Effectiveness of Cleaning and Sanitation of Stable Environment and Riding Equipment Following Contamination With Streptococcus equi Subsp. equi.

Streptococcus equi subsp. equi (S. equi) is transmitted via contact with infected horses or fomites such as equipment or surfaces of the stable environment. Effective cleaning and sanitation is essential to minimize risk of fomite-associated infections. This study assessed the effectiveness of cleaning and sanitation of experimentally S. equi contaminated materials and equipment found in stables. Wood, concrete, plastic, leather halters, leather gloves and polyester webbing halters were inoculated with a 24-hour culture S. equi laboratory strain. In addition, selected materials were inoculated with a clinical strain of S. equi. Three days post inoculation all materials were sampled for retention of viable S. equi and a subset of each material was cleaned and sanitized. After an additional 2 days all treated and untreated materials were sampled for continued retention of viable S. equi. Separate subsets of contaminated polyester halter material were washed at 40°C with or without drying at 70°C, or washed at 60°C. After cleaning and sanitation, all samples except polyester halters were culture negative. Even before cleaning and sanitation leather appears to poorly support survival of S. equi. After washing at 40°C and tumble drying, 14 of 16 halters were culture positive, however culture negative when washed at 60°C. Routine cleaning and sanitation of fomites contaminated with S. equi was generally effective to eliminate viable bacteria. However, survival between materials and strains differed, with leather poorly permissive to S. equi survival even without cleaning, whereas polyester webbing halters retained viable S. equi even after washing at temperatures of 40°C.

Differences in Genotype and Antimicrobial Resistance between Campylobacter spp. Isolated from Organic and Conventionally Produced Chickens in Sweden.

Antibiotic resistance is a major challenge worldwide and increased resistance to quinolones in Campylobacter is being reported. Analysis of antibiotic resistance was performed on 157 Campylobacter strains (123 C. jejuni and 34 C. coli) from conventional and organic chickens produced in Sweden. Susceptibility for tetracycline, ciprofloxacin, erythromycin, nalidixic acid, streptomycin, and gentamycin was determined by microdilution. All 77 isolates from organic chickens were sensitive to all antibiotics, except two C. jejuni that were resistant to tetracycline. Of the 80 isolates from conventional chickens, 22.5% of C. jejuni and 11.1% of C. coli were resistant to quinolones and 5.6% of C. jejuni were resistant to tetracycline. Whole-genome sequencing resulted in 50 different sequence types of C. jejuni and six of C. coli. Nine sequence types were found in both organic and conventional chickens. Two of these (ST-19 and ST-257) included isolates from conventional broilers with different resistance phenotypes to the remaining isolates from conventional and organic broilers. There are management differences between the production systems, such as feed, breed, use of coccidiostats, and access to outdoor area. It is unlikely that quinolone resistance has arisen due to use of antimicrobials, since fluoroquinolones are not permitted in Swedish broiler production.

Reducing Campylobacter jejuni,Enterobacteriaceae, Escherichia coli, and Total Aerobic Bacteria on Broiler Carcasses Using Combined Ultrasound and Steam.

ABSTRACT: Campylobacteriosis is the most frequently reported foodborne illness in Europe and many other parts of the world. Campylobacter can colonize the intestines of broilers, mostly in large amounts. Broilers are usually slaughtered in a high-speed automated system that could cause rupture of the intestines during evisceration, resulting in contamination of carcasses with intestinal bacteria like Campylobacter. This study evaluated the combined effects of ultrasound and steam (SonoSteam) on naturally contaminated chicken carcasses at a large-scale abattoir in Sweden. Ultrasound at 30 to 40 kHz and steam at 84 to 85°C or 87 to 88°C were used at slaughter, with a line speed of 18,000 birds per hour. The amounts of Campylobacter spp., Enterobacteriaceae, Escherichia coli, and total aerobic bacteria on neck skins from 103 chicken carcasses, sampled before and after treatment by ultrasound-steam, were analyzed. Campylobacter spp. were quantified in 58 (56%) of the neck skins, from birds belonging to four of the seven flocks represented. All 58 isolates were identified as Campylobacter jejuni. After the ultrasound-steam treatment, the mean reductions in C. jejuni, Enterobacteriaceae, E. coli and total aerobic bacteria were 0.5 ± 0.8, 0.6 ± 0.6, 0.5 ± 0.6, and 0.4 ± 0.7 log CFU/g, respectively. No significant differences in reduction between the two different treatment temperatures were observed for any of the bacteria. Although the bacterial reductions were significant, large amounts of bacteria remained on the carcasses after treatment. Further studies are needed to identify optimal measures at slaughter to reduce food spoilage bacteria and pathogenic bacteria, which should be considered in a One Health perspective.

Identification of Transmission Routes of Campylobacter and On-Farm Measures to Reduce Campylobacter in Chicken.

An in-depth analysis was performed on Swedish broiler producers that had delivered chickens with Campylobacter to slaughter over several years, in order to identify possible transmission routes and formulate effective measures to prevent chickens being colonized with Campylobacter. Between 2017 and 2019, 626 samples were collected at farm level and Campylobacter was isolated from 133 (21.2%). All C. jejuni and C. coli isolated from these samples were whole-genome sequenced, together with isolates from the corresponding cecum samples at slaughter (n = 256). Core genome multi-locus sequence typing (cgMLST) analysis, using schemes consisting of 1140 and 529 genes for C. jejuni and C. coli, respectively, revealed that nearby cattle, contaminated drinking water, water ponds, transport crates, and parent flocks were potential reservoirs of Campylobacter. A novel feature compared with previous studies is that measures were implemented and tested during the work. These contributed to a nationwide decrease in Campylobacter-positive flocks from 15.4% in 2016 to 4.6% in 2019, which is the lowest ever rate in Sweden. To conclude, there are different sources and routes of Campylobacter transmission to chickens from different broiler producers, and individual measures must be taken by each producer to prevent Campylobacter colonization of chickens.

Presence of pathogenic bacteria in faeces from dogs fed raw meat-based diets or dry kibble.

BACKGROUND: Feeding dogs with raw meat-based diets (RMBD) has increased in popularity in recent years. Proponents claim that RMBD is more natural for dogs, because it is what their ancestors (wolves) eat. Opponents claim that RMBD is a health hazard to both humans and animals, with a risk of spreading zoonotic bacteria and resistant bacterial strains. METHODS: This cross-sectional study investigated differences in bacteria shedding in faeces between dogs fed RMBD and dogs fed dry kibble. Faeces samples from 50 dogs from the same municipality were analysed for the presence of extended-spectrum beta-lactamases (ESBL)-producing Escherichia coli, Campylobacter and Salmonella. RESULTS: For the 25 dogs fed RMBD, ESBL E coli was isolated from 13 faeces samples, Campylobacter from 12 and Salmonella from 1. For the 25 dogs fed dry kibble, ESBL-producing E coli was isolated from one faeces sample and Campylobacter from four, while Salmonella was not detected. CONCLUSION: There was thus a significant difference in excretion of zoonotic and resistant bacteria in faeces between dogs fed RMBD and dogs fed dry kibble. These results confirm that RMBD can pose a microbiological risk not only for dogs, but also for people handling RMBD and faeces from dogs.

Potential for residual contamination by Streptococcus equi subspp equi of endoscopes and twitches used in diagnosis of carriers of strangles.

BACKGROUND: Endoscopic examinations are essential for diagnosis and treatment of strangles (S equi infection) in horses. However, even after disinfection, endoscopes may retain viable bacteria or bacterial DNA. Twitches are commonly used during endoscopic examinations and can thus also potentially transmit the organism to other horses. OBJECTIVES: To evaluate the efficacy of different disinfectant methods to eliminate S equi from experimentally contaminated endoscopes and twitches and the effectiveness of field disinfection of endoscopes used in sampling carriers of S equi. STUDY DESIGN: Experimental contamination and observational field study. METHODS: One endoscope and 30 twitches were contaminated with standardised S equi broth solutions. The endoscope was disinfected following three protocols using various disinfectants for manual disinfection. A fourth protocol used an automated endoscope reprocessor (AER). The twitches (n = 30) were disinfected following eight different disinfecting protocols. Three endoscopes used in sampling for silent carriers were disinfected following a field-based protocol. After each protocol the endoscopes and twitches were sampled for S equi by culture and qPCR. RESULTS: Following experimental contamination all endoscope disinfection protocols, apart from 1/6 of the ethanol protocol were S equi culture negative. However, no endoscope disinfection protocol completely eliminated retention of S equi DNA. Field disinfection of endoscopes after sampling carriers yielded no culture positives and all but one (13/14) were qPCR negative. All twitches disinfected following experimental contamination were culture negative but sodium hypochlorite was the only disinfectant that completely eliminated detection of S equi DNA. MAIN LIMITATIONS: Experimental contamination may not reflect the numbers of S equi transferred to endoscopes or twitches during use on silent carriers and purulent secretions from infected horses may influence survival of S equi. CONCLUSIONS: While most disinfection methods appear to ensure removal of cultivable S equi, residual DNA can remain on both endoscopes and twitches.

Occurrence of Salmonella, Campylobacter, Clostridium and Enterobacteriaceae in raw meat-based diets for dogs.

The practice of feeding raw meat-based diets (RMBD) to dogs has increased in popularity in recent years. However, RMBD are based on offal that has not undergone any type of treatment to reduce the microbial content, so there is a risk of potential pathogenic microorganisms being present. Frozen samples from 60 RMBD packs produced by 10 different manufacturers were analysed for their content of bacteria belonging to the family Enterobacteriaceae, for Clostridium perfringens and for the presence of Salmonella and Campylobacter. Enterobacteriaceae were detected in all 60 samples and in 31 samples exceeded a level of 5000 bacteria/g, which is the threshold for satisfactory microbial hygiene according to EU regulations. In two samples, the amount of C. perfringens exceeded 5000 bacteria/g, which is the maximum level of anaerobic bacteria permitted by Swedish national guidelines. Salmonella species were found in four (7 per cent) and Campylobacter species in three (5 per cent) samples. These results show that it is critical to maintain good hygiene when storing, handling and feeding RMBD, in order to limit the potential health risks to animals and humans, especially young and immunocompromised individuals.

Fate of Listeria monocytogenes , Pathogenic Yersinia enterocolitica , and Escherichia coli O157:H7 gfp+ in Ready-to-Eat Salad during Cold Storage: What Is the Risk to Consumers?

In this study, we investigated the fate of Listeria monocytogenes , pathogenic Yersinia enterocolitica , and Escherichia coli O157:H7 gfp+ inoculated in low numbers into ready-to-eat baby spinach and mixed-ingredient salad (baby spinach with chicken meat). Samples were stored at recommended maximum refrigerator temperature (8°C in Sweden) or at an abuse temperature (15°C) for up to 7 days. Mixed-ingredient salad supported considerable growth when stored at 15°C during shelf life (3 days), with populations of L. monocytogenes , pathogenic Y. enterocolitica , and E. coli O157:H7 gfp+ increasing from less than 2.0 log CFU/g on day 0 to 7.0, 4.0, and 5.6 log CFU/g, respectively. However, when mixed-ingredient salad was stored at 8°C during shelf life, only L. monocytogenes increased significantly, reaching 3.0 log CFU/g within 3 days. In plain baby spinach, only pathogenic Y. enterocolitica populations increased significantly during storage for 7 days, and this was exclusively at an abuse temperature (15°C). Thus, mixing ready-to-eat leafy vegetables with chicken meat strongly influenced levels of inoculated strains during storage. To explore the food safety implications of these findings, bacterial numbers were translated into risks of infection by modeling. The risk of listeriosis (measured as probability of infection) was 16 times higher when consuming a mixed-ingredient salad stored at 8°C at the end of shelf life, or 200,000 times higher when stored at 15°C, compared with when consuming it on the day of inoculation. This indicates that efforts should focus on preventing temperature abuse during storage to mitigate the risk of listeriosis. The storage conditions recommended for mixed-ingredient salads in Sweden (maximum 8°C for 3 days) did not prevent growth of L. monocytogenes in baby spinach mixed with chicken meat. Manufacturers preparing these salads should be aware of this, and recommended storage temperature should be revised downwards to reduce the risk of foodborne disease.

Detection of Campylobacter in human and animal field samples in Cambodia.

Campylobacter are zoonotic bacteria and a leading cause of human gastroenteritis worldwide with Campylobacter jejuni and C. coli being the most commonly detected species. The aim of this study was to detect Campylobacter in humans and livestock (chickens, ducks, pigs, cattle, water buffalo, quail, pigeons and geese) in rural households by routine culturing and multiplex PCR in faecal samples frozen before analysis. Of 681 human samples, 82 (12%) tested positive by PCR (C. jejuni in 66 samples and C. coli in 16), but none by routine culture. Children were more commonly Campylobacter positive (19%) than adult males (8%) and females (7%). Of 853 livestock samples, 106 (12%) tested positive by routine culture and 352 (41%) by PCR. Campylobacter jejuni was more frequent in chickens and ducks and C. coli in pigs. In conclusion, Campylobacter proved to be highly prevalent by PCR in children (19%), ducks (24%), chickens (56%) and pigs (72%). Routine culturing was insufficiently sensitive in detecting Campylobacter in field samples frozen before analysis. These findings suggest that PCR should be the preferred diagnostic method for detection of Campylobacter in humans and livestock where timely culture is not feasible.

Escherichia coli O157:H7 reduction in hamburgers with regard to premature browning of minced beef, colour score and method for determining doneness.

This study investigated the effect of premature browning (PMB) on the survival of Escherichia coli O157:H7 in beef hamburgers after cooking with respect to interior colour of the hamburger and recommendations to cook hamburgers to a core temperature of 71 °C. Assessment of doneness by visual inspection or measurement of internal temperature was compared in terms of survival and the increased relative risk of illness due to PMB was estimated. At the last consume-by-day, hamburgers made from minced meat packaged in 80/20 O2/CO2 (MAP hamburger) and from meat minced at retail packaged in atmospheric condition (control hamburger) were inoculated with a gfp-tagged strain of E. coli O157:H7 (E. coli O157:H7gfp+). Hamburgers were cooked for different times during assessment of the core temperature every 30s and cut in halves after cooking. Doneness was evaluated based on visual judgement of the internal colour using a score chart (C-score) from 'uncooked' (score 1) to 'tan with no evidence of pink' (score 5). An alternative five point score chart (TCC-score) including texture of the meat, clarity of meat juice and internal colour was also developed. Enumeration of viable E. coli O157:H7gfp+ in cooked hamburgers was based on fluorescent colonies recovered from plates. Results showed that MAP hamburgers developed PMB when compared with controls (P=0.0003) and that the shortest cooking time for the highest C-score was 6 and 11 min for MAP and control hamburgers, respectively. The mean temperature in the MAP hamburger was then 60.3 °C. The TCC-score reduced the difference between MAP and control hamburgers. It was also shown that the survival of E. coli O157:H7gfp+ was highest in MAP hamburgers. The predicted absolute risks for illness were highest for MAP hamburgers for all C-scores and the relative risk associated with PMB increased with doneness. For a C-score of 4 (slightly pink) the predicted relative risk for illness was 300 times higher for MAP hamburger than for controls. A variable pathogen reduction was observed when cooking hamburgers to temperatures of 70-76 °C (the 5th and 95th percentile range was around 3.3 log CFU). The lower reductions, at the 5th percentile, may, depending on initial contamination levels, not be enough to ensure sufficient and safe inactivation of E. coli O157:H7. Efforts to inform consumers about PMB in minced meat packaged in high oxygen packages (≥60% O2) are needed with the aim to make consumers use thermometers correctly or at least not determine doneness based only on meat colour.

Salmonella isolated from individual reptiles and environmental samples from terraria in private households in Sweden.

BACKGROUND: This study investigates Salmonella spp. isolated from privately kept reptiles and from environmental samples such as bedding materials or water from the floor of the enclosures (terraria). It also compares isolation of Salmonella using Modified Semisolid Rappaport-Vassiliadis (MSRV) medium or selective enrichment in Rappaport-Vassiliadis-Soya (RVS) pepton broth. Cloacal swabs or swabs from the cloacal area were collected from 63 individual reptiles belonging to 14 households. All reptiles were from different terraria and from 62 of these, environmental samples were also collected. Sampling were done by the reptile owners according to written instructions and sent by mail immediately after sampling. All but three samples were analyzed within 24 h after collection. Colonies suspected for Salmonella were tested for agglutination and serotyped using the White-Kauffmann-Le Minor scheme. The relative sensitivity (se) and specificity (sp) for MSRV compared with RVS, and the agreement coefficient kappa (κ) were calculated. RESULTS: Salmonella was isolated from 50/63 (80%) terraria, either from the reptiles (31/63; 49%) or from bedding material (39/62; 63%). The most common subspecies was Salmonella enterica subspecies enterica followed by S. enterica subspecies diarizonae. In reptiles, the most common S. enterica subspecies enterica serovars were Java (n = 4) and Fluntern (n = 4), compared with the serovars Tennessee (n = 10) and Fluntern (n = 10) in the environmental samples. The exact same set of Salmonella subspecies and serovars were not isolated from the individual reptiles and the environmental samples from any of the households. Isolation using MSRV yielded more Salmonella isolates 61/113 (54%) than enrichment in RVS 57/125 (46%). The se was 97.9% (95% Confidence Interval 93.9-100), the sp 78.5% (95% CI 68.5-88.5) and the κ 0.74, indicating substantial agreement between the tests. CONCLUSIONS: Salmonella can be expected to be present in environments where reptiles are kept. This constitutes public health risks and should be considered during handling of the reptiles and during cleaning and disposal of bedding. A combination of different culturing techniques may be used to increase the isolation rate.

Clostridium difficile in faeces from healthy dogs and dogs with diarrhea.

BACKGROUND: This study was conducted to evaluate the faecal occurrence and characterization of Clostridium difficile in clinically healthy dogs (N = 50) and in dogs with diarrhea (N = 20) in the Stockholm-Uppsala region of Sweden. FINDINGS: Clostridium difficile was isolated from 2/50 healthy dogs and from 2/20 diarrheic dogs. Isolates from healthy dogs were negative for toxin A and B and for the tcdA and tcdB genes. Both isolates from diarrheic dogs were positive for toxin B and for the tcdA and tcdB genes. The C. difficile isolates from healthy dogs had PCR ribotype 009 (SE-type 6) and 010 (SE-type 3) whereas both isolates from dogs with diarrhoea had the toxigenic ribotype 014 (SE-type 21). One of the isolates from healthy dogs was initially resistant to metronidazole. CONCLUSIONS: This study revealed presence of toxigenic C. difficile in faecal samples of diarrheic dogs and low number of non- toxigenic isolates in healthy dogs from Uppsala-Stockholm region in Sweden. However, more comprehensive studies are warranted to investigate the role of C. difficile in gastrointestinal disease in dogs.

Isolation and characterization of Treponema phagedenis-like spirochetes from digital dermatitis lesions in Swedish dairy cattle.

BACKGROUND: Digital dermatitis in cattle is an emerging infectious disease. Ulcerative lesions are typically located on the plantar skin between the heel bulbs and adjacent to the coronet. Spirochetes of the genus Treponema are found in high numbers in the lesions and are likely to be involved in the pathogenesis. The aim of this study was to obtain pure cultures of spirochetes from cattle with digital dermatitis and to describe them further. METHODS: Tissue samples and swabs from active digital dermatitis lesions were used for culturing. Pure isolates were subjected to, molecular typing through 16S rRNA gene sequencing, pulsed-field gel electrophoresis (PFGE), random amplified polymorphic DNA (RAPD) and an intergenic spacer PCR developed for Treponema spp. as well as API-ZYM and antimicrobial susceptibility tests. The antimicrobial agents used were tiamulin, valnemulin, tylosin, aivlosin, lincomycin and doxycycline. RESULTS: Seven spirochete isolates from five herds were obtained. Both 16S rRNA gene sequences, which were identical except for three polymorphic nucleotide positions, and the intergenic spacer PCR indicated that all isolates were of one yet unnamed species, most closely related to Treponema phagedenis. The enzymatic profile and antimicrobial susceptibility pattern were also similar for all isolates. However it was possible to separate the isolates through their PFGE and RAPD banding pattern. CONCLUSION: This is the first report on isolation of a Treponema sp. from cattle with digital dermatitis in Scandinavia. The phylotype isolated has previously been cultured from samples from cattle in the USA and the UK and is closely related to T. phagedenis. While very similar, the isolates in this study were possible to differentiate through PFGE and RAPD indicating that these methods are suitable for subtyping of this phylotype. No antimicrobial resistance could be detected among the tested isolates.