VTEC O157 Research Articles

Untangling the role of environmental and host-related determinants for on-farm transmission of verotoxin-producing Escherichia coli O157

ABSTRACT Background: Cattle colonised by the zoonotic pathogen verotoxin-producing Escherichia coli of serotype O157 (VTEC O157) can shed high levels of the pathogen in their faeces. A suggested key for controlling VTEC O157 is preventing colonisation of individuals. Aim: In this study the role of individual super-shedders and factors related to susceptibility and environmental exposure in the transmission of VTEC O157 among dairy calves are explored. Methods: The association between sex, age, pen hygiene, pen type and stocking density and colonisation of individual calves, established by recto-anal mucosal swabs, on farms where pathogenic VTEC O157 had been confirmed was investigated. In a follow-up sampling, the consistency of previously identified risk factors and the role of shedding pen mates was assessed by studying the risk of new/re-colonisation. Results: The results suggest an important role of stocking density that decreases with age, possibly due to increased resistance to colonisation following exposure. However, previous colonisation did not influence the risk of being colonised in the second sampling. Super-shedders (shedding >103 colony forming units/g faeces) significantly increased the risk of colonisation in peers (OR = 10, CI 4.2–52). In addition, environmental factors associated with survival of the bacteria, affected risk. Conclusion: The results confirm the suggested importance of super-shedders but also emphasises the importance of considering the combined exposure from peers and the environment.

Genetic features of verotoxigenic Escherichia coli O157:H7 isolated from clinical cases of Argentina and Chile

We aimed to compare the genetic diversity existing in VTEC O157:H7 strains isolated from cases of human disease from Argentina and Chile. For it, 76 strains were studied in relation to the distribution of genes encoding virulence factors and subtyped by lineage-specific polymorphisms (LSPA-6), and phylogroups assignment. Our results show the almost exclusive circulation of VTEC O157:H7 isolates belonging to lineage I/II, associated with hypervirulent strains, and to the phylogroup E and, on the other hand, genetic diversity present among Argentinean and Chilean strains analyzed, mainly in relation to putative virulence determinants and nle profiles.

Socially engaged calves are more likely to be colonised by VTEC O157:H7 than individuals showing signs of poor welfare

In cattle herds, the transmission and persistence of VTEC O157:H7 (a serotype of verotoxin-producing Escherichia coli – known for its life threatening complications in humans) is dependent on a small proportion of cattle who become colonised and shed high numbers of the bacteria. Reducing the proportion of these animals is considered key for decreasing the prevalence of VTEC O157:H7. In this study, observations of calf behaviour and animal-based welfare indicators were used to explore individual risk factors and underlying drivers of colonisation in Swedish dairy calves. Interdependencies between variables led to three different approaches being used to visualize and explore the associations. Combining the results of all methods revealed similar patterns and suggest that healthy animals, actively grooming and interacting with others calves in the group have a higher risk of colonisation than small dairy calves in poor condition (diarrhoea, poor ruminal fill, poor body condition score and nasal discharge). This lends no support to the hypothesis that reduced welfare is a risk factor for VTEC O157:H7, but implies that individual differences in calf behaviour affect oral exposure to the bacteria so driving the risk of colonisation. This new finding has important implications for understanding of VTEC O157:H7 transmission within farms.

Molecular subtyping and clonal relatedness of human and cattle verotoxin-producing Escherichia coli O157:H7 isolates

Verotoxin-producing Escherichia coli O157:H7 is the dominant serotype isolated from patients with hemolytic-uremic syndrome (HUS) and, Argentina has the highest rate of HUS in the world. However, not all O157:H7 isolates have the same ability to infect and cause disease in humans. It has been postulated that O157:H7 strains integrate subpopulations related to the origin and virulence. In order to study the population structure and genetic diversity of VTEC O157:H7 from Argentina, a combination of molecular subtyping methods such as multiple loci VNTR analysis (MLVA), single nucleotide polymorphisms (SNP) and phylogroups assignment were used. According to MLVA, high genetic diversity was found among strains isolated from cattle, humans and food. On the other hand, 92% of the isolates presented the allele tir 255 T > A T and 95% were assigned to phylogroup E. We did not find a significant association between the isolates origin and the allele T presence (P > 0,05) postulated as significantly overrepresented in human isolates. Our results show that human and cattle VTEC O157:H7 isolates from Argentina are a homogeneous group and, although it presents high genetic diversity in relation to their MLVA and virulence profiles, it is not possible to distinguish divergent populations. The presence in all the strains of a high number of T3SS effectors genes and the no association of genetic subtypes with strain source, is an alert about the potential risk in public health that VTEC O157:H7 cattle strains possess and, at less, a partial explication about the high incidence of HUS in Argentina.

Spatio-temporal modelling of verotoxigenic Escherichia coli O157 in cattle in Sweden: exploring options for control

A spatial data-driven stochastic model was developed to explore the spread of verotoxigenic Escherichia coli O157 (VTEC O157) by livestock movements and local transmission among neighbouring holdings in the complete Swedish cattle population. Livestock data were incorporated to model the time-varying contact network between holdings and population demographics. Furthermore, meteorological data with the average temperature at the geographical location of each holding was used to incorporate season. The model was fitted against observed data and extensive numerical experiments were conducted to investigate the model’s response to control strategies aimed at reducing shedding and susceptibility, as well as interventions informed by network measures. The results showed that including local spread and season improved agreement with prevalence studies. Also, control strategies aimed at reducing the average shedding rate were more efficient in reducing the VTEC O157 prevalence than strategies based on network measures. The methodology presented in this study could provide a basis for developing disease surveillance on regional and national scales, where observed data are combined with readily available high-resolution data in simulations to get an overview of potential disease spread in unobserved regions.

Data-driven network modelling of disease transmission using complete population movement data: spread of VTEC O157 in Swedish cattle.

European Union legislation requires member states to keep national databases of all bovine animals. This allows for disease spread models that includes the time-varying contact network and population demographic. However, performing data-driven simulations with a high degree of detail are computationally challenging. We have developed an efficient and flexible discrete-event simulator SimInf for stochastic disease spread modelling that divides work among multiple processors to accelerate the computations. The model integrates disease dynamics as continuous-time Markov chains and livestock data as events. In this study, all Swedish livestock data (births, movements and slaughter) from July 1st 2005 to December 31st 2013 were included in the simulations. Verotoxigenic Escherichia coli O157:H7 (VTEC O157) are capable of causing serious illness in humans. Cattle are considered to be the main reservoir of the bacteria. A better understanding of the epidemiology in the cattle population is necessary to be able to design and deploy targeted measures to reduce the VTEC O157 prevalence and, subsequently, human exposure. To explore the spread of VTEC O157 in the entire Swedish cattle population during the period under study, a within- and between-herd disease spread model was used. Real livestock data was incorporated to model demographics of the population. Cattle were moved between herds according to real movement data. The results showed that the spatial pattern in prevalence may be due to regional differences in livestock movements. However, the movements, births and slaughter of cattle could not explain the temporal pattern of VTEC O157 prevalence in cattle, despite their inherently distinct seasonality.

A longitudinal study of risk factors for shedding of VTEC O157 by young cattle in herds with known E. coli O157 carriage.

A longitudinal study in England and Wales of two dairy, five beef-fattener and three beef-suckler herds was carried out to identify risk factors for young cattle excreting verocytotoxin-producing Escherichia coli O157 (VTEC O157). A total of 1383 cattle, selected into cohorts at 0-24 months were sampled between March 2000 and February 2001. Mixed-effects logistic regression was employed to identify significant associations between VTEC O157 isolation from rectal faecal samples and explanatory factors (P < 0·001 unless shown). The results revealed a positive association with feeding root crops and a negative association with animals fed silage, milk (P = 0·001) or grain (P = 0·027). Cattle in suckler herds (P = 0·001) and those changing group between sampling visits were identified as negatively associated with VTEC O157 presence. The recovery of VTEC O157 varied throughout the year. However, the winter period from December to February was a risk factor in the multivariable analysis. Cattle in pens were 4·7 times more likely to shed VTEC O157 than those group-housed or at pasture. VTEC O157 detected in pooled environmental faecal pats and biofilm of the water supply within a group's enclosure were positively associated with an animal's VTEC O157 status in the multivariable logistic regression, as was detection of VTEC O157 in the pooled faecal pats at the previous visit.

Longitudinal observational study over 38 months of verotoxigenic Escherichia coli O157:H7 status in 126 cattle herds

Verotoxigenic Escherichia coli O157:H7 (VTEC O157:H7) is an important zoonotic pathogen capable of causing infections in humans, sometimes with severe symptoms such as hemorrhagic colitis and hemolytic uremic syndrome (HUS). It has been reported that a subgroup of VTEC O157:H7, referred to as clade 8, is overrepresented among HUS cases. Cattle are considered to be the main reservoir of VTEC O157:H7 and infected animals shed the bacteria in feces without showing clinical signs of disease. The aims of the present study were: (1) to better understand how the presence of VTEC O157:H7 in the farm environment changes over an extended period of time, (2) to investigate potential risk factors for the presence of the bacteria, and (3) describe the distribution of MLVA types and specifically the occurrence of the hypervirulent strains (clade 8 strains) of VTEC O157:H7. The farm environment of 126 cattle herds in Sweden were sampled from October 2009 to December 2012 (38 months) using pooled pat and overshoe sampling. Each herd was sampled, on average, on 17 occasions (range=1–20; median=19), at intervals of 64 days (range=7–205; median=58). Verotoxigenic E. coli O157:H7 were detected on one or more occasions in 53% of the herds (n=67). In these herds, the percentage of positive sampling occasions ranged from 6% to 72% (mean=19%; median=17%). Multi-locus variable number tandem repeat analysis (MLVA) typing was performed on isolates from infected herds to identify hypervirulent strains (clade 8). Clustering of MLVA profiles yielded 35 clusters and hypervirulent strains were found in 18 herds; the same cluster was often identified on consecutive samplings and in nearby farms. Using generalized estimating equations, an association was found between the probability of detecting VTEC O157:H7 and status at the preceding sampling, season, herd size, infected neighboring farms and recent introduction of animals. This study showed that the bacteria VTEC O157:H7 were spontaneously cleared from the farm environment in most infected herds over time, and key factors were identified to prevent the spread of VTEC O157:H7 between cattle herds.

Estimates of foodborne illness-related hospitalizations and deaths in Canada for 30 specified pathogens and unspecified agents.

Foodborne illness estimates help to set food safety priorities and create public health policies. The Public Health Agency of Canada estimates that 4 million episodes of foodborne illness occur each year in Canada due to 30 known pathogens and unspecified agents. The main objective of this study was to estimate the number of domestically acquired foodborne illness–related hospitalizations and deaths. Using the estimates of foodborne illness for Canada along with data from the Canadian Hospitalization Morbidity Database (for years 2000–2010) and relevant international literature, the number of hospitalizations and deaths for 30 pathogens and unspecified agents were calculated. Analysis accounted for under-reporting and underdiagnosis. Estimates of the proportion foodborne and the proportion travel-related were incorporated for each pathogen. Monte Carlo simulations were performed to account for uncertainty generating mean estimates and 90% probability intervals. It is estimated that each year there are 4000 hospitalizations (range 3200–4800) and 105 (range 75–139) deaths associated with domestically acquired foodborne illness related to 30 known pathogens and 7600 (range 5900–9650) hospitalizations and 133 (range 77–192) deaths associated with unspecified agents, for a total estimate of 11,600 (range 9250–14,150) hospitalizations and 238 (range 155–323) deaths associated with domestically acquired foodborne illness in Canada. Key pathogens associated with these hospitalizations or deaths include norovirus, nontyphoidal Salmonella spp., Campylobacter spp., VTEC O157 and Listeria monocytogenes. This is the first time Canada has established pathogen-specific estimates of domestically acquired foodborne illness–related hospitalizations and deaths. This information illustrates the substantial burden of foodborne illness in Canada.

The feasibility of testing whether Fasciola hepatica is associated with increased risk of verocytotoxin producing Escherichia coli O157 from an existing study protocol

The parasite Fasciola hepatica is a major cause of economic loss to the agricultural community worldwide as a result of morbidity and mortality in livestock, including cattle. Cattle are the principle reservoir of verocytotoxigenic Escherichia coli O157 (VTEC O157), an important cause of disease in humans. To date there has been little empirical research on the interaction between F. hepatica and VTEC O157. It is hypothesised that F. hepatica, which is known to suppress type 1 immune responses and induce an anti-inflammatory or regulatory immune environment in the host, may promote colonisation of the bovine intestine with VTEC O157. Here we assess whether it is statistically feasible to augment a prospective study to quantify the prevalence of VTEC O157 in cattle in Great Britain with a pilot study to test this hypothesis. We simulate data under the framework of a mixed-effects logistic regression model in order to calculate the power to detect an association effect size (odds ratio) of 2. In order to reduce the resources required for such a study, we exploit the fact that the test results for VTEC O157 will be known in advance of testing for F. hepatica by restricting analysis to farms with a VTEC O157 sample prevalence of >0% and <100%.From a total of 270 farms (mean 27 cows per farm) that will be tested for VTEC O157, power of 87% can be achieved, whereby testing of F. hepatica would only be necessary for an expected 50 farms, thus considerably reducing costs. Pre-study sample size calculations are an important part of any study design. The framework developed here is applicable to the study of other co-infections.

Inter-laboratory comparison of multi-locus variable-number tandem repeat analysis (MLVA) for verocytotoxin-producingEscherichia coliO157 to facilitate data sharing

Multi-locus variable number tandem repeat analysis (MLVA) is used in clinical and reference laboratories for subtyping verocytotoxin-producing Escherichia coli O157 (VTEC O157). However, as yet there is no common allelic or profile nomenclature to enable laboratories to easily compare data. In this study, we carried out an inter-laboratory comparison of an eight-loci MLVA scheme using a set of 67 isolates of VTEC O157. We found all but two isolates were identical in profile in the two laboratories, and repeat units were homogeneous in size but some were incomplete. A subset of the isolates (n = 17) were sequenced to determine the actual copy number of representative alleles, thereby enabling alleles to be named according to international consensus guidelines. This work has enabled us to realize the potential of MLVA as a portable, highly discriminatory and convenient subtyping method.

Detection of Potentially Pathogenic E. coli O157H7 in Buffalo Meat Samples by Conventional and Molecular Methods

Escherichia coli O157 is the classical serotype having zoonotic significance. E. coli O157 has been reported to cause not only diarrheal illness but also hemorrhagic colitis (HC), haemorrhagic uremic syndrome (HUS) and thrombotic thrombocytopaenic purpurea (TTP). In the present study, 150 raw buffalo meat samples were collected from the retail meat market viz. muscle, ground beef, intestine, liver and lung (30 each) from Anand, India. All the samples were screened for VTEC O157, using conventional culture and polymerase chain reaction (PCR). Among 150 buffalo meat samples 3 (2%) were found positive for E. coli O157 by conventional culture as well as for amplification of rfb O157 gene by PCR. The presence of E. coli O157 in the buffalo meat samples of Anand, Gujarat suggests that this enteropathogen may be of public health importance. Hence, routine screening of buffalo meat samples for VTEC O157 may be useful.

Exploration and conservation of bacterial genetic resources as bacteriocin producing inhibitory microorganisms to pathogen bacteria in livestock

Exploration and conservation of microorganisms producing bacteriocin was done as the primary study towards the collection of potential bacteria and its application in improving livestock health condition and inhibit food borne pathogens. Diferent kinds of samples such as beef cattle rectal swab, rumen fluids, cow’s milk, chicken gut content, goat’s milk were collected at Bogor cattle slaughter houses, poultry slaughter houses, dairy cattle and goat farms. A total of 452 bacterial isolates consisted of 73 Gram negative bacteria and 379 Gram positive bacteria were isolated from samples collected and screened for bacteriocin activity. Determination of bacteriocin activity with bioassay using agar spot tests were carried out on liquid and semisolid medium assessing 8 kins of indicators of pathogenic bacteria and food borne pathogens. A total of 51 bacteriocin producing strains were collected and some of the strains had high inhibitory zone such as Lactobacillus casei SS14C (26 mm), Enterobacter cloacae SRUT (24mm), Enterococcus faecalis SK39 (21mm) and Bifidobacterium dent ium SS14T (20mm) respectively, to Salmonella typhimurium BCC B0046/ATCC 13311, E. coli O157 hemolytic BCC B2717, Listeria monocytogenes BCC B2767/ATCC 7764 and E scherichia coli VTEC O157 BCC B2687. Evaluation after conservation ex situ to all bacterocin producing strain at 5 o C for 1 year in freeze drying ampoules in vacuum and dry condition revealed the decreasing viability starting from log 0.8 CFU/ml for Lactococcus and Leuconostoc to log 2.2. CFU/ml for Streptococcus . Result of the study showed that the bacteriocin producing strains obtained were offered a potential resource for preventing disease of livestock and food borne diseases. Key Words: Bacterocin, Bacteria, Exploration, Conservation Ex Situ

Application of multilocus variable number tandem repeat analysis to monitor Verocytotoxin-producingEscherichia coliO157 phage type 8 in England and Wales: emergence of a profile associated with a national outbreak

Evaluation of multilocus variable number tandem repeat analysis (MLVA) to subtype all isolates of Vero cytotoxin-producing Escherichia coli O157 phage type 8 in England and Wales. Over a 13 month period from December 2010, 483 isolates of VTEC O157 PT8 were tested by MLVA; 39% were received in the first 4months of 2011, when infections are generally low. One profile, or single locus variants of it, was present in 249 (52%) isolates but was not common previously. These cases represented a national increase in PT8, associated epidemiologically with soil-contaminated vegetables. Most of the 177 other MLVA profiles were unique to a single isolate. Profiles shared by >1 isolate included cases from two small community, food-borne outbreaks and 11 households. Several shared profiles were found among 23 isolates without known links. Apart from one group, isolates linked to travel abroad had very diverse profiles. Multilocus variable number tandem repeat analysis discriminated apparent sporadic isolates of the same PT and assisted in detection of cases in an emerging national outbreak. Multilocus variable number tandem repeat analysis is an epidemiologically valid complement to surveillance and applicable as a rapid, practical test for large numbers of isolates.

Prevalence of verotoxigenic Escherichia coli O157 (VTEC O157) and compliance with microbiological safety standards in bovine carcasses from an industrial beef slaughter plant

The presence of Salmonella spp. and levels of Enterobacteriaceae and aerobic plate count were determined in 300 bovine carcasses randomly collected in an industrial cattle slaughterhouse in Catalonia (Spain) as part of a control programme to validate good slaughter practices according to Commission Regulation No 2073/2005. The verotoxigenic Escherichia coli O157 (VTEC O157), although not currently legislated, was also investigated in the same carcasses due to the importance of bovines as a reservoir for this micro-organism. Virulence genes (vtx1, vtx2 and eae), the presence of fliCH 7 and antimicrobial susceptibility were studied in E.coli O157 isolates. Levels of Enterobacteriaceae and aerobic colonies and the presence of Salmonella were within the admissible range stipulated by current legislation. However, VTEC O157 was detected in 14·7% of carcasses. Among the VTEC O157 strains tested for antimicrobial susceptibility, 65% were multiresistant. Overall, the results of this study indicate that even with good manufacturing practices, contamination with VTEC O157 can occur and cattle meat can pose a risk to human health. These results confirm the need for a review of the appropriateness of introducing antimicrobial treatments in the processing of cattle carcasses in Europe. This study describes the prevalence of verotoxigenic and multidrug-resistant E.coli O157 strains in bovine carcasses. These results suggest that despite the good manufacturing practices used in the slaughterhouse studied (the largest in Catalonia slaughtering over 81000cattle per year), the absence of verotoxigenic E.coli O157 in bovine carcasses cannot be guaranteed.

IDENTIFICATION OF E. COLI O157 IN A BOVINE MILK FARM BY MULTIPLEX REAL-TIME PCR

Law provisions about direct sell of raw bovine milk require VTEC O157 monitoring in bovine milk farms (milk and faeces). It has been showed that culture-based methods used for this scope, besides being cumbersome and time-consuming, may be also less sensitive, compared to molecular approaches. In this study, a multiplex Real-Time PCR, able to identify VTEC O157, Salmonella spp and Listeria monocytogenes, has been used to analyse milk, filter, sewage and stool samples from a milk farm, in comparison with standard OIE methods. The performances of the molecular protocol have been preliminary assessed with lyophilized samples from proficiency testing VLA, showing 100% accordance. Results from field samples indicated the absence of the pathogen in milk, and the higher sensitivity of Real-Time PCR with other matrices, suggesting its potential use for fast VTEC O157 identification.

Does Levofloxacin Induce Hemolytic Uremic Syndrome in Patients Infected with Verotoxin-Producing Escherichia coli O157 Infections?

Fifteen Japanese colitis patients, aged above 16 years old, infected with verotoxin-producing Escherichia coli O157 (VTEC O157) were divided into 2 treatment groups. Of the 15 patients, 6 (mean ± SD, 41.3 ± 19.0 years old) were treated with levofloxacin (LVFX), while the remaining 9 patients (32.0 ± 10.0 years old) were not treated with any antimicrobial agents. All patients complained of abdominal pain and bloody stool and were not administered antidiarrheals. Hemolytic uremic syndrome (HUS) did not develop in any of the 6 patients treated with LVFX, but developed in 1 of the 9 patients not treated with antimicrobial agents. No statistical difference was found in the occurrence rate of HUS between LVFX-treated patients and patients not treated with antimicrobial agents. Our results suggest that oral administration of LVFX is not associated with risk of HUS in hemorrhagic colitis patients aged above 16 years infected with VTEC O157.

Genetically similar strains of Escherichia coli O157:H7 isolated from sheep, cattle and human patients

BackgroundComparatively little is known about the prevalence or the molecular characteristics of the zoonotic pathogen E. coli O157:H7 in the sheep reservoir. To investigate this and determine the host specificity of subclones of the bacterium, we have conducted a slaughterhouse prevalence study in sheep and compared the collected isolates to O157:H7 previously isolated from cattle and human patients.ResultsVerotoxin-producing O157:H7 was found in 11/597 (1.8%) of samples from sheep in Swedish slaughterhouses, 9/492 faecal (1.8%) and 2/105 ear samples (1.9%). All positive sheep were < 6 months old. Pulsed field gel electrophoresis typing revealed exact matches between isolates from the sheep prevalence study and human patients as well as between isolates from sheep and cattle. In one case, matching isolates were found in sheep, cattle, and a human patient in the same municipality. Identical PFGE profiles generally corresponded to similar but non-identical multi-locus VNTR profiles. In one sheep sample, SNP-typing found the highly virulent clade 8 variant of O157:H7. The virulence gene profiles of sheep isolates from the prevalence study and three sheep farms linked to cases of human illness were investigated by PCR detection (eaeA, hlyA, cdtV-B, vtx1), and partial sequencing of vtx2. The observed profiles were similar to those of cattle strains investigated previously.ConclusionsThe same pathogenic subtypes of VTEC O157:H7, including the highly virulent clade 8, appear to be present in both sheep and cattle in Sweden, suggesting strains can circulate freely between ruminant reservoirs.

Large outbreak of verocytotoxin-producingEscherichia coliO157 infection in visitors to a petting farm in South East England, 2009

SUMMARYIn the summer of 2009, an outbreak of verocytotoxigenic Escherichia coli O157 (VTEC O157) was identified in visitors to a large petting farm in South East England. The peak attack rate was 6/1000 visitors, and highest in those aged <2 years (16/1000). We conducted a case-control study with associated microbiological investigations, on human, animal and environmental samples. We identified 93 cases; 65 primary, 13 secondary and 15 asymptomatic. Cases were more likely to have visited a specific barn, stayed for prolonged periods and be infrequent farm visitors. The causative organism was identified as VTEC O157 PT21/28 with the same VNTR profile as that isolated in faecal specimens from farm animals and the physical environment, mostly in the same barn. Contact with farm livestock, especially ruminants, should be urgently reviewed at the earliest suspicion of a farm-related VTEC O157 outbreak and appropriate risk management procedures implemented without delay.

Investigation of a spatiotemporal cluster of verotoxin-producing Escherichia coli O157 infections in eastern England in 2007

An outbreak of verotoxin-producing Escherichia coli O157 (VTEC O157) infections linked to an open farm occurred in eastern England in April and May 2007. This paper describes the investigation and highlights the importance of multidisciplinary collaboration for successful control of such outbreaks. There was a temporal cluster of 12 confirmed symptomatic cases of VTEC O157 and one asymptomatic carrier, from five families. The investigation revealed that four of these cases formed part of an outbreak involving two families who visited an open farm. The phenotypic and genotypic characteristics of the isolates from the two families and the putative farm animal contacts were indistinguishable, indicating that the animals were the source of the primary infections. No epidemiological link could be established between the remaining three families affected and the open farm or people having visited the farm. Control measures included improved hand washing facilities on the farm, information for visitors and staff, restricted access and suspended petting and feeding of animals, and thorough cleaning and disinfection of affected areas.