Identification of Viruses in Molossus Bats from the Brazilian Amazon: A Descriptive Metagenomic Analysis.

Bats are widely distributed in Brazil, including the Amazon region, and their association with viral pathogens is well-known. This work aimed to evaluate the metavirome in samples of Molossus sp. bats captured in the Brazilian Amazon from 2019 to 2021. Lung samples from 58 bats were divided into 13 pools for RNA isolation and sequencing followed by bioinformatic analysis. The Retroviridae family showed the highest abundance of viral reads. Although no complete genome could be recovered, the Paramyxoviridae and Dicistroviridae families showed the formation of contigs with satisfactory identity and size characteristics for further analysis. One contig of the Paramyxoviridae family was characterized as belonging to the genus Morbillivirus, being grouped most closely phylogenetically to Porcine morbillivirus. The contig related to the Dicistroviridae family was identified within the Cripavirus genus, with 94%, 91%, and 42% amino acid identity with Culex dicistrovirus 2, Rhopalosiphum padi, and Aphid lethal paralysis, respectively. The presence of viruses in bats needs constant updating since the study was able to identify viral sequences related to families or genera still poorly described in the literature in association with bats.

Histopathological and molecular methods as complementary diagnostic in case of lymphadenopathies suggestive of bovine tuberculosis.

Bovine tuberculosis (TB) is a chronically evolving zoonotic infectious disease caused by Mycobacterium bovis. Anatomopathological examination during post mortem inspection in bovines is the main resource engaged in sanitary slaughter; however, it is very troublesome since many granulomatous inflammatory processes have similar morphological characteristics. Thus, this study aims to use complementary diagnosis methods (histopathological and polymerase chain reaction - PCR assays) to confirm the macroscopic assessment of lymphadenopathies indicative of tuberculosis in bovines slaughtered in a refrigerated slaughterhouse in Tailândia city, PA, Brazil. Fifty­one samples were collected from lesions indicative of tuberculosis in pre­scapular and pre­pectoral lymph nodes (or different lymphadenitis) in condemned carcasses. Histological processing employed routine techniques carried out at the Laboratory of Animal Pathology of the Federal Rural University of the Amazon, while the PCR assay was performed at the Bacteriology Laboratory of the Evandro Chagas Institute. Results showed that 1.96% of the histopathology samples corresponded to inflammatory processes typical of TB and that, in PCR, 4.25% of the samples had the amplification profile of the M. bovis species. These results indicate the importance of adding complementary methods to assist the sanitary inspection line and make inspection more efficient in its decisions.

Arboviruses in Free-Ranging Birds and Hematophagous Arthropods (Diptera, Nematocera) from Forest Remnants and Urbanized Areas of an Environmental Protection Area in the Amazon Biome.

The rapid and disorderly urbanization in the Amazon has resulted in the insertion of forest fragments into cities, causing the circulation of arboviruses, which can involve hematophagous arthropods and free-ranging birds in the transmission cycles in urban environments. This study aimed to evaluate the circulation of arboviruses in free-ranging birds and hematophagous arthropods captured in an Environmental Protection Area in the Belem metropolitan area, Brazil. Birds were captured using mist nets, and hematophagous arthropods were collected using a human protected attraction technique and light traps. The birds' sera were subjected to a hemagglutination inhibition test to detect antibodies against 29 arbovirus antigens. Arthropod macerates were inoculated into C6/36 and VERO cell cultures to attempt viral isolation and were tested using indirect immunofluorescence, subsequent genetic sequencing and submitted for phylogenetic analysis. Four bird sera were positive for arbovirus, and one batch of Psorophora ferox was positive for Flavivirus on viral isolation and indirect immunofluorescence. In addition, the Ilheus virus was detected in the sequencing and phylogenetic analysis. The presence of antibodies in sera from free-ranging birds and the isolation of Ilheus virus in Psorophora ferox indicate the circulation of arboviruses in forest remnants in the urban center of Belem.

Aspectos epidemiológicos da infecção por Ehrlichia canis, Anaplasma platys e Mycoplasma spp. em gatos domésticos / Epidemiological aspects of infection by Ehrlichia canis, Anaplasma platys and Mycoplasma spp. in domestic cats

Hemoparasitoses vêm se tornando cada vez mais importantes na clínica médica de pequenos animais. Dentre os agentes causadores encontramos Ehrlichiacanis, Anaplasmaplatys., e Mycoplasma spp., torna-se de grande importância conhecer a epidemiologia nos gatos domésticos. Objetivou-se com esta pesquisa fazer um levantamento retrospectivo de fichas de gatos advindos de consultas no Hospital Veterinário Mário Dias Teixeira (HOVET) que realizaram exame de Reação de Cadeia da polimerase (PCR) no laboratório de biologia molecular, na Universidade Federal Rural da Amazônia, no ano de 2018 e 2019. No total foram 72 amostras de gatos domésticos processadas, sendo 33 machos e 39 fêmeas, 70 animais SRD e 2 Siameses, todos com trombocitopenia, além de outros sinais clínicos que os levaram a precisar de atendimento veterinário, foram categorizados os meses de entrada e processamento das amostras, bairros dos animais e grupos etários. De todos os animais testados, 34,7% obtiveram diagnóstico positivo para uma das enfermidades, sendo o gênero Mycoplasma spp. o que mais prevaleceu em amostras positivas, com maior frequência em fêmeas adultas, bem como foi descrita ocorrência de E. canis apenas nesse sexo, já A. platysfoi descrito com maior frequência em machos, além de achados de infecções concomitantes observado entre os agentes Anaplasmae Mycoplasma. Concluímos que os gatos atendidos no HOVET possuíam parasitismo por diferentes agentes infecciosos.

Hemoparasitosis have become increasingly important in the small animals' internal medicine. Among the causal agents, there are Ehrlichiacanis, Anaplasmaplatys. and Mycoplasma spp., which give the understanding of the epidemiology in domestic cats a great significance. This research aimed to make a retrospective survey of records from cats that came from appointments at the Veterinary Hospital Mário Dias Teixeira (HOVET) and underwent the Polymerase Chain Reaction (PCR) test at the molecular biology laboratory, at the Amazônia Federal Rural University (UFRA), in the years of 2018 and 2019. In total, 72 samples of domestic cats were processed, from which 33 were males and 39 females, 70 of them were mongrel cats and 2 siamese, all of them showed thrombocytopenia amongst other clinical signs that led them to need a veterinary appointment, the months of admission, processing of the samples, districts the animals came from and age group were categorized. 34,7% of all the animals tested showed positive results for one of the diseases, with the genus Mycoplasma spp. being the most prevalent in positive samples, showing a higher rate in adult females, as the occurrence of E. canis was reported only in females, while A. platys was reported with a higher rate in males, as well as concomitant infections following the observation of the agents Anaplasma and Mycoplasma. In conclusion, the cats admitted at HOVET showed parasitism by different infectious agents.

Detecção do vírus da imunodeficiência felina em gatos domésticos (Felis catus) pela técnica de reação em cadeia da polimerase / Detection of feline immunodeficiency virus in domestic cats (Felis catus) by polymerase chain reaction technique

O objetivo deste trabalho foi detectar a presença de DNA do Vírus da Imunodeficiência Felina (FIV) em gatos domesticos (Feliz catus) assintomáticos. Foi realizada a tecnica de reação em cadeia da polimerase (PCR) em 50 animais. Para tal, foram coletadas amostras de sangue, por venopunção da jugular, de forma asséptica para armazenamento de 1-2 mL de sangue total. Os animais que participaram do estudo fizeram parte do projeto de castração "Vida digna" da Universidade Federal Rural da Amazônia. E a escolha dos animais foi realizada de maneira aleatória, sem distinção por sexo ou idade, resultando em 29 foram fêmeas e 21 machos. Para o diagnóstico, foi realizada a extração do DNA, em seguida as amostras foram testadas em duas reações de PCR utilizando- se dois conjuntos de primers do Gene gag de FIV. Achou-se uma prevalência de 2% (1/50), confirmando assim a presença do vírus na cidade de Belém. Assim, evidenciando a importância de testar os felinos mesmo sendo assintomáticos. Desta forma, faz-se necessário a realização de trabalhos futuros que amplie o número amostral dos animais testados para assim elucidar o perfil epidemiológico da doença na região de Belém do Pará, considerando a relevância clínica desta infecção e a correta conduta médica veterinária para evitar novas infecções.

The objective of this work was to detect the presence of Feline Immunodeficiency Virus (FIV) proviral DNA in asymptomatic domestic cats (Feliz catus). The polymerase chain reaction technique was performed from 50 animals. For this, blood samples were collected by jugular venipuncture, aseptically for storage of 1-2 mL of whole blood. The animals that participated in the study were part of the castration project "Vida digna" at the Universidade Federal Rural da Amazônia. And the choice of animals was performed randomly, without distinction by sex or age, resulting in 29 females and 21 males. For diagnosis, DNA extraction was performed, then the samples were tested in two PCR reactions using two sets of FIV gag gene primers. A prevalence of 2% (1/50) was observed, thus confirming the presence of the virus in the city of Belém. Thus, highlighting the importance of testing the felines even if they are asymptomatic. Therefore, it is necessary to carry out future work that expands the sample number of animals tested in order to elucidate the epidemiological profile of the disease in the region of Belém do Pará, considering the clinical relevance of this infection and the correct veterinary medical conduct to avoid new infections.

Factors associated with the prevalence of antibodies against Brucella abortus in water buffaloes from Santarém, Lower Amazon region, Brazil.

We evaluated the factors associated with the prevalence of antibodies against Brucella abortus in buffaloes in the municipality of Santarém, Western Pará, northern Brazil. The study was conducted on 60 farms, representing 25.8% of the total buffalo farms in the region. From those farms, a total of 426 buffaloes were sampled, males of any age and females more than 24 months of age, to avoid a false-positive reaction in the serological test due to vaccination. The Acidified Agglutination Serum Test was carried out on serum samples using B. abortus strain 1,119-3 as the antigen. Univariate and multivariate analysis were performed to investigate the association between brucellosis and potential risk factors. Of the 426 tested buffaloes, 29 were positive, resulting in an overall animal prevalence of antibodies against B. abortus at the animal level of 6.8% (4.6-9.6; 95% confidence interval). The herd level prevalence was 30% (18 of 60) and seroprevalence range within farms was from 0% to 100%. At the animal level, buffaloes raised in the floodplains tended (p = 0.06) to present a higher seroprevalence (9.70%) of antibodies against B. abortus than buffaloes raised in dry land (4.98%) and cows tended (p = 0.054) to have a higher seroprevalence than male buffaloes. Multivariate herd-level analysis revealed association between farm type and brucellosis seroprevalence (p = 0.015); dairy farms were two times more likely to have seropositive buffalo than beef farms. Our survey demonstrated a high farm seroprevalence of B. abortus in buffalo raised in an Amazonian ecosystem with positive animals found in one third of sampled farms.

Molecular epidemiology of mycobacteria among herds in Marajó Island, Brazil, reveals strains genetically related and potential zoonotic risk of clinical relevance.

Mycobacterium bovis is the main causative agent of bovine tuberculosis (bTB) being among the animal-adapted Mycobacterium tuberculosis complex. Herds can also be infected with non-tuberculous mycobacteria (NTM) causing a negative effect on the economy and on animal and human health through zoonotic infections. Molecular tools are required for mycobacteria identification; thus, it is laborious to determine the epidemiological information of mycobacteria among herds. We aimed to describe the mycobacterial pathogens associated with cases of suspected bTB lesions in cattle/buffaloes slaughtered for consumption and to investigate bTB transmission. We evaluated 74 lesion samples from 48 animals (27 bovine/21 buffaloes) from 16 mapped farms. Positives samples from nested-PCR were cultured in Lowenstein-Jensen (LJ), 2% pyruvate (LJ + P), and 2% glycerol (LJ + G) media, followed by Ziehl-Neelsen (ZN) staining technique and partial gene sequencing (hsp65, rpoB, and 16S-rRNA). Spoligotyping and 24-MIRU-VNTR were performed. The LJ + P increased the chance of obtaining bacilli. The respiratory tract and the oral cavity were the most important infection route. In addition, the calcified part of the lesions suggested chronic bTB. Spoligotypes of M. bovis (SIT986/SB0885) differed from others found in South America, and the MIRU-VNTR 24 loci suggested that bTB was associated to highly related strains. The NTM species found are of clinical importance in humans.

Clinical and serological tests for arboviruses in free-living domestic pigeons (Columba livia).

BACKGROUND: In this study, we evaluated the role of free-living domestic pigeons (Columba livia) as a reservoir of arboviruses in the city of Belém, state of Pará, Brazil. We investigated the presence of antibodies against the most prevalent arboviruses. OBJECTIVES: This study was aimed at evaluating some clinical and physical parameters of domestic pigeons, including the presence of antibodies to Amazon-endemic arboviruses. METHODS: Eighty-five healthy pigeons were captured in Mangal das Garças Park, in Belém, and were bled. Upon capture, the birds were subjected to a clinical examination in search of alterations that could indicate the presence of arboviruses. Blood samples were converted to serum and tested using the haemagglutination inhibition (HI) technique with a panel of 19 antigens of arboviruses circulating in the Amazon. The confirmation assay for the positive reactions to the viral species tested by HI was a neutralisation test in new-born Swiss albino mice (Mus musculus) [mouse neutralisation test (MNT)]. FINDINGS: A total of 10 (11.8%) serum samples tested positive for antiflavivirus antibodies by HI. All the samples positive for the HI test were subjected to MNT for detection of viruses and yielded negative results (logarithmic neutralisation index < 1.7). MAIN CONCLUSION: The results represent the first serological detection of antiarbovirus antibodies in domestic pigeons as potential hosts of arboviruses in Brazil. The detection of haemagglutination-inhibiting antibodies against genus Flavivirus indicated that there was recent contact between the analysed domestic pigeons and these arboviruses. Further studies are needed to evaluate the role of free-living pigeons in the maintenance cycle and spread of arboviruses in the Amazon.

Clinical and serological tests for arboviruses in free-living domestic pigeons (Columba livia)

BACKGROUND In this study, we evaluated the role of free-living domestic pigeons (Columba livia) as a reservoir of arboviruses in the city of Belém, state of Pará, Brazil. We investigated the presence of antibodies against the most prevalent arboviruses. OBJECTIVES This study was aimed at evaluating some clinical and physical parameters of domestic pigeons, including the presence of antibodies to Amazon-endemic arboviruses. METHODS Eighty-five healthy pigeons were captured in Mangal das Garças Park, in Belém, and were bled. Upon capture, the birds were subjected to a clinical examination in search of alterations that could indicate the presence of arboviruses. Blood samples were converted to serum and tested using the haemagglutination inhibition (HI) technique with a panel of 19 antigens of arboviruses circulating in the Amazon. The confirmation assay for the positive reactions to the viral species tested by HI was a neutralisation test in new-born Swiss albino mice (Mus musculus) [mouse neutralisation test (MNT)]. FINDINGS A total of 10 (11.8%) serum samples tested positive for antiflavivirus antibodies by HI. All the samples positive for the HI test were subjected to MNT for detection of viruses and yielded negative results (logarithmic neutralisation index < 1.7). MAIN CONCLUSION The results represent the first serological detection of antiarbovirus antibodies in domestic pigeons as potential hosts of arboviruses in Brazil. The detection of haemagglutination-inhibiting antibodies against genus Flavivirus indicated that there was recent contact between the analysed domestic pigeons and these arboviruses. Further studies are needed to evaluate the role of free-living pigeons in the maintenance cycle and spread of arboviruses in the Amazon.

Prevalence of antibodies to equine alphaviruses in the State of Pará, Brazil / Prevalência de anticorpos para alphavirus em equinos do estado do Pará, Brasil

The State of Pará comprises 26% of Brazilian Amazon region, where a large diversity of arboviruses has been described. This study sought to assess the prevalence and distribution of hemagglutination inhibition (HI) antibodies against antigens of four alphaviruses (Togaviridae: Alphavirus ) from the species: Eastern equine encephalitis (EEEV), Western equine encephalitis (WEEV), Mayaro virus (MAYV), and Mucambo virus (MUCV) in 753 serum samples of horses in Pará State, Brazil. All investigated arboviruses were detected and indicate that horses are susceptible to these alphaviruses, and show evidences of their active circulation in farm animals in the Brazilian Amazon.(AU)

O estado do Pará corresponde a 26% da Amazônia brasileira, onde uma grande diversidade de arbovírus foi descrita. Este estudo procurou avaliar a prevalência e a distribuição de anticorpos inibidores da hemaglutinação (IH) contra antígenos de quatro alfavirus (Togaviridae: Alphavirus ), das espécies: Vírus da encefalite equina do leste (EEEV), Vírus da encefalite equina do oeste (WEEV), Vírus mayaro (MAYV) e Vírus mucambo (MUCV), de 753 amostras de soro de equinos no estado do Pará, Brasil. Todos os arbovirus pesquisados foram detectados, indicando que os equinos são suscetíveis a esses Alphavirus e mostrando evidências de sua circulação ativa em animais de fazenda na Amazônia brasileira.(AU)

Diagnosis of arboviruses using indirect sandwich IgG ELISA in horses from the Brazilian Amazon.

BACKGROUND: The Amazon as a whole is the largest reservoir of arboviruses worldwide, while the Brazilian Amazon hosts the largest variety of arboviruses isolated to date. In this study, the results of an indirect sandwich IgG ELISA, standardized for 19 arbovirustypes circulating among horses in Brazilian Amazon, were compared to results of the hemagglutination inhibition test. A screening test assessed the conditional probability distribution and a Pearson linear correlation test determined the correlation strength among the absorbance values recorded for viruses from the same family. FINDINGS: Sensitivity varied between 40.85 and 100%; the specificity was low and ranged from 39.71 to 67.0%; and the accuracy varied between 41 and 65.2%. The test developed in this study yielded a large number of serological cross-reactions. CONCLUSIONS: The test can be employed to detect IgG antibodies within one arbovirus family; however, the hemagglutination test or other more specific techniques, such as the serum neutralization test in mice or the plaque-reduction neutralization test, are essential complementary methods for positive cases.

Diagnosis of arboviruses using indirect sandwich IgG ELISA in horses from the Brazilian Amazon

The Amazon as a whole is the largest reservoir of arboviruses worldwide, while the Brazilian Amazon hosts the largest variety of arboviruses isolated to date. In this study, the results of an indirect sandwich IgG ELISA, standardized for 19 arbovirustypes circulating among horses in Brazilian Amazon, were compared to results of the hemagglutination inhibition test. A screening test assessed the conditional probability distribution and a Pearson linear correlation test determined the correlation strength among the absorbance values recorded for viruses from the same family.

Diagnosis of arboviruses using indirect sandwich IgG ELISA in horses from the Brazilian Amazon

The Amazon as a whole is the largest reservoir of arboviruses worldwide, while the Brazilian Amazon hosts the largest variety of arboviruses isolated to date. In this study, the results of an indirect sandwich IgG ELISA, standardized for 19 arbovirustypes circulating among horses in Brazilian Amazon, were compared to results of the hemagglutination inhibition test. A screening test assessed the conditional probability distribution and a Pearson linear correlation test determined the correlation strength among the absorbance values recorded for viruses from the same family.