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.

Upsurge of human enterovirus 68 infections in patients with severe respiratory tract infections.

BACKGROUND: Enterovirus 68 (EV68) belongs to species Human enterovirus D. It is unique among enteroviruses because it shares properties with human rhinoviruses. After the first isolation in 1962 from four children with respiratory illness, reports of (clusters of) EV68 infections have been rare. During the autumn of 2010, we noticed an upsurge of EV68 infections in the Northern part of the Netherlands in patients with severe respiratory illness. OBJECTIVES: To give a detailed description of the clinical and virological data of patients with EV68 infection identified in 2010, and compare these with data collected in 2009. STUDY DESIGN: We systematically collected clinical data from patients with an EV68 infection detected in 2010. We added four patients with an EV68 infection from 2009. Further characterization of EV68 was performed by partial sequence analysis of the VP1 genomic region. RESULTS: In 2010, EV68 was identified as the only cause of respiratory illness in 24 patients, of which 5 had to be admitted to the intensive care unit. Sequence analysis revealed different lineages in the majority of EV68 detected in 2010 as compared to the 2009 isolates. CONCLUSIONS: We noticed an increase of EV68 infections and present clinical as well as sequence data, in which two distinct phylogenetic clusters could be identified.

European bat lyssaviruses, The Netherlands.

To study European bat lyssavirus (EBLV) in bat reservoirs in the Netherlands, native bats have been tested for rabies since 1984. For all collected bats, data including species, age, sex, and date and location found were recorded. A total of 1,219 serotine bats, Eptesicus serotinus, were tested, and 251 (21%) were positive for lyssavirus antigen. Five (4%) of 129 specimens from the pond bat, Myotis dasycneme, were positive. Recently detected EBLV RNA segments encoding the nucleoprotein were sequenced and analyzed phylogenetically (45 specimens). All recent serotine bat specimens clustered with genotype 5 (EBLV1) sequences, and homologies within subgenotypes EBLV1a and EBLV1b were 99.0%-100% and 99.2%-100%, respectively. Our findings indicate that EBLVs of genotype 5 are endemic in the serotine bat in the Netherlands. Since EBLVs can cause fatal infections in humans, all serotine and pond bats involved in contact incidents should be tested to determine whether the victim was exposed to EBLVs.

Rabies in wildlife in Latvia.

In the Baltic States, lyssaviruses are often detected in wildlife and presumed to constitute an important public health hazard. In order to decrease rabies incidence and eradicate wildlife reservoirs, a national rabies eradication program has been in place. Since 1970 a vaccination program in dogs and cats has been executed, and in 1991 oral vaccination of foxes was started. However, due to an insufficient budget, the latter was not done regularly and nationwide before 2000. Now, the program in force consists of compulsory vaccination of all dogs and cats, and a tetracycline marker vaccine oral vaccination program of foxes in the whole country. In 2001, 151 of 285 (53%) fox jaws were tested positive for tetracycline. All animals showing rabies-like symptoms were killed and tested for rabies. In this way, 250-400 cases of rabies per year were diagnosed in wildlife. To molecularly characterize the prevalent lyssaviruses in wildlife, lyssavirus RNA of 25 recent rabies positive samples, collected in the year 1999, was amplified by RT-PCR. Direct sequencing of the RT-PCR-amplified products of the virus' nucleoprotein encoding region and subsequent sequence analyses resulted in a 99.3-100% homology between isolates and a 99.0-100% similarity with a 1995 genotype I, classical rabies virus (RABV) raccoon dog isolate from Estonia. These results confirmed that RABV is endemic in wildlife in Latvia and should be considered a serious public health threat. To successfully eradicate the wildlife reservoirs, the national rabies eradication program must be continued, and it may need to be intensified.

Study of lyssaviruses of bat origin as a source of rabies for other animal species in the State of Rio De Janeiro, Brazil.

An epidemic-geographic rabies study was carried out in which 72 animal and human brain samples were analyzed for Lyssaviruses by a direct immunofluorescent technique (DIFT) and a reverse transcriptase-polymerase chain reaction (RT-PCR) assay. Fifty-two samples were also tested by a mouse inoculation test. Lyssavirus RNA was detected in 60 of 72 samples. Five DIFT-negative bat samples tested by a nested PCR assay showed evidence of the presence of rabies virus RNA. Sequencing of amplified rabies virus nucleoprotein encoding segments of a selection of the samples resulted in the formation of clusters, corresponding to samples originating from cattle and equines from the same hydrographic basin. Genomically related Lyssavirus strains of bat origin were found in each cluster, most likely because of the role of the bat in the epidemiology of the virus. All samples studied were of genotype 1. With exception of the human sample, all were distinct from the reference sample.

Epidemiology of Norwalk-like virus infections in cattle in The Netherlands.

"Norwalk-like viruses" (NLVs) are the most common cause of acute non-bacterial gastroenteritis in humans. Cattle may be a reservoir of NLVs although never bovine NLVs have been found in humans. To gain more insight into the epidemiology of NLV, infections in cattle in The Netherlands were studied. Individual faecal samples from a large dairy herd and 243 pooled samples from veal calf farms were analysed for NLV by RT-PCR. Calves under 3 months of age in the dairy herd were sampled three to five times with 3-week intervals, whereas dairy cattle were sampled twice with a 2-month interval. In 31.6% (77/243) of the veal calf farm samples and in 4.2% (13/312) of the individual dairy cattle samples NLV was detected. The mean age of virus positive dairy cattle was 2.5 months. The highest numbers of NLV positive veal calf farms in The Netherlands were found in the regions with the highest number of veal calf farms. NLV infected veal calf farms were detected in every month throughout the study period. Cattle appeared to be hosts of NLVs, and virus shedding was weakly associated with diarrhoea. Complete ORF2 sequences were obtained from two calf NLVs and phylogenetic analyses suggested that these strains belong to a distinct cluster (GGIII/2) in between GGI and GGII NLVs of humans. Overall, genetic variation between strains as determined by sequence analysis of the P1/P2 capsid region was limited to 14.6%. Our data shows that NLV is endemic in the cattle population in The Netherlands and genetically distinct from NLVs in humans.