Atypical enteropathogenic Escherichia coli (aEPEC) strains are unable to produce the bundle-forming pilus (BFP), which is responsible for the localized adherence pattern, a characteristic of the pathogenicity of typical EPEC strains. The lack of BFP in aEPEC strains suggests that other fimbrial or non-fimbrial adhesins are involved in their adhesion to the host cells. The aim of this study was to investigate the distribution of major subunit fimbrial genes known to be important adherence factors produced by several E. coli pathotypes in a collection of 72 aEPEC strains. Our results demonstrate that a high percentage (94–100%) of aEPEC strains harbored ecpA, fimA, hcpA, and lpfA fimbrial genes. Other fimbrial genes including pilS, pilV, sfpA, daaC, papA, and sfa were detected at lower frequencies (1–8%). Genes encoding fimbrial subunits, which are characteristic of enteroaggregative E. coli or enterotoxigenic E. coli were not found. No correlation was found between fimbrial gene profiles and adherence phenotypes. Since all aEPEC strains contained ecpA, the major pilin gene of the E. coli common pilus (ECP), a subset of ecpA+ strains was analyzed for transcription of ecpRABCDE and production of ECP upon growth in three different culture conditions at 37°C. Transcription of ecpRABCDE occurred in all conditions; however, ECP production was medium dependent. In all, the data suggest that aEPEC strains are highly heterogeneous in terms of their fimbrial gene profiles. Despite lacking BFP production, other mechanisms of cell adherence exist in aEPEC strains to ensure host colonization, e.g., mediated by other prevalent pili such as ECP. Moreover, the production of ECP by aEPEC strains might be influenced by yet unknown post-transcriptional factors.

RESUMO: Porcine circovirus 2 (PCV2) está associado a vários sinais clínicos que são designados coletivamente como Circovirose e tem grande impacto na suinocultura. O isolamento viral é classicamente realizado em células da linhagem PK-15, contudo outras células têm sido testadas. Apesar dos avanços nos estudos com PCV2, o isolamento ainda é um desafio. Diante da dificuldade de manutenção dessas linhagens celulares comumente utilizadas associadas à necessidade do uso de substâncias tóxicas para o isolamento de PCV2, os objetivos do presente trabalho foram descrever o primeiro isolamento de Porcine circovirus 2b em linhagens de células de macrófago (J744) e verificar a taxa de mutação nesse sistema. Uma amostra de pulmão foi submetida ao sequenciamento e agrupada ao genótipo PCV2b. Essa amostra foi utilizada para inocular uma garrafa de J744 (com 30% de confluência em meio RPMI com 10% de soro fetal bovino) e submetida a cinco passagens, as quais foram acompanhadas por reação em cadeia da polimerase quantitativa (PCRq). As cargas virais inicial e final foram de 2,90 × 103 e de 4,45 × 108 cópias de DNA/µL para PCV2b, respectivamente. O sequenciamento confirmou o isolamento e descartou o coisolamento de mais de um genótipo. Após cinco passagens, o isolado apresentou identidade de 99,7%, com descrição de cinco mutações pontuais, uma sinônima e quatro não sinônimas, observadas nas regiões do gene cap e rep. Os resultados obtidos demonstram que as células J744 apresentam a susceptibilidade, e a instabilidade do vírus em J744 será importante para a compreensão do vírus.

Atypical enteropathogenic Escherichia coli (aEPEC) have emerged as a significant cause of pediatric diarrhea worldwide. Due to the absence of BFP fimbriae (bundle-forming pili), aEPEC are unable to develop the 3 h-localized adhesion pattern observed in typical EPEC. Since some aEPEC show the adherence phenotype observed for other pathotypes of E. coli, the aim of this study was to investigate, in aEPEC, the presence of adhesin genes found in other pathogenic E. coli and to analyze, by comparative proteomic analyses, the protein extracts isolated from aEPEC BA320 (localized-like), Ec292/84 (aggregative) and 9100-83 (diffuse) and BA4013 (non-adherent). Initially, transmission electron microscopy demonstrated the presence of different filamentous structures anchored to the surface in the four EPEC isolates. PCR analyses of aEPEC revealed the presence of the genes fimA, fimH, papA, ecpA, ldaH, pilS, daaC, sfpA, lpfA O113 and polymorphic variants (lpfA1 and lpfA2), which are also present in other pathotypes of E. coli. On the other hand, proteomic analyses of aEPEC extracts obtained with an Omnimixer and precipitated with ammonium sulfate, using 2D gel electrophoresis and LC-MS/MS, identified several proteins involved in different functional processes of bacteria, including proteins involved in the mechanism of bacterial adhesion, namely: a histone-like nucleoid structuring protein (H-NS), an outer membrane protein (OmpX), universal stress proteins (Usp), L-fucose mutarotase (FucU) and galactose-binding transport protein (MglB) associated with the action of adhesins of E. coli fimbriae. The protein extract also revealed "hypothetical" proteins, particularly a putative filamentous protein found in aEPEC with the phenotypes of localized-like, diffuse and aggregative adherence. Therefore, we conclude that the genes encoding type 1 fimbriae and ECP fimbriae are highly prevalent in aEPEC isolates, and that the proteins identified in the proteome of four aEPEC strains, which are related to different bacterial functions, can operate as protein complexes, directly or indirectly involved in adhesion processes.

Porcine parvovirus 4 (PPV4) has been reported in several countries and the high rate of concurrent PCV2 infection with PPV4 may trigger the “porcine circovirus disease” (PCVD). This has awakened the interest in how PPV4 virus behaves. The aim of this study was to show that, like other swine viruses, the elimination of PPV4 in semen is intermittent. The study was conducted in the Unidade de Pesquisa e Desenvolvimento de Itapeva da Agência Paulista de Tecnologia dos Agronegócios Regional (Apta Regional). Over a period of six months, four boars were monitored and the semen was collected every 10 days, totalizing 74 semen samples. Through PCR testing, PPV4 was detected in 32/74 (43.2%) semen samples. Three sequences of 284 nt, showed identity varying from 98.2% to 100%, confirming the detection of the virus in all boars. Semen volume and sperm motility did not indicate significant difference between PPV4 positive or negative semen samples (p > 0.05). There was no significant difference between the monthly distributions of positive and negative samples. This is the first study showing intermittent shedding of PPV4 in naturally infected boars. Absence of typical clinical signs and/or influence in semen quantity was also observed.