Pedro Rubio

Evaluation of a blocking ELISA using monoclonal antibodies for the detection of porcine epidemic diarrhea virus and its antibodies

Two blocking enzyme-linked immunosorbent assays (ELISAs) involving the use of monoclonal antibodies (MAb) as capture and detecting agents and an indirect fluorescence test (IFT) were used for the detection of porcine epidemic diarrhea virus (PEDV) antigen or its antibodies. In the ELISA for viral antigen detection, the blocking step is accomplished by incubation of fecal samples, in duplicate wells, with PEDV-specific positive or negative serum, whereas in the ELISA for antibody detection the blocking step is accomplished by incubation of serum samples with a gut-origin virus suspension. All the methods developed were used to monitor an experimental infection in piglets with the CV-777 strain of PEDV and a natural PED outbreak in a swine fattening unit. The antigen-detection ELISA was able to detect PEDV shedding for a longer time than have previously described methods. The blocking ELISA for antibody detection was able to detect the serum antibody response sooner after PEDV infection than did IFT.

Trends towards Lower Antimicrobial Susceptibility and Characterization of Acquired Resistance among Clinical Isolates of Brachyspira hyodysenteriae in Spain

ABSTRACT The antimicrobial susceptibility of clinical isolates of Brachyspira hyodysenteriae in Spain was monitored, and the underlying molecular mechanisms of resistance were investigated. MICs of tylosin, tiamulin, valnemulin, lincomycin, and tylvalosin were determined for 87 B. hyodysenteriae isolates recovered from 2008 to 2009 by broth dilution. Domain V of the 23S rRNA gene and the ribosomal protein L3 gene were sequenced in 20 isolates for which the tiamulin MIC was ≥4 μg/ml, presenting decreased susceptibility, and in 18 tiamulin-susceptible isolates (MIC ≤ 0.125 μg/ml), and all isolates were typed by multiple-locus variable-number tandem repeats analysis. A comparison with antimicrobial susceptibility data from 2000 to 2007 showed an increase in pleuromutilin resistance over time, doubling the number of isolates with decreased susceptibility to tiamulin. No alteration in susceptibility was detected for lincomycin, and the MIC of tylosin remained high (MIC50 > 128 μg/ml). The decreased susceptibility to tylosin and lincomycin can be explained by mutations at position A2058 of the 23S rRNA gene (Escherichia coli numbering). A2058T was the predominant mutation, but A2058G also was found together with a change of the neighboring base pair at positions 2057 to 2611. The role of additional point mutations in the vicinity of the peptidyl transferase center and mutations in the L3 at amino acids 148 and 149 and their possible involvement in antimicrobial susceptibility are considered. An association between G2032A and high levels of tiamulin and lincomycin MICs was found, suggesting an increasing importance of this mutation in antimicrobial resistance of clinical isolates of B. hyodysenteriae.

Salmonella Derby clonal spread from pork.

The genetic diversity of the Derby serotype of Salmonella enterica in Spain was examined by pulsed-field gel electrophoresis (PFGE). Out of 24 identified PFGE profiles, a major clone was detected in 19% of strains from humans, 52% from food, and 62% from swine. This clone (clone 1) was isolated from pork products, suggesting swine as its source.

Herd-level risk factors for faecal shedding of Salmonella enterica in Spanish fattening pigs

Herd-level risk factors for faecal shedding of Salmonella enterica were investigated in a cross-sectional study on Spanish finishing units. For this purpose, 10 faecal samples were collected from 10 different pens containing pigs close to market weight in a total of 232 fattening units. The total sample size was proportionally distributed according to the fattener census in each of the regions and provinces of the country in order to ensure a sample representative of the entire swine population. All samples were individually examined by culture of 25g of faecal material. Data regarding characteristics and management of each fattening unit were collected by means of a questionnaire. Logistic regression was used to detect relationships between the detection of faecal shedding of S. enterica and potential herd-level risk factors. The feeding of pelleted feed was associated with an increased risk of culture-positive faecal samples (OR=2.28; 95% CI: 1.22, 4.26). The odds of a farm being Salmonella positive were associated with its size. Fattening units that slaughtered more than 3500 pigs per year had a higher risk for Salmonella faecal shedding (OR=1.78; 95% CI: 0.96, 3.31). Interventions at these two points should be considered when designing or managing growing pig facilities to reduce Salmonella faecal shedding by fatteners.

Swine dysentery: aetiology, pathogenicity, determinants of transmission and the fight against the disease.

Swine Dysentery (SD) is a severe mucohaemorhagic enteric disease of pigs caused by Brachyspira hyodysenteriae, which has a large impact on pig production and causes important losses due to mortality and sub-optimal performance. Although B. hyodysenteriae has been traditionally considered a pathogen mainly transmitted by direct contact, through the introduction of subclinically infected animals into a previously uninfected herd, recent findings position B. hyodysenteriae as a potential threat for indirect transmission between farms. This article summarizes the knowledge available on the etiological agent of SD and its virulence traits, and reviews the determinants of SD transmission. The between-herds and within-herd transmission routes are addressed. The factors affecting disease transmission are thoroughly discussed, i.e., environmental survival of the pathogen, husbandry factors (production system, production stage, farm management), role of vectors, diet influence and interaction of the microorganism with gut microbiota. Finally, prophylactic and therapeutic approaches to fight against the disease are briefly described.

Antimicrobial susceptibility testing of Spanish field isolates of Brachyspira hyodysenteriae

This study is the first conducted in Spain to evaluate antimicrobial susceptibility of field isolates of Brachyspira hyodysenteriae. One hundred and eight isolates of the bacterium, recovered from different Spanish swine farms between 2000 and 2007, were investigated. The minimum inhibitory concentrations (MIC) of erythromycin, tylosin, tiamulin, valnemulin, clindamycin and lincomycin were determined using a broth microdilution technique. Most of the isolates showed poor susceptibility to erythromycin (MIC(90)>256 microg/ml), tylosin (MIC(90)>256 microg/ml), clindamycin (MIC(90)>4 microg/ml) and lincomycin (MIC(90)=128 microg/ml). Reduced susceptibility to tiamulin and valnemulin was observed with a MIC>2 microg/ml in 17.6% and 7.41% of the B. hyodysenteriae isolates, respectively. Moreover, a survival analysis permitted the detection of an increasing trend in the MIC values for almost all the antimicrobials used in the treatment of swine dysentery when comparing recent isolates (from 2006 to 2007) with those recovered in earlier years (between 2000 and 2004).

Role of slaughtering in Salmonella spreading and control in pork production.

Salmonella is one of the major foodborne pathogens worldwide. Pork products are among the main sources of Salmonella infection in humans, and several countries have established Salmonella surveillance and control programs. The role of slaughtering in carcass contamination has been indicated by studies focused on the slaughterhouse environment. In this review, we examine and discuss the information available regarding the influence that farm status, pig transport, and lairage have on the carriage of Salmonella by pigs entering the slaughter line. The evolution of carcass contamination throughout the slaughtering process, the main sources of contamination in the dirty and clean zones of the slaughter line, and previously reported prevalence of Salmonella on carcasses and factors affecting this prevalence also are discussed. The importance of implementing interventions at the slaughter level is discussed briefly. Consistent with the information available, pigs from infected farms and newly acquired or recrudescent infections in pigs at the subsequent stages of transport and lairage are important sources of Salmonella at the slaughtering plant. The continuous introduction of Salmonella into the slaughterhouse and the potential for resident flora constitute a risk for carcass contamination. At the slaughterhouse, some dressing activities can reduce carcass contamination, but others are critical control points that jeopardize carcass hygiene. This information indicates the importance of considering slaughter and previous stages in the pork production chain for controlling Salmonella in swine production.

Dissemination of Clonal Groups of Brachyspira hyodysenteriae amongst Pig Farms in Spain, and Their Relationships to Isolates from Other Countries

Background Swine dysentery (SD) is a widespread diarrhoeal disease of pigs caused by infection of the large intestine with the anaerobic intestinal spirochaete Brachyspira hyodysenteriae. Understanding the dynamics of SD, and hence being able to develop more effective measures to counter its spread, depends on the ability to characterise B. hyodysenteriae variants and trace relationships of epidemic strains. Methodology/Principal Findings A collection of 51 Spanish and 1 Portuguese B. hyodysenteriae isolates was examined using a multilocus sequence typing (MLST) scheme based on the sequences of seven conserved genomic loci. The isolates were allocated to 10 sequence types (STs) in three major groups of descent. Isolates in four of the STs were widely distributed in farms around Spain. One farm was infected with isolates from more than one ST. Sequence data obtained from PubMLST for 111 other B. hyodysenteriae strains from other countries then were included in the analysis. Two of the predominant STs that were found in Spain also were present in other European countries. The 73 STs were arranged in eleven clonal complexes (Cc) containing between 2 and 26 isolates. A population snapshot based on amino acid types (AATs) placed 75% of the isolates from 32 of the 48 AATs into one major cluster. The founder type AAT9 included 22 isolates from 10 STs that were recovered in Spain, Australia, Sweden, Germany, Belgium, the UK, Canada, and the USA. Conclusions/Significance This MLST scheme provided sufficient resolution power to unambiguously characterise B. hyodysenteriae isolates, and can be recommended as a routine typing tool that rapidly enables comparisons of isolates. Using this method it was shown that some of the main genetic lineages of B. hyodysenteriae in Spain also occurred in other countries, providing further evidence for international transmission. Finally, analysis of AATs appeared useful for deducing putative ancestral relationships between strains.

Antimicrobial resistance of Salmonella enterica isolates from apparently healthy and clinically ill finishing pigs in Spain.

This study was the first conducted in Spain to evaluate the occurrence of antimicrobial resistance and multi‐resistance in Salmonella isolates recovered from finishing pigs from Spanish swine farms distributed over the whole country. For this purpose, 290 Salmonella isolates recovered from apparently healthy finishing pigs in a farm‐based cross‐sectional study and 192 Salmonella isolates recovered from faecal samples of finishing pigs suffering from diarrhoea were investigated. Resistance to a panel of 17 antimicrobials was determined using a broth microdilution technique. Resistance was a common finding and was detected in 90.3% of the Salmonella isolates from apparently healthy finishing pigs and 95.3% of the Salmonella isolates from clinically diseased finishing pigs. Resistance was particularly high among isolates of serogroup B and serovars Typhimurium and its monophasic variant S. 4,5,12:i:‐. Higher frequencies of resistance were found to tetracycline, sulphamethoxazole, streptomycin, spectinomycin, ampicillin, chloramphenicol and trimethoprim‐sulphamethoxazole. Less than 10% of the isolates were resistant to amoxicillin/clavulanic acid, neomycin, cephalotin, apramycin and gentamicin. Resistance to ciprofloxacin, colistin and ceftiofur was rare (under 1%). Multi‐resistance, defined as resistance to four or more drugs, was detected in more than 50% of the isolates. Although multi‐resistance was particularly frequent among isolates of S. Typhimurium, it was also high among other serovars as Bredeney and the S. Typhimurium monophasic variant. 4,5,12:i:‐.

Critical value of prevalence for vaccination programmes. The case of hepatitis A vaccination in Spain.

Programmes using screening and vaccination are efficient in populations with higher levels of antibodies, while in those with lower levels is more efficient to vaccine the whole target population. The prevalence that makes the cost-effectiveness of vaccination programmes equal to that obtained for programmes using screening and vaccination id defined as the critical value of prevalence p∗. In this study, a mathematical procedure to obtain the critical value of prevalence is developed. The formula obtained is used to decide the best vaccination strategy against hepatitis A in Spain. If V is the vaccination cost, S the screening cost, PV the predictive value of a positive test result, D the mean disease cost, A the attack rate in susceptible individuals, E the vaccine efficacy and C the vaccination compliance, the critical value of prevalence is equal to: p∗ = sV − ((1 − PV)DAEC) = sV − AD The critical value of prevalence obtained for vaccination against hepatitis A in Spain with three doses of vaccine Havrix 720 is 22%. This result show that the optimal decision is to implement vaccination programmes without screening for immunity in individuals aged 15 years.