Seroprevalence of antibodies against SARS-CoV-2 in the school community in Campo Grande, state of Mato Grosso do Sul, Brazil, October 2021-November 2022.

Introduction: With the reopening of schools during the coronavirus disease 2019 (COVID-19) pandemic, it was imperative to understand the role of students and education professionals in the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this paper, we determined the seroprevalence of the SARS-CoV-2 anti-nucleocapsid antibodies in the school community in Campo Grande, the capital and most populous city of the state of Mato Grosso do Sul (Brazil) and evaluated its association with sex, school level, and school type. Materials and methods: The survey was carried out in 20 public and private schools in the urban region of Campo Grande using the TR DPP® COVID-19 immunoglobulin M/immunoglobulin G (IgM/IgG) kit from the Immunobiological Technology Institute (Bio-Manguinhos, Rio de Janeiro, Brazil). Testing was carried out in three periods: from October to December 2021; from March to July 2022; and from August to November 2022. The participants were students aged 6-17 years enrolled in primary or secondary schools and professionals of different ages and roles. Results: During the first testing period, 162 participants were seropositive for the IgM and/or IgG anti-nucleocapsid SARS-CoV-2 antibodies, with an estimated seroprevalence of 19.6% using Bayesian multilevel regression. In the second period, 251 participants were seropositive (estimated seroprevalence, 34.6%), while in the third period, 393 participants were seroconverted (estimated seroprevalence, 56.7%). In 2022, there was an increase in the seroconversion rate compared to that in 2021. The most frequently described acute manifestations in the three periods were fever, headache, sore throat, and runny nose. In terms of the demographic profile, there was no predominance of seropositivity between the sexes, although women represented approximately 70% of the study population. There were also no differences between students and school staff. Discussion: The results made it possible to evaluate the extent of SARS-CoV-2 transmission in the school community through immunity developed against the virus, in addition to providing information about COVID-19 symptoms in children, adolescents, and adults.

Development and validity assessment of ELISA test with recombinant chimeric protein of SARS-CoV-2.

Serological tests developed for COVID-19 diagnostic are based on antibodies specific for SARS-CoV-2 antigens. Most of the antigens consist of a fragment or a whole amino acid sequence of the nucleocapsid or spike proteins. We evaluated a chimeric recombinant protein as an antigen in an ELISA test, using the most conserved and hydrophilic portions of the S1-subunit of the S and Nucleocapsid (N) proteins. These proteins, individually, indicated a suitable sensitivity of 93.6 and 100% and a specificity of 94.5 and 91.3%, respectively. However, our study with the chimera containing S1 and N proteins of SARS-CoV-2 suggested that the recombinant protein could better balance both the sensitivity (95.7%) and the specificity (95.5%) of the serological assay when comparing with the ELISA test using the antigens N and S1, individually. Accordingly, the chimera showed a high area under the ROC curve of 0.98 (CI 95% 0.958-1). Thus, our chimeric approach could be used to assess the natural exposure against SARS-CoV-2 virus over time, however, other tests will be necessary to better understand the behaviour of the chimera in samples from people with different vaccination doses and/or infected with different variants of the virus.

Genomic analysis of an outbreak of bovine tuberculosis in a man-made multi-host species system: A call for action on wildlife in Brazil.

We report on a 15-year-long outbreak of bovine tuberculosis (bTB) in wildlife from a Brazilian safari park. A timeline of diagnostic events and whole-genome sequencing (WGS) of 21 Mycobacterium bovis isolates from deer and llamas were analyzed. Accordingly, from 2003 to 2018, at least 16 animals, from eight species, died due to TB, which is likely an underestimated number. In three occasions since 2013, the deer presented positive tuberculin tests, leading to the park closure and culling of all deer. WGS indicated that multiple M. bovis strains were circulating, with at least three founding introductions since the park inauguration in 1977. Using a previously sequenced dataset of 71 M. bovis genomes from cattle, we found no recent transmission events between nearby farms and the park based on WGS. Lastly, by discussing socio-economic and environmental factors escaping current regulatory gaps that were determinant of this outbreak, we pledge for the development of a plan to report and control bTB in wildlife in Brazil.

Genomic and temporal analyses of Mycobacterium bovis in southern Brazil.

Mycobacterium bovis is a causal agent of bovine tuberculosis (bTB), one of the most important diseases currently facing the cattle industry worldwide. Tracing the source of M. bovis infections of livestock is an important tool for understanding the epidemiology of bTB and defining control/eradication strategies. In this study, whole genome sequencing (WGS) of 74 M. bovis isolates sourced from naturally infected cattle in the State of Rio Grande do Sul (RS), southern Brazil, was used to evaluate the population structure of M. bovis in the region, identify potential transmission events and date the introduction of clonal complex (CC) European 2 (Eu2). In silico spoligotyping identified 11 distinct patterns including four new profiles and two CCs, European 1 (Eu1) and Eu2. The analyses revealed a high level of genetic diversity in the majority of herds and identified putative transmission clusters that suggested that within- and between-herd transmission is occurring in RS. In addition, a comparison with other published M. bovis isolates from Argentina, Brazil, Paraguay and Uruguay demonstrated some evidence for a possible cross-border transmission of CC Eu1 into RS from Uruguay or Argentina. An estimated date for the introduction of CC Eu2 into RS in the middle of the 19th century correlated with the historical introduction of cattle into RS to improve existing local breeds. These findings contribute to the understanding of the population structure of M. bovis in southern Brazil and highlight the potential of WGS in surveillance and helping to identify bTB transmission.

Bovine tuberculosis in safari park in Brazil

ABSTRACT: Bovine tuberculosis (bTB) is an infectious disease caused by Mycobacterium bovis, affecting domestic animals, wild animals and humans. In captivity, for wild animals, bTB represents a risk to animal keepers and zoo visitors, in addition to the possibility of spreading the infection to domestic animals or through the trade of infected wild animals. Sambar (Cervus unicolor), red deer (Cervus elaphus) and fallow deer (Dama dama) from a safari park in the State of Rio Grande do Sul, Brazil, showed a clinical condition of dyspnea and weight loss. Some animals died and showed lesions suggestive of tuberculosis (LST), which were confirmed by histopathology. After the interdiction of the safari park by the state veterinary authorities, 281 deer were euthanized with the authorization of the Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis (IBAMA). Retropharyngeal and submandibular lymph nodes and viscera were collected from 21 animals, which were grown in Stonebrink medium for up to 90 days. After DNA extraction from the bacterial colonies, PCR was performed for targets flanking the region of differentiation 4 (RD4). Of the 21 samples, 14 (66.7%) presented LST with a granulomatous appearance, a whitish coloration, and caseous or calcified consistency, and seven samples (33.3%), showed no lesions. In the culture of 14 samples with LST, 13 (92.8%) presented bacterial growth compatible with M. bovis. In the cultivation of the seven samples without LST, four (57.1%) presented colonies compatible with M. bovis. PCR and DNA sequencing of the PCR amplicons detected as positive all the 17 (100%) bacteriological cultures suggestive of M. bovis, thus confirming the outbreak of bTB in deer. Decisions about positive tested and suspicious animals should be taken based on the evaluation of the risk of transmission to the rest of the zoological animals, animal welfare, conservation considerations and, the zoonotic potential of this pathogen.

RESUMO: A tuberculose bovina (bTB) é uma doença infecciosa causada por Mycobacterium bovis, afetando animais domésticos, animais selvagens e humanos. Para animais selvagens em cativeiro, a bTB representa um risco para os tratadores de animais e visitantes do zoológico, além da possibilidade de espalhar a infecção para animais domésticos ou por meio do comércio de animais silvestres infectados. Cervídeos sambar (Cervus unicolor), veado-vermelho (Cervus elaphus) e gamo (Dama dama) de um parque safári no Estado do Rio Grande do Sul, Brasil, mostraram uma condição clínica de dispneia e perda de peso. Alguns animais morreram e apresentaram lesões sugestivas de tuberculose (LST), as quais foram confirmadas por histopatologia. Após a interdição do parque safári pelas autoridades veterinárias estaduais, 281 veados sofreram eutanásia com a autorização do Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis (IBAMA). Os linfonodos retrofaríngeos e submandibulares e vísceras foram coletados de 21 animais, que foram cultivados em meio Stonebrink por até 90 dias. Após extração de DNA das colônias bacterianas, foi realizada PCR para alvos que flanqueavam a região de diferenciação 4 (RD4). Das 21 amostras, 14 (66,7%) apresentaram LST com aspecto granulomatoso, coloração esbranquiçada e consistência caseosa ou calcificada, e sete amostras (33,3%) não apresentaram lesões. Na cultura de 14 amostras com LST, 13 (92,8%) apresentaram crescimento bacteriano compatível com M. bovis. No cultivo das sete amostras sem LST, quatro (57,1%) apresentaram colônias compatíveis com M. bovis. A PCR e o sequenciamento de DNA dos fragmentos de PCR detectaram como positivo todas as 17 (100%) culturas bacteriológicas sugestivas de M. bovis, confirmando assim o surto de bTB em cervídeos. As decisões sobre animais positivos testados e suspeitos devem ser tomadas com base na avaliação do risco de transmissão para o restante dos animais zoológicos, bem-estar animal, considerações de conservação e no potencial zoonótico desse patógeno.

Bovine tuberculosis in safari park in Brazil / Tuberculose bovina em parque safári no Brasil

Bovine tuberculosis (bTB) is an infectious disease caused by Mycobacterium bovis, affecting domestic animals, wild animals and humans. In captivity, for wild animals, bTB represents a risk to animal keepers and zoo visitors, in addition to the possibility of spreading the infection to domestic animals or through the trade of infected wild animals. Sambar (Cervus unicolor), red deer (Cervus elaphus) and fallow deer (Dama dama) from a safari park in the State of Rio Grande do Sul, Brazil, showed a clinical condition of dyspnea and weight loss. Some animals died and showed lesions suggestive of tuberculosis (LST), which were confirmed by histopathology. After the interdiction of the safari park by the state veterinary authorities, 281 deer were euthanized with the authorization of the "Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis" (IBAMA). Retropharyngeal and submandibular lymph nodes and viscera were collected from 21 animals, which were grown in Stonebrink medium for up to 90 days. After DNA extraction from the bacterial colonies, PCR was performed for targets flanking the region of differentiation 4 (RD4). Of the 21 samples, 14 (66.7%) presented LST with a granulomatous appearance, a whitish coloration, and caseous or calcified consistency, and seven samples (33.3%), showed no lesions. In the culture of 14 samples with LST, 13 (92.8%) presented bacterial growth compatible with M. bovis. In the cultivation of the seven samples without LST, four (57.1%) presented colonies compatible with M. bovis. PCR and DNA sequencing of the PCR amplicons detected as positive all the 17 (100%) bacteriological cultures suggestive of M. bovis, thus confirming the outbreak of bTB in deer. Decisions about positive tested and suspicious animals should be taken based on the evaluation of the risk of transmission to the rest of the zoological animals, animal welfare, conservation considerations and, the zoonotic potential of this pathogen.(AU)

A tuberculose bovina (bTB) é uma doença infecciosa causada por Mycobacterium bovis, afetando animais domésticos, animais selvagens e humanos. Para animais selvagens em cativeiro, a bTB representa um risco para os tratadores de animais e visitantes do zoológico, além da possibilidade de espalhar a infecção para animais domésticos ou por meio do comércio de animais silvestres infectados. Cervídeos sambar (Cervus unicolor), veado-vermelho (Cervus elaphus) e gamo (Dama dama) de um parque safári no Estado do Rio Grande do Sul, Brasil, mostraram uma condição clínica de dispneia e perda de peso. Alguns animais morreram e apresentaram lesões sugestivas de tuberculose (LST), as quais foram confirmadas por histopatologia. Após a interdição do parque safári pelas autoridades veterinárias estaduais, 281 veados sofreram eutanásia com a autorização do Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis (IBAMA). Os linfonodos retrofaríngeos e submandibulares e vísceras foram coletados de 21 animais, que foram cultivados em meio Stonebrink por até 90 dias. Após extração de DNA das colônias bacterianas, foi realizada PCR para alvos que flanqueavam a região de diferenciação 4 (RD4). Das 21 amostras, 14 (66,7%) apresentaram LST com aspecto granulomatoso, coloração esbranquiçada e consistência caseosa ou calcificada, e sete amostras (33,3%) não apresentaram lesões. Na cultura de 14 amostras com LST, 13 (92,8%) apresentaram crescimento bacteriano compatível com M. bovis. No cultivo das sete amostras sem LST, quatro (57,1%) apresentaram colônias compatíveis com M. bovis. A PCR e o sequenciamento de DNA dos fragmentos de PCR detectaram como positivo todas as 17 (100%) culturas bacteriológicas sugestivas de M. bovis, confirmando assim o surto de bTB em cervídeos. As decisões sobre animais positivos testados e suspeitos devem ser tomadas com base na avaliação do risco de transmissão para o restante dos animais zoológicos, bem-estar animal, considerações de conservação e no potencial zoonótico desse patógeno.(AU)

Genomic analysis of an outbreak of bovine tuberculosis in a man-made multi-host species system: a call for action on wildlife in Brazil

We report on a 15-year-long outbreak of bovine tuberculosis (bTB) in wildlife from a Brazilian safari park. A timeline of diagnostic events and whole-genome sequencing (WGS) of 21 Mycobacterium bovis isolates from deer and llamas were analyzed. Accordingly, from 2003 to 2018, at least 16 animals, from eight species, died due to TB, which is likely an underestimated number. In three occasions since 2013, the deer presented positive tuberculin tests, leading to the park closure and culling of all deer. WGS indicated that multiple M. bovis strains were circulating, with at least three founding introductions since the park inauguration in 1977. Using a previously sequenced dataset of 71 M. bovis genomes from cattle, we found no recent transmission events between nearby farms and the park based on WGS. Lastly, by discussing socio-economic and environmental factors escaping current regulatory gaps that were determinant of this outbreak, we pledge for the development of a plan to report and control bTB in wildlife in Brazil.

Matrix Assisted Laser Desorption Ionization-Time-of-Flight mass spectrometry identification of Mycobacterium bovis in Bovinae.

In this study, Matrix Assisted Laser Desorption Ionization-Time-of-Flight (MALDI-TOF) mass spectrometry was used to identify Mycobacterium bovis from cattle and buffalo tissue isolates from the North and South regions of Brazil, grown in solid medium and previously identified by Polymerase Chain Reaction (PCR) based on Region of Difference 4 (RD4), sequencing and spoligotyping. For this purpose, the protein extraction protocol and the mass spectra reference database were optimized for the identification of 80 clinical isolates of mycobacteria. As a result of this optimization, it was possible to identify and differentiate M. bovis from other members of the Mycobacterium tuberculosis complex with 100% specificity, 90.91% sensitivity and 91.25% reliability. MALDI-TOF MS methodology described herein provides successful identification of M. bovis within bovine/bubaline clinical samples, demonstrating its usefulness for bovine tuberculosis diagnosis in the future.

ELISA using a recombinant chimera of ESAT-6/MPB70/MPB83 for Mycobacterium bovis diagnosis in naturally infected cattle.

Bovine tuberculosis (bTB) control programs generally rely on intradermal tuberculin tests for the antemortem diagnosis of Mycobacterium bovis infection in cattle, but these tests detect only a portion of the infected animals. The aim of the present study was to evaluate the diagnostic coverage of a combination of the bTB antemortem techniques known as the comparative intradermal tuberculin test (CITT) and an ELISA based on a recombinant chimera of ESAT-6/MPB70/MPB83 as the antigen in cattle. The results were compared to postmortem findings based on M. bovis culturing and PCR. Paired comparisons of all data (n=92) demonstrated that ELISA and LST results compared to the culturing results did not present significant differences (P=0.27 on McNemar's test and P=0.12 on Fisher's exact test, respectively). Using culturing as the gold standard, the sensitivity and specificity of ELISA were 79.5% (95% CI: 64.5-89.2%) and 75.5% (95% CI: 62.4-85.1%), respectively, whereas LST demonstrated 100% sensitivity (95% CI: 91.03-100%) and 92.5% specificity (95% CI: 82.1-97.0%). The ELISA results did not reveal significant differences in relation to the LST results (P>0.99 on Fisher's exact test). Using the latter as the gold standard, the sensitivity and specificity of ELISA were 79.1% (95% CI: 64.8-88.6%) and 79.6% (95% CI: 66.4-88.5%), respectively. The use of ELISA with the recombinant chimera of ESAT-6/MPB70/MPB83 as the antigen complements the diagnostic coverage provided by CITT and increases the removal of infected animals from herds.

False-negative reactions to the comparative intradermal tuberculin test for bovine tuberculosis / Reações falso-negativas ao teste cervical comparativo para tuberculose bovina

According to the Brazilian National Program for the Control and Eradication of Animal Brucellosis and Tuberculosis (PNCEBT), the routine tests for the diagnosis of bovine tuberculosis in the country are the simple intradermal tuberculin test (SITT) of the Ministry of Agriculture, Livestock and Food Supply (MAPA), the caudal fold test and the comparative intradermal tuberculin test (CITT). The latter is also used as a confirmatory test. A group of 53 animals from three dairy herds in a focal area for bovine tuberculosis, that were submitted to depopulation in the state of Rio Grande do Sul, were submitted to the CITT. Tissues were cultured and the resulting colonies were confirmed by PCR and DNA sequencing. Among the 53 animals analyzed using the CITT, 32 (60.4%) were negative, 14 (26.4%) were positive and seven (13.2%) results were inconclusive. The CITT detected 11 of the 39 animals with culture-confirmed M. bovis infection as positive. Among the total of 14 uninfected animals based on cultures, the CBT detected eight as negative. Thus, the CITT demonstrated sensitivity of 28.2% and specificity of 57.1% for the population sampled. A total of 24/32 (75.0%) of the animals with negative CITT results were culture positive (confirmed by PCR) and were considered false negatives based on the CITT. The maintenance of these false-negative animals in herds has serious implications for the control of the disease, since they can be a source of infection. The addition of complementary tests could help identify such animals and increase the odds of diagnostic success.(AU)

No Brasil, segundo o Programa Nacional de Controle e Erradicação da Brucelose e Tuberculose Animal (PNCEBT), do Ministério da Agricultura, Pecuária e Abastecimento (MAPA), os testes de rotina para o diagnóstico de tuberculose bovina são o teste cervical simples (TCC), o teste da prega caudal (TPC) e o teste cervical comparativo (TCC), sendo que o último também é utilizado como teste confirmatório. Um grupo de 53 animais oriundos de três rebanhos leiteiros de área de foco para tuberculose bovina que foram submetidos a vazio sanitário no Rio Grande do Sul foi submetido ao TCC. Os tecidos destes animais foram cultivados e as colônias resultantes confirmadas por PCR e sequenciamento de DNA. Dos 53 animais analisados no TCC, 32 (60,4%) foram negativos, 14 (26,4%) positivos e sete (13,2%) inconclusivos, com base no PNCEBT. O TCC detectou como positivos 11 dos 39 animais com infecção por M. bovis confirmada por cultivo. Do total de 14 animais não infectados, baseado na cultura, o TCC detectou oito como negativos. Assim, o TCC apresentou, para a população amostrada, sensibilidade de 28,2% e especificidade de 57,1%. Um total de 24/32 (75,0%) dos animais negativos ao TCC foi positivo no cultivo (confirmado por PCR), sendo considerados falso-negativos ao TCC. A manutenção destes animais falso-negativos nos rebanhos tem sérias implicações para o controle da enfermidade, já que os mesmos podem ser fonte de infecção. A adição de testes complementares poderia auxiliar na identificação destes animais, aumentando a cobertura diagnóstica.(AU)