Transboundary Spread of Brucella canis through Import of Infected Dogs, the Netherlands, November 2016-December 2018.

Brucella canis had not been isolated in the Netherlands until November 2016, when it was isolated from a dog imported from Romania. Including this case, 16 suspected cases were notified to the authorities during the following 25 months. Of these 16 dogs, 10 were seropositive; tracking investigations found another 8 seropositive littermates. All seropositive animals were rescue dogs imported from Eastern Europe. B. canis was cultured from urine, blood, and other specimens collected from the dogs. Genotyping of isolates revealed clustering by litter and country. Isolating B. canis in urine indicates that shedding should be considered when assessing the risk for zoonotic transmission. This case series proves introduction of B. canis into a country to which it is not endemic through import of infected dogs from B. canis-endemic areas, posing a threat to the naive autochthonous dog population and humans.

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.

Brucella suis Infection in Dog Fed Raw Meat, the Netherlands.

A Brucella suis biovar 1 infection was diagnosed in a dog without typical exposure risks, but the dog had been fed a raw meat-based diet (hare carcasses imported from Argentina). Track and trace investigations revealed that the most likely source of infection was the dog's raw meat diet.

Coxiella burnetii (Q fever) prevalence in associated populations of humans and small ruminants in The Gambia.

OBJECTIVES: To simultaneously estimate the prevalence of antibodies against Coxiella burnetii (Q fever) among adults and small ruminants, and C. burnetii shedding prevalence among small ruminants in households in the Kiang West district of The Gambia, and to assess associated risk factors. METHODS: Sera of 599 adults and 615 small ruminants from 125 compounds within 12 villages were tested for antibodies against C. burnetii using ELISA. Vaginal swabs and milk samples of 155 small ruminants were tested using PCR to investigate shedding of C. burnetii. RESULTS: A total of 3.8-9.7% of adults, depending on ELISA test cut-off, and 24.9% of small ruminants in Kiang West were seropositive. Having at least one seropositive animal in one's compound was a risk factor for human seropositivity (OR: 3.35, 95% CI: 1.09-14.44). A grazing area within a village was a risk factor for seropositivity in small ruminants (OR: 2.07, 95% CI: 1.26-3.50); others were having lambed (OR: 2.75, 95% CI: 1.37-5.76) and older age of the animals (OR: 2.75, 95% CI: 1.37-5.76 for 1-3 years and OR 5.84, 95% CI: 3.10-11.64 for >3 years); 57.4% of sampled small ruminants were shedding C. burnetii. CONCLUSION: Coxiella burnetii infection is endemic among both humans and small ruminants in this area of The Gambia. Human and animal exposure to C. burnetii were related at compound level. Further research into the clinical relevance of C. burnetii infection in West Africa is needed.

Presence of Coxiella burnetii DNA in inflamed bovine cardiac valves.

BACKGROUND: Bacterial endocarditis is a recognised disease in humans and animals. In humans, infection with Coxiella burnetii can cause endocarditis, but this has not been investigated thoroughly in animals. Endocarditis in cattle is a common post-mortem finding in abattoirs and studies have identified Trueperella pyogenes as a major cause. Despite exposure of cattle to C. burnetii, the significance of this particular bacterium for development and progression of endocarditis has not been studied in detail. Cardiac valves of cattle affected with endocarditis (n = 100) were examined by histology, fluorescence in situ hybridization (FISH) and real time quantitative polymerase chain reaction (PCR). Serum was examined for anti-C. burnetii antibodies by enzyme-linked immunosorbent assay (ELISA). RESULTS: Serology revealed that 70% of the cattle were positive for antibodies to C. burnetii, while PCR analysis identified 25% of endocarditis valve samples as being positive. C. burnetii was not detected by FISH, probably due to the low infection levels. Most cattle had chronic valvular vegetative endocarditis with lesions being characterised by a core of fibrous tissue covered by significant amounts of fibrin, sometimes with areas of liquefaction, and with a coagulum covering the surface. In a few cases, including the case with the highest infection level, lesions were characterized by extensive fibrosis and calcification. Histologically, bacteria other than C. burnetii were observed in most cases. CONCLUSIONS: The presence of C. burnetii DNA is relatively common in cattle affected with valvular endocarditis. The role of C. burnetii remains however unknown as lesions did not differ between C. burnetii infected and non-infected cattle and because T. pyogenes-like bacteria were present in the inflamed valves; a bacterium able to induce the observed lesions. Heart valves of normal cattle should be investigated to assess if C. burnetii may be present without preexisting lesions.

Major differential gene regulation in Coxiella burnetii between in vivo and in vitro cultivation models.

BACKGROUND: Coxiella burnetii is the causative agent of the zoonotic disease Q fever. As it is an intracellular pathogen, infection by C. burnetii requires adaptation to its eukaryotic host and intracellular environment. The recently developed cell-free medium also allows the bacteria to propagate without host cells, maintaining its infection potential. The adaptation to different hosts or extracellular environments has been assumed to involve genome-wide modulation of C. burnetii gene expression. However, little is currently known about these adaptation events which are critical for understanding the intracellular survival of C. burnetii. RESULTS: We studied C. burnetii genome-wide transcriptional patterns in vivo (mice spleen) and in cell and cell-free in vitro culture models to examine its metabolic pathways and virulence associated gene expression patterns that are required to colonize and persist in different environments. Within each model, the gene expression profiles of the Dutch C. burnetii outbreak strain (602) and NM reference strains were largely similar. In contrast, modulation of gene-expression was strongly influenced by the cultivation method, indicating adaptation of the bacterium to available components. Genome-wide expression profiles of C. burnetii from in vitro cell culture were more similar to those seen for in vivo conditions, while gene expression profiles of cell-free culture were more distant to in vivo. Under in vivo conditions, significant alterations of genes involved in metabolism and virulence were identified. We observed that C. burnetii under in vivo conditions predominantly uses glucose as a carbon source (mostly for biosynthetic processes) and fatty acids for energy generation. C. burnetii experienced nutrient limitation and anaerobiosis as major stressors, while phosphate limitation was identified as an important signal for intracellular growth inside eukaryotic host cells. Finally, the in vivo environment significantly induced expression of several virulence genes, including those implicated in LPS synthesis, colonization, host component modulation and DNA repair mechanisms. CONCLUSION: Our study shows that C. burnetii, with its relative small genome, requires only a subset of core gene functions to survive under in vitro conditions, but requires the induction of full repertoire of genes for successful pathogenesis and thriving in harsh environments in vivo.

Q fever in pregnant goats: humoral and cellular immune responses.

Q fever is a zoonosis caused by the intracellular bacterium Coxiella burnetii. Both humoral and cellular immunity are important in the host defence against intracellular bacteria. Little is known about the immune response to C. burnetii infections in domestic ruminants even though these species are the major source of Q fever in humans. To investigate the goat's immune response we inoculated groups of pregnant goats via inhalation with a Dutch outbreak isolate of C. burnetii. All animals were successfully infected. Phase 1 and Phase 2 IgM- and IgG-specific antibodies were measured. Cellular immune responses were investigated by interferon-gamma, enzyme-linked immunosorbent spot test (IFN-γ Elispot), lymphocyte proliferation test (LPT) and systemic cytokines. After two weeks post inoculation (wpi), a strong anti-C. burnetii Phase 2 IgM and IgG antibody response was observed while the increase in IgM anti-Phase 1 antibodies was less pronounced. IgG anti-Phase 1 antibodies started to rise at 6 wpi. Cellular immune responses were observed after parturition. Our results demonstrated humoral and cellular immune responses to C. burnetii infection in pregnant goats. Cell-mediated immune responses did not differ enough to distinguish between Coxiella-infected and non-infected pregnant animals, whereas a strong-phase specific antibody response is detected after 2 wpi. This humoral immune response may be useful in the early detection of C. burnetii-infected pregnant goats.

Reduction of Coxiella burnetii prevalence by vaccination of goats and sheep, The Netherlands.

Recently, the number of human Q fever cases in the Netherlands increased dramatically. In response to this increase, dairy goats and dairy sheep were vaccinated against Coxiella burnetii. All pregnant dairy goats and dairy sheep in herds positive for Q fever were culled. We identified the effect of vaccination on bacterial shedding by small ruminants. On the day of culling, samples of uterine fluid, vaginal mucus, and milk were obtained from 957 pregnant animals in 13 herds. Prevalence and bacterial load were reduced in vaccinated animals compared with unvaccinated animals. These effects were most pronounced in animals during their first pregnancy. Results indicate that vaccination may reduce bacterial load in the environment and human exposure to C. burnetii.

Molecular epidemiology of Coxiella burnetii from ruminants in Q fever outbreak, the Netherlands.

Q fever is a zoonosis caused by the bacterium Coxiella burnetii. One of the largest reported outbreaks of Q fever in humans occurred in the Netherlands starting in 2007; epidemiologic investigations identified small ruminants as the source. To determine the genetic background of C. burnetii in domestic ruminants responsible for the human Q fever outbreak, we genotyped 126 C. burnetii-positive samples from ruminants by using a 10-loci multilocus variable-number tandem-repeat analyses panel and compared them with internationally known genotypes. One unique genotype predominated in dairy goat herds and 1 sheep herd in the human Q fever outbreak area in the south of the Netherlands. On the basis of 4 loci, this genotype is similar to a human genotype from the Netherlands. This finding strengthens the probability that this genotype of C. burnetii is responsible for the human Q fever epidemic in the Netherlands.

A cross sectional study on Dutch layer farms to investigate the prevalence and potential risk factors for different Chlamydia species.

In poultry several Chlamydia species have been detected, but Chlamydia psittaci and Chlamydia gallinacea appear to be most prevalent and important. Chlamydia psittaci is a well-known zoonosis and is considered to be a pathogen of poultry. Chlamydia gallinacea has been described more recently. Its avian pathogenicity and zoonotic potential have to be further elucidated. Within the Netherlands no data were available on the presence of Chlamydia on poultry farms. As part of a surveillance programme for zoonotic pathogens in farm animals, we investigated pooled faecal samples from 151 randomly selected layer farms. On a voluntary base, 69 farmers, family members or farm workers from these 151 farms submitted a throat swab. All samples were tested with a generic 23S Chlamydiaceae PCR followed by a species specific PCR for C. avium, C. gallinacea and C. psittaci. C. avium and psittaci DNA was not detected at any of the farms. At 71 farms the positive result could be confirmed as C. gallinacea. Variables significantly associated with the presence of C. gallinacea in a final multivariable model were 'age of hens,' 'use of bedding material' and 'the presence of horses.' The presence of C. gallinacea was associated with neither clinical signs, varying from respiratory symptoms, nasal and ocular discharges to diarrhoea, nor with a higher mortality rate the day before the visit. All throat swabs from farmers, family members or farm workers tested negative for Chlamydia DNA, giving no further indication for possible bird-to-human (or human-to-bird) transmission.

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.

Genome Plasticity and Polymorphisms in Critical Genes Correlate with Increased Virulence of Dutch Outbreak-Related Coxiella burnetii Strains.

Coxiella burnetii is an obligate intracellular bacterium and the etiological agent of Q fever. During 2007-2010 the largest Q fever outbreak ever reported occurred in The Netherlands. It is anticipated that strains from this outbreak demonstrated an increased zoonotic potential as more than 40,000 individuals were assumed to be infected. The acquisition of novel genetic factors by these C. burnetii outbreak strains, such as virulence-related genes, has frequently been proposed and discussed, but is not proved yet. In the present study, the whole genome sequence of several Dutch strains (CbNL01 and CbNL12 genotypes), a few additionally selected strains from different geographical locations and publicly available genome sequences were used for a comparative bioinformatics approach. The study focuses on the identification of specific genetic differences in the outbreak related CbNL01 strains compared to other C. burnetii strains. In this approach we investigated the phylogenetic relationship and genomic aspects of virulence and host-specificity. Phylogenetic clustering of whole genome sequences showed a genotype-specific clustering that correlated with the clustering observed using Multiple Locus Variable-number Tandem Repeat Analysis (MLVA). Ortholog analysis on predicted genes and single nucleotide polymorphism (SNP) analysis of complete genome sequences demonstrated the presence of genotype-specific gene contents and SNP variations in C. burnetii strains. It also demonstrated that the currently used MLVA genotyping methods are highly discriminatory for the investigated outbreak strains. In the fully reconstructed genome sequence of the Dutch outbreak NL3262 strain of the CbNL01 genotype, a relatively large number of transposon-linked genes were identified as compared to the other published complete genome sequences of C. burnetii. Additionally, large numbers of SNPs in its membrane proteins and predicted virulence-associated genes were identified in all Dutch outbreak strains compared to the NM reference strain and other strains of the CbNL12 genotype. The presence of large numbers of transposable elements and mutated genes, thereof most likely resulted in high level of genome rearrangements and genotype-specific pathogenicity of outbreak strains. Thus, the epidemic potential of Dutch outbreak strains could be linked to increased genome plasticity and mutations in critical genes involved in virulence and the evasion of the host immune system.

First Complete Genome Sequence of the Dutch Veterinary Coxiella burnetii Strain NL3262, Originating from the Largest Global Q Fever Outbreak, and Draft Genome Sequence of Its Epidemiologically Linked Chronic Human Isolate NLhu3345937.

The largest global Q fever outbreak occurred in The Netherlands during 2007 to 2010. Goats and sheep were identified as the major sources of disease. Here, we report the first complete genome sequence of ITALIC! Coxiella burnetiigoat outbreak strain NL3262 and that of an epidemiologically linked chronic human strain, both having the outbreak-related ITALIC! CbNL01multilocus variable-number tandem-repeat analysis (MLVA) genotype.

Low Seroprevalence of Brucellosis in Humans and Small Ruminants in the Gambia.

BACKGROUND: Brucellosis is a worldwide zoonosis with significant impact on rural livelihoods and a potentially underestimated contributor to febrile illnesses. The aim of this study was to estimate the seroprevalence of brucellosis in humans and small ruminants in The Gambia. METHODS: The study was carried out in rural and urban areas. In 12 rural villages in Kiang West district, sera were collected from humans (n = 599) and small ruminants (n = 623) from the same compounds. From lactating small ruminants, milk samples and vaginal swabs were obtained. At the urban study sites, sera were collected from small ruminants (n = 500) from slaughterhouses and livestock markets. Information on possible risk factors for seropositivity was collected through questionnaires. Sera were screened for antibodies against Brucella spp. with the Rose Bengal Test, ELISA and Micro Agglutination Test (human sera only). PCR was performed on 10 percent of the milk samples and vaginal swabs from small ruminants. RESULTS: One human and 14 sheep sera were positive by the Rose Bengal Test. The rest were negative in all serological tests used. The PCR results were all negative. CONCLUSIONS: The results suggest that brucellosis is currently not a generalized problem in humans or small ruminants in The Gambia.

Bacillus anthracis-like bacteria and other B. cereus group members in a microbial community within the International Space Station: a challenge for rapid and easy molecular detection of virulent B. anthracis.

For some microbial species, such as Bacillus anthracis, the etiologic agent of the disease anthrax, correct detection and identification by molecular methods can be problematic. The detection of virulent B. anthracis is challenging due to multiple virulence markers that need to be present in order for B. anthracis to be virulent and its close relationship to Bacillus cereus and other members of the B. cereus group. This is especially the case in environments where build-up of Bacillus spores can occur and several representatives of the B. cereus group may be present, which increases the chance for false-positives. In this study we show the presence of B. anthracis-like bacteria and other members of the B. cereus group in a microbial community within the human environment of the International Space Station and their preliminary identification by using conventional culturing as well as molecular techniques including 16S rDNA sequencing, PCR and real-time PCR. Our study shows that when monitoring the microbial hygiene in a given human environment, health risk assessment is troublesome in the case of virulent B. anthracis, especially if this should be done with rapid, easy to apply and on-site molecular methods.

Test and cull of high risk Coxiella burnetii infected pregnant dairy goats is not feasible due to poor test performance.

A major human Q fever epidemic occurred in The Netherlands during 2007-2009. In response, all pregnant goats from infected herds were culled before the 2010 kidding season without individual testing. The aim of this study was to assess whether high risk animals from recently infected naive herds can be identified by diagnostic testing. Samples of uterine fluid, milk and vaginal mucus from 203 euthanized pregnant goats were tested by PCR or ELISA. The results suggest that testing followed by culling of only the high risk animals is not a feasible method for protecting public health, mainly due to the low specificity of the tests and variability between herds. The risk of massive bacterial shedding during abortion or parturition can only be prevented by removal of all pregnant animals from naive recently infected herds.

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.