A Unique Capsule Locus in the Newly Designated Actinobacillus pleuropneumoniae Serovar 16 and Development of a Diagnostic PCR Assay

Actinobacillus pleuropneumoniae causes pleuropneumonia, an economically significant lung disease of pigs. Recently, isolates of A. pleuropneumoniae that were serologically distinct from the previously characterized 15 serovars were described, and a proposal was put forward that they comprised a new serovar, serovar 16. Here we used whole-genome sequencing of the proposed serovar 16 reference strain A-85/14 to confirm the presence of a unique capsular polysaccharide biosynthetic locus. For molecular diagnostics, primers were designed from the capsule locus of strain A-85/14, and a PCR was formulated that differentiated serovar 16 isolates from all 15 known serovars and other common respiratory pathogenic/commensal bacteria of pigs. Analysis of the capsule locus of strain A-85/14 combined with the previous serological data show the existence of a sixteenth serovar—designated serovar 16—of A. pleuropneumoniae

The effect of a diet with fructan-rich chicory roots on intestinal helminths and microbiota with special focus on Bifidobacteria and Campylobacter in piglets around weaning

The restrictions on the use of antibiotic and anthelmintic treatments in organic pig farming necessitate alternative non-medical control strategies. Therefore, the antibiotic and parasite-reducing effect of a fructan-rich (prebiotic) diet of dried chicory was investigated in free-ranging piglets. Approximately half of 67 piglets from 9 litters were experimentally infected with Ascaris suum and Trichuris suis in the suckling period (1 to 7 weeks of age) and 58 of the piglets were challenged daily with E. coli O138:F8 for 9 days after weaning to induce weaning diarrhoea. The litters were fed either chicory (30% DM) or a control diet. The effect of chicory on intestinal helminths, intestinal microbiota, especially Bifidobacteria and Campylobacter spp., and E. coli post-weaning diarrhoea was assessed. The weight gain of the piglets was not impaired significantly by chicory. The intestinal A. suum worm burden was reduced by 64% (P=0.034) in the chicory-fed piglets, whereas these same piglets had 63% more T. suis worms (P=0.016). Feeding with chicory elicited no changes among the main bacterial groups in ileum according to terminal restriction fragment length polymorphism (T-RFLP) analysis. However, the terminal-restriction fragment (T-RF) 208 bp, which may belong to Lachnospiraceae, was stimulated by the chicory feed (P=0.03), and T-RF 370 bp that matches Enterobacter belonging to the Enterobacteria was reduced (P=0.004). Additionally, chicory increased the level of Bifidobacteria (P=0.001) and the faecal Campylobacter excretion level was transitorily reduced in chicory-fed piglets at 7 weeks of age (P=0.029). Unfortunately, it was not possible to assess the effect of chicory on post-weaning diarrhoea as it did not develop. In conclusion, feeding piglets chicory around the time of weaning caused complex changes of the microbiota and parasite communities within the intestinal tract, and feeding piglets chicory may therefore serve as an animal-friendly strategy to control pathogens

Genetic diversity of Actinobacillus lignieresii isolates from different hosts

Genetic diversity detected by analysis of amplified fragment length polymorphisms (AFLPs) of 54 Actinobacilus lignieresii isolates from different hosts and geographic localities is described. On the basis of variances in AFLP profiles, the strains were grouped in two major clusters; one comprising strains isolated from horses and infected wounds of humans bitten by horses and another consisting of strains isolated from bovine and ovine hosts. The present data indicate a comparatively higher degree of genetic diversity among strains isolated from equine hosts and confirm the existence of a separate genomospecies for A. lignieresi-like isolates from horses. Among the isolates from bovine and ovine hosts some clonal lines appear to be genetically stable over time and could be detected at very distant geographic localities. Although all ovine strains investigated grouped in a single cluster, the existence of distinct genetic lineages that have evolved specificity for ovine hosts is not obvious and needs to be confirmed in other studies

Association between antibodies to Coxiella burnetii in bulk tank milk and perinatal mortality of Danish dairy calves

<p>Abstract</p> <p>Background</p> <p><it>Coxiella burnetii </it>is a well-known cause of placentitis and subsequent abortion in ruminants, but there are no reports on the relationship with perinatal mortality. The study was performed to determine the influence of level and change of bulk tank milk (BTM) antibodies to <it>C. burnetii </it>on two outcomes associated with parturition in cattle: a) stillbirth; and b) stillbirth and neonatal mortality combined (perinatal death).</p> <p>Methods</p> <p>Twenty-four Danish dairy herds were tested repeatedly for antibodies to <it>C. burnetii </it>in BTM using a commercial ELISA. Samples were collected monthly from July 2008 to July 2009. Information on the 2,362 calvings occurring in the study period was obtained from the Danish Cattle Database. Two multilevel logistic regression models were created for the two outcomes stillbirth and perinatal mortality. One model included the level of BTM antibodies in a specified period before or after the outcome had occurred. The other model included the change in antibodies over time. These predictors were included both at herd and animal level. Furthermore, all models included parity and breed.</p> <p>Results</p> <p>The individual monthly BTM antibody levels were highly correlated within herds. Consequently, changes in BTM antibody levels were not found to be associated with neither risk of stillbirth nor the risk of perinatal mortality. However, the risk of stillborn calves and perinatal death was higher with high level of BTM antibodies 8 to 9 months after the incident, but not outside this period.</p> <p>Conclusion</p> <p>We conclude that the level of antibodies to <it>C. burnetii </it>in BTM may be associated with perinatal mortality, but the association was not persistent and should be investigated further.</p

Association between average daily gain, faecal dry matter content and concentration of Lawsonia intracellularis in faeces

<p>Abstract</p> <p>Background</p> <p>The objective of this study was to investigate the association between average daily gain and the number of <it>Lawsonia intracellularis</it> bacteria in faeces of growing pigs with different levels of diarrhoea.</p> <p>Methods</p> <p>A longitudinal field study (<it>n</it> = 150 pigs) was performed in a Danish herd from day 29 to 47 post weaning. Every third day all pigs were weighed, subjected to a clinical examination and faecal samples were obtained. Faecal samples were subjected to dry matter determination and absolute quantification by PCR for <it>L. intracellularis</it> and porcine circovirus type 2 (PCV2). Association between average daily gain, faecal dry matter content, numbers of <it>L. intracellularis</it> bacteria and PCV2 genome copies in faeces was investigated in a multilevel mixed-effects linear model.</p> <p>Results</p> <p>Increasing numbers of <it>L. intracellularis</it> log₁₀ bacteria/g faeces were significantly associated with decreasing average daily gain (<it>P</it> < 0.001). The association was decreasing with increasing faecal dry matter content (<it>P</it> < 0.01). The number of PCV2 log₁₀ copies/g faeces was not significantly associated with average daily gain of the pigs (<it>P</it> > 0.5).</p> <p>Conclusion</p> <p>The results suggest a potential application of a PCR quantifying <it>L. intracellularis</it> in growing pigs. Faecal dry matter content must be taken into consideration in interpretation of such test results.</p

Haemophilus parasuis molecular serotyping assay

Haemophilus parasuis causes Glässer's disease and pneumonia in pigs. Indirect hemagglutination (IHA) is typically used to serotype this bacterium, distinguishing 15 serovars with some nontypeable isolates. The capsule loci of the 15 reference strains have been annotated, and significant genetic variation was identified between serovars, with the exception of serovars 5 and 12. A capsule locus and in silico serovar were identified for all but two nontypeable isolates in our collection of >200 isolates. Here, we describe the development of a multiplex PCR, based on variation within the capsule loci of the 15 serovars of H. parasuis, for rapid molecular serotyping. The multiplex PCR (mPCR) distinguished between all previously described serovars except 5 and 12, which were detected by the same pair of primers. The detection limit of the mPCR was 4.29 × 10(5) ng/μl bacterial genomic DNA, and high specificity was indicated by the absence of reactivity against closely related commensal Pasteurellaceae and other bacterial pathogens of pigs. A subset of 150 isolates from a previously sequenced H. parasuis collection was used to validate the mPCR with 100% accuracy compared to the in silico results. In addition, the two in silico-nontypeable isolates were typeable using the mPCR. A further 84 isolates were analyzed by mPCR and compared to the IHA serotyping results with 90% concordance (excluding those that were nontypeable by IHA). The mPCR was faster, more sensitive, and more specific than IHA, enabling the differentiation of 14 of the 15 serovars of H. parasuis.This work was supported by a BPEX PhD studentship and a Longer and Larger (LoLa) grant from the Biotechnology and Biological Sciences Research Council (grant numbers BB/G020744/1, BB/G019177/1, BB/G019274/1 and BB/G003203/1), the UK Department for Environment, Food and Rural Affairs and Zoetis, awarded to the Bacterial Respiratory Diseases of Pigs-1 Technology (BRaDP1T) consortium. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.This is the author accepted manuscript. The final version is available from American Society for Microbiology via http://dx.doi.org/10.1128/JCM.01991-1

Improving validity of informed consent for biomedical research in Zambia using a laboratory exposure intervention.

BACKGROUND: Complex biomedical research can lead to disquiet in communities with limited exposure to scientific discussions, leading to rumours or to high drop-out rates. We set out to test an intervention designed to address apprehensions commonly encountered in a community where literacy is uncommon, and where complex biomedical research has been conducted for over a decade. We aimed to determine if it could improve the validity of consent. METHODS: Data were collected using focus group discussions, key informant interviews and observations. We designed an intervention that exposed participants to a detailed demonstration of laboratory processes. Each group was interviewed twice in a day, before and after exposure to the intervention in order to assess changes in their views. RESULTS: Factors that motivated people to participate in invasive biomedical research included a desire to stay healthy because of the screening during the recruitment process, regular advice from doctors, free medical services, and trust in the researchers. Inhibiting factors were limited knowledge about samples taken from their bodies during endoscopic procedures, the impact of endoscopy on the function of internal organs, and concerns about the use of biomedical samples. The belief that blood can be used for Satanic practices also created insecurities about drawing of blood samples. Further inhibiting factors included a fear of being labelled as HIV positive if known to consult heath workers repeatedly, and gender inequality. Concerns about the use and storage of blood and tissue samples were overcome by a laboratory exposure intervention. CONCLUSION: Selecting a group of members from target community and engaging them in a laboratory exposure intervention could be a useful tool for enhancing specific aspects of consent for biomedical research. Further work is needed to determine the extent to which improved understanding permeates beyond the immediate group participating in the intervention

Experimental infection of sheep with ovine and bovine Dichelobacter nodosus isolates

AbstractThe aim of this study was, under experimental conditions, to investigate infection of Norwegian White sheep with ovine and bovine isolates of Dichelobacter nodosus of varying virulence. In addition, the efficacy of gamithromycin as a treatment for the experimentally induced infections was examined. The study was performed as a single foot inoculation using a boot. Four groups, each with six lambs, were inoculated with four different challenge strains (Group 1: benign bovine strain; Group 2: virulent bovine strain; Group 3: benign ovine strain; Group 4: virulent ovine strain). The main criterion to determine that infection was transferred was that D. nodosus isolate was obtained by culture. After the trial all lambs were treated with gamithromycin. Clinical symptoms of footrot developed in all groups, and when removing the boots two weeks after challenge, D. nodosus was isolated from 5 of 24 experimental lambs. All lambs tested negative for D. nodosus by PCR within six weeks after treatment with gamithromycin. This study strongly indicates that D. nodosus isolates from both sheep and cattle can be transferred to sheep under experimental conditions. The study also indicates that gamithromycin may be effective against D. nodosus