First molecular evidence of Babesia caballi and Theileria equi infections in horses in Cuba

Parasitology Research - Equine piroplasmosis is a disease of Equidae, including horses, donkeys, mules, and zebras, caused by either Theileria equi or Babesia caballi. This disease represents a...     

Diagnosis of Babesia caballi and Theileria equi infections in horses in Sudan using ELISA and PCR

The purpose of this study was to estimate the prevalence of equine piroplasmosis in Sudan. The presence of antibodies against Babesia caballi and Theileria equi was determined in serum samples obtained from 158 horses raised in different locations in Sudan by enzyme-linked immunosorbent assay (ELISA). The B. caballi 48-kDa and the T. equi EMA-2 purified recombinant proteins were used as antigens in the ELISA test. Results showed that seven (4.4%) were positive for B. caballi and 80 (63.5%) were positive for T. equi. Polymerase chain reaction (PCR) assays have been applied using primers targeting the B. caballi 48-kDa merozoite antigen, the T. equi SSUrRNA and the T. equi EMA-1 genes. PCR performed on 131 blood spots in filter paper revealed that 33 (25.2%) samples were positive for T. equi but no positives were found for B. caballi. It is concluded that equine piroplasmosis is endemic in the country. This is the first study on serological and molecular epidemiological diagnosis on equine piroplasmosis in Sudan.     

Prevalence of Theileria equi, Babesia caballi, and Anaplasma phagocytophilum in horses from the north of Portugal

Piroplasmid protozoa Theileria equi and Babesia caballi and zoonotic rickettsial bacterium Anaplasma phagocytophilum are important agents of equine vector-borne diseases (EVBD). This study aimed at investigating the prevalence of infections with or exposure to these pathogens in horses from the north of Portugal. Blood was randomly collected from 162 horses, living in 72 different stables, to prepare Giemsa-stained slide smears. Additionally, plasma samples were tested for antibodies to T. equi and B. caballi by two competitive enzyme-linked immunosorbent assays and to A. phagocytophilum by an indirect fluorescence antibody test. Five horses were positive to T. equi by microscopy (3.1 %), three to B. caballi (1.9 %), and none to A. phagocytophilum with no horse simultaneously positive for the two piroplasms. Clinically suspect animals had a significantly higher positivity to T. equi by microscopy in comparison with the nonsuspect ones (21.4 vs. 1.4 %). Twenty-nine horses were seropositive to T. equi (17.9 %), 18 to B. caballi (11.1 %), and 21 to A. phagocytophilum (13.0 %). Combined serology and microscopy positive results to T. equi and B. caballi were 19.1 and 11.7 %, respectively, with 33.3 % of the horses found positive to at least one agent. Forty horses were positive to single agents and 14 to more than one agent. An outdoor or mixed outdoor/indoor type of housing was found to be a risk factor for the combined positivity to T. equi. Infections with T. equi, B. caballi, and A. phagocytophilum are endemic in the north of Portugal. In addition to the treatment of positive horses, preventive measures should be put in practice to reduce exposure to and infection with agents of EVBD.     

Culture,isolation and propagation of Babesia caballi from naturally infected horses

Thirteen blood samples of horses from South Africa, five of which were seropositive for Babesia caballi and eight for both B. caballi and Theileria equi, were subjected to in vitro culture to identify carrier animals. None of the animals had a detectable parasitaemia on Giemsa-stained blood smears before culture initiation. Cultures were initiated in L-cysteine-enriched medium, either in an oxygen-reduced gas mixture or in a 5% CO2-in-air atmosphere. All five animals seropositive for B. caballi were identified as carrier animals using an oxygen-reduced atmosphere, whereas only four samples became culture positive under normal atmospheric conditions. Among the eight samples seropositive for both B. caballi and T. equi, two were identified as carriers for both. The remaining six samples were identified as carrying only T. equi.     

Phylogenetic analysis of Theileria equi and Babesia caballi sequences from thoroughbred mares and foals in Trinidad

Parasitology Research - The agents of equine piroplasmosis, Theileria equi and Babesia caballi, are endemic in Trinidad, West Indies. While transmission is mainly by ixodid ticks, transplacental...     

Detection of antibodies to Babesia equi in horses by a latex agglutination test using recombinant EMA-1

A latex agglutination test (LAT) using recombinant equi merozoite antigen 1 (EMA-1) for the detection of antibodies to Babesia equi was developed. The LAT was able to differentiate very clearly between sera from B. equi-infected horses and sera from Babesia caballi-infected horses or from normal horses. The LAT results were identical to those of a previously developed enzyme-linked immunosorbent assay. These results indicate that LAT using recombinant EMA-1 might be very useful as a routine screening method for the diagnosis of B. equi infection.     

Development of an immunochromatographic test with recombinant EMA-2 for the rapid detection of antibodies against Babesia equi in horses

An immunochromatographic test (BeICT) for the rapid detection of antibodies against Babesia equi was developed. It clearly differentiated B. equi-infected horses from B. caballi-infected and uninfected horses. The agreement with enzyme-linked immunosorbent assay results was 96.7% in the detection of field sera. The results suggest that BeICT is rapid, simple, reliable, and suitable for use to detect B. equi infection in the field.     

Rapid detection and identification of Theileria equi and Babesia caballi by high-resolution melting (HRM) analysis

The application of high-resolution melting (HRM) analysis in the differentiation between Theileria equi and Babesia caballi was evaluated using control samples from the United States Department of Agriculture and field samples collected from horses in Sudan and China. A region of the 18S rRNA gene, with four known nucleotide differences between the two parasites, was selected for primer design. HRM analysis successfully allowed the detection and differentiation of T. equi and B. caballi without the necessity of performing time-consuming and expensive post-PCR procedures such as sequencing or restriction digestion. Our results suggest that HRM could be an ideal method for rapid genotyping, which is required to determine a drug of choice or to administer an appropriate vaccine during an outbreak.     

Antibody to a recombinant merozoite protein epitope identifies horses infected with Babesia equi.

Horses infected with Babesia equi were previously identified by the presence of antibodies reactive with a merozoite surface protein epitope (D. P. Knowles, Jr., L. E. Perryman, L. S. Kappmeyer, and S. G. Hennager. J. Clin. Microbiol. 29:2056-2058, 1991). The antibodies were detected in a competitive inhibition enzyme-linked immunosorbent assay (CI ELISA) by using monoclonal antibody 36/133.97, which defines a protein epitope on the merozoite surface. The gene encoding this B. equi merozoite epitope was cloned and expressed in Escherichia coli. The recombinant merozoite protein, designated equi merozoite antigen 1 (EMA-1), was evaluated in the CI ELISA. Recombinant EMA-1 bound antibody from the sera of B. equi-infected horses from 18 countries. The antibody response to EMA-1 was then measured in horses experimentally infected with B. equi via transmission by the tick vector Boophilus microplus or by intravenous inoculation. Anti-EMA-1 antibody was detected 7 weeks post-tick exposure and remained, without reexposure to B. equi, for the 33 weeks of the evaluation period. The data indicate that recombinant EMA-1 can be used in the CI ELISA to detect horses infected with B. equi.     

Detection of Equine Antibodies to Babesia caballi by Recombinant B. caballi Rhoptry-Associated Protein 1 in a Competitive-Inhibition Enzyme-Linked Immunosorbent Assay

A competitive-inhibition enzyme-linked immunosorbent assay (cELISA) was developed for detection of equine antibodies specific for Babesia caballi. The assay used recombinant B. caballi rhoptry-associated protein 1 (RAP-1) and monoclonal antibody (MAb) 79/17.18.5, which is reactive with a peptide epitope of a native 60-kDa B. caballi antigen. The gene encoding the recombinant antigen was sequenced, and database analysis revealed that the gene product is a rhoptry-associated protein. Cloning and expression of a truncated copy of the gene demonstrated that MAb 79/17.18.5 reacts with the C-terminal repeat region of the protein. The cELISA was used to evaluate 302 equine serum samples previously tested for antibodies to B. caballi by a standardized complement fixation test (CFT). The results of cELISA and CFT were 73% concordant. Seventy-two of the 77 serum samples with discordant results were CFT negative and cELISA positive. Further evaluation of the serum samples with discordant results by indirect immunofluorescence assay (IFA) demonstrated that at a serum dilution of 1:200, 48 of the CFT-negative and cELISA-positive serum samples contained antibodies reactive with B. caballi RAP-1. Four of five CFT-positive and cELISA-negative serum samples contained antibodies reactive with B. caballi when they were tested by IFA. These data indicate that following infection with B. caballi, horses consistently produce antibody to the RAP-1 epitope defined by MAb 79/17.18.5, and when used in the cELISA format, recombinant RAP-1 is a useful antigen for the serologic detection of anti-B. caballi antibodies.     

Persistently infected horses are reservoirs for intrastadial tick-borne transmission of the apicomplexan parasite Babesia equi

Tick-borne pathogens may be transmitted intrastadially and transstadially within a single vector generation as well as vertically between generations. Understanding the mode and relative efficiency of this transmission is required for infection control. In this study, we established that adult male Rhipicephalus microplus ticks efficiently acquire the protozoal pathogen Babesia equi during acute and persistent infections and transmit it intrastadially to naïve horses. Although the level of parasitemia during acquisition feeding affected the efficiency of the initial tick infection, infected ticks developed levels of ≥10⁴ organisms/pair of salivary glands independent of the level of parasitemia during acquisition feeding and successfully transmitted them, indicating that replication within the tick compensated for any initial differences in infectious dose and exceeded the threshold for transmission. During the development of B. equi parasites in the salivary gland granular acini, the parasites expressed levels of paralogous surface proteins significantly different from those expressed by intraerythrocytic parasites from the mammalian host. In contrast to the successful intrastadial transmission, adult female R. microplus ticks that fed on horses with high parasitemia passed the parasite vertically into the eggs with low efficiency, and the subsequent generation (larvae, nymphs, and adults) failed to transmit B. equi parasites to naïve horses. The data demonstrated that intrastadial but not transovarial transmission is an efficient mode for B. equi transmission and that persistently infected horses are an important reservoir for transmission. Consequently, R. microplus male ticks and persistently infected horses should be targeted for disease control.     

Continuous in vitro cultivation of Babesia caballi in serum-free medium

Experiments were undertaken to develop a serum-free medium for the in vitro cultivation of Babesia caballi, a tick-borne hemoprotozoan parasite, one of the causative agents of equine piroplasmosis. A modified HL-1 medium supplemented with horse serum, L-glutamine, antibiotics, and hypoxanthine was used. B. caballi organisms were continuously cultivated at 37 °C in microaerophilous stationary-phase culture in a humidified atmosphere containing 5% CO₂ in air before exposure to serum-free culture conditions. For serum-free propagation, lipid-rich bovine serum albumin (LR-BSA), alone or with chemically defined lipids (CDL), were added instead of serum. Media containing LR-BSA alone or LR-BSA and CDL in various amounts supported the in vitro propagation of B. caballi. Growth was maintained for more than 6 months. The growth rates obtained in serum-free media were similar to those previously obtained in traditional serum-containing medium. Received: 30 June 1998 / Accepted: 29 September 1998     

Ultrastructural characteristics of Babesia caballi in equine erythrocytes in vitro

 Babesia caballi cultured continuously in equine erythrocytes was examined by transmission electron microscopy. The use of cultured B. caballi permitted examination of a large number of parasitized cells with various stages of intra erythrocytic development. The piriform merozoites of B. caballi were composed of an outer membrane and an inner double-membrane complex. Numerous micronemes and three rhoptries were found in the pellicle of the merozoite, and a spherical body was seen in the anterior part of the merozoite which usually lay adjacent to the nucleus and the pellicle. These findings were similar to those for merozoites of bovine Babesia parasites such as B. bigemina. The trophozoites were surrounded by a single membrane, were continuously changing their body shape with extension and retraction of the pseudopod. A long pseudopod extended far into the host cell cytoplasm, and was finally completely enclosed in a cell, but did not have hemozoin pigment, the breakdown product of hemoglobin digestion. Received: 23 January 1999 / Accepted: 15 April 1999     

Identification of a specific antigenic region of the P82 protein of Babesia equi and its potential use in serodiagnosis

The efficacy of the Be82 gene product fused with glutathione S-transferase (GST/Be82) in an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of Babesia equi infection was reported previously (H. Hirata et al., J. Clin. Microbiol. 40:1470-1474, 2002). However, the ELISA with the GST/Be82 antigen cross-reacted with Babesia caballi-infected horse sera, despite the high rate of detection of B. equi. These results suggested that GST/Be82 has an antigen in common with B. caballi or antigenicity similar to that of B. caballi. In the present study, we constructed a series of five clones with deletions in the Be82 gene product, each of which was fused with GST, and used them in ELISAs in order to overcome the cross-reactivity seen with B. caballi. One of the deletion clones, a clone with a deletion of the Be82 gene from positions 236 to 381 (Be82/236-381), specifically and sensitively detected B. equi-infected horse sera without cross-reactivity with B. caballi-infected horse sera. Assays with clones from which other gene products were deleted showed decreased sensitivities or remained nonspecific for the detection of B. equi-infected horse sera. These results suggest that the Be82/236-381 gene product is a novel antigen for the diagnosis of B. equi infection in horses.     

Repetitive DNA probes for the detection of Babesia equi

This report describes DNA probes for the identification of Babesia equi. A genomic library of B. equi was constructed in pUC13. Several clones were identified that hybridized strongly to B. equi DNA. Clone pBE33 hybridized specifically to B. equi DNA and did not hybridize to horse DNA nor to DNA from Babesia caballi, Babesia bovis or Babesia bigemina. Two subclones of pBE33 (pSB20 and pEH21) containing B. equi repetitive sequences, could detect 0.49 ng and 0.97 ng B. equi DNA, respectively.     

A dipstick immunobinding enzyme-linked immunosorbent assay for serodiagnosis of hepatitis B and delta virus infections.

A simple, specific and economical dipstick immunobinding enzyme-linked immunosorbent assay (DIA) for detecting hepatitis B surface antigen (HBsAg) and antibodies to hepatitis delta virus (anti-HDV), utilizing cellulose nitrate membrane is described. Screening of 815 serum specimens for HBsAg by DIA and micro ELISA revealed a positivity of 22.69% and 22.94% respectively. In the detection of antibodies to delta antigen, DIA was compared with an indirect immunofluorescence technique using A3 cell line as antigen substrate and a commercial macro ELISA. Of the 143 HBsAg positive sera tested for anti-HDV, 59 (41.25%) were positive by both immunofluorescence and macro ELISA and 61 (42.65%) by DIA. While the positive and negative predictive values of DIA for HBsAg were 100% and 99.6%, for anti-HDV by DIA these were 96.7% and 100% respectively. Based on the simplicity of performance and the economical nature of the test system, DIA is recommended as a diagnostic tool for field surveys and small laboratories in developing countries.     

Immunoblot assay for serodiagnosis of Helicobacter pylori infections.

An immunoblot assay for the serological diagnosis of Helicobacter pylori infection was evaluated. Serum samples from patients whose gastric biopsy specimens were known to be positive or negative for H. pylori on culture were used to establish interpretive criteria for the immunoblot assay. A panel of sera from patients with diseases other than H. pylori infection and sera from healthy blood donors were included to validate these criteria. All sera were initially assessed in an enzyme immunoassay (Ge-EIA), based on acid glycine-extracted cell surface proteins of H. pylori NCTC 11637. The same antigen extract was used in the immunoblot assay. In addition, the Ge-EIA and the immunoblot assay were compared with a commercially available EIA (Seradyn, Color Vue Pylori). Bands of 110/120 kDa and/or two of five low-molecular-mass proteins (26, 29, 30, 31, and 33 kDa, in any combination) showed a strong correlation with the H. pylori culture-positive patients (97.5%) compared to the correlation obtained with the EIA results (Ge-EIA, 87.5%; Seradyn EIA, 92.5%), and the antibody responses to these proteins were considered specific reactions. In 37 of 40 serum samples from culture-negative patients and also in sera from patients with other disorders, a moderate antibody reactivity to the medium-size proteins (43 to 66 kDa) was observed, and these were considered not valuable for a specific immunoblot assay. Among sera from culture-positive patients, 39 of 40 serum samples were defined to be immunoblot positive, and from among sera from culture-negative patients, 3 of 40 serum samples were defined to be immunoblot positive. The use of sera from patients with negative cultures for H. pylori as negative controls may decrease the sensitivity due to sampling error and false-negative culture results. Immunoblot assay-positive results were detected among 10% of sera from patients with other diseases, whereas they were detected among 42.5% of sera by the Ge-EIA and 47.5% of sera by the Seradyn-EIA. The higher number of EIA-positive sera in this group reflects a possible cross-reactivity (false-positive EIA result). Of the blood donors, representing asymptomatic but possibly colonized subjects, 24% were immunoblot positive. In conclusion, our data indicate that immunoblotting is more sensitive as well as more specific than EIA. Moreover, it permits detection of antibody responses to specific antigens, e.g., the cytotoxin-associated CagA protein, which may have pathological implications.     

Parasitological and molecular diagnostic of a clinical Babesia caballi outbreak in Southern Romania

Parasitology Research - Equine piroplasmosis (EP) is a tick-borne disease of equids caused by Babesia caballi and/or Theileria equi, which is endemic in many tropical and temperate areas of the...     

Passive hemagglutination test for measles immunity and serodiagnosis.

A passive hemagglutination (PHA) test for measles was evaluated in comparison with hemagglutination inhibition (HI) and neutralization (NT) tests. The PHA test determines exclusively the level of antibody directed to the hemagglutinin protein of measles virus. The ratio of PHA to HI titer was 1 to 32 (geometric mean, 6.5) for the first 5 weeks of infection but declined to near unity thereafter. It gradually increased again to 4 to 32 (geometric mean, 11.7) over several years. The initial high PHA titer relative to the HI titer was most likely due to the presence of the immunoglobulin M antibody known to be efficient in agglutination, because 2-mercaptoethanol (2ME) treatment of sera reduced the PHA titer to a level similar to that of the HI titer. The PHA titer in sera obtained after the convalescent phase was insensitive to 2ME, and the relative increase in the PHA over the HI titer was presumably a result of increased antibody avidity. In some individuals, the HI titer fell to below detectable levels several years after either natural infection or vaccination, but the PHA as well as the NT titer remained positive. The PHA titer was therefore a more reliable and more sensitive indicator of immune status against measles than the HI titer. The decrease in PHA titer by 2ME treatment provided evidence of a current or very recent infection. PHA was found to be useful both for assessing immunity status and for serodiagnosis.     

Hypotension in acute Babesia bovis (=B. argentina ) infections of splenectomized calves

Systemic blood pressure changes were measured in groups of 4 splenectomized calves on days 3, 6, 7, 8 and 9 after acute infection with Babesia bovis. In addition, changes in five uninfected calves were measured on day 0. Carotid arterial, right atrial, right ventricular pulmonary arterial and ascending vena cava pressures were all lower on day 3 and had fallen dramatically by days 8 to 9. All animals died on days 8 and 9. The PCV began to fall and kallikrein activation started during days 2 to 3. Parasites were first detected on days 2 to 3.These results are discussed in relation to other protozoan diseases of man and animals and to anaphylaxis in cattle. It is concluded that many of the changes observed in acute B. argentina infections are due to kallikrein and kinins, possibly released by the action of parasite secretions during infection.