Nelson Rodrigo da Silva Martins

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.

Does haemosporidian infection affect hematological and biochemical profiles of the endangered Black-fronted piping-guan (Aburria jacutinga)?

Infectious diseases can cause deleterious effects on bird species, leading to population decline and extinction. Haemosporidia can be recognized by their negative effects on host fitness, including reproductive success and immune responses. In captivity, outbreaks of haemosporidian infection have been observed in birds in zoos and aviaries. The endemic Brazilian Atlantic rainforest species Aburria jacutinga is one of the most endangered species in the Cracidae family, and wild populations of this species are currently found mainly in conservation areas in only two Brazilian states. In this study, we aimed to evaluate the effects of avian haemosporidia on hematological and biochemical parameters in two captive populations of A. jacutinga. Forty-two animals were assessed, and the haemosporidian prevalence was similar for males and females. The occurrence of haemosporidian infection in captive A. jacutinga observed in this study was similar to results found in other captive and wild birds in Brazil. We found three different lineages of haemosporidia. Two lineages were identified as Plasmodium sp., one of which was previously detected in Europe and Asia, and the other is a new lineage closely related to P. gallinaceum. A new third lineage was identified as Haemoproteus sp. We found no significant differences in hematological and biochemical values between infected and non-infected birds, and the haemosporidian lineage did not seem to have an impact on the clinical and physiological parameters of A. jacutinga. This is the first report on an evaluation of natural haemosporidian infections diagnosed by microscopic and molecular methods in A. jacutinga by hematology, blood biochemistry, and serum protein values. Determining physiological parameters, occurrence and an estimation of the impact of haemosporidia in endangered avian species may contribute to the management of species rehabilitation and conservation.

Mites affecting hen egg production: some considerations for Brazilian farms

The poultry industry is characterized for its constant search for productivity and profitability, which are based on flock health status. Brazilian Commercial laying hens (Gallus gallus domesticus) have been impacted significantly by mite infestations. This review aims to compile the literature on the occurrence, economic losses, biology, epidemiology and control of mite species considered important for the Brazilian laying poultry industry. The national experience was compared with practices of other countries and a scarcity of studies on this subject in Brazil was evident. The poultry industry has prioritized the use of pesticides to control infestations with little regard for the adverse effects. In this context, the integrated control programs using several strategies simultaneously constitute the best alternative to mite control. Integrated control programs involve measures of chemical, physical and biological nature, as well as attention to cultural aspects. However, studies should be performed aiming at the development of new control methods, evaluating the adequacy of practices developed in other countries to the national reality.

Surto de laringotraqueíte infecciosa em granjas de galinhas poedeiras de múltiplas idades em Minas Gerais, Brasil

A recent (November 2010) outbreak of infectious laryngotracheitis (ILT) in a multi-age laying hen facility in Minas Gerais state, Brazil, is described. Previous ILT outbreak in laying hens was only notified in Sao Paulo state, Brazil, in 2002. In the outbreak described here, the affected population was approximately eight million hens, with flock sizes ranging from 100,000 to 2,900,000 chickens. The average mortality ranged from 1 to 6%, and morbidity was around 90% (most of the twenty seven farms of the area were positive for ILT virus). Three multi-age laying farms from one company were selected for this report. Clinical signs included prostration, dyspnea, conjunctivitis, occasional swelling of the paranasal sinuses and bloody mucous nasal discharge. Severely affected chickens presented with dyspnea, gasping and became cyanotic before death. At necropsy, these chickens had fibrinous exudate blocking the larynx and the lumen of cranial part of the trachea. In addition, conjunctivitis with intense hyperemia, edema and sinuses with caseous exudate were present. On histopathology, there were marked necrosis and desquamation of respiratory ephitelium and conjunctiva with numerous syncytial cells formation and fibrinous exudate. Moderate to marked non suppurative (especially lymphocytes and plasma cells) infiltration in the lamina propria also was observed. Sixteen out of 20 examined chickens, eosinophilic intranuclear inclusion bodies were observed in the syncytial cells. The DNA extracted from larynx and trachea produced positive PCR results for ILT virus (ILTV) DNA using formalin-fixed, paraffin embedded (FFPE) samples. Amplicons from a small region of ICP4 gene were submitted to sequencing and showed 100% identity with ILTV EU104910.1 (USA strain), 99% with ILTV JN596963.1 (Australian strain) and 91% with ILTV JN580316.1 (Gallid herpesvirus 1 CEO vaccine strain) and JN580315.1 (Gallid herpesvirus 1 TCO vaccine strain).

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.

Genotipicação de Clostridium perfringens isolados de frangos de corte através da PCR múltipla

Clostridium perfringens (Cp) is an anaerobic gram-positive bacterium which causes gaseous gangrene and enterotoxaemias in humans and domestic animals, besides being the primary cause of necrotic enteritis in poultry. Cp isolates were preliminary identified according to the lecithinase test on agar TSC-egg yolk, colony with double haemolysis in desfibrinated horse blood agar, Gram staining and biochemical tests. Cp isolates (171) were obtained from the intestinal content of broiler chickens sampled in a slaughterhouse in Para de Minas city, MG, Brazil. Cp was isolated in 62/125 (49.6%) strains from jejunum content and in 109/125 (87.2%) of ileum. Cp strains were classified into five toxigenic types (A-E), using multiplex PCR assay for genotyping of the principal and lethal toxins in the detection of genes coding for toxins alfa, beta, epsilon e iota, nomely genes cpa, cpb, etx e iA genes, beta2 toxin (cpb2) and enterotoxin (cpe). From a total of 62 Cp jejunum isolates obtained 42/62 (67.7%) were type A, 1/62 (1.6%) type A with the amplification of products for beta2 toxin gene (A/B2), 0/62 (0%) type B, 17/62 (27.4%) type C and 1/62 (1.6%) type D. A total of 109 ileum Cp isolates were obtained being 62/109 (56.9%) type A, 3/109 (2.7%) type A/B2 toxin gene, 1/62 (0.9%) type B, 38/109 (34.9%) type C, 1/109 (0.9%) type D. Cp A (60.8%) and Cp C (32.2%) toxigenic types were the most prevalent types in the analyzed intestinal contents of broiler chickens Cp A 104/171 (60.8%) and 55/171 (32.2%) toxigenic types which were the most prevalent types analyzed into two partes of the intestinal content of broiler chickens. Five (2.9%) out of 171 Cp isolates were not typified. The iota toxin (iA) and enterotoxin gene (cpe) codifying genes were not identified.

Avaliação da qualidade da casca dos ovos provenientes de matrizes pesadas com diferentes idades

We conducted a study on eggshell quality from Cobb® broiler breeders at different ages by assessing the relationship between eggshell weight and egg weight and analysis of specific weight, thickness, porosity, strength and electron microscopy. Both treatments were defined by broiler breeder age, considering eggs from young breeders (33 weeks) and eggs from old breeders (63 weeks). It was observed that eggs from breeders at 33 weeks have lower weight and number of pores per cm2 compared with eggs from older birds. 63 weeks broiler breeders had lower percentage of eggshell in relation to egg weight, lower specific weight, strength and thickness, when compared with eggs from young breeders. The proportion of shell membranes in relation to its total thickness was significantly higher in young breeders. It was concluded that eggs from older broiler breeders have lower eggshell quality than the young breeders. The shell membranes in young breeders eggs play a significant role in its structure, making it necessary to focus on this layer in future studies on eggshell quality and their relationship between breeder age and incubation yield.

Vertebral osteomyelitis associated with single and mixed bacterial infection in broilers

ABSTRACT Vertebral osteomyelitis (VO) is a worldwide emerging disease that affects broilers. The objective of this study was to determine the frequency and aetiology of VO in broilers in a highly productive broiler region. For this, 608 broilers with locomotory problems were analysed from 18 farms. Clinical signs were recorded, necropsy was performed and samples were collected from vertebral bodies with gross changes for molecular and histopathological analysis and for bacterial isolation. From broilers with locomotory changes, 5.1% (31/608) had VO and, of these, 93.5% were 40 days old or older and 89.7% were males. The birds with VO presented varying degrees of limited mobility and this was related to the level of compression to the spinal cord. Bacterial species of the genus Enterococcus (DNA detected in 53.6%) were the aetiological agents involved in most VO cases. Enterococcus faecalis was detected most frequently (35.7%), but Enterococcus hirae was also present in some lesions (7.1%). Escherichia coli was detected in 35.7% of vertebral lesions and co-infection with E. faecalis was confirmed in 7.1% cases. Staphylococcus aureus was involved in 14.3% of the cases, being 7.1% in co-infection with Enterococcus spp. or E. hirae. Our study has indicated that, in Brazil, VO in broilers may not be caused by a single infectious agent and has a lower frequency than recently reported in other countries. This study suggests that there are geographical differences between Brazil and other countries concerning the frequency and aetiology of VO.

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.