Salmonella Infection in Nursery Piglets and Its Role in the Spread of Salmonellosis to Further Production Periods.

Few studies have focused on assessing Salmonella infection in the nursery and its role in further pig production periods. Mesenteric lymph nodes, intestinal content, and meat juice from 389 6-week-old male piglets intended for human consumption from five breeding farms and 191 pooled floor fecal samples from gilt development units (GDU) from the same farms were analyzed to estimate and characterize (by pulsed-field gel electrophoresis and antimicrobial resistance analyses) Salmonella infection. The prevalence of infection and shedding among piglets was 36.5% and 37.3%, respectively, shedding being significantly associated with infection (Odds Ratio = 12.7; CI 7.3-22.0). Salmonella Rissen; S. 4,[5],12:i:-; and S. Derby were the most common serotypes. A low level of Salmonella-specific maternal antibodies at the beginning of the nursery period suggested it was a period of high risk of infection. Resistance to 3rd- and 4th-generation cephalosporins was detected in piglet isolates although the piglets never received antibiotics, indicating they could be vectors of antimicrobial resistance. The same Salmonella clones were detected in piglet and GDU isolates, suggesting that infected piglets play a significant role in the infection of gilts and consequently of finishing pigs in the case of production farms. The control of Salmonella infection in nursery piglets may decrease the risk of abattoir and carcass contamination.

Resistance to colistin and production of extended-spectrum ß-lactamases and/or AmpC enzymes in Salmonella isolates collected from healthy pigs in Northwest Spain in two periods: 2008-2009 and 2018.

Salmonellosis is a common subclinical infection in pigs and therefore apparently healthy animals may represent a reservoir of antibiotic-resistant Salmonella for humans. This study estimates and characterizes resistance to two classes of antimicrobials considered of the highest priority within the critically important antimicrobials for humans, i.e. colistin (CR) and 3rd generation cephalosporins (3GC), on a collection of Salmonella isolates from pigs from two periods: between 2008 and 09, when colistin was massively used; and in 2018, after three years under a National Plan against Antibiotic Resistance. Prevalence of CR was low (6 out of 625; 0.96%; 95%CI: 0.44-2.1) in 2008-09 and associated mostly to the mcr-1 gene, which was detected in four S. 4,5,12:i:- isolates. Polymorphisms in the pmrAB genes were detected in a S. 9,12:-:- isolate. No CR was detected in 2018 out of 59 isolates tested. Among 270 Salmonella isolates considered for the assessment of resistance to 3GC in the 2008-2009 sampling, only one Salmonella Bredeney (0.37%; 95%CI: 0.07-2.1) showed resistance to 3GC, which was associated with the blaCMY-2 gene (AmpC producer). In 2018, six isolates out of 59 (10.2%; 95%CI: 4.7-20.5) showed resistance to 3GC, but only two different strains were identified (S. 4,12:i:- and S. Rissen), both confirmed as extended-spectrum ß-lactamases (ESBL) producers. The blaCTX-M-3 and blaTEM-1b genes in S. 4,12:i:- and the blaTEM-1b gene in S. Rissen seemed to be associated with this resistance. Overall, the prevalence of CR in Salmonella appeared to be very low in 2008-2009 despite the considerable use of colistin in pigs at that time, and seemed to remain so in 2018. Resistance to 3GC was even lower in 2008-2009 but somewhat higher in 2018. Resistance was mostly coded by genes associated with mobile genetic elements. Most serotypes involved in these antimicrobial resistances displayed a multidrug resistance pattern and were considered zoonotic.

Biofilms as Promoters of Bacterial Antibiotic Resistance and Tolerance.

Multidrug resistant bacteria are a global threat for human and animal health. However, they are only part of the problem of antibiotic failure. Another bacterial strategy that contributes to their capacity to withstand antimicrobials is the formation of biofilms. Biofilms are associations of microorganisms embedded a self-produced extracellular matrix. They create particular environments that confer bacterial tolerance and resistance to antibiotics by different mechanisms that depend upon factors such as biofilm composition, architecture, the stage of biofilm development, and growth conditions. The biofilm structure hinders the penetration of antibiotics and may prevent the accumulation of bactericidal concentrations throughout the entire biofilm. In addition, gradients of dispersion of nutrients and oxygen within the biofilm generate different metabolic states of individual cells and favor the development of antibiotic tolerance and bacterial persistence. Furthermore, antimicrobial resistance may develop within biofilms through a variety of mechanisms. The expression of efflux pumps may be induced in various parts of the biofilm and the mutation frequency is induced, while the presence of extracellular DNA and the close contact between cells favor horizontal gene transfer. A deep understanding of the mechanisms by which biofilms cause tolerance/resistance to antibiotics helps to develop novel strategies to fight these infections.

Antimicrobial resistance among canine enteric Escherichia coli isolates and prevalence of attaching-effacing and extraintestinal pathogenic virulence factors in Spain.

The aim of this study was to estimate the prevalence of antimicrobial resistance (AMR) in Escherichia coli from a dog population in Spain and assess specific virulence factors. Susceptibility to 22 antimicrobials was tested along with the production of extended-spectrum ß-lactamases (ESBLs) and AmpC in faecal isolates from 100 dogs. Virulence-related genes associated with attaching and effacing E. coli (eae, Stx1, Stx2) and extraintestinal pathogenic E. coli - ExPEC - (papC, hlyA and cnf1) were detected by PCR. At least one kind of AMR was observed in 73% of the isolates. The highest prevalences corresponded to penicillin (45%), aminoglycoside (40%) and non-extended spectrum cephalosporin (39%) classes. Multidrug resistance (MDR) was observed in 53.4% of the resistant isolates. No resistance to colistin was found. Production of ESBL/AmpC enzymes was detected in 5% of E. coli. Shiga toxin-producing E. coli were not observed, enteropathogenic E. coli were identified in only 12% of them, and ExPEC were found in 25%. Dog faeces can be a source of E. coli strains potentially presenting a threat to humans through their virulence factors or AMR. The non-hygienic keeping of animals may increase the risk of colonisation of such pathogens in humans.

Wild griffon vultures (Gyps fulvus) fed at supplementary feeding stations: Potential carriers of pig pathogens and pig-derived antimicrobial resistance?

The carriage of two important pathogens of pigs, that is enterotoxigenic Escherichia coli (ETEC) and Clostridioides difficile, was investigated in 104 cloacal samples from wild griffon vultures (Gyps fulvus) fed on pig carcasses at supplementary feeding stations (SFS), along with their level of antimicrobial resistance (AMR). E. coli was isolated from 90 (86.5%) samples, but no ETEC was detected, likely because ETEC fimbriae confer the species specificity of the pathogen. Resistance to at least one antimicrobial agent was detected in 89.9% of E. coli isolates, with AMR levels being extremely high (>70%) for tetracycline and streptomycin and very high (>50%) for ampicillin and sulfamethoxazole-trimethoprim. Resistance to other critically important antimicrobials such as colistin and extended-spectrum cephalosporins was 2.2% and 1.1%, respectively, and was encoded by the mcr-1 and blaSHV-12 genes. Multidrug resistance was displayed by 80% of the resistant E. coli, and blaSHV-12 gene shared plasmid with other AMR genes. In general, resistance patterns in E. coli from vultures mirrored those found in pigs. Clostridioides difficile was detected in three samples (2.9%); two of them belonged to PCR ribotype 078 and one to PCR ribotype 126, both commonly found in pigs. All C. difficile isolates were characterized by a moderate-to-high level of resistance to fluoroquinolones and macrolides but susceptible to metronidazole or vancomycin, similar to what is usually found in C. difficile isolates from pigs. Thus, vultures may contribute somewhat to the environmental dissemination of some pig pathogens through their acquisition from pig carcasses and, more importantly, of AMR for antibiotics of critical importance for humans. However, the role of vultures would likely be much lesser than that of disposing pig carcasses at the SFS. The monitoring of AMR, and particularly of colistin-resistant and ESBL-producing E. coli, should be considered in pig farms used as sources of carcasses for SFS.

Weaned piglets: another factor to be considered for the control of Salmonella infection in breeding pig farms.

Field studies on Salmonella infection in suckling piglets are scarce due to the intrinsic difficulties of collecting proper samples (i.e. tonsils or mesenteric lymph nodes), and most of them rely on the analysis of rectal swabs that limit their accuracy. We used 495 slaughtered 4-weeks-old male piglets intended for human consumption from 5 Salmonella-seropositive breeding farms to collect gastrointestinal packages and perform a thorough detection of Salmonella on mesenteric lymph nodes and intestinal content. The overall prevalence of both infection and shedding was high (≈ 36%) indicating that piglets played an active role in Salmonella maintenance in the farms. Major serotypes found in piglets included 4,[5],12:i: (35.4%), Rissen (17.1%), Derby (10.9%) and Bovismorbificans (10.3%). In most of the infected animals (72.8%) the same serotype was found in mesenteric lymph nodes and feces. Significant higher ELISA OD% values were found in meat juice samples from non-infected piglets compared to infected ones (median OD% of 12.0 and 17.3, respectively; P = 0.002) suggesting some protective effect of sow's colostrum. Salmonella was also isolated from feces from weaned sows contemporary of the slaughtered piglets, and 89% of the serotypes identified in sows were also detected in piglets. Pulsed field gel electrophoresis analyses showed that 75% of the piglet isolates that were compared to those of sows were related to them, suggesting the circulation of Salmonella strains between sows and piglets. It appears that improving piglet colostrum intake along with the reduction of the shedding in sows may favor the control of Salmonella infection in breeding farms.

Role of wild bird and rodents in the epidemiology of subclinical salmonellosis in finishing pigs.

Wild birds and rodents may play an important role in the dynamics of subclinical pig salmonellosis, either as the introducers of the bacteria into the farm or as receptors of an infection already established in the farm. We tried to gain further insight into the epidemiology of this infection by studying the phenotypic (i.e., serotype and antimicrobial resistance patterns) and molecular characteristics of Salmonella strains isolated from samples collected from pigs and wildlife captured in the vicinity of pig farms. Salmonella-positive pig fecal samples were identified in 56.1% of the 41 farms investigated. Birds shedding Salmonella spp. were detected in 21.4% of the farms despite the low numbers of birds captured in many farms. Most Salmonella isolates from birds (74%) did not show any antimicrobial resistance (AR) pattern and belonged to phage types rarely seen in the pig population (U310, DT56, DT137, DT164), supporting the likely avian source of infection for most birds. The proportion of farms showing Salmonella-infected rodents was higher (46.2%), with Salmonella isolates showing a high homology with those likely originated from pigs. Salmonella-positive environmental samples were found in >50% of the farms, and the characteristics of these Salmonella strains supported the idea of pigs as a major source of Salmonella contamination of the farm environment. Dissemination of Salmonella in pig farms from areas of high Salmonella prevalence appeared to depend to some extent upon rodents and wild birds present in the farm, but the role of rodents in its maintenance seemed to be somewhat more relevant than that of birds. In conclusion, activities aimed at reducing the contact of these wild species with pigs will probably assist in the control of pig salmonellosis. Strict hygienic measures should be considered in areas of high prevalence of infection to lower the high load of environmental contamination.

Assessment of performance of selected serological tests for diagnosing brucellosis in pigs.

Swine brucellosis due to Brucella suis is considered an emerging zoonotic disease whose control is based on serological testing and the subsequent culling of seropositive animals or the full depopulation of affected flocks. Here we assessed the performance of several serological tests (Rose Bengal Test [RBT], indirect ELISA [i-ELISA], blocking ELISA [b-ELISA], and two competitive ELISAs [c-ELISA]) for diagnosing swine brucellosis caused by B. suis biovar 2. Both frequentistic and Bayesian statistical inference were used. A frequentistic analysis, using sera from known gold standard (GS) populations (i.e., from truly infected or brucellosis free animals), resulted in maximum (100%) diagnostic sensitivity (Se) and specificity (Sp) in the RBT, i-ELISA and b-ELISA tests. However, c-ELISAs resulted in lower diagnostic Se (ranging from 68.5% to 92.6%, according to the different cut-offs selected). A Bayesian analysis of tests yielding the best diagnostic performance with GS sera (RBT, i-ELISA and b-ELISA), but using a large collection of field sera, resulted in similar Se among tests but markedly lower (≈ 80%) than that resulting from the frequentistic analysis using the GS serum populations. By contrast, the estimated Sp in the Bayesian analysis was only slightly lower than 100%, thus similar to that obtained frequentistically. Our results show that adequate diagnostic tests for brucellosis in swine are available, but also emphasize the need for more extensive validation studies before applying these tests under field conditions.

The use of meat juice or blood serum for the diagnosis of Salmonella infection in pigs and its possible implications on Salmonella control programs.

Serology is the method of choice for country-scale Salmonella control programs in pigs and can be carried out both on blood serum or meat juice. However, the diagnostic performance of enzyme-linked immunosorbent assays (ELISAs) on these sample matrices has not been sufficiently compared. The agreement between the serum ELISA and meat juice ELISA on samples taken from commercial farms was assessed in 2 pig populations (adult sows and finishers). Results of optical density percentage (OD%) from the serum ELISA were consistently higher than those from the meat juice ELISA (38.5 vs. 27.9; P<0.001), and the mean difference between them was significantly different from zero (P<0.0001). The overall correlation coefficient between serum ELISA and meat juice ELISA results was low (r=0.53). These results indicated an important disagreement between ELISA performed on serum and meat juice matrices and suggested that before implementing a control program to reduce the prevalence of Salmonella in swine the choice of matrix on which to perform the ELISA should be carefully considered.

Survey on Salmonella prevalence in slaughter pigs from Saskatchewan.

A study on slaughter pigs from Saskatchewan detected Salmonella organisms in 12.5% and 5.2% of cecal content and ileocaecal lymph node samples, respectively. Cecal content prevalence was associated with larger farms and longer lairage periods. Antimicrobial resistance was detected in 41.5% of the isolates. Salmonella Enteritidis was the second most prevalent serotype.

Accuracy of two commercial enzyme-linked immunosorbent assays for the detection of antibodies to Salmonella spp. in slaughter pigs from Canada.

Large discrepancies are usually found when different ELISAs for the diagnosis of pig salmonellosis are compared. Thus, our main goal was to estimate the diagnostic accuracy through Bayesian approaches of two commercial assays (Svanovir "test A" and HerdCheck "test B") for the detection of antibodies to Salmonella spp. in slaughter pigs. Previously, we estimated the agreement between both tests and their relative sensitivity (Se) and specificity (Sp) with respect to bacteriology on caecal content and ileocaecal lymph nodes. Test A, at a cut-off OD%>or=20%, indicated higher prevalence than test B (OD%>or=10%) (14.6% vs. 8.6%). Relative Se with respect to overall bacteriology was low (approximately 30%) and similar for both tests, but the relative Sp was significantly lower for test A compared to B (88% vs. 95%). Both tests failed to detect some pigs infected with Salmonella serogroups B and C1, which they were supposed to identify. In general, tests showed only fair-to-moderate agreement when they were compared (kappa: 0.41). In the Bayesian models, Se of test A varied between 63% and 77%, while Se of test B was 73%. Sp of A was always lower than that of test B (89% vs. 95%). The implications derived from the use of these imperfect serological tests will have to be accounted for in large-scale Salmonella-control programs.

Serial blood lactate concentrations in systemically ill dogs.

BACKGROUND: Lactate concentration often is quantified in systemically ill dogs and interpreted based on human data. To our knowledge, there are no published clinical studies evaluating serial lactate concentrations as a prognostic indicator in ill dogs. OBJECTIVES: Our objective was to perform a prospective study, using multivariate analysis, to determine whether serial lactate concentrations were associated with outcome in ill dogs requiring intravenous fluids. METHODS: Eighty sick dogs had lactate concentrations evaluated, using an analyzer that measures lactate in the plasma fraction of heparinized whole blood, at 0 hours and 6 hours after initiation of treatment. Severity of illness and outcome (survivor, nonsurvivor) were determined by reviewing the patient's record 2 weeks after admission. Lactate concentrations, age, body weight, gender, and severity of illness were evaluated using multivariate analysis to determine their effects on outcome. RESULTS: Dogs with lactate concentrations greater than the reference interval at 6 hours were 16 times (95% confidence interval = 2.32-112.71 times, P <.01) more likely not to survive compared to dogs with lactate concentrations within the reference interval. Lactate concentrations above the reference interval at 0 hours were not significantly related to outcome. However, hyperlactatemia that did not improve by > or = 50% within 6 hours was significantly associated with mortality (P = .024). CONCLUSION: Dogs with a lactate concentration higher than the reference interval at 6 hours were more likely not to survive. These results indicate an association between lactate concentration and outcome and emphasize the importance of serial lactate concentrations in evaluating prognosis.

Whole-cell and acellular pertussis vaccination programs and rates of pertussis among infants and young children.

BACKGROUND: The transition from a whole-cell to a 5-component acellular pertussis vaccine provided a unique opportunity to compare the effect that each type of vaccine had on the incidence of pertussis, under routine conditions, among children less than 10 years of age. METHODS: Analyses were based on passive surveillance data collected between 1995 and 2005. The incidence of pertussis by year and birth cohort was compiled according to age during the surveillance period. We determined the association between vaccine type (whole-cell, acellular or a combination of both) and the incidence of pertussis using Poisson regression analysis after controlling for age (< 1 year, 1-4 years and 5-9 years) and vaccination history (i.e., partial or complete). RESULTS: During 7 of the 11 years surveyed, infants (< 1 year of age) had the highest incidence of pertussis. Among children born after 1997, when acellular vaccines were introduced, the rates of pertussis were highest among infants and preschool children (1-4 years of age). Poisson regression analysis revealed that, in the group given either the whole-cell vaccine or a combination of both vaccines, the incidence of pertussis was lower among infants and preschool children than among school-aged children (5-9 years). The reverse was true in the group given only an acellular vaccine, with a higher incidence among infants and preschool children than among school-aged children. INTERPRETATION: These results suggest that current immunization practices may not be adequate in protecting infants and children less than 5 years of age against pertussis. Altering available acellular formulations or adopting immunization practices used in some European countries may increase the clinical effectiveness of routine pertussis vaccination programs among infants and preschool children.

Pertussis in rural populations of Saskatchewan (1995 to 2003): incidence, seasonality, and differences among cases.

BACKGROUND: Few Canadian studies have studied re-emergent pertussis in rural areas. This study described the epidemiology of pertussis in the rural areas of the Saskatoon Regional Health Authority in Saskatchewan, and comparisons were made to the City of Saskatoon. METHODS: Analyses were based on passive surveillance data collected between 1995 and 2003. Estimates of the cumulative incidence (per 10,000 population) measured the occurrence of pertussis. Kaplan-Meier curves were plotted to compare a case's time until disease from their last vaccination by different vaccine types (whole-cell (WCV), or acellular (ACV)) and vaccine histories (complete or partial). Epidemic curves identified peaks in the incidence and checked for seasonal variation in case reporting. RESULTS: Over the 9-year period, 1,135 cases were reported. Rural areas had higher incidence rates and lower proportions of cases vaccinated than urban areas. Overall, the highest age-specific incidence was observed in people aged 10-19 years. Cases aged 0-9 years vaccinated with the ACV, from both rural and urban areas, presented a shorter time to disease (14 months (95% CI: 13-16) and 17 months (95% CI: 11-21), respectively) when compared to cases vaccinated with the WCV (47 months (95% CI: 40-51) and 36 months (95% CI: 31-41), respectively), or with a combination of the two vaccines (40 months (95% CI: 27-47) and 44 months (95% CI: 36-51), respectively, p<0.01). Epidemic curves revealed that reported cases are occurring earlier in successive years (1997, 1999, and 2003) in rural areas. CONCLUSION: Epidemiologic differences among cases from rural areas exist when compared to urban areas. This study further emphasizes the need to better understand age-, vaccine-, and seasonally-related aspects of pertussis epidemiology in rural areas.

Specificity dependence between serological tests for diagnosing bovine brucellosis in Brucella-free farms showing false positive serological reactions due to Yersinia enterocolitica O:9.

When brucellosis false positive serological reactions happen in cattle, the serial use of pairs of specificity-correlated serological tests (rose bengal, complement fixation, competitive ELISA) results in specificities lower than expected. In this situation, highly specific tests, such as the indirect ELISA used alone, may be more adequate than serial testing.

Rough vaccines in animal brucellosis: structural and genetic basis and present status.

Brucellosis control and eradication requires serological tests and vaccines. Effective classical vaccines (S19 in cattle and Rev 1 in small ruminants), however, induce antibodies to the O-polysaccharide of the lipopolysaccharide which may be difficult to distinguish from those resulting from infection and may thus complicate diagnosis. Rough attenuated mutants lack the O-polysaccharide and would solve this problem if eliciting protective immunity; the empirically obtained rough mutants 45/20 and RB51 have been used as vaccines. Strain 45/20 is reportedly unstable and it is not presently used. RB51 is increasingly used instead of S19 in some countries but it is rifampicin resistant and its effectiveness is controversial. Some controlled experiments have found good or absolute protection in adult cattle vaccinated orally (full dose) or subcutaneously (reduced dose) and in one field experiment, RB51 was reported to afford absolute protection to calves and to perform better than S19. Controlled experiments in calves, however, have shown reduced doses of RB51 to be ineffective, full doses only partially effective, and RB51 less effective than S19 against severe challenges. Moreover, other observations suggest that RB51 is ineffective when prevalence is high. RB51 is not useful in sheep and evidence in goats is preliminary and contradictory. Rough mutants obtained by molecular biology methods on the knowledge of the genetics and structure of Brucella lipopolysaccharide may offer alternatives. The B. abortus manBcore (rfbK) mutant seems promising in cattle, and analyses in mice suggest that mutations affecting only the O-polysaccharide result in better vaccines than those affecting both core and O-polysaccharide. Possible uses of rough vaccines also include boosting immunity by revaccination but solid evidence on its effectiveness, safety and practicality is not available.