Analysis of reservoir sources of Campylobacter isolates to free-range broilers in Denmark.

Campylobacter is a common cause of food poisoning in many countries, with broilers being the main source. Organic and free-range broilers are more frequently Campylobacter-positive than conventionally raised broilers and may constitute a higher risk for human infections. Organic and free-range broilers may get exposed to Campylobacter from environmental reservoirs and livestock farms, but the relative importance of these sources is unknown. The aim of the study was to describe similarities and differences between the genetic diversity of the Campylobacter isolates collected from free-range/organic broilers with those isolated from conventional broilers and other animal hosts (cattle, pigs, and dogs) in Denmark to make inferences about the reservoir sources of Campylobacter to free-range broilers. The applied aggregated surveillance data consisted of sequenced Campylobacter isolates sampled in 2015 to 2017 and 2018 to 2021. The data included 1,102 isolates from free-range (n = 209), conventional broilers (n = 577), cattle (n = 261), pigs (n = 30), and dogs (n = 25). The isolates were cultivated from either fecal material (n = 434), food matrices (n = 569), or of nondisclosed origin (n = 99). Campylobacter jejuni (94.5%) dominated and subtyping analysis found 170 different sequence types (STs) grouped into 75 clonal complexes (CCs). The results suggest that CC-21 and CC-45 are the most frequent CCs found in broilers. The relationship between the CCs in the investigated sources showed that the different CCs were shared by most of the animals, but not pigs. The ST-profiles of free-range broilers were most similar to that of conventional broilers, dogs and cattle, in that order. The similarity was stronger between conventional broilers and cattle than between conventional and free-range broilers. The results suggest that cattle may be a plausible reservoir of C. jejuni for conventional and free-range broilers, and that conventional broilers are a possible source for free-range broilers or reflect a dominance of isolates adapted to the same host environment. Aggregated data provided valuable insight into the epidemiology of Campylobacter sources for free-range broilers, but time-limited sampling of isolates from different sources within a targeted area would hold a higher predictive value.

Association between milk yield and milk anti-Fasciola hepatica antibody levels, and the utility of bulk tank milk samples for assessing within-herd prevalence on organic dairy farms.

Fasciola hepatica is an important disease of livestock that is responsible for substantial economic losses worldwide. Estimates of the impact of infection on milk yield vary, likely reflecting different geographical locations, farm-level management, and diagnostic methods. Measuring anti-Fasciola antibodies on bulk tank milk (BTM) by ELISA provides a convenient herd-level diagnosis, but the utility of this test remains unclear. Therefore, we evaluated the utility of BTM ELISA test results in Danish organic dairy farms, including estimating the association between 305 day energy corrected milk yield (305d ECM) and F. hepatica infection both at individual and herd level. BTM samples from 218 organic farms were analysed using IDEXX ELISA and subsequently the farmers were interviewed during spring 2016 with the aim of characterising their management practices. The corresponding farm-level production data covering the period 2014-2017 were collected from the Danish national cattle registry. In the following year, 284 individual milk samples (4-7 per herd) along with BTM samples were collected from a subset of the same herds (n = 55). Linear mixed models were used to estimate the association between milk production and ELISA value at both individual and farm levels, and a generalised additive model was used to assess the relationship between within-herd prevalence and BTM ELISA. A dichotomised BTM result with positive outcome was associated with a reduction of 580.5 kg in average 305d ECM, and a positive outcome on individual-level ELISA was associated with a 919.5 kg reduction in milk yield for cows in their third or later lactations. A strong relationship between quantitative BTM ELISA sample to positive percentage (S/P%) and apparent within-herd prevalence based on dichotomised individual-level milk ELISA was also observed, although this relationship was non-linear in nature. We conclude that a useful indication of the within-herd prevalence of infection can be obtained from BTM ELISA following categorisation as negative, low, medium or high according to S/P% cut-offs of approximately 30, 80, and 150. This approach represents a cheap and useful diagnostic tool for monitoring the long-term success of control strategies for F. hepatica infections on a dairy farm.

Surveillance of important bacterial and parasitic infections in Danish wild boars (Sus scrofa).

BACKGROUND: Similar to the situation in other European countries, Danish wild boars may harbour a wide range of pathogens infectious to humans and domestic pigs. Although wild boars must be kept behind fences in Denmark, hunting and consumption of the meat may cause zoonotic transmission. Moreover, most infections of wild boars are transmissible to domestic pigs, which may have important economic consequences. The aim of this study was to investigate whether Danish wild boars were infected with bacteria and parasites transmissible to humans or domestic pigs: Brucella suis, methicillin-resistant Staphylococcus aureus (MRSA), Salmonella spp., Trichinella spp., lungworms and gastrointestinal parasites, especially Ascaris suum. This is the first study to investigate the prevalence of these important pathogens in Danish wild boars. RESULTS: Wild boars from eight enclosures were analysed over a 5-year period. All tested wild boars were negative for B. suis (n = 240), MRSA (n = 244), Salmonella spp. (n = 115) and Trichinella spp. (n = 232), while eight parasite genera were identified in the faeces (n = 254): Ascaris suum, Capillaria sp., Cystoisospora suis, Eimeria spp., Metastrongylus sp. (lungworm), Strongyloides ransomi, Trichuris suis and strongylid eggs, i.e. strongyles not identified to the genera. Eimeria spp. and Metastrongylus sp. had the highest prevalence (92.3 and 79.5%, respectively) and were identified in wild boars from all eight enclosures, while the remaining parasite genera were present more sporadically. CONCLUSIONS: Wild boars from Denmark constitute a low risk of transmitting B. suis, MRSA, Salmonella spp. and Trichinella spp. to humans or domestic pigs, while economically important parasites transmissible to domestic pigs are highly prevalent in the wild boar population.

Phylogenetic relationships among Toxocara spp. and Toxascaris sp. from different regions of the world.

Toxocara and Toxascaris are parasitic nematodes that infect canids and felids although species of the genus Toxocara also infect humans. This work aimed to establish the phylogenetic and phylogeographic relationship between specimens of T. canis, T. cati, T. malaysiensis and Toxascaris leonina and to evaluate the degree of host specificity. In total, 437 samples (adults and pools of eggs) were collected from canids and felids from eight countries. Parasites were identified by morphology, PCR linked Restriction Fragment Length Polymorphism (PCR-RFLP) and partial sequencing of the mitochondrial gene cox1. Phylogenetic trees were constructed and genetic distance among isolates was estimated. Based on the molecular characterization all worms were identified in agreement with their respective hosts with the exception of three samples; two from cats and one from dogs identified as T. canis and T. cati, respectively. There was no clear geographical clustering of the samples despite this study including parasites from three continents. This is the first study, to our knowledge, to use molecular methods to identify T. canis in cats and T. cati in dogs with host specificity being the most common finding. Our developed PCR-RFLP method was found to be a facile and reliable method for identifying Toxocara species.

Giardia and Cryptosporidium infections in Danish cats: risk factors and zoonotic potential.

Giardia and Cryptosporidium infections are common in cats, but knowledge is limited about their clinical importance, risk factors, and the role of cats as a reservoir for human infections. Here, we collected faeces and questionnaire data from 284 cats from shelters and veterinary clinics in the Copenhagen Metropolitan Region (= study population). Additionally, 33 samples were analysed separately from catteries with gastrointestinal clinical signs (= cases). (Oo-)cysts were quantified by immunofluorescence microscopy. All Giardia (n = 34) and Cryptosporidium (n = 29) positive samples were analysed by sequencing of the 18S rRNA, gdh and hsp70 loci, and co-infections were detected by McMaster/inverted microscopy. In the study population, 7.0% and 6.7% were positive for Giardia and Cryptosporidium respectively; 48.5% and 36.4% of the breeder cats (cases) were infected. Increased odds of diarrhoea were demonstrated in Giardia (p = 0.0008) and Cryptosporidium (p = 0.034) positive cats. For Giardia, the odds were positively correlated with infection intensity. Co-infection with Cryptosporidium (OR 12.79; p < 0.001), parasitic co-infections other than Cryptosporidium (OR 5.22; p = 0.009), no deworming (OR 4.67; p = 0.035), and male sex (OR 3.63; p = 0.025) were risk factors for Giardia. For Cryptosporidium, co-infection with Giardia was the only risk factor (OR 11.93; p < 0.0001). Genotyping revealed G. duodenalis assemblages A and F, and C. felis, all of them previously detected in humans. In conclusion, excretion of Giardia and Cryptosporidium was associated with clinical disease. Although a public health risk is likely, studies including larger sample sizes, more discriminatory markers and samples from other animals and humans are needed to reveal the full zoonotic potential.

Parasite control in organic cattle farming: Management and farmers' perspectives from six European countries.

Organic ruminant production is expanding in the EU, but parasite management remains a constant challenge. Mandatory outdoor access for all age groups can increase exposure to pasture borne parasites, whilst restrictions in the prophylactic use of anthelmintics can limit parasite control. The scientific community has been working to deliver effective parasite control strategies and alternative approaches in order to slow down the development of anthelmintic resistance (AR). However, the current parasite control practices and overall awareness with regards to AR and alternative approaches on farms are largely unknown and may be causing a knowledge gap between the scientific and farming communities. Therefore, a structured survey was conducted in six European countries (Switzerland, Germany, Denmark, Netherlands, Lithuania, Sweden) to provide basic data on practices, management and farmers' perspectives for grazing and parasite control (gastrointestinal worms and liver flukes) on organic cattle farms. Overall, 375 surveys were collected (282 dairy and 93 beef farms) in 2015-2016, and analysed descriptively. Additionally, surveys from the 228 dairy farms were assessed using a double-hurdle adoption model to identify the factors involved in the decision to drench against gastrointestinal parasites. Generally, there are prominent differences between countries, with monitoring methods differing especially, which has important implications in terms of knowledge transfer. For example, media warning was the most common method in DE, while antibody testing in bulk tank milk was the common method in NL. In other countries, clinical signs (diarrhoea, hair coat quality, and reduced weight or yield) and liver condemnation data were used frequently. In general, organic farmers from the six participating countries indicated that they would accept alternative approaches despite greater cost and labour. The likelihood of drenching were higher on farms with smaller farm areas, higher number of young stock and total livestock units and farms where faecal egg counts were used to monitor the parasites. In conclusion, it was evident that grazing and parasite management varied between the countries even though they operate under the same basic principles. Parasite management strategies must therefore be country specific and disseminated with appropriate methods.

Patterns of Fasciola hepatica infection in Danish dairy cattle: implications for on-farm control of the parasite based on different diagnostic methods.

BACKGROUND: Bovine fasciolosis is an economically important livestock disease in Europe, and represents a particular challenge for organic farms, where cattle are grazed extensively and the use of anthelmintic is limited. A two-year longitudinal study was conducted on two conventional and two organic Danish dairy farms to examine the current temporal trend of F. hepatica infection on-farm, and to gather data of practical relevance for parasite control. Data were collected both at the herd and individual level using currently available diagnostic methods: a commercial serum antibody ELISA, a commercial copro-antigen ELISA, faecal egg counts, and monthly bulk tank milk (BTM) ELISA. The temporal patterns (animal age, farm-level temporal trends and seasonality) in the animal-level test results were analysed by generalised additive mixed models (GAMM). RESULTS: Patterns of infection differed substantially between the farms, due to different grazing management and anthelmintic use. However, animals were first infected at the age of 1.5-2 years (heifers), and most at-risk animals sero-converted in autumn, suggesting that summer infections in snails prevail in Denmark. Our results also suggest that the lifespan of the parasite could be over 2 years, as several cows showed signs of low grade infection even after several years of continuous indoor housing without access to freshly-cut grass. The serum antibody ELISA was able to detect infection first, whereas both copro-antigen ELISA and faecal egg counts tended to increase in the same animals at a later point. Decreasing BTM antibody levels were seen on the two farms that started anthelmintic treatment during the study. CONCLUSIONS: While important differences between farms and over time were seen due to varying grazing management, anthelmintic treatment and climatic conditions, the young stock was consistently seen as the main high-risk group and at least one farm also had suspected transmission (re-infection) within the lactating herd. Careful interpretation of test results is necessary for older cows as they can show persistent infections several years after exposure has stopped. Rigorous treatment regimens can reduce BTM ELISA values, but further research is needed to develop a non-medicinal approach for sustainable management of bovine fasciolosis.

Farm-level risk factors for Fasciola hepatica infection in Danish dairy cattle as evaluated by two diagnostic methods.

BACKGROUND: The prevalence of bovine fasciolosis in Denmark is increasing but appropriate guidelines for control are currently lacking. In order to help develop a control strategy for liver fluke, a risk factor study of farm management factors was conducted and the utility of bulk tank milk (BTM ELISA) as a tool for diagnosis in Danish dairy cattle farms was assessed. METHODS: This case-control study aimed to identify farm-level risk factors for fasciolosis in Danish dairy farms (> 50 animals slaughtered in 2013) using two diagnostic methods: recordings of liver condemnation at slaughter, and farm-level Fasciola hepatica antibody levels in BTM. A case farm was defined as having a minimum of 3 incidents of liver condemnation due to liver fluke at slaughter (in any age group) during 2013, and control farms were located within 10 km of at least one case farm and had no history of liver condemnation due to liver fluke during 2011-2013. The selected farmers were interviewed over telephone about grazing and control practices, and BTM from these farms was collected and analysed by ELISA in 2014. The final complete dataset consisting of 131 case and 63 control farms was analysed using logistic regression. RESULTS: Heifers grazing on wet pastures, dry cows grazing on wet pastures, herd size, breed and concurrent beef cattle production were identified as risk factors associated with being classified as a case farm. With the categorised BTM ELISA result as the response variable, heifers grazing on wet pastures, dry cows grazing on wet pastures, and purchase of cows were identified as risk factors. Within the case and control groups, 74.8 and 12.7% of farms were positive for fasciolosis on BTM ELISA, respectively. The differences are likely to be related to the detection limit of the farm-level prevalence by the BTM ELISA test, time span between slaughter data and BTM, and the relatively low sensitivity of liver inspection at slaughter. CONCLUSIONS: Control of bovine fasciolosis in Denmark should target heifers and dry cows through grazing management and appropriate anthelmintic treatment, and BTM ELISA can be a useful diagnostic tool for fasciolosis in Danish dairy farms.

Predictors of Echinococcus multilocularis Prevalence in Definitive and Intermediate Hosts: A Meta-Analysis Approach.

Echinococcus multilocularis (EM) is a pathogenic and potentially fatal cestode causing human alveolar echinococcosis (AE). A meta-analysis was conducted using a generalized estimation equation approach (GEE) to assess the effect of taxonomic, environmental, and diagnostic variables on EM prevalence in different hosts. Red foxes ( Vulpes vulpes ) had significantly higher prevalence of EM than domestic dogs ( Canis lupus familiaris), with the diagnostic method playing an important factor in assessing prevalence. For intermediate hosts genera was significantly associated with EM prevalence, although there was some indication of publication bias in this dataset. This study also highlights the possible importance of temperature and precipitation to EM transmission. This implies the possibility of a changing climate affecting the future distribution of the parasite.