Expression of Marek's Disease Virus MicroRNA MDV1-miR-M4 Increases Susceptibility of Young Chicks to Salmonella Enteriditis

MicroRNAs (miRNAs) are short RNA molecules that can regulate gene expression at the post-transcriptional level, reported Robin W. Morgan of the University of Delaware. They continued that several serotype 1 Marek's disease virus (MDV1) miRNAs map near the meq gene, and among these, MDV1-miR-M4 is particularly interesting.

First, MDV1-miR-M4 is necessary but not sufficient for oncogenicity. Second, MDV1-miR-M4 shares a seed sequence with miR-155, a miR that functions in immune system development and regulation. Third, expression of MDV1-miR-M4 correlates with virulence among MDV1 strains. Finally, chickens inoculated with a recombinant herpesvirus of turkeys (HVT) that expresses MDV1-miR-M4 (rHVT-M4) exhibit increased virus loads in peripheral blood compared to chickens inoculated with parent HVT.

Therefore, Morgan and colleagues hypothesised that one function of MDV1-miR-M4 is to enable infection by suppressing immune responsiveness. To test this, they vaccinated 18-day-old specific-pathogen-free embryos with HVT parent, rHVT-M4, rHVT-meqmiRs (expresses the entire meq miR cluster) or diluent. Then, at hatch, half of the chickens in each group were vaccinated with commercial Salmune® vaccine, and one week later, all chickens were challenged with Salmonella Enteritidis (SE).

Resistance to SE was determined by quantifying the number of chickens in each group from which SE could be isolated from spleens one week post challenge.

The Delaware group's results indicated that chickens vaccinated with rHVT-M4 were more susceptible to SE challenge than chickens vaccinated with the HVT parent. Chickens vaccinated with rHVT-meqmiRs were also more susceptible to SE challenge than HVT parent vaccinates.

From these studies, they concluded that MDV1-miR-M4 functions in immune suppression exhibited by MD V1 strains and contributes to MDV1 virulence.

Evaluation of Marek's Disease Virus-Induced Immunosuppression: Effect of MDV Pathotype

MDV has continuously increased in virulence during the last 50 years, according to Nick Faiz of North Carolina State University. The highly virulent MDVs not only are able to overcome the protection conferred by MD vaccines, but they are also more immunosuppressive.

The group has recently developed a model to study MDV-induced immunosuppression (MDV-IS) under laboratory conditions. In this model, MDV-IS is measured indirectly by evaluating the reduced efficacy of chicken embryo origin (CEO) laryngotracheitis (LT) vaccines against a virulent LT challenge (LT vaccine model).

Using this model, they showed that MDV infection at hatch jeopardised the protection conferred by CEO against LT in commercial meat-type chickens bearing maternal antibodies against MDV. Morever, MD vaccines were not always efficient in controlling MDV-IS.

The objectives of their study were to confirm previous findings, to evaluate if MD vaccines can be immunosuppressive, and to determine if pathotype has an effect on MDV-IS. One experiment was conducted to confirm the role of vaccines on MDV-IS. Our results confirmed that MD vaccines not always protected against MDV-IS induced by vv+MDV 648A. In addition, none of the evaluated vaccines were able to induced immunosuppression by itself.

A second experiment was conducted to evaluate the role of MDV pathotype. The immunosuppressive ability of three MDV strains (GA, Md5, and 648A) belonging to different pathotypes (virulent, very virulent, and very virulent plus) was evaluated. Results will be discussed.

Role of In-ovo Vaccination with HVT on the Maturation of the Chicken Immune System

In-ovo vaccination with HVT has demonstrated to be very efficacious in protecting against early challenge with MDV, according to Isabel Gimeno of North Carolina State University. In a paper with co-authors at NCSU, Pfizer Animal Health and Experimental Pathology Laboratories, she explained that chickens do not achieve complete immune competency until one to two weeks after hatch, therefore chickens embryos receive the vaccine when the immune system is not mature.

Embryo immune responses are based mainly on an innate immune response and on maternal antibodies since they are not exposed to exogenous antigens. Exposure to HVT in-ovo might elicit an adaptive immune response.

The researchers have recently demonstrated that in-ovo vaccination with HVT resulted in an increased percentage of activated T cells by four days of age.

Their hypothesis was that in-ovo vaccination with HVT renders chickens more immunocompetent at hatch. The objective of this study was to evaluate the effect of the administration of HVT at embryonation day 18 (18ED) on the chicken immune system development.

Various immunological parameters (MHC-I and MHC-II expression, immune cells phenotype, cytokine gene expression, and immune responses against various antigens) were evaluated in day-old chickens (HVT and sham-inoculated at 18ED), 7 and 14 day-old chickens (unvaccinated).

Results were discussed at the Convention.

Positive Correlation Between Replication Rate and Pathotype of Marek's Disease Virus Strains in Maternal Antibody-negative Chickens

Pathotyping of new field strains of MDV requires both a long period of time and a large number of birds, according to John Dunn of the USDA ARS Avian Disease and Oncology Laboratory. Confirming a positive correlation of virus replication and pathotype may lead to faster and cheaper alternative pathotyping methods or as a screening assay for choosing isolates to be pathotyped. Past studies have found differences in replication rates between selected vMDV and vv+MDV but this correlation has not been evaluated using a broad selection of virus strains.

A first trial evaluated replication rates of five virus strains from each virulent pathotype (v, vv and vv+) using maternal antibody positive chickens which found very little difference in lymphoid atrophy between groups and mild differences between replication rates by pathotype.

The current trial evaluated differences using maternal antibody-negative chickens.

They found a significant increase in viral load in brain, bursa and lung tissue at days 9 and 11 post-challenge for vvMDV and vv+MDV strains compared to vMDV strains. No significant difference was seen between vvMDV and vv+MDV strains. Similar results were seen comparing lymphoid atrophy between pathotype groups.

Using these results, Dunn suggested that it may be possible to determine a replication rate threshold as a preliminary screen to separate vMDV from vv/vv+MDV field strains.

Immune Response Elicited by Chicken Embryos after Vaccination at 18 Days of Embryonation with Various Marek's Disease Vaccines

In-ovo vaccination against Marek’s disease has become a common practice for the poultry industry. Pathogenesis of herpesvirus of turkey (HVT) in the embryo tissues has been studied in detail, reported Arun Kumar Pandiri from Experimental Pathology Laboratories, Inc. However, albeit the positive effect on protection, little is known about pathogenesis of serotype 1 in the embryonic tissues and the immune response elicited.

In his study, with co-authors from North Carolina State University, chicken embryos at 18 days of embryonation (18ED) were vaccinated via the intra-amniotic route with HVT, bivalent vaccine (HVT plus SB-1) and CVI988.

Replication of the vaccine virus, toll-like receptors and cytokine gene expression were chronologically evaluated (19ED, 20ED and day-old) in spleen and lung.

The results were discussed at the meeting.

Monitoring Serotype 1 Marek’s Disease Vaccine CVI988 Replication in Vaccinated Flocks That Are Exposed to Oncogenic Marek’s Disease Virus

Monitoring replication of serotype 1 MD vaccine CVI988 in vaccinated flocks under field conditions has been difficult due to the lack of an appropriate real time PCR technique that can differentiate between CVI988 and oncogenic MDVs, reported Aneg Lucia Cortes from North Carolina State University.

Recently, a real-time PCR has been developed, based on Mismatch Amplification Mutation Assay (MAMA) primers that can detect unique differences in the pp38 gene of the CVI988 and oncogenic MDV strains.

In this work, the Raleigh-based team has used this new technique to evaluate replication of CVI 988 in the feather pulp of chickens that were vaccinated in-ovo or at day-old and were exposed to oncogenic MDV 648A by contact on day 1.

Cortes reported that they have also optimised the sampling protocols for monitoring CVI988 and for early diagnosis of tumors under field conditions using MAMA primers.

Role of NK Cells in Vaccine-induced Immunity against Marek’s Disease

The antiviral activity of vaccine-induced immunity that markedly reduces the level of early cytolytic infection, production of cell-free infectious virus particles in the FFE, and lymphoma formation by interrupting the normal cascade of pathogenic events is a significant factor in protective efficacy of MD vaccines.

From the USDA-ARS-Avian Disease and Oncology Laboratory, Mohammad Heidari reported that, although MD vaccines have been in use for many decades, the molecular mechanism of their protection is unknown. There is growing evidence that NK cells play a critical role in vaccine-induced protection, genetic resistance and anti-tumor immunity.

Comparative analysis of functional activity of NK cells in vaccinated and naïve chickens revealed that vaccination arms NK cells by increasing cytotoxic granule proteins and IFN-γ production.

Heidari concluded that up-regulation of CD107a, a functional marker for identification of NK cells activity, was suggestive of degranulation of the activated NK cells when mixed with target cells.

Safety and Efficacy of a Back-passaged rMd5-delta-Meq Vaccine Virus in Chickens

MDV EcoR1-Q fragment of the viral genome encodes a meq (MDV EcoQ) gene, explained Lucy F. Lee of the USDA-ARS Avian Disease and Oncology Laboratory. Meq is an unique oncogene, present only in serotype 1 MDV and is consistently expressed in all latent or MDV tumour cells.

The meq gene was deleted from the very virulent rMd5 genome and was designated as rMd5-delta-Meq or meq null virus. This virus was shown to be an effective vaccine candidate in both maternal antibody (MAb) positive (+) and negative (-) chickens. However, the meq null virus induces bursal and thymic atrophy (BTA ) in MAb- chickens but not in MAb+ commercial chickens.

The BTA in MAb- chickens is considered a safety issue and will interfere with commercialisation and licensing of vaccine.

Lee and her colleagues have eliminated BTA at 40th passages in cell cultures and its protective efficacy was similar to the meq null virus at 19th passage (p) in MAb- chickens but not in MAb+ chickens.

Back-passage virus in chickens is another strategy commonly used to improve vaccine efficacy. The researchers have back-passaged p19 meq null virus in chickens for five passages at two-week intervals. The resultant virus showed improved replication in chickens, remained non-pathogenic, and protected at the same level as p19 meq null virus against challenge with very virulent plus 686 strain of MDV.

Differences on Dose Effect of CVI988 Commercial Vaccines Efficacy against an Early Challenge with a Very Virulent Plus Marek’s Disease Virus

MD vaccines are in most cases cell-associated and require special care in the handling, storage and administration. Tarsicio Villalobos of Zoetis Inc. reported that failures in the cold chain improper thawing conditions, and reconstitution in inadequate diluents can severely reduce vaccine titres.

There is no universal agreement regarding the minimal PFU vaccination dose needed to ensure development of protective immunity.

Efficacy of vaccines not only depends on the administered dose. Differences in the protection ability of CVI988 from different manufacturers have being reported. Different levels of attenuation on cell culture or variation on how vaccine seed is handled might have contributed to such differences.

The objective of this study was to evaluate if CVI988 vaccine origin and dose have an effect on the efficacy to protect against an early challenge with a vv+MDV, explained Villalobos.

Two different lines of broilers breeder (SF = slow feathering and FF = fast feathering) were vaccinated in-ovo with two commercial CVI988 vaccines (A and B) at two different doses (HD = manufacturer recommended dose and LD = 2,000 PFU).

Chickens were comingled at hatch with 15-day-old shedder chickens that were inoculated with 500 PFU of vv+MDV 648A strain at day-old.

Vaccine efficacy was evaluated by the MD protection index (PI = %MD in unvaccinated - %MD in vaccinated / %MD vaccinated × 100), for both genetic lines and the different doses of the vaccines.

Within SF chickens, those receiving low dose of vaccine (LD) regardless the type of vaccine had higher mortality than those receiving the manufacturer’s recommended dose (HD). Within FF chickens, differences between HD and LD were less remarkable. The groups receiving Company A vaccine at LD developed significantly higher incidence of MD lesions than other groups. No significant differences were found between groups receiving HD of either CVI988 sources and LD of Company B vaccine.

Company A vaccine at 2,000 PFU per dose gave the worst protection in this study, reported Villalobos. There was no differences in protection against development of MD between Company B and Company A vaccines when the recommended dose (high dose) was used.

November 2013