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.

Proof of concept for multiplex detection of antibodies against Chlamydia species in chicken serum using a bead-based suspension array with peptides as antigens.

The available differentiating tests for Chlamydia are based on detection of genetic material and only give information about the actual infection status, but reveal nothing of past infections. As the use of serological methods increases the window of detection, the goal of this study was to investigate if it is possible to develop a differentiating serological test for antibodies against Chlamydia species in chicken sera. Focus was on C. psittaci, C. gallinacea, and two closely related species, i.e. C. abortus and C. avium. To enable differentiating serology, a bead-based Luminex suspension array was constructed, using peptides as antigens, derived from known immunoreactive Chlamydia proteins. For the majority of these peptides, species-specific seroreactivity in mammalian sera has been reported in literature. The suspension array correctly identified antibodies against various Chlamydia species in sera from experimentally infected mice, and was also able to differentiate between antibodies against C. psittaci and C. gallinacea in sera from experimentally infected chickens. In field sera, signals were difficult to interpret as insufficient sera from experimentally infected chickens were available for evaluating the seroreactivity of all peptides. Nevertheless, results of the suspension array with field sera are supported by published data on the occurrence of C. gallinacea in Dutch layers, thereby demonstrating the proof of concept of multiplex serology for Chlamydial species in poultry.

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.

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.

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.

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.

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.

Effects of Age and Environment on Adaptive Immune Responses to Mycobacterium avium subsp. paratuberculosis (MAP) Vaccination in Dairy Goats in Relation to Paratuberculosis Control Strategies.

Paratuberculosis infection is caused by Mycobacterium avium subsp. paratuberculosis (MAP). In the Netherlands, 75% herd level prevalence of caprine paratuberculosis has been estimated, and vaccination is the principal control strategy applied. Most goat dairy farms with endemic paratuberculosis systematically vaccinate goat kids in the first months of life with a commercially available whole cell MAP vaccine. We hypothesized that the development of adaptive immune responses in goats vaccinated at young age depends on the environment they are raised in, and this has implications for the application of immune diagnostic tests in vaccinated dairy goats. We evaluated the early immune response to vaccination in young goat kids sourced from a MAP unsuspected non-vaccinated herd and raised in a MAP-free environment. Subsequently we compared these with responses observed in birth year and vaccination matched adult goats raised on farms with endemic paratuberculosis. Results indicated that initial adaptive immune responses to vaccination are limited in a MAP-free environment. In addition, adult antibody positive vaccinated goats raised in a MAP endemic environment are less likely to be IS900 PCR-positive as compared to antibody negative herd mates. We conclude that test-and-cull strategies in a vaccinated herd are currently not feasible using available immune diagnostic tests.

Viable Coxiella burnetii Induces Differential Cytokine Responses in Chronic Q Fever Patients Compared to Heat-Killed Coxiella burnetii.

Cytokine responses of chronic Q fever patients to the intracellular bacterium Coxiella burnetii have mostly been studied using ex vivo stimulation of immune cells with heat-killed C. burnetii due to the extensive measures needed to work with viable biosafety level 3 agents. Whether research with heat-killed C. burnetii can be translated to immune responses to viable C. burnetii is imperative for the interpretation of previous and future studies with heat-killed C. burnetii Peripheral blood mononuclear cells (PBMCs) of chronic Q fever patients (n = 10) and healthy controls (n = 10) were stimulated with heat-killed or viable C. burnetii of two strains, Nine Mile and the Dutch outbreak strain 3262, for 24 h, 48 h, and 7 days in the absence or presence of serum containing anti-C. burnetii antibodies. When stimulated with viable C. burnetii, PBMCs of chronic Q fever patients and controls produced fewer proinflammatory cytokines (interleukin-6 [IL-6], tumor necrosis factor alpha, and IL-1ß) after 24 h than after stimulation with heat-killed C. burnetii In the presence of Q fever seronegative serum, IL-10 production was higher after stimulation with viable rather than heat-killed C. burnetii; however, when incubating with anti-C. burnetii antibody serum, the effect on IL-10 production was reduced. Levels of adaptive, merely T-cell-derived cytokine (gamma interferon, IL-17, and IL-22) and CXCL9 production were not different between heat-killed and viable C. burnetii stimulatory conditions. Results from previous and future research with heat-killed C. burnetii should be interpreted with caution for innate cytokines, but heat-killed C. burnetii-induced adaptive cytokine production is representative of stimulation with viable bacteria.

Coxiella burnetii isolates originating from infected cattle induce a more pronounced proinflammatory cytokine response compared to isolates from infected goats and sheep.

Coxiella burnetii is the causative agent of Q fever. Although the prevalence of C. burnetii in cattle is much higher than in goats and sheep, infected cattle are rarely associated with human outbreaks. We investigated whether the immune response of humans differs after contact with C. burnetii isolates from different host origins or with different multilocus variable number of tandem repeat analysis (MLVA) genotypes. Cytokine responses were measured in human peripheral blood mononuclear cells (PBMCs) stimulated with 16 C. burnetii isolates with known MLVA genotype from goats, sheep, cattle, acute and chronic Q fever patients. Coxiella burnetii isolates originating from cattle induce significantly more IL-1ß, TNF-α and IL-22 than the isolates from goats, sheep or chronic Q fever patients. Comparing the cytokine induction of the isolates based on their MVLA genotype did not reveal differences in response between the MLVA genotypes. The proinflammatory cytokine response induced in human PBMCs by C. burnetii isolates from cattle may explain the low incidence of human Q fever outbreaks caused by cattle. The cytokine profile of PBMCs stimulated with C. burnetii isolates from chronic Q fever patients resembles isolates from goats. Furthermore, cytokine responses seem to be depending on host origin than on MLVA genotype.

The effect of C. burnetii infection on the cytokine response of PBMCs from pregnant goats.

In humans, infection with Coxiella burnetii, the causative agent of Q fever, leads to acute or chronic infection, both associated with specific clinical symptoms. In contrast, no symptoms are observed in goats during C. burnetii infection, although infection of the placenta eventually leads to premature delivery, stillbirth and abortion. It is unknown whether these differences in clinical outcome are due to the early immune responses of the goats. Therefore, peripheral blood mononuclear cells (PBMCs) were isolated from pregnant goats. In total, 17 goats were included in the study. Six goats remained naive, while eleven goats were infected with C. burnetii. Toll-like receptor (TLR) and cytokine mRNA expression were measured after in vitro stimulation with heat-killed C. burnetii at different time points (prior infection, day 7, 35 and 56 after infection). In naive goats an increased expression of interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, IL-10 and interferon (IFN)-γ mRNA upon C. burnetii stimulation was detected. In addition, TLR2 expression was strongly up-regulated. In goats infected with C. burnetii, PBMCs re-stimulated in vitro with C. burnetii, expressed significantly more TNF-α mRNA and IFN-γ mRNA compared to naive goats. In contrast, IL-10 mRNA production capacity was down-regulated during C. burnetii infection. Interestingly, at day 7 after inoculation a decreased IFN-γ protein level was observed in stimulated leukocytes in whole blood from infected goats, whereas at other time-points increased production of IFN-γ protein was seen. Our study shows that goats initiate a robust pro-inflammatory immune response against C. burnetii in vitro. Furthermore, PBMCs from C. burnetii infected goats show augmented pro-inflammatory cytokine responses compared to PBMCs from non-infected goats. However, despite this pro-inflammatory response, goats are not capable of clearing the C. burnetii infection.

Q fever in pregnant goats: pathogenesis and excretion of Coxiella burnetii.

Coxiella burnetii is an intracellular bacterial pathogen that causes Q fever. Infected pregnant goats are a major source of human infection. However, the tissue dissemination and excretion pathway of the pathogen in goats are still poorly understood. To better understand Q fever pathogenesis, we inoculated groups of pregnant goats via the intranasal route with a recent Dutch outbreak C. burnetii isolate. Tissue dissemination and excretion of the pathogen were followed for up to 95 days after parturition. Goats were successfully infected via the intranasal route. PCR and immunohistochemistry showed strong tropism of C. burnetii towards the placenta at two to four weeks after inoculation. Bacterial replication seemed to occur predominantly in the trophoblasts of the placenta and not in other organs of goats and kids. The amount of C. burnetii DNA in the organs of goats and kids increased towards parturition. After parturition it decreased to undetectable levels: after 81 days post-parturition in goats and after 28 days post-parturition in kids. Infected goats gave birth to live or dead kids. High numbers of C. burnetii were excreted during abortion, but also during parturition of liveborn kids. C. burnetii was not detected in faeces or vaginal mucus before parturition. Our results are the first to demonstrate that pregnant goats can be infected via the intranasal route. C. burnetii has a strong tropism for the trophoblasts of the placenta and is not excreted before parturition; pathogen excretion occurs during birth of dead as well as healthy animals. Besides abortions, normal deliveries in C. burnetii-infected goats should be considered as a major zoonotic risk for Q fever in humans.

Secreted antigens of Mycobacterium avium subspecies paratuberculosis as prominent immune targets.

We here describe the identification and characterization of three novel secreted Mycobacterium avium subsp. paratuberculosis antigens of 9, 15 and 34 kDa (Map2609, Map2942c and Map0210c, respectively) by screening a genomic expression library with a serum of a naturally infected clinical cow. The 9, 15 and 34 kDa antigens display strong homology to previously described M. tuberculosis antigens, TB8.4, MPT53 and Erp, respectively. Furthermore, these antigens were shown to be recognized by antibodies from infected cattle, when tested with a limited number of sera from subclinical (n=7) and clinical (n=3) infected cattle.