An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against Babesia bovis in cattle

doi:10.1016/S0304-4017(97)00003-4

Veterinary Parasitology

Volume 71, Issue 1, 15 July 1997, Pages 17-26

https://doi.org/10.1016/S0304-4017(97)00003-4 Get rights and content

Abstract

A method for the isolation of Babesia bovis merozoites from infected erythrocytes (Machado et al., 1994) and an enzyme-linked immunosorbent assay (ELISA) for the detection of anti-B. bovis antibodies were developed. This ELISA utilizes a soluble, alkali-digested B. bovis antigen. Sera from calves experimentally infected with B. bovis were screened by this technique from day 9 to day 233 postinfection (PI). Maximum titers were reached between days 29 and 149 PI. Sera from calves (n = 62), heifers (n = 38) and cows (n = 49), raised in tick-infested areas of São Paulo State, showed higher antibody levels in heifers and cows. A higher percentage of negative sera (19.4%) was found among calves. Sodium dodecyl sulphate-polyacrylamide electrophoresis (SDS-PAGE) and immunoblotting have identified proteins of similar molecular mass in the two species. Sera from calves experimentally infected with B. bovis reached with homologous antigens at the level of 95, 66 and 23 kDa. The same serum reacted with the 23 kDa band of heterologous antigen. Sera from calves experimentally infected with B. bigemina recognized 82, 66, 58, 36 and the 23 kDa polypeptides of homologous and heterologous antigens. The experimental ELISA described may prove to be a practical serological test for bovine Babesia infection with the choice of specific test antigen for B. bovis and B. bigemina.

References (29)

M.S. Blake et al.
A rapid sensitive method for detection of alkaline phosphatase-conjugate anti-antibody on Western blots
Anal. Biochem.
(1984)
G.R. Bushell et al.
Babesia bovis host cell recognition proteins
Int. J. Parasitol.
(1991)
T. Fujinaga et al.
Serological relationship between a large Babesia found in Japanese cattle and Babesia major, Babesia bigemina and Babesia bovis
Res. Vet. Sci.
(1980)
B.V. Goodger et al.
Babesia bovis: dextran sulphate as an adjuvant for and precipitant of protective immunogens
Int. J. Parasitol.
(1992)
E.F. Hartree
Determination of protein: a modification of the Lowry method that gives a linear photometric response
Anal. Biochem.
(1972)
R.Z. Machado et al.
Babesia bigemina: isolation and characterization of merozoite rhoptries
Exp. Parasitol.
(1993)
V.S. Mishra et al.
Immunogenicity and sequence analysis of recombinant p58: a neutralization-sensitive, antigenically conserved Babesia bigemina merozoite surface proteins
Mol. Biochem. Parasitol.
(1991)
P. Timms et al.
Immune response of cattle following vaccination with living and non-living Babesia bovis antigens
Vet. Parasitol.
(1984)
D.N. Barry et al.
A microplate enzyme immunoassay for detecting and measuring antibodies to Babesia bovis in cattle serum
Aust. Vet. J.
(1982)
D.E. Bidwell et al.
Comparison of serological tests for babesiosis in British cattle
Vet. Rec.
(1978)

R. Böse et al.

An improved ELISA for the detection of antibodies against Babesia bovis using either a native or a recombinant B. bovis antigen

Parasitol. Res.

(1990)

E. Engvall et al.

Enzyme linked immunosorbent assay (ELISA). III. Quantitation of specific antibodies by enzyme labelled anti-immunoglobulin in antigen coated tubes

J. Immunol.

(1972)

J.V. Figueiroa et al.

Identification of common surface antigens among Babesia bigemina isolates by using monoclonal antibodies

Parasitology

(1990)

W.L. Goff et al.

Identification of Babesia bovis merozoite surface antigens by using immune bovine sera and monoclonal antibodies

Infect. Immun.

(1988)

Cited by (85)

Bovine anaplasmosis as a risk factor for retained placenta, mastitis, and abomasal displacement in dairy cattle
2023, Research in Veterinary Science
This study aimed to evaluate the frequency of IgG antibodies against A. marginale, the occurrence of this bacterium by qPCR, and the effect of bovine anaplasmosis as a risk factor for clinical cases of retained placenta, mastitis, and abomasal displacement in dairy cattle. For that 179 Holstein cows out of three dairy herds, in the municipality of Sertão, Rio Grande do Sul, Brazil. These cows were on farms that were vulnerable to risk factors that are crucial to susceptibility among these animals to this intracellular hemoparasite. The mean seropositivity for A. marginale from the periods evaluated was 54% on farm A, 69.4% on farm B, and 27.3% on farm C. Molecular diagnosis was performed with qPCR and the mean positivity for A. marginale among the cows on farms A, B, and C in December 2017 was 34.6% (67/179). Infected animals showed clinical cases of retained placenta (6.1%), mastitis (6.1%), and abomasal displacement (0.5%). The association between positivity for anaplasmosis and these clinical cases was assessed through the odds ratio. Our results show that females with a positive qPCR assay for A. marginale had 52.48 times increased probability (OR) to develop clinical cases of retained placenta and mastitis (P < 0.001). These clinical cases negatively impact the productivity of positive females. Thus, implementing preventive and prophylactic control measures to ensure the sanitary quality of the herds is needed to avoid losses due to morbidity and mortality and diminish the economic losses suffered by farmers.
Dioctophyme renale: in vitro culture, production of excretory and secretory antigens, and their use in immunodiagnosis
2022, Veterinary Parasitology
The nematode Dioctophyme renale affects mainly the right kidney of domestic and wild mammals, in addition to having zoonotic potential. Therefore, this study aimed to produce excretion and secretion antigens of adult D. renale (DES) to diagnose canine dioctophimosis. To obtain the excretion and secretion antigens (DES), five D. renale adults were maintained for three weeks in supplemented RPMI medium with weekly supernatant collections. After the DES concentration, 2200 mL of the collected supernatant was used, which was centrifuged, followed by two filtrations and dialyzed, resulting in 12.5 mL of DES with a protein concentration of 0.59 mg/mL. The polyacrylamide gel electrophoresis (SDS-PAGE) of the DES antigen showed fractions with molecular weights ranging from 30 to 250 Kda. In the indirect ELISA with the DES antigen, the mean absorbance of the positive sera (38) was 0.839 ± 0.267, and the mean of the negative control sera (7) was 0.208 ± 0.083; the specificity and sensitivity of the ELISA were 100 and 97.4 %, respectively, being as effective as the surgical method in the presence of viable parasites. Thus, for the first time, this study describes the production of excretion and secretion antigens of adult D. renale and standardizes an indirect ELISA to diagnose dioctophimosis.
Anaplasma marginale in goats from a multispecies grazing system in northeastern Brazil
2021, Ticks and Tick-borne Diseases
Anaplasma marginale, a tick-borne α-proteobacterium that causes significant economic losses for the cattle industry worldwide, has been increasingly detected in other animal species. This agent has been previously detected in buffaloes and goats co-grazed with cattle in Brazil. This study aimed to investigate the occurrence of A. marginale in a multispecies (goats, sheep and cattle) grazing farm in the State of Paraíba, northeastern Brazil. A total of 119 goats, 71 sheep, and five cattle were evaluated. An epidemiological questionnaire was applied to the farm owner addressing age, gender, and presence of ticks. Serum samples from goat, sheep and cattle were tested for anti-Anaplasma marginale antibodies by a commercial MSP5-based on indirect enzyme-linked immunosorbent assay (iELISA). EDTA-blood samples were screened for A. marginale- and A. ovis-infection by PCR using primers targeting Anaplasma spp. msp4 gene. Sequencing of the repeat region of the msp1α gene was used for genotyping A. marginale strains found in the present study. A total of 47/119 (39.5 %, 95 % CI: 31.1–48.4 %) goats and 2/71 (3%, 95 % CI: 0.7–9.7 %) sheep were seroreactive for A. marginale rMSP5 by the commercial iELISA. All cattle were seronegative for A. marginale. Anaplasma spp. msp4 PCR results revealed that two out of 119 (1.7 %; 95 % CI: 0.4–5.9 %) goats tested positive and all sheep and cattle samples were negative. It was not possible to sequence one sample. Therefore, the other sequencing sample found tandem repeats of A. marginale msp1α gene demonstrating that goat was infected with the genotype F/91. Rhipicephalus microplus ticks were found parasitizing goats but not on sheep or cattle. Considering that in Brazil A. marginale genotype F/91 and the MSP1a tandem repeat F has only been detected in goats so far, we hypothesized that this genotype may be related to goats.
Occurrence and genetic diversity of hemoplasmas in beef cattle from the Brazilian Pantanal, an endemic area for bovine trypanosomiasis in South America
2019, Comparative Immunology, Microbiology and Infectious Diseases
Hemotropic mycoplasmas (hemoplasmas) are Gram-negative bacteria that parasitize the erythrocyte surface of a wide variety of mammals. The present study aimed at investigating the occurrence of hemoplasmas in beef cattle in the Brazilian Pantanal, an area endemic for bovine trypanosomiasis in South America. Additionally, the objective of this study was to characterize molecularly the genotypes of the found hemoplasmas. For this purpose, blood and serum samples of 400 beef cattle were collected from five properties in Corumbá, Nhecolândia sub-region, Mato Grosso do Sul, in Midwest Brazil. Blood samples underwent DNA extraction and standard 16S rRNA gene-based PCR assays for hemoplasmas. The sequences obtained were submitted to phylogenetic inferences, distance analysis, and genotype diversity. The Indirect Enzyme-Linked Immunoabsorbent Assay (iELISA) indicated the presence of anti-Trypanosoma vivax IgG antibodies in 89.75% of the animals sampled, confirming the endemicity of said agent in the studied region. Among the 400 bovine blood samples tested, 2.25% (9/400) were positive for hemoplasmas in cPCR. The phylogenetic analysis of the obtained sequences confirmed the presence of 'Candidatus Mycoplasma haemobos' and Mycoplasma wenyonii DNA in 0.5% (2/400) and 1.75% (7/400) animals, respectively. Five genotypes of M. wenyonii and one of 'Candidatus M. haemobos' were detected among the sequenced amplicons. The present study showed low molecular occurrence of haemoplasmas in beef cattle sampled in the Brazilian Pantanal, an area endemic for bovine trypanosomiasis. Despite of the conservation of the 16S rRNA gene, there was considerable diversity of hemoplasma genotypes infecting the sampled beef cattle.
Genetic diversity of Anaplasma marginale in beef cattle in the Brazilian Pantanal
2019, Ticks and Tick-borne Diseases
There are few studies on the genetic diversity of Anaplasma marginale in Brazilian cattle herds, especially about beef cattle. The objective of this study was to evaluate the genetic diversity of A. marginale, based on the msp1α gene in Bos taurus indicus sampled from the Brazilian Pantanal. Aliquots of blood with and without EDTA were taken from 400 cattle (200 cows and 200 calves) across five extensive farms. The samples were submitted to the indirect immunoenzymatic assay (iELISA), quantitative real-time PCR (qPCR) for the msp1β gene and to the semi-nested (sn) PCR for the msp1α gene. Positive samples were sequenced by the Sanger method and subjected to diversity analysis using the RepeatAnalyser software. The percentage of positive animals by iELISA, qPCR and (sn) PCR was 72.2% (289/400), 56.7% (227/400) and 23% (52/227), respectively. Cows (154/200) showed to be significantly more seropositive than calves (135/200). In qPCR, the number of calves and average quantification value (138/200; 1.3 × 10⁶) A. marginale msp1α copies per μL proved to be higher when compared to that found for the cows (89/200; 3.9 × 10⁴). The microsatellite analysis of the 26 sequences obtained from the msp1α gene revealed the presence of E (77%), C (15.4%) and B (7.7%) genotypes. Fourteen A. marginale strains were identified in the studied region, with eight that have never before been described in the literature (τ-10-13−13-18; τ-27-18; EV8-EV8-17; α-β-β-β-100; EV7-11−10-15; τ-11-11−27-18; τ-11-10-15; τ-27-13-18). Beef cattle are highly exposed to A. marginale in the Brazilian Pantanal. Moreover, a high genetic diversity of A. marginale, with eight new strains, was found in the studied region. While cows may act as chronic carriers, perpetuating the pathogen within the herd, male beef calves sold to other regions may disperse these strains.
Assessment of equine piroplasmids in the Nhecolândia sub-region of Brazilian Pantanal wetland using serological, parasitological, molecular, and hematological approaches
2019, Ticks and Tick-borne Diseases
Brazilian Pantanal is the world´s largest wetland ecosystem, where cattle’s ranching is the most important economic activity. The objective of this study was to compile some epidemiological features on equine piroplasmids from the Nhecolândia sub-region of Pantanal wetland through the evaluation of the patterns of T. equi and B. caballi infections in different groups of horses; identification of the tick species that infest horses; and to study phylogenetic relationships among Theileria equi 18S rRNA gene sequences. During October 2015, blood and serum samples were collected from 170 horses in four different categories. Ticks, after identification, had their hemolymph and eggs examined for the presence of piroplasmid sporokinets. Also we searched parasites in the peripheral blood smears of the investigated horses. The number of red blood cells (RBCs) and the packed cell volume (PCV) were determined to test for anemia in the infected animals, and exposure to B. caballi and T. equi was evaluated by enzyme-linked immunosorbent assay. “Catch all primers” based on 18S rRNA gene were used in polymerase chain reactions (PCR) to detect equine piroplasmids, followed by three nested PCRs for the phylogenetic analysis. The serological results showed that 61.8% and 52.9% of the horses sampled were exposed to T. equi and B. caballi, respectively. Piroplasmid DNA was detected in 43.5% of the horses analyzed. Our sequencing revealed 98–100% identity with some sequences previously published in GenBank for T. equi, and microheterogeneity among others. We found that 51.2% of the animals sampled were infested with Dermacentor nitens, Amblyomma sculptum, and Rhipicephalus (Boophilus) microplus, singly or co-infested. Since positive and negative animals presented similar RBC and PCV values, and no sporokinets were found on blood smears, hemolymph and eggs of the ticks collected, we suggest that infected equines can act as asymptomatic carriers for piroplasmosis in the studied region. Our results together showed the enzootic characteristic of equine piroplasmids in Pantanal region highlighting the importance of using different methods for detection these parasites. Moreover, breeding mares and foals should be monitored since they displayed the greatest occurrences for molecular test (59.0% and 86.1% respectively) and tick infestations (87.2% and 63.9% respectively).

View all citing articles on Scopus

View full text

Article preview

Veterinary Parasitology

Abstract

References (29)

A rapid sensitive method for detection of alkaline phosphatase-conjugate anti-antibody on Western blots

Anal. Biochem.

Babesia bovis host cell recognition proteins

Int. J. Parasitol.

Serological relationship between a large Babesia found in Japanese cattle and Babesia major, Babesia bigemina and Babesia bovis

Res. Vet. Sci.

Babesia bovis: dextran sulphate as an adjuvant for and precipitant of protective immunogens

Int. J. Parasitol.

Determination of protein: a modification of the Lowry method that gives a linear photometric response

Anal. Biochem.

Babesia bigemina: isolation and characterization of merozoite rhoptries

Exp. Parasitol.

Immunogenicity and sequence analysis of recombinant p58: a neutralization-sensitive, antigenically conserved Babesia bigemina merozoite surface proteins

Mol. Biochem. Parasitol.

Immune response of cattle following vaccination with living and non-living Babesia bovis antigens

Vet. Parasitol.

A microplate enzyme immunoassay for detecting and measuring antibodies to Babesia bovis in cattle serum

Aust. Vet. J.

Comparison of serological tests for babesiosis in British cattle

Vet. Rec.

An improved ELISA for the detection of antibodies against Babesia bovis using either a native or a recombinant B. bovis antigen

Parasitol. Res.

Enzyme linked immunosorbent assay (ELISA). III. Quantitation of specific antibodies by enzyme labelled anti-immunoglobulin in antigen coated tubes

J. Immunol.

Identification of common surface antigens among Babesia bigemina isolates by using monoclonal antibodies

Parasitology

Identification of Babesia bovis merozoite surface antigens by using immune bovine sera and monoclonal antibodies

Infect. Immun.

Cited by (85)

Bovine anaplasmosis as a risk factor for retained placenta, mastitis, and abomasal displacement in dairy cattle

Dioctophyme renale: in vitro culture, production of excretory and secretory antigens, and their use in immunodiagnosis

Anaplasma marginale in goats from a multispecies grazing system in northeastern Brazil

Occurrence and genetic diversity of hemoplasmas in beef cattle from the Brazilian Pantanal, an endemic area for bovine trypanosomiasis in South America

Genetic diversity of Anaplasma marginale in beef cattle in the Brazilian Pantanal

Assessment of equine piroplasmids in the Nhecolândia sub-region of Brazilian Pantanal wetland using serological, parasitological, molecular, and hematological approaches