Porcine respiratory disease complex after the introduction of H1N1/2009 influenza virus in Brazil.

From 2009 to 2015, 74 lungs from suckling (6.8%), nursing (70.3%), fattening (20.3%) pigs and pregnant sows (2.7%) with respiratory signs from pig farms in Southern Brazil were submitted to a diagnostic laboratory for necropsy and/or histologic examination and screening for respiratory agents by RT-qPCR, immunohistochemistry (IHC), virus isolation (VI) and subtyping for influenza A virus (IAV), IHC and nested PCR for Mycoplasma hyopneumoniae (Mhyo), PCR for porcine circovirus 2 (PCV2), RT-qPCR for porcine reproductive and respiratory syndrome virus (PRRSV) and bacterial culture. All lung samples were positive for IAV using RT-qPCR. Seventy-two lungs had histologic lesions associated with acute to subacute IAV infection characterized by necrotizing bronchiolitis/bronchitis or bronchointerstitial pneumonia with lymphocytic peribronchiolitis and bronchiolar/bronchial hyperplasia, respectively. Forty-nine lungs (66.2%) were positive by IHC for IAV nucleoprotein. The H1N1/2009 was the most common subtype and the only IAV detected in 58.1% of lungs, followed by H1N2 (9.5%) and H3N2 (6.8%). Coinfection of IAV and Mhyo was seen in 23 (31%) cases. Although 14.9% of the lungs were positive for PCV2 using PCR, no suggestive lesions of PCV2 disease were observed. Porcine reproductive and respiratory syndrome virus (PRRSV) was not detected, consistent with the PRRS-free status of Brazil. Secondary bacterial infections (8/38) were associated with suppurative bronchopneumonia and/or pleuritis. Primary IAV infection with Mhyo coinfection was the most common agents found in porcine respiratory disease complex (PRDC) in pigs in Southern Brazil.

Genome-wide association study of periweaning failure-to-thrive syndrome (PFTS) in pigs.

Porcine periweaning-failure-to-thrive syndrome (PFTS) is a condition that affects newly weaned piglets. It is characterised by a progressive debilitation leading to death, in the absence of infectious, nutritional, management or environmental factors. In this study, we present the first report of PFTS in South America and the results of a genome-wide association study to identify the genetic markers associated with the appearance of this condition in a crossbred swine population. Four chromosomal regions were associated with PFTS predisposition, one located on SSCX, one on SSC8, and the two other regions on SSC14. Regions on SSC8 and SSC14 harbour important functional candidate genes involved in human depression and might have an important role in PFTS. Our findings contribute to the increasing knowledge about this syndrome, which has been investigated since 2007, and to the identification of the aetiology of this disease.

Avaliação histopatológica de órgãos reprodutivos e bexiga de fêmeas suínas descartadas / Histopathology of reproductive organs and bladder of culled sows

Em um sistema intensivo de produção de suínos, as falhas reprodutivas são uma das principais razões de descarte de matrizes e queda nos índices produtivos. A infecção urinária (cistite) e as endometrites são consideradas importantes causas de descarte em fêmeas suínas, por terem consequências reprodutivas relevantes e elevarem a taxa de reposição do plantel. O presente estudo teve o objetivo de avaliar o aparelho reprodutivo e a bexiga de fêmeas suínas de descarte normal de granjas, bem como investigar a existência de relação entre as patologias encontradas. Foram examinadas 79 matrizes suínas oriundas de 20 rebanhos localizados no Estado de Santa Catarina. De cada fêmea foram coletados os ovários, fragmentos de útero e bexiga. Dentre as fêmeas avaliadas, 32 (40,5%) tinham diferentes graduações de cistite, 24 (30,4%) tinham algum tipo de inflamação uterina, e 9 (11,4%) estavam em anestro, com ovários inativos. Contudo, não foi observada dependência significativa entre cistite e endometrite nas amostras analisadas.

Reproductive failures are the major reasons for removal of sows and decrease of production rates in an intensive swine production system. Urinary infection and endometritis are considered important causes for culling of sows, due to relevant reproductive consequences and increase of the replacement rates. The present study aimed to evaluate the reproductive and urinary system of culled sows, as well as investigate the occurrence of cystitis and endometritis in analyzed sows. Samples, such as ovaries, uterus fragments and bladder were collected from 79 sows originated from 20 farms of Santa Catarina State. Results showed that, 32 (40,5%) analyzed sows presented cystitis in different levels, 24 (30,4%) had some class of uterine inflammation, and 9 (11,4%) were in anestrous, with inactive ovaries. However, unsignificative dependence between cystitis and endometritis in analyzed samples was observed.

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.

Phylogenetic comparison of the carboxy-terminal region of glycoprotein C (gC) of bovine herpesviruses (BoHV) 1.1, 1.2 and 5 from South America (SA).

Different types and subtypes of bovine herpesvirus 1 and 5 (BoHV-1 and BoHV-5) have been associated to different clinical conditions of cattle, in such a way that type/subtype differentiation has become an essential tool for understanding the pathogenesis and epidemiology of BoHV infections. In search for a genomic region that would allow a clear distinction between BoHV-1 and BoHV-5, the carboxy-terminal portion of glycoprotein C (gC), corresponding to residues 321-450 (BoHV-1) and 301-429 (BoHV-5) of 23 South American (SA) isolates (Brazil mostly) was amplified and sequenced. The nucleotide sequence alignments revealed levels of genomic similarity ranging from 98.7 to 99.8% among BoHV-1 isolates, 88.3 to 92% between BoHV-1/BoHV-5 and 96 to 99.7% among BoHV-5 isolates. At the amino acid level, sequence similarity varied ranging from 97.5 to 99.5% among BoHV-1, 77.5 to 84.4% between BoHV-1/BoHV-5 and 92.1 to 99.5% (BoHV-5/BoHV-5). The isolates could be clearly separated into BoHV-1.1, BoHV-1.2 and BoHV-5 after phylogenetic analysis. The results suggest that the phylogenetic analysis performed here can be used as a potential molecular epidemiological tool for herpesviruses.

Coinfecção experimental de circovírus suíno tipo 2 isolado no Brasil e parvovírus suíno em suínos SPF / Experimental coinfection of SPF pigs with porcine circovirus type 2 isolated in Brazil and porcine parvovirus

Avaliou-se a patogenicidade do circovírus suíno tipo 2 (PCV2) isolado no estado de Santa Catarina mediante coinfecção experimental com parvovírus suíno (PPV). Foram utilizados 24 leitões specific pathogen free (SPF) com cinco dias de idade, distribuídos em quatro grupos (G), alojados em salas independentes e inoculados por via intranasal: G1 - controle (n=4); G2 - inoculados com PCV2 (n=7); G3 - inoculados com PPV (n=6); G4 - inoculados com PCV2 e PPV (n=7). Os animais foram monitorados diariamente para avaliação clínica e necropsiados 48 dias após a infecção. As principais lesões anatomopatológicas observadas nos suínos do G2 e G4 foram: aumento do volume dos linfonodos, depleção linfocitária com redução dos folículos linfóides nos órgãos linfocitários e presença de infiltrado eosinofílico nos linfonodos. A técnica de nested-PCR para PCV2 foi utilizada detectando DNA viral em órgãos de todos os animais do G2 e G4. O PCV2 infectou suínos SPF por via intranasal e foi detectado em outros órgãos, com mais lesões histopatológicas e em maior proporção nos animais coinfectados com PPV (G4), quando comparados aos infectados somente com PCV2 (G2).

The virulence of porcine circovirus type 2 (PCV2) isolated in Santa Catarina State by coinfection with porcine parvovirus (PPV) was investigated. Twenty-four, 5-day-old SPF pigs were distributed into four groups, housed in separate rooms and inoculated by intranasal route: G1 - control (n=4); G2 - inoculated with PCV2 (n=7); G3 - inoculated with PPV (n=6); G4 - inoculated with PCV2 and PPV (n=7). The animals were monitored daily for clinical evaluation and were necropsied 48 days after the infection. The pathological lesions seen in G2 and G4 pigs were: enlargement of lymph nodes, mild to moderate lymphoid cell depletion, affecting lymphoid follicles in lymphoid organs and presence of infiltration by eosinophils in lymph nodes. PCV2 DNA was detected by a nested-PCR in all pigs of G2 and G4. These findings confirmed that pigs were successfully infected intranasally with PCV2. The presence of PCV2 DNA in tissue samples and the pathological lesions were more evident in pigs infected with both PCV2 and PPV than in pigs infected with PCV2 alone.

Diagnosis of post-weaning multisystemic wasting syndrome in pigs in Brazil caused by porcine circovirus type 2

Este trabalho descreve a primeira caracterização preliminar de isolados de circovírus suíno tipo 2 (PCV2) a partir de órgãos de suínos acometidos pela síndrome da refugagem multissistêmica (SRM) no Brasil. Leitões doentes foram examinados à necropsia e por histopatologia. Análises macroscópicas e microscópicas demonstraram lesões típicas de SRM, respectivamente, emagrecimento, aumento do volume dos linfonodos, atrofia de timo e pneumonia intersticial, e linfadenite granulomatosa com células sinciciais, entre outras. A presença de antígeno ou DNA do PCV2 foi demonstrada por imunoperoxidase ou reação da polimerase em cadeia nested (nested PCR), respectivamente. Foi possível diferenciar PCV1 e PCV2 por análises de polimorfismo de fragmento de restrição (RFLP) do fragmento amplificado da PCR. O DNA do PCV2 foi detectado em 70 por cento (14/20) das amostras obtidas de suíno com sintomatologia clínica e lesões associadas com SRM. Este estudo apresenta a associação de PCV2 com lesões e sinais clínicos de SRM em suínos e também indica que os isolados do PCV2 brasileiros podem apresentar variações genômicas.

Analysis of fumonisin B1-induced apoptosis.

Fumonisins are mycotoxins produced by Fusarium moniliforme, a prevalent fungus that infects corn and other cereal grains. Fumonisin B1(FB1 is the most common mycotoxin produced by F. moniliforme, suggesting it has toxicologic significance. The structure of FB1 resembles sphingoid bases, and it inhibits ceramide synthase. Because sphingoid bases regulate cell growth, differentiation, transformation, and apoptosis, it is not surprising to find that FB1 can alter growth of certain mammalian cells. Previous studies concluded FB1-induced apoptosis, or cell cycle arrest, in African green monkey kidney fibroblasts (CV-1). In this study we have identified genes that inhibit FB1 induced apoptosis in CV-1 cells and two mouse embryo fibroblasts (MEF). A baculovirus gene, inhibitor of apoptosis (CpIAP), protected these cells from apoptosis. CpIAP blocks apoptosis induced by the tumor necrosis factor (TNF) pathway as well as other mechanisms. Further support for the involvement of the TNF signal transduction pathway in FB1 induced apoptosis was the cleavage of caspase 8. Inhibition of caspases by the baculovirus gene (italic)p35 also inhibited FB1-induced apoptosis. The tumor suppressor gene p53 was not required for FB1 induced apoptosis because p53-/- MEF undergo apoptosis following FB1 treatment. Furthermore, Bcl-2 was not an effective inhibitor of FB1-induced apoptosis in CV-1 cells or p53+/+ MEF. In summary, these results provide new information to help understand the mechanism by which FB1 induces apoptosis.

Fumonisin B1, a mycotoxin contaminant of cereal grains, and inducer of apoptosis via the tumour necrosis factor pathway and caspase activation.

Fumonisins are mycotoxins produced by Fusarium moniliforme, a prevalent fungus which infects corn or other cereal grains. Fumonisin B1 (FB1) is the most common mycotoxin produced by F. moniliforme, suggesting that it has toxicological significance. The structure of FB1 resembles sphingoid bases and it inhibits ceramide synthase. As sphingoid bases regulate cell growth, differentiation, transformation and apoptosis, it is reasonable to hypothesize that FB1 can also regulate these activities. Previous studies concluded that FB1 induced apoptosis or cell-cycle arrest in CV-1 cells (African green monkey kidney fibroblasts). In this study, we have identified genes that inhibit FB1-induced apoptosis in CV-1 cells and in two primary human cell types (lung fibroblasts and neonatal kidney cells). A baculovirus gene. inhibitor of apoptosis (IAP), protected CV-1 and the human cells from apoptosis. IAP blocks apoptosis which is induced by the tumour necrosis factor (TNF) pathway. Inhibition of interleukin converting enzymes (ICE proteases or caspases) by the baculovirus gene p35 also inhibited FB1-induced apoptosis. FB1 treatment led to cleavage of Rb (retinoblastoma protein) at its C-terminus in CV-1 or human lung cells. As the C-terminus of Rb is cleaved by ICE proteases during apoptosis, this supports an active role for ICE proteases in FB1-induced apoptosis. The tumour suppressor gene p53 was not required for FB1-induced apoptosis because p53-/- primary mouse embryo fibroblasts underwent apoptosis following FB1 treatment. Furthermore, Bcl-2 was not an effective inhibitor of FB1-induced apoptosis in CV-1 or IMR-90 cells. In summary, these results demonstrate that the TNF pathway and caspases plays an important role in FB1-induced apoptosis.

Characterization of cell-cycle arrest by fumonisin B1 in CV-1 cells.

Fusarium moniliforme is a widespread fungal pathogen which primarily infects corn, but can also infect rice or wheat. Fusarium moniliforme produce several mycotoxins, the most prominent of which is called fumonisin B1 (FB1). Epidemiological studies have indicated that ingestion of fumonisins correlates with a higher incidence of oesophageal cancer in Africa and China. Fumonisins also cause a neurodegenerative disease in horses, induce hepatic cancer in rats, are nephrotoxic in rats, or cause pulmonary oedema in swine. Structurally, fumonisins resemble sphingolipids and can alter sphingolipid biosynthesis. suggesting that sphingolipid alterations play a role in disease and carcinogenesis. Previous studies determined that FB1 blocked cell-cycle progression in CV-1 cells but not COS-7 cells. Herein, we have examined the effects that FB1 treatment has on cell-cycle regulatory proteins. Our studies established that FB1 treatment of CV-1 cells, but not COS-7 cells, leads to dephosphorylation of the retinoblastoma (Rb) protein. Cyclin dependent kinase 2 (CDK2) activity was repressed five- to 10-fold and cyclin E protein levels were lower in CV-1 cells after fumonisin treatment. Two CDK inhibitors, Kip1 and Kip2, were induced within 3 hours after fumonisin treatment of CV-1 cells, suggesting these two proteins mediate cell-cycle arrest induced by FB1. This mycotoxin caused large increases in sphinganine within 3 hours after addition of FB1. As sphingoid bases are known to induce Rb phosphorylation, this increase in sphinganinie might be the stimulus for the suppression of cyclin dependent kinase activities via Kip1 and Kip2. The ability of FB1 to accumulate sphingosine or sphinganine and arrest the cell cycle in some cells but not others may play an important role in carcinogenesis or disease.