Buffalo Sera Research Articles

Molecular cloning and phylogenetic analysis of Babesia orientalis heat shock protein 70

The heat shock protein 70 (hsp70) gene of Babesia orientalis was obtained from a cDNA expression library by immunoscreening with B. orientalis infected buffalo sera. The nucleotide sequence of the cDNA was 2192bp with an open reading frame (ORF) of 1944bp encoding a polypeptide of 648 amino acid residues. Phylogenetic analysis of the 1944bp sequence together with 30 inter-erythrocytic protozoa hsp70 nucleotide sequences available from GenBank was performed. The results showed that B. orientalis was occurred within the Babesia clade, and most closely related to B. ovis and B. bovis. Similar topologies were obtained from trees based on apicomplexa parasite 18S rRNA sequence. Meanwhile, the BoHsp70 gene was cloned into pET-32a and subsequently expressed in Escherichia coli Rosetta™ strain as a Trx-fusion protein. The recombinant hsp70 of B. orientalis (rBoHsp70) was purified and evaluated as an antigen in the western blot. The serum from B. orientalis infected buffalo recognized the 92kDa rBoHsp70 expressed in E. coli Rosetta™ (DE3) by western blotting. The rabbit antiserum against rBoHsp70 recognized a specific 70kDa band in lysates of B. orientalis infected buffalo erythrocytes. These results suggested that hsp70 gene was well conserved among inter-erythrocytic protozoa and the BoHsp70 might be a diagnostic and candidate vaccine antigen.

A 29-kDa merozoite protein is a prospective antigen for serological diagnosis of Babesia orientalis infection in buffaloes

A gene encoding a 29-kDa protein of Babesia orientalis (BoP29) was isolated by immunoscreening the B. orientalis cDNA expression library using monoclonal antibodies and its nucleotide sequence of the cDNA was 971 bp with an open reading frame of 750 bp. The results of BLASTn and BLASTx in NCBI showed that this cDNA sequence had similarity with Babesia bovis hypothetical protein (BBOV_II005480). Western blotting showed that the recombinant protein (rBoP29) had positive seroreactivity to B. orientalis infected buffalo sera. An indirect enzyme-linked immunosorbent assay (ELISA) was developed using the rBoP29 as antigen. This method could detect specific antibody variation in the sera from the buffaloes experimentally infected with B. orientalis. Analysis of 132 sera samples collected from buffaloes showed that there was no significant difference in relative effectiveness of rBoP29-ELISA and PCR ( χ 2 = 0.910; P = 0.897) in identifying positive samples. These results indicate that the BoP29 is a novel antigen of B. orientalis, and rBoP29 might be a good antigen candidate for diagnosis of B. orientalis infection in buffalo.

The detection of antibody against peste des petits ruminants virus in Sheep, Goats, Cattle and Buffaloes

Monoclonal antibody-based competitive ELISA (C-ELISA) has been used for the specific measurement of antibodies to peste des petits ruminants (PPR) viruses in sheep, goats, cattle and buffalo. Serum samples from sheep (n = 232), goats (n = 428), cattle (n = 43), buffalo (n = 89) were tested. The animals had not been vaccinated against rinderpest or PPR. Findings suggested that the sero-positive cases were significantly higher in sheep (51.29%) than in goats (39.02%) (P = 0.002). The overall sero-prevalence of PPRV in small ruminants was 43.33%. The PPR antibodies seroprevalence was 67.42% in buffalo and 41.86% in cattle which was significantly higher in buffalo (P = 0.005). The overall sero-prevalence of PPRV in large ruminants was 59.09%. Cattle and buffalo sera showed a high prevalence of antibody against PPR virus which may explain the difficulty experienced in achieving high post-vaccination immunity levels against rinderpest. Because antibodies against PPR virus are both cross-neutralizing and cross-protective against rinderpest virus, further vaccination in the presence of antibodies against PPR virus may be a waste of national resources. It was also suggested that antibodies to PPR virus could prevent an immune response to the rinderpest vaccine. This paper presents serological evidence for the transmission of PPR virus from sheep and goats to cattle and buffalo and highlights the need to include PPR serology in the sero-monitoring programme to give a better indication of national herd immunity of sheep and goats against PPR.

Recombinant nucleocapsid-based ELISA for detection of IgG antibody to Rift Valley fever virus in African buffalo

Wild ruminants are thought to serve as natural hosts for Rift Valley fever virus (RVFV) but the role of these animals as reservoirs for RVFV during inter-epidemic periods and as amplifiers during epidemics is not well understood. An indirect enzyme-linked immunoassay (I-ELISA) based on the recombinant nucleocapsid protein (rNp) of RVFV was validated for the detection of specific IgG antibodies in African buffalo. Data sets derived from testing buffalo sera from Kenya ( n = 405) and South Africa ( n = 618) were dichotomised according to the results of a virus neutralisation test. The assay characteristic performance was analysed using threshold values optimised by the two-graph receiver operating characteristics (TG-ROC) analysis, and by mean plus two, as well as by mean plus three standard deviations derived from I-ELISA PP values in uninfected animals. Among 1023 buffalo sera tested, 77 (7.5%) had detectable virus neutralising antibodies. The assay had high intra- and inter-plate repeatability in routine runs. At a cut-off optimised by the TG-ROC at 95% accuracy level, the diagnostic sensitivity of the I-ELISA was 98.7% and diagnostic specificity 99.36% while estimates for the Youden's index ( J) and efficiency (Ef) were 0.98 and 99.31%. When cut-off values determined by traditional statistical approaches were used, the diagnostic sensitivity was 100% but estimates of J, Ef and other combined measures of diagnostic accuracy were lower compared to those based on cut-off value derived from the TG-ROC. Results of the study indicate that the I-ELISA based on the rNp would be useful for seroepidemiological studies of RVFV infections in African buffalo.

Comparison of fluorescence polarization assay with Rose Bengal (RB) test and complement fixation tests for the diagnosis of buffalo (Bubalus bubalis) brucellosis in a high-prevalence area

The fluorescence polarisation assay (FPA) was evaluated for the serological diagnosis of bovine brucellosis in buffalo (Bubalus Bubalis) in southern Italy. This assay uses O-polysaccharide prepared from Brucella Abortus lipopolysaccharide conjugated with fluorescein isothiocyanate as a tracer. It has many methodological advantages over older, more established tests and can be performed in short time. To measure the fluorescence polarization, a Tecan GENios Pro (Prionics) fluorescence-polarization analyzer was used with the procedure described by Nielsen et al. 1996. A cut-off value of 117 millipolarization (mP) units was used for testing 912 buffalo sera from Campania Region (526 positive sera and 386 negative sera according to the complement fixation test; CFT). All samples were tested with the Rose Bengal plate (RB). Sensitivity and specificity (Sn) for RB were 84.0% and 87.8% and for FPA were 92.6% and 88.9 percent. The FPA gave a kappa coefficient of agreement with respect to CFT of 0.755, while RB (relative to the CFT) gave coefficients of 0,715. Finally, ROC analysis suggested a cut-off value which did not agree with the one recommended in the test procedure for cow.

Humoral immune response of water buffalo monitored with three different antigens of Toxocara vitulorum

Humoral immune response of water buffalo naturally infected with Toxocara vitulorum was monitored using three different antigens of this parasite in serum and colostrum of buffalo cows and calves. Soluble extract (Ex) and excretory/secretory (ES) larval antigens and perienteric fluid antigen (Pe) of adult T. vitulorum were used to measure the antibody levels by an indirect ELISA. Serum of 7–12 buffalo cows for the first 365 days and colostrum of the same number of buffalo cows for the first 60 days of parturition, and serum of 8–10 buffalo calves for the first 365 days after birth were assayed. The ELISA detected antibodies against all three T. vitulorum antigens in the colostrum and serum of 100% of buffalo cows and calves examined. The highest antibody levels against Ex, ES and Pe antigens were detected in the buffalo cow sera during the perinatal period and were maintained at high levels through 300 days after parturition. On the other hand, colostrum antibody concentrations of all three antigens were highest on the first day post-parturition, but decreased sharply during the first 15 days. Concomitantly to the monitoring of immune response, the parasitic status of the calves was also evaluated. In calves, antibodies passively acquired were at the highest concentrations 24 h after birth and remained at high levels until 45 days coincidentally with the peak of T. vitulorum infection. The rejection of the worms by the calves occurred simultaneously with the decline of antibody levels, which reached their lowest levels between 76 and 150 days. Thereafter, probably because of the presence of adults/larvae stimulation, the calves acquired active immunity and the antibodies started to increase slightly in the serum and plateaued between the days 211 and 365. All three antigens were detected by the serum antibodies of buffalo calves; however, the concentration of anti-Pe antibody was higher than anti-EX and anti-ES, particularly after 90 days of age. By conclusion, the buffalo cows develop immunity and keep high levels of antibodies against T. vitulorum-Ex, ES and Pe antigens and these antibodies are transferred to their calves through the colostrum. This passively acquired immunity does not protect the calves against the acquisition of the infection, but these antibodies, passively or actively acquired, may have an important role during worm rejection by the calves and prevention of intestinal reinfection.

Avaliação de seis testes sorológicos no diagnóstico da brucelose bubalina

Foram examinados 440 soros bubalinos, selecionados em um outro exame de cerca de 1200 amostras sangüíneas. Utilizaram-se seis diferentes testes sorológicos para o exame dessas amostras: dois de aglutinação, dois de ELISA indireto e dois de ELISA competitivo. Os animais positivos no ELISA competitivo da FAO/IAEA foram considerados como infectados, e a comparação com os resultados dos outros testes aconteceu neste sentido. A sensibilidade relativa foi de 100%, 98,57%, 97,14%, 91,42% e 79,28%, e a especificidade relativa de 99,33%, 97,33%, 95,66%, 94,00% e 86,33% nas provas de ELISA competitivo, ELISA indireto com conjugado antibovino de cadeia leve (anticorpo monoclonal com HRPO), ELISA indireto com conjugado contra IgG bovino total, teste do antígeno acidificado tamponado e aglutinação rápida, respectivamente. Discute-se o valor dos diferentes testes sorológicos no diagnóstico da brucelose.

Detecção de anticorpos contra Neospora caninum e Toxoplasma gondii em soros de bubalinos ( Bubalus bubalis) no Estado de São Paulo, Brasil

Water buffaloes ( Bubalus bubalis), are capable of becoming infected with Neospora caninum and Toxoplasma gondii, contributing to disease dissemination. The incidence of these antibodies was assessed in sera of water buffaloes, using an immunofluorescent antibody technique (IFAT). Any possible influence of breed or age of the buffaloes was investigated, besides assessment of sera antibody titres of any dogs and humans that were in close contact with the tested animals. Serum samples were taken from water buffaloes (n=411), dogs (n=8) and humans (n=14), from buffalo dairy farms in the Sao Paulo State, and tested. Of the samples of buffalo sera tested, 56.0% were reactive ( >1:200) to N. caninum and 49.9% were reactive ( >1:64) to T. gondii. Of all, 33.9% of the buffaloes presented antibody titres for both protozoon parasites. Of the canine serum samples, 25.0% presented titres ( >1:50) for N. caninum and 62.5% for T. gondii ( >1:16). The human serum samples did not show detectable antibodies for N. caninum when using a >100 limit titre for positive findings. The obtained data allows the conclusion that the protozoan parasite N. caninum, as well as T. gondii, are widely disseminated in the water buffalo herds assessed and, apparently, the factors breed and age do not interfere with frequency of N. caninum positives among adult female buffaloes. When concerning T. gondii, female buffaloes of 5 to 6 years of age showed more frequent presence of specific antibodies.

A comparative serological study for the quantification of antibodies against foot-and-mouth disease virus in water buffalo sera.

A comparative serological study for the quantification of antibodies against foot-and-mouth disease virus in water buffalo sera.

Prevalence of antibodies to Neospora caninum and Toxoplasma gondii in cattle and water buffaloes in southern Vietnam

Serum samples from 200 dairy cattle and 200 beef water buffaloes were collected in southern Vietnam during May to September 1995. The sera were analysed for antibodies to Neospora caninum by an enzyme-linked immunosorbent assay and the indirect fluorescent antibody test, and for antibodies to Toxoplasma gondii by the direct agglutination test. Significant levels of N. caninum antibodies were detected in 5.5% of the cattle sera and in 1.5% of the water buffalo sera. 10.5% of the cattle sera and 3% of the water buffalo sera were found to contain T. gondii antibodies. Two of the cattle sera had both T. gondii and N. caninum antibodies. The present communication is the first to report serological evidence of N. caninum infection in the water buffalo.

Liquid-phase blocking sandwich enzyme-linked immunosorbent assay for detection of antibodies against foot-and-mouth disease virus in water buffalo sera

Abstract Objective To develop and apply the liquid-phase blocking sandwich ELISA (BLOCKING-ELISA) for the quantification of antibodies against foot-and-mouth disease virus (FMDV) strains O1 Campos, A24 Cruzeiro, and C3 Indaial. Design Antibody quantification. Sample Population 158 water buffalo from various premises of São Paulo State-Brazil. The sera were collected either from systemically vaccinated or nonvaccinated animals. Procedure The basic reagents of BLOCKING-ELISA (capture and detector antibodies, virus antigens, and conjugate) were prepared and the reaction was optimized and standardized to quantify water buffalo antibodies against FMDV. An alternative procedure based on mathematical interpolation was adopted to estimate more precisely the antibody 50% competition titers in the BLOCKING-ELISA. These titers were compared with the virus-neutralization test (VNT) titers to determine the correlation between these techniques. The percentages of agreement, cutoff points, and reproducibility also were determined. Results The antibody titers obtained in the BLOCKING-ELISA had high positive correlation coefficients with VNT, reaching values of 0.90 for O1 Campos and C3 Indaial, and 0.82 for the A24 Cruzeiro (P <0.0005). The cutoff points obtained by use of the copositivity and conegativity curves allowed determination of high levels of agreement between BLOCKING-ELISA and VNT antibody titers against the 3 FMDV strains analyzed. Conclusions The results characterized by high correlation coefficients, levels of agreement, and reproducibility indicate that the BLOCKING-ELISA may replace the conventional VNT for detection and quantification of antibodies from water buffalo sera to FMDV. (Am J Vet Res 1996;57:840–843)

Observations on the epidemiology of lumpy skin disease in Kenya.

Lumpy skin disease virus strains isolated in Kenya over a period of some 20 years have proved to be serologically identical. They were indistinguishable by indirect fluorescent antibody and serum neutralization test from the South African Neethling and West African serotypes. These two serological methods proved of value in studying the antibody responses to infection. While epizootic spread of LSD has occurred in Kenya, most cases are of a sporadic nature and are thought to be the result of accidental contacts with a maintenance cycle. There is evidence of antibody to LSD in the African buffalo (Syncerus caffer) in those areas where LSD is considered to be enzootic in Kenya, and also in small numbers of domestic cattle. No buffalo or bovine sera contained antibody to cowpox virus. An area enzootic for LSD is proposed and it is suggested that the maintenance cycle involves the buffalo. No antibody was found in the other wild ruminant species examined.

Isolation and characterisation of bovine adenoviruses types 3, 4 and 8 from free-living African buffaloes (Syncerus caffer)

Three virus isolations were made from a group of free-living three-month-old African buffalo calves (Syncerus caffer) in The Zambezi valley of Zimbabwe. All isolates demonstrated characteristic adenovirus cytopathology and fulfilled the criteria set down for adenoviruses. Isolates were identified as bovine adenovirus serotypes 3, 4 and 8 by complement fixation and serum neutralisation tests. Four hundred and twelve buffalo sera were examined and antibodies against all three adenovirus types were found.

Experimental infection of the African buffalo with the virus of Rift Valley fever.

Five African buffalo(Syncercus caffer) were inoculated with pantropic Rift Valley fever virus; 1 of 2 pregnant animals aborted. Four of the 5 developed a viraemia persisting for 48 h with an antibody response comparable to that found in cattle after RVF infection. No neutralising antibody was found in a series of some 114 feral buffalo sera from different parts of East Africa.

Polymorphism of 14C vitamin D3 binding protein in cattle and water buffalo serum

Cattle and water buffalo sera labelled with vitamin D3[14C] (300 and 480 individual samples respectively) were subjected to starch gel electrophoresis followed by autoradiography in an attempt to identify a possible polymorphism of the proteins capable of binding this vitamin. Three phenotypes controlled by two codominant autosomal alleles were identified in cattle while in water buffalo six phenotypes controlled by three codominant autosomal alleles were observed.

Investigations of Allerton-type herpes virus infection in East African game animals and cattle.

Neutralization tests with a strain (BA) of Allerton-type herpes virus, derived from a buffalo (Syncerus caffer) were carried out on 924 sera from 17 species of E. African game animals and on cattle sera from Tanzania (2001), Kenya (792) and Uganda (410).Buffalo populations throughout E. Africa showed a very high rate of infection, with all animals over 2 years of age serologically positive. Antibody was present in some giraffe, waterbuck and hippopotamus sera and, less frequently, in impala, eland, bushbuck and oryx. Data are provided on the titres of positive samples; the mean titre of buffalo sera increased with age.Cattle in many localities of N. Tanzania and S. Kenya showed a very high rate of infection, 85-95% of sera from animals more than 2-years old containing antibody; the titres recorded were lower than those in buffaloes. Very high infection rates were also found in Karamoja and Teso (Uganda) and also in some other areas of Kenya, whilst a considerably lower incidence of infection was detected in W. Nile Province of Uganda and in central Tanzania. Differences in infection rates may have been related to herd size and husbandry practices.It was shown that a wave of infection was probably spreading through cattle in N. Tanzania at about the same time as an outbreak of disease occurred in buffaloes and it is suggested that virus transmission may have been by biting flies.No clinical signs attributable to the virus were reported in cattle but mouth lesions similar to those recorded in buffaloes, or nasal lesions, could have passed undetected. Allerton-type virus probably produces a range of clinical syndromes in cattle, closely resembling those associated with some herpes viruses in primates but infection is seldom related in the field to either pseudo-lumpy skin disease, mammillitis or stomatitis.