Monitoring of ticks and their pathogens from companion animals obtained by the "tekenscanner" application in The Netherlands.

Ticks are vectors for many pathogens of veterinary and medical interest. In order to monitor ticks and tick-borne pathogens, the "Tekenscanner" (Dutch for Tick scanner), a citizen science project, was launched in The Netherlands. It is a smartphone application for pet-owners to get ticks from their dog or cat, identified and checked for pathogens for free. At the same time, information about the pet and the geographic location of tick infestation becomes available for research. The application was launched in 2018, and the results of the first 6 months after launch of the app were reported. Ticks were identified based on morphology, and DNA was extracted and amplified by a panel of tick-borne pathogen-specific primers. Next, the amplicons were subjected to reverse line blot with specific probes for important pathogens to determine their presence or absence. The present paper describes the results of 2019 and 2020. There were 2260 ticks collected from 871 dogs and 255 cats (26 ticks were from an unknown host) and all pet owners were informed about the results. Four species of ticks were collected: Ixodes ricinus (90.0%), Ixodes hexagonus (7.3%), Dermacentor reticulatus (2.8%) and Rhipicephalus sanguineus (0.1%). Ixodes ricinus was the tick with the most divergent pathogens: Anaplasma sp. (1.3%), Babesia sp. (0.8%), Borrelia spp. (4.8%), Neoehrlichia sp. (3.7%) and Rickettsia helvetica (12.6%). In I. hexagonus, R. helvetica (1.8%) and Babesia sp. (0.6%) were detected and Rickettsia raoultii in D. reticulatus (16.2%). One of the two nymphs of R. sanguineus was co-infected with Borrelia and R. helvetica and the other one was uninfected. The high numbers of different pathogens found in this study suggest that companion animals, by definition synanthropic animals, and their ticks can serve as sentinels for emerging tick-borne pathogens.

Theileria sp. OT3 and other tick-borne pathogens in sheep and ticks in Italy: molecular characterization and phylogeny.

PCR Reverse Line Blot (RLB) hybridization and sequencing were used to determine the dynamics of infection with tick-borne pathogens in one hundred apparently healthy sheep in Italy. Blood samples were tested once prior to the onset of the grazing season (June 2010) and once after the end of the grazing season (August 2010). Ticks collected from sheep and from the vegetation were also tested by PCR/RLB. Before grazing, 56% of the sheep harbored several tick-borne pathogens: Anaplasma ovis was the most prevalent (41%), followed by A. ovis co-infected with Theileria sp. OT3 (14%). After grazing, 87% of sheep were positive for A. ovis alone (41%), co-infected with Theileria sp. OT3 (8%) or co-infected with Babesia motasi (5%). Other sheep were infected with Anaplasma phagocytophilum alone (20%), co-infected with B. motasi (7%) or with Theileria sp. OT3 (5%) (p<0.001). After grazing, sheep were significantly more infected with tick-borne pathogens than before grazing. Ticks collected were all Haemaphysalis punctata (n-89) and 36% were positive for A. ovis, Ehrlichia ovina and A. ovis combined with A. phagocytophilum. Phylogenetic analysis including isolates from countries in the Mediterranean Basin show circulation of the same variants of Theileria sp. OT3, whereas two different geographical origins for the isolates of A. ovis and A. phagocytophilum were identified. This is the first report from Italy of Theileria sp. OT3 in sheep, whereas the detection of Ehrlichia ovina in ticks is worth noting, and the presence of A. phagocytophilum in sheep and in ticks poses a potential public health risk.

Ixodid ticks on ruminants, with on-host initiated moulting (apolysis) of Ixodes, Haemaphysalis and Dermacentor larvae.

To screen the host-dependent abundance of hard tick (Acari: Ixodidae) developmental stages on ruminants in South Hungary, red and roe deer, as well as goats and sheep were examined in a season, when larvae and nymphs are active. Altogether 2271 ticks were collected. In the relevant period the prevalence of tick-infestation was significantly higher among goats, than among sheep kept in the very same area, most likely in association with the browsing habit of the former. Roe deer and goats were found to be important hosts for Ixodes ricinus and Haemaphysalis concinna larvae, in contrast to the view that this stage does not usually feed on medium-sized mammals. Interestingly, one third of I. ricinus larvae and one larva of H. concinna and of Dermacentor reticulatus collected from goats in the same herd in August have started the moulting process (showed apolysis) on their host, despite being three-host ticks. This is the first survey involving four species of domestic and wild ruminants in Europe to compare the host-preference of ixodid ticks in the same region.

Defining the concept of 'tick repellency' in veterinary medicine.

Although widely used, the term repellency needs to be employed with care when applied to ticks and other periodic or permanent ectoparasites. Repellency has classically been used to describe the effects of a substance that causes a flying arthropod to make oriented movements away from its source. However, for crawling arthropods such as ticks, the term commonly subsumes a range of effects that include arthropod irritation and consequent avoiding or leaving the host, failing to attach, to bite, or to feed. The objective of the present article is to highlight the need for clarity, to propose consensus descriptions and methods for the evaluation of various effects on ticks caused by chemical substances.

Prevalence of Coxiella burnetii in ticks after a large outbreak of Q fever.

Q fever has emerged as an important human and veterinary public health problem in the Netherlands with major outbreaks in three consecutive years. Goat farms are probably the prime source from which Coxiella burnetii have spread throughout the environment, infecting people living in the vicinity. Coxiella burnetii infection not only spilled over from animal husbandry to humans but could also have spread to neighbouring wildlife and pets forming novel reservoirs and consequently posing another and lingering threat to humans, companion animals and livestock. In these cases, transmission routes other than airborne spread of contaminated aerosols may become significant. Therefore, the role of ticks in the transmission of Coxiella burnetii in the current situation was investigated. A total of 1891 questing Ixodes ricinus ticks and 1086 ticks feeding on pets, wildlife and livestock were tested by a recently developed multiplex Q-PCR. All ticks were negative, except for a few ticks feeding on a herd of recently vaccinated sheep. Coxiella-positive ticks were not detected after resampling this particular herd three months later. Based on these data we conclude that the current risk of acquiring Q fever from questing ticks in the Netherlands is negligible. However, for future risk assessments, it might be relevant to sample more ticks in the vicinity of previously C. burnetii infected goat farms and to assess whether C. burnetii can be transmitted transovarially and transstadially in I. ricinus ticks.

Crimean-Congo hemorrhagic fever in Europe: current situation calls for preparedness.

During the last decade Crimean-Congo hemorrhagic fever (CCHF) emerged and/or re-emerged in several Balkan countries, Turkey, southwestern regions of the Russian Federation, and the Ukraine, with considerable high fatality rates. Reasons for re-emergence of CCHF include climate and anthropogenic factors such as changes in land use, agricultural practices or hunting activities, movement of livestock that may influence host-tick-virus dynamics. In order to be able to design prevention and control measures targeted at the disease, mapping of endemic areas and risk assessment for CCHF in Europe should be completed. Furthermore, areas at risk for further CCHF expansion should be identified and human, vector and animal surveillance be strengthened.

Preliminary evaluation of the BrEMA1 gene as a tool for associating babesia rossi genotypes and clinical manifestation of canine Babesiosis.

Babesia rossi, an intraerythrocytic protozoan, causes a severe, often life-threatening disease of domestic dogs. Dogs treated early for B. rossi infection usually recover from the disease, but dogs left untreated or treated at a later stage of infection seldom survive. Dogs infected with B. rossi have varied clinical manifestations that can be categorized as uncomplicated (with a good prognosis) or complicated (with a poor prognosis). One hundred twenty-one blood samples were collected from dogs presented to the Onderstepoort Veterinary Academic Hospital and diagnosed with babesiosis by the use of a thin blood smear. An additional 20 samples were obtained from Babesia-infected dogs from private clinics around the Onderstepoort, Johannesburg, Durban, White River, and Cape Town areas. The samples were screened by PCR targeting the Babesia rossi erythrocyte membrane antigen gene (BrEMA1) and by sequencing of the polymorphic region (i.e., region with a variable number of hexapeptide repeats). Analysis of PCR products revealed 11 different gene profiles, visualized by gel electrophoresis. Twelve distinct BrEMA1 genotypes were identified by sequencing, but the numbers of hexapeptide repeats varied from 6 to 31 (classified as genotype6 to genotype31). The genotypes were retrospectively compared to the clinical case data. The most frequently encountered B. rossi parasites were those attributed to genotype19 (36.2%), genotype28 and genotype29 (20.6% each), and genotype11 (12.7%). These genotypes were also the ones associated with the poorest prognosis. This preliminary finding suggests clinically important differences between the various B. rossi genotypes identified.

Detection of a Theileria species in dogs in South Africa.

A Theileria species was detected by PCR in blood samples collected from dogs in the Pietermaritzburg area and was also found in dogs presented at the Outpatients Clinic of the Onderstepoort Veterinary Academic Hospital (OVAH), in the Pretoria area, South Africa. In the Pietermaritzburg area, 79 of the 192 samples were positive, while 3 out of 1137 of the Onderstepoort samples were positive. Three positive samples from Pietermaritzburg were co-infected with Ehrlichia canis. PCR positive samples were further analysed by the Reverse Line Blot (RLB) and sequence analysis. Phylogenetic analysis of the 18S rRNA full-length gene sequences of one sample (VT12) from Pietermaritzburg and two samples from OVAH (BC281 and BC295) revealed a close relationship with sequences of Theileria species (sable). Clinical signs of the dogs that were examined at Pietermaritzburg and OVAH included an immune-mediated condition with severe thrombocytopenia. These findings identify a Theileria sp. in dogs for the first time in South Africa and add yet another microorganism to the growing list of haemoprotozoan parasites infecting dogs worldwide. The clinical significance of this infection in dogs is poorly resolved.

Reliminary survey of ticks (Acari: Ixodidae) on cattle in Central Equatoria State, Southern Sudan.

In a preliminary survey conducted in 2005, the species composition and seasonality of ticks infesting cattle in Central Equatoria State, Southern Sudan was determined. Three locations were selected (Gumbo, Khor Rumla and Nyaing) and surveyed every 3 months. Two cattle herds in each of the three locations were visited four times during the study period. Total body collections of ticks were made from each of five cattle (Nilotic Zebu breed) kept in six different herds. Four tick genera and ten species were identified. The tick species identified were Amblyomma lepidum, Amblyomma variegatum, Boophilus annulatus, Boophilus decoloratus, Hyalomma marginatum rufipes, Hyalomma truncatum, Rhipicephalus appendiculatus, Rhipicephalus evertsi evertsi, Rhipicephalus praetextatus and Rhipicephalus sanguineus group. The highest number of ticks was collected in October during the rainy season. A finding of great significance was that R. appendiculatus, vector of East Coast fever, has now firmly established itself throughout the year with possible implications for cattle production in Central Equatoria State.

Repeated high dose imidocarb dipropionate treatment did not eliminate Babesia caballi from naturally infected horses as determined by PCR-reverse line blot hybridization.

Imidocarb treatment of horses infected with Babesia caballi is supposed to eliminate the infection, but data on the efficacy of this treatment is scarce. The study presented here concerns four Paso Fino horses, which were imported into the island of Curacao on the basis of a piroplasmosis negative complement fixation test (CFT). Upon re-testing with an indirect fluorescent antibody test immediately after arrival in Curacao, two horses appeared to have antibodies to B. caballi and all horses had antibodies to Theileria equi. Subsequent testing with polymerase chain reaction combined with a reverse line blot yielded positive results for both agents in all four horses. Treatment with five consecutive doses of imidocarb dipropionate (4.7 mg/kg BW im q 72 h), temporarily resulted in negative results, but B. caballi and T. equi were detected again in the samples taken at 6 and 18 weeks after completion of the treatment. These results confirm that the CFT is not a suitable test for pre-import testing and that even high dose treatment with imidocarb may not be capable of eliminating B. caballi and T. equi infections from healthy carriers.

Host cell-specific protein expression in vitro in Ehrlichia ruminantium.

Ehrlichia ruminantium, a tick-transmitted pathogen, is the causative agent of heartwater in ruminants. In this study, a proteomic approach was used to identify host cell-specific E. ruminantium proteins encoded by the map1 multigene family, expressed in vitro in bovine endothelial and tick cell cultures. Two-dimensional gel electrophoresis combined with mass spectrometry analysis was used to establish the identities of immunodominant proteins. Proteins extracted from E. ruminantium-infected endothelial cells were shown to be products of the map1 gene, whereas tick cell-derived E. ruminantium proteins were products of a different gene, map1-1. The expressed proteins were found to be glycosylated. Differential expression of MAP1 family proteins in vitro in mammalian and tick cell cultures indicates that the map1 multigene family might be involved in the adaptation of E. ruminantium to the mammalian host and vector tick.

Identification of Salp15 homologues in Ixodes ricinus ticks.

The 15-kDa Ixodes scapularis salivary gland protein Salp15 protects Borrelia burgdorferi sensu stricto from antibody-mediated killing and facilitates infection of the mammalian host. In addition, Salp 15 has been shown to inhibit T-cell activation. We determined whether Ixodes ricinus, the major vector for Lyme borreliosis in Western Europe, also express salp15-related genes. We show that engorged I. ricinus express salp15 and we have identified three Salp15 homologues within these ticks by reverse transcriptase-polymerase chain reaction (RT-PCR). One of the predicted proteins showed 80% similarity to I. scapularis Salp15, evenly distributed over the entire amino acid sequence, whereas the two other predicted proteins showed approximately 60% similarity, mainly confined to the signal sequence and C-terminus. Comparison of the DNA and protein sequences with those deposited in several databases indicates that these proteins are part of a Salp15 family of which members are conserved among different Ixodes species, all capable of transmitting B. burgdorferi sensu lato. This suggests that these Salp15 homologues could also play a role in the transmission of diverse Borrelia species and in inhibition of T-cell activation.

Molecular detection of Ehrlichia ruminantium infection in Amblyomma variegatum ticks in The Gambia.

In West Africa, losses due to heartwater disease are not known because the incidence/prevalence has not been well studied or documented. To develop a diagnostic tool for molecular epidemiology, three PCR-based diagnostic assays, a nested pCS20 PCR, a nested map1 PCR and a nested reverse line blot (RLB) hybridization assay, were evaluated to determine their ability to detect infection in vector ticks, by applying them simultaneously to A. variegatum field ticks to detect Ehrlichia ruminantium, the causative agent of heartwater. The nested pCS20 PCR assay which amplified the pCS20 gene fragment showed the highest detection performance with a detection rate of 16.6%; the nested map1 PCR, which amplified the gene encoding the major antigenic protein1 (map1 gene) showed a detection rate of 11% and the RLB, based on the 16S rDNA sequence of anaplasma and ehrlichial species, detected 6.2%. The RLB, in addition, demonstrated molecular evidence of Ehrlichia ovina, Anaplasma marginale and Anaplasma ovis infections in The Gambia. Subsequently, the pCS20 assay was applied to study the prevalence and distribution of E. ruminantium tick infection rates at different sites in five divisions of The Gambia. The rates of infection in the country ranged from 1.6% to 15.1% with higher prevalences detected at sites in the westerly divisions (Western, Lower River and North Bank; range 8.3-15.1%) than in the easterly divisions (Central River and Upper River; range 1.6-7.5%). This study demonstrated a gradient in the distribution of heartwater disease risk for susceptible livestock in The Gambia which factor must be considered in the overall design of future upgrading programmes.

Differential transcription of the major antigenic protein 1 multigene family of Ehrlichia ruminantium in Amblyomma variegatum ticks.

The rickettsial pathogen Ehrlichia ruminantium causes heartwater in ruminants and is transmitted by ticks of the genus Amblyomma. The map1 gene, encoding the major surface protein MAP1, is a member of a multigene family containing 16 paralogs. In order to investigate differential transcription of genes of the map1 multigene family in vivo in unfed and feeding ticks, RNA was extracted from midguts and salivary glands of E. ruminantium-infected adult female Amblyomma variegatum ticks and analysed by RT-PCR using MAP1 paralog-specific primers. In unfed ticks, only transcripts from the map1-1 gene were observed in midguts and no transcripts were detected in salivary glands. In feeding ticks, map1-1 transcripts were more abundant in midguts whereas high levels of map1 transcripts were observed in salivary glands. Our results show that differential transcription of genes of the E. ruminantium map1 cluster occurs in vivo in different tissues of infected ticks before and during transmission feeding, indicating that this multigene family may be involved in functions of biological relevance in different stages of the life cycle of E. ruminantium.

The known distribution and ecological preferences of the tick subgenus Boophilus (Acari: Ixodidae) in Africa and Latin America.

A compilation of the known distribution of Boophilus ticks in Africa and Latin America is presented, together with details on climate preferences. B. annulatus is recorded mainly in the western part of a strip from the equator to parallel 20 degrees N. It associates with woodlands and forests (lowland rain forest and secondary grassland). This species is also present in the Mediterranean region, associated to woodland and open areas. B. decoloratus extends southern to parallel 20 degrees N, in woodland with montane vegetation and Zambezian miombo; some records have been collected in the highveld grassland. B. geigyi is mainly collected in the western range of a stripe extending between parallels 5 degrees N and 18 degrees N, associated with Sudanian woodland, lowland rain forest with secondary grassland and woodland. Confirmed records of microplus in Africa are restricted to Malagasy region and south and eastern Africa, being predominant in the Zambezian miombo, deciduous forest with secondary grassland, and woodland. In Latin America, microplus is abundant in the Mesoamerican corridor to Venezuela and Colombia, and southern in Brazil and Argentina. The tick is mainly associated to the biomes of Chaco and Pampas in Argentina, the North-central moist Andes, the Atlantic forest (southern range) and the moist Meso-American vegetation (northern range). Most collections of B. annulatus and B. geigyi came from areas where winter minimum temperature is above 15 degrees C, maximum temperatures remain between 33 and 36 degrees C and maximum rainfall is recorded between June and September. B. decoloratus and African B. microplus are recorded in sites with low temperatures in May-September. Minimum temperature requirements are similar for both B. decoloratus and African B. microplus, and both are around 4 degrees C less than the value recorded for collections of Latin-American B. microplus. The rainfall pattern observed for decoloratus shows a minimum in May and June. The requirements of total rainfall are highest for B. microplus in Latin America, while records of African B. microplus are concentrated in areas of low rainfall between May and October, and high rainfall between November and March (low rainfall in the same period for B. decoloratus). Statistical analysis revealed the existence of populations (demes) with ecologically different requirements within each tick species. Both B. annulatus and B. decoloratus showed many different demes clearly associated to defined areas. The collections of Latin American B. microplus are very homogeneous according climate preferences and well separated from the African counterpart.

Molecular and serological detection of A. centrale- and A. marginale-infected cattle grazing within an endemic area.

A reverse line blot hybridization (RLB) one-stage nested PCR (nPCR) for Anaplasma centrale and a nested PCR for Anaplasma marginale were used to detect infected cattle grazing within an endemic region in Israel. A novel set of PCR primers and oligonucleotide probes based on a 16S ribosomal RNA gene was designed for RLB detection of both Anaplasma species, and the performance of the molecular assays compared. The immunofluorescent antibody test (IFA) was used to detect antibodies to both Anaplasma species, whereas, a highly sensitive and specific competitive enzyme-linked immunosorbent assay (cELISA) was used to detect antibodies in A. centrale-vaccinated cattle. The RLB and the nested PCR procedures showed bacteremia with sensitivity of 50 infected erythrocytes per milliliter. Up to 93% of the A. centrale vaccinates carried specific antibodies that were detected by cELISA, and up to 71% of the vaccinated cattle were found to be naturally infected with A. marginale according to the PCR and the RLB assays. Nevertheless, no severe outbreaks of A. marginale infection occurred among vaccinated herds in this endemic region. It appears that both, molecular tools and serology are useful for evaluation of the vaccine efficacy. In the light of wide natural field infection with A. marginale, strong recommendations to continue the A. centrale vaccination program regime will continue until a new generation of non-blood-based vaccine will be developed.

Molecular characterization of Theileria species associated with mortality in four species of African antelopes.

Pathogen DNA was isolated from roan antelope (Hippotragus equinus), sable antelope (Hippotragus niger), greater kudu (Tragelaphus strepsiceros), and common gray duiker (Sylvicapra grimmia) in South Africa whose deaths were attributed to either theileriosis or cytauxzoonosis. We developed Theileria species-specific probes used in combination with reverse line blot hybridization assays and identified three different species of Theileria in four African antelope species. The close phylogenetic relationship between members of the genera Theileria and Cytauxzoon, similarities in the morphologies of developmental stages, and confusion in the literature regarding theileriosis or cytauxzoonosis are discussed.

[Autochthonous babesiosis in dogs in The Netherlands associated with local Dermacentor reticulatus ticks]. / Autochtone babesiose bij de hond in Nederland geassocieerd met lokale Dermacentor reticulatus teken.

In the spring and autumn of 2004, 20 respectively 3 cases of autochthonous canine babesiosis were diagnosed in the Netherlands, four of which ended fatally. Adult Dermacentor reticulatus ticks were found on four dogs. Case descriptions and diagnostics of this B. canis outbreak are discussed in more detail.

Borrelia burgdorferi infections with special reference to horses. A review.

This review discusses the literature on B. burgdorferi infections in view of the rising incidence of this infection in general and the increasing concerns of horse owners and equine practitioners. Lyme disease, the clinical expression of Borrelia infections in man is an important health problem. The geographic distribution of B. burgdorferi infections in equidae should resemble that of human cases because the vector tick involved, Ixodes ricinus, feeds on both species and, indeed, the infection has been established many times in horses. However, a definite diagnosis of the disease "Lyme borreliosis" in human beings as well as in horses and other animals is often difficult to accomplish. Although a broad spectrum of clinical signs has been attributed to B. burgdorferi infections in horses, indisputable cases of equine Lyme borreliosis are extremely rare so far, if they exist at all.