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.

Molecular Identification of Babesia and Theileria Infections in Livestock in the Qinghai-Tibetan Plateau Area, China.

The northwestern region of China, known as the Qinghai-Tibet Plateau Area (QTPA), is characterized by unique climate conditions that support the breeding of various highly-adapted livestock species. Tick vectors play a significant role in transmitting Babesia and Theileria species, posing serious risks to animal health as well as the economy of animal husbandry in QTPA. A total of 366 blood samples were collected from Tibetan sheep (n = 51), goats (n = 67), yaks (n = 43), cattle (n = 49), Bactrian camels (n = 50), horses (n = 65), and donkeys (n = 40). These samples were examined using conventional and nested PCR techniques to detect Theileria and Babesia species. The overall infection rates were 0.3% (1/366) for Babesia spp. and 38.2% (140/366) for Theileria spp. Notably, neither Babesia nor Theileria species were detected in donkeys and yaks. The infection rates of Babesia and Theileria species among animals in different prefectures were significantly different (p < 0.05). Furthermore, Babesia bovis, B. bigemina, B. caballi, and B. ovis were not detected in the current study. To our knowledge, this is the first documented detection of Theileria luwenshuni infection in Bactrian camels and goats, as well as T. sinesis in cattle and T. equi in horses on the Qinghai plateau. These novel findings shed light on the distribution of Babesia and Theileria species among livestock species in QTPA.

Molecular survey of canine tick-borne pathogens in ticks and stray dogs in Dhaka city, Bangladesh.

Molecular surveillance of canine tick-borne pathogens (TBPs) in Bangladesh has constantly been undervalued. Therefore, the emergence of new pathogens often remains undetected. This study aimed to screen tick-borne pathogens in stray dogs and ticks in the Dhaka metropolitan area (DMA). Eighty-five dog blood and 53 ticks were collected in six city districts of DMA from September 2022 to January 2023. The ticks were identified by morphology. Screening of TBPs was performed by polymerase chain reaction (PCR), followed by sequencing. The PCR assays were conducted to analyze the 18S rRNA (Babesia gibsoni, B. vogeli, and Hepatozoon canis), 16S rRNA (Anaplasma phagocytophilum, A. platys, and A. bovis), gltA (Ehrlichia canis and Rickettsia spp.), flagellin B (Borrelia spp.) and 16-23S rRNA (Bartonella spp.). Three tick species, Rhipicephalus sanguineus (50/53), R. microplus (1/53), and Haemaphysalis bispinosa (2/53), were identified. Babesia gibsoni (38 out of 85) and A. platys (7 out of 85) were detected in dog blood. In contrast, four pathogens, B. gibsoni (1 out of 53), B. vogeli (1 out of 53), H. canis (22 out of 53), and A. platys (1 out of 53), were detected in the ticks. However, the detection rates of TBPs in dog blood and ticks were not correlated in this study. The phylogenetic analyses suggested that a single genotype for each of the four pathogens is circulating in DMA. This study reports the existence of B. vogeli, H. canis, and A. platys in Bangladesh for the first time.

First report of dog ticks and tick-borne pathogens they are carrying in Malawi.

Ticks are vectors for transmitting tick-borne pathogens (TBPs) in animals and humans. Therefore, tick identification is necessary to understand the distribution of tick species and the pathogens they carry. Unfortunately, data on dog ticks and the TBPs they harbor in Malawi are incomplete. This study aimed to identify dog ticks and the TBPs they transmit in Malawi. One hundred thirty-two ticks were collected from 87 apparently healthy but infested domestic dogs in four districts of Malawi, which were pooled into 128 tick samples. The ticks were morphologically identified under a stereomicroscope using identification keys, and species identification was authenticated by polymerase chain reaction (PCR) through the amplification and sequencing of 12S rRNA and cytochrome c oxidase subunit I (CO1) genes. The tick species identified were Rhipicephalus sanguineus sensu lato (58.3%), Haemaphysalis elliptica (32.6%), and Hyalomma truncatum (9.1%). Screening for TBPs using species-specific PCR assays revealed that 48.4% of the ticks were infected with at least one TBP. The TBP detection rates were 13.3% for Anaplasma platys, 10.2% for Babesia rossi, 8.6% for B. vogeli, 6.3% for Ehrlichia canis, 3.9% for A. phagocytophilum, 3.1% for B. gibsoni, 2.3% for B. canis and 0.8% for Hepatozoon canis. Co-infections of up to three pathogens were observed in 48.4% of the positive samples. This is the first study to identify dog ticks and the TBPs they harbor in Malawi. These findings provide the basis for understanding dog tick distribution and pathogens they carry in Malawi. This study necessitates the examination of ticks from more study locations to have a better picture of tick challenge, and the development of ticks and tick-borne disease control methods in Malawi.

Distribution of Rhipicephalus sanguineus and Heamaphysalis elliptica dog ticks and pathogens they are carrying: A systematic review.

The role of ixodid ticks especially Rhipicephalus sanguineus and Heamaphysalis elliptica in the epidemiology of several diseases of veterinary and public health importance have been documented. This study conducted a systematic review focusing on the distribution of R. sanguineus and H. elliptica, as well as the common tick-borne pathogens they harbour. The Scopus, ScienceDirect, PubMed, and Web of Science databases were used to search for English journal articles published between January 1990 and June 2021. The articles were assessed by following the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. This systematic review was registered on PROSPERO [(ID no: CRD42022327372). Of the studies included in the systematic review, 247 and 19 articles had identified R. sanguineus and H. elliptica respectively, whereas 15 articles had identified both tick species. There is a reported worldwide distribution of R. sanguineus from 64 countries, whereas H. elliptica was only reported in the African continent from 6 countries. In total, 120 articles that were included in this systematic review reported detection of tick-borne pathogens from R. sanguineus (n = 118 articles) and/or H. elliptica (n = 2 articles) ticks. According to the studies tick-borne pathogens harboured by R. sanguineus included protozoa such as Babesia spp., Hepatozoon spp., Leishmania spp., and Theileria spp., as well as bacteria such as Acinetobacter spp. Anaplasma spp., Bacillus spp., Borrelia spp., Brucella spp., Coxiella spp., and Staphylococcus spp. The H. elliptica was reported to be harbouring Babesia spp., Ehrlichia spp. and Rickettsia spp. Most of the studies (50%) used the conventional polymerase chain reaction (PCR) technique for the detection of tick-borne pathogens, followed by real-time PCR (qPCR) (n = 26), and nested PCR (n = 22). This systematic review has shed light on the distribution of two common dog ticks as well as the tick-borne pathogens of veterinary and zoonotic importance they are harbouring. This data will enable surveillance studies that can report whether the distribution of these ticks and their associated tick-borne pathogens is expanding or shrinking or is stable.

Epidemiological Investigation of Tick-Borne Bacterial Pathogens in Domestic Animals from the Qinghai-Tibetan Plateau Area, China.

The Qinghai-Tibetan Plateau area (QTPA) features a unique environment that has witnessed the selective breeding of diverse breeds of domestic livestock exhibiting remarkable adaptability. Nevertheless, Anaplasma spp., Rickettsia spp., Coxiella spp., and Borrelia spp. represent tick-borne bacterial pathogens that pose a global threat and have substantial impacts on both human and animal health, as well as on the economy of animal husbandry within the Qinghai-Tibetan plateau area. In this study, a total of 428 samples were systematically collected from 20 distinct areas within the Qinghai Plateau. The samples included 62 ticks and 366 blood samples obtained from diverse animal species to detect the presence of Anaplasma spp., Rickettsia spp., Coxiella spp., and Borrelia spp. The prevalence of infection in this study was determined as follows: Anaplasma bovis accounted for 16.4% (70/428), A. capra for 4.7% (20/428), A. ovis for 5.8% (25/428), Borrelia burgdorferi sensu lato for 6.3% (27/428), Coxiella burnetii for 0.7% (3/428), and Rickettsia spp. for 0.5% (2/428). Notably, no cases of A. marginale and A. phagocytophilum infections were observed in this study. The findings revealed an elevated presence of these pathogens in Tibetan sheep and goats, with no infections detected in yaks, Bactrian camels, donkeys, and horses. To the best of our knowledge, this study represents the first investigation of tick-borne bacterial pathogens infecting goats, cattle, horses, and donkeys within the Qinghai Plateau of the Qinghai-Tibetan Plateau area. Consequently, our findings contribute valuable insights into the distribution and genetic diversity of Anaplasma spp., Rickettsia spp., Coxiella spp., and Borrelia spp. within China.

Wide bovine tick-borne pathogen spectrum: Predominancy of Theileria annulata and the first molecular detection of Ehrlichia minasensis in Turkey.

Vector-borne diseases indulge in severe economic losses in the livestock industry by adversely affecting cattle breeding in tropical and subtropical zone countries, including Turkey, encompassing a wide land area representing diverse climatic conditions. This study aimed to investigate significant bovine tick-borne piroplasm, rickettsia, and some other bacterial agents by genus- or species-specific PCR and nested PCR techniques in Turkey. A total of 210 cattle blood samples were collected from sixteen provinces in different geographical regions of Turkey. PCR analyses were performed targeting the detection of Babesia/Theileria/Hepatozoon sp. 18S rRNA, Babesia/Theileria sp. 18S rRNA (V4), B. bigemina RAP-1a, B. bovis SBP-4, B. ovata AMA-1, B. naoaki AMA-1, T. annulata Tams-1, T. orientalis MPSP, T. mutans 18S rRNA, Anaplasma/Ehrlichia sp. 16S rRNA, A. marginale MSP4, A. bovis 16S rRNA, A. phagocytophilum 16S rRNA, A. capra 16S rRNA, E. ruminantium pSC20, Mycoplasma sp. 16S rRNA, and Coxiella burnetii 16S rRNA genes. Overall, 133 (63.3%) cattle were found to be infected with at least one of the following protozoan or bacterial pathogens; B. bovis, B. bigemina, B. occultans, T. annulata, T. orientalis, A. marginale, A. phagocytophilum, and Mycoplasma sp. The total prevalence of pathogens was determined as follows; 0.5% B. bovis, 0.5% B. bigemina, 1.4% B. occultans, 41.0% T. annulata, 1.4% T. orientalis, 10.5% A. marginale, 13.8% A. phagocytophilum, 0.5% A. bovis, 2.9% Uncultured Anaplasma sp., 0.5% E. minasensis, 0.5% Uncultured Ehrlichia sp., and 23.3% Mycoplasma sp. Moreover, large part of the total infection (n:133) was composed of single infections (63.9%); however, double (24.8%), triple (7.5%), quadruple (2.3%), and quintuple (1.5%) co-infections were also encountered. In addition to some bovine pathogens such as B. occultans, T. orientalis, A. bovis, M. wenyonii, and Candidatus Mycoplasma haemobos, which were rarely reported in Turkey, sequencing and phylogenetic analysis revealed the first detection of Uncultured Ehrlichia sp. (0.5%), and E. minasensis (0.5%) with 100% nucleotide sequence identities. The study also indicates that the spectrum of pathogens harbored by Turkish cattle is quite wide, and these pathogens cause multiple co-infections with various combinations, and T. annulata stands out as the primary bovine pathogen among them.

Molecular detection and internal transcribed spacer-1 sequence diversity of Trypanosoma evansi in goats from Cavite, Philippines.

Goat production is an important source of livelihood and food. Goats may serve as reservoir of surra affecting livestock production. Here, forty-two free-roaming goats from Cavite, Philippines were screened using two primer sets, Trypanosoma brucei minisatellite chromosome for initial detection and the internal transcribed spacer 1 (ITS-1) to determine phylogeny. Initial PCR detection showed that 19/42 (45%) goats were positive, much higher than the rate previously reported in goats from Cebu (34%). The infectivity rate was higher in male (56%) than in female (42%) and the rate was higher in young ≤1 year old (100%) than in adult >1 year old (43%). Phylogenetic analysis of the ITS-1 sequences between T. evansi goat samples and other isolates indicate potential interspecies transmission.

Anti-babesial activity of a series of 6,7-dimethoxyquinazoline-2,4-diamines (DMQDAs).

Diminazene aceturate (DA), imidocarb dipropionate (ID), atovaquone (ATO), azithromycin (AZI), clindamycin, and quinine have been used to treat animal and human babesiosis for many years, despite their negative effects and rising indications of resistance. Thus, finding anti-babesial compounds that can either treat the infection or lower the dose of drugs given has been a primary objective. Quinazolines are one of the most important nitrogen heterocycles, with a wide range of pharmacological activities including analgesic, anti-inflammatory, sedative-hypnotic, anti-histaminic, anti-cancer, and anti-protozoan properties. The present study investigated the anti-babesial activities of twenty 6,7-dimethoxyquinazoline-2,4-diamines on Babesia spp. One candidate, 6,7-dimethoxy-N4-ethylisopropyl-N2-ethyl(pyridin-4-yl)quinazoline-2,4-diamine (SHG02), showed potent inhibition on Babesia gibsoni in vitro, as well as on B. microti and B. rodhaini in mice. Our findings indicate that the candidate compound SHG02 is promising for further development of anti-babesial drugs and provides a new structure to be explored for developing anti-Babesia therapeutics.

Tick-borne pathogens in camels: A systematic review and meta-analysis of the prevalence in dromedaries.

Published data on tick-borne pathogens (TBPs) in camels worldwide have been collected to provide an overview of the global prevalence and species diversity of camelid TBPs. Several TBPs have been detected in dromedary camels, raising concerns regarding their role as natural or maintenance hosts for tick-borne pathogens. Insubstantial evidence exists regarding the natural infection of camels with Babesia spp., Theileria spp., Anaplasma spp., and Ehrlichia spp., particularly because most of the camels were considered healthy at the time of sampling. Based on polymerase chain reaction (PCR) testing, a pooled prevalence of 35.3% (95% CI: 22.6-48.1%) was estimated for Anaplasma, which was the most frequently tested TBP in dromedaries, and DNA of Anaplasma marginale, Anaplasma centrale, Anaplasma ovis, Anaplasma platys, and A. platys-like were isolated, of which ruminants and dogs are reservoirs. Similarly, the estimated pooled prevalence for the two piroplasmid genera; Babesia and Theileria was approximately equal (10-12%) regardless of the detection method (microscopy or PCR testing). Nevertheless, Babesia caballi, Theileria equi, and Theileria annulata DNA have frequently been detected in camels but they have not yet been proven to be natural hosts. Scarce data detected Babesia microti, Anaplasma phagocytophilum, and Borrelia burgdorferi sensu lato (s.l.) DNA in blood of dromedaries, although ticks of the genus Ixodes are distributed in limited areas where dromedaries are raised. Interestingly, a pooled seroprevalence of 47.7% (26.3-69.2%) was estimated for Crimean-Congo hemorrhagic fever virus, and viral RNA was detected in dromedary blood; however, their contribution to maintain the viral transmission cycles requires further experimental investigation. The substantially low incidence and scarcity of data on Rickettsia and Ehrlichia species could imply that camels were accidentally infected. In contrast, camels may play a role in the spread of Coxiella burnetii, which is primarily transmitted through the inhalation of aerosols emitted by diseased animals and contaminated environments. Bactrian camels showed no symptoms due to the examined TBPs, meanwhile, clinical disease was seen in alpacas infected with A. phagocytophilum. Similar to dromedaries, accidental tick bites may be the cause of TBP DNA found in the blood of Bactrian camels.

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.

Whole genome sequence and diversity in multigene families of Babesia ovis.

Ovine babesiosis, caused by Babesia ovis, is an acute, lethal, and endemic disease worldwide and causes a huge economic loss to animal industry. Pathogen genome sequences can be utilized for selecting diagnostic markers, drug targets, and antigens for vaccine development; however, those for B. ovis have not been available so far. In this study, we obtained a draft genome sequence for B. ovis isolated from an infected sheep in Turkey. The genome size was 7.81 Mbp with 3,419 protein-coding genes. It consisted of 41 contigs, and the N50 was 526 Kbp. There were 259 orthologs identified among eight Babesia spp., Plasmodium falciparum, and Toxoplasma gondii. A phylogeny was estimated on the basis of the orthologs, which showed B. ovis to be closest to B. bovis. There were 43 ves genes identified using hmm model as well. They formed a discriminating cluster to other ves multigene family of Babesia spp. but showed certain similarities to those of B. bovis, B. caballi, and Babesia sp. Xinjiang, which is consistent with the phylogeny. Comparative genomics among B. ovis and B. bovis elucidated uniquely evolved genes in these species, which may account for the adaptation.

Babesia ovis secreted antigen-1 is a diagnostic marker during the active Babesia ovis infections in sheep.

Ovine babesiosis caused by Babesia ovis is an economically significant disease. Recently, a few B. ovis-specific proteins, including recombinant B. ovis secreted antigen-1 (rBoSA1), have been identified. Immunological analyses revealed that rBoSA1 resides within the cytoplasm of infected erythrocytes and exhibits robust antigenic properties for detecting anti-B. ovis antibodies. This protein is released into the bloodstream during the parasite's development. It would be possible to diagnose active infections by detecting this secretory protein. For this purpose, a rBoSA1-specific polyclonal antibody-based sandwich ELISA was optimized in this study. Blood samples taken from the naturally (n: 100) and experimentally (n: 15) infected sheep were analyzed for the presence of native BoSA1. The results showed that native BoSA1 was detectable in 98% of naturally infected animals. There was a positive correlation between parasitemia level in microscopy and protein density in sandwich ELISA. Experimentally infected animals showed positive reactions from the first or second day of inoculations. However, experimental infections carried out by Rhipicephalus bursa ticks revealed the native BoSA1 was detectable from the 7th day of tick attachment when the parasite began to be seen microscopically. Sandwich ELISA was sensitive enough to detect rBoSA1 protein at a 1.52 ng/ml concentration. Additionally, no serological cross-reactivity was observed between animals infected with various piroplasm species, including Babesia bovis, B. bigemina, B. caballi, B. canis, B. gibsoni, Theileria equi, and T. annulata. Taken collectively, the findings show that the rBoSA1-specific polyclonal antibody-based sandwich ELISA can be successfully used to diagnose clinical B. ovis infections in sheep at the early stage.

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.

Molecular Identification of Piroplasmids in Ticks from Infested Small Ruminants in Konya Province, Turkey.

Ticks play a pivotal role in propagating a diverse spectrum of infectious agents that detrimentally affect the health of both humans and animals. In the present study, a molecular survey was executed of piroplasmids in ticks collected from small ruminants in four districts within Konya province, Turkey. Microscopic examination identified 1281 adult ticks, which were categorized into 357 pools based on their species, sexes, host animals, and collection site before DNA extraction. The infection rates were calculated by using a maximum likelihood estimate (MLE) with 95% confidence intervals (CI). Hyalomma detritum, H. excavatum, Rhipicephalus bursa, R. sanguineus, and R. turanicus were identified in this study. Among the five tick species identified here, R. turanicus exhibited the highest infestation rate in both goats and sheep. The presence of Babesia ovis and Theileria ovis based on 18S rRNA was confirmed using molecular assay. The overall MLE of infection rates for B. ovis and T. ovis was 2.49% (CI 1.72-3.46) and 1.46% (CI 0.87-2.23), respectively. The MLE of B. ovis and T. ovis infection rates in R. bursa was 10.80% (CI 7.43-14.90) and 0.33% (CI 0.02-1.42), respectively, while that in R. turanicus was 0.12% (CI 0.01-0.51) and 2.08% (CI 1.25-3.22). This study further confirms that R. turanicus and R. sanguineus can act as vectors for B. ovis, thus advancing our comprehension of tick-borne piroplasmids epidemiology and providing valuable insights for the development of effective control strategies for ticks and tick-borne diseases in Turkey.

Molecular detection of Rickettsia africae from Amblyomma hebraeum ticks in Mafikeng city of North West Province, South Africa.

Rickettsia africae causes zoonotic African tick bite fever, which is a disease of "One Health" importance. There have been reported cases of tourists from Europe and Asia who have been bitten by ticks whilst visiting South Africa's nature reserves, and on their return to their countries, the display African Tick Bite Fever sickness. Hence, the aim of this study was to determine the occurrence of Rickettsia africae in Amblyomma hebraeum ticks infesting livestock in the North West Province. A total of 358 A. hebraeum ticks were collected from 60 ruminants (cattle, sheep and goats) in Mafikeng City of North West Province, South Africa. Ticks were identified morphologically and further confirmed by sequencing of their ITS2 gene. DNA was extracted from 60 pools of ticks which consisted of 5-6 adult ticks that were from the same ruminant host. Infections with Rickettsia spp. were found in 48%, 40%, and 32% of cattle, sheep, and goats, respectively, in amplification by PCR using the ompA gene. The ompA gene sequences showed that the Rickettsia spp. were identified as R. africae. Although the animals from whom the ticks were collected did not exhibit any clinical symptoms, it is well recognised that R. africae is a disease with significant zoonotic potential. Thus, it is important to use the "One Health" approach to formulate prevention and control measures for this pathogen for animal and human health as well as the tourism sector due to the ecotourism importance of the resultant disease.

Draft genome sequence data of Haemaphysalis longicornis Oita strain.

Haemaphysalis longicornis Neumann, 1901 is one of the most well-known hard ticks because of its medical and veterinary importance. Haemaphysalis longicornis transmit a wide range of pathogens among vertebrates, affecting humans and animals in Asia and Oceania. In Japan, the tick species is a major pest of cattle because it can spread a protozoan parasite Theileria orientalis, which causes theileriosis and produces economic losses to the livestock industry (Yokoyama et al. 2012 [1]). Apart from bovine theileriosis, H. longicornis is a vector of bovine babesiosis caused by Babesia ovata, canine babesiosis caused by Babesia gibsoni, and rickettsiosis and viral diseases in humans. Its habitats are mainly Japan, Australia, New Zealand, New Caledonia, the Fiji Islands, Korea, China, and Russia (Oliver et al. 1973 [2]). In the United States, heavy H. longicornis infestations on cattle and white-tailed deer were reported in 2019, making it now one of the tick species to be an increasing threat to livestock animals and humans globally. Ticks reproduce offspring after mating with female and male ticks, however, interestingly, there are two races of H. longicornis: bisexual (diploid) and parthenogenetic (triploid) races [2]. Parthenogenetic H. longicornis is distributed throughout Japan, while the northern limit of the bisexual race is believed to be Fukushima Prefecture on Honshu Island (Fujita et al. 2013 and Kitaoka et al. 1961 [3,4]). This tick species is also considered to be of great scientific importance, and the parthenogenetic race collected in Okayama prefecture has been reared since 1961, while the bisexual race collected in Oita prefecture has been reared since 2008 under laboratory conditions in Japan (Boldbaatar et al. 2010 and Fujisaki et al. 1976 [5,6]). Namely, the "Okayama strain" and "Oita strain" of H. longicornis have been maintained for more than six decades and 15 years, respectively, stably under laboratory conditions. To obtain reference data of bisexual H. longicornis, we sequenced unfed females with haploid genomes using Illumina and MinION Q20 kit then obtained a draft genome consisting of 2.48 Gbp. The number of the contig was 98,529 and N50 was 46.5 Kb. Genome information derived from our laboratory colony of bisexual H. longicornis ticks would provide fundamental insight into understanding how different reproductive lineages occur within the same species of the tick.

Molecular characterization and phylogeny of Anaplasma marginale, A. phagocytophilum and A. bovis in livestock of Bangladesh.

The emergence of Tick-borne Anaplasma spp. poses a significant threat to humans and animals worldwide. Traditional surveys based on examining blood smears overlook the existence of emerging pathogens. This study aimed to screen Anaplasma spp. in livestock species from diverse geographies with molecular tools. We collected 276 blood samples from cattle (Bos indicus), gayals (Bos frontalis) and goats (Capra hircus) in Jhenaidah, Bogura, Sirajganj and Bandarban districts, and Naikhongchari sub-district from June 2021 to March 2022. After that, a molecular screening was conducted through polymerase chain reaction (PCR) and sequencing was done to confirm the PCR results. The PCR assays were performed based on the analyses of groEL (Anaplasma marginale) and 16S rRNA (A. phagocytophilum and A. bovis). The Anaplasma spp. detected in this study were A. marginale (10.51%), A. phagocytophilum (0.72%), and A. bovis (63.77%). However, A. platys was not detected in this study. Among the screened pathogens, the detection of A. bovis (82.86%) was significantly high in the Bandarban district, while A. marginale was found only in cattle in this location. Regarding animal species, the occurrence of A. bovis was significantly higher in cattle. Moreover, the detection rate of A. marginale was significantly higher in adult cattle (≥2 years). The phylogenetic analyses revealed that the groEL sequences of A. marginale and 16S rRNA sequences of A. bovis and A. phagocytophilum were included in a single clade in the respective phylograms, showing a single genotype of each species circulating in Bangladesh. This study reports the existence of A. phagocytophilum in Bangladesh for the first time.

Babesia microti alleviates disease manifestations caused by Plasmodium berghei ANKA in murine co-infection model of complicated malaria.

Malaria remains one of the most significant health issues worldwide, accounting for 2.6% of the total global disease burden, and efforts to eliminate this threat continue. The key focus is to develop an efficient and long-term immunity to this disease via vaccination or therapeutic approach, and innovative strategies would enable us to achieve this target. Previously, using a mouse co-infection disease model, cross-protection was illustrated between Babesia microti and Plasmodium chabaudi. Hence, this study was planned to elucidate the impact of acute B. microti Peabody mjr and Plasmodium berghei ANKA co-infection on the consequence of complicated malaria in the C57BL/6J mouse model of malaria. Furthermore, immune response and pathological features were analyzed, and the course of the disease was compared among experimental groups. Our study established that acute B. microti infection activated immunity which was otherwise suppressed by P. berghei. The immunosuppressive tissue microenvironment was counteracted as evidenced by the enhanced immune cell population in co-infected mice, in contrast to P. berghei-infected control mice. Parasite sequestration in the brain, liver, lung, and spleen of co-infected mice was significantly decreased and tissue injury was ameliorated. Meanwhile, the serum levels of IFN-γ, TNF-α, and IL-12p70 were reduced while the secretion of IL-10 was promoted in co-infected mice. Eventually, co-infected mice showed an extended rate of survival. Hereby, the principal cytokines associated with the severity of malaria by P. berghei infection were TNF-α, IFN-γ, and IL-12p70. Moreover, it was evident from our flow cytometry results that innate immunity is crucial and macrophages are at the frontline of immunity against P. berghei infection. Our study recommended further investigations to shed light on the effects of babesiosis in suppressing malaria with the goal of developing Babesia-based therapy against malaria.