Molecular detection and phylogenetic analysis of tick-borne pathogens in cattle from southern Malawi.

INTRODUCTION: Tick-borne diseases (TBDs) pose a major hindrance to livestock production in countries with limited resources. Effective prevention and management of TBDs require a thorough understanding of disease vectors and pathogens. However, there is limited information on studies of bovine tick-borne pathogens (TBPs) using molecular methods in Malawi. This study aimed to detect TBPs of cattle populations in southern Malawi, which has the largest cattle population in the country. METHODOLOGY: A total of 220 blood samples from apparently healthy cattle were collected in six districts, and were screened for selected TBPs using polymerase chain reaction (PCR). RESULTS: The overall detection rate of TBPs was 72.3%. Among the detected pathogens, Babesia bigemina had the highest detection rate (34.5%), followed by Anaplasma marginale (23.2%), Anaplasma phagocytophilum (22.3%), Theileria taurotragi (22.3%), Theileria parva (15.5%), Anaplasma bovis (9.6%), Babesia bovis (7.3%), Theileria mutans (4.1%), and Babesia naoakii (2.7%). Among the positive samples, 64.2% were found to be co-infected with two or more TBPs, with the highest number of seven pathogens detected in a single sample. The study documents the existence of A. phagocytophilum, B. bovis, and B. naoakii in Malawian cattle for the first time. CONCLUSION: The findings herein demonstrate a significant burden of TBPs on cattle in Malawi, which gives a challenge in combating TBDs. The high TBP burden, along with the high co-infection frequencies in Malawian cattle necessitates the urgency to implement effective control strategies to enhance cattle production in the country.

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.

Molecular Detection and Phylogenetic Analyses of Babesia spp. and Theileria spp. in Livestock in Bangladesh.

Piroplasmosis, caused by Babesia spp. and Theileria spp., poses significant constraints for livestock production and upgradation in Bangladesh. Besides examining blood smears, few molecular reports are available from some selected areas in the country. Therefore, the actual scenario of piroplasmosis in Bangladesh is deficient. This study aimed to screen the piroplasms in different livestock species by molecular tools. A total of 276 blood samples were collected from cattle (Bos indicus), gayals (Bos frontalis) and goats (Capra hircus) in five geographies of Bangladesh. After that, screening was conducted through a polymerase chain reaction, and species were confirmed by sequencing. The prevalence of Babesia bigemina, B. bovis, B. naoakii, B. ovis, Theileria annulata and T. orientalis was 49.28%, 0.72%, 1.09%, 32.26%, 6.52% and 46.01%, respectively. The highest prevalence (79/109; 72.48%) of co-infections was observed with B. bigemina and T. orientalis. The phylogenetic analyses revealed that the sequences of B. bigemina (BbigRAP-1a), B. bovis (BboSBP-4), B. naoakii (AMA-1), B. ovis (ssu rRNA) and T. annulata (Tams-1) were included in one clade in the respective phylograms. In contrast, T. orientalis (MPSP) sequences were separated into two clades, corresponding to Types 5 and 7. To our knowledge, this is the first molecular report on piroplasms in gayals and goats in Bangladesh.

Development of a promising antigenic cocktail for the global detection of Babesia caballi in horse by ELISA.

In this study, we designed novel truncated Babesia caballi (B. caballi) recombinant proteins from the previously used B. caballi proteins; 134-Kilodalton Protein (rBC134) and Merozoite Rhoptry 48 Protein (rBC48). Then, we evaluated the diagnostic performance of the newly designed proteins when used as a single antigen or when used as cocktail antigen consists of rBC134 full length (rBC134f) + newly designed rBC48 (rBC48t) or newly designed rBC134 (rBC134t) + rBC48t for the detection of B. caballi infection in horse using indirect enzyme-linked immunosorbent assay (iELISA). We used one dose and a half of each antigen in the cocktail formulas. The serum samples were collected from different endemic areas in addition to the sera collected from horses experimentally infected with B. caballi were used in the present study. Cocktail antigen in full dose of (rBC134f + rBC48t) exhibited the highest optical density (OD) values with B. caballi-infected sera and showed the lowest OD values with normal equine sera or B. caballi, and Theileria equi mixed infected sera in comparison with the single antigen. Interestingly, the same cocktail antigen exhibited the highest concordance rate (76.74%) and kappa value (0.79) in the screening of 200 field serum samples collected from five B. caballi endemic countries, including South Africa (n = 40), Ghana (n = 40), Mongolia (n = 40), Thailand (n = 40), and China (n = 40) using iELISA and the results were compared to those of indirect fluorescent antibody test (IFAT) as a reference. Moreover, the identified promising cocktail full dose antigen (rBC134f + rBC48t) showed that it can detect the infection as early as the 4th day post-infection in sera collected from experimentally infected horses. The obtained results revealed the reliability of the rBC134f + rBC48t cocktail antigen when used in full dose for the detection of specific antibodies to B. caballi in horses which will be useful for epidemiological surveys and control of equine babesiosis.

Molecular Investigation of Tick-Borne Haemoparasites Isolated from Indigenous Zebu Cattle in the Tanga Region, Tanzania.

Tick-borne diseases (TBDs) are a major hindrance to livestock production in pastoral communities of Africa. Although information on tick-borne infections is necessary for setting up control measures, this information is limited in the pastoral communities of Tanzania. Therefore, this study aimed to provide an overview of the tick-borne infections in the indigenous cattle of Tanzania. A total of 250 blood samples were collected from the indigenous zebu cattle in the Tanga region, Tanzania. Then, we conducted a molecular survey using the polymerase chain reaction (PCR) and gene sequencing to detect and identify the selected tick-borne pathogens. The PCR was conducted using assays, based on Theileria spp. (18S rRNA), Theileria parva (p104), Theileria mutans and T. taurotragi (V4 region of the 18S rRNA), Babesia bigemina (RAP-1a), B. bovis (SBP-2), Anaplasma marginale (heat shock protein groEL) and Ehrlichia ruminantium (pCS20). The PCR screening revealed an overall infection rate of (120/250, 48%) for T. mutans, (64/250, 25.6%) for T. parva, (52/250, 20.8%) for T. taurotragi, (33/250, 13.2%) for B. bigemina and (81/250, 32.4%) for A. marginale. Co-infections of up to four pathogens were revealed in 44.8% of the cattle samples. A sequence analysis indicated that T. parva p104 and A. marginale groEL genes were conserved among the sampled animals with sequence identity values of 98.92−100% and 99.88−100%, respectively. Moreover, the B. bigemina RAP-1a gene and the V4 region of the 18S rRNA of T. mutans genes were diverse among the sampled cattle, indicating the sequence identity values of 99.27−100% and 22.45−60.77%, respectively. The phylogenetic analyses revealed that the T. parva (p104) and A. marginale (groEL) gene sequences of this study were clustered in the same clade. In contrast, the B. bigemina (RAP-1a) and the T. mutans V4 region of the 18S rRNA gene sequences appeared in the different clades. This study provides important basement data for understanding the epidemiology of tick-borne diseases and will serve as a scientific basis for planning future control strategies in the study area.

Discovering the Potent Inhibitors Against Babesia bovis in vitro and Babesia microti in vivo by Repurposing the Natural Product Compounds.

In the present study, we screened 502 natural product compounds against the in vitro growth of Babesia (B.) bovis. Then, the novel and potent identified compounds were further evaluated for their in vitro efficacies using viability and cytotoxicity assays. The in vivo inhibitory effects of the selected compounds were evaluated using B. microti "rodent strain" in mice model. Three potent compounds, namely, Rottlerin (RL), Narasin (NR), Lasalocid acid (LA), exhibited the lowest IC50 (half-maximal inhibitory concentration) as follows: 5.45 ± 1.20 µM for RL, 1.86 ± 0.66 µM for NR, and 3.56 ± 1.41 µM for LA. The viability result revealed the ability of RL and LA to prevent the regrowth of treated parasite at 4 × IC50 and 2 × IC50, respectively, while 4 × IC50 of NR was sufficient to stop the regrowth of parasite. The hematology parameters of B. microti in vivo were different in the NR-treated groups as compared to the infected/untreated group. Interestingly, intraperitoneal administration of NR exhibiting inhibition in the growth of B. microti in mice was similar to that observed after administration of the commonly used antibabesial drug, diminazene aceturate (DA) (76.57% for DA, 74.73% for NR). Our findings indicate the richness of natural product compounds by novel potent antibabesial candidates, and the identified potent compounds, especially NR, might be used for the treatment of animal babesiosis.

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.

Impact of using pyronaridine tetraphosphate- based combination therapy in the treatment of babesiosis caused by Babesia bovis, B. caballi, and B. gibsoni in vitro and B. microti in mice.

The inhibitory efficacies of pyronaridine tetraphosphate (PYR), when used in combination with two novel and potent antibabesial drugs; clofazimine (CF), and MMV396693 were evaluated in the current study against the growth of Babesia bovis, B. caballi, and B. gibsoni in vitro and B. microti in mice. The in vitro study against the selected parasites was performed using combination of PYR with either CF or MMV396693 in ratios ranged from 0.75:0.75 to 0.25:0.25. Combined application of PYR/MMV396693 revealed additive and indifferent interactions against the in vitro growth of all screened Babesia parasites. PYR in combination with CF, achieved indifferent and antagonistic interactions with all used concentration ratios against the in vitro growth of B. bovis and B. caballi. Treatment with PYR-CF combination therapy caused significant inhibition (P < 0.05) of the fluorescence values at days 12, 14, 16, 18, and 22 p.i. in comparison with control mice. Of note, treatment with combination therapy exhibited inhibition in the growth of B. microti (23.16%) greater than those caused by PYR alone. In summary, the obtained results highlight the improvement in the in vivo antibabesial efficacy of PYR when used in combination with CF rather than using PYR alone but such inhibition is still lower than those caused by either DA or CF monotherapies.

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.

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.

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.

Babesia microti Confers Macrophage-Based Cross-Protective Immunity Against Murine Malaria.

Malaria and babesiosis, the two primary intraerythrocytic protozoan diseases of humans, have been reported in multiple cases of co-infection in endemic regions. As the geographic range and incidence of arthropod-borne infectious diseases is being affected by climate change, co-infection cases with Plasmodium and Babesia are likely to increase. The two parasites have been used in experimental settings, where prior infection with Babesia microti has been shown to protect against fatal malarial infections in mice and primates. However, the immunological mechanisms behind such phenomena of cross-protection remain unknown. Here, we investigated the effect of a primary B. microti infection on the outcome of a lethal P. chabaudi challenge infection using a murine model. Simultaneous infection with both pathogens led to high mortality rates in immunocompetent BALB/c mice, similar to control mice infected with P. chabaudi alone. On the other hand, mice with various stages of B. microti primary infection were thoroughly immune to a subsequent P. chabaudi challenge. Protected mice exhibited decreased levels of serum antibodies and pro-inflammatory cytokines during early stages of challenge infection. Mice repeatedly immunized with dead B. microti quickly succumbed to P. chabaudi infection, despite induction of high antibody responses. Notably, cross-protection was observed in mice lacking functional B and T lymphocytes. When the role of other innate immune effector cells was examined, NK cell-depleted mice with chronic B. microti infection were also found to be protected against P. chabaudi. Conversely, in vivo macrophage depletion rendered the mice vulnerable to P. chabaudi. The above results show that the mechanism of cross-protection conferred by B. microti against P. chabaudi is innate immunity-based, and suggest that it relies predominantly upon the function of macrophages. Further research is needed for elucidating the malaria-suppressing effects of babesiosis, with a vision toward development of novel tools to control malaria.

Closing the empty anti-Babesia gibsoni drug pipeline in vitro using fluorescence-based high throughput screening assay.

In the current study, we evaluated the usefulness of a SYBR Green I (SG I) fluorescence assay for evaluation of the inhibitory effects of antibabesial drugs against the in vitro growth of Babesia gibsoni. Linearity and high-throughput screening (HTS) assays exhibited the validity of the SG I fluorescence assay for B. gibsoni parasite when performed at low hematocrits (HCTs) (2.5% and 5%) without daily changing of the medium. Interestingly, 5% HCT showed the highest value of the signal/noise ratio. Of note, there were no significant differences (P > .05) in the IC50s of the commonly used antibabesial drugs (diminazene aceturate and/or imidocarb dipropionate) that calculated by either the SG I fluorescence assay with and without daily medium changing or by the fluorescence and microscopy methods at 2.5% and 5% HCTs. Such results confirmed that both HCTs are valid for mass drug screening against the in vitro growth of B. gibsoni. While the results of the HTS assay add merit to the assay when performed at 5% HCT especially when incubating the plates for 2 h in a dark after adding lysis buffer with SG I stain. Next, nine different drugs were screened to confirm the assay's usefulness. MMV396693, pyronaridine tetraphosphate and nerolidol drugs exhibited the highest effectiveness against the in vitro growth of B. gibsoni, next to diminazene aceturate. In summary, SG I fluorescence assay with 5% HCT without daily changing of the medium for B. gibsoni offers a novel approach for the large-scale screening of huge chemical libraries in in vitro cultures.

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.

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.

Evaluation of the protective effect of a prime-boost strategy with plasmid DNA followed by recombinant adenovirus expressing BmAMA1 as vaccines against Babesia microti infection in hamster.

In the present study, we have investigated the protective effect of a heterologous prime-boost strategy with priming plasmid DNA followed by recombinant adenovirus, both expressing BmAMA1, against Babesia microti infection. Four groups consisting of 3 hamsters per group were immunized with pBmAMA1/Ad5BmAMA1, pNull/Ad5BmAMA1, pBmAMA1/Ad5Null and pNull/Ad5Null, followed by challenge infection with B. microti. Our results showed that hamsters immunized with plasmid and adenovirus expressing BmAMA1 developed a robust IgG and IgG2a antibody response against BmAMA1, suggesting the DNA vaccine or viral vector vaccine tend to induce a Th1-biased response. Compared to the control hamsters, the hamsters vaccinated either with the prime-boost strategy or one of the two "vaccines" exhibited no significant protection against B. microti challenge. Although a slight difference in terms of parasitemia and hematocrit values at days 14-16 post challenge infection was observed, no other statistical difference was detected. Our results indicate that the prime-boost vaccination strategy of injection of plasmid and adenovirus expressing BmAMA1 is not efficient in protecting against B. microti infection.

Detection and molecular characterization of tick-borne pathogens infecting sheep and goats in Blue Nile and West Kordofan states in Sudan.

Tick-borne pathogens (TBPs) are common in livestock of sub-Saharan Africa. However, information regarding TBPs in sheep and goats in Sudan is limited. In this study, 178 blood samples of sheep and goats in Blue Nile and West Kordofan states were investigated for TBPs using PCR. Overall, 110 (61.8%) samples were found to be infected with at least one of the following pathogens: Anaplasma ovis, Theileria ovis, and Ehrlichia ruminantium. Babesia ovis and T. lestoquardi were not identified. A. ovis was the most prevalent pathogen (n = 107, 60.1%), followed by T. ovis (n = 23, 12.9%) and E. ruminantium (n = 1, 0.6%). The prevalence rates of A. ovis and T. ovis were significantly higher in sheep than in goats. Phylogenetic analysis of T. ovis 18S rRNA and A. ovis msp4, groEL, and 16S rRNA, revealed that the pathogens identified in this study are clustered together, indicating similar molecular characteristics. Additionally, phylogenetic analysis of E. ruminantium pCS20 revealed that E. ruminantium in this study belong to the West Africa group, and different to E. ruminantium previously identified in ticks from Sudan. We concluded that TBPs are highly prevalent in the study area and continuous monitoring of TBPs in sheep and goats in Sudan is highly required.

Prevalence, risk factors, and genetic diversity of veterinary important tick-borne pathogens in cattle from Rhipicephalus microplus-invaded and non-invaded areas of Benin.

Babesiosis, theileriosis, anaplasmosis, and heartwater are tick-borne diseases (TBD) that threaten livestock production in sub-Saharan Africa including Benin. This country has been faced with an invasion of Rhipicephalus microplus, a major vector for babesiosis, theileriosis, and anaplasmosis over the last decade. Yet, data on TBD and the impact of the invasive ticks are lacking, making risk level evaluation and disease control arduous. In this study, epidemiological features of Babesia bovis, B. bigemina, Theileria spp., Anaplasma marginale and Ehrlichia ruminantium infections in Benin cattle were investigated in R. microplus-invaded and non-invaded areas. Detection of pathogens was based on species-specific PCR assays and resulting data were used to identify risk factors. Genetic diversity and phylogenies were then evaluated using several markers. Out of 207 samples examined, 170 (82.1%), 109 (52.7%), 42 (20.3%) 24 (11.6%) and 1 (0.5%) were positive for T. mutans, A. marginale, B. bigemina, B. bovis and E. ruminantium, respectively. Animal gender (for B. bovis), exposure to R. microplus (for B. bigemina and A. marginale), animal age (for B. bigemina and A. marginale) and cattle breed and/or antiprotozoal treatment (for T. mutants) significantly modulated pathogen occurrence. In addition, R. microplus exposure was significantly related to co-infection patterns and cases of clinical theileriosis and/or anaplasmosis were recorded among cattle highly exposed to the tick. In the genetic characterization, Theileria spp. and E. ruminantium sequences were conserved. Babesia spp. and A. marginale, however, showed high sequence polymorphisms that indicate the presence of several strains and may be linked to R. microplus invasion. Taken together, these results ascertain the endemicity of tick-borne infections in Benin and suggest that the characteristics of Babesia spp. and A. marginale infections in R. microplus-invaded and non-invaded areas are different.

Identification and characterization of interchangeable cross-species functional promoters between Babesia gibsoni and Babesia bovis.

The development of transgenic techniques has been reported in many protozoan parasites over the past few years. We recently established a successful transient transfection system for Babesia gibsoni based on Bg 5'-ef-1α promoter. This study investigated 6 homologous and 6 heterologous promoters for B. gibsoni and B. bovis and identified novel interchangeable cross-species functional promoters between B. gibsoni and B. bovis. Ten out of twelve promoters had heterologous promoter function. In particular, Bg 5'-ef-1α and Bg 5'-actin heterologous promoters resulted in a significantly higher luciferase activity than Bb 5'-ef-1α homologous promoter in B. bovis. The present study showed that Bg 5'-actin promoted the highest luciferase activity in both B. gibsoni and B. bovis. The study further indicates that heterologous promoter function widely exists between B. gibsoni and B. bovis. This finding is an important step for future stable transfection construct design and for the production of vaccines based on transfected B. gibsoni and B. bovis parasites.