Bigemina Infection Research Articles

Comparative evaluation of real-time polymerase chain reaction, conventional PCR and microscopy for detection of Babesia bigemina in bovines of Punjab (India).

Babesia bigemina is a major cause of bovine babesiosis, an economically devastating tick-borne disease that needs timely diagnosis for precise treatment. In present investigation, the detection efficacy of real-time PCR (qPCR) in comparison to conventional PCR and microscopy targeting 18S ribosomal gene was evaluated on 95 bovines (70 cattle and 25 buffaloes) suspected for babesiosis. Real-time PCR was standardized with the 10-fold serial dilutions in duplication of the given positive control (2 × 106 copy number) ranging from 106 to 100 copy number/µL and mean Ct value of each dilution was taken to extrapolate the curve. The samples with Ct value 36.92 of 10 copy number/µL were considered as positive. Out of 95 samples, 5 (5.26%), 21 (22.10%) and 49 (51.58%) positive by microscopy, conventional PCR and real-time PCR were in corresponding range of > 106-104, 104-103, and 103-<10 copy number/µL, respectively. The concordance of real-time PCR with conventional PCR and microscopy was moderate (Kappa = 0.523) and slight (Kappa = 0.09), respectively. The cows were at four times risk than the buffaloes for B. bigemina infection (Odds ratio:3.85, CI:1.4255-10.4370). This pioneer report from Punjab state (India) of application of real-time PCR to detect B. bigemina in bovines was found to be more sensitive than conventional PCR and microscopy that needs further investigations on a greater number of random samples.

A Comprehensive Review of Tick-Born Blood Protozoan Disease in Cattle: Babesiosis and Vaccination

Babesiosis is a tick-borne blood protozoan cattle disease caused by the genus Babesia worldwide and in tropical and subtropical regions. It is mainly caused by Babesia bovis and Babesia bigemina in cattle. When merozoites enter the erythrocyte, the surface coat of B. bigemina produces many merozoite surface antigens. This study is a comprehensive review of the "Tick-Borne Blood Protozoan Cattle Disease: Babesiosis and Vaccination" survey. There is a need to develop effective vaccines to control bovine babesiosis (BB) under field conditions because of the development of drug resistance against it. B. bigemina live-attenuated vaccine is available in the form of infected red blood cells (RBCs) in Australia and Israel, which has the danger of disseminating exotic DNA to other countries. A recombinant protein-based vaccine is a good choice to be developed, but very few immunogens have been explored yet to launch vaccination against B. bigemina. It is concluded from this study that secretory proteins were characterized, and the 65 kDa protein was found to be immunogenic. Major surface protein (gp45) was found to be antigenic and immunogenic when sera samples of infected and vaccinated calves were screened. The multiepitope-based protein rec-gpME was expressed, and the calves were vaccinated. The vaccinated calves had higher humoral and CTL responses. This rec-gpME could be a potential vaccine candidate against the B. bigemina infection. This rec-gpME would be tested on cattle under field conditions in Pakistan in the future.

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.

An epidemiological survey of vector-borne pathogens infecting cattle in Kyrgyzstan.

Cattle production is a major contributor to the national economy of Kyrgyzstan. Most cattle in Kyrgyzstan are managed via extensive systems and graze in communal pastures. As a result, infestations with ectoparasites are widespread, implying that various vector-borne diseases might be common in cattle. However, methods to control such infectious diseases are not available in Kyrgyzstan because the epidemiology of vector-borne pathogens (VBPs) infecting cattle remains unclear. The present study was therefore designed to survey Kyrgyz cattle for VBPs. We prepared blood DNA samples from 319 cattle in Kyrgyzstan and screened them with specific PCR assays for detecting Babesia bovis, Babesia bigemina, Babesia naoakii, Theileria annulata, Theileria orientalis, Trypanosoma evansi, Trypanosoma theileri, and Anaplasma marginale infections. Our findings indicated that the surveyed cattle were infected with six of the eight pathogens targeted, with the exceptions being B. naoakii and Try. evansi. The most common pathogen was T. orientalis (84.3%), followed by B. bigemina (47.6%), T. annulata (16.6%), A. marginale (11.6%), Try. theileri (7.2%), and B. bovis (2.5%). Additional screening of the B. bovis- and B. bigemina-negative samples with a Babesia genus-specific 18S rRNA PCR identified two positive samples, and sequencing analysis confirmed that each of them was infected with either Babesia major or Babesia occultans. To the best of our knowledge, this is the first report of B. bovis, B. bigemina, B. occultans, Try. theileri, and A. marginale infections in cattle in Kyrgyzstan. Our findings suggest that cattle in Kyrgyzstan are at high risk of infectious diseases caused by VBPs.

Identification of Anaplasma marginale, Babesia bovis and Babesia bigemina resistance alleles in Crioulo Lageano cattle using PCR-SBT and BoLA-DRB3 gene sequencing.

The BoLA-DRB3 gene in cattle is associated with tolerance to several infectious diseases, such as neosporosis, dermatophilosis, leukosis, and mastitis. This study used PCR-SBT and BoLA-DRB3 gene sequencing to determine the association between the presence or absence of Anaplasma marginale, Babesia bovis, and Babesia bigemina infections in 208 Crioulo Lageano cattle and alleles present in the population. The chi-square test and odds ratio analysis were employed to establish the association. Of the BoLA-DRB3 gene alleles present in the population, two alleles were significantly associated with resistance to A. marginale infections: BoLA-DRB3001:01 (p < 0.001; OR = 0.224), which had a frequency of 7.93%, and BoLA-DRB3024:06 (p = 0.007; OR < 0.00001), which had a frequency of 0.72%. Regarding B. bovis infection, the BoLA-DRB3*011:01 allele (p = 0.002; OR = 0.271) had a frequency of 6% in the population and was associated with resistance to the infection. None of the alleles was associated with resistance to infection by B. bigemina. The Crioulo Lageano breed has alleles that may confer resistance against infection by A. marginale and B. bovis.

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.

Molecular prevalence of tick-borne haemoprotozoan diseases in cattle of North Eastern state of Tripura, India.

The tick-borne haemoprotozoan diseases are major constraint in the health and production of cattle and causes substantial losses to the livestock industry throughout the world. Tripura is a northeastern state of India characterized by a warm humid tropical climate and high rainfall which is conducive to the multiplication of vector-ticks of haemoprotozoan diseases. A total of 320 tick-infested cattle (22 male and 298 female) of either sex was selected randomly from different district of Tripura for the detection of carrier cattle infected with tick-borne haemoparasites from July, 2017 to June, 2019. Out of 320 tick-infected cattle, 4.69% prevalence of B. bigemina infection in cattle was found while Theleria spp. was not prevalent in the collected blood sample by using polymerase chain reaction. The prevalence of B. bigemina was significantly (< 0.05) higher in cattle more than 3years of age (6.72%) compared to the 1-3years age group (3.97%) and < 1year age groups (1.66%). Sex-wise B. bigemina infection was significantly (< 0.05) seen only in female (5.03%) cattle infested with ticks. The district-wise B. bigemina prevalence (%) in Tripura was as in cattle; Dhalai (0.63), Sipahijala (0.31), Khowai (0.31), Gomati (0.31), South Tripura (0.63), North Tripura (0.31), Unakoti (0.31) and West Tripura (1.87) respectively. The use of PCR assays for the detection ofB. bigemina and Theileria spp. inthe North Eastern state of Tripura of India showed prevalence of 4.69% and 0%, respectively in tick-infested cattle. This study is the first report about the prevalence of tick-borne haemoprotozoan diseases from cattle of Tripura and suggest that babesiosis is more common disease as compared to theileriosis and is more prevalent in adult female tick-infested cattle in Tripura.

Epidemiological study on Ixodid tick infestation and tick borne haemopathogens on cattle in Awi Zone, northwest Ethiopia.

BackgroundTick and tick borne haemopathogens are the main challenge of livestock production and productivity in Ethiopia particular in northwest Ethiopia due to favourable climate condition.ObjectivesThe objectives of this study was to determining the prevalence of Ixodid tick infestation and tick borne haemopathogens on cattle, identifying the existing Ixodid tick species, assessing seasonal variation and major risk factors associated with tick infestation and tick borne haemopathogens.MethodsA cross‐sectional study was conducted from December 2020 to july 2021 on Ixodid tick infestation and tick borne haemopathogens on cattle in the northwest Ethiopia. A toatal of 384 cattle were used for this study. Tick species were identified using morphological identification keys under a stereomicroscope. Thin blood smear examination were conducted to assess tick borne haemopathogens.ResultsThe overall prevalence of Ixodid tick infestation and tick borne haemopathogens were 45% and 3%, respectively. Babesia bigemina was the only haemoparasite detected in the present survey. Potential risk factors were investigated for their association with tick infestation and B. bigemina using logistic regression and chi‐square test, respectively. Accordingly, age, body condition, agroecological systems and season were significantly (p < 0.05) associated with tick infestation whereas season and body condition were significantly (p < 0.05) associated with B. bigemina infection. A total seven tick species were identified. Amblyomma varigatum (55%) and Boophilus decoloratus (15.7%) were the predominant Ixodid tick species encountered. A total of 128 blood samples were collected from Jawi district and examined using thin blood smear. Of them, 3% were infected by the B. bigemina ConclusionsTick infestation in this study was high and seems to play vital role for the reduction of production, productivity and for the transmission B. bigemina. Therefore, sound and effective tick control and prevention strategies are needed to mitigate the risk factors for tick infestation and B. bigemina infection in Ethiopia.

Bovine babesiosis in India: Estimation of prevalence by systematic review and meta analysis

Bovine babesiosis is a serious threat to the livestock sector especially in tropical countries like India. Understanding the epidemiology of the disease in the country is essentially important in strategizing the available methods to effectively control the disease. Keeping this as the background, the present study was undertaken to estimate the pooled prevalence of bovine babesiosis in India. The relevant literature pertaining to bovine babesiosis was identified and a total of 49 studies published between 1983 and 2018 were included in the final systematic review and meta-analysis. Meta-analysis was conducted using meta-package of R software and prevalence estimates were calculated. Bovine babesiosis was reported from 21 states of India with pooled prevalence estimate of 6% (95% CI = 4%–9%) using random effect model. Zone wise analysis revealed highest pooled prevalence in the west zone and north zone (8%) followed by east zone (7%), central zone (6%), south zone (4%) and northeast zone (4%). The results of meta-analysis indicated high variability between studies. In addition, the pooled seroprevalence was high (29%) compared to prevalence of active infection (5%) of bovine babesiosis in India. Further, the pooled prevalence estimate of B. bigemina infection in India was more (7%) compared to B. bovis infection (1%). The estimation of prevalence of active infection and seroprevalence separately will helps to understand the actual disease prevalence in the country. The study indicated the wide prevalence of bovine babesiosis in India which urges for immediate mitigation strategies.

Incidences of Rhipicephalus (Boophilus) microplus (Canestrini, 1888) Transmitted Pathogens in Cattle in West Africa

Context and PurposeIn a context of recent introduction of Rhipicephalus (Boophilus) microplus tick species in West Africa, the purpose of the authors is to estimate incidence density of cattle babesiosis either caused by Babesia bigemina or Babesia bovis, and cattle anaplasmosis. Incidence density represents how quickly a disease or a condition is occurring amongst a group of individuals at risk.MethodsThe longitudinal and prospective study design took place in south, centre, east, west and north of Côte d’Ivoire. Cattle have been followed for 12 months. At the end of each month, each animal has been RT-PCR tested for new infection by Babesia bovis, Babesia bigemina, and PCR–RFLP tested for new infection by Anaplasma marginale.ResultsFindings show for the study area that incidence densities of Babesia bovis, Babesia bigemina and Anaplasma marginale infections in Côte d’Ivoire are, respectively, 15.3 new infections [95% CI 13.1–17.88] per 100 cattle, 32.2 new infections [95% CI 28.5–36.3] per 100 cattle, and 25.9 new infections [95% CI 22.5–29.6] per 100 cattle.ConclusionFinally, there is increasing of infection incidence density following the region distance from the coast or elevation.

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

BackgroundAmong 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.MethodsA 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.ResultsA 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.ConclusionThese 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.

Molecular survey of cattle ticks in Burundi: First report on the presence of the invasive Rhipicephalus microplus tick.

A recent research study on prevalence of tick-borne pathogens in Burundi reported high prevalence and endemicity of Theileria parva, Anaplasma marginale and Babesia bigemina infections in cattle. Detailed information about tick species infesting animals, their distribution and genetic diversity in Burundi is outdated and limited. This study therefore assessed the prevalence and genetic diversity of tick species infesting cattle across agroecological zones (AEZs) in Burundi. A cross-sectional study on the occurrence of tick species was conducted in 24 districts of Burundi between October and December 2017. Differential identification and characterization of ticks collected was conducted using tick morphological keys and molecular tools (cox1 and 12S rRNA gene). Chi-square test was used to test for association between agroecological zones and the prevalence of tick species. Phylogenetic relationships were inferred using bayesian and maximum likelihood algorithms. A total of 483 ticks were collected from the five AEZs sampled. Six tick species comprising of Rhipicephalus appendiculatus, R. sanguineus, R. evertsi evertsi, R. microplus, R. decoloratus and Amblyomma variegatum were observed. Rhipicephalus appendiculatus were the most prevalent ticks (~45%). A total of 138 specimens (28%) were found to be Rhipicephalus microplus, suggesting an emerging threat for cattle farmers. Twelve R. appendiculatus cox1 haplotypes were obtained from 106 specimens that were sequenced. Two cox1 haplotypes of R. microplus which clustered into previously reported Clade A were observed. Rhipicephalus sanguineus and R. evertsi evertsi ticks, the vectors of numerous zoonotic pathogens, were collected from cattle, which constitute a high risk for public health. These findings reveal an overlapping distribution of tick vectors in Burundi. The design of ticks and tick-borne diseases control strategies should consider the distribution of different vectors across the AEZs particularly the presence of the highly invasive R. microplus tick in Burundi and the potential risk of introducing the pathogenic Babesia bovis.

Cattle anaplasmosis and babesiosis: Major tick-borne diseases affecting the cattle industry in Khartoum State, Sudan.

Tick-borne hemoparasitic (TBH) infections are a major problem affecting livestock industries worldwide, particularly in tropical and subtropical regions. This study was carried out in response to repeated reports from local veterinarians in Khartoum State, Sudan, where TBH infections are prevalent in dairy farms. This cross-sectional study was undertaken from October 2017 to April 2018 with the objective of assessing the prevalence and potential risk factors associated with cattle anaplasmosis and babesiosis in the localities of Omdurman, Khartoum, and Khartoum North, Khartoum State. A total of 292 cattle blood samples collected from apparently healthy animals were examined for the presence of A. marginale, Babesia bigemina, and B. bovis using PCR. The overall prevalence of A. marginale and B. bigemina was found to be 40.41% and 3.42%, respectively, while B. bovis was not detected. Mixed infections with A. marginale and B. bigemina were detected in four (1.37%) cattle. The prevalence of the two pathogens was found to be significantly higher in Khartoum and Omdurman than in Khartoum North. However, no significant difference was observed for the prevalence based on sex, age, breed, and mean packed cell volume values. Our findings indicated that A. marginale is a highly prevalent parasite in Khartoum State, which may be a primary constraint to the cattle industry. Inclusion of this pathogen in the diagnostic protocols, and consequent treatment and tick control are necessary. Moreover, the role of B. bigemina infection may exacerbate the situation to some extent in this region.

Evaluation of cardiovascular biomarkers and histopathological alterations in cattle naturally infected by Babesia bigemina

Hemolytic anemia and secondary hypoxia are characteristics of naturally occurring Babesia bigemina infection in cattle. The anemic phase comes with cardiovascular insufficiency due to hypoxia-induced system dysfunction; but to date there is no description of cardiac damage in the infected animals. Therefore, this study was undertaken to investigate hematological parameters, biomarkers of cardiac function and D-dimer in 13 cattle infected with B. bigemina which were unresponsive to standard treatment. The animals were necropsied and the cardiac tissue was examined for histopathologic alterations. A significant parasitemia burden-dependent increase in the cardiac biomarkers and D-dimer level were recorded in the infected cattle compared to the control animals. Thrombocytes count was also significantly lower in the infected animals than the control. Both macroscopic and microscopic hemorrhage, mononuclear infiltrates, and myocardial necrosis were the evident histopathologic findings. These findings suggest that B. bigemina infection can potentially induce cardiac dysfunction in cattle. Furthermore, mechanistic studies should be conducted to understand the mechanisms beyond cardiac complications.

Comparative evaluation of Babesia bigemina truncated C-terminal rhoptry associated protein-1 and 200 kDa merozoite protein in indirect enzyme-linked immunosorbent assay

Babesia bigemina is an intra-erythrocytic apicomplexan protozoon which causes an acute as well as chronic disease in cattle and is transmitted by ixodid ticks throughout the world. Due to low sensitivity of microscopy for detection of the parasite, there is a need for developing effective diagnostic tests that can be used to identify carrier animals in endemic areas. In the present study, C-terminal fragment of rhoptry associated protein-1 (RAP-1/CT) and 200 kDa merozoite protein (P200/CT) of B. bigemina were cloned into pET-32a(+) expression vector and expressed in Escherichia coli as thioredoxin-fusion proteins for use in an indirect ELISA. The rRAP-1/CT and rP200/CT showed no cross reactivity with plasma from cattle infected with other common parasites namely Theileria annulata, Trypanosoma evansi, Cryptosporidium parvum and Anaplasma marginale in the standardized ELISA. Examination of 116 blood samples collected from cattle suspected for haemoprotozoan infections revealed that 17 (14.6%), 46 (39.6%), 52 (44.8%) and 53 (45.7%) were positive for B. bigemina by microscopy, nested PCR, rRAP-1/CT based and rP200/CT based indirect ELISA, respectively. The diagnostic sensitivities of rRAP-1/CT and rP200/CT indirect ELISAs were 97.8% and 91.3%, while the diagnostic specificities were 90% and 84.3%, respectively, when nested PCR was taken as a reference test. An almost perfect agreement (Kappa value -0.859) between rRAP-1/CT ELISA and nested PCR results, and a substantial agreement (Kappa value -0.737) between rP200/CT ELISA and nested PCR were noticed. The findings of the present study suggest that rRAP-1/CT is a better diagnostic candidate antigen than rP200/CT for diagnosis of B. bigemina infection and it may be used in an ELISA for surveillance or diagnosis of B. bigemina infection in bovines.

Semi-quantitative evaluation of Babesia bovis and B. bigemina infection levels estimated by HRM analysis

Bovine babesiosis is economically the most important arthropod-borne disease of cattle worldwide. The most significant damage caused by bovine babesiosis is attributed to Babesia bovis due to its higher pathogenicity. This study aimed to develop a real-time PCR method followed by HRM (high-resolution melting) analysis for the simultaneous detection of B. bovis and B. bigemina, enabling a semi-quantitative analysis of Babesia levels using a single-tube reaction. The HRM was compared with real-time PCR using species-specific hydrolysis probes. The HRM analysis allowed to differentiate both Babesia species and was sensitive in the detection and differentiation of 10% for each Babesia species in the sample. Our results suggest the use of this method to estimate the prevalence of infections by B. bovis or B. bigemina as an alternative to the methods of absolute quantification by real-time PCR since it neither requires precise estimates of the number of DNA loads nor the construction of calibration curves. The simultaneous detection of the two Babesia species can be used to characterise the infection levels in cattle populations from different geographical regions, allowing a better control of these diseases.

Assessment of hepatic oxidative damage, paraoxonase-1 activity, and lipid profile in cattle naturally infected with Ba7besia bigemina.

Naturally occurring Babesia bigemina infection in cattle is associated with changes in the status of oxidative stress, trace elements, sialic acid, and cholinesterase activity in blood. However, to date there is no description of hepatic damage in the infected animals. More importantly, the majority of the above-mentioned causative factors are synthesized or stored in the liver. Therefore, this study was undertaken to evaluate biomarkers of hepatic function, paraoxonase-1 activity, and lipid profile in 13 cattle infected with B. bigemina which did not respond to standard treatment. The animals were necropsied and the histopathology of the liver and DNA damage of hepatocytes were examined. Blood analysis revealed a significant parasitemia burden-dependent increase in the activities of hepatic enzymes and total bilirubin and a decrease in albumin concentrations in the infected cattle compared to the control ones. Paraoxonase-1 activity was remarkably lower in the infected animals than the control. A significant decrease in the blood concentrations of total cholesterol, low density lipoprotein, and high density lipoprotein and a significant increase in the triglyceride concentration were observed in the infected animals. Severe oxidative damages were also recorded in the haptic tissue evidenced by significant alterations in the activities of antioxidant enzymes, suppression of total antioxidant capacity, and oxidation of biomolecules. Congestion of blood vessels, bile duct hyperplasia, and hepatocyte necrosis were the evident histopathologic findings. Our results revealed significant changes in the indices of liver function in the diseased cattle, leading to the conclusion that the parasite can potentially cause liver dysfunction.

Evaluation of some acute phase proteins in cattle naturally infected with Babesia bigemina

Bovine babesiosis due to Babesia bigemina infection induces systemic inflammation, evidenced by increased sialic acid (SA) levels and declined cholinesterase activity. The current study was undertaken to assess further indicators of the systemic inflammation in the naturally infected cattle. To this end, serum levels of some selected acute phase-proteins (APPs) including serum amyloid A (SAA), haptoglobin (Hp), ceruloplasmin (Cp), and fibrinogen were measured. Additionally, sensitivity and specificity of the APPs were calculated by receiver operating characteristic curve. The correlation among APPs, SA and cholinesterase activity were also assessed. Our previous blood specimens were used to measure APPs. Briefly, the diseased animals were divided into two groups according to the parasitemia: 12 mildly (20 % <) and 8 severely (20 %>) infected animals. Moreover, 10 healthy animals as the control were included. The levels of all APPs were measured to be significantly elevated in a parasitemia burden-dependent fashion as compared to the control. Furthermore, all the APPs showed 100 % specificity, but only SAA and Cp had 100 % sensitivity. A strong and positive correlation was calculated between the APPs and SA; however, cholinesterase activity was inversely correlated with AAPs and SA. In conclusion, inflammatory reactions play a pivotal role in the pathogenesis of bovine babesiosis and APPs can be considered as the potential indicators of inflammation.