Evaluation of three commercial PCR kits for the direct detection of Mycobacterium avium subsp. paratuberculosis (MAP) in bovine faeces.

Mycobacterium avium subsp. paratuberculosis which is more commonly referred to as MAP is the causative agent of Johnes's disease in ruminants. While cultivation of MAP from faecal samples remains the reference standard diagnostic test for the disease, faecal culture is expensive, slow and not widely available in Ireland. The current study evaluated three commercial kits that combine both DNA extraction and real-time PCR amplification of specific targets for direct MAP detection in cattle faeces. In total, 100 positive samples were tested which consisted of 25 high shedders, 25 medium shedders, 25 low shedders and 25 very low shedders. Also included were 100 negative faecal samples obtained from two Irish herds known to be free of Johne's disease. The kits evaluated in this study showed significant differences in identifying MAP DNA from animals shedding various concentrations of the bacterium. Kit C had the highest specificity, followed by kits B and A. Sensitivity in kits A, B and C was 73.5%, 81% and 93%, respectively, and specificity was 99%, 97% and 100%, respectively. Sensitivity decreased with lower concentrations of MAP in faeces but this was more significant for kits A and B than for kit C. The results of this study demonstrate that PCR can provide an accurate and rapid detection of MAP faecal shedding and kit C was selected for further evaluation of the role of PCR in the confirmation of animal infection in cattle.

Corpus callosum shape and morphology in youth across the psychosis Spectrum.

The corpus callosum is the largest white matter tract in the human brain connecting and coordinating homologous regions of the right and left hemispheres and has been strongly implicated in the pathogenesis of psychosis. We investigated corpus callosum morphology in a large community cohort of 917 individuals (aged 8-21), including 267 endorsing subsyndromal or threshold psychotic symptoms (207 on the psychosis spectrum and 60 with limited psychosis based on previously published criteria) and 650 non-psychotic volunteers. We used a highly reliable and previously published algorithm to automatically identify the midsagittal plane and to align the corpus callosum along the anterior and posterior commissures for segmentation, thereby eliminating these sources of error variance in dependent measures, which included perimeter, length, mean thickness and shape (circularity). The parcellation scheme divided the corpus callosum into 7 subregions that consisted of the rostrum, genu, rostral body, anterior midbody, posterior midbody, isthmus, and splenium. Both individuals endorsing psychotic symptoms and those with limited psychosis had significantly (p<.05) smaller area and lower thickness measures compared to healthy volunteers, but did not differ significantly from each other. Findings were relatively widespread indicating a relatively global effect not circumscribed to any particular corpus callosum subregion. These data are consistent with the hypothesis that corpus callosum abnormalities may be evident early in the course of illness and predate the onset of frank psychosis. Given that these measures can be easily obtained and are highly reliable they may assist in the identification of individuals at future risk for psychosis.

Use of multilocus variable-number tandem repeat analysis (MLVA) in eight European countries, 2012.

Genotyping of important medical or veterinary prokaryotes has become a very important tool during the last decades. Rapid development of fragment-separation and sequencing technologies has made many new genotyping strategies possible. Among these new methods is multilocus variable-number tandem repeat analysis (MLVA). Here we present an update on the use of MLVA in eight European countries (Denmark, France, Germany, Ireland, Italy, the Netherlands, Norway and Sweden). Researchers in Europe have been active in developing and implementing a large array of different assays. MLVA has been used as a typing tool in several contexts, from aiding in resolving outbreaks of foodborne bacteria to typing organisms that may pose a bioterrorist threat, as well as in scientific studies.

Application of multiple locus variable number of tandem repeat analysis (MLVA), phage typing and antimicrobial susceptibility testing to subtype Salmonella enterica serovar Typhimurium isolated from pig farms, pork slaughterhouses and meat producing plants in Ireland.

Salmonella enterica subsp. enterica serovar Typhimurium is a common zoonotic pathogen encountered in Irish pigs and the pork industry and its characterisation using highly discriminatory typing methods is necessary for epidemiological studies, outbreak investigation and control. Multiple locus variable number of tandem repeat analysis (MLVA), phage typing and antimicrobial susceptibility testing were applied to characterise 301 S. typhimurium isolates of porcine origin isolated from farms, slaughterhouses and pork meat producing plants in Ireland over a four-year period. 154 MLVA patterns were obtained compared to 19 phage types and 38 AMR patterns, and MLVA was particularly useful for discriminating isolates of the same phage type, e.g. DT104 and DT104b, or isolates that were Untypable or in the category of "react with phage but does not conform to a recognised phage type" (RDNC) by the phage typing method. Cluster analysis of MLVA profiles using a minimum spanning tree (MST) demonstrated two major clusters (I and II), which showed to have a clear association with phage types, cluster I associated to phage types DT104, U302 and DT120 and cluster II associated to DT193 and U288. The results of this present study showed that MLVA is highly discriminatory and permitted the identification of identical profiles among isolates obtained at different points of the pork food chain. The same MLVA profile was observed in some cases among isolates with different phage types. While this can be explained by the fact that some phage types are closely related, it also indicates that combining phage typing and MLVA enhances strain typing of S. typhimurium.

The influence of storing beef aerobically or in vacuum packs on the shelf life of mince.

AIMS: To investigate the influence of aerobic or vacuum pack storage of beef trimmings on the microbiology, colour and odour of subsequently produced mince. METHODS AND RESULTS: Trimmings stored aerobically for 7 or 10 days and in vacuum packs for 7, 10, 14 or 22 days at 0 or 5°C were minced, stored aerobically at 0 or 5°C for up to 7 days and examined daily to determine Total viable, Pseudomonas, Lactic acid bacteria, Brochothrix thermosphacta, and Enterobacteriaceae counts, colour and odour. Mincing reduced counts, particularly of Pseudomonas, B. thermosphacta and Enterobacteriaceae, probably because of the action free radicals released from muscle and bacterial cells. Storage of vacuum-packed trimmings for 22 days resulted in improved mince colour and inhibition of the growth of Pseudomonas. CONCLUSIONS: The shelf life of mince from trimmings is directly influenced by the trimmings storage conditions, and longer-term vacuum storage of trimmings produced improvements in mince quality. SIGNIFICANCE AND IMPACT OF THE STUDY: There appears to be no scientific rationale for limiting the storage of vacuum packaging beef trimmings to 15 days, prior to mince production, as stated in EU 835/2004. This study identifies advantages in storing trimmings in vacuum packs for at least 21 days prior to mincing, in terms of improved mince quality.

Survival of Pseudomonas fluorescens on beef carcass surfaces in a commercial abattoir.

The influence of a commercial chilling process (18 h at 10 degrees C followed by up to 78 h at 2 degrees C) on Pseudomonas fluorescens inoculated on beef carcass surfaces at four sites, neck (NE), outside round (OR), brisket (BR) and foreshank/brisket (FB) before chilling ("hot inoculated") or after chilling for 24h ("cold inoculated") was investigated. Pseudomonas counts increased significantly at all sites on "hot inoculated" carcasses during storage, but on "cold inoculated" carcasses, counts declined or remained unchanged. On hot and cold inoculated carcasses, differences in Pseudomonas growth or survival were demonstrated between sites. No clear relationships were observed between Pseudomonas growth or survival and chiller relative humidity (RH) or surface water activity (a(w)) at the different sites. These results were unexpected, and are discussed in relation to environmental factors that affect the growth/survival of P. fluorescens on carcass surfaces during chilling i.e. temperature, RH, and the relationship of these parameters to surface water activity (a(w)).

Tracking the Salmonella status of pigs and pork from lairage through the slaughter process in the Republic of Ireland.

Salmonella Typhimurium is the predominant serotype isolated from humans in Europe. Pork and pork products are recognized vehicles of Salmonella and are responsible for outbreaks of human salmonellosis. Pigs can become infected with Salmonella on the breeding or fattening farm and during transport, lairage, and slaughter. The aim of this study was to investigate selected points of Salmonella contamination from the time pigs entered the lairage to the time the carcass was processed in the boning hall and to determine the importance of different sources of Salmonella along the Irish pork production chain. A second objective was to evaluate whether the serological status or category of a herd influenced the levels of bacteriological contamination detected on individual carcasses and pork cuts during slaughter and dressing operations. All samples were tested for the presence and numbers of Salmonella. Enterobacteriaceae numbers were also determined. Serotype, phage type, and pulsed-field gel electrophoresis were utilized to determine similarity among Salmonella isolates. Lairage was a major source of cross-contamination with Salmonella as were the hands of evisceration operatives, conveyor belts, and equipment in the boning hall. Cross-contamination within the slaughter plant environment accounted for up to 69 % of Salmonella carcass contamination. In general, herd category reflected the bacteriological status of carcasses and pork cuts. Major findings were a strong association (P < 0.01) between Enterobacteriaceae counts and Salmonella occurrence on prechill carcasses and a significant association (P < 0.05) between Enterobacteriaceae counts and Salmonella occurrence on pork cut samples.

Changes in Escherichia coli O157:H7 numbers during holding on excised lean, fascia and fat beef surfaces at different temperatures.

AIM: To investigate changes in Escherichia coli O157:H7 numbers on excised beef carcass surfaces over 72 h at different temperatures. METHODS AND RESULTS: Excised lean meat, fascia and fat were inoculated with E. coli O157:H7 and held in an environmental chamber for 72 h, at air speed 0.5 m s(-1), relative humidity (RH) 90%, and temperatures 4, 8 and 12 degrees C. On lean, pathogen counts increased significantly at 12 degrees C. On fascia, significant reductions in counts occurred at 4 and 8 degrees C. Pathogen numbers were significantly reduced on fat at 4, 8 and 12 degrees C (64 h). Counts on fat were significantly less at all temperatures, compared to lean or fascia and surface water activity, a(w), decreased significantly over time on fat at 4 degrees C. Significant decreases in surface pH values were recorded on all meat substrates. CONCLUSIONS: The survival of E. coli O157:H7 varied in relation to the meat substrate and the holding temperature. Reductions in counts on fat surfaces appeared to be related to low surface a(w) values. No relationship between pathogen survival and surface pH was established. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of excised meat pieces in an environmental cabinet offers a more flexible approach to determining the use of different chilling regimes in the production of safe meat.

Prevalence, numbers and characteristics of Salmonella spp. on Irish retail pork.

Salmonella is a common contaminant of raw pork and represents a public health risk. Both qualitative and quantitative data on Salmonella in pork at retail are required for assessment of consumer exposure to the pathogen. Pork samples (n=500) were collected at random from butchers' shops and supermarkets in the Republic of Ireland between January and November, 2007 and examined for prevalence and numbers of Salmonella using a PCR screen followed by cultural examination of positive samples. Salmonella numbers were assessed using a three tube most probable number (MPN) technique. Any Salmonella recovered were characterised by serotype, phage type and antibiogram, and subtyped by pulsed-field gel electrophoresis (PFGE). Enterobacteriaceae were also enumerated to see if there was a correlation between the Salmonella status of the pork and hygiene levels at retail outlets. Salmonella spp. were detected on 13/500 (2.6%) pork cuts at numbers between <0.03 and 2.10 MPN/g. The mean Enterobacteriaceae counts was 3.12 log(10) CFU/g (range -0.26-6.52 log(10) CFU/g). Salmonella Typhimurium was the most common serotype and the majority of isolates were multi antibiotic resistant. PFGE analysis showed evidence of persistence of some strains, with an S. Typhimurium U310 recovered from a pork abattoir being identical (100%) to a strain found a year later in a sample from a retail outlet. There was also evidence of cross contamination of Salmonella isolates between samples. There was a direct association between Salmonella contamination of pork and Enterobacteriaceae numbers, which indicates the need for good hygiene practices at retail for control of this pathogen.

The survival of Salmonella enterica serovar Typhimurium DT104 and total viable counts on beef surfaces at different relative humidities and temperatures.

AIMS: The aim of this study was to investigate changes in Salmonella and total viable count (TVC) survival on beef carcass surfaces stored for 72 h under different combinations of relative humidity (i.e. RH 75% or 96%) and temperature (5 degrees C or 10 degrees C). METHODS AND RESULTS: The influence of low water activity (a(w)) and temperature on the survival and growth of Salmonella enterica serovar Typhimurium DT104 and the aerobic mesophilic flora on meat pieces from different sites on beef carcasses was investigated, under controlled conditions (75% or 96% RH; 5 or 10 degrees C) in an environmental cabinet. Salmonella counts declined during storage at low a(w) (75% RH) conditions at 5 degrees C or 10 degrees C. Salmonella counts increased during storage at high a(w) (96% RH) at 10 degrees C only. At 5 degrees C, TVCs increased during storage at high a(w), but not at low a(w). TVCs increased on all samples from carcasses stored at high or low a(w) at 10 degrees C, except those samples taken from areas of surface fat. CONCLUSIONS: This suggests that substrate composition dictates growth rates under low a(w) conditions. The results are discussed in terms of the possible protective effects of substrate osmolyte accumulation in bacterial survival and/or growth. SIGNIFICANCE AND IMPACT OF THE STUDY: The data obtained in this study provides useful insights on the influence of a(w) and temperature on pathogen survival on meat surfaces at chill temperature.

Survival of Escherichia coli O157:H7 and non-pathogenic E. coli on irradiated and non-irradiated beef surfaces.

This study examined changes in numbers of pathogenic (PEC) and non-pathogenic (NPEC) Escherichia coli during storage at 10°C on the surfaces of irradiated (IR) and non-irradiated (NIR) meat pieces excised from the neck, brisket and rump of beef carcasses and in Brain Heart Infusion Broth (BHI) and Maximum Recovery Diluent (MRD). On irradiated meat pieces, there were significant differences between mean PEC and NPEC counts at all sites. Differences in counts were also observed between IR and NIR surfaces and among the three meat sites for both E. coli types. These differences occurred only on IR samples, suggesting that the irradiation associated reductions in normal beef surface flora influenced survival of both E. coli types. PEC and NPEC counts increased during storage in BHI, but only NPEC counts increased in MRD. The results of this study highlight the impact of meat surface type and the presence/absence of the normal beef carcass surface flora on E. coli survival and/or growth during meat storage. Such previously unreported effects, and their precise mechanisms, have direct implications in the development and application of accurate models for the prediction of the safety and shelf life of stored meat.

Prevalence and numbers of Salmonella spp. and Enterobacteriaceae on pork cuts in abattoirs in the Republic of Ireland.

AIMS: This study aimed to determine the numbers and types of Salmonella spp. and Enterobacteriaceae on pork cuts in the meat cutting room environment of four commercial pork abattoirs in the Republic of Ireland. METHODS AND RESULTS: Pork oysters (M. gluteus medius; n = 720) and swabs (n = 56) from equipment and surfaces were screened for Salmonella spp. using a DNA-based PCR method and confirmed by culture. Salmonella numbers were assessed using a three-tube most probable number (MPN) technique. Salmonella spp. was detected on 24/720 (3.3%) pork cuts (range of <0.03-0.36 MPN g(-1)) and in 7/56 (12.5%) environmental swabs (range of <0.03-1.10 MPN cm(-2)). There was significant variation in the prevalence of Salmonella on pork between different abattoirs and days of sampling (range of 0-31.7%). The predominant serotype was Salmonella serotype Typhimurium followed by Salmonella serotype Derby. CONCLUSIONS: Overall prevalence data conceal the key finding that there was considerable variation in the incidence of Salmonella on different days. A direct association between Salmonella contamination of pork cuts and equipment/surfaces was observed. SIGNIFICANCE AND IMPACT OF THE STUDY: Prevalence and numbers of Salmonella were low; however, results clearly demonstrate the potential for cross-contamination from equipment and meat contact surfaces in the cutting room environment.

Survival of Listeria innocua on hot and cold beef carcass surfaces.

AIMS: This study aimed to determine the survival and growth of Listeria innocua on hot and cold beef carcass surfaces. METHODS AND RESULTS: Four sites, the neck, outside round, brisket and foreshank/brisket, were inoculated with L. innocua (i) immediately after dressing while hot and (ii) when cold after chilling. After inoculation, all carcasses were stored at 4 degrees C for 72 h. Survival of L. innocua on cold surfaces declined during storage and was less than on hot carcasses at all times. Data on the survival of L. innocua in broth (maximum recovery diluent) indicated that counts could not be compared with those on carcasses, in particular on cold carcasses. CONCLUSIONS: The results indicate that L. innocua survives on hot carcass surfaces during chilling, but declines over time on cold surfaces. The decrease in L. innocua counts on cold surfaces may be related to a synergy between the combined stresses of low available water (a(w)) and low temperature. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first to determine the effect of chilling on the survival and growth of Listeria on beef carcass surfaces. The information can potentially be used to determine the survival and growth of the pathogen, L. monocytogenes on beef surfaces.

The use of a marked strain of Pseudomonas fluorescens to model the spread of brain tissue to the musculature of cattle after shooting with a captive bolt gun.

AIMS: The aim of this study was to use a marked strain of Pseudomonas fluorescens to model the spread of central nervous system (CNS) tissue in cattle following captive bolt stunning. METHODS AND RESULTS: The marked organism was introduced by injection through the captive bolt aperture immediately after stunning and was subsequently detected in a wide range of derived tissues, including blood, organs, and the musculature of the entire forequarters of test animals. This was dependent on the use of high concentrations of the organism that were recovered sufficiently and rapidly to minimize the bactericidal properties of the circulatory system. These results suggest that a marked organism could potentially be used to model the effects of captive bolt stunning on the dissemination of CNS tissue from the brain. CONCLUSIONS: These results indicate that current commercial methods of captive bolt stunning may induce widespread and significant mobilization of CNS tissue within beef carcasses. This may lead to the widespread dissemination of such materials within meat destined for human consumption. SIGNIFICANCE AND IMPACT OF THE STUDY: In the absence of rapid, simple and sufficiently sensitive methods for the direct detection of prion in commercially slaughtered animals, marked organisms can provide useful models in studies of the dissemination kinetics of prion disease in captive bolt stunned animals.

Dissemination of central nervous system tissue during the slaughter of cattle in three Irish abattoirs.

Sponge samples were taken from the carcases, meat, personnel and surfaces involved in stunning, slaughter and dressing/boning activities at three abattoirs, and from retail beef products. The samples were examined for the presence of central nervous system (CNS)-specific proteins (syntaxin 1B and/or glial fibrillary acidic protein (GFAP), as indicators of contamination with CNS tissue. Syntaxin 1B and GFAP were detected in many of the sponge samples taken along the slaughter line and in the chill rooms of all three abattoirs; GFAP was also detected in one sample of longissimus muscle (striploin) taken in the boning hall of one of the abattoirs but not in the other two abattoirs or in retail meats.

Dissemination of central nervous system tissue from the brain and spinal cord of cattle after captive bolt stunning and carcass splitting.

In the absence of reliable live animal tests for the presence of BSE in cattle, a number of measures have been applied to exclude specified risk materials (SRM) from the human food chain. However, concerns remain that current practices in the stunning and slaughter of cattle may disseminate central nervous system (CNS) tissue to meat and meat contact surfaces. The objective of this study was to establish the particular risks of CNS tissue dissemination associated with captive bolt stunning and carcass splitting. The study applied enzyme linked immunosorbent assays (ELISAs) in the detection and quantification of two CNS proteins, syntaxin 1b and GFAP. The study observed extensive dispersal of both CNS proteins onto equipment, beef hide and personnel. These results demonstrate that despite the rigorous application of current SRM control policies, normal slaughter practices continue to present significant opportunities for CNS material including BSE prion present in the CNS of any sub-clinically infected cattle to contaminate meat entering the human food chain.

Use of a marker organism to model the spread of central nervous system tissue in cattle and the abattoir environment during commercial stunning and carcass dressing.

Due to concerns about a link between variant Creutzfeldt-Jakob disease in humans and similar prion protein-induced disease in cattle, i.e., bovine spongiform encephalopathy (BSE), strict controls are in place to exclude BSE-positive animals and/or specified risk materials including bovine central nervous system (CNS) tissue from the human food chain. However, current slaughter practice, using captive bolt guns, may induce disruption of brain tissues and mobilize CNS tissues into the bovine circulatory system, leading to the dispersion of CNS tissues (including prion proteins) throughout the derived carcass. This project used a marker (antibiotic-resistant) strain of Pseudomonas fluorescens to model the effects of commercial captive bolt stunning procedures on the movement of mobilized CNS material within slaughtered animals and the abattoir environment. The marker organism, introduced by injection through the bolt entry aperture or directly using a cartridge-fired captive bolt, was detected in the slaughter environment immediately after stunning and in the abattoir environment at each subsequent stage of the slaughter-dressing process. The marker organism was also detected on the hands of operatives; on slaughter equipment; and in samples of blood, organs, and musculature of inoculated animals. There were no significant differences between the results obtained by the two inoculation methods (P < 0.05). This study demonstrates that material present in, or introduced into, the CNS of cattle during commercial captive bolt stunning may become widely dispersed across the many animate and inanimate elements of the slaughter-dressing environment and within derived carcasses including meat entering the human food chain.