High serological and molecular prevalence of Ehrlichia canis and other vector-borne pathogens in dogs from Boa Vista Island, Cape Verde.

Despite the high global impacts of canine vector-borne diseases (CVBD) due to their wide distribution and zoonotic potential, the current epidemiological situation of CVBD in many tropical and subtropical regions remains unknown. This study examines the seroprevalence and molecular prevalence of Ehrlichia canis and other pathogens causing CVBDs (Leishmania infantum, Dirofilaria immitis, Babesia spp., Anaplasma spp. and Hepatozoon canis) in dogs living on the island of Boa Vista (Cape Verde Republic). Blood samples and infesting ticks were taken from 150 dogs across the island (stray, shelter, and pet dogs). Serum samples were tested using a rapid immunochromatographic test (Uranotest® Quattro) that detects antibodies against E. canis, L. infantum, Anaplasma spp. and D. immitis antigen. Levels of serum antibodies against E. canis were measured using the immunofluorescence antibody test (IFAT). In addition, tick-borne pathogens in blood samples (Anaplasma spp., Babesia spp., Hepatozoon spp., and Ehrlichia canis) were detected by microscopy observation and/or PCR plus sequencing. The seroprevalence of E. canis was extremely high at 82% (123/150), as revealed by both immunochromatography and IFAT. Most dogs returning a seropositive test result (82.92%; 102/123) had antibody titres > 1:1280 but showed no clinical signs or notable laboratory abnormalities. Of the 123 animals testing seropositive for E. canis, 67 (54.47%) also presented antibodies against Anaplasma spp., and 13 (10.56%) showed the presence of Hepatozoon spp. gamonts in the blood smear. Ehrlichia canis infection was detected in 17.1% (25/146) of dogs tested by direct sequencing of polymerase chain reaction (PCR) products. Co-infections were detected in seven of these dogs: four dogs tested PCR-positive for both E. canis and A. platys, two dogs tested positive for E. canis and Hepatozoon spp., and one dog tested positive for E. canis, A. platys and Hepatozoon spp. Rhipicephalus sanguineus sensu lato was the only tick species found infesting the canine study population. The high prevalence of tick-borne pathogens detected in dogs from Boa Vista Island highlights a need for improved control measures designed to prevent the transmission of these pathogens.

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

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

Tick-Borne pathogens and defensin genes expression: A closer look at Ixodes ricinus and Dermacentor reticulatus.

The immune system of ticks, along with that of other invertebrates, is comparatively simpler than that of vertebrates, relying solely on innate immune responses. Direct antimicrobial defence is provided by the synthesis of antimicrobial peptides (AMPs), including defensins. The aim of this study was to investigate the differences in defensin genes expression between questing and engorged Ixodes ricinus (def1 and def2) and Dermacentor reticulatus (defDr) ticks, in the presence of selected pathogens: Borrelia spp., Rickettsia spp., Babesia spp., Anaplasma phagocytophilum, and Neoehrlichia mikurensis in the natural environment. After pathogen screening by PCR/qPCR, the expression of defensin genes in pathogen positive ticks and ticks without any of the tested pathogens, was analysed by reverse transcription qPCR. The results showed an increased expression of defensin genes in I. ricinus ticks after blood feeding and I. ricinus and D. reticulatus ticks during in cases of co-infection. In particular, the expression of defensins genes was higher in questing D. reticulatus than in questing and engorged I. ricinus ticks, when borreliae were detected. This study contributes to uncovering the expression patterns of defensin genes in the presence of several known tick pathogens, the occurrence of these pathogens and possible regulatory mechanisms of defensins in tick vector competence.

A small proportion of Zebu genetic background in crossbred calves may not be enough to improve resistance against natural bovine Babesia spp. infections.

The main objective of cattle breeders in tropical and subtropical regions is to acquire animals with taurine-productive traits adapted to the broad weather range of these regions. However, one of the main challenges on using taurine genetics in these areas is the high susceptibility of these animals to tick-borne diseases. Consequently, the present study evaluated from 10 November 2021-19 April 2022, the over 13 assessments, the Babesia bovis and Babesia bigemina DNA loads and the IgG anti-B. bovis and anti-B. bigemina levels in Angus (n = 17, 100% Taurine) and Ultrablack (n = 14, ∼82% taurine and 18% Zebu) calves. Data were analyzed using a multivariate mixed model with repeated measures of the same animal including the fixed effects of evaluation, genetic group, sex, Babesia spp., and their interactions. The repeatability values were estimated from the (co)variances matrix and expressed for each species. The correlations between the DNA loads (CNlog) and IgG titers (S/P) values for the two species were also estimated using the same model. Regarding the specific IgG antibody titers for both Babesia spp., no significant differences were observed between the two genetic groups. However, for B. bovis and B. bigemina DNA loads, Ultrablack calves presented significantly higher values than Angus calves. Under the conditions evaluated in this study, our findings suggest that the low percentage of Zebu genetic in the Ultrablack breed was insufficient to improve resistance against babesiosis. Further studies must demonstrate if the low percentages of Zebu genetics in Taurine breeds can modify the susceptibility to babesiosis infections.

Development of a Test Card Based on Colloidal Gold Immunochromatographic Strips for Rapid Detection of Antibodies against Theileria equi and Babesia caballi.

Equine piroplasmosis (EP) is a serious problem in the horse industry, and controlling EP is critical for international horse trading. EP is caused by two apicomplexan protozoan parasites, Theileria equi and Babesia caballi. Rapid and accurate methods that are suitable for detecting these parasites in the field are crucial to control the infection and spread of EP. In this study, we developed a card to detect antibodies against T. equi and B. caballi based on two colloidal gold immunochromatographic strips according to the principle of the double-antigen sandwich. The proteins equi merozoite antigen 1 (EMA1) and rhoptry protein BC48 are commonly used as diagnostic antigens against T. equi and B. caballi, respectively. On the strip, the purified EMA1 or BC48 protein labeled with colloidal gold was used as the detector, and nitrocellulose membranes were coated with EMA1 or BC48 and the corresponding MAb as the test and control lines, respectively. The protocol takes 10 to 15 min and requires no specialized equipment or chemical reagents, and one test can detect two EP pathogens in one card. Specificity tests confirmed there was no cross-reactivity with sera positive for common equine pathogens. Using a commercial competitive enzyme-linked immunosorbent assay (cELISA) kit for comparison, 476 clinical samples were tested with the card. The coincidence rates were 96.43% and 97.90% for T. equi and B. caballi, respectively. The field trial feedback was uniformly positive, suggesting that this diagnostic tool may be useful for controlling the spread of T. equi and B. caballi. IMPORTANCE Equine piroplasmosis (EP), caused by Theileria equi and Babesia caballi, is an important tick-borne disease of equines that is prevalent in most parts of the world. EP is considered a reportable disease by the World Organization for Animal Health (OIE). The accurate diagnosis and differentiation of T. equi and B. caballi are very important for the prevention, control, and treatment of EP. Therefore, we developed a double-antigen sandwich colloidal gold immunochromatography assay (GICG) to detect T. equi and B. caballi. Two GICG strips were assembled side by side on one card for the detection of T. equi and B. caballi, and the two EP pathogens could be detected in one test. This method was simple, rapid, and specific for the detection of EP; therefore, compared to the previous methods, this method is more suitable for pathogen diagnosis in the field.

Seroprevalence and prevalence of Babesia vogeli in clinically healthy dogs and their ticks in Costa Rica.

Canine babesiosis is a disease caused by a parasite of the genus Babesia which destroys red blood cells. Previous studies have shown the presence of Babesia vogeli in rural areas in Costa Rica using molecular techniques. The objective of the present study was to determine the seroprevalence and prevalence of B. vogeli in clinically healthy dogs and their ticks at the national level, both within and outside the Central Valley. Blood samples and ticks from 482 dogs were collected between June 2011 and May 2014, and analyzed by immunofluorescence assay (IFA) and real-time polymerase chain reaction (qPCR); two protocols of endpoint PCR and sequencing were used to confirm qPCR-positive samples. Seroprevalence of canine babesiosis of 5.3% (24/453) was determined at the national level, specifically 2.0% (5/253) within and 9.5% (19/200) outside the Central Valley, respectively. Real-time PCR determined a global prevalence of B. vogeli of 31.3% (125/400): 21.4% (47/220) within the Central Valley and 43.3% (78/180) outside the Central Valley. The endpoint PCR amplified only 10 of the 125 blood samples identified as positive in qPCR. One sample amplified by endpoint PCR was sequenced and identified as B. vogeli. Twelve canines were identified with past infections, seven canines with active infection, and 111 canines with early infection. Two species of ticks were found with B. vogeli: Rhipicephalus sanguineus sensu lato (n = 40) and Amblyomma ovale (n = 1). The prevalence of canine babesiosis at the national level, both within and outside the Central Valley, is reported here for the first time, determining the presence of the piroplasmid throughout the country, with a higher circulation of the agent outside the Central Valley. Only one species, B. vogeli, was detected in the blood of dogs and their ticks. Therefore, veterinarians should consider using qPCR to determine the presence of the parasite in blood donors and before starting treatment of vector-borne disease in dogs.

Identification of a novel variant erythrocyte surface antigen-1 (VESA1) in Babesia orientalis.

Babesia orientalis, belonging to the phylum Apicomplexa, is mainly accountable for water buffalo babesiosis, which adversely affected the livestock industry in China. Variant erythrocyte surface antigen-1 (VESA1), an antigen that helps infected erythrocytes to escape from host immune responses, was first reported in Babesia bovis. Various VESA1 proteins have also been characterized in other Babesia species. Nevertheless, there is no research on the identification and characterization of VESA1 proteins in Babesia orientalis. In this study, the BoVESA1 gene was amplified from both gDNA and cDNA. The results revealed that it is an intronless gene with a full length of 753 bp, encoding a protein of 250 amino acids with a predicted molecular weight of 28 kDa. The coding sequence (CDS) was cloned into the pGEX-6p-1 vector using a homologous recombination kit and expressed as a glutathione-S-transferase (GST)-fusion protein with a molecular weight of 53 kDa. The tertiary structure of BoVESA1 was predicted using the I-TASSER software. The recombinant protein was subjected to western blotting; the immunogenicity of recombinant BoVESA1 (rBoVESA1) was identified by incubating it with B. orientalis-positive serum. The native BoVESA1 was identified using the lysates of B. orientalis-infected water buffalo erythrocytes incubated with the anti-rBoVESA1 mouse serum. The results showed a band of ~ 28 kDa, which is similar to the predicted size. Immunofluorescence assay (IFA) using anti-rBoVESA1 serum probed indicated a strong signal in the infected RBCs, while the negative control showed no signal. In conclusion, the VESA1 protein was first identified in B. orientalis. This study facilitated further investigation of B. orientalis, and the results indicated that BoVESA1 may serve as a potential candidate antigen for diagnosis and detection of B. orientalis infection.

Molecular epidemiology and prevalence of babesial infections in dogs in two hyperendemic foci in Brazil.

Babesial parasites are some of the most ubiquitous blood pathogens and consequently have considerable worldwide veterinary impact. Dogs living in the tropics are highly exposed to babesial parasites, particularly to Babesia vogeli. Limited data on the seroprevalence and molecular prevalence of Babesia spp. in dogs are available in Latin America. We conducted a cross-sectional study combining serological and molecular tests to estimate the seroprevalence and molecular epidemiology of Babesia spp. infections in dogs in two hyperendemic foci in Brazil. A total of 630 privately owned dogs (417 from Goiana municipality, Pernambuco state, north-eastern Brazil, and 213 from São Joaquim de Bicas municipality, Minas Gerais state, south-eastern Brazil) were sampled and molecularly and serologically tested for Babesia spp. Overall, 519 dogs (82.4%) presented detectable IgG antibodies against Babesia spp., and seropositivity was significantly higher in dogs older than 1 year. Molecularly, 34 dogs (5.4%) were positive for a ~ 200 bp fragment of the 18S rRNA gene of Babesia spp. and 88 (14.0%) for a longer fragment (~ 450 bp) of the same gene of Babesia spp. and other protozoa. The 18S rRNA gene sequences generated herein corresponded to B. vogeli (n = 52) or Hepatozoon canis (n = 20). This study confirms a high level of exposure to B. vogeli in two areas of Brazil and highlights that most of the dogs living in these areas are infected during the course of their life, reflected by increased seroprevalence in older dogs. Increased awareness and prevention of tick-borne protozoa infections in dogs from Brazil and Latin America are urgently needed.

Prevalence of serum antibodies of tick-borne diseases and the presence of Rhipicephalus microplus in communal grazing cattle in the north-eastern region of the Eastern Cape Province of South Africa.

A survey to determine the prevalence of tick-borne diseases (TBDs) and ticks infesting cattle was conducted in the communal areas of the north-eastern region of the Eastern Cape Province (ECP) between January 2019 and October 2019. Tick counts, packed cell volume (PCV), body condition scores (BCS), and serological test for TBDs were seasonally conducted in communally grazed cattle (n = 240) in Joe Gqabi district in two local municipalities (Elundini and Senqu). A standard indirect fluorescent antibody test (IFAT) was used to determine antibodies to Babesia bigemina, Babesia bovis, and Ehrlichia ruminantium and indirect enzyme-linked immunosorbent assay (ELISA) was employed for antibodies to Anaplasma marginale detection. The highest tick loads were observed on cattle during the hot-wet and post-rainy seasons and lowest during the cool-dry season. The E. ruminantium prevalence in Elundini was 16% and 14% in post-rainy and hot-dry seasons respectively and 15% at Senqu during the post-rainy season. B. bigemina prevalence was highest at Elundini (18%) and Senqu (16%) during the post-rainy season and hot-wet season respectively. Cattle BCS was negatively correlated with E. ruminantium (P < 0.01; r = - 0.203), B. bovis (P < 0.01; r = - 0.125), and A. marginale (P < 0.01; r = - 0.122) seroprevalence. The PCV was negatively correlated with B. bigemina (P < 0.01; r = - 0.138) seroprevalence. On the other hand, E. ruminantium was positively correlated with Amblyomma hebraeum (P < 0.05; r = 0.112) infestation, while B. bovis (P < 0.05; r = 0.134) and B. bigemina (P < 0.05; r = 0.188) were positively correlated with Rhipicephalus microplus infestation, and B. bigemina (P < 0.05; r = 0.077) was positively correlated with Rhipicephalus decoloratus infestation. Our study reports for the first time the presence of R. microplus in the study area. Further research is, however, needed to better understand seroprevalence and the transmission mode of TBDs to cattle so that effective disease control measures can be developed.

Prevalence of tick-transmitted pathogens in cattle reveals that Theileria parva, Babesia bigemina and Anaplasma marginale are endemic in Burundi.

BACKGROUND: Tick-borne diseases (TBDs) constitute a major constraint for livestock development in sub-Saharan Africa, with East Coast fever (ECF) being the most devastating TBD of cattle. However, in Burundi, detailed information is lacking on the current prevalence of TBDs and on the associated economic losses from mortality and morbidity in cattle as well as the costs associated with TBD control and treatment. The aim of this study was, therefore, to assess the prevalence and spatial distribution of tick-borne pathogens (TBPs) in cattle across the major agro-ecological zones (AEZs) in Burundi. METHODS: In a cross-sectional study conducted in ten communes spanning the five main AEZs in Burundi, blood samples were taken from 828 cattle from 305 farms between October and December 2017. Evidence of Theileria parva infection was assessed by antibody level, measured using a polymorphic immunodominant molecule (PIM) antigen-based enzyme-linked immunosorbent assay (ELISA) and by a T. parva-specific p104 gene-based nested PCR. Antibodies against Theileria mutans infection were detected using the 32-kDa antigen-based indirect ELISA, while the 200-kDa antigen and the major surface protein 5 (MSP5)-based indirect ELISA were used to detect antibodies against Babesia bigemina and Anaplasma marginale, respectively. RESULTS: The prevalence of T. parva across the ten communes sampled ranged from 77.5 to 93.1% and from 67.8 to 90.0% based on the ELISA and PCR analysis, respectively. A statistically significant difference in infection was observed between calves and adult cattle; however, T. parva infection levels were not significantly associated with sex and breed. The seroprevalence indicating exposure to T. mutans, B. bigemina and A. marginale ranged from 30 to 92.1%, 33.7 to 90% and 50 to 96.2%, respectively. Mixed infections of TBPs were detected in 82.91% of cattle sampled, with 11 different combinations of pathogen species detected . CONCLUSIONS: The findings indicate that T. parva, A. marginale and B. bigemina infections are endemic in Burundi. Knowledge of the spatial distribution of TBPs will facilitate the design of effective targeted strategies to control these diseases. There is a need for further investigations of the distribution of tick vectors and the population structure of TBPs in order to identify the key epidemiological factors contributing to TBD outbreaks in Burundi.

Identification and characterization of a Babesia bigemina thrombospondin-related superfamily member, TRAP-1: a novel antigen containing neutralizing epitopes involved in merozoite invasion.

BACKGROUND: Thrombospondin-related anonymous protein (TRAP) has been described as a potential vaccine candidate for several diseases caused by apicomplexan parasites. However, this protein and members of this family have not yet been characterized in Babesia bigemina, one of the most prevalent species causing bovine babesiosis. METHODS: The 3186-bp Babesia bigemina TRAP-1 (BbiTRAP-1) gene was identified by a bioinformatics search using the B. bovis TRAP-1 sequence. Members of the TRAP and TRAP-related protein families (TRP) were identified in Babesia and Theileria through a search of the TSP-1 adhesive domain, which is the hallmark motif in both proteins. Structural modeling and phylogenetic analysis were performed with the identified TRAP proteins. A truncated recombinant BbiTRAP-1 that migrates at approximately 107 kDa and specific antisera were produced and used in Western blot analysis and indirect fluorescent antibody tests (IFAT). B-cell epitopes with neutralizing activity in BbiTRAP-1 were defined by enzyme-linked immunosorbent assays (ELISA) and invasion assays. RESULTS: Three members of the TRAP family of proteins were identified in B. bigemina (BbiTRAP-1 to -3). All are type 1 transmembrane proteins containing the von Willebrand factor A (vWFA), thrombospondin type 1 (TSP-1), and cytoplasmic C-terminus domains, as well as transmembrane regions. The BbiTRAP-1 predicted structure also contains a metal ion-dependent adhesion site for interaction with the host cell. The TRP family in Babesia and Theileria species contains the canonical TSP-1 domain but lacks the vWFA domain and together with TRAP define a novel gene superfamily. A variable number of tandem repeat units are present in BbiTRAP-1 and could be used for strain genotyping. Western blot and IFAT analysis confirmed the expression of BbiTRAP-1 by blood-stage parasites. Partial recognition by a panel of sera from B. bigemina-infected cattle in ELISAs using truncated BbiTRAP-1 suggests that this protein is not an immunodominant antigen. Additionally, bovine anti-recombinant BbiTRAP-1 antibodies were found to be capable of neutralizing merozoite invasion in vitro. CONCLUSIONS: We have identified the TRAP and TRP gene families in several Babesia and Theileria species and characterized BbiTRAP-1 as a novel antigen of B. bigemina. The functional relevance and presence of neutralization-sensitive B-cell epitopes suggest that BbiTRAP-1 could be included in tests for future vaccine candidates against B. bigemina.

Seroprevalence and risk factors associated with Equine piroplasmosis in North Egypt.

Equine piroplasmosis (EP) is caused by Theileria equi and/or Babesia caballi and has economic importance particularly in equines reared in poor management systems. This study is based on cELISA test to study the seroprevalence of EP among 370 horses and 150 donkeys in four Governorates north Egypt. Additionally, its risk factors were studied for the first time. The seroprevalence rates 36.5 %, 20 %, and 5.6 % for T. equi, B. caballi, and mixed infections, respectively. The highest antibody levels against T. equi were detected in Kafr ElSheikh (40 %) and Giza (40.1 %) Governorates, whereas those of B. caballi were detected in Qalyubia (25 %) and Kafr ElSheikh (24.1 %) Governorates. Concerning T. equi, animals >10 years (OR = 2.06) were more likely to be infected with EP than those <5 years old. In addition, the seropositivity increased among grazing (OR = 5.7, 95 % CI: 1.73-19.27) males (OR = 1.8, 95 % CI: 1.23-2.61) infested with ticks (OR = 2.3, 95 % CI: 1.60-3.48) during summer (OR = 4.3, 95 %CI: 2.53-7.46); whereas the seropositivity of animals for B. caballi increased among grazing equines (OR = 7.8, 95 % CI: 1.05-58.25) over 10 years old (OR = 2.08, 95 % CI: 1.10-3.94) and infested with ticks (OR = 2.4, 95 % CI: 1.54-3.76) during summer (OR = 7.12, 95 % CI: 3.15-16.06). Therefore, EP is an important prevalent disease in Egypt and deserves further attention regarding the management system, treatment, and vector control.

Characterization of the variable merozoite surface antigen (VMSA) gene family of Babesia orientalis.

Due to its wide presence in apicomplexan parasites as well as high polymorphism and antigenic diversity, the variable merozoite surface antigen (VMSA) family in Babesia sp. has attracted increasing attention of researchers. Here, all the reported VMSA genes of Babesia spp. were obtained from GenBank, and multiple alignments were performed by using conserved regions to blast the Babesia orientalis genome database (unpublished data). Five MSA genes (named MSA-2a1, MSA-2a2, MSA-2c1, MSA-1, and MSA-2c2, respectively) were identified, sequenced, and cloned from B. orientalis, which were shown to encode proteins with open reading frames ranging in size from 266 (MSA-2c1) to 317 (MSA-1) amino acids. All the five proteins contain an MSA-2c superfamily conserved domain, with an identical signal peptide and glycosyl phosphatidyl inositol (GPI)-anchor for each of them. The five proteins were also predicted to contain B cell epitopes, with only three for BoMSA-2c1, the smallest protein in the BoVMSA family, while at least six for each of the others. Notably, BoMSA-2a1 has 2 identical copies, a specific phenomenon only present in B. orientalis. This research has determined the MSA genes of B. orientalis and provides a genetic basis for further research of functional genes in B. orientalis.

Identification and antigenicity of the Babesia caballi spherical body protein 4 (SBP4).

BACKGROUND: The tick-borne intra-erythrocytic apicomplexan Babesia caballi is one of the etiological agents of equine babesiosis, an economically important disease of equids in most tropical and subtropical areas of the world. Discovering candidate antigens for improved diagnostic tools and vaccines remains needed for controlling equine babesiosis. This study describes the B. caballi sbp4 (Bcsbp4) gene and protein (BcSBP4) and analyzes its antigenicity in infected equids. METHODS: BLAST searches of an uncurated B. caballi assembly genome using the B. bovis SBP4 as a query were carried out, followed by PCR amplification and sequencing of a newly identified BcSBP4. Characterization of this novel gene and protein was performed by bioinformatics analysis, western blots, immunofluorescence (IFA) and an in vitro neutralization test using anti SBP4 peptide antibodies. Antigenicity of recombinant BcSBP4 (rBcSBP4) was tested with sera from field animals (n = 18) using an indirect ELISA (iELISA). RESULTS: Babesia caballi genome searches using B. bovis SBP4 as a query allowed identification of a novel gene termed Bcsbp4. The Bcsbp4 gene encodes for a protein of 30.58 kDa, which is fully conserved among B. caballi isolates from USA and Egypt. Bioinformatics analysis indicates that BcSBP4 contains a signal peptide and lacks additional transmembrane domains. Expression of BcSBP4 in blood stages of B. caballi was confirmed by western blot and IFA using antibodies against synthetic peptides representing putative B-cell epitopes of BcSBP4 predicted by in silico analysis. In vitro neutralization tests using anti-BcSBP4 peptide antibodies showed a marginal, but statistically significant inhibitory effect on the infectivity of B. caballi merozoites in horse red blood cells. Sera from eight B. caballi-infected equids, but none out of ten negative equid control sera, gave a positive signal in an rBcSBP4 based iELISA. CONCLUSIONS: The Bcsbp4 gene is expressed in B. caballi blood stages. The BcSBP4 protein is a potential candidate for developing a novel serological test that could detect B. caballi infection in equids in tropical and subtropical countries worldwide.

A novel 53 kDa protein (BoP53) in Babesia orientalis poses the immunoreactivity using the infection serum.

Babesia orientalis (B. orientalis) is responsible for water buffalo babesiosis, which caused serious economic losses in the south of China. Although the invasion process has been roughly described, there are still some unknown molecules that have not yet been identified. Recently, an invasion-related protein BOV57 has been identified in the Babesia bovis. However, there is no report available about the gene in B. orientalis. B. orientalis P53 (BoP53) sequence was obtained by blast BOV57 sequence in B. orientalis genome database, and the full length of the BoP53 gene is 1599 bp. BoP53 gene was cloned into a pGEX-6P-1 expression vector and expressed as a GST-tag fusion protein. The tertiary structure of BoP53 was predicted with the I-TASSER software. The native BoP53 was identified from of B. orientalis lysate incubation with mouse antiserum against rBoP53. BoP53 as a novel identified protein promotes the study of B. orientalis, the reaction of rBoP53 with the serum of B. orientalis-infected water buffalo but not with healthy buffalo serum indicated its good antigenicity. It may be a candidate antigen for the diagnosis of B. orientalis infection.

Don't let sleeping dogs lie: unravelling the identity and taxonomy of Babesia canis, Babesia rossi and Babesia vogeli.

For most of the 20th century the causative agent of canine babesiosis, wherever it occurred in the world, was commonly referred to as Babesia canis. Early research, from the 1890s to the 1930s, had shown that there were three distinctly different vector-specific parasite entities occurring in specific geographical regions, that host response to infection ranged from subclinical to acute, and that immunity to one stock of the parasite did not necessarily protect against infection with other stocks. This substantial body of knowledge was overlooked or ignored for 50 years. In this review the first records and descriptions of the disease in four geographical regions were traced: sub-Saharan Africa, Europe, North Africa and Asia. Research leading to identification of the specific tick vector species involved is documented. Evidence is given of the growing realisation that there were substantial biological differences between stocks originating from different geographical regions. Etymological provenance for Babesia vogeli is proposed.

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.

Comparison of protectiveness of recombinant Babesia ovis apical membrane antigen 1 and B. ovis-infected cell line as vaccines against ovine babesiosis.

Babesiosis is a disease complex caused by unicellular Babesia parasites and among them, malignant ovine babesiosis caused by B. ovis has a devastating economical impact on the small ruminant industry. The control of disease is mainly based on chemotherapy and preventing animals from tick infestation and to date no vaccine is available against ovine babesiosis. The requirement for vaccination against B. ovis infection in endemically unstable regions is necessary for implementation of effective disease control measures. The aim of the present study was to evaluate the effectiveness of different immunisation protocols against disease in sheep experimentally vaccinated with recombinant B. ovis apical membrane antigen-1 (rBoAMA-1) and/or live, a B. ovis-infected cell line. Sheep were divided into four experimental groups, plus a control group. Animals were immunised either with the B. ovis stabilate, or with rBoAMA-1, or with both rBoAMA-1 and the B. ovis stabilate. Western blots and ELISAs indicated that immunisation with rBoAMA-1 resulted in generation of a specific response against the recombinant protein, but the degree of antibody response did not correlate with the level of induced protection against challenge. The strongest immune response was induced in animals co-immunised with the live B. ovis stabilate plus rBoAMA-1. Both the hematological and parasitological findings indicated that this co-immunisation regimen has vaccine potential to limit losses incurred by ovine babesiosis in endemic countries.

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.