Mycobacterium avium subsp. paratuberculosis is the cause of Johne's disease in cattle and other ruminants. M. avium subsp. paratuberculosis infection of the bovine host is not well understood; however, it is assumed that crossing the bovine intestinal mucosa is important in order for M. avium subsp. paratuberculosis to establish infection. To examine the ability of M. avium subsp. paratuberculosis to infect bovine epithelial cells in vitro, Madin-Darby bovine kidney (MDBK) epithelial cells were exposed to M. avium subsp. paratuberculosis. It was observed that bacteria can establish infection and replicate within MDBK cells. M. avium subsp. paratuberculosis also has been reported to infect mammary tissue and milk, and we showed that M. avium subsp. paratuberculosis infects bovine mammary epithelial cells (MAC-T cell line). Using polarized MAC-T cell monolayers, it was also determined that M. avium subsp. paratuberculosis crosses apical and basolateral surfaces with approximately the same degree of efficiency. Because M. avium subsp. paratuberculosis can be delivered to the naïve host by milk, it was investigated whether incubation of M. avium subsp. paratuberculosis with milk has an effect on invasion of MDBK cells. M. avium subsp. paratuberculosis exposed to milk entered epithelial cells with greater efficiency than M. avium subsp. paratuberculosis exposed to broth medium or water (P < 0.01). Growth of M. avium subsp. paratuberculosis within MAC-T cells also resulted in augmented ability to subsequently infect bovine MDBK cells (P < 0.001). Microarray analysis of intracellular M. avium subsp. paratuberculosis RNA indicates the increased transcription of genes which might be associated with an invasive phenotype.Mycobacterium avium subsp. paratuberculosis is the etiologic agent of Johne's disease in cattle and other ruminants. It is assumed that M. avium subsp. paratuberculosis infects the young calf by crossing the intestinal barrier. Previous work (3, 26) has indicated that the interaction of M. avium subsp. paratuberculosis with bovine epithelial cells is a complex process which might involve participation of several bacterial and host factors. For example, it has been reported that both in calves and in mice, challenge by the gastrointestinal route results in M. avium subsp. paratuberculosis infecting M cells in the Peyer's patches (23,26). Recently, Secott and colleagues have suggested that the invasion of the intestinal mucosa by M. avium subsp. paratuberculosis is secondary to the binding to fibronectin (26). In addition, Bannantine and colleagues demonstrated a role for a 35-kDa M. avium subsp. paratuberculosis protein in the invasion of cultured bovine epithelial cells (5). The 35-kDa protein is exposed in the outer layer of M. avium subsp. paratuberculosis and has also been associated with Mycobacterium avium invasion of human intestinal cells (22).After M. avium subsp. paratuberculosis crosses the intestinal mucosa, the infection spreads to other organs, leading to the advanced stages of disease. Sever...
Administered by intramuscular injection, a DNA vaccine (pIRF1A-G) containing the promoter regions upstream of the rainbow trout interferon regulatory factor 1A gene (IRF1A) driven the expression of the infectious hematopoietic necrosis virus (IHNV) glycoprotein (G) elicited protective immune responses in rainbow trout (Oncorhynchus mykiss). However, less laborious and cost-effective routes of DNA vaccine delivery are required to vaccinate large numbers of susceptible farmed fish. In this study, the pIRF1A-G vaccine was encapsulated into alginate microspheres and orally administered to rainbow trout. At 1, 3, 5, and 7 d post-vaccination, IHNV G transcripts were detected by quantitative real-time PCR in gills, spleen, kidney and intestinal tissues of vaccinated fish. This result suggested that the encapsulation of pIRF1A-G in alginate microparticles protected the DNA vaccine from degradation in the fish stomach and ensured vaccine early delivery to the hindgut, vaccine passage through the intestinal mucosa and its distribution thought internal and external organs of vaccinated fish. We also observed that the oral route required approximately 20-fold more plasmid DNA than the injection route to induce the expression of significant levels of IHNV G transcripts in kidney and spleen of vaccinated fish. Despite this limitation, increased IFN-1, TLR-7 and IgM gene expression was detected by qRT-PCR in kidney of vaccinated fish when a 10 μg dose of the oral pIRF1A-G vaccine was administered. In contrast, significant Mx-1, Vig-1, Vig-2, TLR-3 and TLR-8 gene expression was only detected when higher doses of pIRF1A-G (50 and 100 μg) were orally administered. The pIRF1A-G vaccine also induced the expression of several markers of the adaptive immune response (CD4, CD8, IgM and IgT) in kidney and spleen of immunized fish in a dose-dependent manner. When vaccinated fish were challenged by immersion with live IHNV, evidence of a dose-response effect of the oral vaccine could also be observed. Although the protective effects of the oral pIRF1A-G vaccine after a challenge with IHNV were partial, significant differences in cumulative percent mortalities among the orally vaccinated fish and the unvaccinated or empty-plasmid vaccinated fish were observed. Similar levels of protection were obtained after the intramuscular administration of 5 μg of pIRF1A-G or after the oral administration of a high dose of pIRF1A-G vaccine (100 μg); with 70 and 56 relative percent survival values, respectively. When fish were vaccinated with alginate microspheres containing high doses of the pIRF1A-G vaccine (50 or 100 μg), a significant increase in the production of anti-IHNV antibodies was detected in serum samples of the vaccinated fish compared with that in unvaccinated fish. At 10 days post-challenge, IHNV N gene expression was nearly undetectable in kidney and spleen of orally vaccinated fish which suggested that the vaccine effectively reduced the amount of virus in tissues of vaccinated fish that survived the challenge. In conclusion, our resul...
The outcomes of a coinfection of rainbow trout ( Oncorhynchus mykiss) with Infectious hematopoietic necrosis virus (IHNV) strain S46 and Infectious pancreatic necrosis virus (IPNV) strain S46 was determined after waterborne infection. Trout infected with the IHNV/IPNV.S46 sample, (a mixed sample containing equal infectious titers of the viruses) showed 50% less mortality than fish infected with either of the reference viruses alone. Forty-five days after the coinfection, IPNV antigens were detected by flow cytometry in 49 to 63% of the leukocytes from the surviving trout; whereas, only 9-15.6% of the leukocytes expressed IHNV viral antigens. IPNV was easily detected by reverse transcription-polymerase chain reaction (RT-PCR), whereas, for IHNV, a second step of amplification of a 753 bp fragment corresponding to the internal sequences of the IHNV G gene was necessary to optimize viral detection. The sequence of the IHNV gene involved in virulence, the glycoprotein (G) gene, was determined for the IHNV.S46 and compared with other sequences available in the GenBank. Changes found were not located in the antigenic domains of the glycoprotein and were considered not significant.
Coinfection of farm-reared salmonids involving two viruses has been described, but there is no report on the interactions between viruses. Here we examine whether infectious pancreatic necrosis virus (IPNV) strain Sp interferes with the growth of infectious hematopoietic necrosis virus (IHNV) strain S46, a coinfected isolate from rainbow trout. When BF-2 cell culture was inoculated with S46 the infective titer of the IHNV fraction decreased by 3 log10 units compared to the growth curve of IHNV in the single infection. RT-PCR assay confirmed this reduction, which after successive passages of the co-infected sample led to a decrease in IHNV mRNA and the absence of the specific PCR product for IHNV. Flow cytometry showed that only 13% of the cells inoculated with S46 strain were infected with IHNV at 48-72 h post infection, in contrast to the 50-80% of cells that were positive for IPNV. Exposure of cells to IHNV for 24 h before infection with IPNV did not affect the infective titers of either virus or the PCR results obtained in simultaneous coinfections. Moreover IHNV was not inhibited when the IPNV inoculum was reduced. So, a multiplicity of infection dependence was demonstrated for IPNV-IHNV interference; the RT-PCR assay described here was found to be a suitable technique for identifying and studying dual viral infections.
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