Ixodes ricinus tick bacteriome alterations based on a climatically representative survey in Hungary.

IMPORTANCE: Climate-sensitive disease vectors, such as ticks, respond to the environment with changes in their microbiome. These changes can affect the emergence or re-emergence of various vector-borne pathogens, such as the causative agent of Lyme borreliosis (LB) or tick-borne encephalitis. This aspect is particularly emphasized in light of climate change. The climatically representative assessment of microbiome differences in various developmental stages of the most common Central European tick species, Ixodes ricinus, deepens our understanding of the potential climatic factors behind microbial relative abundance and interaction changes. This knowledge can support the development of novel disease vector control strategies.

First detection of Ixodiphagus hookeri (Hymenoptera: Encyrtidae) in Ixodes ricinus ticks (Acari: Ixodidae) from multiple locations in Hungary.

The parasitoid wasp, Ixodiphagus hookeri (Hymenoptera: Encyrtidae), is the natural enemy of a wide range of hard and soft tick species. While these encyrtid wasps are supposed to be distributed worldwide, only a few studies report on their actual distribution around the globe. Within a shotgun sequencing-based metagenome analysis, the occurrence of I. hookeri was screened at multiple Ixodes ricinus (Acari: Ixodidae) tick sampling points in Hungary to contribute to the assessment of the distribution patterns of the parasitoid wasps in Central Europe. To our knowledge, the first report of the species in Hungary and the description of the southernmost I. hookeri associated geoposition in Central Europe took place within our study. I. hookeri infested I. ricinus nymphs were detected at five sampling points in Hungary. The results show that the exact distribution range of I. hookeri is still barely studied. At the same time, unprecedented public health issues being brought about by climate change might require steps toward the exploitation of the tick biocontrol potential and as an ecological bioindicator role of the parasitoid wasp in the future.

Serodiagnostic difficulties and possibilities of heartworm disease in regions where both Dirofilaria immitis and Dirofilaria repens infections occur.

This retrospective study was performed on 71 dogs which had been admitted for heartworm screening or with clinical suspicion of heartworm disease. The examination methods included polymerase chain reaction (PCR) to identify Dirofilaria immitis and/or Dirofilaria repens infections and a heartworm antigen (Ag) test (VetScan). By using PCR, 26 dogs were found positive only for Dirofilaria immitis (Group 1), while 21 dogs for both D. immitis and D. repens (Group 2). Group 3 included 24 dogs with D. repens infection only according to the PCR results. The sensitivity of the VetScan Ag test for the Group 1 and 2 animals proved to be 97.7% (95% Blaker confidence interval; CI 89.0%-99.9%). The specificity of the VetScan Ag test, calculated from the results of Group 3, was found to be 66.7% (95% CI 45.6%-83.1%), which was lower than that reported from the USA, where D. repens does not occur. In cases when PCR results were positive for D. repens but negative for D. immitis, the occult dirofilariosis was the likely explanation for the positive D. immitis Ag tests. These observations highlight the importance of performing more Ag tests simultaneously in those areas where both Dirofilaria species are present.

Ticks and Tick-Borne Infections of Dogs in Two Jordanian Shelters.

Shelters in Jordan accommodate a huge number of dogs, which are rescued as stray dogs from different cities of the country, but their health receives almost no attention. The aim of this study was to examine tick infestation as well as tick-borne protozoa and bacteria of 80 randomly sampled dogs in two Jordanian shelters. Ticks identified as Rhipicephalus sanguineus sensu lato were found on 14 out of 27 animals in a shelter. No ticks were found on dogs in the other shelter. A total of 42 (52.5% [95% confidence interval: 41.7-63.1]) dogs were infected with one or two pathogens. The DNA of three protozoal (Hepatozoon canis, Babesia vogeli, and Babesia negevi) and two bacterial (Anaplasma platys and Candidatus Bartonella merieuxii) species were detected in the blood samples. To the best of the authors' knowledge, except for H. canis, these species are reported for the first time from Jordan.

Detection of Flea-Borne Pathogens from Cats and Fleas in a Maltese Shelter.

In a sanctuary located on the island of Malta, 23 clinically healthy cats randomly selected were sampled for blood and fleas. Only fleas were collected from 35 cats. All fleas were identified as Ctenocephalides felis, except for one specimen of Ctenocephalides canis. To the best of the authors' knowledge, this may be the first time to establish the occurrence of Bartonella koehlerae and B. clarridgeiae, as well as of Candidatus Mycoplasma haemominutum in the blood samples of 11 cats (47.82% [95% CI: 29.33-67.04]) with conventional PCR assays. One or more pathogens were found in 54 (96.42% [95% CI: 86.74-99.70]) out of 56 pooled flea samples, the most prevalent was Rickettsia felis. The DNA of Bartonella henselae, the commonest etiological agent of cat scratch disease, was detected first time in a pooled flea sample of a cat.

The current situation of canine dirofilariosis in Hungary.

Between April and September 2017, blood samples were collected from 344 randomly selected dogs older than 1 year in 180 settlements of 19 counties in Hungary. The dogs lived exclusively outdoors, had never travelled and had neither been examined for Dirofilaria infection nor treated against mosquitoes with insecticides or/and filarioid worms with macrocyclic lactones. Dirofilaria infection was examined with a modified Knott's test for microfilariae, DiroCHEK®, for the presence of D. immitis antigen, as well as by multiplex and conventional PCR. Altogether, 77 (22.4%) dogs living in 58 settlements of 17 counties were found to be infected with one or both Dirofilaria species based on the PCR techniques. Twenty-eight (8.1%) and 38 (11.1%) dogs were infected with D. immitis and D. repens, respectively. Coinfections were recorded in 11 samples (3.2%) collected in 11 locations of 8 counties. The results confirmed that both dirofilarioses are endemic in dogs and the eastern areas of the country are hyperendemic for heartworm disease. Temperature showed a significant association with the prevalence of D. immitis (OR 2.41, 95% CI 1.24-4.86, p = 0.012) but not with that of D. repens (OR 1.37, 95% CI 0.78-2.47, p = 0.286). The prevalence of neither D. immitis (OR 0.99, 95% CI 0.98-1.00, p = 0.213) nor D. repens (OR 1.01, 95% CI 0.99-1.01, p = 0.094) showed a significant correlation with precipitation. The number of yearly growing degree days (GDD) based on the lifecycle of Dirofilaria in mosquitoes ranged between 3.73 and 7.57 for the Hungarian districts. The GDD showed a significant positive association with the prevalence of D. immitis (OR 2.38, 95% CI 1.43-4.15, p = 0.001) and a non-significant positive relationship with that of D. repens (OR 1.25, 95% CI 0.83-1.95, p = 0.291).

Survey of lungworm infection of domestic cats in Hungary.

From 61 settlements of 12 Hungarian counties, 303 domestic cats were included in this survey. Between autumn 2016 and spring 2018, fresh faecal samples were randomly collected and examined by flotation and by the Baermann-Wetzel method for the presence of lungworm infection. No eggs of Eucoleus aerophilus were detected. Morphological identification of first instar larvae (L1) was also carried out. In the faeces of 60 cats (19.8%) from 17 settlements and Budapest, L1 of Aelurostrongylus abstrusus were found. More than half of the cats were from the western part of the country. The average number of larvae per gram of faeces was 190.2 ± 304.88. These results are in line with the former findings on the prevalence of aelurostrongylosis of domestic cats in Hungary. In addition, Oslerus rostratus was also found for the first time in the faecal samples of three cats from the eastern part of the country, infected also with Ae. abstrusus. The average age (2.51 ± 1.26 years) of infected cats indicates that lungworm infection is more common among younger cats. No relationship was found between the lung-worm infection and the sex of cats. Non-neutered cats had a significantly higher proportion of lungworm infections. Two-thirds of the infected cats were apparently healthy, and only 19 individuals showed clinical signs of respiratory disorders.

The first feline and new canine cases of Thelazia callipaeda (Spirurida: Thelaziidae) infection in Hungary.

BACKGROUND: In Europe, the first Thelazia callipaeda infections were found in the eyes of some dogs in Italy three decades ago. Since that time, this vector-borne nematode species has been diagnosed in domestic and wild carnivores and humans in some western European countries. During the last few years, autochthonous thelaziosis of dogs, red foxes, cats and humans has also been reported from eastern Europe. The first cases of ocular infections caused by T. callipaeda have been described in dogs living in the eastern and southern part of Slovakia and Hungary. METHODS: Whitish parasites found in the conjuctival sac and/or under the third eyelid of one or both eyes of animals were removed and morphologically identified according to species and sex. To confirm the morphological identification with molecular analysis a single step conventional PCR was carried out. RESULTS: A total of 116 adult worms (1-37 per dog, median: 7, IQR: 14.5 and 7 from a cat) were collected from the eyes of 11 animals. Nematodes were identified as T. callipaeda according to the morphological keys and molecular analysis. The sequences of a portion of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene were identical to those representing T. callipaeda haplotype 1, previously reported in neighbouring and other European countries. Since the infected cat and dogs had never travelled abroad, all of the cases were autochthonous thelaziosis. CONCLUSIONS: The present study reports the first case of thelaziosis in a cat and new cases in 10 dogs found in the southern and northern region of Hungary, respectively. Further studies are needed to clarify whether wild carnivores (e.g. red foxes, golden jackals) may act as reservoirs of this eyeworm species in the country.

The effect of weather variables on the flight activity of horseflies (Diptera: Tabanidae) in the continental climate of Hungary.

Although the tabanid species and populations occurring in eastern central Europe (Carpathian Basin) are thoroughly studied, there are only sporadic data about the influence of weather conditions on the abundance and activity of horseflies. To fill in this lack, in Hungary, we performed a 3-month summer survey of horsefly catches registering the weather parameters. Using common canopy traps and polarization liquid traps, we found the following: (i) rainfall, air temperature, and sunshine were the three most important factors influencing the trapping number of tabanids. (ii) The effect of relative air humidity H on tabanids was indirect through the air temperature T: H ≈ 35 % (corresponding to T ≈ 32 °C) was optimal for tabanid trapping, and tabanids were not captured for H ≥ 80 % (corresponding to T ≤ 18 °C). (iii) A fast decrease in the air pressure enhanced the trapping number of both water-seeking and host-seeking horseflies. (iv) Wind velocities larger than 10 km/h reduced drastically the number of trapped tabanids. Our data presented here may serve as a reference for further investigations of the effect of climate change on tabanids in Europe.

Seasonality and daily activity of male and female tabanid flies monitored in a Hungarian hill-country pasture by new polarization traps and traditional canopy traps.

Blood-sucking female tabanid flies cause serious problems for animals and humans. For the control of tabanids, the knowledge about their seasonality and daily activity is of great importance. Earlier, only traditional traps capturing exclusively female tabanids have been used to survey tabanid activity. The data of such temporal trapping do not reflect correctly the activity of male and female tabanid flies. Our major aim was to monitor the trapping numbers of male and female tabanids during a 3-month summer survey in Hungary. We used (i) conventional canopy traps with liquid traps on the ground beneath the canopy and (ii) L-shaped sticky traps with vertical and horizontal components. Our other goal was to compare the efficiencies of the two components of each trap type used. We observed two greater peaks of the trapping number of tabanids. These peaks started with increased catches of female tabanids captured by the canopy traps and the vertical sticky traps and ended with a dominance of male and female tabanids caught by the liquid traps and the horizontal sticky traps. The swarming periods were interrupted by rainy/cool days, when the number of tabanids decreased drastically. Among the 17 species, six dominated and composed 89.4% of the captured tabanids: Haematopota pluvialis, Tabanus tergestinus, Tabanus bromius, Tabanus maculicornis, Tabanus bovinus and Atylotus loewianus. The number of water-seeking male and female tabanids rose up to 12-13 h and then decreased but had a secondary peak at about 17 h. The stochastic weather change and the communities of different species resulted in large standard deviations of the averaged number of tabanids in the course of a day. The horizontally polarizing (liquid and horizontal sticky) traps captured both male and female specimens and were about three times more efficient than the canopy and vertical sticky traps that caught only females. The results of the horizontal sticky traps corresponded to those of the liquid traps, while the catches of the vertical sticky traps corresponded to those of the canopy traps. The catches of the used trap types reflected well the species and water/host-seeking composition of tabanids.

Seroprevalence of some vector-borne infections of dogs in Hungary.

The first comprehensive study on the prevalence of canine vector-borne pathogens (Anaplasma phagocytophilum, Borrelia burgdorferi, Ehrlichia canis, and Dirofilaria immitis) was carried in Hungary because, except for babesiosis and dirofilariosis caused by Dirofilaria repens, there were no data on their regional distribution and prevalence. In 2011 and 2012, 1305 blood samples were collected from randomly selected, apparently healthy pet dogs in 167 localities of 19 counties of Hungary. All sera samples from dogs were screened for simultaneous qualitative detection of circulating antibodies to E. canis and B. burgdorferi sensu lato and A. phagocytophilum and D. immitis antigen using SNAP(®) 4Dx (IDEXX Laboratories). Overall, 170 dogs (13.0%; 95% confidence interval [CI] 11-15) were serologically positive to one or more of the tested pathogens. A. phagocytophilum was the most prevalent pathogen detected in 102 dogs by antibody titers (7.9%, 95% CI 6.5-9.5), followed by D. immitis (2.4%, 95% CI 1.0-4.0, n=64) and B. burgdorferi (0.4%, 95% CI 0.0-1.1, n=11) out of 1305 tested dogs. The least prevalent infection was with E. canis, with only two positive dogs (0.16%, 95% CI 0.03-0.6). Co-infection was found in eight dogs (0.61%, 95% CI 0.29-1.21), of which seven were seropositive to two pathogens (five with A. phagocytophilum and D. immitis, two with A. phagocytophilum and B. burgdorferi). One dog was serologically positive to three pathogens (A. phagocytophilum, B. burgdorferi, and D. immitis). Purebred and crossbred animals did not show significantly different levels of seropositivity. There was no significant association between the gender and the results of diagnostic testing. Logistic regression analysis showed a higher chance of seropositivity in the older dogs.

New kind of polarotaxis governed by degree of polarization: attraction of tabanid flies to differently polarizing host animals and water surfaces.

Aquatic insects find their habitat from a remote distance by means of horizontal polarization of light reflected from the water surface. This kind of positive polarotaxis is governed by the horizontal direction of polarization (E-vector). Tabanid flies also detect water by this kind of polarotaxis. The host choice of blood-sucking female tabanids is partly governed by the linear polarization of light reflected from the host's coat. Since the coat-reflected light is not always horizontally polarized, host finding by female tabanids may be different from the established horizontal E-vector polarotaxis. To reveal the optical cue of the former polarotaxis, we performed choice experiments in the field with tabanid flies using aerial and ground-based visual targets with different degrees and directions of polarization. We observed a new kind of polarotaxis being governed by the degree of polarization rather than the E-vector direction of reflected light. We show here that female and male tabanids use polarotaxis governed by the horizontal E-vector to find water, while polarotaxis based on the degree of polarization serves host finding by female tabanids. As a practical by-product of our studies, we explain the enigmatic attractiveness of shiny black spheres used in canopy traps to catch tabanids.

Polarotactic tabanids find striped patterns with brightness and/or polarization modulation least attractive: an advantage of zebra stripes.

The characteristic striped appearance of zebras has provoked much speculation about its function and why the pattern has evolved, but experimental evidence is scarce. Here, we demonstrate that a zebra-striped horse model attracts far fewer horseflies (tabanids) than either homogeneous black, brown, grey or white equivalents. Such biting flies are prevalent across Africa and have considerable fitness impact on potential mammalian hosts. Besides brightness, one of the likely mechanisms underlying this protection is the polarization of reflected light from the host animal. We show that the attractiveness of striped patterns to tabanids is also reduced if only polarization modulations (parallel stripes with alternating orthogonal directions of polarization) occur in horizontal or vertical homogeneous grey surfaces. Tabanids have been shown to respond strongly to linearly polarized light, and we demonstrate here that the light and dark stripes of a zebra's coat reflect very different polarizations of light in a way that disrupts the attractiveness to tabanids. We show that the attractiveness to tabanids decreases with decreasing stripe width, and that stripes below a certain size are effective in not attracting tabanids. Further, we demonstrate that the stripe widths of zebra coats fall in a range where the striped pattern is most disruptive to tabanids. The striped coat patterns of several other large mammals may also function in reducing exposure to tabanids by similar mechanisms of differential brightness and polarization of reflected light. This work provides an experimentally supported explanation for the underlying mechanism leading to the selective advantage of a black-and-white striped coat pattern.