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.

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.

Influence of different centrifugation protocols on equine semen preservation.

Three experiments were conducted to evaluate the impact of centrifugation on cooled and frozen preservation of equine semen. A standard centrifugation protocol (600 x g for 10 min=CP1) was compared to four protocols with increasing g-force and decreased time period (600 x g, 1200 x g, 1800 x g and 2400 x g for 5 min for CP2, 3, 4, and 5, respectively) and to an uncentrifuged negative control. In experiment 1, the influence of the different CPs on sperm loss was evaluated by calculating the total number of sperm cells in 90% of the supernatant. Moreover, the effect on semen quality following centrifugation was assessed by monitoring several sperm parameters (membrane integrity using SYBR14-PI, acrosomal status using PSA-FITC, percentage total motility (TM), percentage progressive motility (PM) and beat cross frequency (BCF) obtained with computer assisted sperm analysis (CASA)) immediately after centrifugation and daily during chilled storage for 3 d. The use of CP1 resulted in a sperm loss of 22%. Increasing the centrifugation force to 1800 x g and 2400 x g for 5 min led to significantly lower sperm losses (7.4% and 2.1%, respectively; P<0.05). Compared to the uncentrifuged samples, centrifugation of semen resulted in a better sperm quality after chilled storage. There were minimal differences between the CPs although total motility was lower for CP2 than for the other treatments (P<0.005). In experiment 2, the centrifuged samples were cryopreserved using a standard freezing protocol and analyzed immediately upon thawing. Samples centrifuged according to CP2 resulted in a higher BCF (P<0.005), whereas CP3 and CP5 yielded a lower BCF (P<0.05) when compared to CP1. There were no post thaw differences between CP1 and CP4. In experiment 3, DNA integrity of the different samples was analyzed using TUNEL. Although DNA integrity decreased over time, CP had no impact. In conclusion, the loss of sperm cells in the supernatant after centrifugation can be substantially reduced by increasing the g-force up to 1800 x g or 2400 x g for a shorter period of time (5 min) compared to the standard protocol without apparent changes in semen quality, resulting in a considerable increase in the number of insemination doses per ejaculate.

Control of bovine virus diarrhoea at the herd level: reducing the risk of false negatives in the detection of persistently infected cattle.

The need to detect and eliminate cattle persistently infected (PI) with bovine viral diarrhoea virus (BVDV) is key to the control of BVD and has been shown to be very effective in eradicating BVDV from infected herds. However, because of pitfalls in the detection procedures, some PI animals can be missed and, as a result, are not identified and removal is delayed. The high prevalence of BVDV in cattle populations in some countries (such as Belgium and neighbouring countries) means there is a high risk of reinfection of a herd from which BVDV has been eradicated. Based on both practical experience and a literature study, this review considers those points that are critical to minimising the number of false negatives in the detection of PI cattle.

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.

Prevalence and distribution of mastitis pathogens in subclinically infected dairy cows in Flanders, Belgium.

The main objective was to determine the prevalence of intramammary infections (IMI) in dairy cows in Flanders, Belgium. Data were obtained from quarter milk samples of dairy herds subjected to a mandatory yearly screening of all lactating cows. A total of 178,668 quarter milk samples were collected at 1087 cross-sectional dairy herd screenings performed in three consecutive years. Of the dairy cows, 40% had at least one culture-positive quarter. More than 50% of all IMI were caused by non-aureus staphylococci. Streptococcus agalactiae is almost eradicated in Flanders, whereas Staphylococcus aureus was isolated from 18% of the culture-positive quarters. In addition, the distribution of mastitis pathogens in quarter milk samples from selected dairy cows with an elevated somatic cell count (SCC) is described. From 6390 cows with a geometric mean composite SCC 250,000 cells/ml, nearly 65% had at least one culture-positive quarter. The majority of the IMI were caused by non-aureus staphylococci (41.1%), whereas Staph. aureus and aesculin-positive cocci were found in respectively 25% and 18% of the culture-positive milk samples. We conclude that more efforts are needed in the prevention and control of subclinical mastitis in Flanders. Non-aureus staphylococci are the predominant cause of IMI, warranting more research regarding the epidemiology and pathogenicity of those species.

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.

Temporal detection of caspase-3 and -7 in bovine in vitro produced embryos of different developmental capacity.

Embryo quality is most frequently evaluated at the blastocyst stage, although quality parameters further back along the developmental axis, such as early developmental kinetics or oocyte quality, can be equally valuable. Despite the fact that previous studies in bovine have linked oocyte diameter and early developmental kinetics with blastocyst formation and viability, their relation with the incidence of apoptosis during embryo development remains relatively unexplored. Therefore, we related non-invasive parameters of oocyte and embryo quality, such as embryo kinetics, embryo morphology, and oocyte diameter, to the incidence of apoptosis throughout embryo development using fluorescent detection of active caspase-3 and -7. First, bovine in vitro embryos were selected according to developmental kinetics and morphology at four set times during culture and subjected to fluorescent detection of active caspase-3 and -7. Caspase activity was significantly higher in slow developing embryos in comparison with fast cleavers (P < 0.05), but was not related to embryo morphology. Second, bovine oocytes were divided into three groups on the basis of oocyte diameter and the resulting embryos were used for staining at the same four set times. Caspase activity was significantly higher in embryos derived from growing oocytes compared with those of fully grown oocytes at 45, 80, and 117 hours post-insemination (hpi; P < 0.05), but not at 168 hpi.

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.

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.

Comparison of transmission of Mycoplasma hyopneumoniae in vaccinated and non-vaccinated populations.

A transmission experiment was performed to quantify the effect of vaccination on the transmission of Mycoplasma hyopneumoniae (M. hyopneumoniae) in nursery piglets by means of an adjusted reproduction ratio (R(n)). Thirty piglets, vaccinated at 1 week of age, and 30 non-vaccinated piglets, free of M. hyopneumoniae, were housed in six separate pens. In each pen, three animals that were intratracheally inoculated with M. hyopneumoniae, were housed together with seven contact piglets during the conventional nursery period of 6 weeks. At the end of the study, the infectious status of the animals was determined based on results of nPCR performed on bronchoalveolar lavage fluid. The R(n)-value in the vaccinated group was 2.38 (1.07-7.53) while in the non-vaccinated group, an R(n)-value of 3.51 (1.51-9.34) was observed, both not significantly different from each other (p=0.77). Under the actual experimental conditions, transmission of M. hyopneumoniae in nursery piglets was only numerically lower in vaccinated groups. In addition, vaccination with a conventional vaccine could not prevent the establishment of M. hyopneumoniae organisms in the lung.

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%).

Aetiology and pathogenesis of cystic ovarian follicles in dairy cattle: a review.

Cystic ovarian follicles (COF) are an important ovarian dysfunction and a major cause of reproductive failure in dairy cattle. Due to the complexity of the disorder and the heterogeneity of the clinical signs, a clear definition is lacking. A follicle becomes cystic when it fails to ovulate and persists on the ovary. Despite an abundance of literature on the subject, the exact pathogenesis of COF is unclear. It is generally accepted that disruption of the hypothalamo-pituitary-gonadal axis, by endogenous and/or exogenous factors, causes cyst formation. Secretion of GnRH/LH from the hypothalamus-pituitary is aberrant, which is attributed to insensitivity of the hypothalamus-pituitary to the positive feedback effect of oestrogens. In addition, several factors can influence GnRH/LH release at the hypothalamo-pituitary level. At the ovarian level, cellular and molecular changes in the growing follicle may contribute to anovulation and cyst formation, but studying follicular changes prior to cyst formation remains extremely difficult. Differences in receptor expression between COF and dominant follicles may be an indication of the pathways involved in cyst formation. The genotypic and phenotypic link of COF with milk yield may be attributed to negative energy balance and the associated metabolic and hormonal adaptations. Altered metabolite and hormone concentrations may influence follicle growth and cyst development, both at the level of the hypothalamus-pituitary and the ovarian level.

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.

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.

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.

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.

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.