Phylogenetic analysis of Pneumocystis from pig lungs obtained from slaughterhouses in southern and midwestern regions of Brazil / Análise filogenética de Pneumocystis obtidos de pulmões de suínos provenientes de abatedouros da região sul e centro oeste do Brasil

The Pneumocystis genus is comprised of pathogens dwelling in the lungs of terrestrial, aerial, and aquatic mammals. Occasionally they induce severe pneumonitis, particularly in hosts with severe impairment of the immune system and progressively may fill pulmonary alveolar cavities causing respiratory failure. Molecular genetic studies revealed that Pneumocystis gene sequences present a marked divergence with the host species concerned. In the present study, the genetic diversity of Pneumocystis obtained from lungs of swines was examined by analyzing mitochondrial large subunit (mtLSU) and small subunit (mtSSU) rRNA sequences. The samples were obtained from two slaughterhouses located in two Brazilian states. Phylogenetic analysis demonstrated that genetic groupings within Pneumocystis organisms were in accordance with those of the corresponding hosts and that two clusters were formed. In conclusion, these data show that there are genetically distinct porcine Pneumocystis genotypes with at least two separate clusters in Brazil.

O gênero Pneumocystis compreende patógenos que residem em pulmões de animais terrestres, aéreos e aquáticos. Pode ocasionar uma grave pneumonia, particularmente em hospedeiros com o sistema imunológico seriamente comprometido, o que ocorre por meio de uma progressiva disseminação nas cavidades alveolares, causando insuficiência respiratória. Estudos genéticos, baseados em métodos moleculares, revelaram que as sequências dos genes de Pneumocystis apresentam marcante divergência de acordo com a espécie de hospedeiro. Neste estudo, a diversidade genética das amostras obtidas a partir de pulmões de suínos, provenientes de dois abatedouros localizados em dois estados brasileiros, foi examinada por análise das sequencias dos nucleotídeos dos produtos de PCR dos genes mtLSU e mtSSU do rRNA do Pneumocystis. O resultado confirma a tendência registrada em pesquisas com amostras de outros animais e permite concluir que existem, pelo menos, dois grupos filogenéticos distintos de Pneumocystis de suínos no Brasil.

Detection of Pneumocystis spp. in lung samples from pigs in Brazil.

The genus Pneumocystis is composed of opportunistic fungi currently considered as specific pulmonary pathogens in humans and other mammals. In pigs, Pneumocystis pneumonia (PcP) could create significant economical losses due to its detrimental effects on growth, food conversion, and carcass/viscera condemnation. This study revealed that Pneumocystis organisms could be detected by Grocott's staining or immunohistochemistry in 36.9% of 564 slaughtered pigs from two geographic regions of Brazil. The prevalence of positive cases was 39.9% and 33.9% in pigs slaughtered in Rio Grande do Sul (RS) and Mato Grosso (MT) states, respectively. Among the positive cases in RS, Pneumocystis organisms were observed in 41.9% of 33 histologically normal lungs, and in 58.0% of lungs presenting with histological lesions. In contrast, the prevalence in MT in normal and abnormal lungs was 36.3% and 63.5%, respectively. Major histopathological findings in lungs of infected animals were bronchointerstitial pneumonia (47.6%), suggestive of enzootic pneumonia, and interstitial pneumonia (37.9%), compatible with PcP. The results of this survey strengthened the interest of detecting fungal pathogens, in addition to other infectious agents, and evaluating their financial impact on Brazilian pig industry. Preventive and/or therapeutic strategies should be developed in order to minimize the incidence of respiratory fungal infections in pigs and associated economic losses.

Establishment and partial characterization of an ovine synovial membrane cell line obtained by transformation with Simian Virus 40 T antigen.

The small ruminant lentiviruses, namely caprine arthritis encephalitis virus (CAEV) and Maedi Visna virus (MVV) are grown currently in secondary synovial membrane cells. Primary and secondary cell cultures are sometimes difficult to obtain and support a low number of passages and, therefore, permissive cell lines are needed. A transformed cell line was obtained by transfection of ovine synovial membrane secondary cell culture with a plasmid containing the SV40 large T antigen gene. The transformed cell culture described in this paper showed a higher growth rate and a more homogenous population of fibroblast-like cells when compared to the original ovine synovial membrane secondary cell cultures. Karyotype analysis has indicated the induction of many random chromosome changes, leading to a decrease in chromosome number. The SV40 DNA was detected in the nucleus and in the cytoplasm of transformed cells. The putative expression of large T antigen was presumed by the detection of the corresponding mRNA by PCR. Finally, the transformed ovine synovial membrane cells were shown to be permissive to small ruminant lentiviruses, and these are suggested as a cell line for in vitro isolation and propagation of these viruses.

Deteccao da infecçäo pelo virus da artrite - encefalite caprina: imunodifusao em agar e reacao em cadeia da polimerase com primers degenerados / Detection of caprine arthritis-encephalitis virus infection: agar gel immunodiffusion and polymerase chain reaction with degenerated primers

O objetivo deste trabalho foi analisar amostras de soro e de células sangüíneas de caprinos para detecçäo de anticorpos e DNA proviral do vírus da artrite-encefalite caprina (CAEV), respectivamente. Utilizou-se a técnica de imunodifusäo em ágar (AGID) e a reaçäo em cadeia da polimerase (PCR) com "primers" degenerados. Foram analisadas amostras de diferentes procedências: 39 de Mato Grosso do Sul (MS), 19 de Säo Paulo (SP) e 22 do Ceará (CE), dessas últimas, 12 oriundas de animais importados do Canadá. Os resultados de AGID e PCR foram discordantes, pois o primeiro permitiu a detecçäo de 25 animais soropositivos, enquanto a PCR detectou DNA proviral de CAEV em 16 amostras. Pela PCR foi possível identificar animais infectados cujos testes sorológicos foram negativos pela AGID: oito amostras do MS e um do CE. Säo discutidos diferentes aspectos que poderiam estar envolvidos na discordância dos resultados

Phylogenetic analysis of small ruminant lentiviruses from Southern Brazil.

The first lentivirus isolated from sheep in Brazil was analysed phylogenetically. Evolutionary trees of the proviral 597 nucleotide gag and 432 nucleotide pol sequences obtained by the maximum likelihood method demonstrated that the sheep isolate clustered with prototype Maedi Visna virus whereas three lentiviruses isolated from goats in the same geographic region were close to caprine arthritis encephalitis prototypes. A subsequent comparison of sequence data of these viruses with those contained in the EMBL sequence database revealed that, in contrast to caprine prototypic viruses, all prototypic Maedi Visna viruses contain a deletion of six nucleotides in the gag gene resulting in the deletion of two residues in the central region of capsid protein. This deletion may be a useful marker in the analysis of small ruminant lentiviruses, especially when considering possible transmission of lentiviruses between sheep and goats.

Investigation of some Hypericum species native to Southern of Brazil for antiviral activity.

Three plant species, Hypericum connatum, Hypericum caprifoliatum, Hypericum polyanthemum (Guttiferae), growing in Southern of Brazil were chemically investigated and tested for their antiviral activity against feline immunodeficiency virus (FIV). The chemical analysis revealed the presence of polyphenolic compounds such as tannins and flavonoids. Hypericin was not detected in these species. The aqueous extract (AE), the aqueous extract with low tannin concentration (LTCAE) and the methanolic extract (ME) were tested for their cytotoxic properties in concentrations of 50-150 microg/ml. AE was toxic to CRFK for the three species in all concentrations. LTCAE and ME varied between different concentrations being not toxic or allowing 80% of cell growth. LTCAE and ME (10-50 microg/ml) were analyzed for antiviral activity by inhibition of CPE and measuring FIV genome from cell culture supernatant. LTCAE of all species in this work did not cause any inhibition of FIV. Although no difference was seen in CPE, a lower number of viral particles in the supernatant was observed when FIV infected cells were treated with ME of H. connatum. These results suggest that some plants of the genus Hypericum from Southern Brazil contain compounds with potential antiviral activity against lentiviruses.

Caprine arthritis-encephalitis virus: isolation and identification in Rio Grande do Sul, Brazil.

We describe the first report of caprine arthritis-encephalitis virus (CAEV) isolation in Brazil. In December 1989 a four-year old anglonubian arthritic doe was submitted to euthanasia and its synovial membranes were cultured by explantation. This doe was positive for antibodies to the caprine arthritis-encephalitis virus (CAEV). The reverse transcriptase activity detected in culture supernatants, the characteristic cytopathic effect of lentivirus in these cultures, the detection of viral antigens in concentrated supernatants by the agar gel immunodiffusion test, and the results of the fluorescent antibody test and of Northern blot analyses are consistent with the isolation of the CAEV.

Caprine arthritis-encephalitis virus: isolation and identification in Rio Grande do Sul, Brazil

We describe the first report of caprine arthritis-encephalitis virus (CAEV) isolation in Brazil. In December 1989 a four-year old anglonubian arthritic doe was submitted to euthanasia and its synovial membranes were cultured by explantation. This doe was positive for antibodies to the caprine arthritis-encephalitis virus (CAEV). The reverse transcriptase activity detected in culture supernatants, the characteristic cytopathic effect of lentivirus in these cultures, the detection of viral antigens in concentrated supernatants by the agar gel immunodiffusion test, and the results of the fluorescent antibody test and of Northern blot analyses are consistent with the isolation of the CAEV