Håkan Vigre

Molecular and phylogenetic characterization of Cryptosporidium and Giardia from pigs and cattle in Denmark

The genetic diversity of Cryptosporidium spp. and Giardia duodenalis from dairy cattle and pigs in Denmark was determined in the present study. Faecal samples from 1237 pigs and 1150 cattle originating from 50 sow herds and 50 dairy herds, respectively, were analysed for the presence of the two parasites by immunofluorescence microscopy. A large proportion of the (oo)cyst containing samples were selected for molecular characterization. Sequencing and phylogenetic analysis of the 18S rDNA locus and/or the HSP70 gene of 183 pig and 154 cattle isolates of Cryptosporidium revealed the presence of C. suis, pig genotype II, C. parvum (cattle genotype), C. bovis, Cryptosporidium deer-like genotype and a novel C. suis-like genotype. For both cattle and pigs, a host age-related change in distribution of species/genotypes was observed. The zoonotic C. parvum (cattle genotype) was most prevalent in young calves. For Giardia, 82 and 145 isolates from pigs and cattle, respectively, were analysed at the 18S rDNA locus and/or the gdh gene. Giardia isolates belonging to the zoonotic Assemblage A was found in both young and older calves, as well as in weaners and piglets, whereas cows seemed to be infected purely by isolates of the livestock group, Assemblage E.

Using outbreak data for source attribution of human salmonellosis and campylobacteriosis in Europe.

Salmonella and Campylobacter are the most important bacterial causes of foodborne illness in Europe. To identify and prioritize food safety interventions, it is important to quantify the burden of human foodborne illness attributable to specific sources. Data from outbreak investigations are observed at the public health endpoint and can therefore be a direct measure of attribution at the point of exposure. An analysis or summary of outbreak investigations is useful for attributing illnesses to foods, but often the implicated foods in reported outbreaks are complex foods, containing several food items, many of which could be the specific source of the infection. We describe a method that is able to attribute human cases to specific food items contained in complex foods. The model is based on data from investigations of Salmonella and Campylobacter outbreaks in the European Union in 2005 and 2006. The reporting of the causative vehicles for the outbreaks was not harmonized between and within countries. Consequently, we organized the implicated foods in mutually exclusive food categories. We estimated that the most important food sources for salmonellosis cases were eggs (32%) and meat and poultry-meat (15%), and that the majority of the cases of campylobacteriosis were attributed to chicken (10%). For both pathogens, a large proportion of cases could not be linked to any source. Among illnesses that could be attributed to a source, 58% of salmonellosis cases were attributed to eggs, and 29% of campylobacteriosis cases were attributed to chicken. Results also revealed regional differences in the relative importance of specific sources. We assessed the method to be of limited value to attribute human campylobacteriosis due to the limited number of outbreaks. Nevertheless, the presented source attribution approach can be applied to other foodborne pathogens, and is easily adaptable to countries having an appropriate number of reported outbreaks.

A novel strategy to obtain quantitative data for modelling: Combined enrichment and real-time PCR for enumeration of salmonellae from pig carcasses

Salmonella is a major zoonotic pathogen which causes outbreaks and sporadic cases of gastroenteritis in humans worldwide. The primary sources for Salmonella are food-producing animals such as pigs and poultry. For risk assessment and hazard analysis and critical control point (HACCP) concepts, it is essential to produce large amounts of quantitative data, which is currently not achievable with the standard cultural based methods for enumeration of Salmonella. This study presents the development of a novel strategy to enumerate low numbers of Salmonella in cork borer samples taken from pig carcasses as a first concept and proof of principle for a new sensitive and rapid quantification method based on combined enrichment and real-time PCR. The novelty of the approach is in the short pre-enrichment step, where for most bacteria, growth is in the log phase. The method consists of an 8h pre-enrichment of the cork borer sample diluted 1:10 in non-selective buffered peptone water, followed by DNA extraction, and Salmonella detection and quantification by real-time PCR. The limit of quantification was 1.4 colony forming units (CFU)/20 cm(2) (approximately 10 g) of artificially contaminated sample with 95% confidence interval of ± 0.7 log CFU/sample. The precision was similar to the standard reference most probable number (MPN) method. A screening of 200 potentially naturally contaminated cork borer samples obtained over seven weeks in a slaughterhouse resulted in 25 Salmonella-positive samples. The analysis of salmonellae within these samples showed that the PCR method had a higher sensitivity for samples with a low contamination level (<6.7 CFU/sample), where 15 of the samples negative with the MPN method was detected with the PCR method and 5 were found to be negative by both methods. For the samples with a higher contamination level (6.7-310 CFU/sample) a good agreement between the results obtained with the PCR and MPN methods was obtained. The quantitative real-time PCR method can easily be applied to other food and environmental matrices by adaptation of the pre-enrichment time and media.

Pathogenicity of Cryptosporidium parvum—evaluation of an animal infection model

Abstract With the intention of developing a standardised method for assessment of pathogenicity of Cryptosporidium parvum, the CPB-0 isolate was studied by propagation in 1-day-old calves followed by inoculation into specific pathogen free (SPF) piglets. The experiment was repeated. Diarrhoea and shedding of oocysts were seen in all animals infected with the CPB-0 isolate. Clinical signs included depression, inappetence, vomiting (exclusively in the piglets), and death. Histological examination at 17 and 19 days post-infection revealed parasitic stages and microscopic changes primarily restricted to colon and rectum. The unintended presence of rotavirus in some of the experimental animals revealed an additive or synergistic effect between rotavirus and C. parvum as indicated by prolonged diarrhoea, increased oocyst shedding, decreased weight gain and elevated levels of serum haptoglobin and serum amyloid A (SAA) in piglets infected simultaneously with both pathogens. The difference in daily weight gain between infected and control animals was significant only for piglets co-infected with rotavirus. The acute phase response of haptoglobin and SAA was characterised by a large individual variation. In piglets, co-infected with rotavirus, the levels of serum haptoglobin were 3.5 and 4.6 times higher in the infected versus the controls 6 and 9dpi, respectively (mean values: 2411 μg/ml±S.D. 2023 and 1840 μg/ml±S.D. 1697). In the controls infected with rotavirus, peak haptoglobin concentration was seen 3dpi (mean: 1022 μg/ml±S.D. 425). Elevated levels of SAA were seen in 1 of 6 piglets infected with C. parvum, and in 5 of 6 piglets co-infected with rotavirus. Tumour necrosis factor alpha (TNFα) was undetectable in all serum samples from piglets. The obvious advantages of the SPF pig model are the naturally acquired intestinal microflora, the development of distinct clinical signs similar to cryptosporidiosis in humans and calves, the size of the animals, and the accessibility of individuals born within a short time span. This makes the model ideal for dose–response studies, evaluation of therapeutic agents as well as for assessment of differences in the clinical response to isolates of diverse genetic background. In conclusion, it was shown that the CPB-0 isolate was pathogenic to calves and piglets at a dose of 2.5×105 oocysts, and that the clinical signs could be replicated during separate experiments. Moreover, diarrhoea, oocyst shedding, body weight changes, histological alterations, and the acute phase response of haptoglobin and SAA were identified as useful parameters for discrimination of isolate-specific differences of pathogenicity.

Culture-independent quantification of Salmonella enterica in carcass gauze swabs by flotation prior to real-time PCR

To facilitate quantitative risk assessment in the meat production chain, there is a need for culture-independent quantification methods. The aim of this study was to evaluate the use of flotation, a non-destructive sample preparation method based on traditional buoyant density centrifugation, for culture-independent quantification of intact Salmonella in pig carcass gauze swabs (100 cm(2)) prior to quantitative PCR (qPCR). A novel approach was investigated, excluding the homogenization step prior to flotation, to improve the detection limit and speed up the quantification procedure. The buoyant density of two Salmonella strains in different growth conditions was determined to be 1.065-1.092 g/ml. Based on these data, an optimal discontinuous flotation with three different density layers, ~1.200, 1.102 and 1.055 g/ml, was designed for extracting intact Salmonella cells from pig carcass swabs. The method allowed accurate quantification from 4.4 × 10(2) to at least 2.2 × 10(7)CFU Salmonella per swab sample using qPCR (without preceding DNA extraction) or selective plating on xylose lysine deoxycholate agar. Samples with 50CFU could be detected occasionally but fell outside the linear range of the standard curve. The swab samples showed a broad biological diversity; for seven samples not inoculated with Salmonella, the microbial background flora (BGF) was determined to 5.0 ± 2.2 log CFU/ml sample withdrawn after flotation. It was determined that the proceeding PCR step was inhibited by BGF concentrations of ≥ 6.1 × 10(8)CFU/swab sample, but not by concentrations ≤ 6.1 × 10(6)CFU/swab sample. By using the gauze swabs directly in the flotation procedure, the homogenization step normally used for preparation of food-related samples could be excluded, which simplified the culture-independent quantification method considerably.

Transmission of Actinobacillus pleuropneumoniae in pigs under field-like conditions: emphasis on tonsillar colonisation and passively acquired colostral antibodies.

The objectives of this study were to elucidate at which age tonsillar colonisation by Actinobacillus pleuropneumoniae occurs in pigs and relate this occurrence to the presence of colostral antibodies to A. pleuropneumoniae. The infection patterns were studied in an isolated cohort of pigs, which consisted of the offspring from five sows originating from a conventional pig herd. The sows were transferred to isolated research facilities before farrowing. A. pleuropneumoniae was detected on the tonsils of all sows. After a nursing period of 3 weeks, the pigs were weaned and reared isolated from other pigs until slaughter. The pigs were examined repeatedly for the presence of A. pleuropneumoniae on the tonsils and for antibodies to A. pleuropneumoniae using bacteriological and serological techniques, respectively.A. pleuropneumoniae was detected in the tonsils of one pig as early as 11 days after birth, showing that A. pleuropneumoniae can be transmitted from sow to offspring during a 3-week nursing period. The cumulative proportion of pigs carrying A. pleuropneumoniae in their tonsils increased significantly between the age of 4-12 weeks. This age period corresponded to the age at which the proportion of pigs with detectable levels of colostral antibodies to the different serotypes of A. pleuropneumoniae was declining. Since these two events take place in the same age period, we expect a possible biological association between the level of the passive immunity and the degree of tonsillar colonisation. The median duration of tonsillar colonisation was estimated to approximately 7-8 weeks.

Risk factors for the hazard of lameness in Danish Standardbred trotters

A follow-up study focusing on health problems interfering with optimal training of Danish Standardbred trotters was conducted with the participation of seven professional trainers. Our aim was to estimate the incidence of health problems that cause interruptions of optimal training, and to identify associations between the hazard of lameness and selected risk factors. The study population was dynamic and contained data of 265 Standardbred trotters monitored during 5 months in 1997 and 1998. The horses were >or=2 years old. Optimal training was defined as when the horse followed scheduled training including fast-speed trotting. Interruption of optimal training could only be caused by health problems and castration.A total of 123 new events of interruption of optimal training caused by health problems were reported. Lameness (injury located to joints and tendons) was the most-frequent cause of interruption of optimal training: 84 events in 69 horses (0.09 events per horse-month). Respiratory diseases (16 events) and muscular problems (seven events) were the second and third most-frequent causes of interrupted training. The effects of trainer, gender, age-group, time with a trainer, participation in races and current month on the hazard of lameness were estimated in a multivariable Cox proportional-hazard model. The effects of trainer, gender and age-group were modelled as time-independent. The effects of time with a trainer, participation in races and the current month were modelled as time-dependent variables. Trainer affected the hazard of lameness. Geldings had higher hazard than mares, as did 3-year olds (compared to >4-year olds). Compared to the period where horses had been trained by the same trainer for >3 months, horses in the period 1.5-2.5 months after they had entered the training regime had higher risk of lameness (hazard ratio: 3.2; 95% CI: 1.1-9.9). Participation in races increased the hazard of lameness significantly in the 5 days after a races.

Seasonal influence on the prevalence of thermotolerant Campylobacter in retail broiler meat in Denmark.

In Denmark, the incidence of human campylobacteriosis cases, as well as the Campylobacter prevalence in broiler flocks, is strongly influenced by season with a summer peak in July-August. Therefore, it was considered that the prevalence of Campylobacter in broiler meat sold at retail in Denmark might also be influenced by season. A retrospective survey analysis was performed on 2001-2007 national surveillance data of the prevalence of thermotolerant Campylobacter in all conventional broiler flocks at slaughter, and in randomly sampled broiler meat at retail. There was a significant effect of season on the occurrence of Campylobacter in meat at retail; the largest effect was found for domestic chilled meat. Thus, the Campylobacter prevalence in Danish broiler flocks, which fluctuated with season, was found to be a strong predictor for the occurrence of Campylobacter in fresh, chilled, Danish broiler meat. However, besides flock prevalence, there was also a direct effect of season on the occurrence of Campylobacter in Danish broiler meat at retail.

Herd-level risk factors for antimicrobial demanding gastrointestinal diseases in Danish herds with finisher pigs A register-based study

Endemic gastrointestinal (GI) diseases have a substantial negative impact on pig production, because, when present, they reduce animal welfare, productivity and generate high antimicrobial (AM) demand. In Danish legislation, AM can be prescribed only for therapeutic purposes. The objective of the study was to estimate the association between herd-level risk factors and the amount of AM use (AMU) in connection with GI diseases in finisher herds. We conducted a register-based cross-sectional study with repeated measurements from 2004 to 2007. Data were extracted from databases in the Danish Register of Veterinary Medicine, the Central Husbandry Register and the Danish Agriculture and Food Council. In total, 3192 pig herds with 26,973 records (quarters with prescriptions) were included. The outcome was presented as average AM use (measured as Animal Daily Dosage) for GI diseases per finishing pig per quarter per herd. Three potential herd-level risk factors were evaluated: herd size (number of finishers delivered for slaughter); herd health status (herds in the Specific Pathogen Free (SPF) System, conventional herds); and herd type (herds including only finishers, integrated herds). Data were analyzed using general linear mixed models with repeated measurements. Smaller herds had a larger AMU per finisher than larger herds. Integrated herds had lower AMU as compared with herds with only finishers. Herds within the SPF System had a larger decrease in AMU with increasing herd size compared to conventional herds. Significant regional differences in AMU were seen. Additionally, the results showed that other herd factors and veterinarians were more influential than the investigated herd risk factors. This illustrates the difficulties of characterising AM-demanding GI diseases in herds by the use of register data only.

A Quantitative Microbiological Risk Assessment for Salmonella in Pigs for the European Union

A farm-to-consumption quantitative microbiological risk assessment (QMRA) for Salmonella in pigs in the European Union has been developed for the European Food Safety Authority. The primary aim of the QMRA was to assess the impact of hypothetical reductions of slaughter-pig prevalence and the impact of control measures on the risk of human Salmonella infection. A key consideration during the QMRA development was the characterization of variability between E.U. Member States (MSs), and therefore a generic MS model was developed that accounts for differences in pig production, slaughterhouse practices, and consumption patterns. To demonstrate the parameterization of the model, four case study MSs were selected that illustrate the variability in production of pork meat and products across MSs. For the case study MSs the average probability of illness was estimated to be between 1 in 100,000 and 1 in 10 million servings given consumption of one of the three product types considered (pork cuts, minced meat, and fermented ready-to-eat sausages). Further analyses of the farm-to-consumption QMRA suggest that the vast majority of human risk derives from infected pigs with a high concentration of Salmonella in their feces (≥10(4) CFU/g). Therefore, it is concluded that interventions should be focused on either decreasing the level of Salmonella in the feces of infected pigs, the introduction of a control step at the abattoir to reduce the transfer of feces to the exterior of the pig, or a control step to reduce the level of Salmonella on the carcass post-evisceration.