Conservation and variation in the region of the Theileria parva p104 antigen coding gene used for PCR surveillance of the parasite.

The range of the protozoan parasite Theileria parva, which causes East Coast fever in cattle, has been expanding to countries where it has not previously been detected, as a result of cross-border domestic cattle movement. Countries where T. parva has not previously been observed until recently include Cameroon and South Sudan. This raises the issue of the conservation of the p104 antigen gene, on which the nested PCR assay that is widely used for T. parva surveillance in the blood of infected cattle is based. We sampled 40 isolates from six countries widely distributed across the geographical range of the parasite, including eastern, central and southern Africa, for p104 sequence polymorphism. These included parasites from both domestic cattle and the Cape buffalo (Syncerus caffer) wildlife reservoir. The most frequent allelic variants were present in cattle transmissible isolates from multiple widely separated geographical regions in Zambia, Uganda, Kenya, Tanzania, Rwanda and South Africa. These frequent p104 variants were also present in the three component stocks of the Muguga cocktail used for the infection and treatment live immunisation procedure to control T. parva in the field. Other isolates exhibited unique alleles. This includes some of the p104 sequences from Cameroon, which is outside the known range of the Rhipicephalus tick vector and whose origin is therefore unclear. The nested primer oligonucleotides used to generate the amplicons were universally conserved in cattle-derived parasites and a majority of buffalo-derived isolates across the geographical range of the parasite. However, some rare South African buffalo-derived isolates exhibited one or two mismatches with the primer sequences. It therefore remains possible that some p104 alleles may be so divergent that they do not amplify with the current diagnostic primers and are not detectable in surveys, hence the need for increasing knowledge of genetic heterogeneity of diagnostic targets. There was no evidence for positive selection among those p104 mutations that resulted in residue changes. Importantly, the data indicate that the p104-based PCR detection assay should be effective across the majority of the range of T. parva, and if the one or two mismatches are shown in future to result in the primers annealing less efficiently, then the assay can be further improved by introduction of degenerate bases to enable amplification of the less frequent South African buffalo-derived variant p104 genes.

A Culture-Adapted Strain of Babesia bovis Has Reduced Subpopulation Complexity and Is Unable to Complete Its Natural Life Cycle in Ticks.

Babesia bovis natural field strains are composed of several geno-phenotypically distinct subpopulations. This feature, together with possible epigenetic modifications, may facilitate adaptation to variable environmental conditions. In this study we compare geno-phenotypical features among long-term (more than 12 years) (LTCP) and short-term cultured B. bovis parasites (STCP) derived from the B. bovis S74-T3Bo strain. LTCPs intraerythrocytic forms are smaller in size than STCPs and have faster in vitro growth rate. In contrast to its parental strain, the LTCP lack expression of the sexual stage specific 6cysA and 6cysB proteins and are unable to develop sexual forms upon in vitro sexual stage induction. Consistently, in contrast to its parental strain, LTCPs have reduced virulence and are not transmissible to cattle by vector competent Rhipicephalus microplus (R. microplus). Similar to previous comparisons among attenuated and virulent B. bovis strains, the LTCP line has decreased genomic diversity compared to the STCP line. Thus, LTCP may contribute to our understanding of adaptive mechanisms used by the parasites in response to environmental changes, protective immunity, virulence, and transmission by ticks. In addition, LTCPs may be considered as candidates for a non-tick transmissible vaccine against bovine babesiosis.

Isolation of infectious Theileria parva sporozoites secreted by infected Rhipicephalus appendiculatus ticks into an in vitro tick feeding system.

BACKGROUND: Vector-borne diseases pose an increasing threat to global food security. Vaccines, diagnostic tests, and therapeutics are urgently needed for tick-borne diseases that affect livestock. However, the inability to obtain significant quantities of pathogen stages derived from ticks has hindered research. In vitro methods to isolate pathogens from infected tick vectors are paramount to advance transcriptomic, proteomic, and biochemical characterizations of tick-borne pathogens. METHODS: Nymphs of Rhipicephalus appendiculatus were infected with Theileria parva by feeding on a calf during an acute infection. Isolation of sporozoites was accomplished by feeding infected adult ticks on an in vitro tick feeding system. Sporozoite viability was tested using in vitro bovine lymphocytes. RESULTS: We isolated infectious T. parva sporozoites secreted into an in vitro tick feeding system. Infected adult R. appendiculatus ticks attached to and successfully fed on silicone membranes in the in vitro tick feeding system. Bovine blood in the receptacle was replaced with cell-free medium and the ticks were allowed to feed for 3 h to collect secreted T. parva sporozoites. Secreted sporozoites infected in vitro bovine lymphocytes, demonstrating that isolated sporozoites remained viable and infectious. CONCLUSIONS: This work is the first to report the isolation of mature infectious T. parva sporozoites using an in vitro tick feeding system, which represents a significant step towards the development of a more efficient control strategy for T. parva. Isolation of infectious tick-stage parasites will facilitate the examination of the vector-pathogen interface, thereby accelerating the development of next-generation vaccines and treatment interventions for tick-borne pathogens.

Babesiosis as a potential threat for bovine production in China.

Babesiosis is a tick-borne disease with global impact caused by parasites of the phylum Apicomplexa, genus Babesia. Typically, acute bovine babesiosis (BB) is characterized by fever, anemia, hemoglobinuria, and high mortality. Surviving animals remain persistently infected and become reservoirs for parasite transmission. Bovids in China can be infected by one or more Babesia species endemic to the country, including B. bovis, B. bigemina, B. orientalis, B. ovata, B. major, B. motasi, B. U sp. Kashi and B. venatorum. The latter may pose a zoonotic risk. Occurrence of this wide diversity of Babesia species in China may be due to a combination of favorable ecological factors, such as the presence of multiple tick vectors, including Rhipicephalus and Hyalomma, the coexistence of susceptible bovid species, such as domestic cattle, yaks, and water buffalo, and the lack of efficient measures of tick control. BB is currently widespread in several regions of the country and a limiting factor for cattle production. While some areas appear to have enzootic stability, others have considerable cattle mortality. Research is needed to devise solutions to the challenges posed by uncontrolled BB. Critical research gaps include risk assessment for cattle residing in endemic areas, understanding factors involved in endemic stability, evaluation of parasite diversity and pathogenicity of regional Babesia species, and estimation of whether and how BB should be controlled in China. Research should allow the design of comprehensive interventions to improve cattle production, diminish the risk of human infections, and increase the availability of affordable animal protein for human consumption in China and worldwide. In this review, we describe the current state of BB with reference to the diversity of hosts, vectors, and parasite species in China. We also discuss the unique risks and knowledge gaps that should be taken into consideration for future Babesia research and control strategies.

A cross-sectional survey to establish Theileria parva prevalence and vector control at the wildlife-livestock interface, Northern Tanzania.

East Coast fever (ECF) in cattle is caused by the protozoan parasite Theileria parva, transmitted by Rhipicephalus appendiculatus ticks. In cattle ECF is often fatal, causing annual losses >$500 million across its range. The African buffalo (Syncerus caffer) is the natural host for T. parva but the transmission dynamics between wild hosts and livestock are poorly understood. This study aimed to determine the prevalence of T. parva in cattle, in a 30 km zone adjacent to the Serengeti National Park, Tanzania where livestock and buffalo co-exist, and to ascertain how livestock keepers controlled ECF and other vector-borne diseases of cattle. A randomised cross-sectional cattle survey and questionnaire of vector control practices were conducted. Blood samples were collected from 770 cattle from 48 herds and analysed by PCR to establish T. parva prevalence. Half body tick counts were recorded on every animal. Farmers were interviewed (n = 120; including the blood sampled herds) using a standardised questionnaire to obtain data on vector control practices. Local workshops were held to discuss findings and validate results. Overall prevalence of T. parva in cattle was 5.07% (CI: 3.70-7.00%), with significantly higher prevalence in older animals. Although all farmers reported seeing ticks on their cattle, tick counts were very low with 78% cattle having none. Questionnaire analysis indicated significant acaricide use with 79% and 41% of farmers reporting spraying or dipping with cypermethrin-based insecticides, respectively. Some farmers reported very frequent spraying, as often as every four days. However, doses per animal were often insufficient. These data indicate high levels of acaricide use, which may be responsible for the low observed tick burdens and low ECF prevalence. This vector control is farmer-led and aimed at both tick- and tsetse-borne diseases of livestock. The levels of acaricide use raise concerns regarding sustainability; resistance development is a risk, particularly in ticks. Integrating vaccination as part of this community-based disease control may alleviate acaricide dependence, but increased understanding of the Theileria strains circulating in wildlife-livestock interface areas is required to establish the potential benefits of vaccination.

Cattle ticks and tick-borne diseases: a review of Uganda's situation.

Herein we review the epidemiology of ticks and tick-borne diseases (TTBDs), their impact on livestock health and on the economy, control and associated challenges in Uganda. Ticks are leading vectors of economically important pathogens and are widespread in Uganda due to suitable climatic conditions. Besides the physical injury inflicted on the animal host, ticks transmit a number of pathogens that can cause morbidity and mortality of livestock if untreated, resulting in economic losses. Uganda suffers an aggregated annual loss (direct and indirect) of over USD 1.1 billion in the TTBDs complex. East Coast fever (ECF) caused by a protozoan haemoparasite, Theileria parva, is the most prevalent and economically important tick-borne disease (TBD) in Uganda and its vector, the brown ear tick (Rhipicephalus appendiculatus) widely distributed. Other prevalent TBDs in Uganda include anaplasmosis, babesiosis and heartwater. We highlight the role of agro-ecological zones (AEZs) and livestock management system in the distribution of TTBDs, citing warm and humid lowlands as being ideal habitats for ticks and endemic for TBDs. Control of TTBDs is a matter of great importance as far as animal health is concerned in Uganda. Indigenous cattle, which make up over 90% of the national herd are known to be more tolerant to TTBDs and most farms rely on endemic stability to TBDs for control. However, exotic cattle breeds are more capital intensive than indigenous breeds, but the increasing adoption of tick-susceptible exotic cattle breeds (especially dairy) in western and central Uganda demands intensive use of acaricides for tick control and prevention of TBDs. Such acaricide pressure has unfortunately led to selection of acaricide-resistant tick populations and the consequent acaricide resistance observed in the field. Vaccination against ECF, selective breeding for tick resistance and integrated tick control approaches that limit tick exposure, could be adopted to interrupt spread of acaricide resistance. We recommend increasing monitoring and surveillance for TTBDs and for emerging acaricide resistance, improved extension services and sensitization of farmers on tick control measures, appropriate acaricide use and the development and implementation of vaccines for the control of TTBDs as more sustainable and effective interventions. A tick control policy should be developed, taking into account variations of agro-ecological zones, farm circumstances and indigenous technical knowledge, and this should be incorporated into the overall animal health program.

Stray Mexico origin cattle captured crossing into Southern Texas carry Babesia bovis and other tick-borne pathogens.

Cattle fever ticks, Rhipicephalus microplus and R. annulatus have been eradicated from the United States and inspectors from the U.S. Department of Agriculture (USDA), Animal Plant Health Inspection Service (APHIS), Cattle Fever Tick Eradication Program (CFTEP) monitor the quarantine zone along the Texas border to prevent the introduction of livestock carrying cattle fever ticks from Mexico. Stray livestock apprehended by CFTEP in the zone are checked for ticks and tested for infectious disease-causing pathogens but are not evaluated for evidence of infection with tick-borne pathogens. We tested blood samples collected from stray cattle by CFTEP inspectors for evidence of infection with tick-borne pathogens. As a comparison group representing U.S. resident cattle, we tested blood samples that had been sent to the Texas A&M Veterinary Medical Diagnostic Laboratory (TVMDL) for unrelated testing. Both sets of blood samples were evaluated using the same specific and broad-spectrum PCR assays. For the border cattle the overall prevalence of infection with one or more tick-borne pathogen was 58.5 % (79/135) with many co-infections, including 30 cattle positive for Babesia bovis and/or Babesia bigemina (22.2 %) and 77 cattle positive for Anaplasma marginale (57 %), three of these animals were also positive for Borrelia theileri. No resident cattle represented by the TVMDL samples were infected with either of the Babesia spp., or with Borrelia theileri, but three were positive for Theileria orientalis and 7.3 % (7/96) were positive for A. marginale. These data show that cattle originating in Mexico have a higher prevalence of infection with tick-borne pathogens relative to resident U.S. cattle and specifically, a proportion are infected with bovine Babesia, which is absent from U.S. cattle populations. Consequently, these stray cattle may be a reservoir of tick-borne pathogens and if populations of Boophilus ticks become reestablished in areas where they had previously been eradicated, could pose a significant risk to the U.S. Cattle industry.

Cattle ticks and associated tick-borne pathogens in Burkina Faso and Benin: Apparent northern spread of Rhipicephalus microplus in Benin and first evidence of Theileria velifera and Theileria annulata.

Babesiosis, theileriosis, anaplasmosis, and heartwater are tick-borne diseases that threaten livestock production in sub-Saharan Africa including Burkina Faso and Benin. For over a decade, these two bordering countries have been facing an invasion of the livestock by the tick Rhipicephalus microplus, a major vector for babesiosis, accidentally introduced in Benin in 2004. The molecular identification of tick-borne pathogens in this border area is of particular interest due to animals seasonal migration between the two countries. In this survey, epidemiological features of ticks and tick-borne pathogens in cattle were investigated to compare the eastern Burkina Faso, corresponding to a seasonal migration departure zone, and the northern Benin, which represents a seasonal migration arrival zone. Ticks and peripheral blood were collected from a total of 946 cattle in the two areas. Ticks were morphologically identified and the DNA samples from bovine blood and ticks were analysed by Reverse Line Blot (RLB) hybridization process. A total of 2856 ticks were collected on 490 cattle in Burkina Faso, eight tick species were identified, while 3583 ticks were collected on 456 cattle in North Benin with nine tick species identified. The invasive tick, R. microplus was not found in eastern Burkina Faso, but its spread farthest north in Benin is reported. Six tick-borne pathogen species were found in cattle blood both in eastern Burkina Faso and in northern Benin. Ranked in decreasing order of overall prevalences, they are: Theileria mutans (91.1%), Theileria velifera (77.8%), Babesia bigemina (10.9%), Anaplasma marginale (4.2%), Babesia bovis (3.3%), and Theileria annulata (1.8%). To the best of our knowledge, this survey represents the first report of T. velifera and T. annulata in the region. Overall, the TBP prevalences were significantly higher in northern Benin than in eastern Burkina Faso, indicating a higher parasitological risk in this area.

Assessment of Babesia bovis 6cys A and 6cys B as components of transmission blocking vaccines for babesiosis.

BACKGROUND: Babesia bovis reproduces sexually in the gut of its tick vector Rhipicephalus microplus, which involves expression of 6cys A and 6cys B proteins. Members of the widely conserved 6cys superfamily are candidates for transmission blocking vaccines (TBV), but intricacies in the immunogenicity of the 6cys proteins in the related Plasmodium parasites required the identification of transmission blocking domains in these molecules for vaccine design. Hereby, the immunogenic efficacy of recombinant (r) B. bovis 6cys A and B proteins as a TBV formulation was studied. METHODS: The immunogenicity of r6cys A and 6cys B proteins expressed in a eukaryotic system was evaluated in a cattle immunization trial (3 immunized and 3 control calves). A B. bovis sexual stage induction in vitro inhibition assay to assess the ability of antibodies to block the production of sexual forms by the parasite was developed. RESULTS: Immunized cattle generated antibodies against r6cys A and r6cys B that were unable to block sexual reproduction of the parasite in ticks. Additionally, these antibodies also failed in recognizing native 6cys A and 6cys B and peptides representing 6cys A and 6cys B functional domains and in inhibiting the development of sexual forms in an in vitro induction system. In contrast, rabbit antibodies generated against synthetic peptides representing predicted B-cell epitopes of 6cys A and 6cys B recognized recombinant and native forms of both 6cys proteins as well as peptides representing 6cys A and 6cys B functional domains and were able to neutralize development of sexual forms of the parasite in vitro. CONCLUSIONS: These data, combined with similar work performed on Plasmodium 6cys proteins, indicate that an effective 6cys protein-based TBV against B. bovis will require identifying and targeting selected regions of proteins containing epitopes able to reduce transmission.

Combination of entomopathogenic nematodes with acaricides or essential oil of Lippia triplinervis against Rhipicephalus microplus (Acari: Ixodidae).

This study aimed to evaluate the combination effect of Heterorhabditis bacteriophora HP88 and H. indica LPP1, with the acaricides deltamethrin, amitraz and chlorfenvinphos, and the essential oil (EO) of Lippia triplinervis, against engorged females of Rhipicephalus microplus. In order to verify the effect of acaricides and EO, the adult immersion test was used, and in the groups treated only with entomopathogenic nematodes (EPNs), 150 infective juveniles were used per female. In the treatments with nematodes in combination with the acaricides or EO, the females were immersed in the solutions (acaricide or EO) and then transferred to Petri dishes for application of the nematodes. The treatment with acaricides resulted in a control percentage lower than 70%, except in the group treated with chlorfenvinphos in the second experiment (84.3%). The control percentage was 73% for L. triplinervis EO, and greater than 90% in all the groups treated with nematodes. For treatments with EPNs combined with the acaricides or EO, the efficacy was greater than 95% (except for deltamethrin + HP88), and reached 100% in the treatment with LPP1 + amitraz. It can be concluded that the EPNs at the concentrations tested were compatible with the acaricides deltamethrin, amitraz and chlorfenvinphos, and with the EO of L. triplinervis. These combinations enhance the effect of these control agents.

Molecular detection and characterisation of protozoan and rickettsial pathogens in ticks from cattle in the pastoral area of Karamoja, Uganda.

Ticks and tick-borne diseases (TBDs) significantly affect cattle production and the livelihoods of communities in pastoralist areas. Data on protozoan and rickettsial pathogens in ticks infesting cattle in Uganda is scanty; while it is an indicator of the likelihood of disease transmission and occurrence. A cross-sectional study was conducted amongst cattle in the Karamoja Region, northeastern Uganda, from July through September 2017, to determine the tick species diversity, identify protozoan and rickettsial pathogens in the ticks, and characterise pathogenic species by sequence and phylogenetic analyses. About 50 % of the ticks detected from each predilection site on each animal were collected from 100 purposively-selected cattle from 20 randomly-selected herds. Twelve tick species belonging to the genera Amblyomma, Rhipicephalus and Hyalomma were identified, the most abundant being Amblyomma lepidum (93.9 %), followed by Amblyomma variegatum (2.0 %) and Rhipicephalus evertsi evertsi (1.0 %). Tick species that have not been reported in recent studies amongst cattle in Uganda were found, namely Rhipicephalus pravus, Rhipicephalus praetextatus and Rhipicephalus turanicus. The ticks were grouped into 40 pools, by species and location, and the reverse line blot (RLB) hybridisation assay was used to detect pathogens from the ticks. The most frequently detected tick-borne parasites were Theileria mutans, Theileria velifera and Theileria parva, each observed in 25 % (10/40) of the tick pools. Tick-borne pathogens, namely Babesia rossi, Babesia microti and Theileria sp. (sable) that are not common to, or not known to infect, cattle were identified from ticks. The gene encoding Ehrlichia ruminantium pCS20 region, the Ehrlichia and Anaplasma 16S rRNA gene, and T. parva p67 sporozoite antigen gene were amplified, cloned and sequenced. Seven novel E. ruminantium pCS20 variants were identified, and these grouped into two separate clusters with sequences from other parts of Africa and Asia. The T. parva p67 sequences were of the allele type 1, and parasites possessing this allele type are commonly associated with East Coast fever in eastern Africa. Analysis of the Ehrlichia and Anaplasma 16S rRNA gene sequences showed that they were closely related to Rickettsia africae and to a new Ehrlichia species variant recently found in China. Our R. africae 16S rRNA sequences grouped with R. africae isolates from Nigeria, Egypt and Benin. The information on tick species diversity and pathogens in the various tick species provides an indicator of potential transmission amongst cattle populations, and to humans, and can be useful to estimate disease risk and in control strategies.

Rhipicephalus microplus and its vector-borne haemoparasites in Guinea: further species expansion in West Africa.

Rhipicephalus microplus is an ixodid tick with a pantropical distribution that represents a serious threat to livestock. West Africa was free of this tick until 2007, when its introduction into Benin was reported. Shortly thereafter, further invasion of this tick species into other West African countries was identified. In this paper, we describe the first detection of R. microplus in Guinea and list the vector-borne haemoparasites that were detected in the invading and indigenous Boophilus species. In 2018, we conducted a small-scale survey of ticks infesting cattle in three administrative regions of Guinea: N`Zerekore, Faranah, and Kankan. The tick species were identified by examining their morphological characteristics and by sequencing their COI gene and ITS-2 gene fragments. R. microplus was found in each studied region. In the ticks, we found the DNA of Babesia bigemina, Anaplasma marginale, Anaplasma platys, and Ehrlichia sp. The results of this study indicate that R. microplus was introduced into Guinea in association with cows from Mali and/or the Ivory Coast.

Effects of white-tailed deer habitat use preferences on southern cattle fever tick eradication: simulating impact on "pasture vacation" strategies.

BACKGROUND: Rhipicephalus (Boophilus) annulatus and Rhipicephalus (Boophilus) microplus (southern cattle fever tick; SCFT), collectively known as cattle-fever ticks (CFTs), are vectors of protozoal parasites (Babesia bigemina and Babesia bovis) that cause bovine babesiosis (also known as cattle fever). One traditional strategy for CFT eradication involves the implementation of a "pasture vacation," which involves removing cattle (Bos taurus) from an infested pasture for an extended period of time. However, vacated pastures are often inhabited by wildlife hosts, such as white-tailed deer (WTD; Odocoileus virginianus), which can serve as alternate hosts for questing CFTs. We hypothesized that the distribution of host-seeking larvae among habitat types post-pasture vacation would reflect habitat use patterns of WTD, and in turn, affect the subsequent rate of pasture infestation by CFT. METHODS: We adapted a spatially explicit, individual-based model to simulate interactions among SCFT, cattle, and WTD as a tool to investigate the potential effects of WTD habitat use preferences on the efficacy of a pasture vacation. We parameterized the model to represent conditions typical of rangelands in south Texas, USA, simulated a 1-year pasture vacation under different assumptions regarding WTD habitat use preferences, and summarized effects on efficacy through (1) time post-vacation to reach 100% of pre-vacation densities of host-seeking larvae, and (2) the ecological conditions that resulted in the lowest host-seeking larval densities following pasture vacation. RESULTS: Larval densities at the landscape scale varied seasonally in a similar manner over the entire simulation period, regardless of WTD habitat use preferences. Following the removal of cattle, larval densities declined sharply to < 100 larvae/ha. Following the return of cattle, larval densities increased to > 60% of pre-vacation densities ≈ 21 weeks post-vacation, and reached pre-vacation levels in less than a year. Trends in larval densities in different habitat types paralleled those at the landscape scale over the entire simulation period, but differed quantitatively from one another during the pasture vacation. Relative larval densities (highest to lowest) shifted from (1) wood/shrub, (2) grass, (3) mixed-brush during the pre-vacation period to (1) mixed-brush, (2) wood/shrub, (3) grass or (1) wood/shrub, (2) mixed-brush, (3) grass during the post-vacation period, depending on WTD habitat use preferences. CONCLUSIONS: By monitoring WTD-driven shifts in distributions of SCFT host-seeking larvae among habitat types during simulated pasture vacation experiments, we were able to identify potential SCFT refugia from which recrudescence of infestations could originate. Such information could inform timely applications of acaricides to specific refugia habitats immediately prior to the termination of pasture vacations.

Prevalence of serum antibodies of tick-borne diseases and the presence of Rhipicephalus microplus in communal grazing cattle in the north-eastern region of the Eastern Cape Province of South Africa.

A survey to determine the prevalence of tick-borne diseases (TBDs) and ticks infesting cattle was conducted in the communal areas of the north-eastern region of the Eastern Cape Province (ECP) between January 2019 and October 2019. Tick counts, packed cell volume (PCV), body condition scores (BCS), and serological test for TBDs were seasonally conducted in communally grazed cattle (n = 240) in Joe Gqabi district in two local municipalities (Elundini and Senqu). A standard indirect fluorescent antibody test (IFAT) was used to determine antibodies to Babesia bigemina, Babesia bovis, and Ehrlichia ruminantium and indirect enzyme-linked immunosorbent assay (ELISA) was employed for antibodies to Anaplasma marginale detection. The highest tick loads were observed on cattle during the hot-wet and post-rainy seasons and lowest during the cool-dry season. The E. ruminantium prevalence in Elundini was 16% and 14% in post-rainy and hot-dry seasons respectively and 15% at Senqu during the post-rainy season. B. bigemina prevalence was highest at Elundini (18%) and Senqu (16%) during the post-rainy season and hot-wet season respectively. Cattle BCS was negatively correlated with E. ruminantium (P < 0.01; r = - 0.203), B. bovis (P < 0.01; r = - 0.125), and A. marginale (P < 0.01; r = - 0.122) seroprevalence. The PCV was negatively correlated with B. bigemina (P < 0.01; r = - 0.138) seroprevalence. On the other hand, E. ruminantium was positively correlated with Amblyomma hebraeum (P < 0.05; r = 0.112) infestation, while B. bovis (P < 0.05; r = 0.134) and B. bigemina (P < 0.05; r = 0.188) were positively correlated with Rhipicephalus microplus infestation, and B. bigemina (P < 0.05; r = 0.077) was positively correlated with Rhipicephalus decoloratus infestation. Our study reports for the first time the presence of R. microplus in the study area. Further research is, however, needed to better understand seroprevalence and the transmission mode of TBDs to cattle so that effective disease control measures can be developed.

A Trypanosoma species detected in Rhipicephalus (Boophilus) microplus ticks from Argentina.

Specimens of a Trypanosoma sp. were found in a haemolymph sample of Rhipicephalus microplus from Argentina. Polymerase chain reaction (PCR) was done targeting the SSU rRNA gene of Trypanosoma spp. and a fragment of 2300 base pairs (bp) was amplified, subsequently a phylogenetic analysis was conducted, based on an alignment of 905 bp, containing the sequence of the Argentina isolate and sequences of different Trypanosoma species retrieved from GenBank. Phylogenetic analysis revealed that this trypanosome is not related to Trypanosoma theileri as was previously thought, instead the strain of Trypanosoma detected in this study can be provisionally determined as belonging to the recently described organism Trypanosoma rhipicephalis. Furthermore, phylogenetic analysis performed in this work revealed that T. rhipicephalis belongs to a novel clade of tick-related trypanosomes, most with limited genetic data, for which essential aspects of both the vertebrate and invertebrate life cycles are lacking. The lack of basic information restricts the inferences that can be done from the present finding and, in addition, points out a clear knowledge gap in the biology of this group of trypanosomes.

Molecular Diagnosis and Prevalence of Trypanosoma vivax (Trypanosomatida: Trypanosomatidae) in Buffaloes and Ectoparasites in the Brazilian Amazon Region.

Trypanosoma vivax Ziemann is a parasite that affects both wild and domestic ungulates and is transmitted mechanically via tabanids and other blood-sucking insects in the Americas. A total of 621 blood samples from water buffaloes (Bubalus bubalis (Linnaeus) (Artiodactyla: Bovidae), and 184 ectoparasite samples (Amblyomma cajennense (Fabricius) sensu stricto and Rhipicephalus (Boophilus) microplus (Canestrini) (Acari: Ixodidae), and Haematopinus tuberculatus (Burmeister) (Phthiraptera: Haematopinidae)) were obtained from 60 farms in the State of Pará, Brazilian Amazon. Twelve buffalo blood samples (1.89%) and 11 ectoparasites (6%) were positive for T. vivax based on the cathepsin L-like gene. All sequences were 99% similar to T. vivax from northeastern Brazil (EU753788) in amplified PCR assays on each of the hosts tested.

Molecular evidence confirms occurrence of Rhipicephalus microplus Clade A in Kenya and sub-Saharan Africa.

BACKGROUND: The tick vector Rhipicephalus microplus which transmits Babesia spp. and rickettsial pathogens has not been reported in Kenya since 1998. More recently, the pathogenic Babesia bovis has been detected in cattle blood DNA. The status of R. microplus in Kenya remains unknown. This study employed morphological and molecular tools to characterize R. microplus originating from Kenya and assess the genetic relationships between Kenyan and other African R. microplus genotypes. METHODS: Ticks were collected in south-eastern Kenya (Kwale County) from cattle and characterized to investigate the existence of R. microplus. Genetic and phylogenetic relationships between the Kenyan and other annotated R. microplus reference sequences was investigated by analysis of the cytochrome c oxidase subunit 1 (cox1) gene. To further characterize Kenyan ticks, we generated low coverage whole genome sequences of two R. microplus, one R. decoloratus and R. appendiculatus. A B. bovis specific TaqMan probe qPCR assay was used to detect B. bovis in gDNA from R. microplus ticks. RESULTS: Occurrence of R. microplus was confirmed in Kwale County, Kenya. The Kenyan R. microplus cox1 sequences showed very high pairwise identities (> 99%) and clustered very closely with reference African R. microplus sequences. We found a low genetic variation and lack of geographical sub-structuring among the African cox1 sequences of R. microplus. Four complete mitochondrial (mt) genomes for two R. microplus, one R. decoloratus and one R. appendiculatus were assembled from next generation sequence data. The mitochondrial genome sequences of the two Kenyan R. microplus ticks clustered closely with reference genome sequences from Brazil, USA, Cambodia and India forming R. microplus Clade A. No B. bovis was detected in the Kwale R. microplus DNA. CONCLUSIONS: These findings confirm the presence of R. microplus in Kenya and suggest that R. microplus Clade A is prevalent in cattle in sub-Saharan Africa. These and other recent findings of widespread occurrence of R. microplus in Africa provide a strong justification for urgent surveillance to determine and monitor the spread of R. microplus and vector competence of Boophilus ticks for B. bovis in Africa, with the ultimate goal of strategic control.

A tick cell line as a powerful tool to screen the antimicrobial susceptibility of the tick-borne pathogen Anaplasma marginale.

Anaplasma marginale is the causative agent of the severe bovine anaplasmosis. The tick Rhipicephalus microplus is one of the main vectors of A. marginale in tropical and subtropical regions of the world. After the tick bite, the bacterium invades and proliferates within the bovine erythrocytes leading to anemia, impairment of milk production and weight loss. In addition, infection can cause abortion and high mortality in areas of enzootic instability. Immunization with live and inactivated vaccines are employed to control acute bovine anaplasmosis. However, they do not prevent persistent infection. Consequently, infected animals, even if immunized, are still reservoirs of the bacterium and contribute to its dissemination. Antimicrobials are largely employed for the prophylaxis of bovine anaplasmosis. However, they are often used in sublethal doses which may select pre-existing resistant bacteria and induce genetic or phenotypic variations. Therefore, we propose a new standardized in vitro assay to evaluate the susceptibility of A. marginale strains to different antimicrobials. This tool will help health professionals to choose the more adequate treatment for each herd which will prevent the selection and spread of resistant strains. For that, we initially evaluated the antimicrobial susceptibility of two field isolates of A. marginale (Jaboticabal and Palmeira) infecting bovines. The least susceptible strain (Jaboticabal) was used for the standardization of an antimicrobial assay using a culture of Ixodes scapularis-derived tick cell line, ISE6. Results showed that enrofloxacin (ENRO) at 0.25, 1 or 4 µg/mL and oxytetracycline (OTC) at 4 or 16 µg/mL are the most efficient treatments, followed by OTC at 1 µg/mL and imidocarb dipropionate (IMD) at 1 or 4 µg/mL. In addition, this proposed tool has technical advantages compared to the previously established bovine erythrocyte culture. Thereby, it may be used to guide cattle farmers to the correct use of antimicrobials. The choice of the most suitable antimicrobial is essential to eliminate persistent infections, prevent the spread of resistant strains and help controlling of bovine anaplasmosis.

The Rhipicephalus appendiculatus tick vector of Theileria parva is absent from cape buffalo (Syncerus caffer) populations and associated ecosystems in northern Uganda.

Rhipicephalus appendiculatus is the major tick vector of Theileria parva, an apicomplexan protozoan parasite that causes the most economically important and lethal disease of cattle in East and central Africa. The African cape buffalo (Syncerus caffer) is the major wildlife host of T. parva from southern Uganda and Kenya to southern Africa. We show herein that R. appendiculatus appears to be absent from the two largest national parks in northern Uganda. Syncerus caffer is common in both of these national parks, specifically Murchison falls (MFNP) and Kidepo Valley (KVNP). We re-confirmed the previously reported absence of T. parva in buffalo sampled in the two northern parks based on RLB data using a nested PCR based on the T. parva p104 gene. By contrast, T. parva-infected R. appendiculatus ticks and parasite-infected buffalo were present in Lake Mburo (LMNP) in South central Uganda. This suggests that the distribution of R. appendiculatus, which is predicted to include the higher rainfall regions of northern Uganda, may be limited by additional, as yet unknown factors.

Investigation of Ixodid ticks as vectors of Babesia caballi and Theileria equi (Protozoa: Apicomplexa) in central Italy.

Babesia caballi and Theileria equi are widely recognized as causative agents of equine pirolasmosis (EP), an acute, sub-acute, and chronic disease of equines, with relevant economic impact on horse trade worldwide. Although several studies on EP prevalence from central Italy have been published, data on ticks responsible for its transmission are still lacking. In this study, we identified a potential competent vector, investigating main features of its ecology together with EP infection rates. A two-year sampling of questing ticks was carried out for the first time in Italy in an area known for high EP prevalence in horse sera, detecting the association between Rhipicephalus bursa and causative agents of EP. Most of the positive pools harbored a single infection (91.1%); mixed infections were also detected (8.9%). The infection rate for T. equi slightly decreased among years; B. caballi showed a lower, but increasing, infection rate. Tick phenology, climate variables, and peaks of EP prevalence indicated late May and second half of June as periods with the highest risk of new infections, especially during warm and dry days.