Evidence of mixed persistent infections in calves born to cows challenged with a pool of bovine viral diarrhea virus isolates

Pregnant cows infected with noncytopathic (NCP) isolates of bovine viral diarrhea virus (BVDV) between days 40 and 120 days of gestation frequently deliver immunotolerant, persistently infected (PI) calves. We herein report the characterization of PI calves produced experimentally through inoculation of pregnant cows with a pool of Brazilian BVDV-1 (n=2) and BVDV-2 isolates (n=2) between days 60 and 90 of gestation. Two calves were born virus positive, lacked BVDV antibodies, but died 7 and 15 days after birth, respectively. Six other calves were born healthy, seronegative to BVDV, harbored and shed virus in secretions for up to 210 days. Analysis of the antigenic profile of viruses infecting these calves at birth and 30 days later with a panel of monoclonal antibodies indicated two patterns of infection. Whereas three calves apparently harbored only one isolate (either a BVDV-1 or BVDV-2), co-infection by two antigenically distinct challenge viruses was demonstrated in three PI calves. Moreover, testing the viruses obtained from the blood of PI calves by an RT-PCR able to differentiate between BVDV-1 and BVDV-2 confirmed the presence/persistence of two co-infecting viruses of different genotypes (BVDV-1 and BVDV-2) in these animals. These findings indicate that persistent infection of fetuses/calves - a well characterized consequence of fetal infection by BVDV - may be established concomitantly by more than one isolate, upon experimental inoculation. In this sense, mixed persistent infections with antigenically distinct isolates may help in understanding the immunological and molecular basis of BVDV immunotolerance and persistence.

Diarrhea outbreaks in suckling piglets due to rotavirus group C single and mixed (rotavirus groups A and B) infections

Porcine group A rotavirus (PoRVA) is a major cause of neonatal diarrhea in suckling and recently weaned piglets worldwide. The involvement of non-group A rotavirus in cases of neonatal diarrhea in piglets are sporadic. In Brazil there are no reports of the porcine rotavirus group C (PoRVC) as etiologic agent of the diarrhea outbreaks in piglets. The aim of this study was to describe the identification of rotavirus group C in single and in mixed infection with rotavirus groups A and B in three neonatal diarrhea outbreaks in suckling (<21-day-old) piglets, with 70% to 80% and 20% to 25% of morbidity and lethality rates, respectively, in three pig herds located in the state of Santa Catarina, Brazil. The diagnosis of PoRV in the diarrheic fecal samples was performed using polyacrylamide gel electrophoresis (PAGE) to identify the presence of porcine rotavirus groups A, B (PoRVB), and C, and by RT-PCR (PoRVA and PoRVC) and semi-nested (SN)-PCR (PoRVB) to partially amplify the VP4 (VP8*)-VP7, NSP2, and VP6 genes of PoRVA, PoRVB, and PoRVC, respectively. One RT-PCR (PoRVA and PoRVC) and SN-PCR (PoRVB) product of each group of rotavirus of each diarrhea outbreak was submitted to nucleotide (nt) sequence analysis. Based on the PAGE technique, 4 (25%) and 1 (6.25%) of the 16 diarrheic fecal samples evaluated in the first outbreak presented PoRVA and PoRVC electropherotype, respectively, and 11 (68.75%) were negative. In the second outbreak, 3 (42.85%) of the 7 fecal samples evaluated presented PoRVA electropherotype, and in 3 (42.85%) and in 1 (14.3%) fecal samples were detected inconclusive and negative results, respectively. Three (30%) of the 10 fecal samples of the third outbreak presented PoRVC electropherotype; 5 (50%) and 2 (20%) samples showed negative and inconclusive results, respectively. Based on the RT-PCR and SN-PCR assays in the first neonatal diarrhea outbreak, PoRVC was detected in 13 (81.2%) of the 16 diarrheic fecal samples evaluated. PoRVC single infection was identified in 4 (25%) of these samples and mixed infections with PoRVA and PoRVB in 9 (56.2%) fecal samples. All of the seven diarrheic fecal samples evaluated from the second neonatal diarrhea outbreak were positive for PoRVC, whereas its mixed infection with other PoRV groups was detected in 4 (57.2%) samples. In the third outbreak, PoRVC in single infection was detected in all of the 10 diarrheic fecal samples analyzed. In the nt sequence analysis, the PoRVA strains of the first and second outbreaks demonstrated higher nt identity with G4P[6] and G9P[23] genotypes, respectively. The PoRVB strains (first and second outbreaks) and the PoRVC strains (first, second, and third outbreaks) showed higher nt identity and clustered in the phylogenetic tree with PoRVB and PoRVC strains that belong to the N4 and I1 genotypes, respectively. This is the first description in Brazil of the involvement of PoRVC in the etiology of diarrhea outbreaks in suckling piglets. The results of this study demonstrated that PoRVC, in both single and mixed infections, is an important enteropathogen involved in neonatal diarrhea outbreaks in piglets and that the use of more sensitive diagnostic techniques allows the identification of mixed infections involving two or even three groups of PoRV, which may be more common than previously reported.

Parasitic infections in germfree animals

The association of vertebrate hosts with the indigenous microbiota and its effect on the response to infections has long been a subject of scientific curiosity. From the first theory supported by Louis Pasteur that life would be impossible in the absence of associated microorganisms to the development of germfree mammals for research, a lot was learned about how the normal microbiota influences the environment in which pathogens may find themselves. In the present review, we attempt to summarize the more recent results from our group and others on the influence of the normal microbiota on the outcome of parasitic infections. Our results and those of others point to a complex relationship between the mammalian system and its indigenous microbiota, leading to greater resistance to some infections and enhanced susceptibility to others

Detection of cytomegalovirus infections by PCR in renal transplant patients

Cytomegalovirus (CMV) is the single most important infectious agent affecting recipients of organ transplants. To evaluate the incidence and the clinical importance of CMV infection in renal transplants in Brazil, 37 patients submitted to renal allograft transplants were tested periodically for the presence of cytomegalovirus DNA in urine using the polymerase chain reaction (PCR), and for the presence of IgM and IgG antibodies against CMV by enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence (IIF). The PCR-amplified products were detected by gel electrophoresis and confirmed by dot-blot hybridization with oligonucleotide probes. Thirty-two of the 37 patients (86.4%) were positive by at least one of the three methods. In six patients, PCR was the only test which detected the probable CMV infection. Ten patients had a positive result by PCR before transplantation. In general, the diagnosis was achieved earlier by PCR than by serologic tests. Active infection occurred more frequently during the first four months after transplantation. Sixteen of the 32 patients (50%) with active CMV infection presented clinical symptoms consistent with CMV infection. Five patients without evidence of active CMV infection by the three tests had only minor clinical manifestations during follow-up. Our results indicate that PCR is a highly sensitive procedure for the early detection of CMV infection and that CMV infection in renal transplant patients is a frequent problem in Brazil.

Prevalence and risk factors for HBV, HCV and HDV infections among injecting drug users from Rio de Janeiro, Brazil

Viral hepatitis constitutes a major health issue, with high prevalence among injecting drug users (IDUs). The present study assessed the prevalence and risk determinants for hepatitis B, C and D viruses (HBV, HCV and HDV) infections among 102 IDUs from Rio de Janeiro, Brazil. Serological markers and HCV-RNA were detected by enzyme immunoassay and nested PCR, respectively. HCV genotyping was determined by restriction fragment length polymorphism analysis (RFLP). HBsAg, anti-HBc and anti-HBs were found in 7.8, 55.8 and 24.7% of IDUs, respectively. In the final logistic regression, HBV infection was independently associated with male homosexual intercourse within the last 5 years (odds ratio (OR) 3.1; 95% confidence interval (CI) 1.1-8.8). No subject presented anti-delta (anti-HD). Anti-HCV was detected in 69.6% of subjects, and was found to be independently associated with needle sharing in the last 6 months (OR 3.4; 95% CI 1.3-9.2) and with longer duration of iv drug use (OR 3.1; 95% CI 1.1-8.7). These data demonstrate that this population is at high risk for both HBV and HCV infection. Among IDUs from Rio de Janeiro, unprotected sexual intercourse seems to be more closely associated with HBV infection, whereas HCV is positively correlated with high risk injecting behavior. Comprehensive public health interventions targeting this population and their sexual partners must be encouraged.

Fungal infections in marrow transplant recipients under antifungal prophylaxis with fluconazole

Fungal infection is one of the most important causes of morbidity and mortality in bone marrow transplant (BMT) recipients. The growing incidence of these infections is related to several factors including prolonged granulocytopenia, use of broad-spectrum antibiotics, conditioning regimens, and use of immunosuppression to avoid graft-versus-host disease (GvHD). In the present series, we report five cases of invasive mold infections documented among 64 BMT recipients undergoing fluconazole antifungal prophylaxis: 1) A strain of Scedosporium prolificans was isolated from a skin lesion that developed on day +72 after BMT in a chronic myeloid leukemic patient. 2) Invasive pulmonary aspergillosis (Aspergillus fumigatus) was diagnosed on day +29 in a patient with a long period of hospitalization before being transplanted for severe aplastic anemia. 3) A tumoral lung lesion due to Rhizopus arrhizus (zygomycosis) was observed in a transplanted patient who presented severe chronic GvHD. 4) A tumoral lesion due to Aspergillus spp involving the 7th, 8th and 9th right ribs and local soft tissue was diagnosed in a BMT patient on day +110. 5) A patient with a history of Ph1-positive acute lymphocytic leukemia exhibited a cerebral lesion on day +477 after receiving a BMT during an episode of severe chronic GvHD. At that time, blood and spinal fluid cultures yielded Fusarium sp. Opportunistic infections due to fungi other than Candida spp are becoming a major problem among BMT patients receiving systemic antifungal prophylaxis with fluconazole.

Factors associated with penicillin-nonsusceptible pneumococcal infections in Brazil

Resistance of Streptococcus pneumoniae is a worldwide, growing problem. Studies of factors associated with resistance to penicillin have not been conducted in Brazil. The objective of the present study was to evaluate factors associated with infection by S. pneumoniae not susceptible to penicillin. A prevalence study was conducted including all patients with a positive culture for S. pneumoniae in a hospital from July 1991 to December 1992 and the year 1994. Of 165 patients identified, 139 were considered to have clinically relevant infections and 88% of them had invasive infections. All infections were community acquired and consisted of pneumonia (44%) and of central nervous system (19%), pelvic or abdominal (12%), upper airway or ocular (12%), primary bloodstream (9%) and skin and soft tissue (5%) infections. Mortality was 25%. Susceptibility to penicillin was present in 77.6% of the isolates; 21.8% were relatively resistant, and one isolate was resistant (minimal inhibitory concentration = 4 µg/ml). Multivariate analysis showed that age below 4 years (odds ratio (OR): 3.53, 95% confidence interval (95%CI): 1.39-8.96) and renal failure (OR: 5.50, 95%CI: 1.07-28.36) were associated with lack of susceptibility to penicillin. Bacteremia occurred significantly less frequently in penicillin-nonsusceptible infections (OR: 0.34, 95%CI: 0.14-0.84), possibly suggesting that lack of penicillin susceptibility is associated with lower virulence in S. pneumoniae.

Patterns of intracellular cytokines in CD4 and CD8 T cells from patients with mycobacterial infections

Using a short-term bulk culture protocol designed for an intracellular-staining method based on a flow cytometry approach to the frequencies of cytokine-producing cells from tuberculosis and leprosy patients, we found distinct patterns of T cell subset expression. The method also reveals the profile of peak cytokine production and can provide simultaneous information about the phenotype of cytokine-producing cells, providing a reliable assay for monitoring the immunity of these patients. The immune response of Mycobacterium leprae and purified protein derivative (PPD) in vitro to a panel of mycobacteria-infected patients from an endemic area was assessed in primary mononuclear cell cultures. The kinetics and source of the cytokine pattern were measured at the single-cell level. IFN-gamma-, TNF-alpha-, IL-4- and IL-10-secreting T cells were intracytoplasmic evaluated in an attempt to identify M. leprae- and PPD-specific cells directly from the peripheral blood. The analysis by this approach indicated that TNF-alpha was the first (8 h) to be produced, followed by IFN-gamma (16 h), IL-10 (20 h) and IL-4 (24 h), and double-staining experiments confirmed that CD4+ were a greater source of TNF-alpha than of CD8+ T cells (P < 0.05). Both T cell subsets secreted similar amounts of IFN-gamma. We conclude that the protocol permits rapid evaluation of cytokine production by different T cell populations. The method can also be used to define immune status in non-infected and contact individuals.

Etiology of diarrheal infections in children of Porto Velho (Rondonia, Western Amazon region, Brazil)

In the present study, 470 children less than 72 months of age and presenting acute diarrhea were examined to identify associated enteropathogenic agents. Viruses were the pathogens most frequently found in stools of infants with diarrhea, including 111 cases of rotavirus (23.6% of the total diarrhea cases) and 30 cases of adenovirus (6.3%). The second group was diarrheogenic Escherichia coli (86 cases, 18.2%), followed by Salmonella sp (44 cases, 9.3%) and Shigella sp (24 cases, 5.1%). Using the PCR technique to differentiate the pathogenic categories of E. coli, it was possible to identify 29 cases (6.1%) of enteropathogenic E. coli (EPEC). Of these, 10 (2.1%) were typical EPEC and 19 (4.0%) atypical EPEC. In addition, there were 26 cases (5.5%) of enteroaggregative E. coli, 21 cases (4.4%) of enterotoxigenic E. coli, 7 cases (1.4%) of enteroinvasive E. coli (EIEC), and 3 cases (0.6%) of enterohemorrhagic E. coli. When comparing the frequencies of diarrheogenic E. coli, EPEC was the only category for which significant differences were found between diarrhea and control groups. A low frequency of EIEC was found, thus EIEC cannot be considered to be a potential etiology agent of diarrhea. Simultaneous infections with two pathogens were found in 39 diarrhea cases but not in controls, suggesting associations among potential enteropathogens in the etiology of diarrhea. The frequent association of diarrheogenic E. coli strains was significantly higher than the probability of their random association, suggesting the presence of facilitating factor(s).