Identification of a Plasmid‐Mediated Quinolone Resistance Gene in Salmonella Isolates from Texas Dairy Farm Environmental Samples

A recent increase in plasmid‐mediated quinolone resistance (PMQR) has been detected among Salmonella isolated from humans in the United States, and it is necessary to determine the sources of human infection. We had previously isolated Salmonella from dairy farm environmental samples collected in Texas, and isolates were tested for anti‐microbial susceptibility. Two isolates, serotyped as Salmonella Muenster, showed the discordant pattern of nalidixic acid susceptibility and intermediate susceptibility to ciprofloxacin. For this project, whole‐genome sequencing of both isolates was performed to detect genes associated with quinolone resistance. The plasmid‐mediated qnrB19 gene and IncR plasmid type were identified in both isolates. To our knowledge, this is the first report of PMQR in Salmonella isolated from food animals or agricultural environments in the United States.     

Outbreak of Salmonella Typhimurium associated with feeder rodents

In December 2012, an increase in human Salmonella Typhimurium cases was identified in the province of Ontario, Canada launching an outbreak investigation. The outbreak spanned 3 years (2012–2014), with 134 cases reported from five Canadian provinces. There was a substantial burden of illness among children: 45% of cases were children 12 years old or under, and 23% of cases were under 5 years old. Epidemiologic, traceback and laboratory findings linked this outbreak to feeder rodents (used to feed snakes) supplied by a network of rodent breeders in Ontario. Cases likely acquired their illness through either direct or indirect contact with feeder rodents. This investigation not only contributes to the weight of evidence on the risk that feeder rodents pose, but also underscores the importance of investigating indirect animal contact and associated risks, especially for high‐risk individuals.     

应用多重PCR检测人工感染鸡呼吸道疾病的研究

本文报道了IBV，NDV，ILTV，MG人工感染4周龄SPF鸡和非免疫鸡后，用多重PCR检测试验鸡的咽喉棉拭子和器官组织样品，并与传统的病原分离鉴定，血清学方法进行比较，多重PCR无论是对单一感染病原，还是对两种以上混合感染病原，其敏感性和检测速度都优于传统的鉴别诊断方法，具有较高的实用价值，可以直接应用于临床检测，服务于生产。     

Prevalence and Characterization of Salmonella Isolated from Feral Pigs Throughout Texas

Feral pigs are one of the most abundant free‐roaming ungulates in the United States, yet their role in the ecology and transmission of foodborne pathogens is poorly understood. Our objectives were to estimate the prevalence of Salmonella shedding among feral pigs throughout Texas, to identify risk factors for infection, and to characterize the isolates. Faecal samples were collected from feral pigs in Texas from June 2013 through May 2015. Standard bacteriologic culture methods were used to isolate Salmonella from samples, and isolates were characterized via serotyping and anti‐microbial susceptibility testing. The prevalence of faecal Salmonella shedding among sampled pigs was 43.9% (194/442), with positive pigs originating from 50 counties. Pigs sampled during fall and summer were significantly more likely to be shedding Salmonella than pigs sampled during winter. High serovar diversity was evident among the isolates, and many of the detected serovars are leading causes of human salmonellosis. The most common serovars were Montevideo (10.0%), Newport (9.1%), and Give (8.2%). Resistance to anti‐microbial agents was rare. The burgeoning feral pig population in the United States may represent an emerging threat to food safety.     

A four‐season longitudinal study of enterohaemorrhagic Escherichia coli in beef cow–calf herds in Mississippi and Nebraska

Our objective was to describe the probability of detecting seven serogroups of enterohaemorrhagic Escherichia coli (EHEC‐7) of public health importance in faecal samples from beef cow–calf herds and to test for factors associated with their detection. Fresh faecal samples (n = 85) from two Mississippi and two Nebraska herds were collected in each of four seasons. Samples were tested for each EHEC‐7 serogroup by a molecular screening assay. Separate management groups within herds were sampled, and group‐level factors were recorded. To measure the effects of factors on faecal shedding of EHEC‐7, separate multivariable logistic regression models were used, accounting for the random effect of clustering by group within farm. Statistical significance was set α = 0.05. Fifty‐nine samples (4.3%) were positive for EHEC O26, and Nebraska samples were more likely to be positive than Mississippi samples (OR = 12.4, 95% CI: 1.1, 139.2). Forty‐four samples (3.2%) were positive for EHEC O45. Odds for detection were greater in the summer than all other seasons combined (OR = 4.2, 95% CI: 1.3, 14.0), and odds decreased if a precipitation event occurred (OR = 0.07, 95% CI: 0.006, 0.8). EHEC O103 was detected in 66 samples (4.9%) with increased probability to be detected at increased temperature. EHEC O111 was detected in 71 samples (5.2%), and 43 samples (3.2%) were positive for EHEC O145. Both EHEC O111 and O145 were associated separately with season, with greater probability for detection in the summer. Eighteen (1.3%) and 68 (5.0%) samples were positive for EHEC O121 and EHEC O157, respectively. We failed to detect significant explanatory factors associated with probability to detect EHEC O121 or O157. Factors that vary by time and place, such as precipitation, ambient temperature, region and season, are uniquely associated with the probability to detect EHEC‐7 in fresh faeces collected from cow–calf herds.     

Identification and selection of food safety‐related risk factors to be included in the Canadian Food Inspection Agency’s Establishment‐based Risk Assessment model for Hatcheries

Towards the continuous improvement of its inspection system, the Canadian Food Inspection Agency (CFIA) is developing an Establishment‐based Risk Assessment model for Hatcheries to allocate inspection resources based on the food safety risk associated with the Canadian hatcheries falling under its jurisdiction. The objectives of the current study were to identify and select critical food safety‐related risk factors that would be included in this model, with a main focus on Salmonella. A literature review was used to develop a comprehensive list of risk factors that could potentially contribute to the food safety risk attributed to Canadian hatcheries operating in all production streams (breeders, layers, broilers, turkeys, waterfowl and game birds). The development of this list used a selection process that was conducted according to the availability of data sources, the clarity of definition and the measurability of the selected risk factors. A panel of experts reviewed and adjusted the identified risk factors. A final list of 29 risk factors was generated; 20 originated from the scientific literature and nine from the expert panel. Risk factors were grouped in three clusters according to whether they pertained to the inherent risk (nine factors identified), risk mitigation (nine factors identified) or compliance of a hatchery with its preventive control plan and regulatory requirements (11 factors identified). Criteria for assessing each risk factor were defined based on common practices used in the Canadian hatchery industry. This comprehensive list of risk factors and criteria represents useful information to support the design and implementation of a Canadian risk assessment model for hatcheries, but could also be used by like‐minded food safety authorities.     

A randomized controlled trial to evaluate the effects of dietary fibre from distillers grains on enterohemorrhagic Escherichia coli detection from the rectoanal mucosa and hides of feedlot steers

Feeding high levels (≥40% dry matter) of distillers grains may increase the risk for cattle to carry enterohemorrhagic Escherichia coli (EHEC) O157. The mechanism for the increased risk is not known nor whether non‐O157 EHEC are similarly affected. Our objective was to test whether the fibre content or other components of modified distillers grains plus solubles (MDGS) affects the probability for cattle to carry EHEC serogroups of public health importance. A 2 × 2 plus 1 factorial treatment arrangement within a randomized block design was utilized. Within each of four blocks, 25 feedlot pens (n = 8 steers/pen) were assigned randomly to (i) corn‐based control diet; (ii) 20% dry matter (DM) MDGS; (iii) 40% DM MDGS; (iv) corn bran added to corn‐based diet to match fibre of 20% MDGS or (v) 40% MDGS. Rectoanal mucosa swabs (RAMS) were collected on day (d)0, d35, d70 and d105; hide swabs were collected on the last feeding day. Samples were tested for EHEC by a molecular screening assay. The effects of fibre source and fibre level on EHEC carriage were tested using multilevel logistic regression (generalized linear mixed models; α = 0.05). EHEC O45 RAMS detection was associated with fibre level, source and sampling day. EHEC O103 RAMS detection increased by feeding 40% MDGS but not the corresponding corn bran diet. Hide contamination by EHEC O45 or O103 was less likely in cattle fed MDGS compared to corn bran diets. EHEC O111 RAMS detection decreased by feeding 40% MDGS but not by feeding the corresponding corn bran diet. Detection of EHEC O157 or O145 was not associated with dietary factors. Feeding 40% MDGS increased the probability for carriage of some EHEC serogroups but decreased probability of others, which indicated that EHEC serogroups have different risk factors associated with feeding MDGS and little association with dietary fibre.     

Cross‐sectional study to estimate the prevalence of enterohaemorrhagic Escherichia coli on hides of market beef cows at harvest

Cattle hides are an important source of enterohaemorrhagic Escherichia coli (EHEC) carcass contamination at slaughter. Seven EHEC serogroups are adulterants in raw, non‐intact beef: EHEC O26, O45, O103, O111, O121, O145 and O157. The objective of this study was to estimate the probability for hide contamination with EHEC among US market beef cows at slaughter and to test the effects of season and geographic region on prevalence of hide contamination. Hides (n = 800) of market cows were swabbed at slaughter immediately after exsanguination, prior to hide removal. Cows were sampled from two geographically distinct beef packing plants during four seasons of 2015. Cattle source was categorized by northern or southern region. Samples were tested for EHEC by a molecular screening assay. The effects of region, season and their interaction on the probability of hide contamination by each EHEC serogroup were tested in separate multilevel multivariable logistic regression models, accounting for the random effect of clustering by plant. Statistical significance was set α = .05. Of 800 total samples, at least one EHEC was detected on 630 (79%) hides. Enterohaemorrhagic E. coli O26 was detected on 129 (16%) of all hides sampled, EHEC O45 on 437 (55%), EHEC O103 on 289 (36%), EHEC O111 on 189 (24%), EHEC O121 on 140 (18%), EHEC O145 on 171 (21%) and EHEC O157 on 89 (11%). Detection of EHEC O26 and EHEC O121 was associated with season. Season and region were associated with detecting EHEC O45 and EHEC O157. Season‐by‐region interactions were associated with the outcome of detecting EHEC O103, EHEC O111 and EHEC O145. Season, region of origin and the interaction of these factors affect hide contamination of market beef cattle at slaughter by EHEC, and each serogroup responds to these factors uniquely.     

Investigation of the Concurrent Emergence of Salmonella enteritidis in Humans and Poultry in British Columbia,Canada, 2008–2010

An increase in the rate of human infections with Salmonella enteritidis (SE) occurred between 2007 and 2010 in British Columbia (BC). During the same time period, an increase in SE from poultry‐sourced isolates and increased clinical severity in poultry were also observed in BC. This article describes a multi‐sectoral collaboration during a 3‐year investigation, and the actions taken by public health and animal health professionals. Human cases were interviewed, clusters were investigated, and a case–control study was conducted. Environmental investigations were conducted in food service establishments (FSE). Suspect foods were tested. Laboratory data from poultry‐sourced isolates were analysed. Five hundred and eighty‐four human cases of SE with the same pulsed‐field gel electrophoresis pattern were identified between May 2008 and August 2010. Seventy‐three percentage of cases reported consumption of eggs. The odds of egg consumption were 2.4 times higher for cases than controls. Implicated FSE were found to use ungraded eggs, which had been distributed illegally. Investigation suggested that there were multiple suppliers of these eggs. Collaboration between public health and animal health professionals led to data sharing, improved understanding of SE, engagement with the poultry industry and public communication. Multi‐disciplinary, multi‐sectoral and multi‐pronged investigations are recommended to identify the likely source of illness in large, protracted foodborne outbreaks caused by commonly consumed foods.     

Clonal Spread of Salmonella enterica Serovar Infantis in Serbia: Acquisition of Mutations in the Topoisomerase Genes gyrA and parC Leads to Increased Resistance to Fluoroquinolones

Quinolone‐resistant Salmonella Infantis (n = 64) isolated from human stool samples, food and poultry during the years 2006–2011 were analysed for their resistance phenotypes, macrorestriction patterns and molecular mechanisms of decreased susceptibility to fluoroquinolones. Minimum inhibitory concentrations (MICs) of nalidixic acid (NAL) and ciprofloxacin (CIP) were determined by the agar dilution procedure, and the susceptibility to additional antimicrobial agents was determined by the disc diffusion method. To assess the influence of enhanced efflux activity, MICs were determined in the presence and absence of the inhibitor PAβN. The results of pulsed‐field gel electrophoresis (PFGE) typing revealed that quinolone‐resistant S. Infantis in Serbia had similar or indistinguishable PFGE profiles, suggesting a clonal spread. All S. Infantis showed combined resistance to NAL and tetracycline, whereas multiple drug resistance to three or more antibiotic classes was rare (2 isolates of human origin). The MICs ranged between 512 and 1024 μg/mL for NAL and 0.125–2 μg/mL for CIP. A single‐point mutation in the gene gyrA leading to a Ser83→Tyr exchange was detected in all isolates, and a second exchange (Ser80→Arg) in the gene parC was only present in eight S. Infantis isolates exhibiting slightly higher MICs of CIP (2 μg/mL). The inhibitor PAβN decreased the MIC values of CIP by two dilution steps and of NAL by at minimum 3–6 dilution steps, indicating that enhanced efflux plays an important role in quinolone resistance in these isolates. The plasmid‐mediated genes qnr, aac(6′)‐lb‐cr and qepA were not detected by PCR assays.     

Non‐Typhoidal Salmonella Colonization in Chickens and Humans in the Mekong Delta of Vietnam

Salmonellosis is a public health concern in both the developed and developing countries. Although the majority of human non‐typhoidal Salmonella enterica (NTS) cases are the result of foodborne infections or person‐to‐person transmission, NTS infections may also be acquired by environmental and occupational exposure to animals. While a considerable number of studies have investigated the presence of NTS in farm animals and meat/carcasses, very few studies have investigated the risk of NTS colonization in humans as a result of direct animal exposure. We investigated asymptomatic NTS colonization in 204 backyard chicken farms, 204 farmers and 306 matched individuals not exposed to chicken farming, in southern Vietnam. Pooled chicken faeces, collected using boot or handheld swabs on backyard chicken farms, and rectal swabs from human participants were tested. NTS colonization prevalence was 45.6%, 4.4% and 2.6% for chicken farms, farmers and unexposed individuals, respectively. Our study observed a higher prevalence of NTS colonization among chicken farmers (4.4%) compared with age‐, sex‐ and location‐ matched rural and urban individuals not exposed to chickens (2.9% and 2.0%). A total of 164 chicken NTS strains and 17 human NTS strains were isolated, and 28 serovars were identified. Salmonella Weltevreden was the predominant serovar in both chickens and humans. NTS isolates showed resistance (20–40%) against tetracycline, chloramphenicol, sulfamethoxazole‐trimethoprim and ampicillin. Our study reflects the epidemiology of NTS colonization in chickens and humans in the Mekong delta of Vietnam and emphasizes the need of larger, preferably longitudinal studies to study the transmission dynamics of NTS between and within animal and human host populations.     

Effect of laboratory‐isolated Lactobacillus plantarum LGFCP4 from gastrointestinal tract of guinea fowl on growth performance,carcass traits,intestinal histomorphometry and gastrointestinal microflora population in broiler chicken

The study aimed to investigate the effect of feed supplements, viz Lactobacillus plantarum LGFCP4 (laboratory isolate from GIT of Guinea fowl), Lactobacillus acidophilus (NCDC, Karnal) and in‐feed antibiotic bacitracin methylene disalicylate (BMD) on growth performance, FCR, carcass traits and immune organs weight, intestinal histomorphometry and gastrointestinal microflora population in broiler chickens. In a completely randomized design, CARIBRO‐Dhanraja broiler chicks (n = 160) were used with four treatment groups. During the entire experimental duration of 35 days, treatment groups were provided with different dietary treatments (T1 – basal diet (negative control), T2 – antibiotic growth promoter BMD 20 g/100 kg feed (positive control), T3 – 1 × 10⁸ cfu of L. acidophilus/gm‐fermented feed +MOS 1 g/kg feed and T4 – 1 × 10⁸ cfu of laboratory‐isolated L. plantarum LGFCP4/gm‐fermented feed+ MOS 1 g/kg feed. After 35 days of experimental period, no significant results have been observed in different growth performance traits among treatment groups. Cut‐up parts and edible organs' weight remained unaffected by dietary supplementation, whereas weight of immune organs were significantly higher (p < 0.05) in L. plantarum LGFCP4‐supplemented group. At the end of feeding trial, significantly (p < 0.05) lower E. coli count was observed in crop of T4 birds, while in ileum, T2 and T3 showed lower count. In caeca, T2 group showed lowest E. coli count. Salmonella count in crop and ileum was significantly (p < 0.05) low in T3 and T4, while in caeca, T2 group showed lowest count. In terms of histomorphometry, duodenal villous height (VH), crypt depth (CD) and VH:CD ratio were higher for T3 and T4 and lowest values were obtained for T2 group. The results of the study showed that L. plantarum LGFCP4 isolated from GIT of guinea fowl can effectively replace in‐feed antibiotic growth promoters in broiler diets by altering intestinal villi morphology and improving the gut health by reducing the pathogenic microbial load.     

Dietary Bacillus subtilis C-3102 Supplementation Enhances the Exclusion of Salmonella enterica from Chickens

Among the reported probiotic Bacillus strains, B. subtilis C-3102 has the unique potential to improve feed uptake under stress conditions in the broilers, piglets, and cows. In this study, we sought to evaluate the protective effect of feed additive probiotic Bacillus subtilis C-3102 against Salmonella enterica infection of specific pathogen-free (SPF) chicks in floor pens in two experiments. In the experiment-1, the chicks in the control group (n=32) were fed a basal diet and those in the C-3102 group (n=32) were fed a basal diet supplemented with 1×10⁶ CFU/g of feed for 28 days. On day 7 post-challenge with S. enterica, there was no significant change in the body weight between both the groups throughout the test period, whereas detection rates of S. enterica in the C-3102 group were significantly lower in the cecum and liver on days 21 and 14 post-challenge, respectively. In the experiment-2, minimum dosage of C-3102 cells required to protect Salmonella infection was evaluated using 3 dosages. Chicks were divided into four groups, fed with different dosages of C-3102 (1×10⁶, 5×10⁵, 3×10⁵, and 0 CFU/g of feed), and challenged with S. enterica (2.8×10⁸ CFU/chicken). S. enterica infection was completed within 7 days post- challenge and was almost excluded from the liver and spleen on day 21 post- challenge in the control group. Average values showed a trend for higher infection rates in the control group >3×10⁵>5×10⁵>1×10⁶ CFU/g on days 14 and 21 post-challenge. These results suggest that B. subtilis C-3102 supplementation has the potential to reduce S. enterica infection rates and/or to accelerate the exclusion of S. enterica from the chicks.     

Risk Factors Associated with Salmonella Status of Broiler Flocks Delivered to Grow‐Out Farms

In a prospective field observational study in the southeastern USA, we sampled gastrointestinal (GI) tracts from chicks of 65 broiler flocks delivered to conventional grow‐out farms for rearing. The flocks were hatched at seven broiler hatcheries. The mean within‐flock prevalence of Salmonella‐positive samples was 6.5% and ranged from 0% to 86.7%. Of the 65 flocks studied, 25 (38.5%) had at least one Salmonella‐positive sample. Accounting for confounding variability among the hatcheries and broiler companies, we tested whether the probability of detecting Salmonella in GI tracts of the chicks delivered was associated with certain characteristics of parent breeder flocks; hatchery production volume; hatchery ventilation system; hatchery egg‐room conditions; egg incubation, candling, hatching, eggshell and bird separation, and bird‐processing procedures; management of hatchery‐to‐farm transportation; day of week of hatch; weather conditions during transportation; or season of the hatch. Two risk factor models were adopted. The first model indicated that a greater number of parent flocks, manual separation of eggshell and bird, and a greater amount of fluff and feces on tray liners used during hatchery‐to‐farm transportation at delivery were associated with increased probability of detecting Salmonella in chick GI tracts, whereas a greater number of birds in the delivery vehicle was associated with decreased probability. The second model indicated that broiler flocks hatched on Tuesdays versus either Mondays or Thursdays (with no hatches on Wednesdays, Fridays or week‐ends), increased average hatchability of the eggs from the parent flocks, and greater amounts of fluff and feces on the transport tray liners at delivery were all associated with increased probability of detecting Salmonella in chick GI tracts. The results of this study suggest potential management decisions to lessen Salmonella contamination of broilers supplied by commercial hatcheries and areas for further research.     

Purification and Characterization of the Aetiological Agent of Hydropericardium Hepatitis Syndrome from Infected Liver Tissues of Broiler Chickens

Hydropericardium hepatitis syndrome in broiler chickens is an acute, infectious disease characterized by high mortality, excess pericardial fluid and multifocal hepatic necrosis. The aetiological agent was purified to homogeneity from infected liver tissues from field outbreaks. Electron-microscopic and serological confirmation of the virus were undertaken and the disease was reproduced experimentally in broiler chicks. The results indicated that an adenovirus, fowl adenovirus serotype 4, was alone responsible for the disease in the materials studied.     

Estimation Models for the Morbidity of the Horses Infected with Equine Influenza Virus

Estimation formulas for the morbidity of horses infected with equine influenza virus by linear regression, logistic regression and probit transformation were developed, using data from the outbreak at the Sha Tin Racing Track in Hong Kong in 1992. Using these formulas, we estimated the equine influenza virus morbidity rates at training centers belonging to the Japan Racing Association (JRA) in October 1997 and in October 1998. In 1998 JRA started a new vaccination program, and every horse must now be vaccinated twice per year. At that time, the vaccine included two US lineage virus strains, the A/equine/Kentucky/81 strain and the A/equine/La Plata/93 (LP93) strain, against equine type-2 influenza viruses; it did not include any EU lineage virus strains, such as A/equine/Suffolk/89 (SF89). Comparing the geometric mean (GM) values of hemagglutination inhibition (HI) titers between the LP93 strain and the SF89 strain in 1997 and in 1998, they both rose significantly at every age (p<0.05) by Wilcoxon test. Calculations by the simulation models show the morbidity rates for LP93 diminished from 0.439 (linear), 0.423 (logistic) and 0.431 (probit) to 0.276 (linear), 0.265 (logistic) and 0.271 (probit), respectively. On the other hand, the estimated morbidity rates for SF89 diminished only slightly from 0.954 (linear), 0.932 (logistic) and 0.944 (probit) to 0.946 (linear), 0.914 (logistic) and 0.927 (probit), respectively. Our simulation models could estimate the effect of the vaccine on each of the equine virus strains represented by the morbidity of infected horses. Thus, they are useful for vaccine evaluation.