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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

[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.

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.

Expression of genes encoding two major Theileria annulata merozoite surface antigens in Escherichia coli and a Salmonella typhimurium aroA vaccine strain.

The genes, Tams1-1 and Tams1-2, encoding the 30-and 32-kDa major merozoite surface antigens of Theileria annulata (Ta), have recently been cloned and characterized. Both genes encode a protein of 281 amino acids (aa) containing a putative hydrophobic N-terminal signal peptide. Another hydrophobic stretch is predicted at the C terminus which probably functions to anchor the protein in the membrane of the merozoite and piroplasm. Here, we report the successful expression of both Tams1-1 and Tams1-2 in Escherichia coli (Ec) using gene fragments lacking both hydrophobic domains. Attempts to produce high amounts of the entire recombinant (re-) protein, or a fragment containing the N terminus only, were unsuccessful. This is presumably due to the toxicity of these re-proteins. The internal part of both genes was also expressed in Salmonella typhimurium (St) aroA vaccine strain SL3261. We employed a dual-plasmid expression system based on an invertible promoter and selected the most stable St construct in vitro using liquid cultures and a macrophage-like cell line. The re-Tams1-1 protein produced in Ec, as well as in St, was recognized by monoclonal antibody (mAb) 5E1 specific to the 30-kDa protein. Both re-Tams1-1 and re-Tams1-2 were recognized by Ta immune calf serum.

Generation of a mosaic pattern of diversity in the major merozoite-piroplasm surface antigen of Theileria annulata.

The polypeptide Tams1 is an immunodominant major merozoite piroplasm surface antigen of the protozoan parasite Theileria annulata. Generation and selection of divergent antigenic types has implications for the inclusion of the Tams1 antigen in a subunit recombinant vaccine or use in the development of a diagnostic ELISA. In this study a total of 129 Tams1 sequences from parasites isolated in Bahrain, India, Italy, Mauritania, Portugal, Spain, Sudan, Tunisia and Turkey were obtained to estimate the extent of Tams1 diversity throughout a wide geographical range. Significant sequence diversity was found both within and between isolates and many of the sequences were unique. No geographical specificity of sequence types was observed and almost identical sequences occurred in different geographical areas and a panmictic population structure is suggested by our results. A sliding window analysis identified sub-regions of the molecule where selection for amino acid changes may operate. Evidence is also presented for the generation of diversity through intragenic recombination with switching of corresponding variable domains between alleles. Recombination to exchange variable domains appears to occur throughout the length of the gene sequence, and has the potential to generate a mosaic pattern of diversity.

Molecular characterisation of the Theileria buffeli/orientalis group.

Benign bovine Theileria parasites known as either Theileria buffeli, Theileria orientalis or Theileria sergenti are classified on basis of their morphology, vector specificity, pathogenicity and 18S small subunit ribosomal RNA or major piroplasm protein (MPSP) sequences. Since most isolates have been characterized on only some of these criteria and the existing confusion in nomenclature, an analysis was performed on eight different isolates to combine 18S rRNA data with MPSP data and the results were compared with available biological parameters. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) approach for both genes was used in combination with reverse line blot hybridisation for the 18S rRNA gene. Both MPSP and 18S rRNA genes were cloned and sequenced from parasites displaying aberrant MPSP RFLP profiles. Phylogeny based on published and determined 18S rRNA and MPSP sequences did correlate within the same isolate but there was no obvious correlation between molecular and biological data. Based on these findings, we suggest that the appropriate name for all these parasites is Theileria buffeli. A more specific nomenclature should be assigned when new molecular markers may become available.

Characterization of attenuated Theileria annulata vaccines from Spain and the Sudan.

Theileriosis caused by Theileria annulata can be effectively prevented by vaccination with attenuated, cultured schizonts. Although these attenuated vaccines have been applied for a long time, not much is known about the fate of the vaccine strain in the field. Here, two experimental Spanish vaccine strains originating in Cádiz and Cáceres, and one Sudanese strain are studied to address the development of a carrier status and the infectivity for Hyalomma ticks. Moreover, the heterogeneity of the merozoite surface protein, Tams1, was analyzed in search for an attenuation marker. Using the sensitive reverse line blot (RLB) hybridization, the development of a low level carrier status was demonstrated in the Cáceres and Sudanese line vaccinated calves. Although no signal was detected in the Cádiz line vaccinated calves, seroconversion against the schizont stage was observed, as it was in all other calves. The experimental transmission of T. annulata by Hyalomma ticks to naïve calves was unsuccessful for all cell line inoculated calves. Tams1 heterogeneity indicated a clonal selection of parasites during the process of attenuation, but the Tams1 sequence itself has no connection with the attenuation status. In conclusion, a carrier status develops in attenuated schizont culture vaccinated calves, but is not infective for Hyalomma ticks. Based on these data, the risk for spread of the vaccine strains in the field may be very low.

Evaluation of the MAP-1B ELISA for cowdriosis with field sera from livestock in Zimbabwe.

The Map 1B (Senegal) antigen-based indirect ELISA was evaluated in Zimbabwe with field sera from heartwater-free and heartwater-endemic areas. Of 205 sheep sera samples from a heartwater-free area, 34 were negative and 171 were positive by immunoblotting. These 171 samples were classified as false positives. Of the same 205 samples, 199 were negative and only 6 were positive by the MAP 1B ELISA. Of 72 cattle samples tested from a similar area, 71 were negative, with only 1 sample positive by MAP 1B ELISA. By immunoblotting, 43 of 72 cattle sera were negative and 29 were positive. Of the 46 goat samples tested from a heartwater-free area, only 2 were positive by the MAP 1B ELISA. Based on these results, the MAP 1B ELISA was more specific for heartwater than the immunoblotting assay. Of 96 and 282 cattle sera analyzed from heartwater-endemic farms in the lowveld and highveld of Zimbabwe, respectively, approximately 33% were positive by the MAP 1B ELISA. Goat sera from the same farms had a higher sero-prevalence (> 90%). The implications of these results for serodiagnosis of heartwater using the MAP 1B ELISA will be discussed.

Integrated molecular diagnosis of Theileria and Babesia species of cattle in Italy.

A reverse line blot hybridization (RLB) test was developed to specifically identify six Theileria spp. (T. annulata, T. parva, T. mutans, T. velifera, T. taurotragi, and T. buffeli/orientalis) and three Babesia spp. (B. bovis, B. bigemina, and B. divergens). No cross reaction was observed with other livestock pathogens (such as Anaplasma marginale, A. centrale, A. ovis, Cowdria ruminantium, Trypanosoma brucei, T. congolense, and T. vivax). This method was used to test bovine blood samples collected in Sicily in April and November, 1998. Preliminary results indicated that T. annulata and T. buffeli/orientalis were the main species observed in cattle blood. Babesia species represented 1.8% and 23.5% in April and November, respectively.