Paulo Augusto Esteves

High prevalence of co-infections with bovine herpesvirus 1 and 5 found in cattle in southern Brazil.

Based on small scale studies or on little sensitive serological tests, bovines in the south of the state of Rio Grande do Sul, Brazil, are known to be infected with either bovine herpesvirus 1 (BoHV-1) or 5 (BoHV-5). However, whether the prevalence of each of these viruses is high or low is currently still unknown. In order to determine the extent of BoHV (-1 and/or -5) infections in bovines in this region of Brazil, 200 bovines were studied for the presence of BoHV DNA. To this end, first a quantitative PCR was developed that amplified BoHV-1 DNA as well as BoHV-5 DNA. Using this PCR the number of BoHV genomes normally present in latently infected ganglia of naturally infected bovines was estimated. The new PCR was sensitive enough to detect most BoHV DNA in infected ganglia. The results of this first PCR showed that at least 87% of the bovines in the south of Rio Grande do Sul were (latently) infected with either BoHV-1 or BoHV-5. To determine the prevalence of BoHV-1 and BoHV-5 separately, two type-specific PCRs - one for each virus - were developed that used the products of the first PCR as a template. The results of these type-specific PCRs showed that 82.8% of the BoHV positive population was (latently) infected with BoHV-1, 93.1% with BoHV-5 and 75.9% with both BoHV-1 and BoHV-5. This is the first time that such a high frequency of co-infection of BoHV-1 and BoHV-5 in bovines has been demonstrated.

Performance of two swine manure treatment systems on chemical composition and on the reduction of pathogens

Swine effluents must be correctly handled to avoid negative environmental impacts. In this study, the profiles of two swine manure treatment systems were evaluated: a solid-liquid separation step, followed by an anaerobic reactor, and an aerobic step (System 1); and a biodigester followed by serial lagoons (System 2). Both systems were described by the assessment of chemical, bacterial and viral parameters. The results showed that in System 1, there was reduction of chemicals (COD, phosphorus, total Kjeldhal nitrogen - TKN - and NH(3)), total coliforms and Escherichia coli; however, the same reduction was not observed for Salmonella sp. Viral particles were significantly reduced but not totally eliminated from the effluent. In System 2, there was a reduction of chemicals, bacteria and viruses with no detection of Salmonella sp., circovirus, parvovirus, and torque teno virus in the effluent. The chemical results indicate that the treated effluent can be reused for cleaning swine facilities. However, the microbiological results show a need of additional treatment to achieve a complete inactivation for cases when direct contact with animals is required.

Detection of porcine Circovirus type 2 (PCV2) variants PCV2-1 and PCV2-2 in Brazilian pig population.

In the present study whole genome of six Brazilian isolates of PCV2 were sequenced, analyzed and compared with 35 other sequences (24 from other countries and 17 from Brazil). The phylogenetic analysis showed that mostly Brazilian variants of PCV2 were grouped as PCV2-1. Two isolates among the six analyzed here could not be grouped with any other PCV2-2 analyzed in this study. One of these isolates was from an aborted fetus with myocarditis and the other from a PMWS affected pig. The results pointed here showed that both groups of PCV2 are present in Brazilian pig population without any clear geographical correlation.

Detection of circoviruses and porcine adenoviruses in water samples collected from swine manure treatment systems.

Samples collected from two swine manure treatment systems including: swine manure treatment system and demonstrative unit (SMTS and DU), were analyzed by qPCR to quantify the amount of porcine adenovirus (PAdV) and porcine circovirus (PCV2) present. Positive samples were tested for virus integrity using DNase assay. Fifty-six water samples were collected monthly from March 2009 to May 2010. PAdV genome was found 66% of the samples in the SMTS and in 78% of the samples in the DU system. PCV2 was detected in 96% of samples collected from the SMTS system and in 86% of samples from DU. DNase assay revealed that there were undamaged virus particles of both PAdV and PCV2 in all sampling sites in the SMTS. However, undamaged particles of both viruses were detected in samples from the DU system in the affluent and middle sites, though undamaged PCV2 was absent in the effluent samples.

Avian IgY antibodies: characteristics and applications in immunodiagnostic

Immunoglobulin Y (IgY) is the major antibody isotype in birds, reptiles, amphibia, and lungfish, playing a similar biological role as mammal IgG. Due to its phylogenetic distance, immune diversification and presence in the egg yolk, IgY provide a number of advantages in immunodiagnostic compared to IgG from mammals. Moreover, IgY production is in agreement with international efforts to reduce, refine and if possible, to replace animals in experimentation, contributing substantially in favor of animal welfare. This article presents an overview about structural and functional features, production and applications of IgY in immunodiagnostic, as well as the advantages of chicken antibodies use.

Bacterial biodiversity from an anaerobic up flow bioreactor with ANAMMOX activity inoculated with swine sludge

The present study aimed to describe the bacterial community present at an anaerobic up flow bioreactor with ANAMMOX activity, inoculated with the sludge from the anaerobic pond of a swine slurry treatment system. The description was based on the molecular DNA techniques using primers for amplification of complete 16S rRNA gene and also new primers to amplify smaller fragments from 16S rRNA. During the bioreactor operation time, the bacterial community changed significantly, increasing the nitrogen removal efficiency, reaching after 500 days a removal rate of 94%. The complete PCR amplification of 16S rRNA gene generated 17 clones, where three presented similarity with Candidatus Jettenia asiatica (97%), twelve with Janthinobacterium (99%) and two with uncultured clones. The PCR amplification of 436 base pairs had generated 12 clones, of which eight presented 96-100% similarity with Candidatus Anammoxoglobus propionicus, Planctomycete KSU-1 and one with Pseudomonas sp. (99%) and three with uncultured clones.

Variants of the recently discovered avian gyrovirus 2 are detected in Southern Brazil and The Netherlands

A genome of a virus preliminarily named avian gyrovirus 2 (AGV2), a close relative to chicken anemia virus, was recently discovered in a chicken in the state of Rio Grande do Sul, Southern Brazil. To study the occurrence of AGV2 in Rio Grande do Sul and the neighboring state Santa Catarina, a number of adult chickens (n=108 and n=48, respectively) were tested for the presence of AGV2 DNA. An AGV2-specific PCR was developed, optimized and used to analyze DNA extracted from clinical samples. AGV2 DNA was detected in 98/108 (90.7%) of samples collected in the state of Rio Grande do Sul and 29/48 (60.4%) of the samples collected in the state of Santa Catarina. In order to check whether AGV2 DNA would be detected in samples from a geographically distant region, DNA from brain samples of 21 diseased chickens from the Netherlands were tested independently, by the same method. In such specimens, 9/21 (42.9%) brain tissue samples were found to contain AVG2 DNA. Sequence analysis of some of the PCR products demonstrated that the amplified AGV2 sequences could vary up to 15.8% and could preliminarily be divided in three groups. This indicated the occurrence of variants of AGV2, which may reflect differences in geographical origin and/or in biological properties. The data presented here provides evidence that AGV2 seems fairly distributed in chickens in Southern Brazil and that AGV2 also circulates in the Netherlands. Besides, circulating viruses display genetic variants whose significance should be further examined, particularly to determine whether AGV2 would play any role in chicken diseases.

Surveillance of human and swine adenovirus, human norovirus and swine circovirus in water samples in Santa Catarina, Brazil

Animal and human wastewater can potentially contaminate water sources and the treatment of drinking water may not effectively remove all contaminants, especially viruses. The purpose of the present study was to evaluate the viral contamination of water used for human and animal consumption in the city of Concórdia, located in southern Brazil. Porcine circovirus type 2 (PCV2), porcine adenovirus (PAdV), human adenovirus (HAdV) and human norovirus (NoV) were searched for using quantitative polymerase chain reaction (qPCR). HAdV-positive samples were tested for viral infectivity by plaque assay. The qPCR results showed that PAdV, PCV2 and HAdV genetic material were present in all sampling sites. NoV was absent in all samples. The presence of genetic material from PAdV and PCV2 was detected in 30% and 45% of the 36 analyzed samples, respectively, with an average of 10(2) gc mL(-1) for PAdV and 10(4) gc mL(-1) for PCV2. HAdV was present in 100% of the samples, with an average of 10(4) gc mL(-1). However, in plaque assay, only 36% of the samples were positive. As viable particles of HAdV were found in drinking water, these results confirm that swine manure and human sewage impact surface water and groundwater, endangering water quality and indicating a potential risk to public health.

Culturing and molecular methods to assess the infectivity of porcine circovirus from treated effluent of swine manure.

Samples were collected at the effluent of two swine manure treatment systems and were analyzed by qPCR to determine the presence and amounts of porcine circovirus (PCV2) genetic material. ST cells were inoculated with the positive samples to evaluate virus viability and for viral genotyping. Twenty-five water samples were collected monthly from treated effluent (March 2009 to December 2010). The PCV2 genome was identified by qPCR in 60% of the samples, and all of the positive samples were able to infect ST cells in vitro. Positive samples were genotyped and 60% of them were positive for both PCV2a and PCV2b, 20% were positive for genotype 2a, and 20% were positive for genotype 2b. Our results suggest that these viruses were able to resist the regular wastewater treatment, and this finding demonstrates the necessity of adding a virus inactivation step to the treatment system to guarantee the safety of water reuse.

Experimental infection of rabbits with a recombinant bovine herpesvirus type 5 (BoHV-5) gI, gE and US9-negative

Bovine herpesvirus type 5 (BoHV-5) is a major cause of viral meningoencephalitis in cattle. The expression of different viral proteins has been associated with BoHV-5 neuropathogenesis. Among these, gI, gE and US9 have been considered essential for the production of neurological disease in infected animals. To evaluate the role of gI, gE and US9 in neurovirulence, a recombinant from which the respective genes were deleted (BoHV-5 gI-/gE-/US9-) was constructed and inoculated in rabbits of two age groups (four and eight weeks-old). When the recombinant virus was inoculated through the paranasal sinuses of four weeks-old rabbits, neurological disease was observed and death was the outcome in 4 out of 13 (30.7 %) animals, whereas clinical signs and death were observed in 11/13 (84.6%) of rabbits infected with the parental virus. In eight weeks-old rabbits, the BoHV-5 gI-/gE-/US9- did not induce clinically apparent disease and could not be reactivated after dexamethasone administration, whereas wild type BoHV-5 caused disease in 55.5% of the animals and was reactivated. These findings reveal that the simultaneous deletion of gI, gE and US9 genes did reduce but did not completely abolish the neurovirulence of BoHV-5 in rabbits, indicating that other viral genes may also play a role in the induction of neurological disease.