Screening for Taylorella equigenitalis in Equine Semen: An Exploratory Study.

The study examined and compared the sensitivity of culture and a quantitative PCR assay for screening equine semen for the presence of Taylorella equigenitalis (CEMO). Chilled semen samples, both raw and treated with extender, from two stallions were spiked with the organism at seven or 23 days postejaculation and prepared in serial dilutions. Culture of the 7-day raw semen readily detected CEMO at all dilutions, but extended semen yielded counts that were two log cycles lower at equivalent dilutions, with the organism being nearly undetectable at the maximal dilutions. By contrast, PCR sensitivity was not affected by extender, but for 7-day-old raw semen, PCR detection declined abruptly three log dilutions earlier than detection by culture. The more aged 23-day-old semen proved less satisfactory for spiking, with detection of CEMO by culture failing in three of the four samples due to overgrowth with commensal organisms. However, PCR performance was similar in both the 23- and 7-day spiking series. The detection limit by PCR is estimated at between 104 and 105 cfu/mL. Typical CEMO concentrations in the semen of colonized stallions are not widely reported but where natural semen contamination has been investigated, the organism was present at this order of magnitude. The reliability of detecting CEMO infection using semen samples by either method is discussed.

Investigations into Salmonella Contamination in Feed Mills Producing Rations for the Broiler Industry in Great Britain.

Feed-associated Salmonella serovars continue to be reported in poultry flocks. A study was conducted to investigate Salmonella contamination in major commercial feed mills that produce rations for broiler chickens within Great Britain. Dust and large moist gauze swab samples (12,791) were collected from 22 feed mills on 31 visits. Salmonella was isolated from 20 mills, with 15 mills (75%) having fewer than 5% Salmonella-positive samples. Fifty-one Salmonella serovars were isolated, with a large proportion of isolates being Salmonella (S.) Kedougou (29.4%) or S. 13,23:i:- (21.4%). European Union-regulated Salmonella serovars (Enteritidis, Infantis, Typhimurium and its monophasic variants) were isolated from 12 mills, mostly from non-processing areas, accounting for 40 isolates (4.4% of all Salmonella-positive samples). Fifteen Salmonella serovars were only isolated once. In terms of individual sampling locations within the mill, the waste handling locations were significantly more likely to be Salmonella-positive than some other mill locations. When sampling locations were grouped, samples collected from finished product areas were significantly less likely to be Salmonella-positive for Salmonella than some other mill areas. In conclusion, this study found that most mills producing broiler rations showed low-level Salmonella contamination.

Observations on the distribution and control of Salmonella in commercial broiler hatcheries in Great Britain.

Salmonella can enter hatcheries via contaminated eggs and other breaches of biosecurity. The study examined the prevalence and distribution of Salmonella in commercial hatcheries and assessed the effects of providing advice on Salmonella control. Intensive swab sampling was performed throughout 23 broiler hatcheries in Great Britain (GB). Swabs were cultured using a modified ISO6579:2017 method. After each visit, tailored advice on biosecurity and cleaning and disinfection procedures was provided to the hatchery managers. Repeat sampling was carried out in 10 of the 23 hatcheries. Salmonella prevalence ranged between 0% and 33.5%, with the chick handling areas, hatcher areas, macerator area, tray wash/storage areas, external areas and other waste handling areas being more contaminated than the setter areas. Salmonella Senftenberg and Salmonella 13,23:i:- were the most commonly isolated serovars. There was a reduction in Salmonella prevalence at the second visit in eight out of 10 premises, but prevalence values had increased again in all of the improved hatcheries that were visited a third time. One hatchery harboured a difficult-to-control resident Salmonella 13,23:i:- strain and was visited six times; by the final visit, Salmonella prevalence was 2.3%, reduced from a high of 23.1%. In conclusion, the study found low-level Salmonella contamination in some GB broiler hatcheries, with certain hatcheries being more severely affected. Furthermore, it was shown that Salmonella typically is difficult to eradicate from contaminated hatcheries, but substantial reductions in prevalence are possible with improvements to biosecurity, cleaning and disinfection.

Disinfection to control African swine fever virus: a UK perspective.

A review of African swine fever (ASF) was conducted, including manifestations of disease, its transmission and environmental persistence of ASF virus. Findings on infectious doses of contemporary highly-pathogenic strains isolated from outbreaks in Eastern Europe were included. Published data on disinfectant susceptibility of ASF virus were then compared with similar findings for selected other infectious agents, principally those used in the UK disinfectant approvals tests relating to relevant Disease Orders for the control of notifiable and zoonotic diseases of livestock. These are: swine vesicular disease virus, foot and mouth disease virus, Newcastle disease virus and Salmonella enterica serovar Enteritidis. The comparative data thus obtained, presented in a series of charts, facilitated estimates of efficacy against ASF virus for some UK approved disinfectants when applied at their respective General Orders concentrations. Substantial data gaps were encountered for several disinfectant agents or classes, including peracetic acid, quaternary ammonium compounds and products based on phenols and cresols.

Disinfectant testing for veterinary and agricultural applications: A review.

Disinfectants for veterinary and livestock use, plus skin antiseptics, are critical elements for the control of infectious agents, including zoonotic and antimicrobial-resistant micro-organisms, in managed animal species. Such agents impact animal welfare, economic performance and human health. Testing of disinfectants is needed for safety, efficacy and quality control. The present review examines the principal types of test (carrier, suspension, surface and field) that have been developed or attempted, plus the features inherent in the respective tests, particularly with respect to variability. Elements of testing that have to be controlled, or which can be manipulated, are discussed in the context of real-world scenarios and anticipated applications. Current national and international testing regimes are considered, with an emphasis on the UK, continental Europe and North America, and with further detail provided in the Supporting Information. Challenges to disinfectant efficacy include: the nature of the biological targets (bacteria, fungi, yeasts, spores, viruses and prions), the need for economical and safe working concentrations, the physical and chemical nature of contaminated surfaces, constraints on contact times and temperatures, the presence of organic soil and other barrier or neutralising substances (including biofilms), and thoroughness of pre-cleaning and disinfectant application. The principal challenges with veterinary disinfectant testing are the control of test variability, and relating test results to likely performance in variable field conditions. Despite some ambitions to develop standardised field tests for disinfectants, aside from skin antiseptic trials the myriad problems such tests pose with respect to cost, reproducibility and generalisability remain intractable.

Control of Salmonella and Pathogenic E. coli Contamination of Animal Feed Using Alternatives to Formaldehyde-Based Treatments.

This study compared a novel non-formaldehyde combination product developed for pathogen control in animal feed Finio (A), with a panel of three commonly used organic acid feed additive products: Fysal (B), SalCURB K2 (C) and Salgard (D). Products were evaluated for their ability to reduce Salmonella Typhimurium DT104 and avian pathogenic Escherichia coli in poultry feed. A commercial layer-hen mash was treated with each product and then mixed with feed previously contaminated (via inoculated meat and bone meal) with either Salmonella or E. coli. After 24 hours at room temperature, 10 replicate samples were taken from each preparation and plate counts were performed using a selective agar. All concentrations of product A (0.5, 1.0, 1.5, 2.0 and 2.5 kg per metric tonne (MT)) plus the higher concentration of products B and D (6.0 kg MT-1) significantly reduced Salmonella counts compared with those in the untreated control group (p < 0.05). Product C did not significantly reduce levels of Salmonella under these conditions. Because of the poor recovery of E. coli, statistical comparisons for this organism were limited in scope, but only product A at the highest concentration appeared to have eliminated it.

Antimicrobial Resistance on Farms: A Review Including Biosecurity and the Potential Role of Disinfectants in Resistance Selection.

Resistance to therapeutic antimicrobial agents is recognized as a growing problem for both human and veterinary medicine, and the need to address the issue in both of these linked domains is a current priority in public policy. Efforts to limit antimicrobial resistance (AMR) on farms have so far focused on control of the supply and use of antimicrobial drugs, plus husbandry measures to reduce infectious disease. In the United Kingdom and some other countries, substantial progress has been made recently against targets on agricultural antimicrobial drug use. However, evidence suggests that resistant pathogenic and commensal bacteria can persist and spread within and between premises despite declining or zero antimicrobial drug use. Reasons for this are likely complex and varied but may include: bacterial adaptations to ameliorate fitness costs associated with maintenance and replication of resistance genes and associated proteins, horizontal transmission of genetic resistance determinants between bacteria, physical transfer of bacteria via movement (of animals, workers, and equipment), ineffective cleaning and disinfection, and co-selection of resistance to certain drugs by use of other antimicrobials, heavy metals, or biocides. Areas of particular concern for public health include extended-spectrum cephalosporinases and fluoroquinolone resistance among Enterobacteriaceae, livestock-associated methicillin-resistant Staphylococcus aureus, and the emergence of transmissible colistin resistance. Aspects of biosecurity have repeatedly been identified as risk factors for the presence of AMR on farm premises, but there are large gaps in our understanding of the most important risk factors and the most effective interventions. The present review aims to summarize the present state of knowledge in this area, from a European perspective.

Field Interventions Against Colonization of Broilers by Campylobacter.

Poultry accounts for a high proportion of human campylobacteriosis cases, and the problem of Campylobacter colonization of broiler flocks has proven to be intractable. Owing to their broad host range and genetic instability, Campylobacter organisms are ubiquitous and adaptable in the broiler farm environment, colonizing birds heavily and spreading rapidly after introduction into a flock. This review examines strategies to prevent or suppress such colonization, with a heavy emphasis on field investigations. Attempts to exclude Campylobacter via enhanced biosecurity and hygiene measures have met with mixed success. Reasons for this are becoming better understood as investigations focus on houses, ventilation, biosecurity practices, external operators, and compliance, among other factors. It is evident that piecemeal approaches are likely to fail. Complementary measures include feed and drinking water treatments applied in either preventive or suppressive modes using agents including organic acids and their derivatives, also litter treatments, probiotics, prebiotics, and alterations to diet. Some treatments aim to reduce the number of Campylobacter organisms entering abattoirs by suppressing intestinal colonization just before slaughter; these include acid water treatment or administration of bacteriophages or bacteriocins. Experimental vaccines historically have had little success, but some recent subunit vaccines show promise. Overall, there is wide variation in the control achieved, and consistency and harmonization of trials is needed to enable robust evaluation. There is also some potential to breed for resistance to Campylobacter. Good and consistent control of flock colonization by Campylobacter may require an as-yet undetermined combination of excellent biosecurity plus complementary measures.

Co-Selection of Resistance to Antibiotics, Biocides and Heavy Metals, and Its Relevance to Foodborne Pathogens.

Concerns have been raised in recent years regarding co-selection for antibiotic resistance among bacteria exposed to biocides used as disinfectants, antiseptics and preservatives, and to heavy metals (particularly copper and zinc) used as growth promoters and therapeutic agents for some livestock species. There is indeed experimental and observational evidence that exposure to these non-antibiotic antimicrobial agents can induce or select for bacterial adaptations that result in decreased susceptibility to one or more antibiotics. This may occur via cellular mechanisms that are protective across multiple classes of antimicrobial agents or by selection of genetic determinants for resistance to non-antibiotic agents that are linked to genes for antibiotic resistance. There may also be relevant effects of these antimicrobial agents on bacterial community structure and via non-specific mechanisms such as mobilization of genetic elements or mutagenesis. Notably, some co-selective adaptations have adverse effects on fitness in the absence of a continued selective pressure. The present review examines the evidence for the significance of these phenomena, particularly in respect of bacterial zoonotic agents that commonly occur in livestock and that may be transmitted, directly or via the food chain, to human populations.

Assessment of the anti-Salmonella activity of commercial formulations of organic acid products.

Organic acid products are widely used in the UK poultry industry as feed or water additives. Claims for Salmonella control are made for some of these products, but there are few studies comparing the anti-Salmonella effect of the various products in tests that reflect field application. The present studies examined the effects of 13 commercial blends (four water products and nine feed products) on Salmonella Enteritidis and Typhimurium strains. Initial screening, in nutrient broth, of all products with all strains revealed little variation between strains in respect of maximum inhibitory and bactericidal dilutions of each product. However, between the products there was wide and significant variation in the maximum inhibitory and bactericidal dilutions, spanning a 700-fold range in the case of bactericidal dilutions for feed-associated products. Further tests were performed, examining reductions in inoculated Salmonella numbers in various matrices (water, feed, soiled litter, crop and caecal contents) following the addition of the products at recommended inclusion rates. One product, incorporating formaldehyde in addition to organic acid, was consistently most active in all matrices, exceeding reductions associated with other products by 1 to 3 log units at most time points. Many products showed only modest anti-Salmonella activity, amounting to 0 or 1 log unit above negative controls at many time points, and the most active products were not the same in all matrices. Tap water appeared to enhance the ability of products to reduce Salmonella, in comparison with bottled mineral or river water.

Comparison of the environmental survival characteristics of Salmonella Dublin and Salmonella Typhimurium.

To examine possible correlations in bovine Salmonella isolates between environmental survival and serovar-associated epidemiological patterns, bovine field isolates of Salmonella serovars Typhimurium and Dublin (two each) were inoculated into bovine faeces slurry and tested monthly by culture for survival during a six-month period of storage at a variable ambient temperature in a disused animal transporter. Low moisture conditions, where the slurry was dried onto wooden dowels, increased detectable survival of a low-level inoculum by up to five months, compared with wet slurry. A more modest increase of survival time was seen with storage of wet slurry under refrigeration at 4°C. Under both dry and wet conditions, the concentration of culturable Salmonella Typhimurium declined at a slower rate than did that of Salmonella Dublin. Salmonella that was naturally contaminating bovine faeces from farms with Salmonella Typhimurium did not show superior survival times compared with Salmonella Typhimurium that had been artificially inoculated into samples. The differing survival characteristics of the two serovars that was observed in environmental faeces may complement their different modes of infection in cattle. Salmonella Dublin, being a bovine host-adapted strain that establishes chronic infection in some animals, may have less need to survive for a prolonged period outside of its host than does Salmonella Typhimurium.

Evaluation of commonly-used farm disinfectants in wet and dry models of Salmonella farm contamination.

Two experimental models of Salmonella contamination were used in an attempt to mimic the conditions of disinfectant use on farms. A wet model, for conditions such as boot dips, used disinfectant application to a slurry of poultry faeces inoculated with Salmonella Enteritidis or Salmonella Typhimurium. A dry model, for disinfectant application to surfaces and equipment with adherent or residual organic material, used Salmonella-inoculated poultry faeces that were air-dried onto wooden dowels, immersed in disinfectant solution then left in air at room temperature overnight. All samples were subjected to a disinfectant neutralization step and resuscitation in broth, followed by Salmonella culture on semi-solid then indicator media. Disinfectants were tested at 0.5x, 1x and 2x the concentrations specified for the general control of bacterial pathogens on livestock premises in the UK (Defra General Orders rates). Chlorocresol-based disinfectants provided consistently high rates of Salmonella killing in both wet and dry tests. Formaldehyde-containing disinfectants showed very high efficacy in the dry test but were less effective in the shorter wet test, whereas the efficacy of glutaraldehyde without formaldehyde was variable between products. Other chemical classes tested (quaternary ammonium compounds, amphoteric surfactants, iodine preparations, peroxygens and a substituted phenol blend) were only moderately effective. They often required concentrations above General Orders rates to eliminate the test salmonellas, and frequently elimination was not achieved even under maximal conditions of concentration and exposure.

Chemical treatment of animal feed and water for the control of Salmonella.

The control of Salmonella in animal feedstuffs is important, principally to protect the human food chain from contamination by Salmonella derived from infected animals. The transmission of Salmonella from animal feeds to animals, and onward to human food products, has been convincingly documented. This is especially important for chicken breeding and laying flocks and pigs, in view of the consequences of recent or imminent control legislation in the European Union. Animal feed ingredients, particularly animal and plant-derived protein meals, are frequently contaminated with Salmonella either from source or from processing plant, and recontamination in compounding mills is an additional problem. Several complementary strategies have been used to control this feed contamination, and these include a range of chemical treatments. The principal agents used are as follows: organic acids and their salts, formaldehyde, and bacterial membrane disruptors such as terpenes and essential oils. Experimental agents include chlorate compounds. Many products use blends of agents from the same or different chemical groups to achieve synergistic or combination effects. The present review draws upon published and company data to describe the various modes of action and efficacies of different chemical agents delivered in feed or in drinking water against Salmonella occurring in feed or in livestock environments. Reasons for the failure of protection are explored, along with problems in usage such as corrosion and reduced palatability. Given the wide array of products available with contrasting modes of action, the need for standardized tests of efficacy is also discussed.

Escherichia coli O157:H7 colonization in small domestic ruminants.

Enterohaemorrhagic Escherichia coli O157:H7 was first implicated in human disease in the early 1980s, with ruminants cited as the primary reservoirs. Preliminary studies indicated cattle to be the sole source of E. coli O157:H7 outbreaks in humans; however, further epidemiological studies soon demonstrated that E. coli O157:H7 was widespread in other food sources and that a number of transmission routes existed. More recently, small domestic ruminants (sheep and goats) have emerged as important sources of E. coli O157:H7 human infection, particularly with the widespread popularity of petting farms and the increased use of sheep and goat food products, including unpasteurized cheeses. Although the colonization and persistence characteristics of E. coli O157:H7 in the bovine host have been studied intensively, this is not the case for small ruminants. Despite many similarities to the bovine host, the pathobiology of E. coli O157:H7 in small domestic ruminants does appear to differ significantly from that described in cattle. This review aims to critically review the current knowledge regarding colonization and persistence of E. coli O157:H7 in small domestic ruminants, including comparisons with the bovine host where appropriate.

A longitudinal study of environmental Salmonella contamination in caged and free-range layer flocks.

The environmental contamination by salmonella was examined over a 12-month period in 74 commercial layer flocks from eight farms in the UK, which previously had been identified as being contaminated with salmonella. Samples of faeces, dust, litter, egg belt spillage and wildlife vectors were taken, plus swabs of cages, feeders, drinkers, floors, egg belts and boots. Some sampling was performed in each month of the year. Numerous serovars were detected but Salmonella enterica serotype Enteritidis was the only persistent serotype found among single-age flocks. There was a significant correlation between qualitative environmental samples and semi-quantitative faeces samples. The level of environmental contamination increased significantly over time. There were significant temperature and seasonal effects upon contamination. Wildlife vectors proved to be sensitive samples for the detection of salmonella. The efficacy of cleaning and disinfection upon residual salmonella contamination, and upon subsequent flock contamination, was highly variable between and within premises. The variability between detected prevalences over time and between flocks indicates a need for regular, sensitive monitoring of flocks for salmonella to permit targeting of control measures aimed at eliminating contamination of the layer environment by salmonella. There is substantial scope for improvement of cleaning and disinfection procedures.

Assessment of cleaning and disinfection in Salmonella-contaminated poultry layer houses using qualitative and semi-quantitative culture techniques.

Salmonella infection of laying flocks in the UK is predominantly a problem of the persistent contamination of layer houses and associated wildlife vectors by Salmonella Enteritidis. Methods for its control and elimination include effective cleaning and disinfection of layer houses between flocks, and it is important to be able to measure the success of such decontamination. A method for the environmental detection and semi-quantitative enumeration of salmonellae was used and compared with a standard qualitative method, in 12 Salmonella-contaminated caged layer houses before and after cleaning and disinfection. The quantitative technique proved to have comparable sensitivity to the standard method, and additionally provided insights into the numerical Salmonella challenge that replacement flocks would encounter. Elimination of S. Enteritidis was not achieved in any of the premises examined although substantial reductions in the prevalence and numbers of salmonellae were demonstrated, whilst in others an increase in contamination was observed after cleaning and disinfection. Particular problems with feeders and wildlife vectors were highlighted. The use of a quantitative method assisted the identification of problem areas, such as those with a high initial bacterial load or those experiencing only a modest reduction in bacterial count following decontamination.

Non-toxigenic Escherichia coli O157:H7 strain NCTC12900 causes attaching-effacing lesions and eae-dependent persistence in weaned sheep.

Ruminants are regarded as a primary reservoir for Escherichia coli O157:H7, an important human pathogen. Intimin, encoded by the Locus of Enterocyte Effacement by E. coli O157:H7 organisms, has been cited as one bacterial mechanism of colonisation of the gastrointestinal tract. To confirm this and to test whether a non-toxigenic E. coli O157:H7 strain would colonise and persist in a sheep model, E. coli O157:H7 strain NCTC12900, that lacks Shiga toxin (stx) genes, was evaluated for use in a sheep model of persistence. Following oral inoculation of six-week-old sheep, persistent excretion of NCTC12900 was observed for up to 48 days. E. coli O157-associated attaching-effacing (AE) lesions were detected in the caecum and rectum of one six-week-old lamb, one day after inoculation. This is the first recorded observation of AE lesions in orally inoculated weaned sheep. Also, mean faecal excretion scores of NCTC12900 and an isogenic intimin (eae)-deficient mutant were determined from twenty-four six-week-old orally inoculated sheep. The eae mutant was cleared within 20 days and had lower mean excretion scores at all time points after day one post inoculation compared with the parental strain that was still being excreted at 48 days. Tissues were collected post mortem from animals selected at random from the study groups over the time course of the experiment. The eae mutant was detected in only 1/43 samples but the parental strain was recovered from 64/140 samples primarily from the large bowel although rumen, duodenum, jejunum, and ileum were culture positive especially from animals that were still excreting at and beyond 27 days after inoculation.

Variation in the persistence of Escherichia coli O157:H7 in experimentally inoculated 6-week-old conventional lambs.

Six-week-old lambs were inoculated orally with 10(9) cfu of an antibiotic-resistance marked four-strain mixture of enterohaemorrhagic Escherichia coli (EHEC) O157:H7 to investigate faecal excretion and intestinal colonisation. In the first experiment, three E. coli O157:H7 isolates were not detected in the faeces of any lambs beyond day 8 post inoculation (pi), or from any of the tissues derived from inoculated animals. One strain, 140065 Nal(r), was isolated from the caecum and colon of one lamb on day 9 pi, from the rectum of another on day 22 pi and persisted in the faeces for up to 28 days pi. All animals remained clinically normal throughout the study period and histological evidence of adhesion of E. coli O157:H7 to the intestinal mucosa was not found. In a separate experiment, four 6-week-old lambs were inoculated orally with 10(9) cfu of E. coli O157:H7 strain 140065 Nal(r) alone. Faecal samples were positive for this strain until the end of the experiment (day 19 pi). This strain was also recovered from the gastrointestinal tract of lambs on days 6, 18 and 19 pi, but was not isolated at day 17 pi. When sampled separately, rectum and terminal colon contents contained higher numbers of the inoculated strain than the intestinal tissue at these sites. Animals inoculated with O157:H7 strain 140065 Nal(r) alone produced soft faeces from day 5 pi onwards. Although attaching and effacing lesions were observed in the caecum, proximal colon and rectum in one animal on day 18 pi, the adherent bacteria did not stain with antiserum raised against the O157 antigen.