CD4 bovine gene: Differential polymorphisms among cattle breeds and a new tool for rapid identification.

Two mutations in the CD4 bovine gene (G>T/Q306H; A>C/K310N) were identified as causative for altered staining with anti-CD4 mAb #CC8. We developed a HRM qPCR for genotyping these mutations and compare with immunophenotyping in different cattle breeds. The assay distinguished five genotypes, B (homozygous, G/A) and C (heterozygous, G/A and T/C), found in taurine, A (homozygous, G/C) and D (heterozygous, T/C and G/C), found in zebu. The E genotype (homozygous, T/C) was not observed in tested animals. As expected, B and C presented high/very high and intermediate CD4 staining, respectively. The lack/low CD4 staining was mainly related to the A, while the intermediate staining was mainly related to D genotype. The developed HRM qPCR assay accurately identified the altered phenotypes associated with CC8 staining in taurine. However, the assay cannot be applicable in zebu or hybrid breeds, probably due to additional mutations in the CD4 gene from zebu descendant animals.

Comparative Evaluation of Immune Responses and Protection of Chitosan Nanoparticles and Oil-Emulsion Adjuvants in Avian Coronavirus Inactivated Vaccines in Chickens.

Efficient vaccines are the main strategy to control the avian coronavirus (AvCoV), although several drawbacks related to traditional attenuated and inactivated vaccines have been reported. These counterpoints highlight the importance of developing new alternative vaccines against AvCoV, especially those able to induce long-lasting immune responses. This study evaluated and compared two inactivated vaccines formulated with AvCoV BR-I variants, one composed of chitosan nanoparticles (AvCoV-CS) and the second by Montanide oily adjuvant (AvCoV-O). Both developed vaccines were administered in a single dose or associated with the traditional Mass attenuated vaccine. The AvCoV-CS vaccine administered alone or associated with the Mass vaccine was able to induce strong humoral and cell-mediated immune (CMI) responses and complete protection against IBV virulent infection, wherein single administration was characterized by high IgA antibody levels in the mucosa, whereas when associated with the Mass vaccine, the serum IgG antibody was predominantly observed. On the other hand, single administration of the oily vaccine presented poor humoral and CMI responses and consequently incomplete protection against virulent challenge, but when associated with the Mass vaccine, immune responses were developed, and complete protection against infection was observed. Both of our experimental vaccines were able to induce full protection against virulent IBV challenge. A single dose of AvCoV-CS vaccine was sufficient to achieve complete protection, while AvCoV-O required a previous priming by a Mass strain to complete the protection.

Immune responses induced by inactivated vaccine against Aeromonas hydrophila in pacu, Piaractus mesopotamicus.

Aeromonas hydrophila is responsible for outbreaks of a severe infectious disease in fish farms around the world and is one of the major causes of economic losses to the neotropical fish farmers. This study assessed the induction of immune responses and protection against A. hydrophila in pacu, Piaractus mesopotamicus, vaccinated through intraperitoneal and immersion route with inactivated virulent strain. Fish were randomly distributed in three vaccinated groups: intraperitoneal (i.p.) route; immersion; and immersion + booster; and control group (unvaccinated). All vaccination protocols used the concentration of 1.7 × 108 CFU mL-1 of inactivated A. hydrophila., and an oil adjuvant was used for vaccine prepararion for i.p. route vaccination. Blood and skin mucus from 9 fishes per treatment were collected at 14, 28, 42 and 84 days post-vaccination (DPV) for determination of lysozyme concentration in skin mucus, as well as antibodies anti-A. hydrophila in blood serum and skin mucus. Fish were challenged at 84 DPV with homologous and virulent strain of A. hydrophila for evaluation of resistance against bacterial infection. The results demonstrated that vaccination with inactivated A. hydrophila suspension by i.p. or immersion resulted in significant increase of skin mucus lysozyme and specific antibody levels in serum and skin mucus, at 28 and 42 DPV, and this increase in innate and adaptive immunity remained significant in pacu vaccinated through i.p. route up to 84 DPV. Although no significant differences were observed in the survival study, pacu vaccinated through i.p. route presented 31,33% of relative percentage survival (RPS) in LD50-96h when compared unvaccinated fish challenged at 84 DPV. The results observed in this study indicate that vaccination programs with inactivated A. hydrophila, including booster doses by i.p. or immersion routes, could result in more effective protection in pacu against this bacteriosis, by increasing innate and adaptive mucosal and systemic immune responses.

Cross-sectional study of seropositivity, lung lesions and associated risk factors of the main pathogens of Porcine Respiratory Diseases Complex (PRDC) in Goiás, Brazil.

BACKGROUND: The objective of the study was to evaluate the occurrence and severity of Porcine Respiratory Diseases Complex (PRDC) pathogens in the Goiás State, Brazil. Were assessed the serological antibodies occurrency of Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae and swine influenza virus (SIV), as well as the evaluation of pulmonary Mycoplasma-like lung lesions, pleuritis, histopathological lesions and diseases occurrence associated with risk factors, such as management, housing and productive indexes. We conveniently selected 2536 animals for serology testing, and 900 lungs at slaughtering of animals from 30 multisite herds in Goiás State, Brazil. RESULTS: For M. hyopneumoniae, all herds presented seropositive animals at some stage of production. Even though most herds (29/30) vaccinated against this pathogen, 90.0% (27/30) of the herds presented at least 50.0% of seropositive animals in finishing and slaughter. Overall, antibodies against A. pleuropneumoniae were present in lower occurrence, varying from 22.4% of the animals in the nursery phase to 1.3% of the animals at slaughter. Conversely, SIV circulated in most herds, with 29 seropositive herds without vaccination. The occurrence of anti-SIV antibodies was higher at slaughter (74.5% of the animals) than nursery (41.8% of the animals), and at slaughter, 23 herds (76.7%) presented at least 50.0% of seropositive animals. All herds presented animals with pulmonary Mycoplasma-like lung lesions, and of the 900 lungs evaluated in the slaughterhouse, 665 (73.9%) presented an average Mycoplasma-like lung lesions of 7.3%. Evaluations of the pneumonia index (PI) showed that 73.3% of the herds were strongly affected by a pathology that manifested itself in different presentation forms. Microscopically, there was a predominance of bronchopneumonia lesions (74.6% of affected lungs), with a high occurrence of the chronic form (57.1%), and there was a moderate to marked proliferation of bronchial associated lymphoid tissue (BALT) in 64.1% of the affected lungs. Pleuritis were observed in 13.5% of the animals. CONCLUSION: Serological tests evidenced that antibodies against App and SIV were present in the Goiás State herds, and high occurrence of M. hyopneumoniae antibodies in finishing phases and slaughter may be influenced by pathogen circulation in vaccinated herds, leading to respiratory lesions at slaughter. Additionally, swine influenza virus was broadly disseminated in technified herds in Goiás State.

Memory immune responses and protection of chickens against a nephropathogenic infectious bronchitis virus strain by combining live heterologous and inactivated homologous vaccines.

In this study, we evaluated antibody and cell-mediated immune (CMI) responses in the mucosal and systemic compartments and protection against challenge with a nephropathogenic Brazilian (BR-I) strain of infectious bronchitis virus (IBV) in chickens submitted to a vaccination regime comprising a priming dose of heterologous live attenuated Massachusetts vaccine followed by a booster dose of an experimental homologous inactivated vaccine two weeks later. This immunization protocol elicited significant increases in serum and lachrymal levels of anti-IBV IgG antibodies and upregulated the expression of CMI response genes, such as those encoding CD8ß chain and Granzyme homolog A in tracheal and kidney tissues at 3, 7, and 11 days post-infection in the vaccinated chickens. Additionally, vaccinated and challenged chickens showed reduced viral loads and microscopic lesion counts in tracheal and kidney tissues, and their antibody and CMI responses were negatively correlated with viral loads in the trachea and kidney. In conclusion, the combination of live attenuated vaccine containing the Massachusetts strain with a booster dose of an inactivated vaccine, containing a BR-I IBV strain, confers effective protection against infection with nephropathogenic homologous IBV strain because of the induction of consistent memory immune responses mediated by IgG antibodies and TCD8 cells in the mucosal and systemic compartments of chickens submitted to this vaccination regime.

Gonadal pathogenicity of an infectious bronchitis virus strain from the Massachusetts genotype.

An outbreak of infectious bronchitis caused by the IBVPR03 strain of the Massachusetts genotype affected H-120 vaccinated laying hens in South Brazil. We investigated the cross protection of the vaccine by assessing the traqueal ciliostasis, virus recovery, and histopathological changes typically observed in the respiratory tract. Although the IBVPR03 strain is S1-genotyped as Massachusetts with a high genomic similarity to the H-120 vaccine strains, surprisingly, we found no tropism or pathogenicity to the trachea in birds infected with this strain. On the other hand, we observed ovarian and testicle lesions. Here, we show that, despite belonging in the Massachusetts genotype, the IBVPR03 pathotype differs from the expected respiratory pattern, causing instead marked histopathological changes in the gonads, so far not associated with this group.

Anti-influenza A virus antibodies in Tayassuidae from commercial rearing farms in Brazil / Detecção de anticorpos contra vírus influenza A em Tayassuidae de criações comerciais no Brasil

ABSTRACT: Family Tayassuidae in the suborder Suina include two species of peccaries in Brazil: the white-lipped peccary (Tayassu pecari) and the collared peccary (Pecari tajacu). These animals share common pathogens with domestic swine (Sus scrofa); however, their role as potential carrier remains unclear. This study focused on detecting the prevalence of influenza A antibodies in Tayassu pecari and Pecari tajacu from commercial rearing farms from two states in Brazil. A set of 50 blood samples from Pecari tajacu and 55 from Tayassu pecari were analyzed using a commercial indirect ELISA in order to investigate anti influenza A antibodies. Pecari tajacu samples presented 22% (11/50) of seropositivity for the virus. Serological surveillance is an important tool to identify the presence and the spread of the influenza virus in feral pigs.

RESUMO: A família Tayassuidae pertencente a subordem Suina e compreende duas espécies presentes no Brasil: Queixada (Tayassu pecari) e o Caititu (Pecari tajacu). Ambas as espécies compartilham patógenos com o suíno doméstico (Sus scrofa), entretanto o papel destes animais como carreadores destas infecções permanece indefinido. O presente estudo teve como objetivo detectar a ocorrência de anticorpos contra vírus influenza A em amostras de soro de rebanhos comerciais de queixada e caititu, provenientes de dois estados do Brasil. Um total de 50 amostras de soro de Pecari tajacu e 55 amostras de Tayassu pecari foram testadas por meio de ELISA, sendo que 22% (11/50) das amostras de Pecari tajacu foram soropositivas para o agente. Estudos de vigilância sorológica são importantes para identificar a presença e a disseminação do vírus influenza em suínos selvagens.

Experimental inoculation of gilts with bovine viral diarrhea virus 2 (BVDV-2) does not induce transplacental infection.

Bovine viral diarrhea virus (BVDV) belongs to the genus Pestivirus and can cause reproductive problems in cattle. However, there is still a lack of research to clarify its pathogenicity in different gestational periods of sows and its effects in neonates. In this study, 12 gilts divided into groups (G) were experimentally inoculated with the strain BVDV-2 (SV-253) oronasally at a dose of 106·85 TCID50; one group was inoculated 30 days before insemination (G0; n = 2), three groups were inoculated during gestation (first (G1; n = 2), second (G2; n = 3), third (G3; n = 3)), and a fourth was the control group (G4; n = 2). Samples of blood and nasal swabs from the gilts were collected every three days until delivery for a virus neutralization (VN) test, qRT-PCR, and blood count. On the day of delivery, 40% of the neonates were euthanized to obtain tissue and blood samples at necropsy for histopathology and qRT-PCR. The sows were seroconverted between 12 and 33 days after inoculation, and the virus was detected in the blood between 3 and 12 days and on the nasal swab between 6 and 24 days in the G0, G1, G2 and G3 sows but was not detected in piglet tissues, and no significant alterations were found through histopathology. The mean and standard deviation of the mean cycles (Cq) from blood (Cq = 34.87 ± 0.60) and nasal swab (Cq = 34.61 ± 0.87) samples were between 107 and 490 TCID50/ml. Transient infection was demonstrated with a low viral load, but transplacental infection was not possible in gilts.

Inactivated infectious bronchitis virus vaccine encapsulated in chitosan nanoparticles induces mucosal immune responses and effective protection against challenge.

Avian infectious bronchitis virus (IBV) is one of the most important viral diseases of poultry. The mucosa of upper respiratory tract, specially the trachea, is the primary replication site for this virus. However, conventional inactivate IBV vaccines usually elicit reduced mucosal immune responses and local protection. Thus, an inactivated IBV vaccine containing BR-I genotype strain encapsulated in chitosan nanoparticles (IBV-CS) was produced by ionic gelation method to be administered by oculo-nasal route to chickens. IBV-CS vaccine administered alone resulted in markedly mucosal immune responses, characterized by high levels of anti-IBV IgA isotype antibodies and IFNγ gene expression at 1dpi. The association of live attenuated Massachusetts IBV and IBV-CS vaccine also induced strong mucosal immune responses, though a switch from IgA isotype to IgG was observed, and IFNγ gene expression peak was late (at 5 dpi). Efficacy of IBV-CS was evaluated by tracheal ciliostasis analysis, histopathology examination, and viral load determination in the trachea and kidney. The results indicated that IBV-CS vaccine administered alone or associated with a live attenuated heterologous vaccine induced both humoral and cell-mediated immune responses at the primary site of viral replication, and provided an effective protection against IBV infection at local (trachea) and systemic (kidney) sites.

Rapid detection and differentiation of avian infectious bronchitis virus: an application of Mass genotype by melting temperature analysis in RT-qPCR using SYBR Green I.

A method based on Melting Temperature analysis of Hypervariable regions (HVR) of S1 gene within a RT-qPCR was developed to detect different genotypes of avian infectious bronchitis virus (IBV) and identify the Mass genotype. The method was able to rapidly identify the Mass genotype among IBV field isolates, vaccine attenuated strains and reference M41 strain in allantoic liquid and also directly in tissues. The RT-qPCR developed detected the virus in both tracheal and pulmonary samples from M41-infected or H120-infected birds, in a larger post-infection period compared to detection by standard method of virus isolation. RT-qPCR method tested provided a sensitivity and rapid approach for screening on IBV detection and Mass genotyping from IBV isolates.

Assessment of molecular and genetic evolution, antigenicity and virulence properties during the persistence of the infectious bronchitis virus in broiler breeders.

The infectious bronchitis virus (IBV) causes a highly contagious disease [infectious bronchitis (IB)] that results in substantial economic losses to the poultry industry worldwide. We conducted a molecular and phylogenetic analysis of the S1 gene of Brazilian (BR) IBV isolates from a routinely vaccinated commercial flock of broiler breeders, obtained from clinical IB episodes that occurred in 24-, 46- and 62-week-old chickens. We also characterized the antigenicity, pathogenesis, tissue tropism and spreading of three IBV isolates by experimental infection of specific pathogen-free (SPF) chickens and contact sentinel birds. The results reveal that the three IBV isolates mainly exhibited mutations in the hypervariable regions (HVRs) of the S1 gene and protein, but were phylogenetically and serologically closely related, belonging to lineage 11 of the GI genotype, the former BR genotype I. All three isolates caused persistent infection in broiler breeders reared in the field, despite high systemic anti-IBV antibody titres, and exhibited tropism and pathogenicity for the trachea and kidney after experimental infection in SPF chickens and contact birds. In conclusion, BR genotype I isolates of IBV evolve continuously during the productive cycle of persistently infected broiler breeders, causing outbreaks that are not impaired by the current vaccination programme with Massachusetts vaccine strains. In addition, the genetic alterations in the S1 gene of these isolates were not able to change their tissue tropism and pathogenicity, but did seem to negatively influence the effectiveness of the host immune responses against these viruses, and favour viral persistence.

Early immune responses and development of pathogenesis of avian infectious bronchitis viruses with different virulence profiles.

Avian infectious bronchitis virus (IBV) primarily replicates in epithelial cells of the upper respiratory tract of chickens, inducing both morphological and immune modulatory changes. However, the association between the local immune responses induced by IBV and the mechanisms of pathogenesis has not yet been completely elucidated. This study compared the expression profile of genes related to immune responses in tracheal samples after challenge with two Brazilian field isolates (A and B) of IBV from the same genotype, associating these responses with viral replication and with pathological changes in trachea and kidney. We detected a suppressive effect on the early activation of TLR7 pathway, followed by lower expression levels of inflammatory related genes induced by challenge with the IBV B isolate when compared to the challenge with to the IBV A isolate. Cell-mediated immune (CMI) related genes presented also lower levels of expression in tracheal samples from birds challenged with B isolate at 1dpi. Increased viral load and a higher percentage of birds with relevant lesions were observed in both tracheal and renal samples from chickens exposed to challenge with IBV B isolate. This differential pattern of early immune responses developed after challenge with IBV B isolate, related to the downregulation of TLR7, leading to insufficient pro-inflammatory response and lower CMI responses, seem to have an association with a most severe renal lesion and an enhanced capability of replication of this isolate in chicken.

A cross-sectional study of swine influenza in intensive and extensive farms in the northeastern region of the state of São Paulo, Brazil.

Swine influenza (SI) is a seasonal infectious disease highly important to the world pig industry. Loss of daily weight gain, increased costs for the prevention and treatment of secondary infections are the main economic losses associated with the presence of this disease. However, some epidemiological features of SI remain quite unclear. This study focused on assessing the prevalence of swine influenza virus (SIV) infection in intensive and extensive pig herds and associating risk factors. A set of 601 blood samples of five intensive farrow-to-finish farms and 361 blood samples from 56 extensive farms were analyzed using an indirect ELISA kit CIVTEST SUIS INFLUENZA®, Hipra (Amer, Spain), in order to detect anti-SIV antibodies. In total, 24.13 % of samples from intensive herds were positive, while no positive samples were detected in extensive rearing herds. Sow and weaning piglets had the highest prevalence values. In the intensive rearing system, occurrence of reproductive disorders and exposure to recently introduced animals were positively associated with the disease occurrence in swine herds. The findings highlight the importance of sows in the epidemiology of the disease and bring information about risk factors involved in the occurrence of swine influenza in intensive herds.

Polarized M2 macrophages in dogs with visceral leishmaniasis.

The objective of the present study was to analyze the skin (nasal surface and ear regions), lymph nodes (popliteal and pre-scapular), spleen and liver of dogs with visceral leishmaniasis (VL), in order to investigate the relationship between the parasite load measured as DNA copy number of Alpha gene of DNA polymerase of Leishmania infantum by quantitative PCR and the number of M2 macrophages by immunohistochemistry. A set of 29 naturally infected dogs from an endemic area for VL were sampled and another set of six dogs negative for VL and from a non-endemic area were analyzed as the control group (C). The spleen presented the highest number of Leishmania DNA copies, with significant differences between the groups G1 and G2 (with and without skin lesions, respectively). The M2 phenotype immunostaining predominated among the macrophages in granulomas and inflammatory infiltrates of samples from the skin, lymph nodes and spleens examined. The presence of M2 macrophages in dogs from infected group differed significantly from the control group, in all organs analyzed, excepted liver. The highest proportion of M2 macrophages coincided with the highest parasitism loads found in more susceptible organs of VL dogs, even in the skin, considered a more resistant organ, while the liver showed low parasitism load and low immunostaining for M2 macrophages with no significant differences between infected and negative groups. It was concluded that the predominance of M2 phenotype in VL dogs favored the multiplication of Leishmania infantum in organs of dogs that are more susceptible to Leishmania infection, as skin, lymph nodes and spleen.

Inflammatory and cell-mediated immune responses in the respiratory tract of chickens to infection with avian infectious bronchitis virus.

Tracheal mucosa is the primary site of replication of avian infectious bronchitis virus (IBV), which leads to both morphologic and immune modulatory changes in this organ. To increase the understanding of the mechanisms involved in these processes, we focused on the evaluation of local inflammatory and cell-mediated immune responses after challenge with the M41 strain of IBV, associating these responses with pathologic changes in the tracheal mucosa. At 24 h post-infection, inflammatory cytokines related genes were significantly upregulated, including peaks of TNFSF15 and TGFß mRNA production, although no tracheal microscopic alterations were observed and only a slightly increase in viral load occurred. At 3 days post-infection (dpi), we observed that the highest upregulation of IL6, IL1ß, and IFNγ coincided with highest scores of viral load and microscopic lesions, suggesting a role of both these cytokines and virus load on the development of tracheal lesions. Later, at 7 dpi, the most prominent increases of CD8αα mRNA and Granzyme homolog A mRNA were followed by a significant decrease of scores of tracheal lesions and viral load. In conclusion, an early upregulation of expression of proinflammatory cytokines such as IL6, IL1ß, and IFNγ induced by the M41 strain of IBV may be partially implicated in the viral pathogenicity on trachea tissues of nonimmune challenged chickens, in addition to a late induction of a putative protective immune responses by this virus through upregulation of CD8αα and Granzyme homolog A genes in this organ.

A liquid-phase-blocking concanavalin A enzyme-linked immunosorbent assay for the detection of antibodies against Newcastle disease virus in serum of free-ranging pigeons.

A competitive liquid-phase-blocking concanavalin A enzyme-linked immunosorbent assay (LPB-ConA-ELISA) was developed in the current study. The assay used ConA as a capture reagent, and the sera of specific pathogen-free chickens immunized with nonpurified Newcastle disease virus (NDV) suspension as detector antibodies, to detect and quantify specific antiviral antibodies in serum samples from free-ranging pigeons. The comparison between the LPB-ConA-ELISA and the hemagglutination inhibition (HI) test for the detection of antibodies in serum samples from 107 pigeons showed significant correlation between the assays (r = 0.875), a high sensitivity (100%), specificity (95.8%), accuracy (96.3%) for the ELISA, and good agreement (κ = 0.83) between the 2 assays. The results of this study suggest that the LPB-ConA-ELISA could be a useful alternative to HI test in the serodiagnosis of NDV in pigeons, or other species of birds.

Humoral and cell-mediated immune responses to different doses of attenuated vaccine against avian infectious bronchitis virus.

The antibody and cellular immune responses against infectious bronchitis virus (IBV) were evaluated at mucosal sites of chickens after immunization with various doses of an attenuated vaccine at 1 day of age. The correlation of these immune responses with protection of tracheal tissues was evaluated after experimental infection of these birds. Significantly reduced tracheal pathologic effects, measured according to ciliostasis and histology lesions, and reduced viral load were observed only in the full-dose vaccinated group at 5 days post-infection (dpi), while incomplete protection was observed for the subdose vaccinated groups. Moreover, birds of vaccinated groups, especially with full dose, developed higher levels of lachrymal IBV-specific IgG and IgA and increased the expression of cell-mediated immunity (CMI) genes, such as gamma interferon (IFNγ), CD8+ T cell marker, and granzyme homolog A more rapidly. In addition, these humoral and cellular immune responses evaluated at mucosal sites correlated significantly with tracheal protection against homologous IBV challenge in a vaccine dose-dependent manner. The results indicate that IgG, IgA and CD8+ T cell responses developed at mucosal sites after IBV vaccination of day-old chicks, could be taken as good correlates of protection against this virus.

Humoral and cellular immune response generated by different vaccine programs before and after Salmonella Enteritidis challenge in chickens.

The poultry industry has a high demand for Salmonella vaccines in order to generate safer Salmonella-free food for consumers around the world. Vaccination against S. Enteritidis (SE) is vastly undertaken in many countries, although the criteria for the use of live vaccine (LV) or killed vaccine (KV) should also depend on the immune mechanisms triggered by each. In this study, a commercial bacterin (KV) and an attenuated SG mutant (LV) were used in four different vaccine programs (LV; LV+LV; KV; LV+KV). At 1 day before (dbi) and 1, 6 and 9 days after SE challenge (dpi), humoral (IgM, IgG and secretory IgA) and cellular (CD8(+) T cells) immune responses were evaluated along with the production of IL-10, IL-12 and IFN-γ. Although after challenge, all birds from each group had an influx of CD8(+) T cells, birds which received KV had lower levels of these cells in organs and significantly higher levels of immunoglobulins. The expression of the cytokines was up-regulated in all groups post-vaccination, although, after challenge, cytokine expression decreased in the vaccinated groups, and increased in the unvaccinated group A. IL-10 levels were significantly higher at 1 day post-infection in the group that received KV, which may be involved in the weak cellular immune response observed within this group. In caecal tonsils, IFN-γ expression at 1 dbi was higher in birds which received two vaccine doses, and after challenge, the population of CD8(+) T cells constantly increased in birds that were only vaccinated with the LV. This study demonstrated that the development of a mature immune response by CD8(+) T cells, provided by the use of the LV, had better efficacy in comparison to the high antibody levels in the serum stimulated by the KV. However, high secretory IgA levels in the intestinal lumen associated with influx CD8(+) T cells may be indicative of protection as noticed in group E (LV+KV).

Systemic and local antibodies induced by an experimental inactivated vaccine against bovine herpesvirus type 1 / Anticorpos locais e sistêmicos induzidos por uma vacina experimental inativada contra o herpesvírus bovino tipo 1

An experimental inactivated vaccine against bovine herpesvirus-1 (BoHV-1) was produced aiming to evaluate the systemic and local antibody responses in 12 seronegative heifers, after vaccination and revaccination. Serum samples were submitted to virus neutralization assay and to ELISA test for detection of IgG1 and IgG2 isotypes. Nasal secretion samples were submitted to the same ELISA test for detection of IgG1 and IgG2 isotypes. The results showed that moderate to high neutralizing titres and IgG1 and IgG2 antibody responses were induced after the second vaccination in the serum and in nasal secretions up to 114 days post vaccination. IgG2 antibodies were the prevalent isotype for most of the post-vaccination period. The results indicate that BoHV-1 experimental inactivated vaccine elicited potentially protective IgG1 and IgG2 antibody levels, both in the systemic and mucosal compartments.

Uma vacina experimental inativada contra o herpesvírus bovino tipo 1 (BoHV-1) foi produzida com o objetivo de se avaliar a resposta imune humoral local e sistêmica contra o BoHV-1, em 12 novilhas soronegativas, após a vacinação e a revacinação. Os soros foram submetidos à prova de vírus-neutralização para quantificação do título de anticorpos neutralizantes e a um ELISA para detecção de IgG1 e IgG2. Os swabs nasais também foram submetidos ao ELISA para detecção de IgG1 e IgG2 na secreção nasal. Os resultados demonstraram que títulos de anticorpos neutralizantes foram induzidos após a revacinação, em níveis moderados a altos, permanecendo em níveis significativos no soro sanguíneo e na secreção nasal até o dia 114 pós-vacinação. O IgG2 foi o isótipo predominante na maior parte do período pós-vacinação, tanto na secreção nasal, como no compartimento sistêmico. A vacina experimental inativada contra o BoHV-1 estimulou níveis de anticorpos potencialmente protetores dos isótipos IgG1 e IgG2, tanto no compartimento sistêmico, como nas mucosas.

Construção de uma biblioteca de fragmentos de anticorpos monoclonais de galinhas com cadeia única (scFv) por phage display com reatividade cruzada para estirpes heterólogas do vírus da bronquite infecciosa aviária / Construction of an avian single chain monoclonal antibodies (scFv) library by phage display that cross-reacted with heterologous avian infectious bronchitis virus strains

Anticorpos monoclonais constituem a base de vários testes usados na detecção e na identificação de antígenos. Nesse contexto, tais imuno-reagentes têm sido extensivamente empregados na identificação de estirpes virais envolvidas na etiologia de surtos de bronquite infecciosa a campo, permitindo o aperfeiçoamento das técnicas de detecção e caracterização antigênica do vírus da bronquite infecciosa das galinhas (VBI). No presente estudo, uma biblioteca de fragmentos de anticorpos de galinha originalmente preparada por phage display contra a estirpe vacinal (H120) do VBI foi usada para a seleção de fragmentos de anticorpos recombinantes com reatividade cruzada para as estirpes heterólogas IBVPR01 e IBVPR05, isoladas de surtos a campo no Brasil e a estirpe SE-17, isolada nos Estados Unidos. Após três ciclos de panning, foi identificado, pelo ELISA, um conjunto de 15 anticorpos scFv expressos em fagos e com reatividade cruzada para essas mesmas estirpes do VBI. A análise por Western-blotting revelou que dois desses clones apresentavam fagos expressando fragmentos de anticorpos monoclonais com reatividade cruzada para a nucleoproteína N das três estirpes do VBI e também para a forma recombinante dessa nucleoproteína derivada da estirpe M41. Concluindo, os fragmentos de anticorpos monoclonais recombinantes scFv-N produzidos em fagos interagem com um epítopo mais conservado da proteína N do VBI e apresentam um grande potencial para utilização na detecção e no diagnóstico direto desse vírus.

Monoclonal antibodies are the basis of various techniques used for antigen detection or characterization, and their use is specially recommended for the identification of viral strains involved in the etiology of infectious bronchitis outbreaks. These antibodies are homogeneous, highly specific and fully characterizable, allowing the improvement of immunological techniques detection and antigenic characterization of avian infectious bronchitis virus strains (IBV). A phage display library was used, which was prepared previously against the IBV vaccine strain (H120) for the selection of new scFv antibody fragments specific for heterologous IBV strains isolated from outbreaks in Brazil (IBVPR01, IBVPR05) and USA (SE-17). After three cycles of panning, a set of 15 scFv antibodies were expressed in phages and cross-reacted in ELISA with these three viral strains. Western-blotting analysis showed that two of the clones were expressing scFv specific for the nucleoprotein of these IBV strains, as well as to the recombinant form of this protein derived from M41. In conclusion, the recombinant fragments of monoclonal antibodies expressed in phage have a great potential for future use in immunodiagnostic techniques and to study the evolution of infectious bronchitis virus.