F.F. Silva

Tripping over emerging pathogens around the world: A phylogeographical approach for determining the epidemiology of Porcine circovirus-2 (PCV-2), considering global trading

Porcine circovirus-2 (PCV-2) is an emerging virus associated with a number of different syndromes in pigs known as Porcine Circovirus Associated Diseases (PCVAD). Since its identification and characterization in the early 1990s, PCV-2 has achieved a worldwide distribution, becoming endemic in most pig-producing countries, and is currently considered as the main cause of losses on pig farms. In this study, we analyzed the main routes of the spread of PCV-2 between pig-producing countries using phylogenetic and phylogeographical approaches. A search for PCV-2 genome sequences in GenBank was performed, and the 420 PCV-2 sequences obtained were grouped into haplotypes (group of sequences that showed 100% identity), based on the infinite sites model of genome evolution. A phylogenetic hypothesis was inferred by Bayesian Inference for the classification of viral strains and a haplotype network was constructed by Median Joining to predict the geographical distribution of and genealogical relationships between haplotypes. In order to establish an epidemiological and economic context in these analyses, we considered all information about PCV-2 sequences available in GenBank, including papers published on viral isolation, and live pig trading statistics available on the UN Comtrade database (http://comtrade.un.org/). In these analyses, we identified a strong correlation between the means of PCV-2 dispersal predicted by the haplotype network and the statistics on the international trading of live pigs. This correlation provides a new perspective on the epidemiology of PCV-2, highlighting the importance of the movement of animals around the world in the emergence of new pathogens, and showing the need for effective sanitary barriers when trading live animals.

The Paradox of Feline Coronavirus Pathogenesis: A Review

Feline coronavirus (FCoV) is an enveloped single-stranded RNA virus, of the family Coronaviridae and the order Nidovirales. FCoV is an important pathogen of wild and domestic cats and can cause a mild or apparently symptomless enteric infection, especially in kittens. FCoV is also associated with a lethal, systemic disease known as feline infectious peritonitis (FIP). Although the precise cause of FIP pathogenesis remains unclear, some hypotheses have been suggested. In this review we present results from different FCoV studies and attempt to elucidate existing theories on the pathogenesis of FCoV infection.

A genome-wide association study reveals a novel candidate gene for sperm motility in pigs

Sperm motility is one of the most widely used parameters in order to evaluate boar semen quality. However, this trait can only be measured after puberty. Thus, the use of genomic information appears as an appealing alternative to evaluate and improve selection for boar fertility traits earlier in life. With this study we aimed to identify SNPs with significant association with sperm motility in two different commercial pig populations and to identify possible candidate genes within the identified QTL regions. We performed a single-SNP genome-wide association study using genotyped animals from a Landrace-based (L1) and a Large White-based (L2) pig populations. For L1, a total of 602 animals genotyped for 42,551 SNPs were used in the association analysis. For L2, a total of 525 animals genotyped for 40,890 SNPs were available. After the association analysis, a false discovery rate q-value ≤0.05 was used as the threshold for significant association. No SNPs were significantly associated with sperm motility in L1, while six SNPs on Sus scrofa chromosome 1 (position 117.26-119.56Mb) were significant in L2. The mitochondrial methionyl-tRNA formyltransferase (MTFMT) gene, which affects translation efficiency of proteins in sperm cells, was identified as a putative candidate gene. The significant markers identified in this study may be useful to enhance the genetic improvement of sperm motility by selection of boars at an earlier age under a marker assisted selection strategy.

Porcine Circovirus-2 Viral Load versus Lesions in Pigs: Perspectives for Post-weaning Multisystemic Wasting Syndrome

Porcine circovirus-2 (PCV-2) is the main agent related to post-weaning multisystemic wasting syndrome (PMWS) and it is also associated with other syndromes affecting pigs. Not all pigs infected with PCV-2 will develop PMWS and the incidence of PMWS is higher when coinfecting viral and bacterial pathogens are present. In this study, PCV-2 viral loads were evaluated in the tissues of animals with and without PMWS in order to investigate the relationship between viral load and microscopical lesions. Lymph nodes had the highest average viral load, but there was no significant difference between lesion severity and the viral load in these structures. There was no significant difference between the average viral load in inguinal lymph nodes of animals with and without PMWS. However, samples from pigs with PMWS had more severe lesions compared with samples from non-PMWS animals. These findings suggest that other infectious and non-infectious cofactors may be important in the pathogenesis of PMWS.

Tracking the molecular epidemiology of Brazilian Infectious bursal disease virus (IBDV) isolates.

Infectious bursal disease is a highly contagious disease of young chickens caused by Infectious bursal disease virus (IBDV). Genome segment A encodes the capsid protein (VP2), while segment B encodes the RNA-dependent RNA polymerase (VP1). In the present study, we trace the molecular epidemiology of IBDV in Brazil by analyzing 29 isolates collected in the major regions of poultry production. To genetically characterize the isolates, phylogenetic and population dynamic analyses were conducted using 68 VP1 (2634 nt) and 102 VP2 (1356 nt) coding sequences from IBDV isolates from different regions of the world. Furthermore, the evolution of IBDV was analyzed by characterizing the selective forces that operated during the diversification of viral isolates. We show that IBDV isolates were introduced into Brazil mainly from the Netherlands and the USA. These introductions were associated with all Brazilian poultry production regions analyzed in this work. In addition, we show that the evolution of IBDV has been shaped by a combination of very low recombination rates and relatively high rates of nucleotide substitution (2.988×10(-4) for VP1 and 3.2937×10(-4) for VP2), which themselves are a function of purifying selection operating on VP1 and VP2. Furthermore, our extended Bayesian skyline plot suggests that the increase in the effective population size of isolates of IBDV is consistent with its epidemiological history, with a large increase during the emergence of acute outbreaks of IBD in the 1980s.

Supervised independent component analysis as an alternative method for genomic selection in pigs

The objective of this work was to evaluate the efficiency of the supervised independent component regression (SICR) method for the estimation of genomic values and the SNP marker effects for boar taint and carcass traits in pigs. The methods were evaluated via the agreement between the predicted genetic values and the corrected phenotypes observed by cross-validation. These values were also compared with other methods generally used for the same purposes, such as RR-BLUP, SPCR, SPLS, ICR, PCR and PLS. The SICR method was found to have the most accurate prediction values.

Use of molecular markers to improve relationship information in the genetic evaluation of beef cattle tick resistance under pedigree-based models

The selection of genetically superior individuals is conditional upon accurate breeding value predictions which, in turn, are highly depend on how precisely relationship is represented by pedigree. For that purpose, the numerator relationship matrix is essential as a priori information in mixed model equations. The presence of pedigree errors and/or the lack of relationship information affect the genetic gain because it reduces the correlation between the true and estimated breeding values. Thus, this study aimed to evaluate the effects of correcting the pedigree relationships using single-nucleotide polymorphism (SNP) markers on genetic evaluation accuracies for resistance of beef cattle to ticks. Tick count data from Hereford and Braford cattle breeds were used as phenotype. Genotyping was carried out using a high-density panel (BovineHD - Illumina® bead chip with 777xa0962 SNPs) for sires and the Illumina BovineSNP50 panel (54xa0609 SNPs) for their progenies. The relationship between the parents and progenies of genotyped animals was evaluated, and mismatches were based on the Mendelian conflicts counts. Variance components and genetic parameters estimates were obtained using a Bayesian approach via Gibbs sampling, and the breeding values were predicted assuming a repeatability model. A total of 460 corrections in relationship definitions were made (Tablexa01) corresponding to 1018 (9.5%) tick count records. Among these changes, 97.17% (447) were related to the sires information, and 2.8% (13) were related to the dams information. We observed 27.2% (236/868) of Mendelian conflicts for sire-progeny genotyped pairs and 14.3% (13/91) for dam-progeny genotyped pairs. We performed 2174 new definitions of half-siblings according to the correlation coefficient between the coancestry and molecular coancestry matrices. It was observed that higher-quality genetic relationships did not result in significant differences of variance components estimates; however, they resulted in more accurate breeding values predictions. Using SNPs to assess conflicts between parents and progenies increases certainty in relationships and consequently the accuracy of breeding value predictions of candidate animals for selection. Thus, higher genetic gains are expected when compared to the traditional non-corrected relationship matrix.

Development and evaluation of a recombinant DNA vaccine candidate expressing porcine circovirus 2 structural protein

Porcine circovirus 2 (PCV2) is generally associated with the porcine circovirosis syndrome, which is considered an important disease of swine and has potentially serious economic impact on the swine industry worldwide. This article describes the construction of a recombinant plasmid expressing the PCV2 structural protein and the evaluation of cellular and humoral immune responses produced by this recombinant vaccine in BALB/c mice. The vaccine candidate was obtained and analyzed in vivo, in an effort to determine the ability to induce a specific immune response in mice. DNA was extracted from a Brazilian PCV2 isolate and the gene coding for Cap protein was amplified by PCR and inserted into an expression plasmid. Groups of BALB/c mice were inoculated intra-muscularly and intradermally in a 15-day interval, with 100 µg and 50 µg of the vaccine construct, respectively. Another group was inoculated intramuscularly with 100 µg of empty plasmid, corresponding to the control group. Seroconversion and cellular response in BALB/c mice were compared and used for vaccine evaluation. Seroconversion was analyzed by ELISA. After a series of 3 immunizations the spleen cells of the immunized animals were used to perform lymphocyte proliferation assays. Seroconversion to PCV2 was detected by ELISA in the animals inoculated with the vaccine construct when compared with control groups. Lymphocyte proliferation assays showed a stronger cell proliferation in the inoculated animals compared with the control group. Thus, the vaccine candidate construct demonstrated to be able to induce both humoral and cellular responses in inoculated mice.

Detection of Mycoplasma hyopneumoniae in lungs and nasal swabs of pigs by nested PCR

Fifty-four samples were collected from growing and finishing pigs for the molecular diagnosis of enzootic porcine pneumonia. Nineteen lung fragments were obtained from pigs that showed signs of respiratory disease and 35 nasal swabs were obtained from clinically healthy pigs. For the detection of the bacterial genome in the samples, the nested PCR technique was used to amplify a fragment of 706bp. This fragment was subsequently cloned and sequenced. The sequence of obtained nucleotides was compared with six other sequences of Mycoplasma hyopneumoniae and 11 sequences of other bacteria available in the Genbank. To measure the sensitivity of the nested PCR, serial dilutions (10-1 to 10-15) of cloned fragments were conducted based on the concentration of 300ng. Ten lung fragments and eight nasal swabs showed positive for M. hyopneumoniae and the limit of detection was estimated to be 0.3fg DNA cloned. The sequence of nucleotides obtained showed 99.1% homology with the other sequences of M. hyopneumoniae, demonstrating that the nested PCR used in this study may provide an important diagnostic tool for the detection of this agent.

Retrospective study on Porcine circovirus-2 by nested pcr and real time pcr in archived tissues from 1978 in Brazil

Porcine circovirus-2 (PCV-2) infection is currently considered an important disease of swine. The pathogenic agent was first described in Brazil in 2000. This study detected the PCV-2 DNA in four Brazilian pig tissues collected between 1978 and 1979. This observation is the oldest description of this virus in Brazil.