Transmission of Brucella canis in a canine kennel following introduction of an infected dog.

Brucella canis is a zoonotic pathogen and the main causative agent of canine brucellosis. In the Netherlands, B. canis had previously only been detected in individual cases of imported dogs. However, an outbreak of B. canis occurred for the first time in a cohort of autochthonous dogs in a breeding kennel in 2019. The outbreak began with a positive serological test result of an imported intact male dog showing clinical symptoms of brucellosis. Consequently, urine and blood samples were collected and tested positive for B. canis by culture, matrix-assisted laser desorption/ionization - time of flight mass spectrometry (MALDI-TOF MS) and whole-genome-sequencing (WGS). Screening of the contact dogs in the kennel where the index case was kept, revealed that antibodies against B. canis could be detected in 23 out of 69 dogs (34 %) by serum agglutination test (SAT). Of the 23 seropositive dogs, B. canis could be cultured from the urine and/or heparin samples of 19 dogs (83 %). This outbreak represents the first documented case of transmission of B. canis to autochthonous contact dogs in the Netherlands. WGS revealed all B. canis isolates belonged to the same cluster, which means the transmission of B. canis in the breeding kennel was most likely caused by the introduction of one infected dog. Comparing this cluster with data from other B. canis isolates, it also appears that characteristic clusters of B. canis are present in several endemic countries. These clusters seem to remain stable over time and may help in locating the origin of new isolates found. This outbreak showed that the international movement of dogs from endemic countries poses a threat to the canine population, while serological screening and WGS proved to be valuable tools for respectively screening and the epidemiological investigation.

Genome-wide epitope mapping across multiple host species reveals significant diversity in antibody responses to Coxiella burnetii vaccination and infection.

Coxiella burnetii is an important zoonotic bacterial pathogen of global importance, causing the disease Q fever in a wide range of animal hosts. Ruminant livestock, in particular sheep and goats, are considered the main reservoir of human infection. Vaccination is a key control measure, and two commercial vaccines based on formalin-inactivated C. burnetii bacterins are currently available for use in livestock and humans. However, their deployment is limited due to significant reactogenicity in individuals previously sensitized to C. burnetii antigens. Furthermore, these vaccines interfere with available serodiagnostic tests which are also based on C. burnetii bacterin antigens. Defined subunit antigen vaccines offer significant advantages, as they can be engineered to reduce reactogenicity and co-designed with serodiagnostic tests to allow discrimination between vaccinated and infected individuals. This study aimed to investigate the diversity of antibody responses to C. burnetii vaccination and/or infection in cattle, goats, humans, and sheep through genome-wide linear epitope mapping to identify candidate vaccine and diagnostic antigens within the predicted bacterial proteome. Using high-density peptide microarrays, we analyzed the seroreactivity in 156 serum samples from vaccinated and infected individuals to peptides derived from 2,092 open-reading frames in the C. burnetii genome. We found significant diversity in the antibody responses within and between species and across different types of C. burnetii exposure. Through the implementation of three different vaccine candidate selection methods, we identified 493 candidate protein antigens for protein subunit vaccine design or serodiagnostic evaluation, of which 65 have been previously described. This is the first study to investigate multi-species seroreactivity against the entire C. burnetii proteome presented as overlapping linear peptides and provides the basis for the selection of antigen targets for next-generation Q fever vaccines and diagnostic tests.

Brucella suis biovar 1 infection in a dog with orchitis in Germany.

In 2021, a case of canine brucellosis diagnosed in a dog with orchitis was presented to a veterinary practice in Germany. Serological testing excluded Brucella (B.) canis as a causative agent, but molecular analysis revealed the presence of B. suis biovar 1. Since biovar 1 is not endemic in Europe and the dog had no history of travel to endemic areas, a comprehensive epidemiological investigation was conducted using whole genome sequence data to determine the source of infection. We describe the clinical progress of the animal and the potential infection of a veterinary clinic employee. The findings highlight the importance of considering less common Brucella species as possible causes of canine brucellosis. The data also emphasize that it is quite challenging to identify Brucella species in a routine diagnostic laboratory and to conduct epidemiological investigations to unveil possible transmission routes.

The emergence of Brucella canis as a public health threat in Europe: what we know and what we need to learn.

The zoonotic bacteria, Brucella canis, is becoming the leading cause of canine brucellosis in Europe. In dogs, it causes reproductive problems as well as non-specific lameness or discospondilitis. In humans, B. canis can be origin of chronic debilitating conditions characteristic to its genus such as undulant fever, splenomegaly, and lymphadenopathy. Although B. canis shows some pathogenic characteristics similar to B. abortus and B. melitensis, it lacks surface O-polysaccharide, like nonzoonotic B. ovis. This review shows that host-B. canis interactions are still poorly understood, with many knowledge and capability gaps, causing relatively poor sensitivity and specificity of existing diagnostic tools. Currently, there is no vaccine for this rough Brucella species. Besides, antimicrobial therapy does not guarantee bacterial elimination, and infection relapses are frequently reported, increasing the risks of antibiotic resistance development. B. canis has been detected in dogs in almost all European countries which increased human exposure, but currently there is no systematic surveillance. Moreover, B. canis caused brucellosis is not included in Animal Health Law, and therefore there is no legal framework to tackle this emerging infectious disease. To map out the diagnostic strategies, identify risks for human infections and propose management scheme for infected pet and kennel dogs, we present current understanding of canine B. canis caused brucellosis, outline major knowledge gaps and propose future steps. To address and highlight challenges veterinary and public health services encounter in Europe, we developed two B. canis infection scenarios: of a single household pet and of a kennel dog in larger group.

Efficacy of Phase I and Phase II Coxiella burnetii Bacterin Vaccines in a Pregnant Ewe Challenge Model.

The bacterium Coxiella burnetii can cause the disease Q-fever in a wide range of animal hosts. Ruminants, including sheep, are thought to play a pivotal role in the transmission of C. burnetii to humans; however, the only existing livestock vaccine, namely, Coxevac® (Ceva Animal Health Ltd., Libourne, France), a killed bacterin vaccine based on phase I C. burnetii strain Nine-Mile, is only approved for use in goats and cattle. In this study, a pregnant ewe challenge model was used to determine the protective effects of Coxevac® and an experimental bacterin vaccine based on phase II C. burnetii against C. burnetii challenge. Prior to mating, ewes (n = 20 per group) were vaccinated subcutaneously with either Coxevac®, the phase II vaccine, or were unvaccinated. A subset of pregnant ewes (n = 6) from each group was then challenged 151 days later (~100 days of gestation) with 106 infectious mouse doses of C. burnetii, Nine-Mile strain RSA493. Both vaccines provided protection against C. burnetii challenge as measured by reductions in bacterial shedding in faeces, milk and vaginal mucus, and reduced abnormal pregnancies, compared to unvaccinated controls. This work highlights that the phase I vaccine Coxevac® can protect ewes against C. burnetii infection. Furthermore, the phase II vaccine provided comparable levels of protection and may offer a safer and cost-effective alternative to the currently licensed vaccine.

Screen the unforeseen: Microbiome-profiling for detection of zoonotic pathogens in wild rats.

Wild rats can host various zoonotic pathogens. Detection of these pathogens is commonly performed using molecular techniques targeting one or a few specific pathogens. However, this specific way of surveillance could lead to (emerging) zoonotic pathogens staying unnoticed. This problem may be overcome by using broader microbiome-profiling techniques, which enable broad screening of a sample's bacterial or viral composition. In this study, we investigated if 16S rRNA gene amplicon sequencing would be a suitable tool for the detection of zoonotic bacteria in wild rats. Moreover, we used virome-enriched (VirCapSeq) sequencing to detect zoonotic viruses. DNA from kidney samples of 147 wild brown rats (Rattus norvegicus) and 42 black rats (Rattus rattus) was used for 16S rRNA gene amplicon sequencing of the V3-V4 hypervariable region. Blocking primers were developed to reduce the amplification of rat host DNA. The kidney bacterial composition was studied using alpha- and beta-diversity metrics and statistically assessed using PERMANOVA and SIMPER analyses. From the sequencing data, 14 potentially zoonotic bacterial genera were identified from which the presence of zoonotic Leptospira spp. and Bartonella tribocorum was confirmed by (q)PCR or Sanger sequencing. In addition, more than 65% of all samples were dominated (>50% reads) by one of three bacterial taxa: Streptococcus (n = 59), Mycoplasma (n = 39) and Leptospira (n = 25). These taxa also showed the highest contribution to the observed differences in beta diversity. VirCapSeq sequencing in rat liver samples detected the potentially zoonotic rat hepatitis E virus in three rats. Although 16S rRNA gene amplicon sequencing was limited in its capacity for species level identifications and can be more difficult to interpret due to the influence of contaminating sequences in these low microbial biomass samples, we believe it has potential to be a suitable pre-screening method in the future to get a better overview of potentially zoonotic bacteria that are circulating in wildlife.

Metabolomic changes in polyunsaturated fatty acids and eicosanoids as diagnostic biomarkers in Mycobacterium avium ssp. paratuberculosis (MAP)-inoculated Holstein-Friesian heifers.

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative organism of Johne's disease, a chronic granulomatous enteritis of ruminants. We have previously used naturally MAP-infected heifer calves to document metabolomic changes occurring in MAP infections. Herein, we used experimentally MAP-inoculated heifer calves to identify biomarkers for MAP infections. At 2-weeks of age, 20 Holstein-Friesian (HF) calves were experimentally inoculated with MAP. These calves, along with 20 control calves, were sampled biweekly up to 13-months of age and then monthly up to 19-months of age. Sera were assessed using flow infusion electrospray high-resolution mass spectrometry (FIE-HRMS) on a Q Exactive hybrid quadrupole-Orbitrap mass spectrometer for high throughput, sensitive, non-targeted metabolite fingerprinting. Partial least squares-discriminate analysis (PLS-DA) and hierarchical cluster analysis (HCA) discriminated between MAP-inoculated and control heifer calves. Out of 34 identified metabolites, six fatty acyls were able to differentiate between experimental groups throughout the study, including 8, 11, 14-eicosatrienoic acid and cis-8, 11, 14, 17-eicosatetraenoic acid which were also detected in our previous study and so further suggested their value as biomarkers for MAP infection. Pathway analysis highlighted the role of the alpha-linoleic acid and linoleic acid metabolism. Within these pathways, two broad types of response, with a rapid increase in some saturated fatty acids and some n-3 polyunsaturated fatty acids (PUFAs) and later n-6 PUFAs, became predominant. This could indicate an initial anti-inflammatory colonisation phase, followed by an inflammatory phase. This study demonstrates the validity of the metabolomic approach in studying MAP infections. Nevertheless, further work is required to define further key events, particularly at a cell-specific level.

First Case of Human Brucella canis Infection in the Netherlands.

A patient was diagnosed with Brucella canis following exposure to infected dogs in her breeding facility. Transboundary spread of B. canis through (illegal) import of infected dogs to non-endemic countries in Europe suggest that B. canis infection should be considered in European patients with occupational exposure to dogs.

Defining Fatty Acid Changes Linked to Rumen Development, Weaning and Growth in Holstein-Friesian Heifers.

After birth, as effectively monogastric animals, calves undergo substantial physiological changes to become ruminants by 3 months of age and reach sexual maturity at approximately 15 months of age. Herein, we assess longitudinal metabolomic changes in Holstein-Friesian (HF) heifers from birth until sexual maturity during this developmental process. Sera from 20 healthy, HF heifers were sampled biweekly from 2 weeks of age until 13 months of age and then monthly until 19 months of age. Sera were assessed using flow infusion electrospray high-resolution mass spectrometry (FIE-HRMS) on a Q Exactive hybrid quadrupole-Orbitrap mass spectrometer for high-throughput, sensitive, non-targeted metabolite fingerprinting. Partial least squares discriminant analysis (PLS-DA) and unsupervised hierarchical clustering analysis (HCA) of the derived metabolomes indicated changes detectable in heifers' sera over time. Time series analyses identified 30 metabolites that could be related to rumen development and weaning at ~3 months of age. Further time series analysis identified 40 metabolites that could be correlated with growth. These findings highlight the role of acetic acid and 3-phenylpropionate (3-PP) in rumen development and growth, suggest that weaning induces elevated levels of fatty acyls in response to a post-weaning stress-induced innate immune response and demonstrate the utilization of fatty acyls in growth. The identified metabolites offer serum metabolites which could inform the nutrition and healthy development of heifers.

Effect of Type of Cow-Calf Contact on Health, Blood Parameters, and Performance of Dairy Cows and Calves.

Prolonged cow-calf contact (CCC) could potentially improve dairy calf welfare. However, it is currently unknown how different types of CCC affect animals' biological functions. We evaluated health and performance parameters of dairy calves and their dams, where calves: (i) had no contact with their dam (NC), in which the calf was removed from the dam directly after birth (n = 10); (ii) were allowed to have partial contact (PC) with their dam, in which the calf was housed in a calf pen adjacent to the cow area allowing physical contact on the initiative of the dam but no suckling (n = 18); (iii) were allowed to have full contact (FC) with their dam, including suckling, in which calves were housed together with their dams in a free-stall barn (n = 20). Throughout the first 7 weeks postpartum, data were collected on the health status, fecal microbiota, hematological profile, immune and hormonal parameters, and growth rates of calves, and on the health status, metabolic responses, and performance of dams. Overall, FC calves had more health issues (P = 0.02) and a tendency for higher antibiotic usage (P = 0.07) than NC calves. Additionally, FC calves showed elevated levels of erythrocytes, hematocrit, hemoglobin, and leukocytes on day 49 compared to NC calves (P < 0.001). Calf fecal microbiota changed over time, and we found preliminary evidence that fecal microbiota is affected by the type of CCC, as reflected by differences in relative abundances of taxa including Lactobacillus in FC calves compared to NC and PC calves except on days 7 and 66. The FC calves had a greater average daily gain in body weight than NC and PC calves (P = 0.002). Cow health was not affected by the type of CCC, although in the first 7 weeks of lactation FC cows had a lower machine-gained milk yield accompanied by a lower fat percentage than NC and PC cows (P < 0.001). These results indicate that full contact posed a challenge for calf health, presumably because the housing conditions of FC calves in this experimental context were suboptimal. Secondly, ad libitum suckling leads to higher weight gains and negatively affected milk fat content besides machine-gained yields. More research into strategies to improve cow-calf housing and management in CCC systems is warranted.

The effectiveness of bovine tuberculosis surveillance in Dutch badgers.

Countries survey wildlife for bovine tuberculosis (bTB) to ensure case detection or to ascertain a high probability of freedom from bTB in wildlife. The Eurasian badger (Meles meles) is a potential bTB reservoir host. Between 2008 and 2019, 282 badgers were examined post-mortem in the context of general wildlife disease and targeted bTB surveillance programmes in the Netherlands, and no bTB cases were detected. However, it was unclear how effective this surveillance effort was to demonstrate freedom from Mycobacterium bovis infection in the badger population of ±6000 or to detect cases if present. Therefore, surveillance effectiveness was assessed using scenario tree modelling. For lack of standards for wildlife, the models were run against three assumed levels of disease in the population called design prevalence P*: 0.1%, 0.5%, and 3%. A small risk of introduction (0.015/year) was applied, because the Netherlands are officially free from bTB in cattle, with rare import of bTB-infected cattle and no bTB-infected wildlife reported along the Belgian and German borders with the Netherlands. Surveillance more readily picks up bTB presence in badgers when case detection sensitivity tends towards 100% and demonstrates freedom best when the probability of freedom tends towards 100%. For P* 0.1%, 0.5% and 3%, respectively, maximum case detection sensitivity during 2008-2019 was 8%, 35% and 94% and the probability of freedom in 2019 was 46%, 67%, and 95%. At P* = 3%, performing targeted surveillance on 300 badgers in a year would make it extremely unlikely to miss a case (case detection sensitivity > 99.9%); and if no cases are detected, the adjusted probability of freedom would then reach nearly 98.5%. Stakeholders should be made aware that at P* = 3%, one case detected implies around 3% infected badgers. Additional surveillance system components to assess bTB in wildlife and its economics are to be explored further.

Experimental Chlamydia gallinacea infection in chickens does not protect against a subsequent experimental Chlamydia psittaci infection.

Chlamydia psittaci was considered the predominant chlamydial species in poultry until Chlamydia gallinacea was discovered in 2009. C. psittaci is a zoonotic obligate intracellular bacterium reported in more than 465 bird species including poultry. In poultry, infections can result in asymptomatic disease, but also in more severe systemic illness. The zoonotic potential of C. gallinacea has yet to be proven. Infections in poultry appear to be asymptomatic and in recent prevalence studies C. gallinacea was the main chlamydial species found in chickens. The high prevalence of C. gallinacea resulted in the question if an infection with C. gallinacea might protect against an infection with C. psittaci. To investigate possible cross protection, chickens were inoculated with C. gallinacea NL_G47 and subsequently inoculated with either a different strain of C. gallinacea (NL_F725) or C. psittaci. Chickens that had not been pre-inoculated with C. gallinacea NL_G47 were used as a C. gallinacea or C. psittaci infection control. In the groups that were inoculated with C. psittaci, no difference in pharyngeal or cloacal shedding, or in tissue dissemination was observed between the control group and the pre-inoculated group. In the groups inoculated with C. gallinacea NL_F725, shedding in cloacal swabs and tissues dissemination was lower in the group pre-inoculated with C. gallinacea NL_G47. These results indicate previous exposure to C. gallinacea does not protect against an infection with C. psittaci, but might protect against a new infection of C. gallinacea.

Classification and prediction of Mycobacterium Avium subsp. Paratuberculosis (MAP) shedding severity in cattle based on young stock heifer faecal microbiota composition using random forest algorithms.

BACKGROUND: Bovine paratuberculosis is a devastating infectious disease caused by Mycobacterium avium subsp. paratuberculosis (MAP). The development of the paratuberculosis in cattle can take up to a few years and vastly differs between individuals in severity of the clinical symptoms and shedding of the pathogen. Timely identification of high shedding animals is essential for paratuberculosis control and minimization of economic losses. Widely used methods for detection and quantification of MAP, such as culturing and PCR based techniques rely on direct presence of the pathogen in a sample and have little to no predictive value concerning the disease development. In the current study, we investigated the possibility of predicting MAP shedding severity in cattle based on the faecal microbiota composition. Twenty calves were experimentally infected with MAP and faecal samples were collected biweekly up to four years of age. All collected samples were subjected to culturing on selective media to obtain data about shedding severity. Faecal microbiota was profiled in a subset of samples (n = 264). Using faecal microbiota composition and shedding intensity data a random forest classifier was built for prediction of the shedding status of the individual animals. RESULTS: The results indicate that machine learning approaches applied to microbial composition can be used to classify cows into groups by severity of MAP shedding. The classification accuracy correlates with the age of the animals and use of samples from older individuals resulted in a higher classification precision. The classification model based on samples from the first 12 months of life showed an AUC between 0.78 and 0.79 (95% CI), while the model based on samples from animals older than 24 months showed an AUC between 0.91 and 0.92 (95% CI). Prediction for samples from animals between 12 and 24 month of age showed intermediate accuracy [AUC between 0.86 and 0.87 (95% CI)]. In addition, the results indicate that a limited number of microbial taxa were important for classification and could be considered as biomarkers. CONCLUSIONS: The study provides evidence for the link between microbiota composition and severity of MAP infection and shedding, as well as lays ground for the development of predictive diagnostic tools based on the faecal microbiota composition.

Metabolomic Changes in Naturally MAP-Infected Holstein-Friesian Heifers Indicate Immunologically Related Biochemical Reprogramming.

Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP), causes weight loss, diarrhoea, and reduced milk yields in clinically infected cattle. Asymptomatic, subclinically infected cattle shed MAP bacteria but are frequently not detected by diagnostic tests. Herein, we compare the metabolite profiles of sera from subclinically infected Holstein-Friesian heifers and antibody binding to selected MAP antigens. The study used biobanked serum samples from 10 naturally MAP-infected and 10 control heifers, sampled monthly from ~1 to 19 months of age. Sera were assessed using flow infusion electrospray-high-resolution mass spectrometry (FIE-HRMS) on a Q Exactive hybrid quadrupole-Orbitrap mass spectrometer for high-throughput, sensitive, non-targeted metabolite fingerprinting. Partial least-squares discriminant analyses (PLS-DA) and hierarchical cluster analysis (HCA) of the data discriminated between naturally MAP-infected and control heifers. In total, 33 metabolites that differentially accumulated in naturally MAP-infected heifers compared to controls were identified. Five were significantly elevated within MAP-infected heifers throughout the study, i.e., leukotriene B4, bicyclo prostaglandin E2 (bicyclo PGE2), itaconic acid, 2-hydroxyglutaric acid and N6-acetyl-L-lysine. These findings highlight the potential of metabolomics in the identification of novel MAP diagnostic markers and particular biochemical pathways, which may provide insights into the bovine immune response to MAP.

Genetic and phenotypic analysis of the pathogenic potential of two novel Chlamydia gallinacea strains compared to Chlamydia psittaci.

Chlamydia gallinacea is an obligate intracellular bacterium that has recently been added to the family of Chlamydiaceae. C. gallinacea is genetically diverse, widespread in poultry and a suspected cause of pneumonia in slaughterhouse workers. In poultry, C. gallinacea infections appear asymptomatic, but studies about the pathogenic potential are limited. In this study two novel sequence types of C. gallinacea were isolated from apparently healthy chickens. Both isolates (NL_G47 and NL_F725) were closely related to each other and have at least 99.5% DNA sequence identity to C. gallinacea Type strain 08-1274/3. To gain further insight into the pathogenic potential, infection experiments in embryonated chicken eggs and comparative genomics with Chlamydia psittaci were performed. C. psittaci is a ubiquitous zoonotic pathogen of birds and mammals, and infection in poultry can result in severe systemic illness. In experiments with embryonated chicken eggs, C. gallinacea induced mortality was observed, potentially strain dependent, but lower compared to C. psittaci induced mortality. Comparative analyses confirmed all currently available C. gallinacea genomes possess the hallmark genes coding for known and potential virulence factors as found in C. psittaci albeit to a reduced number of orthologues or paralogs. The presence of potential virulence factors and the observed mortality in embryonated eggs indicates C. gallinacea should rather be considered as an opportunistic pathogen than an innocuous commensal.

A Scoping Review of On-Farm Colostrum Management Practices for Optimal Transfer of Immunity in Dairy Calves.

Newborn calves are agammaglobulinemic and rely for their first immune protection almost completely on the transfer of immune constituents via colostrum. Inadequate colostrum management practices such as on-farm colostrum storage practices and colostrum feeding methods could affect immune components in colostrum and subsequently immune status of the newborn calf. We conducted a scoping review to identify all literature on the interactions between several colostrum management factors and immunological colostrum quality and passive transfer of immunity. Three major stages were defined: milking methods, colostrum treatment and storage, and administration procedures. Separate CAB Abstracts searches were performed for each of the subjects of interest. The search process was completed on November 9, 2020. Colostrum should be milked as soon as possible, as IgG concentration diminishes over time, probably due to dilution. To minimize bacterial contamination, it is advised to pasteurize colostrum in small batches at maximal 60°C for 30 or 60 min. Freeze/thawing of colostrum does not or only slightly affect IgG concentrations, as long as thawing is done au bain-marie and temperature does not exceed 40°C. In on-farm situations, it is difficult to determine the volume that should be fed as the variables contributing to the absorption of IgG by the newborn calf are many and include the quality of the colostrum, the bacterial contamination, the time interval between birth and first moment of feeding and the weight of the calf. Despite all knowledge regarding optimal colostrum management strategies, it remains challenging to predict the effects of certain colostrum management choices in field conditions. Therefore, we recommend measuring the colostral quality, weighing the newborn calf, adjusting the feeding volume accordingly to ensure optimal colostrum intake for each calf.

Pathogenicity of Chlamydia gallinacea in chickens after oral inoculation.

Chlamydia gallinacea is a recently discovered and widespread obligate intracellular bacterium in chickens. In chickens, infections appear to be asymptomatic, but can result in reduced weight gain in broilers. Molecular typing revealed C. gallinacea is genetically diverse which might lead to differences in pathogenic potential between strains. However, studies about the pathogenesis of different C. gallinacea strains are still limited. In this study, the pathogenesis of C. gallinacea strain NL_G47 was investigated in three consecutive animal experiments. The first experiment served as a pilot in which a maximum culturable dose was administered orally to 13 chickens. Excretion of chlamydial DNA in cloacal swabs was measured during 11 days post infection, but no clinical signs were observed. The second and third experiment were a repetition of the first experiment, but now chickens were sacrificed at consecutive time points to investigate tissue dissemination of C. gallinacea. Again excretion of chlamydial DNA in cloacal swabs was detected and no clinical signs were observed in line with the results of the first experiment. PCR and immunohistochemistry of tissue samples revealed C. gallinacea infected the epithelium of the jejunum, ileum and caecum. Furthermore, C. gallinacea could be detected in macrophages in the lamina propria and in follicular dendritic cells (FDCs) of the B cell follicles in the caecal tonsil. Results of serology showed a systemic antibody response from day seven or eight and onward in all three experiments. The experiments with strain NL_G47 confirmed observations from field studies that C. gallinacea infection does not result in acute clinical disease and mainly resides in the epithelium of the gut. Whether the presence of C. gallinacea results in chronic persistent infections with long term and less obvious health effects in line with observations on other infections caused by Chlamydiae, needs further investigation.

Survey on Colostrum Management by Dairy Farmers in the Netherlands.

Colostrum feeding is essential for the transfer of passive immunity and health of newborn calves. Information on current colostrum management practices to reduce calf morbidity and mortality is important but lacking for Dutch dairy herds. We therefore conducted a survey to investigate colostrum management strategies on Dutch dairy farms. The survey was specifically focused on the most recently born calf and was returned by 107 respondents (response rate of 13.4%). The mean amount of colostrum fed at first feeding was 2.9 liters. Overall, 79% of farmers provided the calf with at least 6 liters of colostrum in up to three feedings. The majority of respondents (84%) claimed to provide the calf with colostrum for the first time within 2 h post-partum. Using ordinal logistic regression and Wilcoxon rank sum test, we found no differences in time to first colostrum feeding or total amount of colostrum fed between bull calves and heifer calves, respectively. Ordinal logistic regression showed no significant differences in time to first colostrum feeding or time between calving and removing the calf from the dam between AMS and conventional milking herds. Two sample T-test comparing the total volume of colostrum showed no significant difference between AMS and conventional milking herds. Time of day at which a calf was born affected both volume fed at first colostrum feeding and time until first colostrum feeding. Calves born between 00.00 and 06.00 were significantly at risk of receiving the first colostrum later as compared to calves born at other times. Calves born in the evening received on average a lower amount of colostrum at first feeding. Survey results on colostrum management on most Dutch dairy farms are in agreement with the advice to feed as soon as possible after parturition and to provide at least 6 liters within 24 h of age. The current study points at time of calving as a potential risk factor for sub-optimal colostrum feeding. Further research is necessary to determine the consequences of this observation.

Isotype-specific Antibody Responses to Mycobacterium avium paratuberculosis Antigens Are Associated With the Use of Biologic Therapy in Inflammatory Bowel Disease.

BACKGROUND: The role of Mycobacterium avium paratuberculosis [MAP] in inflammatory bowel disease [IBD], especially Crohn's disease [CD] is controversial due conflicting results and lack of reproducibility and standardised tests. The current study focuses on the role of MAP in disease progression and genetic susceptibility, as MAP is likely one of many factors involved in the complex pathogenesis of IBD, potentially affecting a subgroup depending on genetic susceptibility. METHODS: Serum from 812 patients was evaluated with seven immunoglobulin [Ig] isotype-specific serology tests assessing humoral response to three different MAP antigens. For each of these in total 21 tests, the intra-assay and inter-assay coefficients were used to evaluate test accuracy. Reliable assays were subsequently analysed in relation to disease characteristics and need for biologic therapy/surgery. Genome-wide genotyping was available for all participants. Genetic determinants of humoral response to MAP antigens were evaluated using genome-wide association analysis and polygenic risk scores [PRS]. RESULTS: High IgA or IgM response to MAP2609 was associated with increased use of biologic therapy in CD and ulcerative colitis [UC] [odds ratios 2.69; 95% confidence interval 1.44-5.01; and 2.60, 1.46-4.64, respectively]. No associations were seen for risk of surgery [p-values > 0.29]. We could not identify genetic determinants nor polygenic risk scores for MAP response with genome-wide significance. CONCLUSIONS: Extensive assays for serological response to MAP were evaluated using stringent criteria for reliability. Increased IgA and IgM response to MAP antigens was seen in patients exposed to biologic therapy, but no genetic determinants underlying this humoral response were found.

Experimental Coxiella burnetii infection in non-pregnant goats and the effect of breeding.

Q fever is a zoonosis caused by the intracellular bacterium Coxiella burnetii. In Europe, small ruminants are the main source of human Q fever. Small ruminant herds can be infectious during several lambing seasons. However, it is not clear how infection is maintained in a herd and what role non-pregnant animals play in the transmission of C. burnetii. We therefore inoculated nulliparous goats with C. burnetii, isolated from the outbreak of Q fever in the Netherlands, to gain a better understanding of the role of non-pregnant goats. Seroconversion and excretion of C. burnetii were monitored after inoculation. To study the effect of breeding on the excretion of C. burnetii, the goats were naturally bred and monitored during gestation and after lambing. Our results indicate that C. burnetii infection prior to breeding did not result in infection of the placenta nor did it affect the gestation length or the number of kids born. However, one of the ten does did excrete C. burnetii in the colostrum post-partum and the bacterium was detected in the mammary gland and associated lymph nodes at necropsy. This result indicates that non-pregnant goats might play a role in maintaining Q fever in a goat herd as persistent carriers of infection.