Epidemiological assessment of Anaplasma marginale, Babesia bigemina, and Babesia bovis infections in Colombian creole cattle breeds: A molecular survey in northeastern Colombia.

Anaplasmosis and babesiosis are globally distributed arthropod-borne diseases known for causing substantial economic losses due to their high morbidity and mortality rates. This study aims to assess the frequency and epidemiological features associated with the infection of Anaplasma marginale, Babesia bigemina, and Babesia bovis in three Creole cattle breeds (Chino Santandereano (Chino), Casanareño (CAS), and Sanmartinero (SM)) in northeastern Colombia. Between June 2019 and March 2020, a total of 252 Creole cattle were sampled, with Chino, CAS, and SM accounting for 42.8%, 29.5%, and 29.5% of the samples, respectively. Blood samples were subjected to molecular analysis to detect the DNA of A. marginale, B. bigemina, and B. bovis, using species-specific primers. Additionally, Packed Cell Volume (PCV), total serum proteins, and body condition were evaluated. Molecular analyses revealed the presence of B. bigemina, A. marginale, and B. bovis in 83.7% (211/252; 95% CI = 79.1%-88.3%), 59.9% (151/252; 95% CI = 53.8%-66.1%), and 40.9% (103/252; 95% CI = 34.7%-46.9%) of the samples, respectively, with 69% (174/252; 95% CI = 57.8%-80.3%) exhibiting coinfections. Notably, in infected animals, no significant alterations in PCV, total serum proteins, or body condition were observed. Multivariate analyses indicated a statistically significant association between the frequency of A. marginale infection and the breed and season, with a higher frequency in SM during the rainy season (P < 0.05). To our knowledge, this is the first molecular survey that evaluates multiple arthropod-borne pathogens in Colombian Creole breeds. The results revel a high frequency of B. bigemina and A. marginale infections, coupled with a notable frequency of coinfections, all without significant alteration in the PCV, total serum proteins and body conditions. Our findings enhance the understanding of the epidemiological aspects of arthropod-borne pathogens in Colombian Creole breed and contribute to the improvement of sanitary programs for these animals.

Expression of sex-specific molecular markers by Babesia bovis gametes.

BACKGROUND: Bovine babesiosis caused by Babesia bovis is one of the most important tick-borne diseases of cattle in tropical and subtropical regions. Babesia bovis parasites have a complex lifecycle, including development within the mammalian host and tick vector. In the tick midgut, extracellular Babesia parasites transform into gametes that fuse to form zygotes. To date, little is known about genes and proteins expressed by male gametes. METHODS AND RESULTS: We developed a method to separate male gametes from in vitro induced B. bovis culture. Separation enabled the validation of sex-specific markers. Collected male gametocytes were observed by Giemsa-stained smear and live-cell fluorescence microscopy. Babesia male gametes were used to confirm sex-specific markers by quantitative real-time PCR. Some genes were found to be male gamete specific genes including pka, hap2, α-tubulin II and znfp2. However, α-tubulin I and ABC transporter, trap2-4 and ccp1-3 genes were found to be upregulated in culture depleted of male gametes (female-enriched). Live immunofluorescence analysis using polyclonal antibodies confirmed surface expression of HAP2 by male and TRAP2-4 by female gametes. These results revealed strong markers to distinguish between B. bovis male and female gametes. CONCLUSIONS: Herein, we describe the identification of sex-specific molecular markers essential for B. bovis sexual reproduction. These tools will enhance our understanding of the biology of sexual stages and, consequently, the development of additional strategies to control bovine babesiosis.

Detection and quantification of Babesia bovis and Babesia bigemina using different target genes.

Molecular assays have been widely used for the detection and quantification of bovine babesiosis due to their high sensitivity and specificity. However, variations in the sensitivity of pathogen detection may occur depending on the selected target gene. Thus, this study aimed to compare the detection sensitivity (DS) of Babesia bovis and B. bigemina infection levels in artificially and naturally infected cattle using quantitative PCR (qPCR) and six target genes. For B. bovis, the merozoite surface antigen genes 2b and 2c (msa-2b and msa-2c), and the mitochondrial cytochrome b gene (cybmt) were used. For B. bigemina, the genes encoding the proteins associated with rhoptry 1c (rap-1c), rap-1a, and cybmt were used. Six bovines, free of babesiosis, were artificially infected with 1 × 10-8 red blood cells infected (iRBC) with B. bovis (n = 3) or 1 × 10-6B. bigemina iRBC (n = 3). The animals were evaluated daily until parasitemia was confirmed (≥ 2.0%). The quantity of iRBC present in each animal was determined by examining blood smears. Blood samples were then subjected to DNA extraction, serial dilution, and qPCR analysis to determine the DS of each target gene. In addition, 30 calves naturally infected by Babesia spp. were also evaluated using the same six target genes. Regarding the artificial infection, B. bovis cybmt showed 25-fold higher sensitivity than the msa-2b and msa-2c genes, while the B. bigemina cybmt exhibited 5-fold and 25-fold higher sensitivity than the rap-1a and rap-1c genes, respectively. The rap-1a gene was found to be 5 times more sensitive than the rap-1c gene, while the B. bovis msa-2b and msa-2c genes exhibited similar DS. The positive frequencies of naturally infected calves for the target cybmt, msa-2b, and msa-2c genes (B. bovis) were: 100%, 33.3% and 50%, while cybmt, rap-1a, and rap-1c genes (B. bigemina) were 90%, 83.3%, and 63.3%, respectively. This study may contribute to the selection of suitable genes for molecular monitoring of bovine babesiosis. Mitochondrial genes could be considered as an alternative to improve the sensitivity of B. bovis and B. bigemina detection using qPCR.

Genetic Diversity of Merozoite Surface Antigens in Global Babesia bovis Populations.

Cattle can be severely infected with the tick-borne protozoa Babesia bovis, giving rise to serious economic losses. Invasion of the host's RBCs by the parasite merozoite/sporozoites depends largely on the MSA (merozoite surface antigens) gene family, which comprises various fragments, e.g., MSA-1, MSA-2a1, MSA-2a2, MSA-2b and MSA-2c, highlighting the importance of these antigens as vaccine candidates. However, experimental trials documented the failure of some developed MSA-based vaccines to fully protect animals from B. bovis infection. One reason for this failure may be related to the genetic structure of the parasite. In the present study, all MSA-sequenced B. bovis isolates on the GenBank were collected and subjected to various analyses to evaluate their genetic diversity and population structure. The analyses were conducted on 199 MSA-1, 24 MSA-2a1, 193 MSA-2b and 148 MSA-2c isolates from geographically diverse regions. All these fragments displayed high nucleotide and haplotype diversities, but the MSA-1 was the most hypervariable and had the lowest inter- and intra-population gene flow values. This fragment also displayed a strong positive selection when testing its isolates for the natural selection, which suggests the potential occurrence of more genetic variations. On the contrary, the MSA-2c was the most conserved in comparison to the other fragments, and displayed the highest inter- and intra-population gene flow values, which was evidenced by a significantly negative selection and negative neutrality indices (Fu's Fs and Tajima's D). The majority of the MSA-2c tested isolates had two conserved amino acid repeats, and earlier reports have found these repeats to be highly immunogenic, which underlines the importance of this fragment in developing vaccines against B. bovis. Results of the MSA-2a1 analyses were also promising, but many more MSA-2a1 sequenced isolates are required to validating this assumption. The genetic analyses conducted for the MSA-2b fragment displayed borderline values when compared to the other fragments.

Disruption of a DNA fragment that encodes the microneme adhesive repeat domain-containing region of the BBOV_III011730 does not affect the blood stage growth of Babesia bovis in vitro.

Babesia bovis, an intraerythrocytic hemoprotozoan parasite, causes the most pathogenic form of bovine babesiosis, negatively impacting the cattle industry. Comprehensive knowledge of B. bovis biology is necessary for developing control methods. In cattle, B. bovis invades the red blood cells (RBCs) and reproduces asexually. Micronemal proteins, which bind to sialic acid of host cells via their microneme adhesive repeat (MAR) domains, are believed to play a key role in host cell invasion by apicomplexan parasites. In this study, we successfully deleted the region encoding MAR domain of the BBOV_III011730 by integrating a fusion gene of enhanced green fluorescent protein-blasticidin-S-deaminase into the genome of B. bovis. The transgenic B. bovis, lacking the MAR domain of the BBOV_III011730, invaded bovine RBCs in vitro and grew at rates similar to the parental line. In conclusion, our study revealed that the MAR domain is non-essential for the intraerythrocytic development of B. bovis in vitro.

Detection of Babesia bovis using loop-mediated isothermal amplification (LAMP) with improved thermostability, sensitivity and alternative visualization methods.

Bovine babesiosis is one of the most economically important tick-borne diseases in tropical and subtropical countries. A conventional microscopic diagnosis is typically used because it is inexpensive and expeditious. However, it is highly dependent on well-trained microscopists and tends to be incapable of detecting subpatent and chronic infections. Here, we developed a novel nucleic acid-based amplification method using loop-mediated isothermal amplification (LAMP) in conjunction with a colori-fluorometric dual indicator for the rapid and accurate detection of Babesia bovis based on the mitochondrial cytochrome b gene. We aimed to improve the thermostability, sensitivity, specificity, and alternative visualization of LAMP-based methods. We assessed its diagnostic performance compared to two conventional PCR agarose gel electrophoresis (PCR-AGE) methods. The thermostability of LAMP reaction mixtures and DNA templates in variable conditions was also assessed. In addition, we evaluated alternative visualization methods using different light sources including neon, LED, and UV lights. We found that the LAMP-neon was ten times more sensitive than the PCR-AGE, while the LAMP-LED and LAMP-UV were 1,000 times more sensitive. The current LAMP method showed no cross-amplification with uninfected cattle DNA or other common blood parasites in cattle, including Babesia bigemina, Theileria orientalis, Anaplasma marginale, and Trypanosoma evansi. In addition, the developed LAMP method has good thermostability and the potential for on-site utility as B. bovis DNA could still be detected up to 72 h after initial preparation. Our findings suggested that the developed LAMP method provides an alternative approach for B. bovis detection with sensitivity higher than PCR-AGE diagnostics, high specificity, and the flexibility to use neon, LED, and UV light sources for positive signal observations.

Genetic diversity in Babesia bovis from southern Africa and estimation of B. bovis infection levels in cattle using an optimised quantitative PCR assay.

Babesia bovis is a causal agent of bovine babesiosis, a disease which leads to mortality and morbidity and impacts the cattle industry worldwide. We amplified, cloned and sequenced the B. bovis merozoite surface antigen-2b (msa-2b) gene (∼940 bp) and the near full-length 18S rRNA gene (∼1600 bp) from cattle samples from South Africa and Mozambique to determine sequence variation between B. bovis parasites in the region. A TaqMan quantitative real-time PCR (qPCR) assay (18S rRNA gene) was optimised for the detection of B. bovis and estimation of parasitaemia in field samples from cattle from southern Africa. Phylogenetic analysis grouped the Msa-2b sequences in six clades and these were 59.7 to 99.6% identical to reference sequences. Sequence variation amongst B. bovis 18S rRNA sequences was found at 2 to 36 positions, and the sequences were 97 to 99% identical to published sequences. Mismatches between the B. bovis 18S rRNA sequences and a previously published qPCR forward primer (BoF) were observed; therefore, we developed a new forward primer (BoF2), and optimised the qPCR assay. Six 10-fold dilution series of B. bovis infected erythrocytes (2 × 108 to 2 × 103 infected red blood cells [iRBC]/ml) were analysed in triplicate in each of six separate qPCR runs, to determine the efficiency of the assay. The qPCR assay amplified the B. bovis 18S rRNA gene with 92.0 to 94.9% efficiency. The detection limit of the qPCR assay was approximately 6 iRBCs/µl. The performance of the optimised assay to diagnose B. bovis in field samples was assessed by testing DNA from 222 field samples of cattle from South Africa and Mozambique using three methods: the optimised qPCR assay, the reverse line blot (RLB) hybridisation assay, and the previously published qPCR assay. The detection rate of B. bovis using the optimised qPCR assay (31.1%, 69/222) was significantly higher (p<0.001) than both that using RLB (20.7%, 46/222) and the previously published qPCR assay (5.4%; 12/222). The B. bovis parasitaemia in samples from infected cattle ranged from 6 iRBCs/µl to 101,852 iRBCs/µl of blood. Our study revealed marked sequence variation between B. bovis parasites from southern Africa. The optimised qPCR assay will be useful in epidemiological studies and clinical diagnosis of B. bovis in southern Africa, and can be used to determine parasitaemia and potential carrier status in cattle populations, which is essential in the control of babesiosis.

Prevalence and risk factors associated with Babesia bovis infection in Crioula Lageana cattle.

INTRODUCTION: Bovine babesiosis caused by the protozoan Babesia bovis is a worldwide disease and causes great economic damage to livestock. There are no studies on the epidemiology of this disease in native breeds such as Crioula Lageana cattle raised in the South of Brazil. METHODOLOGY: DNA samples from 311 animals were amplified by polymerase chain reaction (PCR) for the identification of the gene rap-1 (Rhoptry Associated Protein 1) from B. bovis. An epidemiological questionnaire was used to determine the risk factors associated with infection. RESULTS: The prevalence of B. bovis infection was 72% (224/311). Age and tick infestation affected infection. The factors associated with infection were the breeding objective (p = 0.042; CI = 0.746-0.995; OR = 0.861), contact of cattle with other animal species (p = 0.002; CI = 0.517-0.860; OR = 0.484), absence of tick control (p = < 0.001; CI = 0.074-0.480; OR = 0.188) and timing of tick treatment (p = 0.026; CI = 0.673-0.975; OR = 0.810), and these were considered to be factors that can protect against the disease. CONCLUSIONS: The Crioula Lageana cattle breed has near enzootic stability with regards to B. bovis infection.

Molecular characterization and genetic diversity of Babesia bovis and Babesia bigemina of cattle in Thailand.

Babesia bovis and B. bigemina are the most common tick-borne parasites that cause bovine babesiosis which effects livestock production, leading to economic losses in tropical and subtropical areas of the world. The aims of this study were to determine the molecular detection, genetic diversity and antigenicity prediction of B. bovis based on spherical body protein 2 (sbp-2) gene and B. bigemina based on rhoptry-associated protein 1a (rap-1a) gene in cattle in Thailand. By PCR assay, the molecular detection of B. bovis and B. bigemina infection revealed levels of 2.58% (4/155) and 5.80% (9/155), respectively. The phylograms showed that B. bovis sbp-2 and B. bigemina rap-1a sequences displayed 5 and 3 clades with similarity ranging between 85.53 to 100% and 98.28 to 100%, respectively, when compared within Thailand strain. Diversity analysis of sbp-2 and rap-1a sequences showed 18 and 4 haplotypes, respectively. The entropy analysis illustrated 104 and 7 polymorphic sites of sbp-2 and rap-1a nucleic acid sequences, respectively, while those of sbp-2 and rap-1a amino acid sequences showed 46 and 4 high entropy peaks, respectively. Motifs analysis exhibited the distribution and conservation among sbp-2 and rap-1a sequences. The continuous and discontinuous B-cell epitopes have also been evaluated in this work. Therefore, our findings may be used to ameliorate the understanding inputs of molecular phylogeny, genetic diversity and antigenicity of B. bovis and B. bigemina Thailand stains.

Identification of Babesia bovis MSA-1 functionally constraint regions capable of binding to bovine erythrocytes.

Merozoite surface antigen-1 is a glycoprotein expressed by Babesia bovis and is considered a vaccine candidate given that antibodies against it are able to partially block in vitro invasion of bovine erythrocytes. Despite this, no study to date has confirmed the target cell binding properties of the full MSA-1 or its fragments. This research has thus been focused on identifying protein regions playing a role in erythrocyte attachment, based on genetic diversity and natural selection analysis. Two regions under functional constraint (nucleotides 134-428 and 464-629) having a preponderance of negatively-selected signals were identified in silico. Three non-overlapping peptides derived from functionally constraint regions (42422 (39PEGSFYDDMSKFYGAVGSFD58), 42424 (91NALIKNNPMIRPDLFNATIV110) and 42426 (150TDIVEEDREKAVEYFKKHVY169)) were able to specifically bind to a sialoglycoprotein located on the bovine erythrocyte surface as confirmed by sensitive and specific peptide-cell interaction competition assays using both enzymatically treated and untreated red blood cells. Interestingly, it was predicted that peptides 42422 and 42426 have a helical structure and conserved motifs in all strain/isolates. These findings provide evidence, for the first time, related to B. bovis MSA-1 short regions used by the parasite in erythrocyte binding which could be predicted using natural selection analysis. Future work focused on evaluating these peptides' antigenic ability during natural infection, and their ability to induce protection in immunisation assays are needed to confirm their usefulness as synthetic vaccine candidates.

Detection of Babesia and the associated factors in cattle and humans from Magdalena Medio region, Colombia.

This study aimed to determine the frequency of Babesia spp. infection in cattle, livestock farmers, and patients with acute febrile illness (AFI) from the Magdalena Medio region in Colombia using molecular and serological methods. PCR detected Babesia in 83.9 % (161/192) of cattle and 14.8 % (21/143) of farmers tested. Molecular analysis based on eight DNA sequences from the 18S rRNA identified Babesia bovis and Babesia bigemina in cattle and Babesia bigemina in farmers. There was no molecular detection in the patients with acute febrile illness; nonetheless, the serological test in the AFI population yielded 10.7 % (23/215) seropositivity for Babesia microti. Our findings suggest natural infection by this hemoparasite in this livestock region, and it is, therefore, essential to continue determining the role of this parasite as an etiological agent of diseases in the area, not only because of its veterinary relevance but also because of its zoonotic potential.

Molecular epidemiology and characterization of Babesia bovis in cattle of Khyber Pakhtunkhwa province, Pakistan.

Babesiosis is a tick-borne disease found globally but most prominent in tropical and subtropical regions. It is responsible for huge mortality and morbidity, especially in developing countries like Pakistan. The current study was designed to determine the molecular epidemiology and characterization of Babesia bovis (B. bovis) infection in cattle populations of districts Mardan, Kohat and Swat of Khyber Pakhtunkhwa (KP) province of Pakistan. A total of 434 tick-infested animals were sampled. Blood samples were collected, processed and then examined initially by microscopy for the presence of Babesia and were later confirmed through PCR by targeting cytochrome b gene, and the PCR products were sequenced. Phylogenetic analysis of sequenced isolates of the current study showed close sequence similarity with the reported strain of China. A non-significant association (p > 0.05) was observed between the prevalence of infections and risk factors. The overall prevalence of infection in all three districts was 10.11%. In district Swat (12.61%), the prevalence was recorded as the highest for B. bovis infection followed by district Mardan (10.60%) and district Kohat (06.90%). The Friesian breed of cattle, females and adult animals were highly susceptible to B. bovis infection. The prevalence of infection was recorded highest during the summer season and lowest during the winter season. This study concludes that B. bovis infection is prevalent in three studied districts of KP province and the sequenced isolates of the current study showed close sequence similarity with the reported strain of China.

Molecular survey of Babesia parasites in Kenya: first detailed report on occurrence of Babesia bovis in cattle.

BACKGROUND: Among protozoan parasites in the genus Babesia, Babesia bigemina is endemic and widespread in the East African region while the status of the more pathogenic Babesia bovis remains unclear despite the presence of the tick vector, Rhipicephalus microplus, which transmits both species. Recent studies have confirmed the occurrence of R. microplus in coastal Kenya, and although B. bovis DNA has previously been detected in cattle blood in Kenya, no surveillance has been done to establish its prevalence. This study therefore investigated the occurrence of B. bovis in cattle in Kwale County, Kenya, where R. microplus is present in large numbers. METHODS: A species-specific multiplex TaqMan real-time PCR assay targeting two Babesia bovis genes, 18S ribosomal RNA and mitochondrially-encoded cytochrome b and B. bigemina cytochrome b gene was used to screen 506 cattle blood DNA samples collected from Kwale County for presence of Babesia parasite DNA. A sub-set of 29 B. bovis real-time PCR-positive samples were further amplified using a B. bovis-specific spherical body protein-4 (SBP-4) nested PCR and the resulting products sequenced to confirm the presence of B. bovis. RESULTS: A total of 131 animals (25.8%) were found to have bovine babesiosis based on real-time PCR. Twenty-four SBP4 nucleotide sequences obtained matched to B. bovis with a similarity of 97-100%. Of 131 infected animals, 87 (17.2%) were positive for B. bovis while 70 (13.8%) had B. bigemina and 26 (5.1%) were observed to be co-infected with both Babesia species. A total of 61 animals (12.1%) were found to be infected with B. bovis parasites only, while 44 animals (8.7%) had B. bigemina only. Babesia bovis and B. bigemina infections were detected in the three Kwale sub-counties. CONCLUSION: These findings reveal high prevalence of pathogenic B. bovis in a Kenyan area cutting across a busy transboundary livestock trade route with neighbouring Tanzania. The Babesia multiplex real-time PCR assay used in this study is specific and can detect and differentiate the two Babesia species and should be used for routine B. bovis surveillance to monitor the spread and establishment of the pathogen in other African countries where B. bigemina is endemic. Moreover, these findings highlight the threat of fatal babesiosis caused by B. bovis, whose endemic status is yet to be established. GRAPHICAL ABTRACT.

Lactate Dehydrogenase as a Potential Therapeutic Drug Target to Control Babesia bigemina.

Babesia bigemina is a tick-borne apicomplexan hemoprotozoan responsible for bovine babesiosis. The current drugs used for bovine babesiosis treatment have several drawbacks, including toxicity, the lack of effectiveness to clear the parasite, and potential to develop resistance. Identifying compounds that target essential and unique parasite metabolic pathways is a rational approach toward finding alternative drug treatments. Based on the genome sequence and transcriptomics analysis, it can be inferred that anaerobic glycolysis is the dominant adenosine triphosphate (ATP) supply for Babesia, and lactate dehydrogenase (LDH) is one of the essential enzymes in this pathway. Furthermore, the Babesia LDH sequence is distinct from its bovine homologue and thus a potential chemotherapeutic target that would result in decreasing the ATP supply to the parasite but not to the host. Gossypol is a known efficient specific inhibitor of LDH in the sensu stricto B. bovis and the sensu lato B. microti, among other related parasites, but no such data are currently available in the sensu stricto B. bigemina parasites. Hereby, we show that the LDH amino acid sequence is highly conserved among sensu stricto but not in sensu lato Babesia spp. A predictive structural analysis of B. bigemina LDH showed the conservation of the key amino acids involved in the binding to gossypol compared to B. bovis. Gossypol has a significant (P < 0.0001) inhibitory effect on the in vitro growth of B. bigemina, with IC50 of 43.97 mM after 72 h of treatment. The maximum IC (IC98) was observed at 60 mM gossypol. However, a significant effect on the viability of cattle PBMC was observed when the cells were cultured with 60 mM (IC98) gossypol compared with DMSO-exposed control cells. Interestingly, B. bigemina cultured at 3% oxygen expresses significantly higher levels of LDH and is more resistant to gossypol than the parasites maintained at ambient conditions containing ~20% oxygen. Altogether, the results suggest the potential of gossypol as an effective drug against B. bigemina infection, but the risk of host toxicity at therapeutic doses should be further evaluated in in vivo studies.

International interlaboratory validation of a nested PCR for molecular detection of Babesia bovis and Babesia bigemina, causative agents of bovine babesiosis.

Babesia bovis and B. bigemina are tick-transmitted parasites causing bovine babesiosis, characterized by significant morbidity and mortality leading to economic losses to the livestock industry in tropical and subtropical regions worldwide. Animals that recover from acute infection remain carriers with low parasitemia acting as a source of transmission, and often escape detection. An improved diagnosis of a B. bovis and/or B. bigemina infection of carrier animals is enabled by the availability of detection methods with high sensitivity. To this end, two nested PCR assays targeting the cytochrome b (cytb) genes of B. bovis and B. bigemina (cytb-nPCR), have been recently developed and an increased sensitivity with respect to reference protocols has been shown (Romero-Salas et al., 2016). In this study, the specificity against a panel of hemoparasites that potentially co-occur with B. bovis and B. bigemina was demonstrated to ensure applicability of the cytb-nPCR assays in a wide range of regions where bovine babesiosis is endemic. Furthermore, we compared both reported cytb-nPCR assays with reference nPCR and qPCR protocols for (i) their capability to detect carrier animals in the field, and (ii) their reproducibility when performed in different laboratories by independent operators. We show that, in a panel of bovine field samples (n = 100), the cytb-nPCR assays detected a considerably higher number of 25% B. bovis and 61% B. bigemina-positive animals compared to 7% and 20% B. bovis and 55% and 49% B. bigemina-positive animals when tested by reference nPCR and qPCR protocols, respectively. Cytb-nPCRs were also found superior in the detection of carrier animals when field samples from Africa were analyzed. In addition, both the B. bovis and B. bigemina cytb-nPCR assays were independently validated in a single blinded study in three laboratories. Importantly, no significant differences in the number/percentage of infected animals was observed using cytb-nPCR assays. In summary, the cytb-nPCR assays detected a considerably higher number of chronically infected B. bovis and B. bigemina carrier animals compared to reference nPCR and qPCR protocols, when applied in different epidemiological field situations. Furthermore, a high reproducibility between laboratories could be demonstrated.

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

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

Establishment and application of a qPCR diagnostic method for Theileria annulata.

Bovine theileriosis caused by several Theileria species including Theileria annulata, Theileria parva, Theileria orientalis, Theileria mutans, and Theileria sinensis is a significant hemoprotozoan tick-borne disease. Among these, Theileria species, T. annulata, which causes tropical theileriosis (TT), is regarded as one of the most pathogenic and is responsible for high mortality. At present, most conventional diagnostic methods for tropical theileriosis are time-consuming and laborious and cannot distinguish newfound T. sinensis in China. Therefore, a high sensitivity and specificity real-time quantitative PCR method based on the TA19140 target molecule was developed, and the method was found to be specific for T. annulata. No cross-reaction was observed with T. sinensis, T. orientalis, Babesia bovis, Babesia bigemina, or Hyalomma anatolicum which is negative for T. annulata. A total of 809 field samples from different regions of China were analyzed by using the developed qPCR and conventional PCR. The positive samples for T. annulata detected by real-time qPCR and conventional PCR were 66/809 (8.16%) and 20/809 (2.47%), respectively, and all positive amplicons by qPCR were confirmed by Sanger sequencing. The results showed that the developed qPCR for the T. annulata 19,140 gene was more sensitive than conventional PCR. In addition, we first discovered that TA19140 was mainly expressed at the schizont and merozoite stages of T. annulata by relative quantification. The protein encoded by the TA19140 gene may be used as a potential diagnostic antigen for tropical theileriosis. In conclusion, a real-time quantitative PCR diagnostic method targeting the TA19140 gene was successfully established and could be used for both the quantitative and qualitative analysis of T. annulata infection from cattle and vector ticks, which will greatly help to control and diagnosis of tropical theileriosis.

Comparison of high throughput RNA sequences between Babesia bigemina and Babesia bovis revealed consistent differential gene expression that is required for the Babesia life cycle in the vertebrate and invertebrate hosts.

Bovine babesiosis caused by Babesia bigemina and Babesia bovis is an economically important disease that affects cattle worldwide. Both B. bigemina and B. bovis are transovarially transmitted by Rhipicephalus ticks. However, little is known regarding parasite gene expression during infection of the tick vector or mammalian host, which has limited the development of effective control strategies to alleviate the losses to the cattle industry. To understand Babesia gene regulation during tick and mammalian host infection, we performed high throughput RNA-sequencing using samples collected from calves and Rhipicephalus microplus ticks infected with B. bigemina. We evaluated gene expression between B. bigemina blood-stages and kinetes and compared them with previous B. bovis RNA-seq data. The results revealed similar patterns of gene regulation between these two tick-borne transovarially transmitted Babesia parasites. Like B. bovis, the transcription of several B. bigemina genes in kinetes exceeded a 1,000-fold change while a few of these genes had a >20,000-fold increase. To identify genes that may have important roles in B. bigemina and B. bovis transovarial transmission, we searched for genes upregulated in B. bigemina kinetes in the genomic datasets of B. bovis and non-transovarially transmitted parasites, Theileria spp. and Babesia microti. Using this approach, we identify genes that may be potential markers for transovarial transmission by B. bigemina and B. bovis. The findings presented herein demonstrate common Babesia genes linked to infection of the vector or mammalian host and may contribute to elucidating strategies used by the parasite to complete their life cycle.

Molecular survey of bovine Babesia species in Bactrian camels (Camelus bactrianus) in Mongolia.

Bovine babesiosis, which is caused by species of genus Babesia, is a leading cause of considerable economic losses to the cattle industry each year. Bovine Babesia species have frequently been detected in non-cattle hosts, such as water buffalo (Bubalus bubalis), from which the parasites can be transmitted by ticks to cattle. Therefore, Babesia infections should be minimized not only in cattle but also in non-cattle carriers. In the present study, we surveyed the Bactrian camels (Camelus bactrianus) in Mongolia for three clinically significant bovine Babesia species, including Babesia bovis, B. bigemina, and Babesia sp. Mymensingh, which had been detected previously in Mongolian cattle. We screened blood DNA samples from 305 Bactrian camels in six Mongolian provinces for these species, using parasite-specific PCR assays. Our findings showed that the Bactrian camels in Mongolia were infected with all three Babesia species surveyed. The overall positive rates of B. bovis, B. bigemina, and Babesia sp. Mymensingh were 32.1%, 21.6%, and 24.3%, respectively, whereas 52.5% of the surveyed animals were infected with at least one parasite species. We also found that the female Bactrian camels and the Mongolian native camel breed had significantly higher Babesia positive rates than the male Bactrian camels and the Hos Zogdort breed. In Mongolia, cattle and Bactrian camels usually share common pasture lands for grazing; furthermore, tick species infesting cattle also infest Bactrian camels. Our findings, together with these observations, suggest that the tick transmission of bovine Babesia species might be possible between cattle and Bactrian camels. Therefore, strategies for the control of bovine babesiosis in Mongolia should include methods to minimize bovine Babesia species infections in Bactrian camels.