Expression of Grapevine leafroll-associated virus 3 coat protein gene in Escherichia coli and production of polyclonal antibodies

Grapevine leafroll-associated virus 3 (GLRaV-3), the main viral species of the grapevine leafroll complex, causes yield and quality reduction in grapes (Vitis spp.). The coat protein gene was RT-PCR-amplified from total RNA extracted from infected grapevine leaves and the amplified fragment was cloned and completely sequenced. The fragment was subsequently subcloned into the pRSET-C expression vector. The recombinant plasmid was used to transform Escherichia coli BL21:DE3 and express the capsid protein. The coat protein, fused to a 6 His-tag, was purified by affinity chromatography using an Ni-NTA resin. The identity of the purified protein was confirmed by SDS-PAGE and Western blot. The in vitro-expressed protein was quantified and used for rabbit immunizations. The antiserum was shown to be sensitive and specific for the detection of GLRaV-3 in grapevine extracts in Western blot and DAS-ELISA assays, with no unspecific or heterologous reactions against other non-serologically related viruses being observed.

Natural history of celiac disease-associated antibodies and progression to overt disease in children at increased genetic risk

Background: Celiac disease is a lifelong, gluten-sensitive, autoimmune-mediated chronic enteropathy, tightly associated with risk alleles at the HLA class II genes. Aims: This study was carried out as a part of the population-based Type 1 Diabetes Prediction and Prevention (DIPP) Project. The first aim was to study the natural history of celiac disease-associated antibodies before the diagnosis of celiac disease was made. The second aim was to describe when and in which order celiac disease-associated and type 1 diabetes-associated antibodies appeared in children with genetic risk for both diseases. Subjects and Methods: Antibodies against tissue transglutaminase (TGA) and other celiac disease-associated antibodies were measured in serum samples collected at 3- to 12-month intervals of children at genetic risk for celiac disease who participated in the DIPP project. Celiac disease was confirmed by duodenal biopsy. Type 1 diabetes-associated antibodies were measured in all samples that had been collected. Overt disease was diagnosed according to World Health Organization criteria. Follow-up continued until a diagnosis of type 1 diabetes or until the end of a defined follow-up period. Results: TGA appeared in children at genetic risk for celiac disease only after the first year of life, but anti-gliadin antibodies often emerged significantly earlier, at age 6 months. The data show that spontaneous disappearance of celiac disease-associated antibodies, transient or persisting, is a common phenomenon, at least in prepubertal children. In children with genetic susceptibility to type 1 diabetes and celiac disease, celiac disease-associated antibodies usually develop earlier than the type 1 diabetes-associated antibodies. Conclusions: The transient nature of celiac disease-associated antibodies emphasizes the significance of establishing seropositivity repeatedly in screening detected celiac disease before gastroscopy and duodenal biopsy are considered and emphasized the importance of duodenal biopsy for diagnosing celiac disease.

Evaluation of an enzyme-linked immunosorbent assay to detect antibodies against Anaplasma marginale

A rapid indirect enzyme-linked immunosorbent assay (ELISA) was developed for measuring antibodies against Anaplasma marginale using a partially soluble antigen prepared from semi-purified initial bodies from erythrocytes with 80.0% of rickettsiaemia. This technique utilized alkaline phosphatase and p-nitrophenyl phosphate as reaction indicators. The high sensitivity (100.0%) was confirmed with sera from 100 calves experimentally-infected with A. marginale. All of these animals showed seroconversion before or at the same time of the first rickettsiaemia or even when it was not detected. Also the elevated specificity (94.0%) was confirmed by the low percentage of cross-reactions with sera from animals experimentally-infected with Babesia bigemina and Babesia bovis (1.4 and 6.6%, respectively). Performances of ELISA and indirect fluorescent antibody test (IFAT) with 324 sera from enzootically stable area did not show statistical difference (P>0.05), since the former showed 96.9% and the latter 97.2% of positive reactions. The advantage of this ELISA is a shorter execution time than others developed until now, allowing more samples to be analyzed.

A conglutination test for rapid detection of antibodies against Babesia bigemina

A rapid conglutination test (RCT) with performance comparable to the indirect fluorescent antibody technique (IFAT) was developed to detect antibodies against Babesia bigemina (B. bigemina-RCT). The B. bigemina-RCT is a sensitive, specific, economical, and rapidly performed serological test suitable for field application or minimally equipped laboratories. This test had a sensitivity of 90.9%, and specificity of 97.6%, compared to IFAT, which showed for the same parameters respectively, 98.3% and 99.7%. The early detection of anti- B. bigemina immunoglobulins by RCT in experimental infections was nearly parallel to that of IFAT. Cross reactions were observed with sera from calves experimentally infected with Babesia bovis (1.8%) and with Anaplasma marginale (1.2%). RCT antigen prepared with non parasitized erythrocytes (negative antigen) showed 1.5%, 3.5% and 2.2% of positive reactions with sera from animals experimentally infected with B. bigemina, B. bovis and A. marginale. However, none of the sera from animals of endemic areas for babesia infection resulted in positive reactions with the negative antigen. Considering these results and shelf life over six months, the B. bigemina-RCT could be used for epidemiological surveys and evaluation of control measures against this species of Babesia.

Prevalence of antibodies against chicken anaemia virus (CAV) in broiler breeders in Southern Brazil

Chicks infected during the first two weeks of life with chicken anaemia virus (CAV) manifest clinical disease that can be avoided if the breeder hens transfer enough antibodies to their progeny. The objective of the present work was to establish the prevalence and titer of anti-CAV antibodies in some Brazilian broiler hen breeder flocks and verify in which phase of life the birds were infected. A total of 1,709 serum samples from 12 broiler hen flocks vaccinated against CAV and 64 unvaccinated flocks were analyzed for CAV antibodies with an enzyme-linked immunosorbent assay (ELISA). All non-vaccinated breeder flocks were found to be infected with CAV, with 89% of the hens tested presenting antibodies, 52% of these with titers considered high enough to protect their progeny against CAV infection. Likewise, all vaccinated hens had antibody titer to CAV capable of conferring protection to their progeny. Thus, vaccination of hens seems capable of conferring protection to chicks against clinically apparent CAV-associated disease.

Evaluation of the indirect fluorescent antibody test and modified agglutination test for detection of antibodies against Toxoplasma gondii in experimentally infected pigs

The study determined the sensitivity and specificity of the indirect fluorescent antibody test (IFAT) and modified agglutination test (MAT) for anti-Toxoplasma gondii antibody detection by analyzing sera from 46 experimentally infected pigs. Values for sensitivity were 95.7% (confidence interval 95%: 84.0-99.2%) and for specificity 97.8% (confidence interval 95%: 87.0-99.9%) in both tests. There was an optimum agreement of results between IFAT and MAT evidenced by a Kappa test of 0.86. These results validate these tests for the detection of T. gondii infection in pigs. IFAT and MAT despite methodologies with different characteristics and readings have similar accuracy in pig serum samples.

An enzyme-linked immunosorbent assay (ELISA) for the detection of IgM antibodies against Leishmania chagasi in dogs

Visceral leishmaniasis is an emergent zoonosis with an increasing number of new cases in Brazil where the domestic dog is an important parasite reservoir in the infectious cycle of Leishmania chagasi. An enzyme-linked immunosorbent assay (ELISA), based upon the use of a total soluble antigenic preparation of L. chagasi, was adapted for the detection of IgM antibodies in the serum of infected dogs. Optimal dilutions of the antigen, using positive and negative reference sera, were determined by checkboard titrations. The specificity and sensitivity of the ELISA were 100 %. A total of 110 serum samples were taken from dogs in Belo Horizonte, Minas Gerais, Brazil, and examined for anti-L. chagasi IgM antibody by ELISA and indirect fluorescent antibody test (IFAT). About 25% (n=27) of all the dogs tested were found serologically positive for L. chagasi by IFAT, while 89.09% (n=98) were seropositive by ELISA. The results obtained by ELISA and IFAT were significantly different (P<0.01). The combined use of ELISA and IFAT is recommended in order to enable veterinary services to more efficiently detect canine visceral leishmaniasis.

Development of IgY antibodies in chickens and IgG in rabbits immunized against proteins of Pythium insidiosum isolated from horses in the state of Rio de Janeiro

Pythiosis is caused by Pythium insidiosum and the occurrence of disease in horses was described in the North and Northwest State of Rio de Janeiro, Brazil. The disease was described in cattle, sheep, humans, and horses in different states and regions across the country. This paper describes the development of IgY and IgG polyclonal antibodies, in chicken and rabbits, respectively against proteins extracted from kunkers and hyphae of P. insidiosum from affected horses. The proteins were recognized by chicken, rabbit and horse antibodies by immunodiffusion and Western blot against majority bands of 27 and 43 KDa, and titrated by ELISA. The antibodies IgY developed by the first time against Brazilian strains of P. insidiosum may represent a valuable tool in the detection of antigens of the pathogen and contribute to further studies aimed at immunotherapy and knowledge about this disease in endemic areas in Rio de Janeiro and in Brazil.

Risk factors and presence of antibodies to Toxoplasma gondii in dogs from the coast of São Paulo State, Brazil

Toxoplasmosis is caused by the obligate intracellular protozoan parasite Toxoplasma gondii and affects warm-blooded vertebrates, including pets and man. Dogs are epidemio-logically important since they act as sentinels for the infection in humans. The present study aimed to determine the presence of antibodies to T. gondii in 205 serum samples from dogs in Ubatuba, Sao Paulo state, Brazil, through indirect immunofluorescence reaction (IFAT), as well as the risk factors related to toxoplasmosis in the animals such as breed, age, sex, access to outdoors, homemade food ingestion, access to untreated water, and contact with rodents. Toxoplasmosis-positive samples accounted for 52/205 (25.4%), with titers ranging from 16 to 256. The serological results presented significant association (P<0.05) with homemade food ingestion (45/118; 38.1%; CI95% 29.9%-47.2%) (OR=7.0; CI95% 3.0-16.6), and with access to outdoors where those that do not have access to the street were prevalent (37/121; 30.6%; CI95% 23.1%-39.3%) (OR=0.5; CI95% 0.2-1.0). These results show that toxoplasmosis in this region is related to problems of sanitary education, mainly concerning the appropriate cooking of foods, since most positive animals did not show significant association with the presence of rodents or untreated water consumption but showed, instead association with ingestion of homemade food. Thus, toxoplasmosis is a public health problem in the studied region, and sanitary measures are needed to control the infection due to the strict relationship between man and dog and the presented risk factors

Evaluation of a recombinant p24 antigen for the detection of Feline Immunodeficiency Virus-specific antibodies

Feline Immunodeficiency Virus is a worldwide infection and is considered a significant pathogen. The diagnosis of FIV infections is mainly based on commercially available rapid tests that are highly expensive in Brazil, hence it is rarely performed in the country. Furthermore, lentiviruses grow slowly and poorly in tissue cultures, making the production of viral antigen by classic means and thus the establishment of FIV immunodiagnosis impracticable. In order to deal with this, recombinant DNA techniques were adopted to produce the protein p24, a viral capsid antigen. The protein's reactivity evaluation analyzed by Western blot indicated that this recombinant antigen can be a useful tool for the immunodiagnostic of FIV infections.

Risk factors associated with the frequency of antibodies against Babesia bovis and Babesia bigemina in cattle in southern Mozambique

The study aimed to evaluate the risk factors associated with the frequency of IgG antibodies against Babesia bovis and B. bigemina in cattle in southern Mozambique. Eight hundred and nine serum samples were collected from cattle in three provinces namely Maputo, Gaza and Inhambane, and tested by indirect enzyme-linked immunosorbent assay (i-ELISA) to assess the humoral immune response towards B. bovis and B. bigemina. The chi-square test at 5% significance was used to determine whether there was an association between gender, age and geographic origin of seropositive animals. The overall prevalence was 78.8% (548/695) for B. bovis and 76.0% (528/695) for B. bigemina. The origin of the animals showed a significant association (p<0.05) with seropositivity to both agents, while gender and age was not associated (p>0.05). Maputo province had the highest rate of positive animals, with 93.7% (118/126) for B. bovis and 97.6% (123/126) for B. bigemina. In Gaza province 77.3% (321/415) of the animals were positive for B. bovis and 67.5% (280/415) for B. bigemina, while in the province of Inhambane the levels of seropositivity were 70.8% (109/154) and 81.2% (125/154) for B. bovis and B. bigemina respectively. In the present study, the frequency of cattle positive for B. bovis and B. bigemina was shown to increase among older age groups, suggesting that infection and re-infection persisted even after the primary infection. Thus, this region is considered to be in a state of enzootic stability with regards to B. bovis and B. bigemina.

Anti-Rickettsia spp. antibodies in free-ranging and captive capybaras from southern Brazil

Capybaras (Hydrochaeris hydrochaeris) are among the main hosts of Amblyomma spp. ticks, which is able to transmit Rickettsia species to human beings and animals. Since they are often infested with potential vector ticks, capybaras may be used as sentinels for rickettsiosis, such as the Brazilian Spotted Fever. The aim of the present study was to determine the prevalence of antibodies against Rickettsia spp. using the indirect immunofluorescence assay (IFA) in 21 free-ranging and 10 captive animals from the Zoological Park of the 'Bela Vista Biological Sanctuary' (BVBS), Itaipu Binational, Foz do Iguaçu, Southern Brazil. Antigens of six rickettsial species already identified in Brazil (Rickettsia rickettsii, R. parkeri, R. bellii, R. rhipicephali, R. amblyommii and R. felis) were used for IFA. Ticks from each capybara were collected for posterior taxonomic identification. A total of 19 (61.3%) samples reacted to at least one of tested species. Seropositivity was found in 14 (45.2%), 12 (38.7%), 5 (16.1%), 4 (12.9%), 3 (9.7%) and 3 (9.7%) animals for R. rickettsii, R. bellii, R. parkeri, R. amblyommii, R. felis and R. rhipicephali, respectively. Two captive capybaras presented suggestive titers of R. rickettsii infection and one sample showed homologous reaction to R. parkeri. Only one free-ranging capybara presented evidence R. bellii infection. Ticks collected on capybaras were identified as Amblyomma dubitatum e Amblyomma sp. Results evidenced the rickettsial circulation in the area, suggesting a potential role of capybaras on bacterial life cycle.

Production and characterization of monoclonal antibodies against Campylobacter fetus subsp. venerealis

Myeloma cells Sp2/0-Ag14 and spleen cells from BALB/c mouse immunized with sonicated Campylobacter fetus subsp. venerealis NCTC 10354 were fused with polyethylene glycol (PEG) for the selection of clones producing antibodies. Clones were obtained by limiting dilution and screened for the production of specific antibodies to C. fetus subsp. venerealis NCTC 10354 by indirect ELISA and western blot against a panel of bacteria: C. fetus subsp. venerealis NCTC 10354, C. fetus subsp fetus ADRI 1812, C. sputorum biovar sputorum LMG 6647, C. lari NCTC 11352, and Arcobacter skirrowii LMG 6621 for the ELISA and C. fetus subsp. venerealis NCTC 10354 and C. sputorum biovar sputorum LMG 6647 for the western blotting. Fifteen clones producing monoclonal antibodies (MAbs) anti-C. fetus subsp. venerealis of the IgM (1) and IgG (14) classes were further screened for species-specificity. Four clones of the 15 obtained were producers of species-specific monoclonal antibodies (MAbs): two were specific for C. fetus subsp. venerealis and two were specific for C. fetus subsp. fetus. None of the clones were reactive against C. sputorum biovar sputorum LMG 6647. All clones recognized a protein with molecular mass of approximately 148 kDa from lysed C. fetus subsp. venerealis NCTC 10354.

Prokaryotic expression of a truncated form of bovine herpesvirus 1 glycoprotein E (gE) and its use in an ELISA for gE antibodies

This article describes the expression of a truncated form of bovine herpesvirus 1 (BoHV-1) glycoprotein E (gE) for use as immunodiagnostic reagent. A 651 nucleotide fragment corresponding to the amino-terminal third (217 amino acids) of BoHV-1 gE - that shares a high identity with the homologous BoHV-5 counterpart - was cloned as a 6×His-tag fusion protein in an Escherichia coli expression vector. A soluble protein of approximately 25 kDa purified from lysates of transformed E. coli was recognized in Western blot (WB) by anti-6xHis-tag and anti-BoHV-1 gE monoclonal antibodies. In addition, the recombinant protein was specifically recognized in WB by antibodies present in the sera of cattle seropositive to BoHV-1 and BoHV-5. An indirect ELISA using the expressed protein as coating antigen performed comparably to a commercial anti-gE ELISA and was able to differentiate serologically calves vaccinated with a gE-deleted BoHV-5 strain from calves infected with BoHV-1. Thus, the truncated gE may be useful for serological tests designed to differentiate BoHV-1/BoHV-5 infected animals from those vaccinated with gE-negative marker vaccines.

Frequency of Toxoplasma gondii antibodies in tufted capuchin monkeys (Cebus apella nigritus) from an ecological station in the State of São Paulo, Brazil

Toxoplasmosis is a worldwide zoonosis caused by Toxoplasma gondii, an obligate intracellular parasite protozoan. A large percentage of animals presents specific antibodies caused by a previous exposition, resulting in a chronic infection. Felides are the definitive hosts and the other warm-blooded animals, including primates, are the intermediate hosts. This study was aimed to determine the prevalence of T. gondii infection in free-living tufted capuchin monkeys (Cebus apella nigritus) from an ecological station located on Mata de Santa Teresa, Ribeirão Preto, SP, Brazil. T. gondii antibodies were analyzed by modified agglutination test (MAT) in serum samples of 36 tufted capuchin monkeys, considering eight as cut-off titer. From the studied animals, 3/36 (8.33%; CI95% 3.0-21.9%) presented T. gondii antibodies, all with titer 32. No significative difference was observed relating to the sex (1/3 male and 2/3 female), and to the age (1/3 young and 2/3 adult) (P>0.05). Thus, these results demonstrate the presence of T. gondii antibodies in primates from São Paulo state.