Histopathological and immunohistochemical findings of swine with spontaneous influenza A infection in Brazil, 2009-2010

Swine influenza (SI) is caused by the type A swine influenza virus (SIV). It is a highly contagious disease with a rapid course and recovery. The major clinical signs and symptoms are cough, fever, anorexia and poor performance. The disease has been associated with other co-infections in many countries, but not in Brazil, where, however, the first outbreak has been reported in 2011. The main aim of this study was to characterize the histological features in association with the immunohistochemical (IHC) results for influenza A (IA), porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) in lung samples from 60 pigs submitted to Setor de Patologia Veterinária at the Universidade Federal do Rio Grande do Sul (SPV-UFRGS), Brazil, during 2009-2010. All of these lung samples had changes characterized by interstitial pneumonia with necrotizing bronchiolitis, never observed previously in the evaluation of swine lungs in our laboratory routine. Pigs in this study had showed clinical signs of a respiratory infection. Swine samples originated from Rio Grande do Sul 31 (52%), Santa Catarina 14 (23%), Paraná 11 (18%), and Mato Grosso do Sul 4 (7%). Positive anti-IA IHC labelling was observed in 45% of the cases, which were associated with necrotizing bronchiolitis, atelectasis, purulent bronchopneumonia and hyperemia. Moreover, type II pneumocyte hyperplasia, alveolar and bronchiolar polyp-like structures, bronchus-associated lymphoid tissue (BALT) hyperplasia and pleuritis were the significant features in negative anti-IA IHC, which were also associated with chronic lesions. There were only two cases with positive anti-PCV2 IHC and none to PRRSV. Therefore, SIV was the predominant infectious agent in the lung samples studied. The viral antigen is often absent due to the rapid progress of SI, which may explain the negative IHC results for IA (55%); therefore, IHC should be performed at the beginning of the disease. This study has shown how important a careful histological evaluation is for the diagnosis. Since 2009, a new histological feature of swine pneumonia in animals with respiratory clinical signs has been observed in samples from pigs with clinical respiratory disease submitted to SPV-UFRGS. In addition, the results proved the importance of histological evaluation for swine herd health management.

Diagnosis and clinic-pathological findings of influenza virus infection in Brazilian pigs

Influenza A virus (IAV) is a respiratory pathogen of pigs and is associated with the porcine respiratory disease complex (PRDC), along with other respiratory infectious agents. The aim of this study was to diagnose and to perform a clinic-pathological characterization of influenza virus infection in Brazilian pigs. Lung samples from 86 pigs in 37 farrow-to-finish and two farrow-to-feeder operations located in the States of Minas Gerais, São Paulo, Paraná, Rio Grande do Sul, Santa Catarina, and Mato Grosso were studied. Virus detection was performed by virus isolation and quantitative real time reverse-transcription PCR (qRT-PCR). Pathologic examination and immunohistochemistry (IHC) were performed in 60 lung formalin-fixed paraffin-embedded tissue fragments. Affected animals showed coughing, sneezing, nasal discharge, hyperthermia, inactivity, apathy, anorexia, weight loss and growth delay, which lasted for five to 10 days. Influenza virus was isolated from 31 (36.0%) lung samples and 36 (41.9%) were positive for qRT-PCR. Thirty-eight (63.3%) lung samples were positive by IHC and the most frequent microscopic lesion observed was inflammatory infiltrate in the alveoli, bronchiole, or bronchi wall or lumen (76.7%). These results indicate that influenza virus is circulating and causing disease in pigs in several Brazilian states.

Orientações para o diagnóstico de influenza em suínos

Este trabalho descreve a colheita adequada de amostras, as técnicas/procedimentos disponíveis para o diagnóstico de influenza A em suínos, assim como os resultados e suas respectivas interpretações, para auxiliar médicos veterinários de campo na identificação dessa doença. Em suínos vivos, as amostras adequadas são: secreção nasal, fluido oral e sangue (soro). Para suínos mortos, colher preferencialmente amostras de pulmão com consolidação cranioventral. Secreção nasal e fragmentos de pulmão refrigerado são utilizados para detectar partícula viral viável (isolamento viral - IV) ou ácido nucleico viral (RT-PCR convencional e RT-PCR em tempo real). As amostras não devem ser congeladas, pois o vírus é inativado a -20°C. A caracterização molecular dos isolados é feita pela análise filogenética obtida pelo sequenciamento de DNA. O soro é utilizado para a detecção de anticorpos (Acs) por meio do teste da inibição da hemaglutinação e ELISA. O fluido oral pode ser utilizado para detecção de anticorpo (ELISA) ou de vírus. Fragmentos de pulmão fixados em formol a 10% são examinados microscopicamente para identificar pneumonia broncointersticial e para detecção de antígeno viral pela imuno-histoquímica (IHQ). Para o sucesso do diagnóstico, as amostras devem ser colhidas de suínos que estão preferencialmente na fase aguda da doença, para aumentar as chances de detecção viral. As melhores opções para o diagnóstico de influenza A em suínos vivos são RT-PCR e isolamento viral de amostras de swab nasal ou fluido oral. Pulmão para análise por RT-PCR, isolamento viral ou IHQ é a amostra de escolha em suínos mortos. Testes sorológicos têm valor diagnóstico limitado e são utilizados apenas para determinar o estado imune do rebanho, não indicando doença clínica, pois os Acs são detectados 7-10 dias pós-infecção (fase subaguda). O diagnóstico de influenza é importante para avaliar o envolvimento desse agente no complexo de doença respiratória suína. Além disso, o isolamento do vírus influenza é essencial para o monitoramento dos principais subtipos circulantes em uma determinada região ou país, assim como para a detecção de novos rearranjos virais, já que influenza é considerada uma zoonose.

Efficacy of bacterin-, outer membrane protein- and fimbriae extract-based vaccines for the control of Salmonella Enteritidis experimental infection in chickens

The efficacy of three vaccines was evaluated in chickens for the control of experimental infection with Salmonella Enteritidis (SE) phage type 4. The vaccines were produced with bacterin, outer membrane proteins (OMP) and fimbriae crude extract (FE). The chickens were vaccinated intramuscularly with two doses of each vaccine at 12 and 15 weeks of age. The chickens were then orally challenged with 10(9) CFU/chicken Salmonella Enteritidis phage type 4 at 18 weeks of age. Fecal swabs were performed for the recovery of shedding SE, and SE was recovered from the liver and spleen. Additionally, antibody titers were measured in the serum by micro-agglutination test. The results indicated that the vaccine produced with bacterin yielded better results and resulted in reduction of fecal shedding and organ invasion by SE after oral challenge, although no vaccine was 100% effective for the control of SE experimental infection.

Application of various antioxidants in the treatment of influenza

We determined the effect of the antioxidants superoxide dismutase, desferrioxamine and allopurinol on the survival of male CBA mice infected intranasally with 2-5 LD50 lung influenza virus A/Aichi/2/68. Survival for at least 20 days was observed for 45% of the mice that received 1000 U/day superoxide dismutase prepared from red blood cells on days 5, 6, 7 and 8 after infection, and 75% survival was observed for mice that received the same dose on days 4, 5, 6, 7 and 8. Desferrioxamine, 25 mg/kg per day and 100 mg/kg per day injected subcutaneously, resulted in survival rates of 5 and 0%, respectively, compared to 10% survival observed for saline-injected controls. Allopurinol at doses of 5 to 50 mg/kg per day had no effect on mouse survival. These data demonstrate the efficacy of superoxide dismutase for the protection of mice against hemorrhagic lung edema. The ineffectiveness of allopurinol suggests that the xanthine oxidase system does not play a major role in hemorrhage or lung edema and that caution is necessary when desferrioxamine is administered during an acute inflammatory process accompanied by erythrocyte lysis

Analysis of viral and cellular parameters which affect the fusion process of influenza viruses

In the present investigation we studied the fusogenic process developed by influenza A, B and C viruses on cell surfaces and different factors associated with virus and cell membrane structures. The biological activity of purified virus strains was evaluated in hemagglutination, sialidase and fusion assays. Hemolysis by influenza A, B and C viruses ranging from 77.4 to 97.2%, from 20.0 to 65.0%, from 0.2 to 93.7% and from 9.0 to 76.1% was observed when human, chicken, rabbit and monkey erythrocytes, respectively, were tested at pH 5.5. At this pH, low hemolysis indexes for influenza A, B and C viruses were observed if horse erythrocytes were used as target cells for the fusion process, which could be explained by an inefficient receptor binding activity of influenza on N-glycolyl sialic acids. Differences in hemagglutinin receptor binding activity due to its specificity to N-acetyl or N-glycolyl cell surface oligosaccharides, density of these cellular receptors and level of negative charges on the cell surface may possibly explain these results, showing influence on the sialidase activity and the fusogenic process. Comparative analysis showed a lack of dependence between the sialidase and fusion activities developed by influenza B viruses. Influenza A viruses at low sialidase titers (<2) also exhibited clearly low hemolysis at pH 5.5 (15.8%), while influenza B viruses with similarly low sialidase titers showed highly variable hemolysis indexes (0.2 to 78.0%). These results support the idea that different virus and cell-associated factors such as those presented above have a significant effect on the multifactorial fusion process

Immunization against the colonization factor antigen I of enterotoxigenic Escherichia coli by administration of a bivalent Salmonella typhimurium aroA strain

An expression plasmid (pCFA-1) carrying the cfaB gene that codes for the enterotoxigenic Escherichia coli (ETEC) fimbrial adhesin colonization factor antigen I (CFA/I) subunit was constructed and used to transform a derivative of the attenuated Salmonella typhimurium aroA vaccine strain SL3261 carrying an F'lacIq. Treatment of the transformed strain with isopropyl-ß-D-thiogalactopyranoside (IPTG) resulted in elevated in vitro expression of the CFA/I subunit. Although flagellar function and lipopolysaccharide (LPS) synthesis were similar in both the parental and the recombinant strains, spleen colonization was reduced in the recombinant strain. All BALB/c mice parenterally inoculated with the recombinant strain developed significant anti-CFA/I and anti-LPS serum antibody titers (P<0.05). Moreover, 2 of 5 mice orally inoculated with the engineered Salmonella strain developed anti-CFA/I intestinal IgA (P>0.05) while 4/5 of the same mice developed anti-LPS IgA (P<0.05). The results indicate that the vaccine strain elicited an antibody response against the bacterial host both after oral and intravenous immunization while the response against the CFA/I antigen was significant only after inoculation by the intravenous route

Pituitary glycoprotein hormone a-subunit secretion by cirrhotic patients

Secretion of the a-subunit of pituitary glycoprotein hormones usually follows the secretion of intact gonadotropins and is increased in gonadal failure and decreased in isolated gonadotropin deficiency. The aim of the present study was to determine the levels of the a-subunit in the serum of patients with cirrhosis of the liver and to compare the results obtained for eugonadal cirrhotic patients with those obtained for cirrhotic patients with hypogonadotropic hypogonadism. Forty-seven of 63 patients with cirrhosis (74.6%) presented hypogonadism (which was central in 45 cases and primary in 2), 7 were eugonadal, and 9 women were in normal menopause. The serum a-subunit was measured by the fluorimetric method using monoclonal antibodies. Cross-reactivity with LH, TSH, FSH and hCG was 6.5, 1.2, 4.3 and 1.1%, respectively, with an intra-assay coefficient of variation (CV) of less than 5% and an interassay CV of 5%, and sensitivity limit of 4 ng/l. The serum a-subunit concentration ranged from 36 to 6253 ng/l, with a median of 273 ng/l. The median was 251 ng/l for patients with central hypogonadism and 198 ng/l for eugonadal patients. The correlation between the a-subunit and basal LH levels was significant both in the total sample (r = 0.48, P<0.01) and in the cirrhotic patients with central hypogonadism (r = 0.33, P = 0.02). Among men with central hypogonadism there was a negative correlation between a-subunit levels and total testosterone levels (r = 0.54, P<0.01) as well as free testosterone levels (r = -0.53, P<0.01). In conclusion, although the a-subunit levels are correlated with LH levels, at present they cannot be used as markers for hypogonadism in patients with cirrhosis of the liver.

Liposomes: from biophysics to the design of peptide vaccines

Liposomes (lipid-based vesicles) have been widely studied as drug delivery systems due to their relative safety, their structural versatility concerning size, composition and bilayer fluidity, and their ability to incorporate almost any molecule regardless of its structure. Liposomes are successful in inducing potent in vivo immunity to incorporated antigens and are now being employed in numerous immunization procedures. This is a brief overview of the structural, biophysical and pharmacological properties of liposomes and of the current strategies in the design of liposomes as vaccine delivery systems.

MHC-restricted antigen presentation and recognition: constraints on gene, recombinant and peptide vaccines in humans

The target of any immunization is to activate and expand lymphocyte clones with the desired recognition specificity and the necessary effector functions. In gene, recombinant and peptide vaccines, the immunogen is a single protein or a small assembly of epitopes from antigenic proteins. Since most immune responses against protein and peptide antigens are T-cell dependent, the molecular target of such vaccines is to generate at least 50-100 complexes between MHC molecule and the antigenic peptide per antigen-presenting cell, sensitizing a T cell population of appropriate clonal size and effector characteristics. Thus, the immunobiology of antigen recognition by T cells must be taken into account when designing new generation peptide- or gene-based vaccines. Since T cell recognition is MHC-restricted, and given the wide polymorphism of the different MHC molecules, distinct epitopes may be recognized by different individuals in the population. Therefore, the issue of whether immunization will be effective in inducing a protective immune response, covering the entire target population, becomes an important question. Many pathogens have evolved molecular mechanisms to escape recognition by the immune system by variation of antigenic protein sequences. In this short review, we will discuss the several concepts related to selection of amino acid sequences to be included in DNA and peptide vaccines.

Immunological properties of gene vaccines delivered by different routes

Gene vaccines represent a new and promising approach to control infectious diseases, inducing a protective immune response in the appropriate host. Several routes and methods of genetic immunization have been shown to induce antibody production as well as T helper (Th) cell and cytotoxic T lymphocyte activation. However, few studies have compared the nature of the immune responses generated by different gene vaccination delivery systems. In the present study we reviewed some aspects of immunity induced by gene immunization and compared the immune responses produced by intramuscular (im) DNA injection to gene gun-mediated DNA transfer into the skin of BALB/c mice. Using a reporter gene coding for ß-galactosidase, we have demonstrated that im injection raised a predominantly Th1 response with mostly IgG2a anti-ßgal produced, while gene gun immunization induced a mixed Th1/Th2 profile with a balanced production of IgG2a and IgG1 subclasses. Distinct types of immune responses were generated by different methods of gene delivery. These findings have important implications for genetic vaccine design. Firstly, a combination between these two systems may create optimal conditions for the induction of a broad-based immune response. Alternatively, a particular gene vaccine delivery method might be used according to the immune response required for host protection. Here, we describe the characteristics of the immune response induced by gene vaccination and the properties of DNA involved in this process.

DNA vaccines for viral diseases

DNA plasmids encoding foreign proteins may be used as immunogens by direct intramuscular injection alone, or with various adjuvants and excipients, or by delivery of DNA-coated gold particles to the epidermis through biolistic immunization. Antibody, helper T lymphocyte, and cytotoxic T lymphocyte (CTL) responses have been induced in laboratory and domesticated animals by these methods. In a number of animal models, immune responses induced by DNA vaccination have been shown to be protective against challenge with various infectious agents. Immunization by injection of plasmids encoding foreign proteins has been used successfully as a research tool. This review summarizes the types of DNA vaccine vectors in common use, the immune responses and protective responses that have been obtained in animal models, the safety considerations pertinent to the evaluation of DNA vaccines in humans and the very limited information that is available from early clinical studies.

New vaccine strategies against enterotoxigenic Escherichia coli: I: DNA vaccines against the CFA/I fimbrial adhesin

Stimulation of the mammalian immune system by administration of plasmid DNA has been shown to be an important approach for vaccine development against several pathogens. In the present study we investigated the use of DNA vaccines to induce immune responses against an enteric bacterial pathogen, enterotoxigenic Escherichia coli (ETEC). Three plasmid vectors encoding colonization factor antigen I (CFA/I), an ETEC fimbrial adhesin, were constructed. Eukaryotic cells transfected with each of these plasmids expressed the heterologous antigen in different compartments: bound to the cytoplasmic membrane (pRECFA), accumulated in the cytoplasm (pPolyCFA) or secreted to the outside medium (pBLCFA). BALB/c mice were intramuscularly (im) inoculated with purified plasmid DNA and the systemic, cellular and secreted CFA/I-specific immune responses were analyzed. The results showed that all three DNA vaccine formulations could elicit CFA/I-specific immune responses. Moreover, cellular location of the plasmid-encoded CFA/I seems to have an important role in the induced immune response. Taken together, these results indicate that DNA vaccines also represent a promising approach against enteric bacterial pathogens.

Expression and biological activity of two recombinant polypeptides related to subunit 1 of the interferon-a receptor

Abnormal production of interferon alpha (IFN-a) has been found in certain autoimmune diseases and can be also observed after prolonged therapy with IFN-a. IFN-a can contribute to the pathogenesis of allograft rejection in bone marrow transplants. Therefore, the development of IFN-a inhibitors as a soluble receptor protein may be valuable for the therapeutic control of these diseases. We have expressed two polypeptides encoding amino acids 93-260 (P1) and 261-410 (P2) of the extracellular domain of subunit 1 of the interferon-a receptor (IFNAR 1-EC) in E. coli. The activities of the recombinant polypeptides and of their respective antibodies were evaluated using antiproliferative and antiviral assays. Expression of P1 and P2 polypeptides was achieved by transformation of cloned plasmid pRSET A into E. coli BL21(DE3)pLysS and by IPTG induction. P1 and P2 were purified by serial sonication steps and by gel filtration chromatography with 8 M urea and refolded by dialysis. Under reducing SDS-PAGE conditions, the molecular weight of P1 and P2 was 22 and 17 kDa, respectively. Polyclonal anti-P1 and anti-P2 antibodies were produced in mice. P1 and P2 and their respective polyclonal antibodies were able to block the antiproliferative activity of 6.25 nM IFN-aB on Daudi cells, but did not block IFN-aB activity at higher concentrations (>6.25 nM). On the other hand, the polypeptides and their respective antibodies did not inhibit the antiviral activity of IFN-aB on Hep 2/c cells challenged with encephalomyocarditis virus.

Reimmunization after bone marrow transplantation: current recommendations and perspectives

Autologous and allogeneic bone marrow transplantation (BMT) recipients lose immune memory of exposure to infectious agents and vaccines accumulated through a lifetime and therefore need to be revaccinated. Diphtheria toxoid, tetanus toxoid, pertussis vaccine (children <7 years old), Haemophilus influenzae type b conjugate, 23-valent pneumococcal polysaccharide, inactivated influenza vaccine, inactivated polio vaccine and live-attenuated measles-mumps-rubella vaccine are the currently recommended vaccines to be included in a vaccination program after BMT. For most of them, the best time to vaccinate, the number of vaccine doses and/or the duration of immunity after vaccination have not been established. Vaccination protocols vary greatly among BMT centers, suggesting that the lack of sufficient data has not permitted the formulation of reliable recommendations. The use of other vaccines and the perspectives for different vaccination protocols are analyzed in this review.