Babesia bigemina and Theileria annulata infections in cattle: molecular detection, phylogenetic analysis, and assessment of risk factors.

Babesia bigemina and Theileria annulata are tick-borne protozoans that cause piroplasmosis in cattle, resulting in huge damages to the livestock industry. The prevalence of these infections depends on various intrinsic and extrinsic risk factors. In Pakistan, there is no information regarding the molecular characterization of Babesia bigemina and the risk factors associated with piroplasmosis. This study aimed to molecularly characterize Babesia spp. and Theileria spp. infecting various cattle breeds in Khyber Pakhtunkhwa, Pakistan, and to shed light on risk factors associated with these infections. Altogether, 219 blood samples were collected from various symptomatic cattle breeds, including Holstein Friesian (65.3%; 143/219), Jersey (21.5%; 47/219) and Sahiwal (13.2%; 29/219). Isolated genomic DNA from these blood samples was used in PCR for the amplification of the 18S rRNA fragment of apicomplexan parasites. Obtained 18S rDNA sequences from cattle hosts showed 99.5% identity with B. bigemina, or 100% with T. annulata. Having an overall infection rate of 61.6% (135/219), the highest infection rate was recorded for T. annulata (43.8%; 95/219), followed by B. bigemina (18.3%; 40/219). Phylogenetic analysis of 18S rDNA sequences revealed that B. bigemina clustered with corresponding species reported from Bolivia, and South Africa, while T. annulata grouped with same species from Italy, India, and Turkey. Among the different risk factors, the breed, season, and tick infestation were found to have a significant (P < 0.05) association with the piroplasmid infections. The information obtained in this study can be employed for effective surveillance and control of babesiosis and theileriosis in Pakistan. In addition to confirming our previous molecular detection of T. annulata in cattle, this study provides the first molecular surveillance and phylogenetic position of B. bigemina and associated risk factors in the study region.

RON2, a novel gene in Babesia bigemina, contains conserved, immunodominant B-cell epitopes that induce antibodies that block merozoite invasion.

Bovine babesiosis is the most important protozoan disease transmitted by ticks. In Plasmodium falciparum, another Apicomplexa protozoan, the interaction of rhoptry neck protein 2 (RON2) with apical membrane antigen-1 (AMA-1) has been described to have a key role in the invasion process. To date, RON2 has not been described in Babesia bigemina, the causal agent of bovine babesiosis in the Americas. In this work, we found a ron2 gene in the B. bigemina genome. RON2 encodes a protein that is 1351 amino acids long, has an identity of 64% (98% coverage) with RON2 of B. bovis and contains the CLAG domain, a conserved domain in Apicomplexa. B. bigemina ron2 is a single copy gene and it is transcribed and expressed in blood stages as determined by RT-PCR, Western blot, and confocal microscopy. Serum samples from B. bigemina-infected bovines were screened for the presence of RON2-specific antibodies, showing the recognition of conserved B-cell epitopes. Importantly, in vitro neutralization assays showed an inhibitory effect of RON2-specific antibodies on the red blood cell invasion by B. bigemina. Therefore, RON2 is a novel antigen in B. bigemina and contains conserved B-cell epitopes, which induce antibodies that inhibit merozoite invasion.

Molecular characterization of South Indian field isolates of bovine Babesia spp. and Anaplasma spp.

Ticks and tick-borne diseases (TTBDs) are considered major causes of economic loss in the livestock sector which incur an annual control cost estimated at US$ 498.7 million in India. Among these diseases, babesiosis, theileriosis and anaplasmosis are listed among the top ten livestock diseases in India and cause significant mortality and morbidity among cattle. However, molecular characterization of bovine Babesia and Anaplasma species are scant; thus, the aim of this study is to perform molecular characterization of field isolates of Babesia spp. and Anaplasma spp. infecting bovines in Kerala, South India. Blood smears and whole blood samples were collected from a total of 199 apparently healthy adult female cattle in Kerala. Based on microscopy, Babesia spp., Theileria orientalis and Anaplasma spp. organisms were detected in 9 (4.5%), 40 (20%) and 6 (3%) samples, respectively. Genus-specific polymerase chain reactions for amplification of 18S rRNA of Babesia spp. and 16S rRNA of Anaplasma spp. revealed positive results with 18 (9%) and 14 (7%) samples. The phylogenetic analysis of 18S rRNA gene sequences of Babesia spp. confirmed the existence of two different populations of Babesia spp. circulating in the blood of infected cattle viz., Babesia bigemina and a Babesia sp. genetically related to Babesia ovata. Further phylogenetic analysis using rap-1a sequences of isolates of B. bigemina revealed higher levels of genetic heterogeneity. However, the field isolates of B. bigemina displayed only slight heterogeneity when the rap-1c gene was examined. Polymerase chain reaction followed by sequencing and phylogenetic analysis of 16S rRNA gene of Anaplasma spp. revealed the existence of Anaplasma marginale, Anaplasma bovis and Anaplasma platys in bovines in South India. Based on msp4 gene sequences, all the field isolates of A. marginale from Kerala were clustered in a single clade with others isolated from around the world. To our knowledge, this study forms the first report on occurrence of B. ovata-like parasites and A. platys in cattle from India.

PCR detection of Ehrlichia ruminantium and Babesia bigemina in cattle from Kwara State, Nigeria: unexpected absence of infection.

Ticks and tick-borne diseases (TBDs) continue to pose an insidious and ever-present threat to livestock and livelihoods across the globe. Two of the most significant TBDs of cattle in Africa are heartwater and babesioisis, caused by Ehrlichia ruminantium and Babesia bigemina respectively. Both pathogens are endemic in Nigeria. However, to date, little data has been published regarding the number of cattle infected. In this study, blood samples were collected from cattle of the Kwara State, north-central Nigeria. Probe-based quantitative PCR (qPCR) and semi-nested PCR were used to investigate the presence of both pathogens, respectively. Our study found all samples (n = 157) to be surprisingly negative for both B. bigemina and E. ruminantium. These results contribute new information on the current burden of these two pathogens in Kwara State and may be helpful in informing more effective targeting of control strategies in Nigeria.

Comparisons of the topographic characteristics and electrical charge distributions among Babesia-infected erythrocytes and extraerythrocytic merozoites using AFM.

Tick-borne Babesia parasites are responsible for costly diseases worldwide. Improved control and prevention tools are urgently needed, but development of such tools is limited by numerous gaps in knowledge of the parasite-host relationships. We hereby used atomic force microscopy (AFM) and frequency-modulated Kelvin probe potential microscopy (FM-KPFM) techniques to compare size, texture, roughness and surface potential of normal and infected Babesia bovis, B. bigemina and B. caballi erythrocytes to better understand the physical properties of these parasites. In addition, AFM and FM-KPFM allowed a detailed view of extraerythrocytic merozoites revealing shape, topography and surface potential of paired and single parasites. B. bovis-infected erythrocytes display distinct surface texture and overall roughness compared to noninfected erythrocytes. Interestingly, B. caballi-infected erythrocytes do not display the surface ridges typical in B. bovis parasites. Observations of extraerythrocytic B. bovis, B. bigemina and B. caballi merozoites using AFM revealed differences in size and shape between these three parasites. Finally, similar to what was previously observed for Plasmodium-infected erythrocytes, FM-KPFM images reveal an unequal electric charge distribution, with higher surface potential above the erythrocyte regions that are likely associated with Babesia parasites than over its remainder regions. In addition, the surface potential of paired extraerythrocytic B. bovis Mo7 merozoites revealed an asymmetric potential distribution. These observations may be important to better understand the unique cytoadhesive properties of B. bovis-infected erythrocytes, and to speculate on the role of differences in the distribution of surface charges in the biology of the parasites.

Babesiosis prevalence in malaria-endemic regions of Colombia.

BACKGROUND & OBJECTIVES: : The presence of Babesia spp in humans, bovine cattle and ticks (the transmitting vector) has not been well characterized in Colombia. Babesia infection in humans can be overlooked due to similarity of the disease symptoms with malaria specially in the regions where malaria is endemic. The aim of the present work was to study the frequency of Babesia infection in humans, bovines and ticks in a malaria endemic region of Colombia, and explore the possible relationship of infection with host and the environmental factors. METHODS: : A cross-sectional study was carried out between August 2014 and March 2015 to determine the frequency of B. bovis and B. bigemina infection in a sample of 300 humans involved in cattle raising, in 202 bovines; and in 515 ticks obtained from these subjects, using molecular (PCR), microscopic and serological methods. In addition, the demographic, ecological and zootechnical factors associated with the presence of Babesia, were explored. RESULTS: : In the bovine population, the prevalence of infection was 14.4% (29/202); the highest risk of infection was found in cattle under nine months of age (OR = 23.9, CI 8.10-94.30, p = 0.0). In humans, a prevalence of 2% (6/300) was found; four of these six cases were positive for B. bovis. Self-report of fever in the last seven days in the positive cases was found to be associated with Babesia infection (Incidence rate ratio = 9.08; CI 1.34-61.10, p = 0.02). The frequency of B. bigemina infection in the collected ticks was 18.5% (30/162). INTERPRETATION & CONCLUSION: : The study established the presence of Babesia spp in humans, bovines and ticks. The most prevalent species responsible for babesiosis in humans and bovines was B. bovis, while B. bigemina was the species most frequently found in the tick population. The results contribute to the knowledge of the epidemiology of babesiosis in the country and can provide guidelines for the epidemiological surveillance of this non-malarial febrile illness in humans as well as cattle.

Rhipicephalus microplus (Acarina, Ixodidae) in Angola: evidence of its establishment and expansion.

Livestock constitutes a fundamental asset for the livelihood of rural communities in Angola. Rhipicephalus microplus (Acarina, Ixodidae) is considered to be the most important external parasite impacting the cattle industry in the world. The present work used a cross-sectional study on tick species infesting cattle in smallholder and commercial farms, to investigate the presence of R. microplus in Angola. This species was found to be recently established in certain areas of Cuanza-Sul and Benguela provinces. Furthermore, taking the current understanding of the pattern of dispersal of R. microplus into account, it is probable that this species is already present in other areas of the country. Animal movement without the necessary control measures facilitates the dispersal of this tick species. Consequently, R. microplus is causing rapid displacement of the native tick species, Rhipicephalus decoloratus. The presence of R. microplus in Angola constitutes a serious problem and poses a potential threat to the livestock sector as well as being an important risk factor for the sustainability of the livelihood strategies developed by smallholders.

Histopathological changes and oxidative damage in hepatic tissue of rats experimentally infected with Babesia bigemina.

The present study was aimed to investigate oxidative stress, DNA damage, and histopatholog- ical alterations in hepatic tissues of splenectomized Wistar rats experimentally infected with Ba- besia bigemina. Rats were challenged with 5x106 infected erythrocytes. Babesia infection was con- firmed both with Giemsa's staining blood smears and nested-PCR amplified region of apical membrane antigen-1 (AMA-1) gene. Parasitemia reached approximately 10 % at day 5 post-in- fection. Livers of infected rats were enlarged and darker in color, became extremely brittle with marked congestion. Microscopic evaluation showed cytoplasmic clearing of hepatocytes and se- vere hydropic changes with significantly dilated sinusoids containing macrophages and also intra- sinosoidal parasitized erythrocytes. Severe infiltration of lymphoplasma cells was also present throughout the liver parenchyma. Furthermore, Kupffer cells were enlarged and, occasionally, containing Babesia-parasitized erythrocytes. The activity of Glutathione (GSH) and catalase (CAT), and total antioxidant capacity (TAC) were also significantly decreased (p ⟨ 0.05) after infection of rats with B. bigemina. B. bigemina infection also induced a significant increase (p ⟨ 0.05) in hepatic malondialdehyde (MDA) and nitric oxide-derived products (NOx) concentra- tions as well as amount of endogenous hepatocytes DNA damage. Hepatic damage was also re- flected through the measurement of lactic acid dehydrogenase (LDH) and protein carbonyl con- tent (PCO) in liver cells. These two indices of liver injury were also significantly elevated (p ⟨ 0.5) during B. bigemina infection. Evaluation of correlation between assayed variables in infected rats revealed that MDA levels were positively correlated with PCO, NOx, LDH and DNA damage in the infected group and negatively correlated with GSH, CAT and TAC. There was also an inverse relationship between the antioxidant enzymes activities of GSH, CAT and TAC with PCO, NOx and DNA damage in infected rats. However, NOx showed positive correlation with PCO and DNA damage in infected rats. On the basis of the above results it can be concluded that the Ba- besia infection increases oxidative stress markers, protein carbonyl content and DNA damage and decreases antioxidant enzymes activities in the liver. These results suggest that B. bigemina infec- tion could alter the liver histopathology and causes DNA damage following oxidative stress in hepatic tissue. Further studies are needed to precisely define how hepatic tissue damage takes place in B. bigemina infection.

Characterization of tabanid flies (Diptera: Tabanidae) in South Africa and Zambia and detection of protozoan parasites they are harbouring.

Tabanids are haematophagous flies feeding on livestock and wildlife. In the absence of information on the relationship of tabanid flies and protozoan parasites in South Africa and Zambia, the current study was aimed at characterizing tabanid flies collected in these two countries as well as detecting protozoan parasites they are harbouring. A total of 527 tabanid flies were collected whereby 70·2% were from South Africa and 29·8% were from Zambia. Morphological analysis revealed a total of five different genera collected from the sampled areas namely: Ancala, Atylotus, Haematopota, Philoliche and Tabanus. DNA extracted from South African Tabanus par and Tabanus taeniola tested positive for the presence of Trypanosoma congolense (Savannah) and Trypanosoma theileri whilst one member from T. par was positive for Trypanosoma brucei species. DNA extracted from Zambian tabanid flies tested positive for the presence of Besnoitia species at 1·27% (2/157), Babesia bigemina 5·73% (9/157), Theileria parva 30·11% (30/157) and 9·82% (14/157) for Trypanosoma evansi. This study is the first to report on relationship of Babesia and Theileria parasites with tabanid flies. Further investigations are required to determine the role of tabanids in transmission of the detected protozoan parasites in livestock and wildlife in South Africa and Zambia.

Seroprevalence of Anaplasma marginale and Babesia bigemina infections and associated risk factors in Machakos County, Kenya.

Anaplasma marginale and Babesia bigemina are important tick-borne pathogens of cattle. A cross-sectional survey was undertaken to determine the seroprevalence of A. marginale and B. bigemina infections and identify associated risk factors on traditional smallholder farms in Machakos County, Kenya. A total of 421 cattle from 127 farms from four divisions in the county were sampled and visited between September and November 2007. The farms were selected by a proportional allocation approach based on the number of farms in the four divisions previously selected by stratified random sampling method. Information on animal and individual farm management variables was obtained using standardized questionnaires. Prevalence of serum antibodies due to A. marginale and B. bigemina pathogens was determined using the enzyme-linked immunosorbent assay (ELISA) technique. The relationship between the seropositivity and associated risk factors was assessed by multivariable analyses using standard logistic regression models. The overall estimation (and their 95% confidence intervals) of A. marginale and B. bigemina seropositivity at the animal level was 53.4% (48.5%, 58.2%) and 40.6% (35.8%, 45.4%), respectively. Two variables, "animal age" and "administrative division," were significantly associated with the A. marginale seroresponse. Three variables, "animal age" "grazing system" and "administrative division" were significantly associated with the B. bigemina seroresponse. These findings suggest possible indicators of existence of endemic instability for the two infections. The study identifies characterization of environmental suitability for the vectors and how they interact with grazing systems to cause the infections as an area for further studies, for improved understanding of the infections and in designing disease control programs.

Typification of virulent and low virulence Babesia bigemina clones by 18S rRNA and rap-1c.

The population structure of original Babesia bigemina isolates and reference strains with a defined phenotypic profile was assessed using 18S rRNA and rap-1c genes. Two reference strains, BbiS2P-c (virulent) and BbiS1A-c (low virulence), were biologically cloned in vitro. The virulence profile of the strains and clones was assessed in vivo. One fully virulent and one low-virulence clone were mixed in identical proportions to evaluate their growth efficiency in vitro. Each clone was differentiated by two microsatellites and the gene gp45. The 18S rRNA and rap-1c genes sequences from B. bigemina biological clones and their parental strains, multiplied exclusively in vivo or in vitro, were compared with strain JG-29. The virulence of clones derived from the BbiS2P-c strain was variable. Virulent clone Bbi9P1 grew more efficiently in vitro than did the low-virulence clone Bbi2A1. The haplotypes generated by the nucleotide polymorphism, localized in the V4 region of the 18S rRNA, allowed the identification of three genotypes. The rap-1c haplotypes allowed defining four genotypes. Parental and original strains were defined by multiple haplotypes identified in both genes. The rap-1c gene, analyzed by high-resolution melting (HRM), allowed discrimination between two genotypes according to their phenotype, and both were different from JG-29. B. bigemina biological clones made it possible to define the population structure of isolates and strains. The polymorphic regions of the 18S rRNA and rap-1c genes allowed the identification of different subpopulations within original B. bigemina isolates by the definition of several haplotypes and the differentiation of fully virulent from low virulence clones.

First evaluation of an outbreak of bovine babesiosis and anaplasmosis in Southern Brazil using multiplex PCR.

Outbreaks of tick-borne disease cases in Santa Catarina, Brazil are known, but the presence of the pathogen DNA has never been determined. In this study, the first survey of Anaplasma marginale, Babesia bigemina, and Babesia bovis DNA on blood samples of 33 cattle from an outbreak in Ponte Alta Municipality, Santa Catarina, Brazil, has been carried out. A multiplex PCR detected 54.5% of animals were co-infected with 2 or 3 parasites, while 24.2% were infected with only 1 species. The most prevalent agent was B. bigemina (63.6%) followed by A. marginale (60.6%). This is the first report of tick-borne disease pathogens obtained by DNA analysis in Southern Brazil.

Babesia bigemina (smith and Kilbourne, 1893) detection in Amblyomma sculptum (Berlese, 1888) ticks in the Mato Grosso do Sul state, Brazil.

Ticks parasitize various hosts, including humans, and are known to transmit pathogens that can be harmful not only to animals but also to humans. To evaluate the possible presence of pathogens in ticks, we aimed to collect and identify tick fauna specimens in Lagoa Comprida Municipal Natural Park, an anthropogenic urban area located in Aquidauana, Mato Grosso do Sul, Brazil. A total of 1216 ticks, of which 51.2% were Amblyomma sculptum, 1.2% were Amblyomma dubitatum, and 41% were Amblyomma spp. were collected. These results show that the prevalence of A. sculptum is significantly higher than that of A. dubitatum across all four seasons. Molecular analyses revealed positive samples for the genus Babesia, including the confirmation of Babesia bigemina in an A. sculptum specimen, marking the first record of this relationship. This unexpected finding demands greater attention and deeper analysis in the context of the epidemiology of tick-borne diseases.

Evaluation of different supportive treatments during packed cell volume replacement in calves naturally infected with tick fever agents.

This study evaluated the effect of different supportive treatments on PCV replacement of dairy calves naturally infected with tick fever (TF) agents, and treated with diminazene and enrofloxacin. Five products were tested as supportive treatments in four experiments. In these experiments, we used Girolando female calves (Gyr × Holstein, genetic ratio of 15/16 and 31/32 Holstein) four to six months old, raised in pasture, naturally infected with TF agents, and infested with R. microplus. Supportive treatment was administered once on day 0 of the study concurrently with specific treatment targeting TF agents. The animals were observed on days 0, 3, and 7. Oral or intravenous administration of a vitamin complex and mineral salts enhanced the increase in PCV and biochemical analytes present in the serum of calves naturally infected with TF agents. No positive effect on PCV values was observed with the administration of (1) invigorating tonic: calcium, casein-peptides and vitamin B12, (2) iron-based stimulant tonic and (3) metabolic tonic: vitamin A, vitamin D, and a fraction of polyunsaturated fatty acids. Supplementation by injection with Type III iron resulted in increased hemoglobin and PCV in treated animals. However, these results did not occur with iron citrate. Therefore, more studies with Type III iron need to be performed. Supportive treatment conferred no advantage in animals with no history of reduced PCV.

Sero epidemiological study on bovine babesiosis in cattle and buffaloes in Sharkia Governorate, Egypt.

Background: Bovine babesiosis represents a serious challenge for animal health, herd production, and profitability. Understanding the epidemiology and risk factors associated with babesiosis is critical to reduce their negative impacts. Aim: Investigation of the seroprevalence and risk factors associated with Babesia bigemina (B. bigemina) and Babesia bovis (B. bovis) in five districts in Sharkia governorate using ELISA. Methods: Across-sectional research was conducted to determine the seropositivity of babesiosis by collecting a total of 352 blood samples from 250 cattle and 102 buffaloes. A multivariate logistic regression model was implemented to evaluate the strength of the risk factors associated with both Babesia species infection. Results: The seroprevalence of B. bigemina and B. bovis was 42.6% and 17.0 %, respectively. The prevalence of babesiosis in cattle was found to be 48.8% for B. bigemina and 16.8% for B. bovis. Inclusive, in buffaloes, the prevalence was 27.5% for B. bigemina and 17.6% for B. bovis. Adult animals were more vulnerable to infection with babesia than young animals by 3-5 times, respectively. Males were more susceptible to B. bigemina and B. bovis than females by 3.7 and 3.5 times. Similarly, the odds of infection in infested animals with ticks were 2-4 times higher than in animals without ticks. Conclusion: The obtained results revealed that age, sex of the animal, and tick infestation were major risk factors for the seropositivity of both Babesia species. Inclusive, there was no evidence to support the premise that seroprevalence of babesiosis is correlated with the season and species.

Molecular prevalence of Anaplasma marginale, Babesia bigemina, and Theileria orientalis and their associations with Mafriwal cattle's age groups.

Objective: This study was designed to determine the molecular prevalence of hemoparasites and their associations with Mafriwal cattle's age groups. Materials and Methods: Blood samples were taken from the coccygeal veins of calves (n = 92), yearlings (n = 95), lactating (n = 90), and dry (n = 94) cows, which were subjected to microscopic and molecular identification of hemoparasites. The prevalence rate was determined based on the proportion of infected samples in the observed samples. Associations between hemoparasitism and different age groups of Mafriwal cattle were determined by the odds ratio and Fisher's exact test. Results: Babesia bigemina was the most prevalent hemoparasite in monospecies infection (20.8%), while the co-infection of Anaplasma marginale and B. bigemina (36.4%) had the highest molecular prevalence. Highly significant associations of hemoparasitism were observed between calves and yearlings (p < 0.001, Odds ratio = 21.340, 95% CI = 3.200-907.871), lactating (p < 0.01, Odds ratio = 6.600, 95% CI = 1.808-36.516), and dry (p < 0.001, Odds ratio = 10.457, 95% CI = 2.363-96.242) cows. Nevertheless, calves and yearlings were 2-4 times more likely to be co-infected with multiple hemoparasite species in comparison to older age groups. Conclusion: Mafriwal cattle were more susceptible to hemoparasitism with advancing age, but the younger calves were more prone to be co-infected with multiple hemoparasite species.

Molecular detection and characterization of Theileria annulata, Babesia bovis, and Babesia bigemina infecting cattle and buffalo in southern Egypt.

Tick-borne diseases have a major adverse effect on livestock worldwide, causing enormous economic losses in meat and milk production as well threatening animal and public health. In this study, we aimed to detect and characterize piroplasms isolated from cattle and buffalo in southern Egypt, using molecular techniques. Three hundred blood samples were collected from cattle and buffalo in two governorates in southern Egypt. All 300 samples (100%) were confirmed to contain DNA, as they exhibited bands of bovine ß-actin gene at the expected 227 bp for cattle and buffalo. The samples were analyzed by PCR for the presence of piroplasms, specifically Babesia bovis, Babesia bigemina, and Theileria annulata. Samples positive for the piroplasma 18S ribosomal RNA gene were further examined for two additional genes, spherical body protein 4 gene, to provide an enhanced degree of specificity for the identification of B. bovis and B. bigemina, and the major merozoite surface antigen gene for T. annulata. The infection rate for piroplasma spp. was 60/300 (20%). The positivity rates were 10.7% (32/300) for T. annulata, 5.3% (16/300) for B. bovis, and 4% (12/300) for B. bigemina. By host species, 42/150 (28%) cattle and 18/150 (12%) buffalo were positive for piroplasms. None of the isolates sequenced for the B. bovis isolates from buffalo in this study showed 100% identity with any sequence deposited in GenBank for the small subunit ribosomal RNA gene (maximum identity value = 99.74%). Similarly, no T. annulata small subunit ribosomal RNA gene sequence identified in this study exhibited 100% identity with any sequence deposited in GenBank (maximum identity value = 99.89%). The current study provides a partial sequence of the T. annulata merozoite-piroplasm surface antigen gene, as well as the B. bovis and B. bigemina spherical body protein 4 genes, in cattle and buffalo in southern Egypt, and is the first report on these piroplasma genes in cattle and buffalo in southern Egypt.

Comparative Transcriptome Analysis of Babesia bigemina Attenuated Vaccine and Virulent Strains of Mexican Origin.

Bovine babesiosis, caused by the protozoan Babesia bigemina, is one of the most important hemoparasite diseases of cattle in Mexico and the world. An attenuated B. bigemina strain maintained under in vitro culture conditions has been used as a live attenuated vaccine; however, the biological mechanisms involved in attenuation are unknown. The objective of this study was to identify, through a comparative transcriptomics approach, the components of the B. bigemina virulent parasites that are differentially expressed in vivo, as opposed to those expressed by B. bigemina attenuated vaccine parasites when inoculated into naïve cattle. The biological material under study was obtained by inoculating spleen-intact cattle with infected erythrocytes containing either the attenuated strain or a virulent field strain. After RNA extraction, transcriptomic analysis (RNA-seq) was performed, followed by bioinformatic Differential Expression (DE) analysis and Gene Ontology (GO) term enrichment. The high-throughput sequencing results obtained by analyzing three biological replicates for each parasite strain ranged from 9,504,000 to 9,656,000, and 13,400,000 to 15,750,000 reads for the B. bigemina attenuated and virulent strains, respectively. At least 519 differentially expressed genes were identified in the analyzed strains. In addition, GO analysis revealed both similarities and differences across the three categories: cellular components, biological processes, and molecular functions. The attenuated strain of B. bigemina derived from in vitro culture presents global transcriptomic changes when compared to the virulent strain. Moreover, the obtained data provide insights into the potential molecular mechanisms associated with the attenuation or pathogenicity of each analyzed strain, offering molecular markers that might be associated with virulence or potential vaccine candidates.

Detection of tick-borne pathogens in Rhipicephalus bursa ticks collected from the autochthonous Garrano breed of horses in Portugal.

The Garrano is a semi-feral horse breed native to several mountains in the northern Iberian Peninsula. Despite being endangered, this unique breed of pony has managed to survive in the wild and continues to be selectively bred, highlighting their remarkable resilience and adaptability to harsh environments. Wildlife plays a critical role in the survival of tick vectors in their natural habitats and the transfer of tick-borne pathogens, as they can serve as reservoir hosts for many agents and amplifiers for these vectors. The semi-feral lifestyle of the Garrano horses makes them particularly vulnerable to exposure to numerous tick species throughout the year. Therefore, the aim of this study was to investigate the occurrence of Anaplasma, Ehrlichia, Babesia, Theileria, and spotted fever rickettsiae in the Garrano horse ticks to obtain a knowledge of circulating agents in this host population. The collected ticks (n = 455) were identified as Rhipicephalus bursa. DNA specimens were organized in pools of 5 ticks, for molecular screening. Pools PCR results confirmed the presence of Candidatus Rickettsia barbariae (n = 12 for the ompB gene, n = 11 for the ompA gene and n = 6 for the gltA gene), Babesia bigemina (n = 1), Babesia caballi (n = 3), Theileria equi (n = 15) and Theileria haneyi (n = 1).These results confirm the circulation of an emerging rickettsial spotted fever group member, Candidatus R. barbariae, in R. bursa ticks. Our findings demonstrated that Candidatus R. barbariae co-circulates with B. bigemina and T. equi, which are vectored by R. bursa. We are reporting for the first time, the detection of T. haneyi among R. bursa ticks feeding in the Garrano horses in Portugal. Surveillance studies for tick-borne infections are essential to provide information that can facilitate the implementation of preventive and control strategies.

Artemisone inhibits in vitro and in vivo propagation of Babesia bovis and B. bigemina parasites.

Artemisone was evaluated, in in vitro and in vivo, for control of bovine babesiosis caused by Babesia bigemina and Babesia bovis parasites. In vitro, artemisone reduced parasitemia in a dose-dependent manner: the inhibitory effects increased gradually, reaching a maximum inhibition of 99.6% and 86.4% for B. bigemina and B. bovis, respectively 72 h after initiation of treatment with initial parasitemia of 0.5%. In calves infected with either B. bigemina or B. bovis artemisone treatment was well tolerated and prevented development of acute babesiosis in all animals except for one B. bovis-infected calf. The treatment did not eliminate all blood parasites, and recovered animals carried a persistent low-level infection. Treatment with artemisone may be useful as an alternative drug for preventing the pathology that results from babesiosis, without interfering with acquired immune protection following recovery from an acute babesiosis infection or vaccination.