J. S Resende

Molecular Analysis of Brazilian Infectious Bronchitis Field Isolates by Reverse Transcription–Polymerase Chain Reaction, Restriction Fragment Length Polymorphism, and Partial Sequencing of the N Gene

Abstract Molecular analysis of 15 Brazilian infectious bronchitis virus (IBV) isolates, obtained from clinical outbreaks of the disease in chickens (broilers or layers) in the state of Minas Gerais (Brazil) between 1972 and 1989, is reported. Using the N protein gene as target, IBVs were analyzed by reverse transcription–polymerase chain reaction/restriction fragment length polymorphism (RT-PCR/RFLP) with the restriction enzymes AvaII, HphI, Sau96I, and Tsp509I and cDNA sequencing. Results obtained from those isolates were compared to 19 sequences available in GenBank. N gene RFLP profiles, cDNA sequences, and predicted amino acid composition were used for the construction of dendrograms. Brazilian isolates were grouped into one distinct group. Identity of predicted N protein amino acid composition varied from 45% (between isolates G and 208) up to 99% (PM1 and PM2), and, when compared to the other IBVs, the amino acid identity was from 42% (Q3/88 and G) up to 97% (D41 and PM1). The great genetic diversity was shown to occur before the official use of vaccination in Brazil and has remained thereafter.

Genotyping of Infectious Bursal Disease Virus Strains by Restriction Fragment Length Polymorphism Analysis of the VP1, VP2, and VP3 Genes

Abstract This study aimed to genotype infectious bursal disease virus (IBDV) isolates from the Minas Gerais state poultry industry. RNA was extracted from bursae obtained from field cases without passage or commercial vaccines. Genetic subtyping of IBDV isolates and vaccine strains was carried out by the reverse transcriptase–polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP) analysis. A 588-bp fragment in the VP1 gene, an 847-bp fragment in the VP2 gene, and a 320-bp fragment in the VP3 gene were amplified by PCR and digested with restriction enzymes PstI and ScaI (VP1); BamHI, BstEII, and PstI (VP2); and NcoI, ScaI, and XbaI (VP3). Our work shows that complementing the clinical history of the outbreaks with RT-PCR followed by RFLP analysis using PstI for VP1, BamHI for VP2, and XbaI for VP3 allowed an accurate classification of a causative agent as a very virulent IBDV.

Occurrence of chicken anemia virus in backyard chickens of the metropolitan region of Belo Horizonte, Minas Gerais

The occurrence of CAV in backyard chickens in the metropolitan area of Belo Horizonte, Brazil, was evaluated. The spleen and thymus of chickens from different origins were collected for DNA extraction and nested-PCR. CAV genome was detected in 30% of the flocks (n=20) examined. CAV origin for backyard chickens is speculated, taking into consideration its widespread incidence in the chicken industry, the contamination of live vaccines with CAV prior to its eradication from SPF flocks, and the use of attenuated CAV vaccines.

Molecular Characterization of Contaminating Infectious Anemia Virus of Chickens in Live Commercial Vaccines Produced in the 1990s

SUMMARY. The presence of infectious chicken anemia virus (CAV) was detected in a previous study by nested-PCR as a contaminant in seven commercial vaccines, produced in the 1990s by three different manufacturers, prepared against the most relevant virus etiologies. In order to phylogenetically characterize the genome and compare it to CAV isolates from Brazil and other parts of the world, sequences of approximately 675 bp of the gene encoding the hypervariable region of VP1 protein of three CAV vaccine contaminant strains were studied. The CAV genome in contaminated vaccines showed high similarity (>98.9%) with the Brazilian BR91/99 and Argentinian ArgA001028 (>99%) strains. However, the comparison with the Cuxhaven-1 vaccine strain showed a lower identity of between 96.8% and 97.7%, and comparing it with the CAV26P4 vaccine strain showed an identity between 97.2% and 98.2%; both are available in Brazil. Such differences might be relevant for the highly conserved CAV genome. CAV contaminants were positioned in the same genetic group (clusters) with the Brazilian strain BR91/99 and Argentinian strain ArgA001028. Results indicated that the contamination of live vaccines by CAV may have influenced CAV epidemiology in the Brazilian and Argentinian poultry industry. RESUMEN. Caracterización molecular de los virus de la anemia infecciosa del pollo detectados como contaminantes en vacunas comerciales producidas en la década de los noventas. En un estudio previo, se detectó mediante un método de PCR anidado la presencia del virus de la anemia infecciosa del pollo (CAV) como contaminantes en siete vacunas comerciales contra etiologías variadas, que fueron producidas en los años noventas por tres laboratorios diferentes. Con el objetivo de caracterizar filogenéticamente el genoma y compararlo con aislamientos del virus de la anemia de Brasil y de otras partes del mundo, se estudiaron las secuencias de aproximadamente 675 pares de bases que codificaban para la región hipervariable de la proteína VP1 de tres cepas del virus de la anemia que fueron contaminantes de vacunas. El genoma del virus de la anemia que se encontró en las vacunas contaminadas mostro una similitud alta (>98.9%), con la cepa brasileña BR91/99 y con la estirpe argentina ArgA001028 (>99%). Sin embargo, la comparación con la cepa vacunal Cuxhaven-1, mostró una similitud de entre 96.8 a 97.7% y de 97.2 a 98.2% con la estirpe vacunal CAV26P4, ambas cepas son usadas en Brasil para producir vacunas comerciales contra el virus de la anemia. Estas diferencias pueden ser relevantes debido a que el genoma del virus de la anemia infecciosa es altamente conservado. Los virus contaminantes de la anemia infecciosa se agruparon en el mismo grupo genético (clúster) con la cepa brasileña BR91/99 y de Argentina ArgA001028. Estos resultados indican que la contaminación de las vacunas vivas con el virus de la anemia infecciosa pudo haber influenciado la epidemiología del virus en la industria avícola de Brasil y Argentina.

Estudo comparativo da virulência de amostras de vacina do vírus da doença de Newcastle em galinhas SPF por meio da análise morfométrica da espessura traqueal

Virulence of three vaccinal lentogenic strains (La Sota, Ulster and VG-GA) of the Newcastle disease virus (NDV) was determined by morphometric analysis of tracheal thickness, in 45-day-old SPF chickens (n=12), free from NDV antibodies. Tracheal thickness was evaluated 1, 2, 3, 4, 6, and 8 days after intratracheal vaccination. La Sota strain induced higher tracheal swelling than the others, during most of the time of the experiment. Maximum swelling of tracheal mucosa occurred at the third day after vaccination. At that time, La Sota and Ulster had the same virulence, and both caused higher swelling of tracheal mucosa than VG-GA strain.

Detection of Mycoplasma gallisepticum in dead captive psittacines in Belo Horizonte, Brazil

Mycoplasma gallisepticum (Mg) infection of wild native Brazilian psittacines (Psittaciformes) which died of any cause during sorting, rehabilitation, or conservation, was investigated by PCR. Two previously described PCR methodologies using Mg specific primers were employed for the analyses of 140 swab samples (cloaca, trachea, or palatine cleft). Average positive Mg detection in cloacal swabs was 51.9%, with 80.0% (n=5) of Blue-and-yellow Macaws (Ara ararauna), 60.0% (n=3) Dusky Parrots (Pionus fuscus), 52.5% (n=59) Amazon Parrots (Amazona aestiva), 50.0% (n=2) Orange-winged Parrots (Amazona amazonica), 50.0% (n=2) Jandaya Parakeetsor Jandaya Conures (Aratinga jandaya), 0% (n=2) Golden Conures or Golden Parakeets (Guarouba guarouba), and 0% (n=2) Hyacinth Macaws (Anodorhynchus hyacinthinus). Palatine cleft swab sampling was more sensitive to detect Mg, with 85.4% (n=17) detection rate, as compared to 67.4% (n=46) obtained with tracheal samples, and 53.5% (n=77) with cloacal swabs. The surprisingly high Mg incidence in psittacines kept in conservation or triage environments is possibly due to the proximity or cohabitation with several bird species during confinement and housing psittacines of different origins together. The implementation of biosecurity measures and species-specific facilities is recommended.

Cryopreservation of an avian spirochete strain in liquid nitrogen

Soros de aves experimentalmente infectadas, contendo espiroquetas viaveis, foram submetidos a dois procedimentos antes da criopreservacao: glicerol na diluicao de 1/2 (v/v), designado como soro com glicerol a 50% (GS), e dimetilsulfoxido na proporcao de 1/10 (v/v), designado como soro com DMSO a 10% (DS). Apos 15 meses de estocagem em nitrogenio liquido, amostras dos tratamentos GS e DS foram descongeladas e suas infectividades foram testadas em frangos susceptiveis. Apesar de ambos os procedimentos terem mantidos a infectividade da bacteria, DMSO a 10% no soro de frango apresentou-se mais satisfatorio como criopreservante.

HEALTH ASSESSMENT OF CAPTIVE TINAMIDS (AVES, TINAMIFORMES) IN BRAZIL

Ninety-five (95) captive tinamids (Aves, Tinamiformes) of species Crypturellus obsoletus (brown tinamou), Crypturellus parvirostris (small-billed tinamou), Crypturellus tataupa (Tataupa tinamou), Crypturellus undulatus (undulated tinamou), Rhynchotus rufescens (red-winged tinamou), and Tinamus solitarius (solitary tinamou) were evaluated for diseases of mandatory control in the Brazilian Poultry Health Program (PNSA). Antibodies were detected by serum agglutination test (SAT) in 4 birds for Mycoplasma gallisepticum (MG) and in 27 birds for Salmonella Pullorum (SP) and Salmonella Gallinarum (SG). However, by hemagglutination inhibition (HI), sera were negative to MG and Mycoplasma synoviae (MS). Bacteriology was negative for SP and SG. No antibody was detected by HI to avian paramyxovirus type 1. However, antibodies to infectious bursal disease virus were detected in 9.4% (9/95) by ELISA. Fecal parasitology and necropsy revealed Capillaria spp. in 44.2% (42/95), Eimeria rhynchoti in 42.1% (40/95), Strongyloides spp. in 100% (20/20), Ascaridia spp., and unknown sporozoa in small-billed tinamou. Ectoparasites were detected in 42.1% (40/95) by inspection, and collected for identification. The louse Strongylocotes lipogonus (Insecta: Phthiraptera) was found on all Rhynchotus rufescens. An additional four lice species were found on 14 individuals. Traumatic lesions included four individual R. rufescens (4/40, 10%) with rhinotheca fracture, one with mandible fracture and three with posttraumatic ocular lesions (3/40, 7.5%). One C. parvirostris had phalangeal loss, another had tibiotarsal joint ankylosis and another had an open wound on the foot. Results suggest that major poultry infections/ diseases may not be relevant in tinamids, and that this group of birds, as maintained within distances for biosecurity purposes, may not represent a risk to commercial poultry. Ecto- and endoparasites were common, disseminated, and varied; regular monitoring of flocks is recommended for best performance.

A retrospective PCR investigation of avian Orthoreovirus, chicken infectious anemia and fowl Aviadenovirus genomes contamination in commercial poultry vaccines in Brazil

Vacinas avicolas vivas comerciais produzidas entre 1991 e 2005 foram examinadas para a presenca de genomas dos virus da anemia infecciosa das galinhas (Gyrovirus CAV), da hepatite por corpusculo de inclusao (Aviadenovirus FAdV) e da artrite viral/sindrome da ma absorcao (Orthoreovirus aviario ARV). Vinte e seis partidas de vacinas vivas liofilizadas de oito fabricantes com lacre original foram examinadas. As extracoes de DNA e PCR de CAV e FAdV, e de RNA e RT-PCR para ARV, foram descritas previamente. Contaminacoes triplas de ARV, CAV e FAdV foram detectadas em vacinas de mesmo fabricante, produzidas em 1991 e 1992 contra a doenca de Newcastle (DN), e para a encefalomielite aviaria, produzida em 1994. ARV e CAV em co-infeccao foram encontrados em vacinas contra a doenca de Marek liofilizadas produzidas em 1996 por dois fabricantes diferentes. Genoma de ARV foi detectado em vacinas contra a bronquite infecciosa de setembro e dezembro de 1998, doenca infecciosa bursal, de dezembro de 1998 e DN de janeiro de 1998. Tres dos oito fabricantes apresentaram vacinas com contaminacao e cinco nunca apresentaram vacinas contaminadas. Nenhuma vacina produzida a partir de 2001 apresentou contaminacao. Cogita-se um papel epidemiologico para vacinas vivas, como fonte de infeccao para ARV, CAV e FAdV e, potencialmente determinante da atual alta disseminacao destes.

The occurrence of Orthoreovirus, Rotavirus and chicken anemia virus in chickens of the poultry industry in Minas Gerais, Brazil

Fifty-four fecal samples taken from broiler chickens from 1 to 45 days of age, and of pullets from 10 to 13 weeks of age, original from eight different poultry regions in the state of Minas Gerais, Brazil, were collected from March 2008 to January 2010 for avian Orthoreovirus (ARV) and avian Rotavirus (AvRV) analyses. For the assay of ARV, RNA was immediately extracted (Trizolâ) and transcribed into cDNA for assaying in a nested-PCR with ARV-specific primers. For AvRV, polyacrylamide gel electrophoresis (PAGE) was performed with RNA extracts obtained by phenol-chloroform extraction. CAV was additionally investigated through a nested-PCR of thymus and spleen. Results found 5.55% positive for ARV and 9.25% for AvRV. Also, CAV and ARV genomes were detected in co-infection, in a highly prostrated and claudicating chicken flock. No ARV or AvRV infections were detected in pullets. Material of a clinically affected flock was inoculated into SPF embryos, resulting in embryonic hemorrhage, whitish foci in the chorio-allantoic membrane and death. Sequencing of ARV amplicons and isolate cDNA grouped local strains with the ARV S1133 strain, historically used in live vaccines, suggesting the continued circulation of this vaccine virus strain in intensive poultry regions. Detection rates for ARV and AvRV, as well as the presence of CAV, were additionally indicative of failing biosecurity strategies for the intensive poultry regions examined.