Robert M. Friendship

Antimicrobial Resistance and Virulence Genes of Escherichia coli Isolates from Swine in Ontario

ABSTRACT A total of 318 Escherichia coli isolates obtained from diarrheic and healthy pigs in Ontario from 2001 to 2003 were examined for their susceptibility to 19 antimicrobial agents. They were tested by PCR for the presence of resistance genes for tetracycline, streptomycin, sulfonamides, and apramycin and of 12 common virulence genes of porcine E. coli. Antimicrobial resistance frequency among E. coli isolates from swine in Ontario was moderate in comparison with other countries and was higher in isolates from pigs with diarrhea than in isolates from healthy finisher pigs. Resistance profiles suggest that cephamycinases may be produced by ≥8% of enterotoxigenic E. coli (ETEC). Resistance to quinolones was detected only in enterotoxigenic E. coli (≤3%). The presence of sul3 was demonstrated for the first time in Canada in porcine E. coli isolates. Associations were observed among tetA, sul1, aadA, and aac(3)IV and among tetB, sul2, and strA/strB, with a strong negative association between tetA and tetB. The paa and sepA genes were detected in 92% of porcine ETEC, and strong statistical associations due to colocation on a large plasmid were observed between tetA, estA, paa, and sepA. Due at least in part to gene linkages, the distribution of resistance genes was very different between ETEC isolates and other porcine E. coli isolates. This demonstrates that antimicrobial resistance epidemiology differs significantly between pathogenic and commensal E. coli isolates. These results may have important implications with regards to the spread and persistence of resistance and virulence genes in bacterial populations and to the prudent use of antimicrobial agents.

Prevalence of antibodies to the hepatitis E virus in pigs from countries where hepatitis E is common or is rare in the human population.

Hepatitis E virus (HEV) is a very important public health concern in many developing countries where epidemics of hepatitis E are common. Sporadic cases of clinical hepatitis E not only occur in these countries but also occur uncommonly in patients with no known epidemiological exposure to HEV in industrialized countries. The source of infection in industrialized countries is unknown but it has been suggested that animals might serve as a reservoir for HEV in both settings. We recently identified and characterized an HEV strain (swine HEV) that infects large numbers of pigs in the United States. To assess the potential of pigs to serve as a global reservoir of HEV, we measured the prevalence of HEV antibodies in pigs in two countries where hepatitis E is endemic and two countries where it is not. Swine herds in all four countries contained many pigs that were seropositive for IgG anti‐HEV, although the percentage of seropositive pigs varied greatly from herd to herd. A very limited number of pig handlers in the two endemic countries were also tested and most of them were found to be seropositive for HEV. The results from this study suggest that hepatitis E is enzootic in pigs regardless of whether HEV is endemic in the respective human population. J. Med. Virol. 59:297–302, 1999. Published 1999 Wiley‐Liss, Inc.

Prevalence of Hepatitis E Virus Antibodies in Canadian Swine Herds and Identification of a Novel Variant of Swine Hepatitis E Virus

ABSTRACT Swine hepatitis E virus is a newly identified potentially zoonotic virus from pigs of particular concern for possible direct transmission to a human xenotransplant recipient by organ transplantation. In the present study, prevalence of serum antibodies to hepatitis E virus was examined in Canadian swine herds. A total of 998 serum samples collected from 6-month-old healthy slaughter hogs were examined by enzyme immunoassay and Western blot analysis for antibodies to the recombinant open reading frame 3 (ORF3) protein of hepatitis E virus expressed in Escherichia coli. These samples represented more than 80 different swine production units from five major swine-producing provinces across Canada. From this study, 594 samples (59.4%) were found to be positive for hepatitis E virus antibody. The seroprevalence was higher in Quebec (88.8%) and Ontario (80.1%) than in Alberta and Saskatchewan (38.3%). By PCR using a pair of oligonucleotide primers deduced from the ORF2 sequence of human hepatitis E virus, a specific hepatitis E virus sequence was recovered from feces of pigs. The nucleotide sequence identity between the U.S. swine hepatitis E virus and the Canadian isolate (SK3) was only 85.8%, suggesting that genotypic variations may exist in swine hepatitis E virus in North America. Among 165 serum samples collected from humans in Saskatchewan, 2.4% were found to be positive for antibodies to the hepatitis E virus ORF3 protein. Our data indicate that hepatitis E virus is highly prevalent in commercial swine populations in Canada and support the suggestion that the swine hepatitis E virus may be an important zoonotic agent for humans.

Associations among antimicrobial drug treatments and antimicrobial resistance of fecal Escherichia coli of swine on 34 farrow-to-finish farms in Ontario, Canada

Logistic regression was used to model associations between antimicrobial treatment and resistance among fecal Escherichia coli of finisher pigs at the farm level. Four sets of potential risk factors representing different levels of refinement of antimicrobial use on farms were modelled on resistance to antimicrobials. Final models for each antimicrobial were constructed from treatment and management variables significant on initial screening, and corrections for overdispersion were made. In general, in-feed antimicrobial treatment of pigs was more consistently associated with an increased risk of resistance than individual-animal treatment. Antimicrobial treatment in starter rations was significant in final models of resistance to ampicillin, carbadox, nitrofurantoin, sulfisoxizole, and tetracycline. Treatment in grower-finisher rations was significantly associated with resistance to ampicillin, spectinomycin, sulfisoxizole, and tetracycline. There was little evidence that in-feed antimicrobials increased the risk of resistance to gentamicin, which is a drug used only for individual-pig treatment in this study population. These results suggest that antimicrobial medication of rations of post-weaning pigs selects for and maintains antimicrobial resistance among E. coli of finisher pigs. Although resistance was common on farms that did not medicate rations of post-weaning pigs, the results indicate that antimicrobial use does increase the risk of resistance to the antimicrobials studied.

INDIVIDUAL AND GROUP ANTIMICROBIAL USAGE RATES ON 34 FARROW-TO-FINISH SWINE FARMS IN ONTARIO, CANADA

Antimicrobial drug-use was assessed on 34 farrow-to-finish operations that marketed at least 500 hogs/yr. These operations either did not use any antimicrobials or used narrow-spectrum or broad-spectrum antimicrobials in rations of post-weaning pigs. Total antimicrobial use was measured for two months after obtaining inventories and records of all antimicrobials used. The collection of empty medication bottles and inventories of drugs on hand was convenient for producers and useful for estimating or validating recorded treatment rates, particularly for antimicrobials that were used only in one class of pig. Treatment records, however, underestimated by approximately 35% the amounts used for 27/29 farm-antimicrobial combinations. Rates of individual-pig treatment varied from 0-24.1 pigs treated/1000 pig-days, with a median of 5.29. Most individual animal treatments were given to piglets and sows at parturition and penicillin was the most commonly used antimicrobial. Gentamicin was administered to suckling piglets on 19 of the farms.

Longitudinal Investigation of Methicillin‐Resistant Staphylococcus aureus in Piglets

Methicillin‐resistant Staphylococcus aureus (MRSA) has emerged as an important public health concern and pigs have been implicated in human infections. Cross‐sectional studies have demonstrated that MRSA can be commonly found in pigs internationally, but little is known about age‐related changes in MRSA colonization. This study evaluated MRSA colonization in piglets in a longitudinal manner. Serial nasal swabs were collected from piglets born to 10 healthy sows. The prevalence of MRSA colonization on days 1, 3, 7, 14 and 21 was 1% (1/100), 6.2% (3/97), 8.5% (8/94), 4.4% (4/91) and 20% (18/91) respectively, with an overall pre‐weaning prevalence of 34.5%. The prevalence on days 28, 42, 56 and 70 was 34% (31/91), 65% (57/88), 50% (44/88) and 42% (36/87) respectively, with an overall post‐weaning prevalence of 85%. Eighty‐four percent of piglets from negative sows and 100% of piglets from positive sows that survived at least until the time of weaning were colonized with MRSA at one or more times during the study. There was a significant association between sow and piglet colonization. The age of the piglet was significantly associated with the probability of colonization. No piglets or sows received antimicrobials during the study period. These results indicate that age must be considered when designing surveillance programmes and interpreting results of different studies on MRSA.

Occurrence of Salmonella, Campylobacter, Yersinia enterocolitica, Escherichia coli O157 and Listeria monocytogenes in Swine

The objective of this study was to investigate the occurrence of major bacterial foodborne pathogens in swine. In total, 359 samples from manure storage tanks (91) and fresh pooled faeces (268) obtained from finisher (110), sows (78) and weanlings (80) were collected and tested. Campylobacter, Salmonella, Yersinia enterocolitica, Escherichia coli O157 and Listeria monocytogenes were isolated from 36.5%, 31.5%, 5.8%, 3.3% and 3.3% of samples respectively. All E. coli O157 isolates found on 10 farms were tested but none was determined to be E. coli O157:H7. Salmonella and Campylobacter were more likely to be detected from stored manure rather than from fresh faecal samples. Yersinia enterocolitica tended to be detected more commonly from fresh samples than from manure pits. Listeria monocytogenes was not recovered from manure pits or from sow faecal samples and only infrequently found in the faeces of weanling pigs and finisher pigs. The proportion of positive samples showed a seasonal change. Salmonella was twice as likely not be recovered in winter, whereas the chance of culturing Campylobacter was higher in winter. The 113 Salmonella isolates recovered on 24 farms and the four most common serovars were Salmonella Typhimurium var. Copenhagen (31.0%), Salmonella Derby (12.4%), S. Typhimurium (10.6%) and Salmonella Agona (10.6%). Of 131 Campylobacter isolates recovered on 21 farms, 118 isolates were Campylobacter coli and 13 isolates could not be speciated. Fifteen of 21 Y. enterocolitica isolates found on 15 farms were detected in finisher pigs. The sero/biogroups of Y. enterocolitica were O3/biotype 4 (16 isolates), O6,30/biotype 1A (three isolates), O5/biotype 1A (one isolate) and O8/biotype 1B (one isolate). These findings provide baseline information on the distribution of important zoonotic pathogens in swine and indicate that pigs should be considered as a possible source of foodborne diseases in humans.

The negative effects of exogenous DNA binding on porcine spermatozoa are caused by removal of seminal fluid

Sperm-mediated gene transfer (SMGT) might become the most efficient and cost effective technique to generate transgenic animals, which will significantly increase their application in biomedical research and in commercial production. Despite some successes, the technique has remained controversial for almost 20 years and despite number of studies the reasons for poor reproducibility of this promising technology has not been understood. We suggest that the reason for poor reproducibility is the presence of natural defences against exogenous DNA invasion acting in spermatozoa or in embryo. Based on previous reports we have investigated the effect of foreign DNA binding on spermatozoa by monitoring motility, viability and genomic DNA damage. Evaluation of DNA binding in sperm collected from 16 boars demonstrated that 28-45% of the added pEGFP plasmid was bound to spermatozoa with 9-32% being internalized in sperm nucleus. In agreement with previous reports, our results demonstrated that the pEGFP-treated sperm show an average a 2-fold decrease in motility (p<0.05), 5-fold decrease in progressive motility (p<0.05), and 1.4-fold increase in number of sperm with highly damaged DNA (p<0.05) as detected by Comet assay. In contrast with previous reports, we demonstrate that all such changes were associated with the removal of seminal plasma during the washing step and not with foreign DNA binding per se. We suggest that poor reproducibility of SMGT most likely result from selection against DNA-loaded sperm at later stages of fertilization.

Prevalences of resistance to seven antimicrobials among fecal Escherichia coli of swine on thirty-four farrow-to-finish farms in Ontario, Canada.

Fecal specimens were composited and a hydrophobic-grid membrane-filter method was used to measure antimicrobial resistance to ampicillin 16 micrograms/ml, carbadox 30 micrograms/ml, gentamicin 4 mu/ml, nitrofurantoin 32 micrograms/ml, spectinomycin 16 micrograms/ml, sulfisoxazole 32 micrograms/ml and tetracycline 8 micrograms/ml among 8119 Escherichia coli isolates from 68 fecal samples collected on 34 farrow-to-finish swine farms marketing over 500 hogs/yr. The overall prevalences of resistance to antimicrobials among these isolates were: ampicillin 29%, carbadox 3.5%, gentamicin 0.6%, nitrofurantoin 27%, spectinomycin 28%, sulfasoxizole 38% and tetracycline 71%. Thirty to seventy-six per cent of the variations in prevalences were explained by between-farm differences.

The effects on litter size of previous lactation length and previous weaning-to-conception interval in Ontario swine

Lactation length and the weaning-to-conception interval are two key features of reproductive efficiency. A retrospective study of 112 Ontario swine farms was conducted to determine the association between litter size and previous lactation lengths up to 40 days and previous weaning-to-conception intervals up to 14 days. Threshold dummy variables were created for lactation lengths between 14 days and 40 days and for weaning-to-conception intervals up to 14 days. The litter size increased progressively at previous lactation lengths of 25, 27 and 33 days (P<0.05). Sows bred on Days 2–4 post weaning had essentially the same litter size. Litter size decreased progressively for sows bred on Days 5, 6, and 7 post weaning and then reached a plateau until it increased again for sows bred on Day 11 or later (P<0.05).