 2002. On arrival the bulls were approximately six months old. They were divided into groups, based on breed and body weight and placed in ten adjacent semi-outdoor pens under the same roof. The bulls were weighed every second week throughout the testing period (September-March) and at the end of the period, an individual growth index was calculated. Bulls with fast growth rates were sold at livestock auc- tion and bulls with growth indexes below the threshold, stated by the breeders’ organisations, were either slaugh- tered or returned to their owners. In total, 43 of the beef bulls that were sent to slaughter were included in this study (23 Charolaise, 7 Hereford, 6 Simmental, 3 Aberdeen Angus, 3 Limousine and 1 Blonde d’Aqui- taine). The daily growth rates of these bulls were some- what lower than the bulls sold at auction, but were still higher than the growth rates of non-tested beef sires in Sweden [26]. Because of co-operation with another study and their definite criteria [27], only bulls with clinically normal reproductive organs and scrotal cir- cumference above 30 cm were included. Testes and epididymides were removed at the time of slaughter and immediately put in a container with crushed ice and transported refrigerated to the laboratory where they arrived the next day. An incision (approx. 1.5 cm long and 0.5 cm deep) was made with a scalpel blade in the middle, distal part of the cauda epididymides [28], and a sample of 0.5 × 0.5 cm was taken and stored at -70°C until used for DNA preparation. In addition, blood samples for serum preparation were taken on arrival (6 month of age) and at departure (1 year of age) from the testing station. Sera were stored at -20°C. Dairy bulls Semen samples (0.2 ml payettes) and sera from 21 dairy bulls about 1 year old (Swedish Holstein and Swedish Red) in service, were submitted to the laboratory from one of the only two semen producing companies in Page 2 of 4 Sweden, Svensk Avel http://www.vikinggenetics.com. Semen samples were stored at -70°C prior to prepara- tion for analysis by real-time polymerase chain reaction (PCR). Sera for serology were stored at -20°C until analysed. Detection of Chlamydiaceae by real-time polymerase chain reaction DNA was extracted from semen and cauda epididymidis samples for PCR analysis using a High Pure Template Preparation kit, following manufacturer’s instructions (Roche Diagnostics, Basel, Switzerland) and stored at -20°C. Analyses were performed using a Chlamydiaceae- specific real-time PCR protocol developed by Everett and others [29], targeting the 23S ribosomal DNA. Briefly, the primers used were TQF (5’-GAA AAG AAC CCT TGT TAA GGG AG-3’) and TQR (5’-CTT AAC TCC CTG GCT CAT CAT G-3’). The sequence of the fluorescent FAM-labelled probe was 5’-CAA AAG GCA CGC CGT CAA C-3’. An internal amplification control (mimic) was con- structed and used to detect false negative PCR results, as previously described [30]. The primers used in the mimic producing PCR were TQFActin (5’-GAA AAG AAC CCT TGT TAA GGG AGC CAT GTA CCC TGG CAT TG-3’) and TQRActin (5’-CTT AAC TCC CTG GCT CAT CAT GGA TCC ACA CGG AGT ACT TGC-3’). The sequence of the ROX-labelled mimic probe used in real-time PCR was 5’-CCG ACA GGA TGC AGA AGG AGA TCA-3’. The 25-μl PCR mixture comprised 2.5 μl of 10× PCR- buffer II (Applied Biosystems, Foster City, CA, USA), 2.5 mM MgCl2, 0.2 mM of each of the four dNTP, 0.15 μM of each of the primer TQF and TQR, 0.25 μl (1.25 U) of AmpliTaq Gold DNA polymerase (Applied Biosys- tems), and 0.1 μM of each probe. Reaction mixtures were placed in a Rotor-Gene 3000 (Corbett Research, Cambridge, UK) and amplification was performed according to the protocol of Everett and others [29]. The results were analysed with the Rotor-Gene software version 5.0. The sensitivity of the PCR was estimated to one inclu- sion forming unit (IFU) per PCR by spiking semen and tissue samples prior to DNA extraction with ten-fold dilutions of Cp. abortus (inactivated strain S26/3 in ori- ginal concentration of 3 × 108 IFU/ml, kindly provided by D. Longbottom, Moredun Research Institute, UK). Detection of antibodies to Cp. abortus For detection of antibodies the Pourquier® ELISA Chla- mydophila abortus serum verification kit (Montpellier, France) was applied. The ELISA uses a recombinant fragment of an 80-90 kDa polymorphic outer membrane protein and detects antibodies against Cp. abortus. The assay was used according to the manufacturer’s instruc- tion with S/P% values ≥ than 100 as positive for cattle.