Imperfection of Commercial Inactivated Salmonella Vaccine Against Salmonella Infantis During Induced Molting in Chickens and Proposed Evaluation Method.

In the present study, we evaluated the continuance and efficacy of inactivated vaccine against Salmonella Infantis (SI) in chickens raised on a commercial farm. Chickens (88-days-old) were inoculated with 1 or 0.5 doses of commercially available trivalent inactivated Salmonella vaccine; anti-SI antibody titer was examined continuously for 11 mo thereafter. Molting was induced 11 mo after vaccination, and SI was administered orally. SI colony-forming units (CFUs) were measured in cecal feces, cecal contents, liver, and spleen samples. Anti-SI antibodies in the 1 dose vaccination group could be detected in at least 90% of cases until the end of testing. SI discharge was significantly reduced in birds treated with either dose of vaccine. However, SI CFUs were elevated in the induced molting group, regardless of vaccination dose, particularly in the cecal feces, cecal contents, and spleen. Thus, the vaccine provided remarkable protection against SI infection under ordinary rearing methods but not during induced molting. To achieve sufficient SI protective efficacy, we recommend inoculation with 1 dose of vaccine. Moreover, the efficacy of inactivated Salmonella vaccine is recommended to be evaluated by challenging chickens with live Salmonella in addition to Salmonella antibody titration.

Limitaciones de la vacuna comercial contra Salmonella inactivada contra Salmonella infantis durante la muda inducida en pollos y propuesta de un para evaluación. En el presente estudio, se evaluó la continuidad y la eficacia de la vacuna inactivada contra Salmonella Infantis (SI) en pollos criados en una granja comercial. Los pollos (de 88 días de vida) se inocularon con una o media dosis de una vacuna trivalente de Salmonella inactivada disponible comercialmente. El título de anticuerpos contra S. Infantis se examinó de forma continua durante once meses. La muda se indujo a los once meses después de la vacunación y S. Infantis se administró por vía oral. Se contaron las unidades formadoras de colonias (UFC) de S. Infantis de muestras de heces cecales, contenido cecal, hígado y bazo. Los anticuerpos contra S. Infantis en el grupo que recibió una dosis de la vacuna pudieron detectarse en al menos el 90% de los casos hasta el final de la prueba. La eliminación de descarga de S. Infantis se redujo significativamente en las aves tratadas con cualquiera de las dosis de vacuna. Sin embargo, las unidades formadoras de colonias de S. Infantis se elevaron en el grupo al que se le indujo la de muda, independientemente de la dosis de vacunación, particularmente en las heces del ciego, el contenido cecal y el bazo. Por lo tanto, la vacuna proporcionó una protección importante contra la infección por S. Infantis bajo los métodos de cría ordinarios, pero no durante la muda inducida. Para lograr suficiente eficacia protectora contra S. Infantis se recomienda la inoculación con 1 dosis de vacuna. Además, se recomienda evaluar la eficacia de la vacuna de Salmonella inactivada mediante desafío de los pollos con Salmonella viva además de la titulación de anticuerpos contra Salmonella.

Cytotoxicity associated with prolonged room temperature storage of serum and proposed methods for reduction of cytotoxicity.

Canine serum preserved at room temperature (25°C) for longer than 24h is known to exhibit significant cytotoxicity. This phenomenon is one of the major reasons for the failure of virus neutralization tests. In this study, a method for reducing this cytotoxicity was investigated by applying several treatments to dog, cat and human serum prior to room temperature storage. Additionally, the identity of the cytotoxic factor generated during room temperature storage was investigated. Heat-inactivation at 56°C or 65°C and the addition of protease inhibitor prior to storage were found to be effective for reducing cytotoxicity in the serum. Furthermore, heat-inactivation at 65°C reduced the cytotoxicity that was induced under room temperature storage. Several protein factors in serum were suspected to play a role in the observed cytotoxicity. According to this study, the membrane-attack-complex in serum was not involved in the cytotoxicity. This study provides useful information for development and improvement of cell culture and virus neutralization tests.

Neutralizing antibody response in dogs and cats inoculated with commercial inactivated rabies vaccines.

In Japan, the import quarantine regulation against rabies has required from 2005 that dogs and cats should be inoculated with the rabies vaccine and that the neutralizing antibody titer should be confirmed to be at least 0.5 international units (IU)/ml. The fluorescent antibody virus neutralization (FAVN) test is used as an international standard method for serological testing for rabies. To achieve proper immunization of dogs and cats at the time of import and export, changes in the neutralizing antibody titer after inoculation of the rabies vaccine should be understood in detail. However, few reports have provided this information. In this study, we aimed to determine evaluated, such changes by using sera from experimental dogs and cats inoculated with the rabies vaccine, and we tested samples using the routine FAVN test. In both dogs and cats, proper, regular vaccination enabled the necessary titer of neutralizing antibodies to be maintained in the long term. However, inappropriate timing of blood sampling after vaccination could result in insufficient detected levels of neutralizing antibodies.

Determination of critical factors causing cytotoxicity in the virus neutralization test.

The fluorescent antibody virus neutralization (FAVN) test, an international standard method for serological testing for rabies, has been adopted by many countries. However, some dog serum samples inhibit the formation of cell monolayers by BHK-21 cells used in the test, resulting in failures to determine antibody titers. This inhibition of cell monolayer formation was defined as cytotoxicity. In this study, critical factors that induce cytotoxicity of the dog serum in BHK-21 cells were determined, and the effective ways to prevent cytotoxicity were also established. Specifically, some anticoagulants, anti-BHK-21 cell IgG antibodies, and serum storage at temperatures of >25°C were found to induce cytotoxicity. On the other hand, several treatments of the dog serum, including the absorption by BHK-21 cells or kaolin, incubation with trypsin-EDTA, and the use of collagen- or gelatin-coated plates, were shown to reduce cytotoxicity. Based on these results, the FAVN test may be modified to enhance its performance.

Comparison of commercial enzyme-linked immunosorbent assay kits with agar gel precipitation and hemagglutination-inhibition tests for detecting antibodies to avian influenza viruses.

We evaluated the utility of 5 commercial enzyme-linked immunosorbent assay (ELISA) kits for detecting antibodies to avian influenza viruses. The sensitivities and specificities of the ELISA kits were compared with those of the agar gel precipitation (AGP) and hemagglutination-inhibition (HI) tests. The results suggest that some ELISA kits might not be suitable for monitoring during the early stages of avian influenza virus infections. Therefore, ELISA kits should only be used in conjunction with a profound knowledge about monitoring of avian influenza.

Detection of highly pathogenic avian influenza virus infection in vaccinated chicken flocks by monitoring antibodies against non-structural protein 1 (NS1).

H5 and H7 highly pathogenic avian influenza virus (HPAIV) represent a major global concern in poultries and human health. Avian influenza (AI) vaccines are available but not preferred for field applications, primarily because vaccination interferes with sero-surveillances of AIV infection. To overcome the problem, ELISA systems using non-structural protein 1 (NS1) of AIV as antigens (NS1-ELISA) have been developed to measure anti-NS1 antibodies that are raised in AIV-infected but not in vaccinated chickens. However, some AI-vaccinated chickens having a weak anti-virus immune response may subsequently be infected with AIV and spread the virus. This raises a concern for the validity of NS1-ELISA to detect AIV infection in previously vaccinated chickens. In this study, we developed NS1-ELISA and assessed its feasibility to detect HPAIV infection in chickens previously immunized with H5 or H7 AI vaccines. The results indicated that the NS1-ELISA could identify HPAIV infection in both unvaccinated and vaccinated chickens at 1 week after infection in correlation with results from time-consuming virus isolation tests. Taken together, the NS1-ELISA system would be valuable tool to define HPAIV infection when AI vaccine program is in place.

The respiratory burst activity of bottlenose dolphin neutrophils elicited by several stimulants.

To investigate the early host defense function in aquatic animals, the respiratory burst activity of bottlenose dolphin neutrophils against soluble and particulate stimulants was measured by luminol-dependent chemiluminescence assays and compared with those of bovine and human. Dolphin neutrophils generated the respiratory burst in response to phorbol 12-myristate 13-acetate (PMA), concanavalinA (ConA), heated-plasma (HP), and homologous-plasma opsonized zymosan except N-formyl-Met-Leu-Phe (fMLP). However, the respiratory burst of dolphin neutrophils stimulated by lipopolysaccharide and Staphylococcus aureus was inferior to those of bovine and human. Furthermore, DP-OZ also induced the respiratory burst of bovine and human neutrophils. In conclusion, dolphin neutrophils responded to several soluble and particulate stimulants as well as human neutrophils, but were refractory or slightly responded to bacterial agents.

Priming of dolphin neutrophil respiratory burst by recombinant tumor necrosis factor alpha.

We studied the effects of recombinant dolphin tumor necrosis factor alpha (rdoTNFalpha) on the respiratory burst activity of dolphin neutrophils. rdoTNFalpha enhanced the luminol-dependent chemiluminescence response of dolphin neutrophils induced by concanavalin-A, opsonized zymosan, and heated plasma, but not that induced by phorbol myristate acetate. The TNF-associated priming activity was concentration- and preincubation time-dependent, and heat-instable. These data suggest that, as in human neutrophils, TNFalpha enhances the respiratory burst in dolphin neutrophils that follows short-term incubation with various receptor-mediated agonists.