Transmission of Bluetongue Virus Serotype 8 by Artificial Insemination with Frozen-Thawed Semen from Naturally Infected Bulls.

Transmission of bluetongue (BT) virus serotype 8 (BTV-8) via artificial insemination of contaminated frozen semen from naturally infected bulls was investigated in two independent experiments. Healthy, BT negative heifers were hormonally synchronized and artificially inseminated at oestrus. In total, six groups of three heifers received semen from four batches derived from three bulls naturally infected with BTV-8. Each experiment included one control heifer that was not inseminated and that remained BT negative throughout. BTV viraemia and seroconversion were determined in 8 out of 18 inseminated heifers, and BTV was isolated from five of these animals. These eight heifers only displayed mild clinical signs of BT, if any at all, but six of them experienced pregnancy loss between weeks four and eight of gestation, and five of them became BT PCR and antibody positive. The other two infected heifers gave birth at term to two healthy and BT negative calves. The BT viral load varied among the semen batches used and this had a significant impact on the infection rate, the time of onset of viraemia post artificial insemination, and the gestational stage at which pregnancy loss occurred. These results, which confirm unusual features of BTV-8 infection, should not be extrapolated to infection with other BTV strains without thorough evaluation. This study also adds weight to the hypothesis that the re-emergence of BTV-8 in France in 2015 may be attributable to the use of contaminated bovine semen.

Methicillin-resistant Staphylococcus aureus in poultry.

Methicillin-resistant Staphylococcus aureus (MRSA) has been detected in several species and animal-derived products. To determine whether MRSA is present in poultry, we sampled 50 laying hens and 75 broiler chickens. MRSA was found in some broiler chickens but no laying hens. In all samples, spa type t1456 was found.

Teaching transrectal palpation of the internal genital organs in cattle.

In this article, a simulation model for rectal palpation teaching in cows, Breed'n Betsy, is evaluated. Furthermore, the learning process of rectal palpation is depicted during a training period in live cows. In experiment 1, eight students were trained in live cows (group A) and nine students were trained using Breed'n Betsy (group B). After 25 palpations, their ability to localize and evaluate structures was evaluated in practical tests in live cows. Group A had higher results than group B (p<0.001) and were more skilled at localizing the uterus and localizing and evaluating the ovaries (p<0.05). Group B was better at pregnancy diagnosis (nonsignificant). Results suggest that Breed'n Betsy cannot fully replace training in live cows, but may be a valuable addition to the classical teaching method. Suggestions for future improvement are made. In experiment 2, 10 students were intensely trained in live cows throughout the year and evaluated in practical tests at three time points (September, January, and March). Results were analyzed as a function of time point and the category of experience (1: 0-50 cows; 2: 50-100 cows; 3: 100-150 cows; 4: 150-200 cows; 5: >200 cows). Results increased in time (p<0.05) and were higher in categories 3, 4, and 5 than in category 1 (p<0.05). Although all of the students in the higher categories successfully localized the cervix, uterus, and ovaries, they had difficulties in interpreting these structures, suggesting that palpation of 200 cows is insufficient to reach a consistent level of expertise.

Accuracy of susceptibility testing of Pasteurella multocida and Mannheimia haemolytica.

The accuracy of antimicrobial susceptibility determinations relies on the bacterial species identification and the test methodology that is being used. In the present study, both aspects were investigated for 141 Pasteurella multocida and 34 Mannheimia haemolytica sensu lato isolates recovered from the upper respiratory tract of healthy calves. This was performed on the one hand by comparing of classical phenotyping with tDNA-PCR genotyping and on the other hand by pairwise comparison of disk diffusion with agar dilution results for seven antimicrobial compounds (ampicillin, ceftiofur, oxytetracycline, gentamicin, florfenicol, enrofloxacin, and the combination trimethoprim-sulfonamides). Phenotyping and genotyping correlated well (>90%). The pairwise comparisons of the susceptibility methods were investigated traditionally by means of error binding rates and sensitivity and specificity test characteristics. Obtained sensitivities (indication for absence of false susceptible results) were often lower than 85%, especially for older antimicrobial agents (oxytetracycline, gentamicin, trimethoprim-sulfonamides) and when M. haemolytica sensu lato was considered. Specificities (indication for absence of false-resistant results) exceeded 90% for almost all antimicrobial-bacterial combinations. The calculated test characteristics (sensitivities and specificities) were subsequently used in a second dataset of Pasteurellaceae from intensively (n = 99) and extensively housed calves (n = 196), to modify the apparent prevalence of antimicrobial resistance based upon disk diffusion results into an estimated true prevalence. It was concluded that the disk diffusion method is reliable in epidemiological studies like surveillance programs if resistance is sparse, whereas it needs to be interpreted with caution in situations where resistance is abundantly present.

Antimicrobial resistance patterns of Escherichia coli through the digestive tract of veal calves.

The aim of the present study was to evaluate differences in resistance patterns of Escherichia coli in different parts of the digestive tract of veal calves. Therefore, after slaughter, the lower intestinal tract of 19 calves was sampled at five locations (duodenum, jejunum, cecum, colon, and rectum), and up to three E. coli isolates per sample underwent susceptibility testing for seven antimicrobial agents (gentamicin, amoxycillin + clavulanic acid, tetracycline, trimethoprim + sulfamethoxazole, ampicillin, nalidixic acid, and enrofloxacin), using the Kirby-Bauer disk diffusion method. Multiresistance (resistance to more than two compounds) was present in 93.5% of all isolates (n = 179). For gentamicin, nalidixic acid, and enrofloxacin, the percentage of resistant E. coli isolates was significantly lower in the duodenum and jejunum than in the cecum, colon, and rectum. For ampicillin, the percentage of resistance was significantly lower in the jejunum, compared to the other segments of the intestinal tract. For the other antimicrobials tested, no significant differences in the percentage of resistant isolates throughout the intestinal tract were detected. In conclusion, resistance among enteric E. coli from veal calves can reach high levels and prevalence depends on localization of sampling. These considerations should be taken into account when further fine-tuning sampling protocols for indicator bacteria.

Recovery of Moraxella ovis from the bovine respiratory tract and differentiation of Moraxella species by tDNA-intergenic spacer PCR.

The purpose of the present study was to identify Moraxella (M.)--like organisms recovered from calves suffering from respiratory disease down to species level by means of tDNA-intergenic spacer length polymorphism analysis (tDNA-PCR), and to perform antimicrobial susceptibility testing of these isolates using an agar dilution technique. A total of 16 isolates originating from 12 unrelated occasions were identified as Moraxella ovis, and tDNA fingerprinting showed clear delineation from other Moraxella species. The minimal inhibitory concentrations (in microg/mL) for 90% of the investigated isolates were < or =0.03 for ampicillin; 0.25 for ceftiofur; 0.5 for oxytetracycline; 8 for gentamicin; 64 for spectinomycin; 0.5/9.5 for the combination trimethoprim-sulfonamides; 4 for erythromycin; 8 for tilmicosin; 1 for florfenicol and 0.125 for enrofloxacin.

Effect of culture-medium supplementation with alpha-mannosidase and/or beta-N-acetyloglucosaminidase on in vitro bovine embryonic development.

Glycosidases are enzymes with a potential role in embryonic development. The objectives of this study were to assess: (a) whether in vitro bovine embryonic development is affected by the addition of beta-N-acetyloglucosaminidase (beta-NAGASE) and/or alpha-mannosidase to the culture medium and (b) whether these enzymes are utilized by bovine embryos during their development in vitro. Bovine embryos were produced using standard methods of IVM, IVF and IVC. Presumptive zygotes were cultured in groups of 20 in 50 microl drops of SOF medium (plus 5% FBS after 24 h culture) incubated in 5% CO2, 5% O2 and 90% N2 at 38.5 degrees C. The groups of zygotes were allocated to four treatments in which the culture medium was supplemented with: (1) beta-NAGASE, (2) alpha-mannosidase, (3) beta-NAGASE plus alpha-mannosidase, and (4) control (no supplement). Embryos were evaluated and samples of culture medium collected and frozen prior to assay for glycosidases at day 7 of culture. The experimental design was a randomised block arrangement of 4 treatments x 7 replicates with 20 zygotes per plot (culture droplet). Data were analysed by ANOVA and presented as mean +/- S.E.M. The osmolarity of the control culture medium was 272 mOsm. This was increased to 279 mOsm by the addition of alpha-mannosidase, 424 mOsm by beta-NAGASE and 337 mOsm with a combination of the two enzymes. The beta-NAGASE supplemented medium and the combined supplement reduced (0%) the development of zygotes to morula or blastocyst stages (P < 0.002) relative to control medium (35.7 +/- 8.4%). Embryo development was also reduced to 21.9 +/- 3.2 (P< 0.002), relative to control, by alpha-mannosidase supplementation. The reduced embryo development in the beta-NAGASE-supplemented medium was attributed to increased osmolarity of the culture medium. Embryos appeared to utilize alpha-mannosidase because its concentration decreased from 600.95 +/- 174.03 IU/l in drops without zygotes/embryos to 211.01 +/- 71.59 IU/l in drops with zygotes/embryos. Other culture media supplementation showed no significant differences between droplets, with or without zygotes/embryos. It was concluded that beta-NAGASE increased medium osmolarity, embryos utilized alpha-mannosidase and both glycosidases (singly or in combination) inhibited the development of bovine zygotes to morulae/blastocysts.

Comparison of molecular techniques for the typing of Mycoplasma hyopneumoniae isolates.

In this study, we compared the potential of amplified fragment length polymorphism (AFLP), random amplified polymorphic DNA (RAPD) analysis, restriction fragment length polymorphism (RFLP) of the gene encoding lipoprotein P146, and the variable number of tandem repeats (VNTR) of the P97 encoding gene, as possible methods for typing an international collection of Mycoplasma hyopneumoniae isolates. All techniques showed a typeability of 100% and high intraspecific diversity. However, the discriminatory power of the different techniques varied considerably. AFLP (>0.99) and PCR-RFLP of the P146 encoding gene (>0.98) were more discriminatory than RAPD (0.95) and estimation of the VNTR of P97 (<0.92). Other, preferentially well spread, tandem repeat regions should be included in order for this latter technique to become valuable for typing purposes. RAPD was also found to be a less interesting typing technique because of its low reproducibility between different runs. Nevertheless, all molecular techniques showed overall more resemblance between strains isolated from different pigs from the same herd. On the other hand, none of the techniques was able to show a clear relationship between the country of origin and the fingerprints obtained. We conclude that AFLP and an earlier described PFGE technique are highly reliable and discriminatory typing techniques for outlining the genomic diversity of M. hyopneumoniae isolates. Our data also show that RFLP of a highly variable gene encoding P146 may be an equally useful alternative for demonstrating intraspecific variability, although the generation of sequence variability of the gene remains unclear and must be further examined.

Quantification and evaluation of antimicrobial drug use in group treatments for fattening pigs in Belgium.

To control the emergence of antimicrobial resistance, knowledge of antimicrobial drug consumption is essential. Because consumption data are not available in Belgium, a study was conducted between March and October 2003 to investigate the antimicrobial drug consumption in pigs, using the treatment incidence based on the animal daily dose pig (ADDpig), the treatment incidence based on the used daily dose pig (UDDpig) (number of ADDpig or UDDpig/1,000 pigs at risk/day), and the ratio UDDpig/ADDpig. The sampling frame consisted of 821 pig herds that (a) used a closed or semi-closed production system, (b) were located in the most dense pig areas of Belgium, and (c) had at least 150 sows and 600 fattening pigs each. Of 50 randomly selected herds, all group treatments with antimicrobial drugs, applied to fattening pigs that were within 2 weeks of slaughter (median age 187 days), were collected retrospectively. The treatment incidence based on ADDpig for all oral and injectable antimicrobial drugs was 178.1 per 1,000 pigs at risk per day. The treatment incidence based on UDDpig shows that in reality fewer pigs were treated, namely 170.3 per 1,000 pigs at risk per day. Proportionally, the most often applied oral antimicrobial drugs were: doxycycline, amoxicillin, combination trimethoprim-sulphonamides and polymyxin E. The most often applied injectable antimicrobial drugs were long-acting amoxicillin and ceftiofur. The distribution of the UDDpig/ADDpig ratio per antimicrobial drug shows that 50-75% of the oral formulations were underdosed. Injectable formulations were almost always overdosed (>90%).

Is apoptosis in bovine in vitro produced embryos related to early developmental kinetics and in vivo bull fertility?

Although several studies have indicated a paternal effect on bovine embryo development, no conclusive data exist on the effect of in vivo bull fertility on apoptosis. Therefore, it was the main objective of this study to compare the apoptotic cell ratio (ACR) in embryos originating from bulls with different in vivo fertility. However, since it is has been demonstrated before that bulls with different in vivo fertility differ in timing of first cleavage, it was necessary to investigate first the effect of timing of development on apoptosis in vitro in order to get an unbiased insight in the contribution of in vivo bull fertility on apoptosis in bovine blastocysts. In the first experiment, bovine embryos (n = 939) were allocated to different groups according to cleavage rate at 30, 36 and 48 hpi and blastocysts were selected at 7 and 8 dpi. The blastocyst rate at 7 dpi was significantly lower in embryos which had first cleaved at 48 hpi than in embryos from the 30 and 36 hpi group (P < 0.05). The ACR after TUNEL in day 7 blastocyst was significantly lower in the 30 hpi group in comparison with the 36 and 48 hpi group (P < 0.05) and lower in day 7 blastocysts than in day 8 blastocysts. In the second experiment, sperm of eight bulls with different non return rates was used for in vitro bovine embryo production (n = 3820 oocytes). Cleavage rates (30, 36 and 48 hpi) and blastocyst rate (7 dpi) were determined. Only very low negative correlations could be found between in vivo and in vitro bull fertility and ACR did not differ between groups derived from sires with either low or normal fertility (P > 0.05). Further research in serum free conditions is needed to confirm that the lower ACR in early cleaved embryos could be mediated by the cooperative interaction of embryos of good quality cultured in group. In vivo bull fertility could hardly be correlated with in vitro blastocyst yield and could not be correlated with appearance of apoptosis.

Effect of Antimicrobial Consumption and Production Type on Antibacterial Resistance in the Bovine Respiratory and Digestive Tract.

The aim of this study was to investigate the relationship between antimicrobial use and the occurrence of antimicrobial resistance in the digestive and respiratory tract in three different production systems of food producing animals. A longitudinal study was set up in 25 Belgian bovine herds (10 dairy, 10 beef, and 5 veal herds) for a 2 year monitoring of antimicrobial susceptibilities in E. coli and Pasteurellaceae retrieved from the rectum and the nasal cavity, respectively. During the first year of observation, the antimicrobial use was prospectively recorded on 15 of these farms (5 of each production type) and transformed into the treatment incidences according to the (animal) defined daily dose (TIADD) and (actually) used daily dose (TIUDD). Antimicrobial resistance rates of 4,174 E. coli (all herds) and 474 Pasteurellaceae (beef and veal herds only) isolates for 12 antimicrobial agents demonstrated large differences between intensively reared veal calves (abundant and inconstant) and more extensively reared dairy and beef cattle (sparse and relatively stable). Using linear mixed effect models, a strong relation was found between antimicrobial treatment incidences and resistance profiles of 1,639 E. coli strains (p<0.0001) and 309 Pasteurellaceae (p≤0.012). These results indicate that a high antimicrobial selection pressure, here found to be represented by low dosages of oral prophylactic and therapeutic group medication, converts not only the commensal microbiota from the digestive tract but also the opportunistic pathogenic bacteria in the respiratory tract into reservoirs of multi-resistance.

Characterization of In Vivo acquired resistance of Mycoplasma hyopneumoniae to macrolides and lincosamides.

Macrolides and related antibiotics are used to control mycoplasma infections in the pig industry worldwide. Some porcine mycoplasmas, however, survive these treatments by acquiring resistance. The mechanism of acquired resistance to macrolides and lincosamides was studied in more detail for Mycoplasma hyopneumoniae by comparing both the phenotype and genotype of a resistant field isolate to five susceptible isolates. The MICs were significantly higher for the resistant strain for all antibiotics tested. The MICs for the 16-membered macrolide tylosin ranged from 8 to 16 microg for the resistant strain and from 0.03 to 0.125 microg/ml for the five susceptible strains. The MICs for the 15-membered macrolides and lincosamides were higher than 64 microg/ml for the resistant strain while only 0.06 to 0.5 microg/ml for the susceptible strains. Mycoplasma hyopneumoniae strains are intrinsically resistant to the 14-membered macrolides due to a G 2057 A transition (E. coli numbering) in their 23S rDNA. Therefore, high MICs were observed for all strains, although the MICs for the resistant strain were clearly increased. An additional, acquired A 2058 G point mutation was found in the 23S rRNA gene of the resistant strain. No differences linked to resistance were found in the ribosomal proteins L4 and L22. The present study showed that 23S rRNA mutations resulting in resistance to macrolides and lincosamides as described in other Mycoplasma spp. also occur under field conditions in M. hyopneumoniae.

Evaluation of amplified rDNA restriction analysis (ARDRA) for the identification of Mycoplasma species.

BACKGROUND: Mycoplasmas are present worldwide in a large number of animal hosts. Due to their small genome and parasitic lifestyle, Mycoplasma spp. require complex isolation media. Nevertheless, already over 100 different species have been identified and characterized and their number increases as more hosts are sampled. We studied the applicability of amplified rDNA restriction analysis (ARDRA) for the identification of all 116 acknowledged Mycoplasma species and subspecies. METHODS: Based upon available 16S rDNA sequences, we calculated and compared theoretical ARDRA profiles. To check the validity of these theoretically calculated profiles, we performed ARDRA on 60 strains of 27 different species and subspecies of the genus Mycoplasma. RESULTS: In silico digestion with the restriction endonuclease AluI (AG--CT) was found to be most discriminative and generated from 3 to 13 fragments depending on the Mycoplasma species. Although 73 Mycoplasma species could be differentiated using AluI, other species gave undistinguishable patterns. For these, an additional restriction digestion, typically with BfaI (C--TAG) or HpyF10VI (GCNNNNN--NNGC), was needed for a final identification. All in vitro obtained restriction profiles were in accordance with the calculated fragments based on only one 16S rDNA sequence, except for two isolates of M. columbinum and two isolates of the M. mycoides cluster, for which correct ARDRA profiles were only obtained if the sequences of both rrn operons were taken into account. CONCLUSION: Theoretically, restriction digestion of the amplified rDNA was found to enable differentiation of all described Mycoplasma species and this could be confirmed by application of ARDRA on a total of 27 species and subspecies.

The diversity of Mycoplasma hyopneumoniae within and between herds using pulsed-field gel electrophoresis.

Over the years, pulsed-field gel electrophoresis (PFGE) has been proven a robust technique to type isolates with a high resolution and a good reproducibility. In this study, a PFGE protocol is described for the typing of Mycoplasma hyopneumoniae isolates. The potential of this technique was demonstrated by comparing M. hyopneumoniae isolates obtained from the same as well as from different herds. The use of two different restriction enzymes, SalI and ApaI, was evaluated. For each enzyme, the resulting restriction profiles were clustered using the unweighted pair group method with arithmetic means (UPGMA). For both obtained dendrograms, the included isolates of the related M. flocculare species clustered separately from all M. hyopneumoniae isolates, forming the root of the dendrograms. The PFGE patterns of the M. hyopneumoniae isolates of different herds were highly diverse and clustered differently in both dendrograms, illustrated by a Pearson's correlation coefficient of only 0.33. A much higher similarity was observed with isolates originating from different pigs of a same herd. The PFGE patterns of these isolates always clustered according to their herd and this for both dendrograms. In conclusion, the results indicate a closer relationship of M. hyopneumoniae isolates within a herd compared to isolates from different herds and this for both restriction enzymes used. Since the described PFGE technique was shown to be highly discriminative and reproducible, it will be a helpful tool to further elucidate the epidemiology of M. hyopneumoniae.

Distribution of salmonella strains in farrow-to-finish pig herds: a longitudinal study.

The aims of this study were to investigate patterns of Salmonella shedding in finishing pigs and to study the role of the sow in the transmission of Salmonella to her offspring. In each of the three herds (A, B, and C), one cohort of sows (n = 34, n = 40, n = 32, respectively) together with three piglets of their offspring (n = 102, n = 120, n = 96, respectively) were selected. Individual fecal and blood samples were taken from the sows at different times during one production cycle and from the piglets from weaning until slaughter. At slaughter, contents from the jejunum, colon, and mesenteric lymph nodes were collected. Fecal samples, as well as the jejunum, colon, and mesenteric lymph node samples collected at slaughter, were submitted to a qualitative Salmonella analysis. Isolates were characterized by random amplified polymorphic DNA, and if necessary, further characterization was done by pulsed-field gel electrophoresis. In herds A and B, Salmonella shedding began in the nursery. A significant increase in the number of Salmonella shedders was seen after transferring pigs to the growing unit in herd B (P = 0.003) and to the finishing unit in herds A (P < 0.001) and B (P = 0.013). None of the fattening pigs in herd C were shedding Salmonella. This study reveals that transferring pigs is an important trigger to induce Salmonella shedding, leading to horizontal spread. Direct transmission of Salmonella from the sows to their piglets could not be demonstrated, but the similarities between the isolates found in the sows and those found during the nursery and finishing periods and at slaughter suggested indirect transmission.

Comparison of three diluents for the storage of fresh bovine semen.

In New Zealand, 95% of the semen used for artificial insemination in cattle is processed as liquid semen. Storage of liquid semen for up to 3 days in Caprogen) diluent enables a 10-fold reduction of the insemination dose, compared to frozen-thawed semen, without a reduction in fertility. In this Caprogen) diluent spermatozoa are stored under N2 gas in the presence of catalase. However, a new diluent (CEP-2), which was originally based on the biochemical composition of bovine cauda epididymal plasma, could become an appropriate alternative to Caprogen. In this study, the effect of addition of catalase to bovine spermatozoa stored for 6 days in CEP-2 diluent under aerobic and anaerobic conditions was evaluated and compared with a Tris diluent. Additionally, the quality and in vitro fertilizing capacity of fresh bovine semen stored for 6 days at 5 degrees C in the Triladyl, CEP-2 (without catalase and N2 gas) and Caprogen diluent were compared. Addition of 4.5 mg/mL catalase to CEP-2 diluent under aerobic and anaerobic conditions had no effect on sperm quality. Spermatozoa stored in CEP-2 diluent moved faster and straighter than spermatozoa stored in Triladyl or Caprogen diluent. The in vitro fertilization and polyspermy rates did not differ significantly between spermatozoa stored for 6 days at 5 degrees C in CEP-2 and Caprogen diluent, but were significantly lower for spermatozoa stored in Triladyl diluent. We can conclude that based on the in vitro results, the CEP-2 diluent is a better diluent than Triladyl and a good alternative to the Caprogen diluent for long term storage of fresh bovine semen at 5 degrees C. To confirm these promising in vitro results further in vivo experiments are required.

Low dose insemination in cattle with the Ghent device.

A new artificial insemination device for semen deposition near the uterotubal junction (UTJ) in cattle (Ghent device) was developed at Ghent University (Belgium). In this study, UTJ insemination of dairy cows with the Ghent device was compared with the conventional insemination technique to evaluate the effect on pregnancy rates after insemination with different doses of semen. In each of three field trials, the cows (n=795, 659, 360) and heifers (n=253, 182, 231) were randomly assigned to receive 12 million sperm deposited in the uterine body using conventional techniques (control) or a reduced sperm dose (RSD) deposited in the same manner as the control or bilateral deposition near the uterotubal junction using the Ghent device (Ghent). Sperm dosages for RSD and Ghent inseminations were 8, 4, and 2 million sperm for field trials 1-3, respectively. In the multivariable analysis, the pregnancy rates were significantly affected by the parity of the cow (p

Use of a homologous radioimmunoassay (RIA) to evaluate the effect of maternal and foetal parameters on pregnancy-associated glycoprotein (PAG) concentrations in sheep.

Early pregnancy detection and prediction of the number of lambs would be profitable for sheep breeders because it enables them to adjust nourishment of pregnant ewes according to the individual needs in order to prevent health problems around parturition. The concentration of ovPAG has previously been reported to be related with maternal parameters (farm, breed and age) as well as foetal parameters (number of lambs, their sex and birth weight), but contradictory results were obtained in different small-scale studies. This large-scale study evaluates the effect of these parameters on the ovPAG concentration, determined by a homologous radioimmunoassay (RIA), and it further investigates the possibility to predict the number of lambs by means of homologous ovPAG determination. Eighty-three and ninety-five ewes of the Suffolk and Texel breed, respectively, housed on four different farms (experiment 1) and 68 ewes of the Suffolk breed, housed on two different farms (experiment 2) were included in this study, and their estrous cycles were synchronised using a progesterone analogue. On the day of synchronisation (D-14) and at 25 (D25), 35 (D35) and 45 (D45) days after insemination, blood samples were taken and a homologous radioimmunoassay (RIA) was used to determine the ovPAG concentrations. At parturition, age of the ewe, number and sex of the lambs (experiment 1) and birth weight (experiment 2) were registered. OvPAG concentrations were not affected by age of the ewe and sex of the lambs. Farm and breed of the ewes, number and birth weight of the lambs had a significant effect on ovPAG concentrations at all time points (P<0.05). The odds of multiple lambs increased significantly with increasing ovPAG concentration, although prediction of litter size based on ovPAG concentration at the individual ewe level was not useful due to small sensitivity and/or specificity whatever the cutoff value used. In conclusion, the ovPAG concentration is affected by farm and breed of the ewes, and number and birth weight of the lambs.

Analytical performance of several classical swine fever laboratory diagnostic techniques on live animals for detection of infection.

The diagnostic properties of several assays on live animals were studied using data from different experiments. These experiments involved 128 classical swine fever virus (CSFV) infected pigs (weaner pigs, fatteners and sows). Since all pigs in the study were infected with CSFV, only the proportion of test positive results and the time until a test positive result is obtained were evaluated. The RT-nPCR detected the highest proportion of infected pigs (98.9%), whereas the Antigen ELISA gave the worst detection results (74.7%). Within the group of test positive animals, infection was detected earliest using the leukocyte count and latest using Antigen ELISA. Using the virus neutralisation test, antibodies against CSFV were detectable on average 7.6 days after the onset of viraemia in virus isolation in whole blood. Using survival analysis, the time until the first positive diagnosis and the proportion of detected animals were combined in one test. Results showed that RT-nPCR performed significantly better than either virus isolation in different blood fractions or antigen ELISA. It is concluded that the RT-nPCR technique is the best diagnostic tool available for early detection of a classical swine fever infection.

Hormonal regulation of bovine secretory proteins derived from caput and cauda epididymal epithelial cell cultures.

The goal of this study was to investigate the effect of hormones (testosterone, dihydrotestosterone [DHT], and hydrocortisone) on the protein secretion of caput and cauda epididymal epithelial cells cultured in principal cell medium (PCM). A confluent monolayer of caput and cauda epididymal epithelial cells was obtained from serum-containing PCM in the presence or absence of hormones after 7 days of culture at 38.5 degrees C (5% CO(2) in air). The protein secretion of epididymal epithelial monolayers incubated in serum-free PCM for 3 days was examined. The secreted proteins were separated by 2-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D SDS-PAGE). A comparison of the different protein patterns showed 61 spots, of which 11 were secreted only in the presence of hormones, 3 appeared to show hormone-related changes, and 25 were region-specific. Most of these secreted proteins were low-molecular-weight acidic proteins. To obtain evidence of the epididymal origin of the secreted proteins, proteins present in caput and cauda epididymal plasma were analyzed. In conclusion, our data indicate that hormones influence the synthesis of a number of caput and cauda epididymal proteins. Some of these proteins could be important for improving our understanding of spermatozoa maturation and storage and their acquisition of fertilizing ability.