Campylobacter presence on Dutch broiler farms and associated risk factors.

Campylobacter is the most reported zoonotic pathogen in humans in the European Union. Poultry is a major source of human infection with Campylobacter. Although many studies are done on the presence of Campylobacter in broilers and theoretically effective control measures are known, their relative importance at broiler farms remains poorly understood. Therefore, the aim of this study was to investigate the presence of Campylobacter on selected broiler farms in the Netherlands, to determine the moment of introduction, and associated risk factors. A longitudinal study on 25 broiler farms was carried out between June 2017 and December 2020. Fecal samples were collected weekly from 43 broiler houses. In total 497 flocks were sampled. Putative variables on flock and farm characteristics for a risk factor analysis were gathered through questionnaires. Risk factors associated with the presence of Campylobacter in a broiler flock were determined using regression models. In total 30% of the flocks included in the study were positive for Campylobacter. Factors associated with presence of Campylobacter at slaughter age included: season, mowing lawns and presence of agricultural side activities. While summer/autumn and mowing lawns were associated with an increase in Campylobacter presence in flocks, the farmer having agricultural side activities other than poultry production was associated with a decrease. Analysis of the age at which flocks first tested Campylobacter positive revealed that slower growing breeds became positive on average 1 wk later compared to regular growers. This study revealed a delayed introduction of Campylobacter in slower grower vs. regular grower broiler flocks reared indoors. In addition, it confirmed importance of season as major risk factor. The relevance of mowing and preceding positive flocks as risk factors needs further investigation.

Environmental Sampling Methods for Detection of Salmonella Infections in Laying Hens: A Systematic Review and Meta-Analysis.

Salmonellosis is the second most commonly reported foodborne gastrointestinal infection in humans in the European Union (EU). Most outbreaks are caused by Salmonella Enteritidis, present in contaminated food products, particularly in egg and egg products. In recent years, an increase in the prevalence of Salmonella in laying hen flocks in the EU has been observed. For the effective control of infection, adequate detection is key. In laying hen flocks, the occurrence of Salmonella in the EU is monitored by the culture of environmental samples (dust, faeces, and boot swabs). The performance of sampling procedures described in the literature for the detection of Salmonella in laying hens was reviewed. In total, 924 abstracts were screened, resulting in the selection of 87 abstracts and 18 publications for qualitative and quantitative analyses, respectively. Sample sizes and sampling locations of faecal material and dust were variable and poorly described. Microbiological culture methods used to detect Salmonella were variably described in the literature and were often incomplete. Overall, the available literature indicates higher sensitivity of environmental versus individual hen matrices and points to differences in sensitivity between environmental matrices. For non-cage housing systems, boot swabs are the preferred samples, while for cage housing systems dust might be a more reliable sample.

Combining a parsimonious mathematical model with infection data from tailor-made experiments to understand environmental transmission.

Although most infections are transmitted through the environment, the processes underlying the environmental stage of transmission are still poorly understood for most systems. Improved understanding of the environmental transmission dynamics is important for effective non-pharmaceutical intervention strategies. To study the mechanisms underlying environmental transmission we formulated a parsimonious modelling framework including hypothesised mechanisms of pathogen dispersion and decay. To calibrate and validate the model, we conducted a series of experiments studying distance-dependent transmission of Campylobacter jejuni in broilers. We obtained informative simultaneous estimates for all three model parameters: the parameter of C. jejuni inactivation, the diffusion coefficient describing pathogen dispersion, and the transmission rate parameter. The time and distance dependence of transmission in the fitted model is quantitatively consistent with marked spatiotemporal patterns in the experimental observations. These results, for C. jejuni in broilers, show that the application of our modelling framework to suitable transmission data can provide mechanistic insight in environmental pathogen transmission.

Monitoring Wind-Borne Particle Matter Entering Poultry Farms via the Air-Inlet: Highly Pathogenic Avian Influenza Virus and Other Pathogens Risk.

Wind-supported transport of particle matter (PM) contaminated with excreta from highly pathogenic avian influenza virus (HPAIv)-infected wild birds may be a HPAIv-introduction pathway, which may explain infections in indoor-housed poultry. The primary objective of our study was therefore to measure the nature and quantity of PM entering poultry houses via air-inlets. The air-inlets of two recently HPAIv-infected poultry farms (a broiler farm and a layer farm) were equipped with mosquito-net collection bags. PM was harvested every 5 days for 25 days. Video-camera monitoring registered wild bird visits. PM was tested for avian influenza viruses (AIV), Campylobacter and Salmonella with PCR. Insects, predominantly mosquitoes, were tested for AIV, West Nile, Usutu and Schmallenberg virus. A considerable number of mosquitoes and small PM amounts entered the air-inlets, mostly cobweb and plant material, but no wild bird feathers. Substantial variation in PM entering between air-inlets existed. In stormy periods, significantly larger PM amounts may enter wind-directed air-inlets. PM samples were AIV and Salmonella negative and insect samples were negative for all viruses and bacteria, but several broiler and layer farm PM samples tested Campylobacter positive. Regular wild (water) bird visits were observed near to the poultry houses. Air-borne PM and insects-potentially contaminated with HPAIv or other pathogens-can enter poultry air-inlets. Implementation of measures limiting this potential introduction route are recommended.

Low seroprevalence of equine piroplasmosis in horses exported from the Netherlands between 2015 and 2021.

Equine piroplasmosis (EP) is a tick-borne disease affecting horses, donkeys, mules and zebras, caused by the intracellular apicomplexan protozoa Babesia caballi and Theileria equi. The geographical distribution of EP is closely related to the distribution of its vector tick species belonging to the genera of Dermacentor, Rhipicephalus and Hyalomma. Since the discovery of Dermacentor reticulatus ticks in 2007 and the first reported autochthonous cases in the South of the Netherlands in 2012, no data on the (sero)prevalence of EP in horses in the Netherlands have been reported and it remains unclear whether B. caballi and T. equi have been able to establish themselves in the Netherlands. This study aims to give an update on the current status of EP in horses in the Netherlands using data from serological tests performed in the context of export and screening of 12,881 horses from 2015 through 2020. Horses were categorized as "Dutch," "Foreign," or "Unknown" based on microchip number. The overall seroprevalence of EP in Dutch horses was found to be 0.5% (95% exact CI [0.4-0.7]), compared to 1.9% (95% exact CI [1.3-2.6]) in horses in the category "Foreign" and 1.7% (95% exact CI [1.2-2.3]) in horses in the category "Unknown." In addition, the seroprevalence per country in the category "Foreign" ranged from 0% (0.95% exact CI [0-2.8]) for Ireland to 6.0% (0.95% exact CI [3.5-9.3]) for Spain. In light of the reports on the seroprevalence during the outbreak of autochthonous EP reported in 2012 and on seroprevalences of EP in other countries in Northwestern Europe, the seroprevalence of EP in horses exported from the Netherlands is very low. However, the higher seroprevalence of EP in horses from abroad warrants the need for the monitoring of EP, as tick vectors are present in the Netherlands and the import of horses from endemic areas increases the chances of EP becoming more prevalent in the Netherlands.

Tularemia Transmission to Humans, the Netherlands, 2011-2021.

We used national registry data on human cases of Francisella tularensis subspecies holarctica infection to assess transmission modes among all 26 autochthonous cases in the Netherlands since 2011. The results indicate predominance of terrestrial over aquatic animal transmission sources. We recommend targeting disease-risk communication toward hunters, recreationists, and outdoor professionals.

BRUCELLA PINNIPEDIALIS IN GREY SEALS ( HALICHOERUS GRYPUS) AND HARBOR SEALS ( PHOCA VITULINA) IN THE NETHERLANDS.

Brucellosis is a zoonotic disease with terrestrial or marine wildlife animals as potential reservoirs for the disease in livestock and human populations. The primary aim of this study was to assess the presence of Brucella pinnipedialis in marine mammals living along the Dutch coast and to observe a possible correlation between the presence of B. pinnipedialis and accompanying pathology found in infected animals. The overall prevalence of Brucella spp. antibodies in sera from healthy wild grey seals ( Halichoerus grypus; n=11) and harbor seals ( Phoca vitulina; n=40), collected between 2007 and 2013 ranged from 25% to 43%. Additionally, tissue samples of harbor seals collected along the Dutch shores between 2009 and 2012, were tested for the presence of Brucella spp. In total, 77% (30/39) seals were found to be positive for Brucella by IS 711 real-time PCR in one or more tissue samples, including pulmonary nematodes. Viable Brucella was cultured from 40% (12/30) real-time PCR-positive seals, and was isolated from liver, lung, pulmonary lymph node, pulmonary nematode, or spleen, but not from any PCR-negative seals. Tissue samples from lung and pulmonary lymph nodes were the main source of viable Brucella bacteria. All isolates were typed as B. pinnipedialis by multiple-locus variable number of tandem repeats analysis-16 clustering and matrix-assisted laser desorption ionization-time of flight mass spectrometry, and of sequence type ST25 by multilocus sequence typing analysis. No correlation was observed between Brucella infection and pathology. This report displays the isolation and identification of B. pinnipedialis in marine mammals in the Dutch part of the Atlantic Ocean.

Evaluation of a multiplex real-time PCR for detection of four bacterial agents commonly associated with bovine respiratory disease in bronchoalveolar lavage fluid.

BACKGROUND: Pasteurella multocida, Mannheimia haemolytica, Histophilus somni and Trueperella pyogenes are four bacterial agents commonly associated with bovine respiratory disease (BRD). In this study a bacterial multiplex real-time PCR (the RespoCheck PCR) was evaluated for the detection in bronchoalveolar lavage fluid (BALF) of these four bacterial agents. RESULTS: The analytical sensitivity of the multiplex real-time PCR assay determined on purified DNA and on bacterial cells of the four target pathogens was one to ten fg DNA/assay and 4 × 10-1 to 2 × 100 CFU/assay. The analytical specificity of the test was, as evaluated on a collection of 118 bacterial isolates, 98.3% for M. haemolytica and 100% for the other three target bacteria. A set of 160 BALF samples of calves originating from ten different herds with health problems related to BRD was examined with bacteriological methods and with the RespoCheck PCR. Using bacteriological examination as the gold standard, the diagnostic sensitivities and specificities of the four bacterial agents were respectively between 0.72 and 1.00 and between 0.70 and 0.99. Kappa values for agreement between results of bacteriological examination and PCRs were low for H. somni (0.17), moderate for P. multocida (0.52) and M. haemolytica (0.57), and good for T. pyogenes (0.79). The low and moderate kappa values seemed to be related to limitations of the bacteriological examination, this was especially the case for H. somni. CONCLUSION: It was concluded that the RespoCheck PCR assay is a valuable diagnostic tool for the simultaneous detection of the four bacterial agents in BALF of calves.

Mycoplasma detection by triplex real-time PCR in bronchoalveolar lavage fluid from bovine respiratory disease complex cases.

BACKGROUND: In this study we evaluated the RespoCheck Mycoplasma triplex real-time PCR for the detection in bronchoalveolar lavage fluid (BALF) of Mycoplasma (M.) dispar, M. bovis and M. bovirhinis, all three associated with bovine respiratory disease (BRD). Primers and probes of the RespoCheck Mycoplasma triplex real-time PCR are based on the V3/V4 region of the 16S rRNA gene of the three Mycoplasma species. RESULTS: The analytical sensitivity of the RespoCheck triplex real-time PCR was, as determined by spiking experiments of the Mycoplasma strains in Phosphate Buffered Saline, 300 colony forming units (cfu)/mL for M. dispar, and 30 cfu/mL for M. bovis or M. bovirhinis. The analytical sensitivity of the RespoCheck Mycoplasma triplex real-time PCRwas, as determined on purified DNA, 10 fg DNA per assay for M. dispar and 100 fg fo rM. bovis and M. bovirhinis. The analytical specificity of the RespoCheck Mycoplasma triplex real-time PCR was, as determined by testing Mycoplasmas strains (n = 17) and other bacterial strains (n = 107), 100, 98.2 and 99.1% for M. bovis, M. dispar and M. bovirhinis respectively. The RespoCheck Mycoplasma triplex real-time PCR was compared with the PCR/DGGE analysis for M. bovis, M. dispar and M. bovirhinis respectively by testing 44 BALF samples from calves. CONCLUSION: In conclusion, the RespoCheck PCR assay can be a valuable tool for timely and accurate detection of three Mycoplasma species associated with in bovine respiratory disease.

Invasive behavior of Campylobacter jejuni in immunosuppressed chicken.

Campylobacter jejuni is a predominant cause of gastroenteritis in humans but rather harmless in chickens. The basis of this difference is unknown. We investigated the effect of the chicken immune defense on the behavior of C. jejuni using glucocorticoid (GC)-treated and mock-treated 17-day old Ross 308 chicken bearing in mind that GCs have immunosuppressive effects and dampen the innate immune response. The effect of GC administration on the behavior of C. jejuni was compared with that on infection with Salmonella Enteritidis to address possible microbe-associated differences. Our results revealed that GC treatment fastened the intestinal colonization of C. jejuni (p < 0.001) and enhanced its dissemination to the liver (p = 0.007). The effect of GC on intestinal colonization of S. Enteritidis was less pronounced (p = 0.033) but GC did speed up the spread of this pathogen to the liver (p < 0.001). Cytokine transcript analysis showed an up to 30-fold reduction in baseline levels of IL-8 mRNA in the cecal (but not spleen) tissue at Day 1 after GC treatment (p < 0.005). Challenge with C. jejuni strongly increased intestinal IL-8, IL-6, and iNOS transcript levels in the non-GC treated animals but not in the GC-treated birds (P < 0.005). In vitro assays with chicken macrophages showed that GC dampened the TLR agonist- and C. jejuni induced-inflammatory gene transcription and production of nitric oxide (P < 0.005). Together, the results support the hypothesis that C. jejuni has the intrinsic ability to invade chicken tissue and that an effective innate immune response may limit its invasive behavior.

Serum protein profiles as potential biomarkers for infectious disease status in pigs.

BACKGROUND: In veterinary medicine and animal husbandry, there is a need for tools allowing the early warning of diseases. Preferably, tests should be available that warn farmers and veterinarians during the incubation periods of disease and before the onset of clinical signs. The objective of this study was to explore the potential of serum protein profiles as an early biomarker for infectious disease status. Serum samples were obtained from an experimental pig model for porcine circovirus-associated disease (PCVAD), consisting of Porcine Circovirus type 2 (PCV2) infection in combination with either Porcine Parvovirus (PPV) or Porcine Reproductive and Respiratory Syndrome virus (PRRSV). Sera were collected before and after onset of clinical signs at day 0, 5 and 19 post infection. Serum protein profiles were evaluated against sera from non-infected control animals. RESULTS: Protein profiles were generated by SELDI-TOF mass spectrometry in combination with the Proteominer™ technology to enrich for low-abundance proteins. Based on these protein profiles, the experimentally infected pigs could be classified according to their infectious disease status. Before the onset of clinical signs 88% of the infected animals could be classified correctly, after the onset of clinical sigs 93%. The sensitivity of the classification appeared to be high. The protein profiles could distinguish between separate infection models, although specificity was moderate to low. Classification of PCV2/PRRSV infected animals was superior compared to PCV2/PPV infected animals. Limiting the number of proteins in the profiles (ranging from 568 to 10) had only minor effects on the classification performance. CONCLUSIONS: This study shows that serum protein profiles have potential for detection and identification of viral infections in pigs before clinical signs of the disease become visible.

Escherichia coli concentrations in feces of geese, coots, and gulls residing on recreational water in The Netherlands.

Contamination of recreational water by bird feces is a main concern of water managers. It is important to understand the sources of Escherichia coli contamination since the organism is frequently used as a water hygiene parameter. Here, we address presence and levels of E. coli in fecal shedding from several waterfowl (25 geese, 20 coots, and 40 gulls) and demonstrate that there is a bird species variation. Results indicate that gull feces contain a greater average concentration of E. coli per gram than do geese or coot feces. However, contamination risks also depends on bird abundance. These are important aspects for effective water bird management.

Strain variation within Campylobacter species in fecal samples from dogs and cats.

To investigate the incidence of co-colonization of different strains of Campylobacter species present in canine and feline stool samples, isolates were recovered by culture from 40 samples from dogs (n=34) and cats (n=6). Animals were of different ages, with diarrhoea or without clinical signs. Three isolation procedures were used: two selective agars and a filtration method. In each stool sample, multiple colonies were identified to the species level by PCR, subsequently genotyped by Amplified Fragment Length Polymorphism (AFLP) and pattern similarities (451 isolates) were calculated to investigate their phylogenetic relationships. Genetic heterogeneity of strains in individual stool samples was detected within the species Campylobacter jejuni, C. upsaliensis and C. helveticus, though to a different degree in dogs and cats. In 3 of the 34 (9%) canine samples, more than one genotype of the same Campylobacter species was present, while strain variation was detected in four of the six feline samples. The results show that preferably, multiple colonies should be analyzed in molecular epidemiological and aetiological studies.