André Salvador Kazantzi Fonseca

Prevalence of hepatitis C virus in alcoholic patients: role of parenteral risk factors

BACKGROUND The prevalence of hepatitis C virus (HCV) infection is elevated in alcoholic patients, but the risk factors are unclear. The role of parenteral risk factors are indeterminated in this population. AIMS To determine the prevalence of hepatitis C virus infection in alcoholic patients admitted to a detoxification unit and to evaluate the presence of underlying parenteral risk factors. METHODS A total of 114 consecutive unselected alcoholic patients admitted to a single chemical dependency unit during 14 month were included. Epidemiological data and history of parenteral risk factors for hepatitis C virus infection were obtained with a standardized questionnaire. Blood was collected for determination of aminotransferases and anti-hepatitis C virus antibodies (ELISA-3). Positive samples were confirmed by polymerase chain reaction and tested for genotype. RESULTS Among the 114 alcoholics, 17 (15%) were anti-hepatitis C virus positive. Of these, 12 (71%) had detectable serum HCV-RNA by PCR. Genotype 1 was found in six cases and genotype 3 in five (one patient was undetermined). Forty-nine (43%) patients had elevated serum ALT and/or AST at baseline. The comparison between the 17 positive and the 97 negative patients showed significant differences in mean serum ALT levels (42 +/- 41 IU/L vs. 22 +/- 20 IU/L), rate of elevated ALT (65% vs. 34%), and presence of parenteral risk factors (94% vs. 10%). Comparison between alcoholic patients with and without elevated aminotransferases showed significant difference only in the rate of positive anti-hepatitis C virus antibodies (24% vs. 7%). Furthermore, among the 17 anti-hepatitis C virus positive patients, the rate of detectable HCV-RNA was significantly higher in the 12 with elevated aminotransferases versus the 5 with normal aminotransferases (92% vs. 20%). CONCLUSIONS There was a high prevalence of anti-hepatitis C virus antibodies in alcoholics and the majority was confirmed by the presence of detectable HCV-RNA. Intravenous drug use was the main risk factor for hepatitis C virus infection in this population.

Emergence of a New Genotype of Avian Infectious Bronchitis Virus in Brazil

SUMMARY Infectious bronchitis virus (IBV) is the agent of a highly contagious disease that affects domestic fowl (Gallus gallus). Recent reports showed a high prevalence of one main IBV genotype (Brazil or BR-I) with low genetic diversity in commercial poultry flocks from Brazil. This research analyzed IBV positive poultry flocks from different rearing regions to verify the S1 gene variability and geographic distribution of variant IBV strains in recent years (2010 and 2011). Samples of IBV-positive flocks were obtained from 60 different farms. Forty-nine partial S1 gene sequences were determined and aligned for phylogenetic and amino acid similarity analyses. Eleven samples (22.4%) were similar to Massachusetts vaccine strains (Mass genotype) and 34 samples (69.4%) to the previously characterized Brazilian BR-I genotype. Interestingly, the remaining four samples (8.2%) clustered into a new IBV variant genotype (Brazil-II or BR-II), divergent from the BR-I. A unique nucleotide sequence insertion coding for five amino acid residues was observed in all the Brazilian variant viruses (BR-I and BR-II genotypes). These results show a higher genetic diversity in Brazilian IBV variants than previously described. RESUMEN Aparición de un nuevo genotipo del virus de la bronquitis infecciosa aviar en Brasil. El virus de la bronquitis infecciosa (IBV) es el agente de una enfermedad altamente contagiosa que afecta a las gallinas domésticas (Gallus gallus). Los reportes recientes muestran una alta prevalencia de un genotipo principal de este virus (Brasil o BR-I) con baja diversidad genética en las parvadas avícolas comerciales de Brasil. Esta investigación analiza las parvadas avícolas positivas a la presencia de este virus en diferentes regiones, para verificar la variabilidad del gene S1 y la distribución geográfica de las cepas variantes de este virus en los últimos años (2010 y 2011). Se recolectaron muestras del virus de bronquitis de parvadas positivas en 60 granjas diferentes. Se determinaron 49 secuencias parciales del gene S1 y se alinearon para llevar a cabo análisis filogenéticos y de similitud de aminoácidos. Once muestras (22.4%) fueron similares a la cepa vacunal Massachusetts (Mass) (genotipo Mass) y 34 muestras (69.4%) fueron similares a la cepa brasileña previamente caracterizada, genotipo BR-I. De manera interesante, las cuatro muestras restantes (8.2%) se agruparon en un nuevo genotipo variante (Brasil-II o BR-II), que es divergente de la BR-I. Una inserción única en la secuencia de nucleótidos que codifica para cinco residuos de aminoácidos se observó en todos los virus variantes de Brasil (genotipos BR-I y BR-II). Estos resultados muestran una mayor diversidad genética de las cepas variantes brasileñas del virus de la bronquitis infecciosa a diferencia de lo que se había descrito anteriormente.

Infectious bronchitis virus in different avian physiological systems—A field study in Brazilian poultry flocks

Abstract Avian infectious bronchitis is a highly contagious viral disease with economic effects on poultry agribusiness. The disease presents multi-systemic clinical signs (respiratory, renal, enteric, and reproductive) and is caused by one coronavirus (infectious bronchitis virus, IBV). Infectious bronchitis virus is classified into different serotypes and genotypes (vaccine strains and field variants). This study aimed to evaluate the occurrence of IBV in commercial poultry flocks from 3 important producing regions in Brazil and to determine the tropism of the main circulating genotypes to 3 different avian physiological systems (respiratory, digestive, urinary/reproductive). Clinical samples with suggestive signs of IBV infection were collected from 432 different poultry commercial flocks (198 from broilers and 234 from breeders). The total number of biological samples consisted of organ pools from the 3 above physiological systems obtained of farms from 3 important producing regions: midwest, northeast, and south. Infectious bronchitis virus was detected by reverse-transcription, real-time PCR of the 5′ untranslated region. The results showed 179 IBV-positive flocks (41.4% of the flocks), with 107 (24.8%) from broilers and 72 (16.8%) from breeders. There were similar frequencies of IBV-positive flocks in farms from different regions of the country, most often in broilers (average 54%) compared with breeders (average 30.8%). reverse-transcription was more frequently detected in the digestive system of breeders (40%), and in the digestive (43.5%) and respiratory (37.7%) systems of broilers. Infectious bronchitis virus genotyping was performed by a reverse-transcription nested PCR and sequencing of the S1 gene from a selection of 79 IBV-positive flocks (45 from broilers and 34 from breeders). The majority of the flocks were infected with Brazilian variant genotype than with Massachusetts vaccine genotype. These results demonstrate the predominance of the Brazilian variant (mainly in the enteric tract) in commercial poultry flocks from 3 important producing regions in Brazil.

Detection and differentiation of field and vaccine strains of canine distemper virus using reverse transcription followed by nested real time PCR (RT-nqPCR) and RFLP analysis.

Abstract Canine distemper virus (CDV) is the cause of a severe and highly contagious disease in dogs. Practical diagnosis of canine distemper based on clinical signs and laboratory tests are required to confirm CDV infection. The present study aimed to develop a molecular assay to detect and differentiate field and vaccine CDV strains. Reverse transcription followed by nested real time polymerase chain reaction (RT-nqPCR) was developed, which exhibited analytical specificity (all the samples from healthy dogs and other canine infectious agents were not incorrectly detected) and sensitivity (all replicates of a vaccine strain were positive up to the 3125-fold dilution – 100.7 TCID50). RT-nqPCR was validated for CDV detection on different clinical samples (blood, urine, rectal and conjunctival swabs) of 103 animals suspected to have distemper. A total of 53 animals were found to be positive based on RT-nqPCR in at least one clinical sample. Blood resulted in more positive samples (50 out of 53, 94.3%), followed by urine (44/53, 83.0%), rectal (38/53, 71%) and conjunctival (27/53, 50.9%) swabs. A commercial immunochromatography (IC) assay had detected CDV in only 30 conjunctival samples of these positive dogs. Nucleoprotein (NC) gene sequencing of 25 samples demonstrated that 23 of them were closer to other Brazilian field strains and the remaining two to vaccine strains. A single nucleotide sequences difference, which creates an Msp I restriction enzyme digestion, was used to differentiate between field and vaccine CDV strains by restriction fragment length polymorphism (RFLP) analysis. The complete assay was more sensitive than was IC for the detection of CDV. Blood was the more frequently positive specimen and the addition of a restriction enzyme step allowed the differentiation of vaccine and Brazilian field strains.

Caracterização antigênica e fenotípica de cepas de Pasteurella multocida isoladas de pulmões de suínos com pneumonia e/ou pleurite

The antigenic and phenotypic variation among 22 strains of Pasteurella multocida isolated from pig lungs with pneumonia and/or pleuritic lesions was studied. Phenotypic tests consisted of biochemical assays and antimicrobial sensitivity tests. All isolates fermented mannitol and sorbitol, but none fermented arabinose. Fourteen fermented xylose, 4 trehalose, 2 dulcitol and 1 maltose. Analyzing these characteristics, 5 different biochemical groups were revealed. There was a wide variation in the results of antimicrobial sensitivity testing to 9 products, with 50% of isolates showing sensitivity at least to 7. No antimicrobial agent was able to inhibit all strains. The highest efficiency (72% sensitivity) was observed with amoxycillin (30 mg). Spectinomycin (100 mg) presented the lowest efficiency, 45.5%. Antigenic characterization consisted in capsular serotyping and assessment of variability of an outer membrane protein gene (ompH), using polymerase chain reaction (PCR) and digestion with 5 endonuclease enzymes (restriction fragment length polymorphism, RFLP). Out of 22 strains, 21 were classified as capsular type A and one as type D. Characterization of ompH revealed 7 different patterns. They corresponded well to previously established biochemical groups.

Development and Application of Reverse Transcriptase Nested Polymerase Chain Reaction Test for the Detection of Exogenous Avian Leukosis Virus

SUMMARY. A polymerase chain reaction (PCR) assay that utilizes nested primers to amplify a fragment of the long terminal repeat of exogenous avian leukosis virus (ALV) was developed and evaluated for detection of ALV subgroup J directly from clinical samples. Compilation of sequence data from different endogenous and exogenous ALVs allowed the selection of a conserved set of nested primers specific for the amplification of exogenous ALV subgroups A, B, C, D, and J and excluded amplification of endogenous viruses or endogenous viral sequences within the chicken genome. The nested primers were successfully used in both PCR and reverse transcriptase (RT)-PCR assays to detect genetically diverse ALV-J field isolates. Detection limits of ALV-J isolate ADOL-Hc1 DNA by nested PCR and RNA by RT–nested PCR were superior to detection of group-specific antigen by enzyme-linked immunosorbent assay (ELISA) in cell culture. Detection of ALV-J in cloacal swabs by RT–nested PCR was compared with direct detection by antigen-capture (ac)-ELISA; RT–nested PCR detected fewer positive samples than ac-ELISA, suggesting that RT–nested PCR excluded detection of endogenous virus in clinical samples. Detection of ALV-J in plasma samples by RT–nested PCR was compared with virus isolation in C/E chicken embryo fibroblasts; the level of agreement between both assays as applied to plasma samples ranged from low to moderate. The main disagreement between both assays was observed for a group of plasma samples found positive by RT–nested PCR and negative by virus isolation, suggesting that RT–nested PCR detected ALV-J genome in plasma samples of transiently or intermittently infected birds. ALV-J transient and intermittent infection profiles are characterized by inconsistent virus isolation responses throughout the life of a naturally infected flock.

A Multiplex real-time PCR for detection of Mycoplasma gallisepticum and Mycoplasma synoviae in clinical samples from Brazilian commercial poultry flocks

Mycoplasma gallisepticum (MS) and Mycoplasma synoviae (MS) are important avian pathogens and cause economic losses to the poultry industry. Molecular biology techniques are currently used for a rapid detection of these pathogens and the adoption of control measures of the diseases. The aim of this study was to develop and validate a technique for simultaneous detection of MG and MS by multiplex real time polymerase chain reaction (PCR). The complete assay (Multiplex MGMS) was designed with primers and probes specific for each pathogen and developed to be carried out in a single tube reaction. Vaccines, MG and MS isolates and DNA from other Mycoplasma species were used for the development and validation of the method. Further, 78 pooled clinical samples from different poultry flocks in Brazil were obtained and used to determine the sensitivity and specificity of the technique in comparison to 2 real time PCR assays specific for MG (MG PCR) and MS (MS PCR). The results demonstrated an agreement of 100% (23 positive and 44 negative samples) between Multiplex MGMS and MG PCR in the analysis of 67 samples from MG positive and negative poultry flocks, and an agreement of 96.9% between Multiplex MGMS and MS PCR in the analysis of 64 samples from MS positive and negative poultry flocks. Considering the single amplification tests as the gold standard, the Multiplex MGMS showed 100% of specificity and sensitivity in the MG analysis and 94.7% sensitivity and 100% specificity in the MS analysis. This new assay could be used for rapid analysis of MG and MS in the poultry industry laboratories.

Isolation of Arcobacter spp from poultry carcasses, in Brazil

Fourty eight isolates of Arcobacter spp were obtained from 37 poultry carcasses, from abattoir, among 80 carcasses examined. Attempts for culturing were made from the skin and muscle, resulting on 25 positive cultures from muscle and 23 from skin. Classification was achieved by phenotypic characterization and PCR and multiplex PCR, resulting 41 samples of Arcobacter butzleri and 07 Arcobacter sp. This is the first report on the occurrence of Arcobacter in animal carcasses in Brazil.

Virulence gene content in Escherichia coli isolates from poultry flocks with clinical signs of colibacillosis in Brazil

Escherichia coli is a commensal bacterium of the birds intestinal tract, but it can invade different tissues resulting in systemic symptoms (colibacillosis). This disease occurs only when the E. coli infecting strain presents virulence factors (encoded by specific genes) that enable the adhesion and proliferation in the host organism. Thus, it is important to differentiate pathogenic (APEC, avian pathogenic E. coli) and non-pathogenic or fecal (AFEC, avian fecal E. coli) isolates. Previous studies analyzed the occurrence of virulence factors in E. coli strains isolated from birds with colibacillosis, demonstrating a high frequency of the bacterial genes cvaC, iroN, iss, iutA, sitA, tsh, fyuA, irp-2, ompT and hlyF in pathogenic strains. The aim of the present study was to evaluate the occurrence and frequency of these virulence genes in E. coli isolated from poultry flocks in Brazil. A total of 138 isolates of E. coli was obtained from samples of different tissues and/or organs (spleen, liver, kidney, trachea, lungs, skin, ovary, oviduct, intestine, cloaca) and environmental swabs collected from chicken and turkey flocks suspected to have colibacillosis in farms from the main Brazilian producing regions. Total DNA was extracted and the 10 virulence genes were detected by traditional and/or real-time PCR. At least 11 samples of each gene were sequenced and compared to reference strains. All 10 virulence factors were detected in Brazilian E. coli isolates, with frequencies ranging from 39.9% (irp-2) to 68.8% (hlyF and sitA). Moreover, a high nucleotide similarity (over 99%) was observed between gene sequences of Brazilian isolates and reference strains. Seventy-nine isolates were defined as pathogenic (APEC) and 59 as fecal (AFEC) based on previously described criteria. In conclusion, the main virulence genes of the reference E. coli strains are also present in isolates associated with colibacillosis in Brazil. The analysis of this set of virulence factors can be used to differentiate between APEC and AFEC isolates in Brazil.

Oseltamivir-resistant influenza A(H1N1)pdm09 virus in southern Brazil.

The neuraminidase (NA) genes of A(H1N1)pdm09 influenza virus isolates from 306 infected patients were analysed. The circulation of oseltamivir-resistant viruses in Brazil has not been reported previously. Clinical samples were collected in the state of Rio Grande do Sul (RS) from 2009-2011 and two NA inhibitor-resistant mutants were identified, one in 2009 (H275Y) and the other in 2011 (S247N). This study revealed a low prevalence of resistant viruses (0.8%) with no spread of the resistant mutants throughout RS.