Dust sprinkling as an effective method for infecting layer chickens with wild-type Salmonella Typhimurium and changes in host gut microbiota.

Role of dust in Salmonella transmission on chicken farms is not well characterised. Salmonella Typhimurium (ST) infection of commercial layer chickens was investigated using a novel sprinkling method of chicken dust spiked with ST and the uptake compared to a conventional oral infection. While both inoculation methods resulted in colonisation of the intestines, the Salmonella load in liver samples was significantly higher at 7 dpi after exposing chicks to sprinkled dust compared to the oral infection group. Infection of chickens using the sprinkling method at a range of doses showed a threshold for colonisation of the gut and organs as low as 1000 CFU/g of dust. Caecal content microbiota analysis post-challenge showed that the profiles of chickens infected by the sprinkling and oral routes were not significantly different; however, both challenges induced differences when compared to the uninfected negative controls. Overall, the study showed that dust sprinkling was an effective way to experimentally colonise chickens with Salmonella and alter the gut microbiota than oral gavage at levels as low as 1000 CFU/g dust. This infection model mimics the field scenario of Salmonella infection in poultry sheds. The model can be used for future challenge studies for effective Salmonella control.

Evaluation of the safety and efficacy of the novel Mycoplasma gallisepticum vaccine, Vaxsafe MG304, after spray-vaccination of 1-day-old specific pathogen-free chicks.

Mycoplasma gallisepticum causes chronic respiratory disease in poultry. A novel vaccine, Vaxsafe MG304 (the ts-304 strain), has greater protective efficacy in chickens than the Vaxsafe MG (strain ts-11) vaccine when delivered by eye drop at 3 weeks of age. Applying this vaccine in the hatchery to 1-day-old birds, using mass administration methods, would improve animal welfare and reduce labour costs associated with handling individual birds. This study assessed the protection provided by vaccination with Vaxsafe MG304 after administration to 1-day-old chicks. Chicks were administered a single dose of the vaccine to assess the efficacy of either a high dose (107.0 colour changing units, CCU) or a low dose (105.7 CCU) after eye drop or spray (in water or gel) administration against experimental challenge with virulent M. gallisepticum strain Ap3AS at 7 weeks of age. The vaccine was able to colonise the palatine cleft of chicks after vaccination by eye drop (at both doses) or by spray (in water or gel) (at the high dose). The high dose of vaccine, when delivered by eye drop or spray, was shown to be safe and induced a serological response and protective immunity (as measured by tracheal mucosal thickness and air sac lesion scores) against challenge. Vaccination of 1-day-old chicks with Vaxsafe MG304 by eye drop induced protective immunity equivalent to vaccination at 3 weeks of age. Vaxsafe MG304 was also protective when applied by both coarse- and gel spray methods at the higher dose and is therefore a suitable live attenuated vaccine for use in 1-day-old chicks.

A live attenuated Salmonella Typhimurium vaccine dose and diluent have minimal effects on the caecal microbiota of layer chickens.

Among the Salmonella reduction strategies in poultry production, one option is to use a Salmonella vaccine. The aim of vaccinating layer flocks is to reduce the shedding of wild-type Salmonella in the poultry environment, thereby reducing the contamination of poultry products (eggs and meat). Nutritive diluent and a higher dose of vaccine may enhance its colonization potential in the gut of chickens. In this study, a commercially available live attenuated vaccine (Vaxsafe® ST) was reconstituted in different media and delivered orally to day-old chicks at three different doses (107, 108, and 109 CFU/chick). Gut colonization of the vaccine strain and the effects of vaccination on gut microbiota were assessed in commercial-layer chickens. The vaccine diluent and dosage minimally affected microbiota alpha diversity. Microbiota beta diversity was significantly different (P < 0.05) based on the vaccine diluent and dose, which indicated that the vaccinated and unvaccinated chickens had different gut microbial communities. Differences were noted in the abundance of several genera, including Blautia, Colidextribacter, Dickeya, Enterococcus, Lactobacillus, Pediococcus, and Sellimonas. The abundance of Colidextribacter was significantly lower in chickens that received vaccine reconstituted in Marek's and water diluents, while Lactobacillus abundance was significantly lower in the water group. The highest vaccine dose (109 CFU/chick) did not significantly alter (P > 0.05) the abundance of microbial genera. Chicken age affected the microbiota composition more significantly than the vaccine dose and diluent. The abundance of Lactobacillus, Blautia, Caproiciproducens, Pediococcus, and Colidextribacter was significantly higher on day 14 compared with day 7 post-vaccination. The Salmonella Typhimurium vaccine load in the caeca was not significantly affected by diluent and vaccine dose; however, it was significantly lower (P < 0.0001) on day 14 compared with day 7 post-vaccination. Overall, the S. Typhimurium vaccine minimally affected the gut microbiota structure of layer chicks, whereas changes in microbiota were more significant with chicken age.

Obg plays a significant role in temperature sensitivity of Mycoplasma synoviae live attenuated vaccine strain MS-H.

The MS-H vaccine strain (Vaxsafe MS®; Bioproperties Pty. Ltd., Australia) is a live attenuated temperature sensitive derivative of a virulent strain of M. synoviae, 86079/7NS, and is used to prevent diseases from M. synoviae challenges in poultry farms. The genome sequence of MS-H includes 32 single nucleotide polymorphisms (SNPs) compared to that of 86079/7NS. To investigate the nature of mutations responsible for temperature sensitivity, MS-H strain was subjected to thermal adaptation in vitro and in vivo. The only observed variation detected in the MS-H culture following sequential passages with incremental incubation temperature from 33 °C to 39.5 °C was an Ala210Val variation in Obg protein, associated with loss of temperature sensitivity phenotype. An identical variation was detected in the MS-H culture reisolated from one out of five bird 28 days after inoculation with MS-H. These findings suggest that M. synoviae is capable of thermoadaptive evolution and Obg plays a significant role in this trait.

Reversion of mutations in a live mycoplasma vaccine alters its metabolism.

The live attenuated temperature sensitive vaccine strain MS-H (Vaxsafe® MS, Bioproperties Pty. Ltd., Australia) is widely used to control disease associated with M. synoviae infection in commercial poultry. MS-H was derived from a field strain (86079/7NS) through N-methyl-N'-nitro-N-nitrosoguanidine (NTG)-induced mutagenesis. Whole genomic sequence analysis of the MS-H and comparison with that of the 86079/7NS have found that MS-H contains 32 single nucleotide polymorphisms (SNPs). Three of these SNPs, found in the obgE, oppF and gapdh genes, have been shown to be prone to reversion under field condition, albeit at a low frequency. Three MS-H reisolates containing the 86079/7NS genotype in obgE (AS2), obgE and oppF (AB1), and obgE, oppF and gapdh (TS4), appeared to be more immunogenic and transmissible compared to MS-H in chickens. To investigate the influence of these reversions in the in vitro fitness of M. synoviae, the growth kinetics and steady state metabolite profiles of the MS-H reisolates, AS2, AB1 and TS4, were compared to those of the vaccine strain. Steady state metabolite profiling of the reisolates showed that changes in ObgE did not significantly influence the metabolism, while changes in OppF was associated with significant alterations in uptake of peptides and/or amino acids into the M. synoviae cell. It was also found that GAPDH plays a role in metabolism of the glycerophospholipids as well as an arginine deiminase (ADI) pathway. This study underscores the role of ObgE, OppF and GAPDH in M. synoviae metabolism, and suggests that the impaired fitness arising from variations in ObgE, OppF and GAPDH contributes to attenuation of MS-H.

Investigation of a gel-based delivery method for the administration of a live, attenuated Salmonella Typhimurium vaccine.

Poultry vaccines are often administered using water as a suspension media and applied using an oral or coarse spray method. Gel-based vaccine diluents have been developed as an alternative vaccine delivery method. Gels are more viscous, and droplets adhere more effectively to feathers giving the vaccine a longer time to be ingested (through preening). Application of gel diluents with live bacterial vaccines, however, is limited. The present study tested a gel diluent prepared in various media, using a live, attenuated Salmonella Typhimurium vaccine, Vaxsafe ST. Reconstitution in gel diluent did not negatively affect vaccine viability or motility. The invasive capacity of vaccine suspended in gel diluent into cultured intestinal epithelial cells was also tested. Results demonstrated that vaccine suspended in gel diluent retained invasiveness. Day old chicks were orally administered with Vaxsafe ST suspended in gel diluent to characterize in vivo colonization capacity of the vaccine. The results revealed that the VaxSafe ST suspended in gel diluent could efficiently colonize the caeca of chicks, which is needed for the development of effective immunity.

Effects of administration of tylosin on the duration of protective immunity induced by the novel Mycoplasma gallisepticum vaccine strain, Vaxsafe MG ts-304, in chickens.

Prophylactic use of antimicrobials after administration of live vaccines is a common practice in the poultry industry, but the impact of this on the efficacy and duration of protection induced by the vaccines is unknown. The effect of treatment with tylosin on the efficacy of vaccination with the live attenuated M. gallisepticum strain, Vaxsafe MG ts-304, was examined. This vaccine has previously been shown to provide protection for at least 57 weeks. Ten-week-old specific-pathogen-free chickens were vaccinated with Vaxsafe MG ts-304 and then treated with tylosin at a therapeutic dose in drinking water from 6 weeks after vaccination. Tylosin was withdrawn 5 days before challenge with M. gallisepticum strain Ap3AS at 6, 10, 14, 18 or 22 weeks after vaccination. Air sac lesions, tracheal mucosal thickening and the concentrations of serum antibodies against M. gallisepticum were assessed at 2 weeks after challenge. The protection induced by the vaccine in the 6 weeks before initiation of tylosin treatment persisted for 18 weeks after vaccination, with lesions only observed in the air sacs of vaccinated birds that had been treated with tylosin after challenge at 22 weeks after vaccination. Concentrations of serum antibodies against M. gallisepticum began to decrease in vaccinated birds that had been treated with tylosin from 16 weeks after vaccination. This study has suggested that treatment of chickens with tylosin after vaccination with a live attenuated mycoplasma vaccine reduces the duration of protective immunity afforded by the vaccine.

Virulence factors of Mycoplasma synoviae: Three genes influencing colonization, immunogenicity, and transmissibility.

Infections caused by Mycoplasma synoviae are major welfare and economic concerns in poultry industries worldwide. These infections cause chronic respiratory disease and/or synovitis in chickens and turkeys leading to reduced production and increased mortality rates. The live attenuated vaccine strain MS-H (Vaxsafe® MS), commonly used for protection against M. synoviae infection in many countries, contains 32 single nucleotide variations compared to its wildtype parent strain, 86079/7NS. Genomic analysis of vaccine strains reisolated from flocks following the administration of MS-H has identified reversions to the original 86079/7NS sequence in the obgE, oppF and gapdh genes. Here, three MS-H field reisolates containing the 86079/7NS genotype in obgE (AS2), obgE and oppF (AB1), and obgE, oppF and gapdh (TS4), as well as the vaccine MS-H and the parental strain 86079/7NS were experimentally inoculated to chickens. The strains were assessed for their ability to infect and elicit immune responses in the recipient chickens, as well as in naïve in-contact chickens. Despite the loss of temperature sensitivity phenotype and colonization of the reisolates in the lower respiratory tract, there was no significant differences detected in the microscopic mucosal thickness of the middle or lower trachea of the inoculated chickens. Concurrent reversions in ObgE, OppF and GAPDH proteins were associated with higher gross air sac lesion scores and increased microscopic upper-tracheal mucosal thickness in chickens directly inoculated with the reisolates following intratracheal administration of a virulent strain of infectious bronchitis virus. The gross air sac lesions of the chickens in-contact with those inoculated with reisolates were not significantly different to those of chickens in-contact with MS-H inoculated chickens, suggesting that horizontal transmission of the reisolates in the poultry flock will not lead to higher pathogenicity or clinical signs. These results suggest a significant role of GAPDH and/or cumulative effect of ObgE, OppF and GAPDH on M. synoviae pathogenicity. Future experiments will be required to investigate the effect of single mutations in gapdh or oppF gene on pathogenicity of M. synoviae.

Genomic Diversity of a Globally Used, Live Attenuated Mycoplasma Vaccine.

The Mycoplasma synoviae live attenuated vaccine strain MS-H (Vaxsafe MS; Bioproperties Pty., Ltd., Australia) is commonly used around the world to prevent chronic infections caused by M. synoviae in birds and to minimize economic losses in the poultry industry. MS-H is a temperature-sensitive strain that is generated via the chemical mutagenesis of a virulent M. synoviae isolate, 86079/7NS. 32 single nucleotide polymorphisms have been found in the genome of MS-H compared to that of 86079/7NS, including 25 in predicted coding sequences (CDSs). There is limited information on the stability of these mutations in MS-H in vitro during the propagation of the vaccine manufacturing process or in vivo after the vaccination of chickens. Here, we performed a comparative analysis of MS-H genomes after in vitro and in vivo passages under different circumstances. Studying the dynamics of the MS-H population can provide insights into the factors that potentially affect the health of vaccinated birds. The genomes of 11 in vitro laboratory passages and 138 MS-H bird reisolates contained a total of 254 sequence variations. Of these, 39 variations associated with CDSs were detected in more than one genome (range = 2 to 62, median = 2.5), suggesting that these sequences are particularly prone to mutations. From the 25 CDSs containing previously characterized variations between MS-H and 86079/7NS, 7 were identified in the MS-H reisolates and progenies examined here. In conclusion, the MS-H genome contains individual regions that are prone to mutations that enable the restoration of the genotype or the phenotype of wild-type 86079/7NS in those regions. However, accumulated mutations in these regions are rare. IMPORTANCE Preventative measures, such as vaccination, are commonly used for the control of mycoplasmal infections in poultry. A live attenuated vaccine strain (Vaxsafe MS; MS-H; Bioproperties Pty. Ltd., Australia) is used for the prevention of disease caused by M. synoviae in many countries. However, information on the stability of previously characterized mutations in the MS-H genome is limited. In this study, we performed a comparative analysis of the whole-genome sequences of MS-H seeds used for vaccine manufacturing, commercial batches of the vaccine, cultures minimally passaged under small-scale laboratory and large-scale manufacturing conditions, MS-H reisolated from specific-pathogen-free (SPF) chickens that were vaccinated under controlled conditions, and MS-H reisolated from vaccinated commercial poultry flocks around the world. This study provides a comprehensive assessment of genome stability in MS-H after in vitro and in vivo passages under different circumstances and suggests that most of the mutations in the attenuated MS-H vaccine strain are stable.

Measures of tracheal lesions are more discriminatory and reproducible indications of chronic respiratory disease caused by Mycoplasma gallisepticum in poultry.

Mycoplasma gallisepticum is the primary causative agent of chronic respiratory disease in poultry, and vaccination is the measure most commonly used for its control. Pathological changes caused by M. gallisepticum are mainly observed in the trachea and air sacs, but assessment of air sac lesions is subjective. Standardized parameters for evaluation of pathological changes, and their reproducibility and discrimination in uninfected and infected groups, are critical when assessing the efficacy of M. gallisepticum vaccination. This study reviewed and critically appraised the published literature on evaluation of vaccine efficacy against pathological changes caused by M. gallisepticum in poultry in the trachea and air sacs. A search of four electronic databases, with subsequent manual filtering, identified 23 eligible papers published since 1962 describing the assessment of histopathological changes in the trachea using tracheal lesion scores and/or measurement of tracheal mucosal thicknesses and assessment of gross air sac lesions using lesion scores. Measurement of tracheal lesions proved a more reliable and robust method of assessing disease induced by M. gallisepticum when compared to assessment of air sac lesions, highlighting the importance of including assessment of tracheal lesions as the primary outcome variable in vaccine efficacy studies. In addition, this study also identified the necessity for use of a standardized model for evaluation and reporting on M. gallisepticum vaccines to minimize variations between vaccine efficacy studies and to allow direct comparisons between them.RESEARCH HIGHLIGHTS Tracheal and air sac lesions have been used to assess M. gallisepticum vaccine efficacy.The specific parameters and statistical tests used to compare tracheal and air sac lesions vary greatly.Measures of tracheal lesions are more discriminatory than measures of air sac lesions.A standardized model is needed to evaluate vaccines against infection with M. gallisepticum.

Effects of immunosuppression on the efficacy of vaccination against Mycoplasma gallisepticum infection in chickens.

Immunosuppression can increase the susceptibility of chickens to other disease-causing pathogens and interfere with the efficacy of vaccination against those pathogens. Chicken anaemia virus (CAV) and infectious bursal disease virus (IBDV) are common causes of immunosuppression in chickens. Immunosuppression was induced by experimental infection with either CAV or IBDV to assess the effect of immunosuppression on the efficacy of vaccination with Mycoplasma gallisepticum strain ts-304 against infection with virulent M. gallisepticum, a common bacterial pathogen of chickens worldwide. Birds were experimentally infected with either CAV or IBDV at 1 week of age, before vaccination and challenge with M. gallisepticum to examine the effect of immunosuppression at the time of vaccination, or at 6 weeks of age, after vaccination against M. gallisepticum but before challenge with virulent M. gallisepticum, to investigate the effect of immunosuppression at the time of challenge. All birds were vaccinated with a single dose of the ts-304 vaccine at 3 weeks of age and experimentally challenged with the virulent M. gallisepticum strain Ap3AS at 8 weeks of age. In immunosuppressed chickens there was a reduction in protection offered by the ts-304 vaccine at two weeks after challenge, as measured by tracheal mucosal thicknesses, serum antibody levels against M. gallisepticum, air sac lesion scores and virulent M. gallisepticum load in the trachea. Immunosuppressed birds with detectable serum antibodies against M. gallisepticum were less likely to have tracheal lesions. This study has shown that immunosuppression caused by infection with CAV or IBDV can interfere with vaccination against mycoplasmosis in chickens.

Challenges in Vaccinating Layer Hens against Salmonella Typhimurium.

Salmonella Typhimurium is among the most common causes of bacterial foodborne gastrointestinal disease in humans. Food items containing raw or undercooked eggs are frequently identified during traceback investigation as the source of the bacteria. Layer hens can become persistently infected with Salmonella Typhimurium and intermittently shed the bacteria over the course of their productive lifetime. Eggs laid in a contaminated environment are at risk of potential exposure to bacteria. Thus, mitigating the bacterial load on farms aids in the protection of the food supply chain. Layer hen producers use a multifaceted approach for reducing Salmonella on farms, including the all-in-all-out management strategy, strict biosecurity, sanitization, and vaccination. The use of live attenuated Salmonella vaccines is favored because they elicit a broader host immune response than killed or inactivated vaccines that have been demonstrated to provide cross-protection against multiple serovars. Depending on the vaccine, two to three doses of Salmonella Typhimurium vaccines are generally administered to layer hens within the first few weeks. The productive life of a layer hen, however, can exceed 70 weeks and it is unclear whether current vaccination regimens are effective for that extended period. The objective of this review is to highlight layer hen specific challenges that may affect vaccine efficacy.

Duration of protective immunity induced by Mycoplasma gallisepticum strain ts-304 vaccine in chickens.

Mycoplasma gallisepticum (MG) is an important pathogen of poultry worldwide, causing chronic respiratory disease in chickens and turkeys. MG ts-304 is a GapA positive clone recovered from Vaxsafe MG (strain ts-11) that has been shown to be safe in chickens when delivered by the eye drop route to 3-week-old specific-pathogen-free chickens and to confer protection against challenge at 4 weeks after vaccination, as measured by tracheal mucosal thickness and air sac lesion scores. In this study, specific pathogen-free chickens (SPF) were vaccinated with a single dose of the MG ts-304 vaccine (106.0 colour changing units) at 3 weeks of age and experimentally challenged by aerosol with the virulent M. gallisepticum strain Ap3AS at 40, 48 and 57 weeks after vaccination. There were no significant differences in tracheal mucosal thickness 2 weeks after challenge between chickens challenged at the three time points, or between the vaccinated birds after challenge and unvaccinated/unchallenged control birds. Thus there was clear evidence that the immunity conferred by vaccination with the MG ts-304 vaccine resulted in significant protection against tracheitis in chickens that extended to, but was highly likely to exceed, 57 weeks after vaccination and that similar long term protective immunity could be expected to be conferred by a vaccine dose lower than that used in this study.

Mycoplasma gallisepticum strain ts-304 is a safe and effective live attenuated vaccine for use in chickens.

Vaxsafe MG (strain ts-11) is a live attenuated vaccine against the important poultry pathogen Mycoplasma gallisepticum that has been used globally to improve poultry health. However, the majority of the bacterial cells in Vaxsafe MG do not express the GapA cytadhesin, reducing their capacity to colonise the respiratory tract. Vaxsafe MG (strain ts-304) is a GapA positive clone recovered from Vaxsafe MG (strain ts-11) that has been shown to be safe and efficacious in turkeys, and preliminary studies have suggested that Vaxsafe MG (strain ts-304) may have greater efficacy in chickens than Vaxsafe MG (ts-11). The studies described here aimed to meet the international regulatory requirements for safety and efficacy in chickens. The vaccine colonised the trachea of 3-week-old chickens without inducing signs of respiratory disease or significant lesions in the respiratory tract, and was safe at a tenfold overdose and after repeated administration. It was transmissible from vaccinated to naïve chickens with no evidence of reversion to virulence following multiple in vivo passages. Finally, the superiority of Vaxsafe MG (strain ts-304) was demonstrated by its capacity to induce similar protection against infection with wild type M. gallisepticum at a 40 fold lower dose than the end of shelf life titre dose of Vaxsafe MG (ts-11). The lower effective dose of Vaxsafe MG (strain ts-304) allows it to be freeze-dried, enhancing its stability, making it easier to transport and store the vaccine and increasing its shelf life. Vaxsafe MG (strain ts-304) is, therefore, a highly efficacious and promising live attenuated vaccine candidate suitable for use in chickens.

Pathogenesis and tissue tropism of natural field recombinants of infectious laryngotracheitis virus.

Infectious laryngotracheitis virus (ILTV) is an economically significant respiratory pathogen of poultry. Novel recombinant strains of ILTV have emerged in Australia during the last decade and currently class 9 (CL9) and class 10 (CL10) ILTV are the most prevalent circulating strains. This study conducted a comprehensive investigation of the pathogenesis of these two viral strains. Commercial broiler and specific pathogen free (SPF) chickens were inoculated with varying doses of CL9 or CL10 ILTV and subsequently evaluated for clinical and pathological signs of infection. While no difference in the levels of acute viral replication were observed across the different challenge doses, the severity of clinical signs, tracheal pathology and mortality were dose dependent. Both strains of virus persisted in the respiratory tract for up to 14 days post inoculation (dpi) and could be detected in the lung and feathers with sporadic detection in the liver, spleen or bursa. Given the prevalence of CL9 and CL10 in Australian poultry flocks, this study provides an important foundation for the development of diagnostic and therapeutic approaches for the detection and prevention of ILTV.

Evaluation of Mycoplasma gallisepticum (MG) ts-304 vaccine as a live attenuated vaccine in turkeys.

Mycoplasma gallisepticum (MG) is an important pathogen of poultry worldwide that causes chronic respiratory disease (CRD) in chickens and infectious sinusitis in turkeys. Vaxsafe MG (strain ts-11) is a live attenuated temperature sensitive vaccine that has been proven to be effective in controlling CRD in chickens, but it is not efficacious in turkeys. The gapA gene, which encodes a mature cytadhesin protein with a molecular weight of approximately 105 kDa, is not expressed in strain ts-11 because a 20 base pair reiterated sequence introduces a frame shift and causes premature truncation of the translated peptide. A GapA positive clone, MG ts-304, isolated from strain ts-11 has been shown to have enhanced efficacy in chickens. Here we describe studies we conducted to assess the safety and efficacy of the MG ts-304 vaccine candidate in turkeys. We found that MG ts-304 was able to colonise the trachea of 3-week-old turkeys and was safe, even at a tenfold overdose, inducing no adverse clinical signs of respiratory disease or significant gross lesions in the respiratory tract (air sacs or trachea), and was poorly transmissible to in-contact birds. We also showed that it was efficacious when administered to 3-week-old turkeys, inducing protective immunity against challenge with the M.gallisepticum wild-type strain Ap3AS. MG ts-304 is therefore a promising live attenuated vaccine candidate for use in turkeys.

Kinetics of Marek's disease virus (MDV) infection in broiler chickens 1: effect of varying vaccination to challenge interval on vaccinal protection and load of MDV and herpesvirus of turkey in the spleen and feather dander over time.

Two experiments in commercial broiler chickens vaccinated with herpesvirus of turkeys (HVT) and challenged with Marek's disease virus (MDV) investigated the effects of the vaccination-to-challenge interval (VCI) on vaccinal protection against Marek's disease, and the kinetics of MDV and HVT load in the spleen and feather dander determined using real-time quantitative polymerase chain reaction. Experiment 1 in isolators tested VCI of 2, 4 and 7 days, while Experiment 2 in floor pens tested VCI of 0, 2, 4, 7 and 10 days. MDV challenge induced gross Marek's disease lesions in 14% to 74% of chickens by 56 days post-challenge. Vaccinal protection increased from approximately 40% to approximately 80% with increasing VCI between days 2 and 7 in both experiments, but not thereafter. MDV was detected in both the spleen and dander at 7 days post-challenge and increased rapidly to approximately 21 days post-challenge, after which levels plateaued, rose or fell gradually depending on treatment. HVT was also shed in significant amounts, 1 to 2 logs lower than for MDV, with a clear peak around 14 to 21 days post-vaccination. Vaccination significantly reduced the log(10)MDV load in the spleen (vaccinated, 2.99+/-0.20/10(6) spleen cells; unvaccinated, 4.60+/-0.23/10(6) spleen cells) and dander (vaccinated, 5.28+/-0.13/mg; unvaccinated, 6.00+/-0.18/mg) from infected chickens. The MDV load had a significant negative association with the VCI and the level of vaccinal protection. Measurement of dander production in Experiment 1 and the dust content of air in Experiment 2, combined with determination of the MDV load in these, enabled estimation of total daily shedding rates of MDV per chicken and of the MDV load in air for the first time.

Relationship between Marek's disease virus load in peripheral blood lymphocytes at various stages of infection and clinical Marek's disease in broiler chickens.

Vaccination with herpesvirus of turkey (HVT) vaccine provides protection against clinical Marek's disease (MD) but does not preclude infection with wild-type MD virus (MDV). The quantity of MDV detected in circulating lymphocytes during the early period after infection may be a useful predictor of subsequent clinical MD later in the life. A study was designed to quantify MDV and HVT copy number in peripheral blood lymphocytes (PBL) using real-time polymerase chain reaction between days 5 and 35 post-challenge and to relate this to subsequent development of gross MD lesions. Female commercial broiler chickens were vaccinated with HVT or were sham-vaccinated at hatch, then challenged with MDV strain MPF-57 at day 2 post-vaccination and reared in positive-pressure isolators up to 56 days post-challenge, when all survivors were euthanized. All dead and euthanized chickens were examined post mortem for gross MD lesions. Birds were scored for MD lesions and mortality. MDV and HVT genome copy numbers were determined for each PBL sample. There was an increase in HVT load in PBL between days 7 and 37 post-vaccination, with marked increases between days 7 and 16 and again between days 30 and 37. There was a steady increase in MDV load to 35 days post-challenge. The mean MDV copy number (log(10)) was greater in chickens subsequently exhibiting gross MD lesions (5.05 +/- 0.21) than in those that did not (2.88 +/- 0.223), with the largest difference at 14 and 21 days post-challenge (P < 0.001). Quantification of MDV during early infection is therefore a potential tool for monitoring MD in broiler flocks.