Bovine Piroplasma Populations in the Philippines Characterized Using Targeted Amplicon Deep Sequencing.

Molecular assays and capillary electrophoresis sequencing have been used to identify parasites in livestock. The low sample capacity, which increases labor and processing time, is one drawback. Targeted amplicon sequencing (Ampliseq) uses the fast and large sample capacity platform to identify parasites in the target host, overcoming this limitation. DNA was extracted from 162 whole blood samples collected from cattle in three provinces in the Philippines. Using Illumina's Miseq platform, the V4 hypervariable region of the piroplasma 18S rRNA gene was amplified and sequenced. The AMPtk pipeline was used to obtain distinct amplicon sequence variants (ASVs) and the NCBI BLAST non-redundant database was used to assign taxonomy. In total, 95 (58.64%) samples were positive for piroplasma. Using the AMPTk pipeline, 2179 ASVs were obtained. A total of 79 distinct ASVs were obtained after clustering and filtering, which belonged to genera Babesia (n = 58), Theileria (n = 17), Hepatozoon (n = 2), and Sarcocystis (n = 2). The ASV top hits were composed of 10 species: Babesia bovis, B. bigemina, Theileria orientalis, Babesia sp., Hepatozoon canis, Sarcocystis cruzi, T. annulata, T. equi, T. mutans, and Theileria sp. Thung Song. The results generated in this study demonstrated the applicability of Ampliseq in detecting piroplasmid parasites infecting cattle in the Philippines.

Tick-borne Pathogen Detection and Its Association with Alterations in Packed Cell Volume of Dairy Cattle in Thailand.

Tick-borne diseases (TBDs) massively impact bovine production. In endemic countries, animals are often subclinically infected, showing no signs of the illness. Anemia is a hallmark of TBDs, but there is inadequate information on its presence in infected Thai cattle. In the present study, 265 cattle from four provinces in Thailand were surveyed to identify tick-borne pathogens (TBPs) and to evaluate the changes in the packed cell volume (PCV) values associated with detection. Microscopy and polymerase chain reaction (PCR) were also compared for TBP detection. Babesia/Theileria/Hepatozoon was detected in 33.58% (89/265) of the cattle samples. Specifically, Babesia bovis (9/265), B. bigemina (12/265), Theileria orientalis (62/265), and Anaplasma marginale (50/265) were identified using species-specific assays. Significant decreases in the mean PCV levels were observed in cattle that were positive for at least one TBP (p < 0.001), Babesia/Theileria/Hepatozoon (p < 0.001), T. orientalis (p < 0.001), and A. marginale (p = 0.049). The results of PCR and microscopy for the detection of TBPs suggested slight and fair agreement between the two detection tools. The present findings contribute to a better understanding of TBDs in the field and shall facilitate the formulation of effective control for TBDs in Thailand.

Identification and Characterization of Rhipicephalus microplus ATAQ Homolog from Haemaphysalis longicornis Ticks and Its Immunogenic Potential as an Anti-Tick Vaccine Candidate Molecule.

Although vaccines are one of the environmentally friendly means to prevent the spread of ticks, there is currently no commercial vaccine effective against Haemaphysalis longicornis ticks. In this study, we identified, characterized, localized, and evaluated the expression patterns, and tested the immunogenic potential of a homologue of Rhipicephalus microplus ATAQ in H. longicornis (HlATAQ). HlATAQ was identified as a 654 amino acid-long protein present throughout the midgut and in Malpighian tubule cells and containing six full and one partial EGF-like domains. HlATAQ was genetically distant (homology < 50%) from previously reported ATAQ proteins and was expressed throughout tick life stages. Its expression steadily increased (p < 0.001) during feeding, reached a peak, and then decreased slightly with engorgement. Silencing of HlATAQ did not result in a phenotype that was significantly different from the control ticks. However, H. longicornis female ticks fed on a rabbit immunized with recombinant HlATAQ showed significantly longer blood-feeding periods, higher body weight at engorgement, higher egg mass, and longer pre-oviposition and egg hatching periods than control ticks. These findings indicate that the ATAQ protein plays a role in the blood-feeding-related physiological processes in the midgut and Malpighian tubules and antibodies directed against it may affect these tissues and disrupt tick engorgement and oviposition.

PLK:Δgra9 Live Attenuated Strain Induces Protective Immunity Against Acute and Chronic Toxoplasmosis.

Toxoplasmosis is a zoonotic parasitic disease caused by the obligate intracellular protozoa Toxoplasma gondii, which threatens a range of warm-blooded mammals including humans. To date, it remains a challenge to find safe and effective drug treatment or vaccine against toxoplasmosis. In this study, our results found that the development of a mutant strain based on gene disruption of dense granule protein 9 (gra9) in type II PLK strain decreased parasite replication in vivo, severely attenuated virulence in mice, and significantly reduced the formation of cysts in animals. Hence, we developed an immunization scheme to evaluate the protective immunity of the attenuated strain of Δgra9 in type II PLK parasite as a live attenuated vaccine against toxoplasmosis in the mouse model. Δgra9 vaccination-induced full immune responses characterized by significantly high levels of pro-inflammatory cytokine interferon gamma (IFN-γ) and interleukin-12 (IL-12), maintained the high T. gondii-specific immunoglobulin G (IgG) level, and mixed high IgG1/IgG2a levels. Their levels provided the complete protective immunity which is a combination of cellular and humoral immunity in mouse models against further infections of lethal doses of type I RH, type II PLK wild-type tachyzoites, or type II PLK cysts. Results showed that Δgra9 vaccination proved its immunogenicity and potency conferring 100% protection against acute and chronic T. gondii challenges. Together, Δgra9 vaccination provided safe and efficient immune protection against challenging parasites, suggesting that PLK:Δgra9 is a potentially promising live attenuated vaccine candidate.

A Survey of Tick Infestation and Tick-Borne Piroplasm Infection of Cattle in Oudalan and Séno Provinces, Northern Burkina Faso.

In this study, cattle farms located in Oudalan and Séno, two provinces in the Sahel region, northern Burkina Faso, were surveyed. Cattle owners were interviewed, cattle were examined for tick infestation, and ticks as well as blood samples were collected during the dry season (October). Blood DNA samples were tested for Babesia and Theileria infections using nested PCRs and sequencing. A total of 22 herds, 174 Zebu cattle were investigated at 6 different sites. Overall, 76 cattle (43.7 %) from 18 farms (81.8%) were found infested with ticks. Cattle in Séno, adult cattle (>5 years) and those owned by the Fulani ethnic group were significantly (p < 0.05) more likely to be tick-infested. A total of 144 adult ticks belonging to five species namely: Hyalomma impeltatum, Hyalomma impressum, Hyalomma rufipes, Rhipicephalus evertsi evertsi, and Rhipicephalus guilhoni were collected from the animals. Piroplasms were detected in the blood DNA of 23 (13.2%) cattle. The cattle in Séno and adult cattle were significantly more likely to be piroplasm-positive. Five pathogens diversely distributed were identified. Theileria mutans (12/174), Babesia bigemina (5/174), Theileria annulata (3/174), and Theileria velifera (3/174) were detected for the first time in northern Burkina Faso, whereas Babesia occultans (1/174) was found for the first time in cattle in West Africa. The analysis of the sequences, including B. bigemina RAP-1a, T. annulata Tams1 genes, and the 18S rRNA genes of all the five protozoa, revealed identities ranging from 98.4 to 100% with previously published sequences. Phylogenetic analysis based on the 18S rRNA gene sequences located north Burkina Faso piroplasms in the same clade as isolates from Africa and other regions of the world. Notably, T. mutans sequences were distributed in two clades: the T. mutans Intona strain clade and the Theileria sp. (strain MSD)/ Theileria sp. B15a clade, suggesting the presence of at least two strains in the area. These findings indicate that the control of ticks and tick-borne diseases should be taken into account in strategies to improve animal health in the Sahel region.

Molecular detection of Borrelia burgdorferi (sensu lato) and Rickettsia spp. in hard ticks distributed in Tokachi District, eastern Hokkaido, Japan.

Ticks transmit various pathogens, including parasites, bacteria and viruses to humans and animals. To investigate the ticks and the potentially zoonotic pathogens that they may carry, questing ticks were collected in 2017 from 7 sites in Tokachi District, eastern Hokkaido, Japan. A total of 1563 ticks including adults (male and female), nymphs and larvae were collected. Four species of ticks were identified: Ixodes ovatus, Ixodes persulcatus, Haemaphysalis japonica and Haemaphysalis megaspinosa. Of the 1563 ticks, 1155 were used for DNA extraction. In total, 527 individual tick DNA samples prepared from adults (n = 484), nymphs (n = 41) and larvae (n = 2); and 67 pooled tick DNA samples prepared from larval stages (n = 628) were examined using PCR methods and sequencing to detect Borrelia burgdorferi (sensu lato) and Rickettsia spp. The phylogenetic analysis of Borrelia spp. flaB gene sequences showed the presence of the human pathogenic B. burgdorferi (s.l.) species (Borrelia garinii, Borrelia bavariensis and Borrelia afzelii) in I. persulcatus, whereas the non-pathogenic species Borrelia japonica was found only in I. ovatus. In I. persulcatus, B. garinii and/or its closely related species B. bavariensis was detected in both adults and nymphs at a prevalence of 21.9% whereas B. afzelii was found only in adults (1.8%). The prevalence of B. japonica in adult I. ovatus was 21.8%. Rickettsia species were identified through phylogenetic analysis based on gltA, 16S rRNA, ompB and sca4 genes. Four genotypes were detected in the samples which were classified into three species. The prevalence of human pathogenic Rickettsia helvetica was 26.0% in I. persulcatus adults and nymphs, 55.6% in I. persulcatus larval pools, and 1.7% in H. megaspinosa larval pools. The prevalence of "Candidatus R. tarasevichiae" was 15.4% in I. persulcatus adults and nymphs and 33.3% in I. persulcatus larval pools. The prevalence of "Candidatus R. principis" in H. megaspinosa adults and nymphs was 11.1% whereas it was detected in 3.4% of the H. megaspinosa larval pools. These results indicate that most of the risks of Lyme borreliosis and spotted fever group rickettsiosis infection in eastern Hokkaido, Japan, are restricted to I. persulcatus.

First Molecular Detection of Babesia ovis, Theileria spp., Anaplasma spp., and Ehrlichia ruminantium in Goats from Western Uganda.

Ticks and tick-borne diseases are major impediments to livestock production. To date, there have been several studies on the prevalence of tick-borne pathogens (TBPs) in cattle, but very few studies have documented TBPs in goats in Uganda. In this study, polymerase chain reaction assays and sequence analysis of different molecular markers were used to assess the presence and genetic characteristics of TBPs in 201 goats from Kasese district in western Uganda. The risk factors associated with TBP infections were also analyzed. We detected Theileria spp. (13.4%), Anaplasma phagocytophilum (10.9%), Anaplasma ovis (5.5%), Babesia ovis (5.5%), and Ehrlichia ruminantium (0.5%). The sequences of B. ovis ssu rRNA and A. ovismsp4 genes showed some degree of diversity among the parasite isolates in this study. The E. ruminantium pCS20 sequence formed a well-supported clade with isolates from Amblyomma variegatum ticks from Uganda. Wildlife interaction, sampling location, low body condition score, tick infestation, and herd size were significantly associated with TBP infections in the goats. The findings in this study provide important information on the epidemiology of tick-borne pathogens in Uganda, and show that goats could be potential reservoirs for tick-borne pathogens.

Porin Expression Profiles in Haemaphysalis longicornis Infected With Babesia microti.

The porin gene is widely disseminated in various organisms and has a pivotal role in the regulation of pathogen infection in blood-sucking arthropods. However, to date, information on the porin gene from the Haemaphysalis longicornis tick, an important vector of human and animal diseases, remains unknown. In this study, we identified the porin gene from H. longicornis and evaluated its expression levels in Babesia microti-infected and -uninfected H. longicornis ticks at developmental stages. We also analyzed porin functions in relation to both tick blood feeding and Babesia infection and the relationship between porin and porin-related apoptosis genes such as B-cell lymphoma (Bcl), cytochrome complex (Cytc), caspase 2 (Cas2), and caspase 8 (Cas8). The coding nucleotide sequence of H. longicornis porin cDNA was found to be 849 bp in length and encoded 282 amino acids. Domain analysis showed the protein to contain six determinants of voltage gating and two polypeptide binding sites. Porin mRNA levels were not significantly different between 1-day-laid and 7-day-laid eggs. In the nymphal stage, higher porin expression levels were found in unfed, 12-h-partially-fed (12 hPF), 1-day-partially-fed (1 dPF), 2 dPF nymphs and nymphs at 0 day post-engorgement (0 dAE) vs. nymphs at 2 dAE. Cytc and Cas2 mRNA levels were higher in 2 dPF nymphs in contrast to nymphs at 2 dAE. Porin expression levels appeared to be higher in the infected vs. uninfected nymphs during blood feeding except at 1 dPF and 0-1 dAE. Especially, the highest B. microti burden negatively affected porin mRNA levels in both nymphs and female adults. Porin knockdown affected body weight and Babesia infection levels and significantly downregulated the expression levels of Cytc and Bcl in H. longicornis female ticks. In addition, this study showed that infection levels of the B. microti Gray strain in nymphal and female H. longicornis peaked at or around engorgement from blood feeding to post engorgement. Taken together, the research conducted in this study suggests that H. longicornis porin might interfere with blood feeding and B. microti infection.

First detection of Anaplasma ovis in sheep and Anaplasma platys-like variants from cattle in Menoufia governorate, Egypt.

Tick-borne diseases are of global economic importance, especially due to the costs associated with disease treatment and productivity losses in livestock. In this study, 244 livestock animals (cattle N = 92, buffaloes N = 86 and sheep N = 66) from Menoufia, Egypt were tested for Anaplasma, Ehrlichia and Babesia species using PCR. Results revealed detection of A. ovis (9.1%) in sheep while Anaplasma spp. (14.1%), A. marginale (15.2%), B. bigemina (6.5%) and B. bovis (5.4%) in cattle. On the other hand, Anaplasma spp. (1.2%), A. marginale (1.2%) and B. bovis (1.2%), were detected in buffaloes. Significantly higher detection rates were observed in cattle for Anaplasma spp. (P = .020), A. marginale (P = .001) and B. bigemina (P = .022) than in buffaloes. Sequence analysis of Anaplasma spp. isolates from cattle, revealed A. platys-like strains. Phylogenetic analyses of the A. platys-like isolates revealed variation among the strains infecting cattle. The A. marginale buffalo isolate, on the other hand, showed some level of divergence from the cattle isolates. This study reports the first detection of A. ovis in sheep and A. platys-like strains in cattle in Menoufia and Egypt at large. The results of the current study provide valuable information on the epidemiology and genetic characteristics of tick-borne pathogens infecting livestock in Egypt.

Molecular detection and characterization of tick-borne haemoparasites among cattle on Zanzibar Island, Tanzania.

Tick-borne diseases (TBDs) are serious constraints to livestock production in Tanzania and other tropical and subtropical countries and impact the livelihoods of resource-poor farming communities in the region. In Tanzania, detailed studies on tick-borne pathogens (TBPs) in cattle using sensitive molecular detection methods are scarce. The objective of this study was to investigate the occurrence and species composition of bovine TBPs in cattle kept in Zanzibar Island. A total of 236 blood samples were randomly collected in cattle population in June and July 2019. We used polymerase chain reaction (PCR) and gene sequencing to detect and identify pathogens. PCR screening of all 236 samples revealed that 64.5% of animals were infected by TBPs, including Theileria parva (34.3%), T. mutans (38.1%), T. taurotragi (30.9%), Anaplasma marginale (10.2%), Babesia bigemina (5.1%), T. velifera (3.4%) and B. bovis (2.1%). Overall a total of 86 animals (36.4%) were co-infected with up to five pathogens including T. parva, T. mutans, T. taurotragi, A. marginale and B. bigemina. The pathogens mostly involved in the co-infection were T. parva, T. taurotragi and T. mutans. Sequence analysis indicated that T. parva p104 and B. bigemina RAP1a genes are diverse among the sampled animals in Zanzibar Island, with 99.64%-100% and 99.51%-100% nucleotide sequence identity value respectively. In contrast, the A. marginale MSP-5, T. mutans 18S rRNA V4 region and B. bovis SBP-2 genes are conserved, with 100%, 99.05%-100% and 99.66%-100% nucleotide sequence identity values respectively. The phylogenetic analyses revealed that T. parva p104 and B. bigemina RAP1a gene sequences showed significant differences of genotypes, as they appear in different clades. Meanwhile, A. marginale MSP-5, T. mutans 18S rRNA V4 region and B. bovis SBP-2 gene sequences appear in the same clade with other sequences extracted from the NCBI GenBank. The epidemiological findings revealed in this study will provide important information on tick-borne diseases in Tanzania and will be used as scientific basis for planning future control strategies.

Molecular survey of tick-borne pathogens infecting backyard cattle and water buffaloes in Quezon province, Philippines.

Tick-borne diseases (TBD) cause enormous losses for farmers. Backyard raising comprises majority of the livestock population in the Philippines, but TBD information in backyard livestock is scarce. In this study, 48 cattle and 114 water buffalo samples from Quezon province, Philippines were molecularly screened for tick-borne pathogens. Anaplasma marginale (16.67%) and hemoplasma (20.99%) were detected in the samples. A. marginale infection (P=0.0001) was significantly higher in cattle, while hemoplasma infection (P=0.011) was significantly higher in water buffaloes. A. marginale isolates from this study were highly similar to previous isolates from the Philippines while Mycoplasma wenyonii and Candidatus Mycoplasma haemobos were the identified hemoplasma species. Our findings reveal additional information on the TBD situation of Philippine backyard livestock.

Identification of Haemaphysalis longicornis Genes Differentially Expressed in Response to Babesia microti Infection.

Haemaphysalis longicornis is a tick and a vector of various pathogens, including the human pathogenetic Babesia microti. The objective of this study was to identify female H. longicornis genes differentially expressed in response to infection with B. microti Gray strain by using a suppression subtractive hybridization (SSH) procedure. A total of 302 randomly selected clones were sequenced and analyzed in the forward subtracted SSH cDNA library related to Babesia infection, and 110 clones in the reverse cDNA library. Gene ontology assignments and sequence analyses of tick sequences in the forward cDNA library showed that 14 genes were related to response to stimulus or/and immune system process, and 7 genes had the higher number of standardized sequences per kilobase (SPK). Subsequent real-time PCR detection showed that eight genes including those encoding for Obg-like ATPase 1 (ola1), Calreticulin (crt), vitellogenin 1 (Vg1) and Vg2 were up-regulated in fed ticks. Compared to uninfected ticks, infected ticks had six up-regulated genes, including ola1, crt and Vg2. Functional analysis of up-regulated genes in fed or Babesia-infected ticks by RNA interference showed that knockdown of crt and Vg2 in infected ticks and knockdown of ola1 in uninfected ticks accelerated engorgement. In contrast, Vg1 knockdown in infected ticks had delayed engorgement. Knockdown of crt and Vg1 in infected ticks decreased engorged female weight. Vg2 knockdown reduced B. microti infection levels by 51% when compared with controls. The results reported here increase our understanding of roles of H. longicornis genes in blood feeding and B. microti infection.

Human Spotted Fever Group Rickettsia Infecting Yaks (Bos grunniens) in the Qinghai-Tibetan Plateau Area.

The Qinghai-Tibetan Plateau Area (QTPA) is a plateau with the highest average altitude, located in Northwestern China. There is a risk for interspecies disease transmission, such as spotted fever rickettsioses. However, information on the molecular characteristics of the spotted fever group (SFG) Rickettsia spp. in the area is limited. This study performed screenings, and detected the DNA of human pathogen, SFG Rickettsia spp., with 11.3% (25/222) infection rates in yaks (Bos grunniens). BLASTn analysis revealed that the Rickettsia sequences obtained shared 94.3-100% identity with isolates of Rickettsia spp. from ticks in China. One Rickettsia sequence (MN536161) had 100% nucleotide identity to two R. raoultii isolates from Chinese Homo sapiens, and one isolate from Qinghai Dermacentor silvarum. Meanwhile, another Rickettsia sequence (MN536157) shared 99.1-99.5% identity to one isolate from Dermacentor spp. in China. Furthermore, the phylogenetic analysis of SFG Rickettsia spp. ompA gene revealed that these two sequences obtained from yaks in the present study grouped with the R. slovaca and R. raoultii clades with isolates identified from Dermacentor spp. and Homo sapiens. Our findings showed the first evidence of human pathogen DNA, SFG Rickettsia spp., from animals, in the QTPA.

Transient Transfection of the Zoonotic Parasite Babesia microti.

The development of genetic manipulation techniques has been reported in many protozoan parasites over the past few years. However, these techniques have not been established for Babesia microti. Here, we report the first successful transient transfection of B. microti. The plasmids containing the firefly luciferase reporter gene were transfected into B. microti by an AMAXA 4D Nucleofection system. Twenty-four-hour synchronization, the 5'-actin promoter, program FA100, and 50 µg of plasmid DNA constituted the best conditions for the transient transfection of B. microti. This finding is the first step towards a stable transfection method for B. microti, which may contribute to a better understanding of the biology of the parasite.

Molecular evidence of hemotropic mycoplasmas in goats from Cebu, Philippines.

Hemotropic mycoplasma (hemoplasma), a neglected vector-borne pathogen in goats, causes extensive economic damage to farmers due to production losses. In this study, 107/295 (36.27%) goats sampled from 4 farms (Barili, Danao City, Dumanjug and Minglanilla) in Cebu, Philippines tested positive for PCR targeting the 16S rRNA gene of Mycoplasma. All hemoplasma-positive goats were from Barili and no clinical sign was observed. Sex (P=0.0005) and age (P=0.03) were found associated with hemoplasma infection. Mycoplasma ovis, Candidatus Mycoplasma haemobos, Candidatus Mycoplasma haemominutum and 3 Uncultured Mycoplasma sp. sequences were identified by sequencing analysis. This is the first report of molecular detection and genetic characterization of hemoplasmas in goats in the Philippines.

First molecular detection and characterization of tick-borne pathogens in water buffaloes in Bohol, Philippines.

The water buffalo industry is a vital part of the Philippine livestock economy and is an essential contributor to the developing local dairy industry. Although relatively less susceptible to diseases, water buffaloes can still be infected and can act as reservoirs of tick-borne pathogens (TBPs). However, limited information is available regarding the prevalence of tick-borne infections in water buffaloes in the Philippines. This study was conducted to identify TBPs harbored by water buffaloes and to characterize these pathogens molecularly. One hundred water buffalo blood samples collected from three areas in Bohol, Visayas region, Philippines were screened for various TBPs using pathogen-specific PCR assays. TBPs were detected in 46% of the samples (39% singly infected, 7% coinfected). The pathogens detected were Anaplasma marginale (29%), Babesia bovis (21%), and B. bigemina (3%). None of the blood samples were positive for Theileria annulata, T. orientalis, and B. ovata. A. marginale infection rates were significantly higher (37.5%) among water buffaloes aged ≤6 years (P = 0.046) than those >6 years old (18.2%) and was detected only in Bulgarian Murrah (36.1%) and US Murrah (25.9%) breeds. Phylogenetic analyses revealed that groEL sequences of A. marginale were 100% identical with isolates from the Philippines (Batangas and Cebu) and China. Two B. bigemina RAP-1a gene sequences were identical to each other and were homologous with previous isolates from Thailand, Indonesia, Uruguay, and the Philippines. Moreover, four B. bovis SBP-2 partial sequences obtained in this study had 92.4-99.7% identities. This study is the first molecular detection and characterization of A. marginale, B. bigemina and B. bovis in water buffaloes in the Visayas region, and the first molecular confirmation of B. bovis infection in water buffaloes in the country. The findings presented in this study may serve as baseline data for crafting effective tick-borne disease surveillance and prevention programs in Bohol and in the Philippines.

Genetic characterization of tick-borne pathogens in ticks infesting cattle and sheep from three South African provinces.

Ticks are involved in the transmission of many public health and veterinary important pathogens. Although tick-borne pathogens are widely distributed in South Africa, information on tick-pathogen relationship needs to be updated particularly using modern molecular techniques. This study used PCR and sequencing to confirm the identity of the tick species collected from cattle and sheep from KwaZulu-Natal, Free State and Eastern Cape. Furthermore, presence of Babesia spp., Theileria spp., Anaplasma marginale, Rickettsia spp., Ehrlichia ruminantium and Coxiella burnetii was detected from tick DNA using species-specific PCR or nested PCRs. The study samples consisted of 390 adult ticks (male and female) which were pooled according to species, host animal and sampling site (three ticks per pool) for DNA extraction. The PCR results revealed that out of 130 tick DNA pools, 30 (23.1%) were positive for at least one pathogen. The most frequent pathogen was C. burnetii (9.2%), followed by Rickettsia spp. (7.7%), A. marginale (3.8%), T. mutans (3.1%), T. taurotragi (2.3%) and E. ruminantium (1.5%). The highest prevalence of pathogens was observed in ticks collected from cattle in Eastern Cape (16/42) and the lowest was in ticks obtained from sheep in Free State (1/21). Infected ticks were identified as Rhipicephalus evertsi evertsi (n = 13), R. appendiculatus (n = 3), R. decoloratus (n = 7) and Amblyomma hebraeum (n = 7). Coinfection with two pathogens was found in 21% of pathogen-positive pools. Analysis of Theileria taurotragi 18S rRNA, T. mutans 18S rRNA, C. burnetii htpB, Rickettsia spp. gltA, Rickettsia spp. ompA, E. ruminantium pCS20 and A. marginale Msp5 sequences showed that the pathogens detected in this study were genetically related to isolates previously reported in Africa. These findings provide important information on distribution of ticks and tick-borne pathogens of ruminants and will contribute in the formulation of future control strategies in South Africa.

Inhibitory effects of the phytohormone inhibitors fluridone and inabenfide against Babesia gibsoni in vitro.

Pharmacological options to treat canine babesiosis caused by Babesia gibsoni, are limited. To address this challenge, screening for novel drug candidates and drug targets against B. gibsoni is urgently needed. In this study, we explored the inhibitory effects of two phytohormone inhibitors, fluridone (FLU) and inabenfide (INA), against B. gibsoni in vitro. The half-maximal inhibitory concentration (IC50) values of FLU and INA against B. gibsoni were 60.6 ± 3.4 and 4.3 ± 0.3 µM, respectively. Parasitemia and viability at 24, 48, and 72 h after FLU and INA treatments were significantly lower than those in the control group. The cytotoxicity of FLU and INA was evaluated using the dog-derived Madin-Darby canine kidney (MDCK) cell line; both FLU and INA were less toxic to the MDCK cells than to the control cells. The selectivity index of FLU and INA were higher than 16.5 and 232.6, respectively. In summary, the present study demonstrated that FLU and INA were effective against B. gibsoni infection in vitro and that these compounds might have potential as candidate drugs for the treatment of B. gibsoni.

Molecular detection and characterization of tick-borne protozoan and rickettsial pathogens isolated from cattle on Pemba Island, Tanzania.

Tick-borne diseases cause significant losses to livestock production in tropical and subtropical regions. In Tanzania, detailed studies on tick-borne pathogens in cattle using sensitive molecular detection methods are scarce. In this study, we investigated the occurrence of Theileria spp., Babesia spp., Anaplasma spp. and Ehrlichia spp. in 245 blood samples collected from cattle on Pemba Island, Tanzania. We used polymerase chain reaction (PCR) and gene sequencing to detect and identify pathogens. PCR screening revealed overall infection rates of 62.4% for Theileria spp., 17.6% for Babesia bigemina, 15.9% for Anaplasma marginale, 7.4% for Ehrlichia ruminantium and 4.5% for Babesia bovis. Further analysis using sequences of Theileria spp. 18S rRNA revealed infection of cattle with Theileria mutans (68.6%), Theileria taurotragi (48.4%), Theileria parva (41.2%), and Theileria ovis (1.9%). Co-infections of cattle, with up to six tick-borne pathogens, were revealed in 46.9% of the samples. Sequence analysis indicated that T. parva p104, E. ruminantium pCS20 and A. marginale MSP-5 genes are conserved among cattle blood samples in Pemba, with 99.3%-100%, 99.6%-100% and 100% sequence identity values, respectively. In contrast, the B. bigemina RAP-1a and B. bovis SBP-2 gene sequences were relatively diverse with 99.5%-99.9% and 66.4%-98.7% sequence identity values respectively. The phylogenetic analyses revealed that T. parva p104, E. ruminantium pCS20 and A. marginale MSP-5 gene sequences clustered in the same clade with other isolates from other countries. In contrast, the B. bigemina RAP-1 and B. bovis SBP-2 gene sequences showed significant differences in the genotypes, as they appeared in separate clades. This study provides important data for understanding the epidemiology of tick-borne diseases, and is expected to improve the approach for diagnosis and control of tick-borne diseases in Tanzania.

Molecular survey and characterization of Theileria annulata and Ehrlichia ruminantium in cattle from Northwest China.

Theileriosis and ehrlichiosis are two important tick-borne diseases affecting cattle farming in China. However, limited information is available regarding prevalence and molecular characterization of Theileria annulata and Ehrlichia ruminantium in cattle in Xinjiang Uygur Autonomous Region (XUAR), northwestern China. In this study, a total of 176 blood samples of cattle from three rural areas of XUAR were collected in June 2017 and were tested by nested-PCR. A total of 34 (19.3%) samples were found to be infected with one or two pathogens. The overall prevalence rates of T. annulata and E. ruminantium were 18.2% and 1.7%, respectively. Phylogenetic analyses revealed that the E. ruminantium isolates from XUAR were located in the same clade but diverged from the isolates from African countries using pCS20 gene while T. annulata isolates from XUAR revealed differences in the genotypes using Tams1 sequences. To our knowledge, this is the first report of E. ruminantium infection in cattle in China. It also provides the first genetic characterization of T. annulata in cattle in XUAR. The current findings are important for understanding the distribution of agents of theileriosis and ehrlichiosis and in designing measures for the prevention and control of tick-borne diseases in cattle, other animals, and humans.