Use of recombinant malate dehydrogenase (MDH) and superoxide dismutase (SOD) [CuZn] as antigens in indirect ELISA for diagnosis of bovine brucellosis.

The objective of this study was to validate an indirect enzyme-linked immunoassay (iELISA) using the recombinant proteins, malate dehydrogenase (MDH) and superoxide dismutase (SOD) [CuZn], as antigens and to evaluate its ability to discriminate antibodies produced by vaccination from those induced by infection in the diagnosis of bovine brucellosis. Sera from six groups were evaluated: G1 - culture-positive animals (52 serum samples) (naturally infected); G2 - non-vaccinated animals (28 serum samples) positive in RBT (Rose Bengal test) and 2ME (2-mercaptoethanol test) selected from brucellosis-positive herds; G3 - animals from a brucellosis-free area (32 serum samples); G4 - S19 vaccinated heifers (114 serum samples); G5 - RB51 vaccinated heifers (60 serum samples); G6 - animals inoculated with inactivated Yersinia enterocolitica O:9 (42 serum samples). Diagnostic sensitivity (DSe) and diagnostic specificity (DSp) were estimated using the frequentist approach and the confidence interval (CI) (95%) calculated by the Clopper-Pearson (exact) method. The DSe for iELISA_MDH in the G1 group was 71.7% (CI 95%: 57.6-83.2%) and for the G2 100.0% (CI 95%: 87.7-100.0%), whereas the DSp was 84.4% in the G3 (CI 95%: 67.2-94.7%). For the iELISA_SOD the DSe was estimated 67.3% for the G1 (CI 95%: 52.9-79.7%) and 71.4% for G2 (CI 95%: 51.3-86.8%), while the DSp for G3 was 87.5% (CI 95%: 71.0-96.5%). iELISA_MDH and iELISA_SOD showed potential to be used in the diagnosis of infected animals, increasing the range of serological tests available for the diagnosis of bovine brucellosis, with the advantage of being S-LPS-free. However, none of the tests could differentiate between infection and vaccination.

Staphylococcus aureus Protection-Related Type 3 Cell-Mediated Immune Response Elicited by Recombinant Proteins and GM-CSF DNA Vaccine.

Staphylococcus aureus mastitis remains a major challenge for dairy farming. Here, 24 mice were immunized and divided into four groups: G1: control; G2: Granulocyte Macrophage Colony-Stimulating Factor (GM-CSF) DNA vaccine; G3: F0F1 ATP synthase subunit α (SAS), succinyl-diaminopimelate (SDD), and cysteinyl-tRNA synthetase (CTS) recombinant proteins; and G4: SAS+SDD+CTS plus GM-CSF DNA vaccine. The lymphocyte subpopulations, and the intracellular interleukin-17A (IL-17A) and interferon-γ production in the draining lymph node cells were immunophenotyped by flow cytometry. The immunophenotyping and lymphocyte proliferation was determined in spleen cells cultured with and without S. aureus stimulus. Immunization with S. aureus recombinant proteins generated memory cells in draining lymph nodes. Immunization with the three recombinant proteins plus GM-CSF DNA led to an increase in the percentage of IL-17A+ cells among overall CD44+ (memory), T CD4+, CD4+ T CD44+ CD27-, γδ TCR, γδ TCR+ CD44+ CD27+, and TCRVγ4+ cells. Vaccination with S. aureus recombinant proteins associated with GM-CSF DNA vaccine downregulated TH2 immunity. Immunization with the three recombinant proteins plus the GM-CSF DNA led to a proliferation of overall memory T, CD4+, and CD4+ TEM cells upon S. aureus stimulus. This approach fostered type 3 immunity, suggesting the development of a protective immune response against S. aureus.

Risk of Trypanosoma cruzi transmission in southern Minas Gerais, Brazil - Data from 2014 to 2020.

Trypanosoma cruzi, the etiologic agent of Chagas disease, is widely distributed in the Americas and is transmitted through vectorial, transfusional, and oral routes. This study aimed to evaluate the risk of vectorial transmission of Chagas disease in municipalities located in southern Minas Gerais, Brazil, by analyzing triatomine specimens collected from 2014 to 2020. All 1522 hematophagous triatomines were identified as Panstrongylus megistus, and were subjected to parasitological and molecular examinations. From 2014 to 2016, approximately 10% of insects were positive in the microscopic analysis of intestinal content, and 27% were positive as detected by the quantitative polymerase chain reaction (qPCR) of the same sampling. However, in the last investigated years, an increase in infected triatomines was observed in microscopic analysis (22%) and qPCR methods (41%). This corroborates the findings of acute human Chagas disease cases, which have increased in the study area from a maximum of 2 cases in previous years to 20 cases in 2019, and 17 cases in 2020 through June. Additionally, bloodmeal sources of infected triatomines were investigated; human blood was detected in up to 85.7% of the samples. Moreover, canine blood was also detected in triatomine intestinal content in recent years, reaching 91% of analyzed insects in 2018. Data on bloodmeal sources have demonstrated human-vector contact and have suggested the participation of dogs in the parasite transmission cycle. These results indicate the risk of T. cruzi vectorial transmission in Southern Minas Gerais and São Paulo owing to the boundary between these states. Thus, enhanced surveillance and vector control of Chagas disease are highly recommended in these areas.

Immunoproteomics of Brucella abortus reveals potential of recombinant antigens for discriminating vaccinated from naturally infected cattle.

Brucellosis serodiagnosis is still a challenge and vaccination is the main measure used to control bovine brucellosis, being S19 and RB51 the most currently used vaccines. So, in order to contribute to brucellosis control, a bidimensional (2D) immunoblot-based approach was used to find immunogenic proteins to be used in serodiagnosis, particularly with ability to be employed in DIVA (Differentiating Infected from Vaccinated Animals) strategy. Immunoproteomic profile of Brucella abortus 2308 was analyzed in 2D western blotting using pooled sera from S19 vaccinated animals, RB51 vaccinated animals, B. abortus naturally infected animals and non-vaccinated seronegative animals. Evaluation of the antigens differentially immunoreactive against the groups of sera showed three proteins of particular importance: MDH (malate dehydrogenase) immunoreactive for S19-vaccinated animals, SOD (superoxide dismutase) reactive for infected animals and ABC transporter (multispecies sugar ABC transporter) reactive against sera from vaccinated animals (S19 and RB51). These three proteins were produced in E. coli and tested in an indirect ELISA (I-ELISA). For MDH, comparison between the vaccinated animals (independent of the vaccine used) and the seropositive and seronegative animals in I-ELISA showed significant differences. Data on the I-ELISA using SOD showed that sera from non-vaccinated naturally infected animals exhibited significant difference in comparison with all other groups. Otherwise, sera from vaccinated animals (S19 and RB51) and from non-vaccinated naturally infected animals did not show significant difference in OD values, but they were all significant different from non-vaccinated seronegative animals using ABC transporter as antigen in I-ELISA. In conclusion, together the 2D western blot analysis and the preliminary I-ELISA results suggest that the combined use of MDH and SOD could be successful employed in a LPS-free protein based serodiagnosis approach to detect bovine brucellosis and to discriminate vaccinated from naturally infected animals, in early post-vaccination stages.

Atualização em coccidioses intestinais: uma abordagem crítica / Intestinal coccidia: a critical review

Os coccídios intestinais compreendem um grupo de protozoários emergentes e oportunistas. As manifestações clínicas causadas podem variar desde diarreia autolimitada até quadros crônicos associados a distúrbios eletrolíticos. Com a pandemia do HIV/ AIDS, essas infecções ganharam notoriedade devido ao quadro agressivo nesses pacientes. Segundo dados da literatura, em pacientes HIV positivos foram encontradas taxas de infecção média de 24%. Dentre os coccídios intestinais, Cryptosporidium sp. possui maior destaque devido ao seu caráter zoonótico. Há registros da recuperação frequente de oocistos em cães e gatos, tendo sido encontrados em 100% dos animais analisados em um hospital veterinário. Além disso, bovinos também podem atuar como reservatórios, com prevalências no Brasil variando de 0,6% a 72,13%. Atualmente, há variadas técnicas para diagnóstico desses coccídios, desde métodos microscópicos que apresentam limitações técnicas até moleculares com elevado custo associado. Além da importância médica, a prevalência de coccidioses intestinais é relevante como um indicador do status socioeconômico de determinada população e da intensidade de transmissão seja ela antroponótica ou zoonótica. Entretanto, ainda são necessárias melhorias no sentido de tornar mais acessíveis novas técnicas diagnósticas, para se detectarem com mais facilidade e confiabilidade os coccídios intestinais.

Intestinal coccidia comprise a group of emerging and opportunistic protozoa. The manifestations caused may range from diarrhea to chronic variables associated with electrolytic disturbances. With an HIV / AIDS pandemic, these infections can be reported because of the aggressive picture in patients. The data of the literature, in HIV positive patients were 24 mm of average of 24%. Among the intestinal coccidia, Cryptosporidium sp. is more prominent due to its zoonotic nature. Recovery records of dogs and cats were found in 100% of treated animals in a veterinary hospital. In addition, cattle may also act as reservoirs, with prevalences in Brazil varying from 0.6% to 72.13%. Currently, there are several methodologies for the diagnosis of these coccidians, from the microscopic methods that present the main techniques for the diagnosis of these coccidians. In addition to the medical importance, the prevalence of intestinal coccidioses is relevant as an indicator of the socioeconomic status of a given population and the intensity of energy is an anthropopathic or zoonotic. However, improvements are still needed to make new diagnostic techniques more accessible, in order to detect intestinal coccidia more easily and reliably.

Novel recombinant multiepitope proteins for the detection of anti-Cryptococcus antibodies.

AIM: To produce and test recombinant multiepitope proteins as an alternative assay for the serological diagnosis of cryptococcosis. MATERIALS & METHODS: Previously, synthetic peptides were used to detect anti-Cryptococcus antibodies, and in silico analyses showed that the union of peptides would improve the results. Here, the coding sequences of these peptides were assembled into synthetic genes. Four genes have been cloned and expressed in Escherichia coli, producing recombinant multiepitope proteins: proteins A, B, C and D. RESULTS: All constructs yielded good results; however, protein D showed the best results, with a sensitivity of 88.57% and specificity of 100%. CONCLUSION: The multiepitope proteins were shown to be potential antigens for the diagnosis of cryptococcosis in an attempt to detect anti-Cryptococcus antibodies.

Canine visceral leishmaniasis follow-up: a new anti-IgG serological test more sensitive than ITS-1 conventional PCR.

Visceral leishmaniasis (VL) is a neglected tropical disease with dogs serving as reservoirs for one of its etiological agents, Leishmania infantum. In Brazil, VL control involves culling of seropositive dogs, among other actions. However, the most employed serological tests lack accuracy, and are not able to detect canine visceral leishmaniasis (CVL) during the early stages of infection. Early detection of CVL is highly desirable in order to shorten the contact time between the infected reservoirs and the vectors. In this study, we investigated the ability of two multiepitope proteins, PQ10 and PQ20, to detect CVL at earlier stages than currently employed methods, including ITS-1 conventional PCR. Using serum samples from naturally infected dogs, we observed that ELISA-PQ10 and ELISA-PQ20 were able to detect Leishmania infection at earlier time points as compared with kDNA PCR-RFLP in anti-IgG and anti-IgM assays. Using sera from experimentally infected dogs, we monitored seroconversion using multiepitope proteins, ELISA-crude antigen, as well as ITS-1 conventional and real-time PCR. While seroconversion was detected by ELISA-crude antigen in 16.6% of the dogs, multiepitope proteins were able to detect seroconversion in more than 80% of them. Moreover, the ability of ELISA-PQ10 and ELISA-PQ20 to detect Leishmania infection at earlier time points as compared with conventional PCR was also confirmed in experimental infection dogs' sera. Immunofluorescence to Babesia canis and Ehrlichia canis did not show cross-reactions with ELISA-PQ10/PQ20 positive samples. Results of real-time PCR and ELISA with multiepitope proteins were very similar, with concordances between 80 and 100%. Furthermore, our findings indicated that PQ10 and PQ20 immunoassays can be related to parasite load. ELISA-PQ10 and ELISA-PQ20 are more sensitive diagnostic tools for early CVL detection as compared with other methods They could potentially be used in screening tests due to easy execution and low costs facilities.

Novel recombinant multiepitope proteins for the diagnosis of asymptomatic leishmania infantum-infected dogs.

BACKGROUND: Visceral leishmaniasis is the most severe form of leishmaniasis. Worldwide, approximately 20% of zoonotic human visceral leishmaniasis is caused by Leishmania infantum, also known as Leishmania chagasi in Latin America. Current diagnostic methods are not accurate enough to identify Leishmania-infected animals and may compromise the effectiveness of disease control. Therefore, we aimed to produce and test two recombinant multiepitope proteins as a means to improve and increase accuracy in the diagnosis of canine visceral leishmaniasis (CVL). METHODOLOGY/PRINCIPAL FINDINGS: Ten antigenic peptides were identified by CVL ELISA in previous work. In the current proposal, the coding sequences of these ten peptides were assembled into a synthetic gene. Furthermore, other twenty peptides were selected from work by our group where good B and T cell epitopes were mapped. The coding sequences of these peptides were also assembled into a synthetic gene. Both genes have been cloned and expressed in Escherichia coli, producing two multiepitope recombinant proteins, PQ10 and PQ20. These antigens have been used in CVL ELISA and were able to identify asymptomatic dogs (80%) more effectively than EIE-LVC kit, produced by Bio-Manguinhos (0%) and DPP kit (10%). Moreover, our recombinant proteins presented an early detection (before PCR) of infected dogs, with positivities ranging from 23% to 65%, depending on the phase of infection in which sera were acquired. CONCLUSIONS/SIGNIFICANCE: Our study shows that ELISA using the multiepitope proteins PQ10 and PQ20 has great potential in early CVL diagnosis. The use of these proteins in other methodologies, such as immunochromatographic tests, could be beneficial mainly for the detection of asymptomatic dogs.

Immunoreactivity of synthetic peptides derived from proteins of Cryptococcus gattii.

AIM: To determine the immunoreactivity of synthetic Cryptococcus-derived peptides. MATERIALS & METHODS: A total of 63 B-cell epitopes from previously identified Cryptococcus gattii immunoreactive proteins were synthesized and evaluated as antigens in ELISAs. The peptides were first evaluated for their ability to react against sera from immunocompetent subjects carrying cryptococcal meningitis. Peptides that yielded high sensitivity and specificity in the first test were then retested with sera from individuals with other fungal pathologies for cross-reactivity determination. RESULTS: Six of 63 synthetic peptides were recognized by antibodies in immunoassays, with a specificity of 100%, sensitivity of 78% and low cross-reactivity. CONCLUSION: We successfully determined the immunoreactivity of selected synthetic peptides of C. gattii derived proteins.

Evaluation of three recombinant Leishmania infantum antigens in human and canine visceral leishmaniasis diagnosis.

Visceral leishmaniasis (VL) is a neglected disease and is fatal if untreated. Dogs serve as reservoirs for Leishmania infantum (syn. L. chagasi) due to their susceptibility to infection and high skin parasitism. Therefore, VL control in Brazil involves the elimination of seropositive dogs, among other actions. However, the most frequently used serological tests have limitations regarding sensitivity and specificity. In this study, we have selected three Leishmania antigens (C1, C8 and C9) and have produced them as recombinant proteins using pET-28a-TEV vector and Escherichia coli BL-21 as expression system. When tested in ELISA with human samples, the C9 antigen was the one showing the most promising results, with 68% sensitivity and 78% specificity. When testing canine samples, the C1, C8 and C9 antigens showed a sensitivity range from 70% to 80% and specificity range from 60% to 90%. The C1 antigen presented higher sensitivity (80%) and the C8 antigen presented higher specificity (90%). Due to it, we decided to mix and test C1 and C8 antigens together, resulting in the C18 antigen. The mix also yielded high percentages of detected symptomatic and asymptomatic dogs however it did not improve the performance of the diagnostic. Comparison of our tests with the tests recommended by the Brazilian Ministry of Health revealed that our antigens' sensitivities and the percentage of detected asymptomatic dogs were much higher. Our results suggest that the C1, C8, C18 and C9 recombinant proteins are good antigens to diagnose canine visceral leishmaniasis and could potentially be used in screening tests. To diagnose human visceral leishmaniasis, the C9 antigen presented reasonable results, but more optimization must be performed for this antigen to provide better performance.