Optimizing protocols for monitoring in vivo replication of a novel chimeric Marek's disease vaccine with an insertion of the long terminal repeat of reticuloendotheliosis virus in the CVI988 strain genome (CVI-LTR).

RESEARCH HIGHLIGHTS: CVI-LTR replicates in the thymus and spleen earlier than CVI988.CVI-LTR replicates in lymphoid organs but it cannot be detected in feather pulp.CVI-LTR reduced the load of oncogenic MDV DNA more efficiently than CVI988.

Salmonella in native "village" chickens (Gallus domesticus): prevalence and risk factors from farms in South-Central Peninsular Malaysia.

Village chicken or Ayam Kampung, common to Southeast Asian countries, has always been regarded as superior in comparison to commercial broiler chicken in terms of wholesomeness and health benefits. The current study investigates the prevalence and risk factors of Salmonella among village chicken flocks from the central and southern states of Peninsular Malaysia. A total of 35 village flocks were sampled from Selangor (n = 19), Melaka (n = 10), Johor (n = 4), and Negeri Sembilan (n = 2). In total, 1,042 samples were collected; these included cloacal swabs (n = 675), eggs (n = 62), pooled drinking water (n = 175), pooled feeds (n = 70), and pooled flies (n = 60). Isolation of Salmonella from cloacal swabs, poultry drinking water, and feeds was carried out according to the protocols and recommendations of the World Organization for Animal Health (OIE) terrestrial manual. The prevalence of Salmonella at an individual bird-level was 2.5% (17/675, 95% CI: 1.6 to 4.0). All eggs screened were negative; in the case of environmental samples, however, Salmonella was detected in 5.14% (9/175), 7.14% (5/70), and 5.0% (3/60) for water, feed, and flies, respectively. A total of 34 isolates and 8 Salmonella serotypes were identified. Weltevreden (20.6%) was the most common, followed by Typhimurium and Agona (17.6%), Albany and Enteritidis (8.8%), Molade (5.9%), Corvallis and Schleissheim (2.9%), and others grouped as Salmonella spp. (11.8%). Multivariable logistic regression models revealed that Salmonella positivity among flocks could be strongly predicted by storage of feeds (uncovered feeds; OR = 10.38; 95% CI: 1.25 to 86.39; p = 0.030) and uncovered water tanks (uncovered tank; OR = 6.43; 95% CI: 1.02 to 40.60; p = 0.048). The presence of Salmonella in village chickens in the study area was lower than that of commercial chickens in Malaysia.

Differential attenuation of Marek's disease virus-induced tumours and late-Marek's disease virus-induced immunosuppression.

Marek's disease virus (MDV) is a herpesvirus that induces lymphoma and a variety of non-neoplastic syndromes in chickens. Furthermore, very virulent plus (vv+) MDVs induce a form of immunosuppression (late-MDV-IS) that might involve both neoplastic and non-neoplastic mechanisms. The objective of this study was to evaluate whether the attenuation of MDV-induced tumours and late-MDV-IS occurs simultaneously or can be dissociated. The immunosuppressive ability of three viruses derived from vv+ MDV strain 686 (wild-type 686, the somewhat attenuated molecular clone 686-BAC, and the nononcogenic molecular clone lacking the two copies of the oncogene meq 686-BACΔMEQ) was evaluated. Late-MDV-IS was evaluated indirectly by assessing the negative effect of MDV strains on the protection conferred by infectious laryngotracheitis (ILT) vaccines. Our results showed that the ability to induce late-MDV-IS was attenuated before the ability to induce tumours. Strain 686 induced both tumours and late-MDV-IS, 686-BAC induced tumours but did not induce late-MDV-IS and 686-BACΔMEQ did not induce either tumours or late-MDV-IS. Further comparison of strains 686 and 686-BAC revealed that strain 686 reduced the humoral immune responses to ILTV (1132 vs 2167) more severely, showed higher levels of meq transcripts (2.1E+09 vs 4.98E+8) and higher expression of MDV microRNAs (mdv1-miR-M4-5p and mdv1-miR-M2-3p) in the spleen, and further reduced the percentage of CD45+-MHC-I+splenocytes (13 vs32 %) compared to molecular clone 686-BAC. This study suggests that the immunosuppressive ability of MDV might follow a continuous spectrum and only the most virulent MDVs can overcome a certain threshold level and induce clinical MDV-IS in the ILT model.

Evaluation of factors influencing the development of late Marek's disease virus-induced immunosuppression: virus pathotype and host sex.

Marek's disease virus (MDV) is a herpesvirus that induces lymphoma and immunosuppression in chickens. MDV-induced immunosuppression (MDV-IS) is complex and can be divided into two phases: early-MDV-IS associated with cytolytic infection in the lymphoid organs in chickens lacking maternal antibodies against MDV (MAbs) and late-MDV-IS that appears later in the pathogenesis and occurs even in chickens bearing MAbs. We have recently developed a model to reproduce late-MDV-IS under laboratory conditions. This model evaluates late-MDV-IS indirectly by assessing the effect of MDV infection on the efficacy of infectious laryngotracheitis (ILT) vaccines against challenge with ILT virus. In the present study, we have used this model to investigate the role of two factors (MDV pathotype and host sex) on the development of late-MDV-IS. Five MDV strains representing three different pathotypes: virulent (vMDV; 617A, GA), very virulent (vvMDV; Md5), and very virulent plus (vv+MDV; 648A, 686), were evaluated. Only vv+ strains were able to induce late-MDV-IS. An immunosuppression rank (IS-rank) was established based on the ability of MDV to reduce the efficacy of chicken embryo origin vaccine (values go from 0 to 100, with 100 being the highest immunosuppressive ability). The IS-rank of the evaluated MDV strains ranged from 5.97 (GA) to 20.8 (617A) in the vMDV strains, 5.97 to 16.24 in the vvMDV strain Md5, and 39.08 to 68.2 in the vv+ strains 648A and 686. In this study both male and female chickens were equally susceptible to MDV-IS by vv+MDV 686. Our findings suggest that late-MDV-IS is a unique feature of vv+ strains.

Efficacy of various Marek's disease vaccines protocols for prevention of Marek's disease virus-induced immunosuppression.

Marek's disease virus (MDV) induces tumors and severe immunosuppression in chickens. MDV-induced immunosuppression (MDV-IS) is very complex and difficult to study. In particular, the late MDV-IS (late-MDV-IS) is of great concern since it can occur in the absence of lymphoid organ atrophy or gross tumors. We have recently developed a model to reproduce late-MDV-IS under laboratory conditions. This model measures MDV-IS indirectly by assessing the effect of MDV infection on the efficacy of infectious laryngotracheitis (ILT) vaccination; hence the name late-MDV-IS ILT model. In this study, we have used the late-MDV-IS ILT model to evaluate if MD vaccination can protect against late-MDV-IS. One experiment was conducted to determine whether serotype 1 MD vaccines (CVI988 and Md5ΔMEQ) could induce late-MDV-IS by themselves. Three additional experiments were conducted to evaluate efficacy of different MD vaccines (HVT, HVT+SB-1, CVI988, and Md5ΔMEQ) and different vaccine protocols (day-old vaccination, in ovo vaccination, and double vaccination) against late-MDV-IS. Our results show that none of the currently used vaccine protocols (HVT, HVT+SB-1, or CVI988 administered at day of age, in ovo, or in double vaccination protocols) protected against late-MDV-IS induced by vv+MDV strains 648A and 686. Experimental vaccine Md5ΔMEQ administered subcutaneously at one day of age was the only vaccine protocol that significantly reduced late-MDV-IS induced by vv+MDV strain 686. This study demonstrates that currently used vaccine protocols confer high levels of protection against MDV-induced tumors (protection index=100), but do not protect against late-MDV-IS; thus, commercial poultry flocks could suffer late-MDV-IS even in complete absence of tumors. Our results suggest that MDV-IS might not be related to the development of tumors and novel control methods are needed. Further evaluation of the experimental vaccine Md5ΔMEQ might shed light on protective mechanisms against late-MDV-IS.

Early infection with Marek's disease virus can jeopardize protection conferred by laryngotracheitis vaccines: a method to study MDV-induced immunosuppression.

Marek's disease virus (MDV) is a herpesvirus that induces lymphomas and immunosuppression in chickens. MDV-induced immunosuppression (MDV-IS) is divided into two phases: early-MDV-IS occurring mainly in chickens lacking maternal antibodies (MAb) against MDV and associated with lymphoid organ atrophy; and late-MDV-IS occurring once MDV enters latency and during tumour development. Our objectives were to document the impact of late-MDV-IS on commercial poultry (meat-type chickens bearing MAb against MDV and that were vaccinated or unvaccinated against MD) and to optimize a model to study late-MDV-IS under laboratory conditions. The impact of late-MDV-IS was evaluated by assessing the effect of early infection (day of age) with a very virulent plus MDV (vv+MDV) on the efficacy of chicken-embryo-origin (CEO) infectious laryngotracheitis (ILT) virus vaccine against ILT challenge. The CEO ILT vaccine was administered in water at 14 days of age and ILT virus (ILTV) challenge was done intratracheally at 30 days of age. Development of ILT was monitored by daily evaluation of clinical signs, development of gross and histological lesions in trachea, and quantification of ILTV transcripts in trachea. Infection with vv+MDV strain 648A resulted in total abrogation of protection conferred by the CEO vaccine against ILTV challenge even in chickens vaccinated at 1 day of age with either HVT, HVT+SB-1, or CVI988. Chickens exposed to vv+MDV prior to vaccination with CEO ILTV vaccine had similar (P < 0.05) clinical scores, gross lesions, histopathologic lesion scores, and load of ILTV transcripts in trachea after ILTV challenge, as chickens that were not vaccinated with CEO ILTV vaccine.

In Ovo Vaccination with Turkey Herpesvirus Hastens Maturation of Chicken Embryo Immune Responses in Specific-Pathogen-Free Chickens.

Administration of Marek's disease (MD) vaccines in ovo has become a common practice for the poultry industry. Efficacy of MD vaccines is very high, even though they are administered to chicken embryos that are immunologically immature. We have recently demonstrated that in ovo vaccination with turkey herpesvirus (HVT) results in increased activation of T cells at hatch. Our previous results suggested that in ovo vaccination with HVT might have a positive impact not only on MD protection but also on the overall maturity of the developing immune system of the chicken (Gallus gallus domesticus). The objective of this study was to evaluate the effect of administration of HVT at 18 days of embryonation (ED) on the maturation of the embryo immune system. Four experiments were conducted in Specific-Pathogen-Free Avian Supplies (SPAFAS) chickens to evaluate the effect of administration of HVT at 18 ED on the splenic cell phenotypes at day of age (experiment 1) and on the ability of 1-day-old chickens to respond to various antigens compared with older birds (experiments 2 and 3). In addition, a fourth experiment was conducted to elucidate whether administration of other serotype's MD vaccines (CVI988 and SB-1) at 18 ED had the same effect as HVT on the spleen cell phenotypes at day of age. Our results demonstrated that 1-day-old chickens that had received HVT in ovo (1-day HVT) had higher percentages of CD45+, MHC-I+, CD45+MHC-I+, CD3+, MHC-II+, CD3+MHC-II+, CD4+, CD8+, and CD4+CD8+ cells in the spleen than 1-day-old sham-inoculated chickens (1-day sham). Moreover, spleens of 1-day HVT chickens had greater percentages of CD45+MHC-I+ cells and equal or greater numbers of CD4+CD8- and CD4-CD8+ cells than older unvaccinated chickens. In addition, administration of HVT at 18 ED rendered chicks at hatch more responsive to unrelated antigens such as concavalin A, phytohemagglutinin-L, and keyhole limpet hemocyanin. Administration of MD vaccines of other serotypes had an effect, although less remarkable than HVT, on the spleen cell phenotypes at hatch. Vaccines of all three serotypes resulted in an increased percentage of MHC-I+, CD45-MHC-I+, CD4-CD8+, and CD8+ cells, but only HVT resulted in a higher percentage of CD45+, CD45+MHC-I+, CD3+MHC-II+, and CD4+CD8- cells. Results of this study show that it is possible to hasten maturation of the chicken embryo immune system by administering HVT in ovo and open new avenues to optimize the procedure to improve and strengthen the immunocompetency of commercial chickens at hatch.

Evaluation of Factors Influencing Efficacy of Vaccine Strain CVI988 Against Marek's Disease in Meat-Type Chickens.

Marek's disease (MD) strain CVI988 is the most-protective commercially available vaccine against very virulent plus (vv+) Marek's disease virus (MDV). However, its use in meat-type chickens has been controversial. While several countries have been using CVI988 for more than 40 yr, others do not authorize its use or it is restricted mainly to layers. The use of CVI988 in meat-type chickens will be necessary in the future in areas where other vaccine protocols fail. The objective of this study was to evaluate factors (vaccine dose, vaccine origin, chicken genetics, age and route of vaccination, and combination with other MD vaccines) influencing the efficacy of CVI988 against MD in meat-type chickens. Three animal experiments were conducted in which various vaccine protocols using CVI988 were tested for their protection against challenge with vv+ strain 648A by contact at day of age. Experiments 1 and 2 were to compare the efficacy of CVI988 vaccines from three different origins (CVI988-A, CVI988-B, and CVI988-C) and evaluate the effect of vaccine dose and chicken genetics. Experiment 3 was to evaluate the effect of adding CVI988 vaccine to various vaccine protocols using other MD vaccines of serotypes 2 (SB-1) and 3 (rHVT). Our results show that, regardless of the origin of the vaccine, protection against early challenge with 648A was good when vaccines were administered at a high dose (>3000 plaque-forming units [PFU]). Differences among vaccines, however, were detected even when using a high dose in experiment 2 (vaccine CVI988-B conferred higher protection than did CVI988-C) but not in Experiment 1 (CVI988-B was compared to CVI988-A). The use of a fixed low dose (2000 PFU) of vaccine resulted in reduction in protection, and such reduction was more remarkable when using CV1988-A. No statistically significant differences were found when we compared the efficacy of CVI988 in two different genetic lines of broiler chickens (G1 and G2). Vaccination protocols that included CVI988 had better protection than protocols that only included MD vaccines of serotypes 2 and 3. This was true regardless of the vaccine protocol used (CVI988/rHVT+SB-1; CVI988+rHVT+SB-1/None; rHVT+SB-1/CVI988; wherein the vaccine before the slash (/) was administered in ovo at embryonation day 18 and the vaccine after the slash was administered at day of age, subcutaneously). When only vaccines of serotypes 2 and 3 were used, protection against early challenge with vv+MDV was higher when vaccines were administered in ovo (rHVT+SB-1/None) than if vaccines were administered at hatch (None/rHVT+SB-1). Monitoring vaccine DNA load in feather pulp (FP) samples at 1 wk was used to monitor vaccination, and results showed that differences in vaccine replication exist among vaccines but such differences were not necessarily related to protection (r = 0.41, P > 0.05). Monitoring load of challenge MDV DNA in FP at 21 days was conducted, and results correlated (r = 0.85, P < 0.05) with the percentage of chickens with MD lesions at the termination of the study, confirming that early diagnosis is a very powerful tool with which to evaluate protection.

Evaluation of the Protection Efficacy of a Serotype 1 Marek's Disease Virus-Vectored Bivalent Vaccine Against Infectious Laryngotracheitis and Marek's Disease.

Laryngotracheitis (LT) is a highly contagious respiratory disease of chickens that produces significant economic losses to the poultry industry. Traditionally, LT has been controlled by administration of modified live vaccines. In recent years, the use of recombinant DNA-derived vaccines using turkey herpesvirus (HVT) and fowlpox virus has expanded, as they protect not only against the vector used but also against LT. However, HVT-based vaccines confer limited protection against challenge, with emergent very virulent plus Marek's disease virus (vv+MDV). Serotype 1 vaccines have been proven to be the most efficient against vv+MDV. In particular, deletion of oncogene MEQ from the oncogenic vvMDV strain Md5 (BACδMEQ) resulted in a very efficient vaccine against vv+MDV. In this work, we have developed two recombinant vaccines against MD and LT by using BACδMEQ as a vector that carries either the LT virus (LTV) gene glycoprotein B (gB; BACΔMEQ-gB) or LTV gene glycoprotein J (gJ; BACδMEQ-gJ). We have evaluated the protection that these recombinant vaccines confer against MD and LT challenge when administered alone or in combination. Our results demonstrated that both bivalent vaccines (BACΔMEQ-gB and BACδMEQ-gJ) replicated in chickens and were safe to use in commercial meat-type chickens bearing maternal antibodies against MDV. BACΔMEQ-gB protected as well as a commercial recombinant (r)HVT-LT vaccine against challenge with LTV. However, BACδMEQ-gJ did not protect adequately against LT challenge or increase protection conferred by BACΔMEQ-gB when administered in combination. On the other hand, both BACΔMEQ-gB and BACδMEQ-gJ, administered alone or in combination, protected better against an early challenge with vv+MDV strain 648A than commercial strains of rHVT-LT or CVI988. Our results open a new avenue in the development of recombinant vaccines by using serotype 1 MDV as vectors.