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.

Association between chronic Anaplasma marginale and Babesia spp. infection and hematological parameters of taurine heifers.

The aim of this study was to investigate the association between chronic Anaplasma marginale and Babesia spp. infection and hematological parameters of pregnant and non-pregnant taurine heifers. Blood samples from 94 females were collected on the first day (D-10) of timed artificial insemination (TAI) protocol and on pregnancy diagnosis (D+34). Hematological parameters were determined and compared between pregnant (PG) and non-pregnant (NPG) heifers, and within group at different sampling days. Real-time PCR (qPCR) was used to determine A. marginale and Babesia bovis infection, and for absolute quantification of Babesia spp. between PG and NPG groups. Correlation analysis was performed between the number of gDNA copies (CN) of Babesia spp. and hematological parameters. On D-10, mean hemoglobin concentration was higher for NPG, and hematocrit and total plasma protein were higher on D+34 for both groups. There was no difference in Babesia spp. CN between groups. In the first qPCR, all heifers were positive for A. marginale and B. bovis. Significant correlations were found between hemoglobin and erythrocyte and between hemoglobin and hematocrit (r = 0.8082 and r = 0.3009, respectively). Low levels of A. marginale and Babesia spp. did not affect hematological parameters of chronically infected pregnant and non-pregnant taurine heifers.

Developing Anti-Babesia bovis Blood Stage Vaccines: A New Perspective Regarding Synthetic Vaccines.

Bovine babesiosis is caused by the Apicomplexa parasites from the genus Babesia. It is one of the most important tick-borne veterinary diseases worldwide; Babesia bovis being the species associated with the most severe clinical signs of the disease and causing the greatest economic losses. Many limitations related to chemoprophylaxis and the acaricides control of transmitting vectors have led to the adoption of live attenuated vaccine immunisation against B. bovis as an alternative control strategy. However, whilst this strategy has been effective, several drawbacks related to its production have prompted research into alternative methodologies for producing vaccines. Classical approaches for developing anti-B. bovis vaccines are thus discussed in this review and are compared to a recent functional approach to highlight the latter's advantages when designing an effective synthetic vaccine targeting this parasite.

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.

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.

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.

Babesia bovis AMA-1, MSA-2c and RAP-1 contain conserved B and T-cell epitopes, which generate neutralizing antibodies and a long-lasting Th1 immune response in vaccinated cattle.

Vaccines against bovine babesiosis must, ideally, induce a humoral immune response characterized by neutralizing antibodies against conserved epitopes and a cellular Th1 immune response. In Babesia bovis, proteins such as AMA-1, MSA-2c, and RAP-1 have been characterized and antibodies against these proteins have shown a neutralizing effect, demonstrating the implication of B and T-cell epitopes in the immune response. There is evidence of the existence of B and T-cell epitopes in these proteins, however, it remains to be defined, the presence of conserved peptides in strains from around the world containing B and T-cell epitopes, and their role in the generation of a long-lasting immunity. The aim in this paper was to identify peptides of Babesia bovis AMA-1, MSA-2c, and RAP-1 that elicit a neutralizing and long-lasting Th1 immune response. Peptides containing B-cell epitopes of AMA-1, MSA-2c and RAP-1, were identified. The immune response generated by each peptide was characterized in cattle. All peptides tested induced antibodies that recognized intraerythrocytic parasites, however, only 5 peptides generated neutralizing antibodies in vitro: P2AMA-1 (6.28%), P3MSA-2c (10.27%), P4MSA-2c (10.42%), P1RAP-1 (32.45%), and P4RAP-1 (36.98%). When these neutralizing antibodies were evaluated as a pool, the inhibition percentage of invasion increased to 52.37%. When the T cellular response was evaluated, two peptides: P3MSA2c and P2AMA1 induced a higher percentage (>70%) of activated CD4 +/CD45RO+ T cells than unstimulated cells. Additionally, both peptides induced the production of gamma interferon (IFN-) in PBMCs from vaccinated cattle after one year proving the implication of a long-lasting Th1 immune response. In conclusion, we identified conserved peptides containing B and T-cell epitopes in antigens of B. bovis that elicit a Th1 immune response and showed evidence that peptides from the same protein elicit different immune responses, which has implication for vaccine development in bovine babesiosis.

Babesia bovis in large ruminants in Pakistan - molecular detection and haemato-biochemical alterations

Background: Babesiosis is endemic in Pakistan and is one of the most important bovine diseases that causes huge economic losses and high mortality in young animals. A hematobiochemical study was conducted to unveil the difference between diseased and healthy animals in selected districts i.e., Faisalabad (31° 25' 7.3740'' N and 73° 4' 44.7924'' E), Toba Tek Singh (30° 58' 9.7392'' N and 72° 27' 40.7484'' E) and Jhang (31° 16' 40.9656'' N and 72° 18' 42.3360'' E) of Punjab, Pakistan. Materials, Methods & Results: A total of 518 (Cattle = 360, Buffalo = 158) blood samples were collected. The samples were analyzed by polymerase chain reaction (PCR) targeting apocytochrome b-gene (Babesia bovis-gene) (CYTb) followed by haemato-biochemical analysis. Chi-square test for univariate analysis was used to analyze the data. In summer the PCR-based prevalence was 29.4 (53/180) and 24.05% (19/79) in cows and buffaloes, respectively. On the other hand, in winter results showed that 12.7 (23/180), 13.92 % (11/79) samples positive for Babesia genus from cows and buffaloes, respectively. The positive samples were further investigated for hematological and biochemical analysis. The results revealed that, the mean value of hematological parameters like RBCs, Hb, PCV, MCV and MCHC was significantly (P < 0.05) decreased in infected animals (cows and buffaloes) as compared to the non-infected ones. While the biochemical parameters like Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), cholesterol and Lactate dehydrogenase were significantly (P < 0.05) increased in infected animals as compared to healthy animals. This study is the first molecular and hematobiochemical evidence of Babesia bovis in dairy herds of Punjab province, Pakistan. Discussion: Bovine babesiosis is one of the important tick-borne diseases (TBD) affecting dairy industry. In bovines, among 3 Babesia species that cause the disease B. bovis is more pathogenic with high mortality and morbidity. Pakistan is situated in tropical and sub-tropical region where the humidity is high in some part of countries. This high humidity mostly favors the reproduction of the ticks thus higher prevalence of TBDs in this region. Initially the babesiosis was diagnosed by light microscopy using thin blood smear stained with Giemsa stain. Many studies verified that PCR is a more specific and sensitive tool than conventional techniques for the detection of carrier / asymptomatic ruminants. The haemato-biochemical profile is another valuable footprint to track the disease. Keeping in view the above-mentioned fact the present project has been planned to evaluate the haemato-biochemical alteration between health and Babesia infected cattle along with the molecular detection of Babesia species involved in bovine babesiosis. The mean values of haematobiochemical parameters in clinically ill and healthy animals revealed that the mean values of hematological parameters like RBCs, Hb, PCV, and HCT were significantly decreased in diseased animals as compared to the healthy ones. All these might be due the fact that the parasite is intra-erythrocytic in nature and destruction of red blood cells results in significant (P < 0.05) decrease level of all the hematological parameters. The mean value of ALT in babesiosis infected cattle was significantly higher as compared to healthy cattle. The mean values of AST and LDH in babesiosis infected cows was significantly higher as compared to that in healthy cows. The elevation in liver enzymes in babesiosis may be due to the hepatic damage and lesions induced by the parasite during its multiplication in the blood followed by disturbed liver function. These enzymes are present in high concentrations in the muscles and liver. High level of these enzymes in the blood is indicator of organ necrosis or damage.

Development of highly sensitive one step-PCR tests for improved detection of B. bigemina and B. bovis.

Bovine babesiosis caused by Babesia bigemina and B. bovis is an economically relevant tick-borne disease distributed over tropical and subtropical world regions. Animals that recover from the clinical disease can remain persistently infected, and those carriers are epidemiologically relevant since they can act as a source of infection to other animals through the tick bite. According to the manual of the World Organisation for Animal Health (OIE), the recommended molecular diagnosis test for both parasites is a nested polymerase chain reaction (nPCR) based on an amplification of a fragment of the rap-1 gene. Since nPCRs are time consuming, have a higher cost and risk of contamination, we propose a single step PCR for B. bigemina (BbiVESA) and B. bovis (BboVESA) based on the amplification of the multi-copy ves-1α gene. We developed these methods and we achieved a detection limit of 1 × 10-12 % parasitemia for B. bigemina and of 1 × 10-6 % for B. bovis using reference strains, which compared to the reference OIE tests, results in an improvement in sensitivity of six orders for B. bigemina. Finally, we tested 48 field samples from a babesiosis enzootic region where we were able to detect a higher proportion of positive animals with both VESA methods than with the reference rap-1 nPCRs. This difference was statistically significant for each Babesia species. Concordance between both diagnostic schemes based on Cohen's kappa coefficient showed minimal to non-agreement (κ = 0.32) for B. bigemina and non-agreement (κ = 0.16) for B. bovis since BbiVESA and BboVESA PCR tests showed a significantly higher detection capacity. In conclusion, the high sensitivity of the assay, together with the lower demand of time and reagents make the VESA PCR methods developed here a valuable diagnostic tool for the molecular detection and epidemiological survey of both Babesia pathogens.

The key to egress? Babesia bovis perforin-like protein 1 (PLP1) with hemolytic capacity is required for blood stage replication and is involved in the exit of the parasite from the host cell.

Bovine babesiosis is a tick-borne disease caused by apicomplexan parasites of the Babesia genus that represents a major constraint to livestock production worldwide. Currently available vaccines are based on live parasites which have archetypal limitations. Our goal is to identify candidate antigens so that new and effective vaccines against Babesia may be developed. The perforin-like protein (PLP) family has been identified as a key player in cell traversal and egress in related apicomplexans and it was also identified in Babesia, but its function in this parasite remains unknown. The aim of this work was to define the PLP family in Babesia and functionally characterize PLP1, a representative member of the family in Babesia bovis. Bioinformatic analyses demonstrate a variable number of plp genes (four to eight) in the genomes of six different Babesia spp. and conservation of the family members at the secondary and tertiary structure levels. We demonstrate here that Babesia PLPs contain the critical domains present in other apicomplexan PLPs to display the lytic capacity. We then focused on the functional characterization of PLP1 of B. bovis, both in vitro and in vivo. PLP1 is expressed and exposed to the host immune system during infection and has high hemolytic capacity under a wide range of conditions in vitro. A B. bovis plp1 knockout line displayed a decreased growth rate in vitro compared with the wild type strain and a peculiar phenotype consisting of multiple parasites within a single red blood cell, although at low frequency. This phenotype suggests that the lack of PLP1 has a negative impact on the mechanism of egression of the parasite and, therefore, on its capacity to proliferate. It is possible that PLP1 is associated with other proteins in the processes of invasion and egress, which were found to have redundant mechanisms in related apicomplexans. Future work will be focused on unravelling the network of proteins involved in these essential parasite functions.

Proteínas importantes para la invasión de Babesia bovis a las células huésped / Important Proteins for Babesia bovis Invasion to Host Cells / Proteínas importantes para a invasão das células hospedeiras por Babesia bovis

Introducción: La babesiosis bovina es causada por parásitos Apicomplexa del género Babesia, siendo la Babesia bovis la especie asociada con cuadros clínicos más graves de la enfermedad. La invasión de B. bovis a los eritro-citos bovinos implica la interacción entre moléculas de los merozoítos del parásito con receptores de las células huésped. Por ende, conocer las proteínas involucradas en este proceso supone un importante paso para entender la biología del parásito. Objetivo: Describir las principales moléculas implicadas en el proceso de invasión de B. bovis a eritrocitos bovinos. Metodología: Se realizó una búsqueda en NCBI, Medline, LILACS y SciELO usando los términos: "Babesia bovis AND invasion process", "MSA-1", "RON2", "AMA-1", "moving junction", "B. bovis AND Vaccine candidates". Con corte en mayo de 2020, había 61 publicaciones disponibles en inglés que describen el estudio de las anteriores proteínas y su participación en la invasión.Resultados: Por ser clave el proceso de invasión a eritrocitos bovinos para la patogénesis de la babesiosis bovina, la revisión encontró 3 proteínas de B. bovis que participan en el reconocimiento e invasión a las células diana: MSA-1, AMA-1 y RON2. Sin embargo, los detalles a nivel molecular para las interacciones inter e intramoleculares aún no se han dilucidado por completo. Conclusiones: Conocer las moléculas involucradas en las interacciones parásito-hospedero permitirá entender cómo ocurre el proceso de invasión de B. bovis a los eritrocitos y, así, evaluar su futura utilidad como componente de una estrategia de control efectiva contra esta parasitosis

Introduction: Bovine babesiosis is caused by Apicomplexas parasites of the genus Babesia, Babesia bovis being the species associated with the most serious clinical conditions of the disease. B. bovisinvasion into the bovine erythrocytes involves the interaction between the parasites merozoites mo-lecules with host cell receptors. Therefore, knowing the proteins involved in the invasion process will enable understanding the parasite biology. Objective: To describe the important molecules involved in the B. bovis invasion process to bovine erythrocytes.Methodology: A search was made on NCBI, Medline, LILACS and SciELO databases using keywords as "Babesia bovis AND invasion process", "MSA-1", "RON2", "AMA-1", "moving junction", "B. bovis AND Vaccine candidates". 61 studies written in English describing the study for proteins that take place during invasion process which have been published until mayo were completely revised. Results: Given that the bovine erythrocyte invasion process is key for the pathogenesis of bovine babesiosis, a review was made where 3 proteins were found to be associated to the recognition and invasion processes of target cells: MSA-1, AMA-1 and RON2. However, the details at molecular level for the inter an intramolecular interaction have not yet been fully elucidated. Conclusions: Study the molecules involved in host-parasite interactions will allow understanding how the B. bovis invasion process to erythrocytes occurs and evaluating their future utility as a component of an effective control strategy for this parasitosis

Introdução: A babesiose bovina é causada por parasitas Apicomplexa do gênero Babesia, sendo a Babesia bovis a espécie associada com os sinais clínicos mais graves da doença. A invasão de B. bovis em eritrócitos bovinos envolve a interação entre moléculas dos merozoítos parasitas com receptores nas células hospedeiras. Por conseguinte, o conhecimento das proteínas envolvidas neste processo é um passo importante para a compreensão da biologia do parasita. Objetivo: Descrever as principais moléculas envolvidas no processo de invasão de B. bovis em eritró-citos bovinos. Metodologia: Foi realizada uma pesquisa no NCBI, Medline, LILACS e SciELO utilizando os termos: "Babesia bovis AND invasion process", "MSA-1", "RON2", "AMA-1", "moving junction", "B. bovis AND Vaccine candidates". Até maio de 2020 estavam disponíveis 61 publicações em inglês, que descreviam o estudo das proteínas acima referidas e o seu envolvimento na invasão. Resultados: Como o processo de invasão de eritrócitos bovinos é fundamental para á patogênese da babesiose bovina, a revisão encontrou 3 proteínas de B. bovis envolvidas no reconhecimento e invasão de células alvo: MSA-1, AMA-1 e RON2. No entanto, os detalhes a nível molecular para as interações Inter e intramoleculares ainda não foram completamente elucidados. Conclusões: A compreensão das moléculas envolvidas nas interações parasita-hospedeiro permitirá entender como ocorre o processo da invasão de B. bovis em eritrócitos e, assim, avaliar sua utilidade futura como componente de uma estratégia efetiva de controle contra esta parasitose

Genetic diversity of Babesia bovis studied longitudinally under natural transmission conditions in calves in the state of Rio de Janeiro, Brazil.

Serum and DNA samples from 15 naturally infected calves in Seropédica, Brazil, were obtained quarterly from birth to 12 months of age, in order to longitudinally evaluate their humoral immune response against Babesia bovis and the merozoite surface antigen diversity of B. bovis. Anti-B. bovis IgG antibodies were detected by an indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA). Using DNA amplification, sequencing and phylogenetic analysis, the genetic diversity of B. bovis was assessed based on the genes that encode merozoite surface antigens (MSA-1, MSA-2b and MSA-2c). The serological results demonstrated that up to six months of age, all the calves developed active immunity against B. bovis. Among the 75 DNA samples evaluated, 0, 3 and 5 sequences of the msa-1, msa-2b and msa-2c genes were obtained, respectively. The present study demonstrated that the msa-2b and msa-2c gene sequences amplified from blood DNA of B. bovis-positive calves were genetically diversified. These data emphasize the importance of conducting deeper studies on the genetic diversity of B. bovis in Brazil, in order to design diagnostic antigens and vaccines in the future.

Genomic Study of Babesia bovis Infection Level and Its Association With Tick Count in Hereford and Braford Cattle.

Bovine babesiosis is a tick-borne disease caused by intraerythrocytic protozoa and leads to substantial economic losses for the livestock industry throughout the world. Babesia bovis is considered the most pathogenic species, which causes bovine babesiosis in Brazil. Genomic data could be used to evaluate the viability of improving resistance against B. bovis infection level (IB) through genomic selection, and, for that, knowledge of genetic parameters is needed. Furthermore, genome-wide association studies (GWAS) could be conducted to provide a better understanding of the genetic basis of the host response to B. bovis infection. No previous work in quantitative genetics of B. bovis infection was found. Thus, the objective of this study was to estimate the genetic correlation between IB and tick count (TC), evaluate predictive ability and applicability of genomic selection, and perform GWAS in Hereford and Braford cattle. The single-step genomic best linear unbiased prediction method was used, which allows the estimation of both breeding values and marker effects. Standard phenotyping was conducted for both traits. IB quantifications from the blood of 1,858 animals were carried using quantitative PCR assays. For TC, one to three subsequent tick counts were performed by manually counting adult female ticks on one side of each animal's body that was naturally exposed to ticks. Animals were genotyped using the Illumina BovineSNP50 panel. The posterior mean of IB heritability, estimated by the Bayesian animal model in a bivariate analysis, was low (0.10), and the estimations of genetic correlation between IB and TC were also low (0.15). The cross-validation genomic prediction accuracy for IB ranged from 0.18 to 0.35 and from 0.29 to 0.32 using k-means and random clustering, respectively, suggesting that genomic predictions could be used as a tool to improve genetics for IB, especially if a larger training population is developed. The top 10 single nucleotide polymorphisms from the GWAS explained 5.04% of total genetic variance for IB, which were located on chromosomes 1, 2, 5, 6, 12, 17, 18, 16, 24, and 26. Some candidate genes participate in immunity system pathways indicating that those genes are involved in resistance to B. bovis in cattle. Although the genetic correlation between IB and TC was weak, some candidate genes for IB were also reported in tick infestation studies, and they were also involved in biological resistance processes. This study contributes to improving genetic knowledge regarding infection by B. bovis in cattle.

Kinetic characterization of a novel cysteine peptidase from the protozoan Babesia bovis, a potential target for drug design.

C1A cysteine peptidases have been shown to play an important role during apicomplexan invasion and egress of host red blood cells (RBCs) and therefore have been exploited as targets for drug development, in which peptidase specificity is deterministic. Babesia bovis genome is currently available and from the 17 putative cysteine peptidases annotated four belong to the C1A subfamily. In this study, we describe the biochemical characterization of a C1A cysteine peptidase, named here BbCp (B. bovis cysteine peptidase) and evaluate its possible participation in the parasite asexual cycle in host RBCs. The recombinant protein was obtained in bacterial inclusion bodies and after a refolding process, presented typical kinetic features of the cysteine peptidase family, enhanced activity in the presence of a reducing agent, optimum pH between 6.5 and 7.0 and was inhibited by cystatins from R. microplus. Moreover, rBbCp substrate specificity evaluation using a peptide phage display library showed a preference for Val > Leu > Phe. Finally, antibodies anti-rBbCp were able to interfere with B. bovis growth in vitro, which highlights the BbCp as a potential target for drug design.

Correlations and repeatability between Babesia spp. infection levels using two dairy cattle breeding systems.

Babesia bovis and Babesia bigemina are tick-transmitted piroplasms that cause severe damage to the livestock industry in tropical regions of the world. Recent studies demonstrated differences in infection levels of these haemoparasites among bovine breeds and variation between individual cows regarding resistance to these diseases. This study aimed to estimate the repeatability and correlations between B. bovis and B. bigemina using two cattle breeding systems, an individual system (IS) and a collective paddock system (CPS). All animals were Holstein breed, and the levels of B. bovis and B. bigemina in blood samples were estimated by quantitative polymerase chain reaction (qPCR). The estimated correlations for the B. bigemina and B. bovis DNA copy number for IS and CPS were moderate and high, respectively, whereas repeatability estimates for both systems and both Babesia species were moderate. Although we cannot infer that the type of rearing system directly influenced the correlation and repeatability coefficients, it appears that the bovine parasitemia burden may be dependent on (or determine) the parasitemia burden on ticks because the bovines remained in the same place for a longer time in both systems. Thus, the babesiosis infection levels of the ticks may have been uniform, a phenomenon that also ensures greater uniformity in cattle infection. This factor may have favored the occurrence of infected ticks leading to higher repeatability estimates and correlations. Our study confirms high variability in resistance/susceptibility between breeds, and the high correlations found may be linked to this characteristic and the most intensive breeding type of dairy cattle. Besides, under the present study conditions, the estimated correlations suggest that measuring an infection level of one Babesia species can predict the level of infection of the other.

Bovine abortion, stillbirth and neonatal death associated with Babesia bovis and Anaplasma sp. infections in southern Brazil.

Anaplasmosis and babesiosis are tick-borne diseases widely disseminated in cattle herds in many parts of the world. These diseases represent important causes of death and economic losses in several countries, including Brazil, and are characterized by hemolytic disease and anemia. Animals of all ages may be affected. Although transplacental infections are known to occur, abortion, stillbirth and neonatal death directly associated with Anaplasma marginale and especially Babesia spp. infections have rarely been documented in cattle. The objective of the present study is to describe the pathological and molecular findings of two cases of bovine abortion, two cases of stillbirth and two cases of neonatal death associated with intrauterine anaplasmosis and/or babesiosis in southern Brazil. All cases occurred in beef farms in the state of Rio Grande do Sul, between 2017 and 2019. Angus and crossbred calves were affected. At the necropsy, the main gross lesions observed included different degrees of splenomegaly, enlarged and yellow liver, thick and grumous bile, pallor or jaundice of mucous membranes and carcass, and dark kidneys. Four calves also presented cherry-pink discoloration of the central nervous system. Cytological slides enabled the observation of intraerythrocytic organisms consistent with Babesia bovis (3/6) and A. marginale (2/6). Through PCR assays, it was possible to detect three cases of Babesia sp. infection alone, and one case of Anaplasma sp. infection alone. Co-infections with Anaplasma sp. and Babesia sp. were detected in two cases. These findings reaffirm that anaplasmosis and babesiosis should be considered as an important differential diagnosis of fetal loss, stillbirth and neonatal death in cattle in areas where these diseases occur.

Validation of an indirect ELISA using recombinant proteins as antigen to identify animals exposed to Babesia bigemina.

The objective of this study was to instrument a serological assay for the epidemiological diagnosis of bovine babesiosis in Mexico, using the Babesia bigemina recombinant protein RAP-1 (rRAP-1α) as antigen. rRAP-1α, r12d3 and rGP45 were the three recombinant antigens initially tested. Based on the highest titres obtained in the indirect ELISA (iELISA) with the positive control serum, using similar antigen concentrations, rRAP-1α was selected for further use. The diagnostic sensitivity and specificity rates estimated for the iELISA with rRAP-1α as antigen were 89.9% and 86.5%, respectively, while for the Indirect Fluorescent Antibody Test (IFAT), the gold standard assay, the sensitivity was 86.66% and the specificity was 95%. The ĸ agreement value determined was 0.52, indicating a moderate agreement between the iELISA and IFAT assays. The instrumented iELISA with rRAP-1α as antigen shows an excellent specificity rate and an acceptable sensitivity that allows for the detection of antibodies to B. bigemina in cattle naturally exposed to the vector tick Rhipicephalus microplus. By using the iELISA-rRAP-1α, along with an iELISA with recombinant Merozoite Surface Antigen (rMSA-1) for antibody determination against Babesia bovis in the serum samples collected from cattle at 'La Posta' experimental station in Mexico, a seroprevalence of 20.3% was estimated for B. bigemina and 19.4% for B. bovis, while 36.89% of samples were positive for both Babesia species. The iELISA test promises to be a safe and low-cost type of diagnosis available to cattle producers in Mexico and would facilitate the definition of herd immunity status to implement measures of control adapted for the prevention of bovine babesiosis outbreaks.

Identification of conserved peptides containing B-cell epitopes of Babesia bovis AMA-1 and their potential as diagnostics candidates.

The apical membrane antigen 1 (AMA-1) is a protein of the micronemes that is present in all organisms of the phylum Apicomplexa; it has been shown that AMA-1 plays an essential role for parasite invasion to target cells. It has been reported that AMA-1 is conserved among different isolates of Babesia; however, it is unknown whether the protein contains conserved B-cell epitopes and whether these epitopes are recognized by antibodies from cattle in endemic areas. In this research, using an in silico analysis, four peptides were designed containing exposed and conserved linear B-cell epitopes from the extracellular region of Babesia bovis AMA-1. The selected peptides were chemically synthesized, and then each peptide was emulsified and used to immunize two bovines per peptide. The antibodies produced against these peptides were able to recognize intra-erythrocytic parasites in an IFAT, except peptide 4, which was insoluble. The synthetic peptides were covalently fixed to the wells of an ELISA plate and incubated with sera from B. bovis naturally infected cattle. Peptides P2AMA and P3AMA were recognized by the sera of naturally infected cattle from different regions of Mexico. Statistical analysis showed that the ELISA test for peptides P2AMA and P3AMA had a concordance of 91.2% and 61.1% compared to the IFAT, a sensitivity of 94.56% and 71.74%, and a specificity of 76.19% and 14.2%, respectively. The presence of antibodies in bovine sera from endemic areas that bind to the identified peptides indicates that AMA-1 from B. bovis has conserved B-cell epitopes involved in the immune response under natural conditions. However, to propose their use as vaccine or diagnostics candidates, a further characterization of the humoral immune response elicited in cattle by these peptides is needed.

Highly sensitive nested PCR and rapid immunochromatographic detection of Babesia bovis and Babesia bigemina infection in a cattle herd with acute clinical and fatal cases in Argentina.

Bovine babesiosis is a tick-transmitted haemoparasitic disease caused by Babesia bovis and B. bigemina affecting cattle of tropical and subtropical regions around the world. Pathogens are transmitted by the tick vector Rhipicephalus microplus displaying a widespread distribution in northeastern Argentina. The disease is characterized by significant animal morbidity and mortality resulting in considerable economic loss. In this study, B. bovis and B. bigemina infection was investigated in a cattle herd of 150 adult bovines of pure Braford breed raised in a tick-hyperendemic field using molecular and serum antibody tests. A highly sensitive nested polymerase chain reaction (nPCR) assay targeting a species-specific region of the apocytochrome b gene resulted in direct B. bovis and B. bigemina detection in 27.3% and 54.7% of bovines, respectively. A recently developed immunochromatographic strip test (ICT) based on recombinant forms of spherical body protein 4 and the C-terminal region of rhoptry-associated protein 1 showed that 71.3% and 89.3% of bovines were seropositive for B. bovis and B. bigemina, respectively. The mixed infection rate as observed by direct (19.3%) and indirect detection (65.3%) coincided with those expected, respectively. Importantly, four months after sampling, nine bovines of the studied herd showed clinical signs of bovine babesiosis of which six animals eventually died. Microscopic detection of infected erythrocytes in Giemsa-stained blood smears confirmed B. bovis infection. Our study demonstrates that although animals showed a relatively high and very high rate of immunity against infection with B. bovis (71.3%) and B. bigemina (89.3%) parasites, respectively, clinical cases and fatalities due to the infection with B. bovis were observed. It is proposed that the most adequate control measure in the studied epidemiological situation is to vaccinate animals to prevent losses and/or an outbreak of bovine babesiosis.