Vaccinating parent flocks against colibacillosis reduces broiler mortality - A retrospective observational study from 2016 to 2019 in Finland.

Colibacillosis is one of the most important infectious diseases in modern poultry production. The complex nature of colibacillosis has made it challenging to produce an effective vaccine. As a control measure for colibacillosis outbreak in Finland, a vaccination program with a commercial colibacillosis vaccine and later also an autogenous vaccine was started for parent flocks in 2017. In this retrospective observational study from years 2016-2019, we evaluated first week and total mortality of broiler flocks (n= 6969) originating from parents with different colibacillosis vaccination status. Broiler flocks were divided into three groups according to vaccination status of their parent flocks. First group were flocks from parents with no colibacillosis vaccines; second group was flocks from parents vaccinated with commercial vaccine only; and third group was flocks from parents with both commercial and autogenous vaccine. Bayesian modelling was used to predict posterior distributions of first week mortality and total mortality of the broiler flocks. Results of the modelling revealed that broiler flocks from unvaccinated parents had the highest mortality rates (mean first week mortality 1.40 % and mean total mortality 4.33 %, respectively) whereas flocks from parents with a combination of commercial and autogenous vaccinations had the lowest mortality rates (mean first week mortality 0,91 % and mean total mortality 3,14 %). The mortalities from broilers flocks from parents with only commercial vaccine fell in between these groups. Also, standard deviations of mortality rates were lower in broilers from parents with commercial or both vaccines. This demonstrates that in addition to lowering the mean mortality rates, the vaccinations made high mortality broiler flocks less common. Best performance was obtained when autogenous vaccine was combined to the commercial vaccine. The autogenous vaccine consists of the same type of Escherichia coli strain that was causing most colibacillosis cases during the study period in Finland. This study adds to the evidence of benefits of colibacillosis vaccines during outbreaks. It also demonstrates the importance of the knowledge of the types of APEC strains causing outbreaks to produce effective autogenous vaccines.

Core genome multilocus sequence typing analysis of Finnish Taylorella equigenitalis isolates.

In Finland, Taylorella equigenitalis, the causative agent of contagious equine metritis (CEM), was first detected in 1992. The aim of this study was to genotype Finnish T. equigenitalis isolates to investigate the epidemiology of the infection in the Finnish horse population. A total of 34 T. equigenitalis isolates from 24 horses obtained during 1992-2021 were subjected to whole genome sequencing (WGS) and subsequent local ad hoc core genome multi-locus sequence typing (cgMLST) targeting 1259 loci. Classical MLST profiles were extracted from the whole-genome sequence data. Three novel MLST types, ST81, ST82 and ST83, and four previously described sequence types, ST16, ST17, ST50 and ST63 were detected among the isolates. cgMLST minimum spanning tree analysis using 12 allele difference as threshold, resulted in five clusters and three singletons. cgMLST clusters were congruent with the MLST-defined groups, except for the ST83 isolates which were divided into two clusters. However, the high discriminatory power cgMLST allowed differentiation between isolates of the same MLST type as each isolate had a unique core genome ST. Our study suggests that cgMLST has the prospective for being a standardised typing method for T. equigenitalis in the future, and further contributes to worldwide phylogenetic and spatio-temporal analyses needed to better understand the epidemiology of the bacterium.

Pilot testing the EARS-Vet surveillance network for antibiotic resistance in bacterial pathogens from animals in the EU/EEA.

Introduction: As part of the EU Joint Action on Antimicrobial Resistance (AMR) and Healthcare-Associated Infections, an initiative has been launched to build the European AMR Surveillance network in veterinary medicine (EARS-Vet). So far, activities included mapping national systems for AMR surveillance in animal bacterial pathogens, and defining the EARS-Vet objectives, scope, and standards. Drawing on these milestones, this study aimed to pilot test EARS-Vet surveillance, namely to (i) assess available data, (ii) perform cross-country analyses, and (iii) identify potential challenges and develop recommendations to improve future data collection and analysis. Methods: Eleven partners from nine EU/EEA countries participated and shared available data for the period 2016-2020, representing a total of 140,110 bacterial isolates and 1,302,389 entries (isolate-antibiotic agent combinations). Results: Collected data were highly diverse and fragmented. Using a standardized approach and interpretation with epidemiological cut-offs, we were able to jointly analyze AMR trends of 53 combinations of animal host-bacteria-antibiotic categories of interest to EARS-Vet. This work demonstrated substantial variations of resistance levels, both among and within countries (e.g., between animal host species). Discussion: Key issues at this stage include the lack of harmonization of antimicrobial susceptibility testing methods used in European surveillance systems and veterinary diagnostic laboratories, the absence of interpretation criteria for many bacteria-antibiotic combinations of interest, and the lack of data from a lot of EU/EEA countries where little or even surveillance currently exists. Still, this pilot study provides a proof-of-concept of what EARS-Vet can achieve. Results form an important basis to shape future systematic data collection and analysis.

Transmission of Mycoplasma bovis infection in bovine in vitro embryo production.

Mycoplasma bovis (M. bovis) causes several costly diseases in cattle and has a negative effect on cattle welfare. There is no effective commercial vaccine, and antimicrobial resistance is common. Maintaining a closed herd is the best method to minimize the risk of introduction of M. bovis. Assisted reproduction is crucial in a closed herd to make genetic improvements. M. bovis has been found in commercial semen, and contaminated semen has been the source of disease in naïve dairy herds. The objective of this study was to evaluate M. bovis transmission in bovine in vitro embryo production (IVP) using several possible exposure routes. We used a wild-type M. bovis strain isolated from semen at a final concentration of 106 CFU/mL to infect cumulus-oocyte complexes, spermatozoa, and 5-day-old embryos. We also used naturally contaminated semen in fertilization. Blastocysts were collected on day 7-8 and zona pellucida (ZP)-intact embryos were either washed 12 times, including trypsin washes as recommended by the International Embryo Technology Society (IETS), or left unwashed. Washed and unwashed embryos, follicular fluids, maturation medium, cumulus cells, fertilization medium, and G1 and G2 culture media, as well as all wash media were analyzed using enrichment culture followed by real-time PCR detection of M. bovis. Altogether, 76 pools containing 363 unwashed embryos and 52 pools containing 261 IETS washed embryos were analyzed after oocytes, spermatozoa, or 5-day-old embryos were infected with M. bovis or naturally contaminated semen was used in fertilization. We could not detect M. bovis in any of the embryo pools. M. bovis was not found in any of 12 wash media from different exposure experiments. M. bovis did not affect the blastocyst rate, except when using experimentally infected semen. Contrary to an earlier study, which used a cell co-culture system, we could not demonstrate M. bovis in embryo wash media or tight adherence of M. bovis to ZP-intact embryos. Naturally infected semen did not transmit M. bovis to embryos. We conclude that by using our IVP system, the risk of M. bovis transmission via IVP embryos to recipient cows is very low.

Review and Analysis of National Monitoring Systems for Antimicrobial Resistance in Animal Bacterial Pathogens in Europe: A Basis for the Development of the European Antimicrobial Resistance Surveillance Network in Veterinary Medicine (EARS-Vet).

The monitoring of antimicrobial resistance (AMR) in bacterial pathogens of animals is not currently coordinated at European level. To fill this gap, experts of the European Union Joint Action on Antimicrobial Resistance and Healthcare Associated Infections (EU-JAMRAI) recommended building the European Antimicrobial Resistance Surveillance network in Veterinary medicine (EARS-Vet). In this study, we (i) identified national monitoring systems for AMR in bacterial pathogens of animals (both companion and food-producing) among 27 countries affiliated to EU-JAMRAI, (ii) described their structures and operations, and (iii) analyzed their respective strengths, weaknesses, opportunities and threats (SWOT). Twelve countries reported having at least one national monitoring system in place, representing an opportunity to launch EARS-Vet, but highlighting important gaps in AMR data generation in Europe. In total, 15 national monitoring systems from 11 countries were described and analyzed. They displayed diverse structures and operations, but most of them shared common weaknesses (e.g., data management and representativeness) and common threats (e.g., economic vulnerability and data access), which could be addressed collectively under EARS-Vet. This work generated useful information to countries planning to build or improve their system, by learning from others' experience. It also enabled to advance on a pragmatic harmonization strategy: EARS-Vet shall follow the European Committee on Antimicrobial Susceptibility Testing (EUCAST) standards, collect quantitative data and interpret AMR data using epidemiological cut-off values.

Suitability of Nasal and Deep Nasopharyngeal Swab Sampling of Calves in the Mycoplasma bovis Control Program.

Mycoplasma bovis is an important cattle pathogen affecting animal health, welfare, and productivity. The main disease syndromes are mastitis, pneumonia, and otitis media in young stock, as well as arthritis. Response to antibiotic treatment is poor and no effective vaccine is available. Asymptomatic carriers are common and usually harbor the organism in the airways or mammary glands. Purchase of carrier animals is a major risk for the introduction of infection into naive herds. Following the detection of M. bovis in Finland in 2012, a voluntary control program was established. It aims to prevent the spread of the infection and to help farms attain certification of a low M. bovis risk. Among the diagnostic tools in the program, nasal swabs (NS) from young calves have been tested for M. bovis to indicate the infection status of the herd. In this study, we assessed the suitability of this test method. We analyzed the effectiveness of NS and deep nasopharyngeal swabs (NP) to detect M. bovis in pneumonic and healthy calves in dairy herds recently infected with M. bovis. In pneumonic calves, NP sampling followed by culture and real-time PCR demonstrated a proportion of positive agreement (PPA) of 0.91 compared with bronchoalveolar lavage (BAL), whereas NS showed only 0.5 PPA compared with BAL. Among healthy dairy calves, overall M. bovis prevalence in NS was 29.6%. The highest rate of shedding (43%) occurred in calves 31-60 days old. At the calf level, M. bovis prevalence in NP samples was 47% compared with 33% in NS samples among the 284 studied calves. However, at the herd level, NS sampling classified 51 out of 54 herds with a positive infection status as infected, whereas in NP sampling, the respective figure was 43 out of 54 herds (p = 0.061). In conclusion, NS sampling from calves under 6 months of age and analyzed by real-time PCR is a cost-efficient method for a control program to detect M. bovis in dairy herds, even if no M. bovis mastitis has been detected in the herd. For pneumonic calves, we recommend only NP or BAL sampling.

Mycoplasma bovis infection in dairy herds-Risk factors and effect of control measures.

As Mycoplasma bovis spreads to new countries and becomes increasingly recognized as a disease with major welfare and economic effects, control measures on dairy farms are needed. To minimize the risk of infection spread to naive herds, all possible risk factors for M. bovis infection should be identified and controlled. Mycoplasma bovis was first diagnosed in dairy cattle in Finland in 2012, and by January 2020, 86 Finnish dairy farms (<1.5%) supporting M. bovis infections were identified. We evaluated risk factors for M. bovis infection using a questionnaire provided to 40 infected and 30 control dairy farms. Control measures were advised for 19 of the infected dairy farms during visits by a veterinarian. The course of the infection on those farms was followed by analyzing calf nasal swabs with PCR for presence of M. bovis 4 times at 6-mo intervals. Control measures included culling of M. bovis mastitic cows, isolation of new calves from older animals after initial M. bovis mastitic cows had been culled, prevention of nose-to-nose contact with infected animals, early detection of mastitis cases using M. bovis PCR, and hygiene measures mainly related to milking, calf pens, feeding buckets, and teats. Farms implemented the control measures related to the isolation of calves or avoidance of nose-to-nose contact in various ways, according to farm structures and financial circumstances.In our study, the control measures recommended to the dairy farms appeared effective, such that 13 of 19 farms reached a low risk level during at least 3 consecutive negative samplings from calves, with no M. bovis mastitis detected subsequently. Among risk factors, insemination with an M. bovis-positive bull indicated a trend of increasing the odds of M. bovis infection on the farm in a multivariable logistic model. In contrast, higher herd average milk yield had an association with lower odds for M. bovis infection. Occurrence of other infectious diseases affecting several animals on the dairy farm in the previous 6 mo before M. bovis infection were more frequent on M. bovis-infected farms.

Efficacy of Two Antibiotic-Extender Combinations on Mycoplasma bovis in Bovine Semen Production.

Mycoplasma bovis is an important bovine pathogen. Artificial insemination (AI) using contaminated semen can introduce the agent into a naïve herd. Antibiotics, most often gentamycin, tylosin, lincomycin, spectinomycin (GTLS) combination are added to semen extender to prevent transmission of pathogenic bacteria and mycoplasmas. In a commercial AI straw production system with industrial scale procedures, we analyzed the mycoplasmacidal efficacy of GTLS and ofloxacin on M. bovis ATCC and wild type strain isolated from commercial AI straws. The strains were spiked at two concentrations (106 and 103 CFU/mL) into semen. Viable M. bovis in frozen semen straws was detected by enrichment culture and real-time PCR. We also compared different protocols to extract M. bovis DNA from spiked semen. None of the antibiotic protocols had any effect on the viability of either of the M. bovis strains at high spiking concentration. At low concentration, the wild type was inhibited by all other protocols, except low GTLS, whereas the ATCC strain was inhibited only by high GTLS. The InstaGene™ matrix was the most effective method to extract M. bovis DNA from semen. When there is a low M. bovis contamination level in semen, GTLS used at high concentrations, in accordance with Certified Semen Services requirements, is more efficient than GTLS used at concentrations stated in the OIE Terrestrial Code.

Mycoplasma bovis in Nordic European Countries: Emergence and Dominance of a New Clone.

Mycoplasma (M.) bovis is an important pathogen of cattle implicated in a broad range of clinical manifestations that adversely impacts livestock production worldwide. In the absence of a safe, effective commercial vaccine in Europe, the reported reduced susceptibility to antimicrobials for this organism has contributed to difficulties in controlling infection. Despite global presence, some countries have only recently experienced outbreaks of this pathogen. In the present study, M. bovis isolates collected in Denmark between 1981 and 2016 were characterized to determine (i) genetic diversity and phylogenetic relationships using whole genome sequencing and various sequence-based typing methods and (ii) patterns of antimicrobial resistance compared to other European isolates. The M. bovis population in Denmark was found to be highly homogeneous genomically and with respect to the antimicrobial resistance profile. Previously dominated by an old genotype shared by many other countries (ST17 in the PubMLST legacy scheme), a new predominant type represented by ST94-adh1 has emerged. The same clone is also found in Sweden and Finland, where M. bovis introduction is more recent. Although retrieved from the Netherlands, it appears absent from France, two countries with a long history of M. bovis infection where the M. bovis population is more diverse.

Dynamics of the within-herd prevalence of Mycoplasma bovis intramammary infection in endemically infected dairy herds.

We aimed to identify the dynamics of the within-herd prevalence of Mycoplasma (M.) bovis intramammary infection (IMI) in four dairy herds, estimate prevalence of M. bovis in colostrum and clinical mastitis cases and compare M. bovis strains from calves' respiratory and cow clinical mastitis samples. Within a six-month study period, cow composite milk samples (CMS) were collected three times during routine milk recording, first milking colostrum samples from all calving cows and udder quarter milk samples from clinical mastitis cases. Calf respiratory samples were collected from calves with respiratory disease. Pooled milk samples were analysed for M. bovis with the Mastitis 4B polymerase chain reaction (PCR) test kit (DNA Diagnostic A/S). Prevalence estimates were calculated with Bayesian framework in R statistical programme. cg-MLST was used for M. bovis genotyping. In Herd I and II first testing M. bovis IMI within-herd prevalence (95 % credibility interval (CI)) was 4.7 % (2.9; 6.8) and 3.4 % (2.3; 4.6), changing to 1.0 % (0.1; 1.7) and 0.8 % (0.1; 1.4) in Herd I and 0.4 % (0.0; 0.7) in Herd II at the next samplings. In Herd III and IV first testing M. bovis IMI within-herd prevalence was 12.3 % (9.7; 15.2) and 7.8 % (6.2; 9.5), changing to 4.6 % (3.0; 6.4) and 3.2 % (1.9; 4.8) in Herd III and to 2.8 % (1.9; 3.8) and 4.9 % (3.6; 6.4) in Herd IV at the next samplings. The estimated prevalence of M. bovis in colostrum ranged between 1.7 % (0.2; 2.8) and 4.7 % (2.7; 7.1) and in clinical mastitis cases between 3.7 % (1.7; 6.4) and 11.0 % (7.5; 15.2) in the study herds. M. bovis strains isolated from cows and calves clustered within herds indicating possible transmission of M. bovis between dairy cows and calves. Prevalence of M. bovis in colostrum and clinical mastitis cases as well as the within-herd prevalence of M. bovis IMI was low in endemically infected dairy herds.

A European interlaboratory trial to evaluate the performance of different PCR methods for Mycoplasma bovis diagnosis.

BACKGROUND: Several species-specific PCR assays, based on a variety of target genes are currently used in the diagnosis of Mycoplasma bovis infections in cattle herds with respiratory diseases and/or mastitis. With this diversity of methods, and the development of new methods and formats, regular performance comparisons are required to ascertain diagnostic quality. The present study compares PCR methods that are currently used in six national veterinary institutes across Europe. Three different sample panels were compiled and analysed to assess the analytical specificity, analytical sensitivity and comparability of the different PCR methods. The results were also compared, when appropriate, to those obtained through isolation by culture. The sensitivity and comparability panels were composed of samples from bronchoalveolar fluids of veal calves, artificially contaminated or naturally infected, and hence the comparison of the different methods included the whole workflow from DNA extraction to PCR analysis. RESULTS: The participating laboratories used i) five different DNA extraction methods, ii) seven different real-time and/or end-point PCRs targeting four different genes and iii) six different real-time PCR platforms. Only one commercial kit was assessed; all other PCR assays were in-house tests adapted from published methods. The analytical specificity of the different PCR methods was comparable except for one laboratory where Mycoplasma agalactiae was tested positive. Frequently, weak-positive results with Ct values between 37 and 40 were obtained for non-target Mycoplasma strains. The limit of detection (LOD) varied from 10 to 103 CFU/ml to 103 and 106 CFU/ml for the real-time and end-point assays, respectively. Cultures were also shown to detect concentrations down to 102 CFU/ml. Although Ct values showed considerable variation with naturally infected samples, both between laboratories and tests, the final result interpretation of the samples (positive versus negative) was essentially the same between the different laboratories. CONCLUSION: With a few exceptions, all methods used routinely in the participating laboratories showed comparable performance, which assures the quality of diagnosis, despite the multiplicity of the methods.

Semen as a source of Mycoplasma bovis mastitis in dairy herds.

Mycoplasma bovis infections are responsible for substantial economic losses in the cattle industry, have significant welfare effects and increase antibiotic use. The pathogen is often introduced into naive herds through healthy carrier animals. In countries with a low prevalence of M. bovis, transmission from less common sources can be better explored as the pathogen has limited circulation compared to high prevalence populations. In this study, we describe how M. bovis was introduced into two closed and adequately biosecure dairy herds through the use of contaminated semen during artificial insemination (AI), leading to mastitis outbreak in both herds. Epidemiological analysis did not reveal an infection source other than semen. In both farms the primary clinical cases were M. bovis mastitis in cows inseminated with the semen of the same bull four weeks before the onset of the disease. One semen straw derived from the semen tank on the farm and other semen lots of this bull were positive for M. bovis. In contrast, semen samples were negative from other bulls that had been used for insemination in previous or later oestrus to those cows with M. bovis mastitis. Furthermore, cgMLST of M. bovis isolates supported the epidemiological results. To our knowledge this is the first study describing the introduction of M. bovis infection into a naive dairy herd via processed semen. The antibiotics used in semen extenders should be re-evaluated in order to provide farms with M. bovis-free semen or tested M. bovis-free semen should be available.

Spread of avian pathogenic Escherichia coli ST117 O78:H4 in Nordic broiler production.

BACKGROUND: Escherichia coli infections known as colibacillosis constitute a considerable challenge to poultry farmers worldwide, in terms of decreased animal welfare and production economy. Colibacillosis is caused by avian pathogenic E. coli (APEC). APEC strains are extraintestinal pathogenic E. coli and have in general been characterized as being a genetically diverse population. In the Nordic countries, poultry farmers depend on import of Swedish broiler breeders which are part of a breeding pyramid. During 2014 to 2016, an increased occurrence of colibacillosis on Nordic broiler chicken farms was reported. The aim of this study was to investigate the genetic diversity among E. coli isolates collected on poultry farms with colibacillosis issues, using whole genome sequencing. METHODS: Hundred and fourteen bacterial isolates from both broilers and broiler breeders were whole genome sequenced. The majority of isolates were collected from poultry with colibacillosis on Nordic farms. Subsequently, comparative genomic analyses were carried out. This included in silico typing (sero- and multi-locus sequence typing), identification of virulence and resistance genes and phylogenetic analyses based on single nucleotide polymorphisms. RESULTS: In general, the characterized poultry isolates constituted a genetically diverse population. However, the phylogenetic analyses revealed a major clade of 47 closely related ST117 O78:H4 isolates. The isolates in this clade were collected from broiler chickens and breeders with colibacillosis in multiple Nordic countries. They clustered together with a human ST117 isolate and all carried virulence genes that previously have been associated with human uropathogenic E. coli. CONCLUSIONS: The investigation revealed a lineage of ST117 O78:H4 isolates collected in different Nordic countries from diseased broilers and breeders. The data indicate that the closely related ST117 O78:H4 strains have been transferred vertically through the broiler breeding pyramid into distantly located farms across the Nordic countries.

Species distribution and in vitro antimicrobial susceptibility of coagulase-negative staphylococci isolated from bovine mastitic milk.

BACKGROUND: Coagulase-negative staphylococci (CoNS) are the most common bovine mastitis causing bacteria in many countries. It is known that resistance for antimicrobials is in general more common in CoNS than in Staphylococcus aureus but little is known about the antimicrobial resistance of specific CoNS species. In this study, 400 CoNS isolates from bovine mastitic milk samples were identified to species level using ribotyping and MALDI-TOF MS, and their antimicrobial susceptibility was determined using a commercially available microdilution system. The results were interpreted according to the epidemiological cut-off values by the European Committee on Antimicrobial Susceptibility testing. RESULTS: The most common CoNS species were S. simulans, S. epidermidis, S. chromogenes and S. haemolyticus. Penicillin resistance was the most common type of antimicrobial resistance. Staphylococcus epidermidis was the most resistant among the four major species. Almost one-third of our S. epidermidis isolates were resistant to >2 antimicrobials and close to 7 % were multidrug resistant. The majority of S. epidermidis isolates were resistant to benzylpenicillin. On the contrary, only few S. simulans isolates were penicillin-resistant. Phenotypic oxacillin resistance was found in all four main species, and 34 % of the isolates were oxacillin resistant. However, only 21 isolates (5 %) were positive for the mecA gene. Of these, 20 were S. epidermidis and one S. sciuri. mecC positive isolates were not found. CONCLUSION: Staphylococcus epidermidis differed from the three other major CoNS species as resistance to the tested antimicrobials was common, several isolates were multidrug resistant, and 19 % of the isolates carried the mecA gene encoding methicillin resistance.

Performance of a real-time PCR assay in routine bovine mastitis diagnostics compared with in-depth conventional culture.

Reliable identification of the aetiological agent is crucial in mastitis diagnostics. Real-time PCR is a fast, automated tool for detecting the most common udder pathogens directly from milk. In this study aseptically taken quarter milk samples were analysed with a real-time PCR assay (Thermo Scientific PathoProof Mastitis Complete-12 Kit, Thermo Fisher Scientific Ltd.) and by semi-quantitative, in-depth bacteriological culture (BC). The aim of the study was to evaluate the diagnostic performance of the real-time PCR assay in routine use. A total of 294 quarter milk samples from routine mastitis cases were cultured in the national reference laboratory of Finland and examined with real-time PCR. With BC, 251 out of 294 (85.7%) of the milk samples had at least one colony on the plate and 38 samples were considered contaminated. In the PCR mastitis assay, DNA of target species was amplified in 244 samples out of 294 (83.0%). The most common bacterial species detected in the samples, irrespective of the diagnostic method, was the coagulase negative staphylococci (CNS) group (later referred as Staphylococcus spp.) followed by Staphylococcus aureus. Sensitivity (Se) and specificity (Sp) for the PCR assay to provide a positive Staph. aureus result was 97.0 and 95.8% compared with BC. For Staphylococcus spp., the corresponding figures were 86.7 and 75.4%. Our results imply that PCR performed well as a diagnostic tool to detect Staph. aureus but may be too nonspecific for Staphylococcus spp. in routine use with the current cut-off Ct value (37.0). Using PCR as the only microbiological method for mastitis diagnostics, clinical relevance of the results should be carefully considered before further decisions, for instance antimicrobial treatment, especially when minor pathogens with low amount of DNA have been detected. Introducing the concept of contaminated samples should also be considered.

Extraintestinal pathogenic Escherichia coli in poultry meat products on the Finnish retail market.

BACKGROUND: Extraintestinal pathogenic Escherichia coli bacteria (ExPEC) exist as commensals in the human intestines and can infect extraintestinal sites and cause septicemia. The transfer of ExPEC from poultry to humans and the role of poultry meat as a source of ExPEC in human disease have been discussed previously. The aim of the present study was to provide insight into the properties of ExPEC in poultry meat products on the Finnish retail market with special attention to their prevalence, virulence and phylogenetic profiles. Furthermore, the isolates were screened for possible ESBL producers and their resistance to nalidixic acid and ciprofloxacin was tested. METHODS: The presence of ExPEC in 219 marinated and non-marinated raw poultry meat products from retail shops has been analyzed. One E. coli strain per product was analyzed further for phylogenetic groups and possession of ten virulence genes associated with ExPEC bacteria (kpsMT K1, ibeA, astA, iss, irp2, papC, iucD, tsh, vat and cva/cv) using PCR methods. The E. coli strains were also screened phenotypically for the production of extended-spectrum ß-lactamase (ESBL) and the susceptibility of 48 potential ExPEC isolates for nalidixic acid and ciprofloxacin was tested. RESULTS: E. coli was isolated from 207 (94.5%) of 219 poultry meat products. The most common phylogenetic groups were D (50.7%), A (37.7%), and B2 (7.7%). Based on virulence factor gene PCR, 23.2% of the strains were classified as ExPEC. Two ExPEC strains (1%) belonged to [O1] B2 svg+ (specific for virulent subgroup) group, which has been implicated in multiple forms of ExPEC disease. None of the ExPEC strains was resistant to ciprofloxacin or cephalosporins. One isolate (2.1%) showed resistance to nalidixic acid. CONCLUSIONS: Potential ExPEC bacteria were found in 22% of marinated and non-marinated poultry meat products on the Finnish retail market and 0.9% were contaminated with E. coli [O1] B2 svg+ group. Marinades did not have an effect on the survival of ExPEC as strains from marinated and non-marinated meat products were equally often classified as ExPEC. Poultry meat products on the Finnish retail market may have zoonotic potential.

Acute phase protein changes in calves during an outbreak of respiratory disease caused by bovine respiratory syncytial virus.

Bovine acute phase proteins (APPs), lipopolysaccharide binding protein (LBP), serum amyloid A (SAA), haptoglobin (Hp) and alpha(1)-acid glycoprotein (AGP) were evaluated as inflammatory markers during an outbreak of bovine respiratory disease (BRD) caused by bovine respiratory syncytial virus (BRSV). Calves (n = 10) presented mild to moderate signs of respiratory disease. Secondary bacterial infections, Pasteurella multocida and Mycoplasma dispar as major species, were detected in tracheobronchial lavage samples. Concentrations of SAA and LBP increased at week 1 had the highest values at week 3 and decreased at week 4 of outbreak. Some calves had high Hp concentrations at week 3, but AGP concentrations did not rise during respiratory disease. Higher SAA, LBP and Hp concentrations at a later stage of BRD (week 3) were associated with the low BRSV-specific IgG(1) production, suggesting that these calves had enhanced inflammatory response to the secondary bacterial infection. In conclusion, APPs (especially SAA and LBP) are sensitive markers of respiratory infection, and they may be useful to explore host response to the respiratory infections in clinical research.

Phylogeny, virulence factors and antimicrobial susceptibility of Escherichia coli isolated in clinical bovine mastitis.

The aim of this study was to identify specific phylogeny groups, virulence genes or antimicrobial resistance traits of Escherichia coli isolated in bovine mastitis associated to clinical signs, persistence of intramammary infection in the quarter and recovery from mastitis. A total of 154 E. coli isolates from bovine clinical mastitis, 144 from the acute stage and 10 from follow-up samples 3 weeks later, originating from 144 cows in 65 dairy herds in Southern Finland were investigated. Phylogeny groups and virulence genes of the isolates were determined using polymerase chain reaction, and antimicrobial susceptibility using the VetMIC™ microdilution method. In ten cows (11.8%), infection persisted, confirmed by re-isolation of the same pulsed-field gel electrophoresis type from the affected quarter at 3 weeks post-treatment. The majority of isolates, 119 (82.6%), belonged to phylogeny group A, which mainly consisted of commensal strains. Altogether 56 isolates (38.9%) had at least one virulence gene detected. Most common virulence genes detected were irp2, iucD, papC iss; genes svg, stx1, stx2, cnf1 and hlyA were not found. Combinations of virulence genes varied greatly. Forty (27.8%) of the 144 E. coli isolates showed resistance to at least one antimicrobial tested. None of the studied phylogeny groups, virulence factors or antimicrobial resistance traits was associated with clinical signs, persistence of intramammary infection or clinical recovery from mastitis. The results support the conclusion that mastitis-causing E. coli bacteria are typical commensals.

Tracing shigatoxigenic Escherichia coli O103, O145, and O174 infections from farm residents to cattle.

Severe diarrheal infections caused by Shigatoxigenic Escherichia coli O103:H2:stx(1):eae-epsilon:ehx, O145:H28:stx(1):eae-gamma:ehx (two cases in a family), and O174:H21:stx(2c) in farm residents were traced to cattle. Molecular methods were applied to the isolation and characterization of the strains. The causative strains were also isolated from cattle samples 1 or 4 months later.

Characterisation of atypical Aeromonas salmonicida infection in Arctic charr Salvelinus alpinus and European grayling Thymallus thymallus.

Cultured stocks of Arctic charr Salvelinus alpinus and European grayling Thymallus thymallus are vulnerable to infection by achromogenic atypical Aeromonas salmonicida (AAS). In Finland, natural stocks of both fish species have to be supported by restocking, and AAS infection poses a threat to successful restocking because no preventive means are available. In this study, we analysed AAS isolates from Arctic charr and European grayling and from other sources genetically, and characterised the signs and pathology of AAS infection in Arctic charr and European grayling both under farming conditions and after experimental challenge. AAS outbreaks were recorded in 1 fish farm over an 8 yr period. Among various salmonid fishes under farming conditions, only Arctic charr and European grayling were susceptible to AAS infection. The disease caused by AAS could be reproduced in both species using the same AAS strain in an experimental challenge. The course of the disease and pathology of natural and experimental AAS infection differed between the 2 species, even though only 1 strain was used for challenge. Isolates of AAS from Arctic charr and European grayling were genetically identical within a single river water basin. However, genetic heterogeneity was observed among the isolates from different water basins. In both species, AAS caused systemic infection. The results suggest that the same AAS strain could be used to develop a vaccine to protect both Arctic charr and European grayling from AAS infection.