Diversity of Hepatozoon species in wild mammals and ticks in Europe.

BACKGROUND: Hepatozoon spp. are tick-borne parasites causing subclinical to clinical disease in wild and domestic animals. Aim of this study was to determine Hepatozoon prevalence and species distribution among wild mammals and ticks in Europe. METHODS: Samples of wild mammals and ticks, originating from Austria, Bosnia and Herzegovina, Croatia, Belgium and the Netherlands, were tested with PCR to amplify a ~ 670-bp fragment of the small subunit ribosomal RNA gene. RESULTS: Of the 2801 mammal samples that were used for this study, 370 (13.2%) tested positive. Hepatozoon canis was detected in samples of 178 animals (3 Artiodactyla, 173 Carnivora, 1 Eulipotyphia, 1 Lagomorpha), H. martis in 125 (3 Artiodactyla, 122 Carnivora), H. sciuri in 13 (all Rodentia), Hepatozoon sp. in 47 (among which Hepatozoon sp. Vole isolate, all Rodentia) and H. ayorgbor in 4 (all Rodentia). Regarding origin, 2.9% (6/208) tested positive from Austria, 2.8% (1/36) from Bosnia and Herzegovina, 14.6% (173/1186) from Croatia and 13.9% (190/1371) from Belgium/the Netherlands. Of the 754 ticks collected, 0.0% (0/35) Hyalomma sp., 16.0% (4/25) Dermacentor spp., 0.0% (0/23) Haemaphysalis spp., 5.3% (24/50) Ixodes and 1.4% (3/221) Rhipicephalus spp. tested positive for Hepatozoon (4.2%; 32/754), most often H. canis (n = 22). CONCLUSIONS: Hepatozoon canis is most present in mammals (especially in Carnivora such as gray wolves and golden jackals) and ticks, followed by H. martis, which was found merely in stone martens and pine martens. None of the rodent-associated Hepatozoon spp. were detected in the ticks, suggesting the possible implication of other arthropod species or non-vectorial routes in the transmission cycle of the hemoprotozoans in rodents. Our findings of H. canis in ticks other than R. sanguineus add to the observation that other ticks are also involved in the life cycle of Hepatozoon. Now that presence of Hepatozoon has been demonstrated in red foxes, gray wolves, mustelids and rodents from the Netherlands and/or Belgium, veterinary clinicians should be aware of the possibility of spill-over to domestic animals, such as dogs.

Signalling and responding to zoonotic threats using a One Health approach: a decade of the Zoonoses Structure in the Netherlands, 2011 to 2021.

In the Netherlands, the avian influenza outbreak in poultry in 2003 and the Q fever outbreak in dairy goats between 2007 and 2010 had severe consequences for public health. These outbreaks led to the establishment of an integrated human-veterinary risk analysis system for zoonoses, the Zoonoses Structure. The aim of the Zoonoses Structure is to signal, assess and control emerging zoonoses that may pose a risk to animal and/or human health in an integrated One Health approach. The Signalling Forum Zoonoses (SO-Z), the first step of the Zoonoses Structure, is a multidisciplinary committee composed of experts from the medical, veterinary, entomology and wildlife domains. The SO-Z shares relevant signals with professionals and has monthly meetings. Over the past 10 years (June 2011 to December 2021), 390 different signals of various zoonotic pathogens in animal reservoirs and humans have been assessed. Here, we describe the Zoonoses Structure with examples from signals and responses for four zoonotic events in the Netherlands (tularaemia, Brucella canis, West Nile virus, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)). This may serve as an example for other countries on how to collaborate in a One Health approach to signal and control emerging zoonoses.

Imported Hyalomma ticks in the Netherlands 2018-2020.

BACKGROUND: Ticks of the genus Hyalomma, which are vectors for several tick-borne diseases, are occasionally found in areas outside their endemic range including northern parts of Europe. The objective of this study was to analyse adult Hyalomma ticks that were recently found in the Netherlands. METHODS: Hyalomma ticks were morphologically identified. Cluster analysis, based upon sequence data (cox1 barcoding) for molecular identification, and pathogen detection were performed. Additionally, a cross-sectional survey of horses was conducted to actively search for Hyalomma ticks in summer 2019. Analysis of temperature was done to assess the possibility of (i) introduced engorged nymphs moulting to adults and (ii) establishment of populations in the Netherlands. RESULTS: Seventeen adult Hyalomma ticks (one in 2018, eleven in 2019, five in 2020) were found by citizens and reported. Fifteen ticks were detected on horses and two on humans. Twelve were identified as H. marginatum, one as H. rufipes and four, of which only photographic images were available, as Hyalomma sp. No Crimean-Congo haemorrhagic fever virus or Babesia/Theileria parasites were detected. One adult tick tested positive for Rickettsia aeschlimannii. In the cross-sectional horse survey, no Hyalomma ticks were found. Analysis of temperatures showed that engorged nymphs arriving on migratory birds in spring were able to moult to adults in 2019 and 2020, and that cumulative daily temperatures in the Netherlands were lower than in areas with established H. marginatum populations. CONCLUSIONS: Our results show that Hyalomma ticks are regularly introduced in the Netherlands as nymphs. Under the Dutch weather conditions, these nymphs are able to develop to the adult stage, which can be sighted by vigilant citizens. Only one human pathogen, Rickettsia aeschlimannii, was found in one of the ticks. The risk of introduction of tick-borne diseases via Hyalomma ticks on migratory birds is considered to be low. Establishment of permanent Hyalomma populations is considered unlikely under the current Dutch climatic conditions.

Giardia duodenalis multi-locus genotypes in dogs with different levels of synanthropism and clinical signs.

BACKGROUND: In dogs, infections with Giardia duodenalis are mainly caused by assemblages C and D, but also by the potentially zoonotic assemblages A and B. The aims of this study were to assess differences in assemblages (i) between dogs living mainly in close proximity to humans (synanthropic dogs) versus dogs living mainly among other dogs, (ii) between samples of dogs with or without loose stool, and (iii) related to the amount of cysts shedding. METHODS: One hundred eighty-nine qPCR Giardia positive fecal samples of dogs originating from four groups (household, sheltered, hunting, and dogs for which a veterinarian sent a fecal sample to a diagnostic laboratory) were used for genotyping. For this, multi-locus genotyping of beta-giardin, triose phosphate isomerase, and glutamate dehydrogenase and genotyping of SSU rDNA gene fragments were performed. Fecal consistency was scored (loose or non-loose stool), and cysts per gram of feces were determined with qPCR. RESULTS: Assemblage D was the most prevalent in all groups, followed by the other canid assemblage C. Also, mixed C/D was common. In two (synanthropic) household dogs, the potentially zoonotic assemblage AI was present. Although occurrence of assemblage AI in household dogs was not significantly different from dogs living among other dogs (sheltered and hunting dogs), it was significantly higher compared to dogs for which a sample was sent to a diagnostic laboratory. Dogs with assemblage D shed significantly more cysts than dogs with other assemblages (except for mixed C/D results) or dogs in which no assemblage could be determined. None of the assemblages was significantly associated with loose stool. CONCLUSION: Not only do dogs mainly shed the canid Giardia duodenalis assemblages D and/or C, the numbers of cysts per gram for the canid assemblage D were also higher than for the potential zoonotic assemblage AI. Based on the assemblages shed by dogs, the risk to public health posed by dogs is estimated to be low, even though the dogs that shed AI were synanthropic household dogs. Loose stool in infected dogs was not associated with any particular Giardia assemblage.

Host factors associated with Giardia duodenalis infection in dogs across multiple diagnostic tests.

BACKGROUND: The aim of this study was to assess potential associations between Giardia duodenalis infection in dogs, as determined by three diagnostic tests, and dog's group of origin, fecal consistency, age, sex, neuter status, and co-infections with other gastrointestinal parasites. METHODS: Fecal samples from 1291 dogs from four groups (household, shelter, hunting and clinical dogs) were tested with qPCR, rapid enzyme immunochromatographic assay (IDEXX SNAP® Giardia), and direct immunofluorescence (DFA, Merifluor) for presence of G. duodenalis. Moreover, fecal samples were tested with centrifugation sedimentation flotation (CSF) coproscopical analysis for presence of gastrointestinal parasites. Associations were expressed as odds ratios (ORs). RESULTS: Several significant associations were found, of which a few were consistent for all three tests and Giardia positivity in general (positive with at least one of these tests). Dogs older than one year were significantly less likely to test positive for Giardia than younger dogs. Group-housed dogs, especially hunting dogs, were significantly more likely to test positive for Giardia compared to household and clinical dogs. A consistently significant association with Trichuris appeared to be driven by the high prevalence in hunting dogs. Although there was no significant association between loose stool and Giardia infection in the overall population, household dogs were significantly more likely to test Giardia-positive when having loose stool. Overall, Giardia-positive dogs with loose stool shed significantly more cysts, both determined semi-quantitatively with CSF and quantitatively by qPCR, than positive dogs with no loose stool. When other gastrointestinal parasites were present, significantly fewer cysts were detected with CSF, but this was not confirmed with qPCR. CONCLUSION: Giardia is the most common gastrointestinal parasite in Dutch dogs, except for hunting dogs, in which Trichuris and strongyle-type eggs (hookworms) prevailed. Giardia infection was not significantly associated with loose stool, except for household dogs. Young dogs and group-housed dogs were significantly more often Giardia-positive. These associations were consistent across diagnostic tests. Young dogs, clinical dogs and dogs with loose stool shed Giardia cysts in the highest numbers. If another gastrointestinal parasite was present lower numbers of cysts were observed by microscope (CSF), but not with a molecular method (qPCR).

Comparing four diagnostic tests for Giardia duodenalis in dogs using latent class analysis.

BACKGROUND: To accurately diagnose giardiosis in dogs, knowledge of diagnostic test characteristics and expected prevalence are required. The aim of this work was to estimate test characteristics (sensitivity and specificity) of four commonly used diagnostic tests for detection of Giardia duodenalis in dogs. METHODS: Fecal samples from 573 dogs originating from four populations (household dogs, shelter dogs, hunting dogs and clinical dogs) were examined with centrifugation sedimentation flotation (CSF) coproscopical analysis, direct immunofluorescence assay (DFA, Merifluor Cryptosporidium/Giardia®), a rapid enzyme immunochromatographic assay (IDEXX SNAP Giardia®) and qPCR (SSU rDNA) for presence of G. duodenalis. Bayesian latent class analysis was used to determine test performance characteristics and to estimate G. duodenalis prevalence of each of the four dog populations. RESULTS: All tests were highly specific. IDEXX SNAP Giardia® showed the highest specificity (99.6%) and qPCR the lowest (85.6%). The sensitivities were much more variable, with qPCR showing the highest (97.0%) and CSF the lowest (48.2%) sensitivity. DFA was more sensitive than IDEXX SNAP Giardia®, but slightly less specific. Prevalences of G. duodenalis differed substantially between populations, with the hunting dogs showing the highest G. duodenalis prevalence (64.9%) and the household dogs the lowest (7.9%). CONCLUSIONS: This study identifies qPCR as a valuable screening tool because of its high sensitivity, whereas methods using microscopy for cyst identification or cyst wall detection should be used in situations where high specificity is required. G. duodenalis is a prevalent gastro-intestinal parasite in Dutch dogs, especially in dogs living in groups (hunting and shelter dogs) and clinical dogs.