Assessment of genotyping array performance for genome-wide association studies and imputation in African cattle.

BACKGROUND: In cattle, genome-wide association studies (GWAS) have largely focused on European or Asian breeds, using genotyping arrays that were primarily designed for European cattle. Because there is growing interest in performing GWAS in African breeds, we have assessed the performance of 23 commercial bovine genotyping arrays for capturing the diversity across African breeds and performing imputation. We used 409 whole-genome sequences (WGS) spanning global cattle breeds, and a real cohort of 2481 individuals (including African breeds) that were genotyped with the Illumina high-density (HD) array and the GeneSeek bovine 50 k array. RESULTS: We found that commercially available arrays were not effective in capturing variants that segregate among African indicine animals. Only 6% of these variants in high linkage disequilibrium (LD) (r2 > 0.8) were on the best performing arrays, which contrasts with the 17% and 25% in African and European taurine cattle, respectively. However, imputation from available HD arrays can successfully capture most variants (accuracies up to 0.93), mainly when using a global, not continent-specific, reference panel, which partially reflects the unusually high levels of admixture on the continent. When considering functional variants, the GGPF250 array performed best for tagging WGS variants and imputation. Finally, we show that imputation from low-density arrays can perform almost as well as HD arrays, if a two-stage imputation approach is adopted, i.e. first imputing to HD and then to WGS, which can potentially reduce the costs of GWAS. CONCLUSIONS: Our results show that the choice of an array should be based on a balance between the objective of the study and the breed/population considered, with the HD and BOS1 arrays being the best choice for both taurine and indicine breeds when performing GWAS, and the GGPF250 being preferable for fine-mapping studies. Moreover, our results suggest that there is no advantage to using the indicus-specific arrays for indicus breeds, regardless of the objective. Finally, we show that using a reference panel that better represents global bovine diversity improves imputation accuracy, particularly for non-European taurine populations.

Cutaneous Immunoprofiles of Three Spotted Fever Group Rickettsia Cases.

Spotted fever group rickettsia (SFGR) can cause mild to fatal illness. The early interaction between the host and rickettsia in skin is largely unknown, and the pathogenesis of severe rickettsiosis remains an important topic. A surveillance of SFGR infection by PCR of blood and skin biopsy specimens followed by sequencing and immunohistochemical (IHC) detection was performed on patients with a recent tick bite between 2013 and 2016. Humoral and cutaneous immunoprofiles were evaluated in different SFGR cases by serum cytokine and chemokine detection, skin IHC staining, and transcriptome sequencing (RNA-seq). A total of 111 SFGR cases were identified, including 79 "Candidatus Rickettsia tarasevichiae," 22 Rickettsia raoultii, 8 Rickettsia sibirica, and 2 Rickettsia heilongjiangensis cases. The sensitivity to detect SFGR in skin biopsy specimens (9/24, 37.5%) was significantly higher than that in blood samples (105/2,671, 3.9%) (P < 0.05). As early as 1 day after the tick bite, rickettsiae could be detected in the skin. R. sibirica infection was more severe than "Ca Rickettsia" and R. raoultii infections. Increased levels of serum interleukin-18 (IL-18), IP10, and monokine induced by gamma interferon (MIG) and decreased levels of IL-2 were observed in febrile patients infected with R. sibirica compared to those infected with "Ca Rickettsia." RNA-seq and IHC staining could not discriminate between SFGR-infected and uninfected tick bite skin lesions. However, the type I interferon (IFN) response was differently expressed between R. sibirica and R. raoultii infections at the cutaneous interface. It is concluded that skin biopsy specimens were more reliable for the detection of SFGR infection in human patients although the immunoprofile may be complicated by immunomodulators induced by the tick bite.

Canine Leishmaniasis Control in the Context of One Health.

Dogs are the main reservoir of Leishmania infantum and in some countries have been regularly culled as part of government policy to control visceral leishmaniasis. At the 13th Symposium of the Companion Vector-Borne Diseases World Forum in Windsor, UK, March 19-22, 2018, we consolidated a consensus statement regarding the usefulness of dog culling as a means of controlling visceral leishmaniasis. The statement highlighted the futility of culling infected dogs, whether healthy or sick, as a measure to control the domestic reservoir of L. infantum and reduce the risk for visceral leishmaniasis.

Influence of the spatial heterogeneity in tick abundance in the modeling of the seasonal activity of Ixodes ricinus nymphs in Western Europe.

The seasonal weather-driven activity of the tick Ixodes ricinus is frequently explored using multisite surveys. This study aimed to investigate the statistical modeling of seasonal trends in the activity of I. ricinus nymphs when both the influence of abiotic factors and spatial heterogeneity were taken into account. Time series data of abiotic covariates (temperature, relative humidity, rainfall and photoperiod) and nymphal tick counts were recorded on several sites in The Netherlands, Belgium and in France in 2008 and 2009. The sites were divided into two subsets which were used for model construction or model validation. A generalized linear mixed model was set up, with aggregated abiotic covariates as fixed effects, and the collection site as a random effect to account for the site-varying density in nymphs. A linear regression model was developed to estimate the site effect against the observed local abundance on each site. The activity patterns simulated from the weather and photoperiod covariates realistically reproduced the observed seasonal trends in nymphal tick activity. The fit between observed and simulated nymphal count time series was greatly improved when the site-specific local abundance in nymphs was included. Our modeling approach allows indicators of local tick abundance and the temporal modeling of I. ricinus activity to be combined. The model presented here can also be used to study scenarios on the temporal patterns of I. ricinus activity in the present and in the context of climate change.

Diagnostic performance of a rapid immunochromatographic test for the simultaneous detection of antibodies to Theileria equi and Babesia caballi in horses and donkeys.

BACKGROUND: Equine piroplasmosis is caused by two tick-borne protozoan parasites, Theileria equi and Babesia caballi,, which are clinically relevant in susceptible horses, donkeys, and mules. Moreover, equine piroplasmosis significantly constrains international trading and equestrian events. Rapidly diagnosing both parasites in carrier animals is essential for implementing effective control measures. Here, a rapid immunochromatographic test for the simultaneous detection of antibodies to T. equi and B. caballi was evaluated using samples from horses and donkeys collected in Greece, Israel, and Italy. The results were compared with an improved competitive enzyme-linked immunosorbent assay (cELISA) for detecting antibodies to both parasites using the same panel of samples. METHODS: Blood samples were collected from 255 horses and donkeys. The panel consisted of 129 horses sampled at four locations in northern Greece, 105 donkeys sampled at four locations in Sicily, and 21 horses sampled at two locations in Israel. The rapid test and the cELISA were performed according to the manufacturer's instructions, and the results were subjected to a statistical analysis to determine the sensitivity and specificity of both tests and their association. RESULTS: The immunochromatographic test provided a result within 15 min and can be performed in the field, detecting both pathogens simultaneously. The overall coincidence rate between the rapid test and the cELISA for detecting antibodies against T. equi was 93% and 92.9% for B. caballi. The rapid test's sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for T. equi were above 91.5%. Sixteen samples were positive for both parasites in the rapid test and eight in the cELISA. Either test had no significant association between T. equi and B. caballi detection. The detection rates of both parasites were significantly higher in Italy than in Greece or Israel and in donkeys than in horses. The agreement for T. equi between the results of both tests was high in Greece (93.8%) and Italy (95.2%) and moderate in Israel (76.2%). For B. caballi, the specificity and NPV of the rapid test were high (94.2% and 98.3%, respectively), although the sensitivity and PPV were moderate (69.2% and 39.1%, respectively) due to the small sample size. However, for B. caballi, the sensitivity was higher with the rapid test. CONCLUSIONS: The rapid test detected T. equi and B. caballi simultaneously in the field, potentially replacing laborious cELISA testing and is recommended for import/export purposes. The test can also be helpful for the differential diagnosis of clinical cases, since seropositivity may rule out equine piroplasmosis since it does not indicate current or active infection.

Acaricide resistance status of livestock ticks from East and West Africa and in vivo efficacy of acaricides to control them.

Through a collaborative effort across six Sub-Saharan African countries, using recognized international assessment techniques, 23 stocks of three tick species (Rhipicephalus microplus, Rhipicephalus appendiculatus and Amblyomma variegatum) of economic importance for rural small holder farming communities from East and West Africa were collected from cattle, and evaluated in in vitro larval packet tests (LPT). The results demonstrated medium to high resistance to chlorfenvinphos and amitraz across species. Rhipicephalus microplus demonstrated high level alpha-cypermethrin and cypermethrin resistance. Stocks of A. variegatum (West Africa) and R. appendiculatus (Uganda) demonstrated medium level ivermectin resistance. The four least susceptible stocks (East and West African R. microplus, A. variegatum and R. appendiculatus) were taken into in vivo controlled cattle studies where fipronil was found effective against West and East African R. microplus isolates although persistent efficacy failed to reach 90%. Cymiazole and cypermethrin, and ivermectin based acaricides were partially effective against R. microplus without persistent efficacy. Flumethrin spray-on killed A. variegatum within 72 h for up to 10 days posttreatment, however product application was directly to tick attachment sites, which may be impractical under field conditions. A flumethrin pour-on formulation on goats provided persistent efficacy against A. variegatum for up to one-month. Therapeutic control was achieved against R. appendiculatus through weekly spraying cattle with flumethrin, amitraz or combined cymiazole and cypermethrin. A fipronil pour-on product offered four-week residual control against R. appendiculatus (with slow onset of action). Few studies have assessed and directly compared acaricidal activity in vitro and in vivo. There was some discordance between efficacy indicated by LPT and in vivo results. This observation calls for more research into accurate and affordable assessment methods for acaricide resistance. No single active or product was effective against all three tick species, emphasising the need for the development of alternative integrated tick management solutions.

Association of environmental traits with the geographic ranges of ticks (Acari: Ixodidae) of medical and veterinary importance in the western Palearctic. A digital data set.

We compiled information on the distribution of ticks in the western Palearctic (11°W, 45°E; 29°N, 71°N), published during 1970-2010. The literature search was filtered by the tick's species name and an unambiguous reference to the point of capture. Records from some curated collections were included. We focused on tick species of importance to human and animal health, in particular: Ixodes ricinus, Dermacentor marginatus, D. reticulatus, Haemaphysalis punctata, H. sulcata, Hyalomma marginatum, Hy. lusitanicum, Rhipicephalus annulatus, R. bursa, and the R. sanguineus group. A few records of other species (I. canisuga, I. hexagonus, Hy. impeltatum, Hy. anatolicum, Hy. excavatum, Hy. scupense) were also included. A total of 10,280 records was included in the data set. Almost 42 % of published references are not adequately referenced (and not included in the data set), host is reported for only 61 % of records and a reference to time of collection is missed for 84 % of published records. Ixodes ricinus accounted for 44.3 % of total records, with H. marginatum and D. marginatus accounting for 7.1 and 8.1 % of records, respectively. The lack of homogeneity of the references and potential pitfalls in the compilation were addressed to create a digital data set of the records of the ticks. We attached to every record a coherent set of quantitative descriptors for the site of reporting, namely gridded interpolated monthly climate and remotely sensed data on vegetation (NDVI). We also attached categorical descriptors of the habitat: a standard classification of land biomes and an ad hoc classification of the target territory from remotely sensed temperature and NDVI data. A descriptive analysis of the data revealed that a principal components reduction of the environmental (temperature and NDVI) variables described the distribution of the species in the target territory. However, categorical descriptors of the habitat were less effective. We stressed the importance of building reliable collections of ticks with specific references as to collection point, host and date of capture. The data set is freely downloadable.

Molecular evidence of Ehrlichia canis and Rickettsia massiliae in ixodid ticks of carnivores from South Hungary.

To monitor the emergence of thermophilic, Mediterranean ixodid tick species and tick-borne pathogens in southern Hungary, 348 ticks were collected from shepherd dogs, red foxes and golden jackals during the summer of 2011. Golden jackals shared tick species with both the dog and the red fox in the region. Dermacentor nymphs were collected exclusively from dogs, and the sequence identification of these ticks indicated that dogs are preferred hosts of both D. reticulatus and D. marginatus nymphs, unlike previously reported. Subadults of three ixodid species were selected for reverse line blot hybridisation (RLB) analysis to screen their vector potential for 40 pathogens/groups. Results were negative for Anaplasma, Babesia and Theileria spp. Investigation of D. marginatus nymphs revealed the presence of Ehrlichia canis, Rickettsia massiliae and Borrelia afzelii for the first time in this tick species. These findings broaden the range of those tick-borne agents, which are typically transmitted by Rhipicephalus sanguineus, but may also have Dermacentor spp. as potential or alternative vectors. Ehrlichiacanis was also newly detected in Ixodes canisuga larvae from red foxes. In absence of transovarial transmission in ticks this implies that Eurasian red foxes may play a reservoir role in the epidemiology of canine ehrlichiosis.

De novo assembly and annotation of the Amblyomma hebraeum tick midgut transcriptome response to Ehrlichia ruminantium infection.

The South African bont tick Amblyomma hebraeum is a hematophagous vector for the heartwater disease pathogen Ehrlichia ruminantium in southern Africa. During feeding, the tick's enterocytes express proteins that perform vital functions in blood digestion, including proteins that may be involved in E. ruminantium acquisition, colonization or immunity. To delineate the molecular mechanism of midgut response to E. ruminantium infection, we performed comparative analyses of midgut transcriptomes of E. ruminantium infected engorged A. hebraeum nymphs, and infected adult male and female ticks with their corresponding matched uninfected controls, before and during feeding. A total of 102,036 unigenes were annotated in public databases and their expression levels analyzed for engorged nymphs as well as unfed and partly-fed adult ticks. There were 2,025 differentially expressed genes (DEGs) in midguts, of which 1,225 unigenes were up-regulated and 800 unigenes were down-regulated in the midguts of infected ticks. Annotation of DEGs revealed an increase in metabolic and cellular processes among E. ruminantium infected ticks. Notably, among the infected ticks, there was up-regulation in the expression of genes involved in tick immunity, histone proteins and oxidative stress responses. We also observed up-regulation of glycoproteins that E. ruminantium could potentially use as docking sites for host cell entry. Insights uncovered in this study offer a platform for further investigations into the molecular interaction between E. ruminantium and A. hebraeum.

First clinical case of tick-borne encephalitis (TBE) in a dog in Greece.

Tick-borne encephalitis virus (TBEV) causes tick-borne encephalitis (TBE), affecting human health in Europe and Asia. Reports on canine clinical cases of TBE are rare, although dogs are used as sentinels for assessing human health risks. The first canine clinical TBE case in Greece is reported in this case report. The dog had a history of tick infestation and displayed neurological symptoms, particularly tetraparesis, neck hyperalgesia, and a sudden behavior change. Serum samples were obtained and examined in a commercial ELISA to detect anti-TBEV specific IgG and IgM antibodies. The dog tested seropositive for both IgG and IgM, and based on its history and compatible clinical signs, the diagnosis of TBE infection was reached. The prognosis was initially poor, and treatment included the administration of fluids, corticosteroids, and antibiotics, followed by physical therapy. After a 10-day hospitalization, the dog had a much better prognosis. This case highlights that TBEV does emerge in new locations, increasing human and animal infection risk. Veterinarians should include TBE in their differential diagnosis of canine patients with a history of tick infestations, progressive neurological symptoms, and abnormal behavior.

Correlation between Babesia Species Affecting Dogs in Taiwan and the Local Distribution of the Vector Ticks.

The objective of our study was to survey Babesia infection rates by PCR and tick species on stray dogs to correlate the distribution of Babesia with the distribution of ticks infesting dogs in Taiwan. Three hundred eighty-eight blood samples and 3037 ticks were collected from 388 roaming, and free-ranging owned dogs at residential sites in Taiwan between January 2015 and December 2017. The prevalence of B. gibsoni and B. vogeli was 15.7% (61/388) and 9.5% (37/388), respectively. Most positive B. gibsoni dogs were found in the northern part of the country 56/61 (91.8%), whereas a few were found in the middle 5/61 (8.2%). Babesia vogeli infection rates were 10%, 3.6%, and 18.2% in the northern, central, and southern regions, respectively. Five species of ticks were found: Rhipicephalus sanguineus (throughout Taiwan), Rhipicephalus haemaphysaloides (in the north), Haemaphysalis hystricis (in the north and middle of Taiwan), and Amblyomma testidunarium and Ixodes ovatus (both in the north). None of the dogs in the south were infected with B gibsoni, which correlated with the absence of H. hystricis, a tick recently identified as the local vector for B gibsoni. Babesia vogeli was more equally distributed, coinciding with R. sanguineus, a tick that is present throughout Taiwan. Anaemia was detected in 86.9% of infected dogs; among these dogs, approximately 19.7% showed severe anaemia (HCT < 20). These findings provide useful advice for owners regarding outdoor activities with their dogs and local veterinarians with a regional differential diagnosis of babesiosis in Taiwan.

Tick communities of cattle in smallholder rural livestock production systems in sub-Saharan Africa.

BACKGROUND: The majority of the African population lives in rural areas and depends on agriculture for their livelihoods. To increase the productivity and sustainability of their farms, they need access to affordable yield-enhancing inputs of which parasite control is of paramount importance. We therefore determined the status of current tick species with the highest economic impact on cattle by sampling representative numbers of animals in each of seven sub-Saharan countries. METHODS: Data included tick species' half-body counts from approximately 120 cattle at each of two districts per country, collected four times in approximately 1 year (to include seasonality). Study sites were chosen in each country to include high cattle density and tick burden. RESULTS: East Africa (Ethiopia, Uganda and Tanzania) showed overall a higher diversity and prevalence in tick infestations compared to West African countries (Benin, Burkina Faso, Ghana and Nigeria). In East Africa, Amblyomma variegatum (vector of Ehrlichia ruminantium), Rhipicephalus microplus (Babesia bovis, B. bigemina, Anaplasma marginale), R. evertsi evertsi (A. marginale) and R. appendiculatus (Theileria parva) were the most prevalent tick species of economic importance. While the latter species was absent in West Africa, here both A. variegatum and R. microplus occurred in high numbers. Rhipicephalus microplus had spread to Uganda, infesting half of the cattle sampled. Rhipicephalus appendiculatus is known for its invasive behaviour and displacement of other blue tick species, as observed in other East and West African countries. Individual cattle with higher body weights, as well as males, were more likely to be infested. For six tick species, we found reduced infestation levels when hosts were treated with anti-parasiticides. CONCLUSIONS: These baseline data allow the determination of possible changes in presence and prevalence of ticks in each of the countries targeted, which is of importance in the light of human-caused climate and habitat alterations or anthropogenic activities. As many of the ticks in this study are vectors of important pathogens, but also, as cattle may act as end hosts for ticks of importance to human health, our study will help a wide range of stakeholders to provide recommendations for tick infestation surveillance and prevention.

Tick-borne pathogens and body condition of cattle in smallholder rural livestock production systems in East and West Africa.

BACKGROUND: The majority of the African population lives in rural areas where they heavily depend on crop and livestock production for their livelihoods. Given their socio-economic importance, we initiated a standardized multi-country (Benin, Burkina Faso, Ghana, Nigeria, Ethiopia Tanzania and Uganda) surveillance study to assess the current status of important tick-borne haemoparasites (TBHPs) of cattle. METHODS: We assessed pathogen prevalences (Anaplasma marginale, Anaplasma centrale, Babesia bigemina, Babesia bovis, Ehrlichia ruminantium, and Theileria parva) in the blood of 6447 animals spread over fourteen districts (two districts per country). In addition, we screened for intrinsic (sex, weight, body condition) and extrinsic (husbandry, tick exposure) risk factors as predictors of infections with TBHPs. RESULTS: There was a large macro-geographic variation observed in A. marginale, B. bigemina, B. bovis and E. ruminantium prevalences. Most correlated with the co-occurrence of their specific sets of vector-competent ticks. Highest numbers of infected cattle were found in Ghana and Benin, and lowest in Burkina Faso. While T. parva was seldomly found (Uganda only: 3.0%), A. marginale was found in each country with a prevalence of at least 40%. Babesia bovis infected individuals had lower body condition scores. Age (as estimated via body weight) was higher in A. marginale infected cattle, but was negatively correlated with B. bigemina and E. ruminantium prevalences. Ehrlichia ruminantium infection was more often found in males, and A. marginale more often in transhumance farming. High levels of co-infection, especially the combination A. marginale × B. bigemina, were observed in all countries, except for Uganda and Burkina Faso. Babesia bigemina was more or less often observed than expected by chance, when cattle were also co-infected with E. ruminantium or A. marginale, respectively. CONCLUSIONS: Tick-borne pathogens of cattle are ubiquitous in African's smallholder cattle production systems. Our standardized study will help a wide range of stakeholders to provide recommendations for TBHP surveillance and prevention in cattle, especially for B. bovis which heavily impacts production and continues its spread over the African continent via the invasive Rhipicephalus microplus tick.

Control of ticks and tick-borne diseases in Africa through improved diagnosis and utilisation of data on acaricide resistance.

A meeting, sponsored by the Bill and Melinda Gates Foundation (BMGF) and organised by Clinglobal, was held at The International Livestock Research Institute (ILRI) in Nairobi, Kenya, from 19th - to 21st October 2022. The meeting assembled a unique group of experts on tick control in Africa. Academia, international agencies (FAO and ILRI), the private Animal Health sector and government veterinary services were represented. The significant outcomes included: (i) a shared commitment to standardisation and improvement of acaricide resistance bioassay protocols, particularly the widely used larval packet test (LPT); (ii) development of novel molecular assays for detecting acaricide resistance; (3) creation of platforms for disseminating acaricide resistance data to farmers, veterinary service providers and veterinary authorities to enable more rational evidence-based control of livestock ticks. Implementation of enhanced control will be facilitated by several recently established networks focused on control of parasites in Africa and globally, whose activities were presented at the meeting. These include a newly launched community of practice on management of livestock ticks, coordinated by FAO, an African module of the World Association for the Advancement of Veterinary Parasitology (WAAVP-AN) and the MAHABA (Managing Animal Health and Acaricides for a Better Africa) initiative of Elanco Animal Health.

Development of multiple-locus variable-number tandem-repeat analysis for rapid genotyping of Ehrlichia ruminantium and its application to infected Amblyomma variegatum collected in heartwater endemic areas in Uganda.

The rickettsial bacterium Ehrlichia ruminantium is the causative agent of heartwater, a serious tick-borne disease in ruminants. The genetic diversity of organisms in the field will have implications for cross-protective capacities of any vaccine developed, and for an effective vaccine design strategy proper genotyping and understanding of existing genetic diversity in the field is necessary. We searched for variable-number tandem-repeat (VNTR) loci for use in a multi-locus VNTR analysis (MLVA). Sequencing analysis of 30 potential VNTRs using a panel of 17 reference strains from geographically diverse origins identified 12 VNTRs with allelic profiles differing between strains. Application of MLVA to 38 E. ruminantium-infected Amblyomma variegatum collected from indigenous cattle in 6 different districts of Uganda identified 21 MLVA types. The discriminatory power of MLVA was greater than that of map1 PCR-restriction fragment length polymorphism analysis, with which only 6 genotypes were obtained. The high discriminatory power as well as cost- effective performance of MLVA provide the potential for this technique to be applied in the future with respect to optimizing vaccine trials by identifying local strain diversity, and also raise the possibility of exploring the association between E. ruminantium genotypes and phenotypes such as pathological outcome in the ruminant host.

The Scenario of Ticks and Tick-Borne Pathogens of Sheep on a Mediterranean Island.

Ticks and transmitted pathogens constitute a major concern for livestock health/welfare and productivity for the Mediterranean region, often posing an important zoonotic threat. The aim of this study was to investigate the presence, infection intensity, and seasonality of ticks and tick-borne pathogens on the island of Lesvos in Greece, which was selected as a potential hotspot for their circulation. To this end, 101 sheep farms were visited over a tick activity season, and ticks, blood samples, and questionnaire data were collected. Ticks were identified by species, and DNA from both ticks and blood samples was further investigated using the polymerase chain reaction-reverse line blot (PCR-RLB) technique. In 72.3% of the farms, sheep were found to be infected by 9 ixodid species, with Rhipicephalus turanicus being the most common during the spring/early summer period. As regards tick-borne pathogens (TBPs), 84.9% of the animals were found to be infected with at least one pathogen, the most common being genera of Anaplasma and Theileria, alone or in co-infections. To further characterize the Anaplasma species found, selected samples were sequenced, revealing isolates of A. ovis, A. capra, A. marginale, and A. phagocytophilum. Of the 169 female R. turanicus ticks analyzed by PCR-RLB, 89.9% were harboring at least one TBP belonging to the genera Anaplasma, Ehrlichia, Babesia, Theileria, or Rickettsia. Overall, the data presented in this study revealed a high burden of ticks and TBPs in sheep, including zoonotic species, stressing the need for applying effective monitoring and control programs using a more holistic One Health approach.

Vector-borne and other pathogens of potential relevance disseminated by relocated cats.

Large populations of unowned cats constitute an animal welfare, ecological, societal and public health issue worldwide. Their relocation and homing are currently carried out in many parts of the world with the intention of relieving suffering and social problems, while contributing to ethical and humane population control in these cat populations. An understanding of an individual cat's lifestyle and disease status by veterinary team professionals and those working with cat charities can help to prevent severe cat stress and the spread of feline pathogens, especially vector-borne pathogens, which can be overlooked in cats. In this article, we discuss the issue of relocation and homing of unowned cats from a global perspective. We also review zoonotic and non-zoonotic infectious agents of cats and give a list of practical recommendations for veterinary team professionals dealing with homing cats. Finally, we present a consensus statement consolidated at the 15th Symposium of the Companion Vector-Borne Diseases (CVBD) World Forum in 2020, ultimately to help veterinary team professionals understand the problem and the role they have in helping to prevent and manage vector-borne and other pathogens in relocated cats.

Vectra 3D (dinotefuran, pyriproxyfen and permethrin) prevents acquisition of Borrelia burgdorferi sensu stricto by Ixodes ricinus and Ixodes scapularis ticks in an ex vivo feeding model.

BACKGROUND: We evaluated the efficiency of an ex vivo feeding technique using a silicone membrane-based feeding chamber to (i) assess the anti-feeding and acaricidal efficacy of a spot-on combination of dinotefuran, pyriproxyfen and permethrin (DPP, Vectra® 3D) against adult Ixodes scapularis and Ixodes ricinus ticks, and to (ii) explore its effect on blocking the acquisition of Borrelia burgdorferi sensu stricto. METHODS: Eight purpose-bred dogs were randomly allocated to two equal-size groups based on body weight assessed on day 2. DPP was administered topically, as spot-on, to four dogs on day 0. Hair from the eight dogs was collected individually by brushing the whole body on days 2, 7, 14, 21, 28 and 35. On each day of hair collection, 0.05 g of sampled hair was applied on the membrane corresponding to each feeding unit (FU). Seventy-two FU were each seeded with 30 adults of I. scapularis (n = 24 FU) or I. ricinus ticks (n = 48 FU). Bovine blood spiked with B. burgdorferi sensu stricto (strain B31) was added into each unit and changed every 12 h for 4 days. Tick mortality was assessed 1 h after seeding. One additional hour of incubation was added for live/moribund specimens and reassessed for viability. All remaining live/moribund ticks were left in the feeders and tick engorgement status was recorded at 96 h after seeding, and the uptake of B. burgdorferi s.s. was examined in the collected ticks by applying quantitative real-time PCR. RESULTS: Exposure to DPP-treated hair was 100% effective in blocking B. burgdorferi s.s. acquisition. The anti-feeding efficacy remained stable (100%) against both Ixodes species throughout the study. The acaricidal efficacy of DPP evaluated at 1 and 2 h after exposure was 100% throughout the study for I. ricinus, except the 1-h assessment on day 28 (95.9%) and day 35 (95.3%). The 1-h assessment of acaricidal efficacy was 100% at all time points for I. scapularis. CONCLUSIONS: The ex vivo feeding system developed here demonstrated a protective effect of DPP against the acquisition of B. burgdorferi without exposing the animals to the vectors or to the pathogen.

Differential expression of genes in salivary glands of male Rhipicephalus (Boophilus)microplus in response to infection with Anaplasma marginale.

BACKGROUND: Bovine anaplasmosis, caused by the rickettsial tick-borne pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae), is vectored by Rhipicephalus (Boophilus)microplus in many tropical and subtropical regions of the world. A. marginale undergoes a complex developmental cycle in ticks which results in infection of salivary glands from where the pathogen is transmitted to cattle. In previous studies, we reported modification of gene expression in Dermacentor variabilis and cultured Ixodes scapularis tick cells in response to infection with A. marginale. In these studies, we extended these findings by use of a functional genomics approach to identify genes differentially expressed in R. microplus male salivary glands in response to A. marginale infection. Additionally, a R. microplus-derived cell line, BME26, was used for the first time to also study tick cell gene expression in response to A. marginale infection. RESULTS: Suppression subtractive hybridization libraries were constructed from infected and uninfected ticks and used to identify genes differentially expressed in male R. microplus salivary glands infected with A. marginale. A total of 279 ESTs were identified as candidate differentially expressed genes. Of these, five genes encoding for putative histamine-binding protein (22Hbp), von Willebrand factor (94Will), flagelliform silk protein (100Silk), Kunitz-like protease inhibitor precursor (108Kunz) and proline-rich protein BstNI subfamily 3 precursor (7BstNI3) were confirmed by real-time RT-PCR to be down-regulated in tick salivary glands infected with A. marginale. The impact of selected tick genes on A. marginale infections in tick salivary glands and BME26 cells was characterized by RNA interference. Silencing of the gene encoding for putative flagelliform silk protein (100Silk) resulted in reduced A. marginale infection in both tick salivary glands and cultured BME26 cells, while silencing of the gene encoding for subolesin (4D8) significantly reduced infection only in cultured BME26 cells. The knockdown of the gene encoding for putative metallothionein (93 Meth), significantly up-regulated in infected cultured BME26 cells, resulted in higher A. marginale infection levels in tick cells. CONCLUSIONS: Characterization of differential gene expression in salivary glands of R. microplus in response to A. marginale infection expands our understanding of the molecular mechanisms at the tick-pathogen interface. Functional studies suggested that differentially expressed genes encoding for subolesin, putative von Willebrand factor and flagelliform silk protein could play a role in A. marginale infection and multiplication in ticks. These tick genes found to be functionally relevant for tick-pathogen interactions will likely be candidates for development of vaccines designed for control of both ticks and tick-borne pathogens.

Subolesin expression in response to pathogen infection in ticks.

BACKGROUND: Ticks (Acari: Ixodidae) are vectors of pathogens worldwide that cause diseases in humans and animals. Ticks and pathogens have co-evolved molecular mechanisms that contribute to their mutual development and survival. Subolesin was discovered as a tick protective antigen and was subsequently shown to be similar in structure and function to akirins, an evolutionarily conserved group of proteins in insects and vertebrates that controls NF-kB-dependent and independent expression of innate immune response genes. The objective of this study was to investigate subolesin expression in several tick species infected with a variety of pathogens and to determine the effect of subolesin gene knockdown on pathogen infection. In the first experiment, subolesin expression was characterized in ticks experimentally infected with the cattle pathogen, Anaplasma marginale. Subolesin expression was then characterized in questing or feeding adult ticks confirmed to be infected with Anaplasma, Ehrlichia, Rickettsia, Babesia or Theileria spp. Finally, the effect of subolesin knockdown by RNA interference (RNAi) on tick infection was analyzed in Dermacentor variabilis males exposed to various pathogens by capillary feeding (CF). RESULTS: Subolesin expression increased with pathogen infection in the salivary glands but not in the guts of tick vector species infected with A. marginale. When analyzed in whole ticks, subolesin expression varied between tick species and in response to different pathogens. As reported previously, subolesin knockdown in D. variabilis infected with A. marginale and other tick-borne pathogens resulted in lower infection levels, while infection with Francisella tularensis increased in ticks after RNAi. When non-tick-borne pathogens were fed to ticks by CF, subolesin RNAi did not affect or resulted in lower infection levels in ticks. However, subolesin expression was upregulated in D. variabilis exposed to Escherichia coli, suggesting that although this pathogen may induce subolesin expression in ticks, silencing of this molecule reduced bacterial multiplication by a presently unknown mechanism. CONCLUSIONS: Subolesin expression in infected ticks suggested that subolesin may be functionally important for tick innate immunity to pathogens, as has been reported for the akirins. However, subolesin expression and consequently subolesin-mediated innate immunity varied with the pathogen and tick tissue. Subolesin may plays a role in tick innate immunity in the salivary glands by limiting pathogen infection levels, but activates innate immunity only for some pathogen in the guts and other tissues. In addition, these results provided additional support for the role of subolesin in other molecular pathways including those required for tissue development and function and for pathogen infection and multiplication in ticks. Consequently, RNAi experiments demonstrated that subolesin knockdown in ticks may affect pathogen infection directly by reducing tick innate immunity that results in higher infection levels and indirectly by affecting tissue structure and function and the expression of genes that interfere with pathogen infection and multiplication. The impact of the direct or indirect effects of subolesin knockdown on pathogen infection may depend on several factors including specific tick-pathogen molecular interactions, pathogen life cycle in the tick and unknown mechanisms affected by subolesin function in the control of global gene expression in ticks.