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Centre for Animal Health and Welfare, School of Rural Science and Agriculture, University of New England, Armidale, NSW 2351, Australia
Correspondence
Aminul Islam
Aminul.Islam{at}hnehealth.nsw.gov.au
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: Division of Microbiology, Hunter Area Pathology Service (HAPS), Locked Bag 1, Hunter Region Mail Centre, NSW 2310, Australia. 
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Antigenically, they are closely related and their classification into three serotypes, namely serotype 1 (MDV1), 2 (MDV2) and 3 (MDV3 or herpesvirus of turkeys, HVT) is widely used. MDV1 is oncogenic and the aetiological agent of Marek's disease (MD), a lymphoproliferative and neoplastic disease of poultry (Churchill & Biggs, 1967; Nazerian et al., 1968) with an estimated worldwide economic impact of US$1–2 billion (Morrow & Fehler, 2004). MDV1 isolates vary widely in their pathogenic and oncogenic potential (Witter, 1997). The other two serotypes (MDV2 and HVT) are non-oncogenic and are used in live vaccines against MD, either alone, in combination or in combination with attenuated MDV1 strains (Witter & Schat, 2003). Attenuated strains of MDV1 and naturally occurring vaccine strains of MDV2 or HVT produce non-sterile immunity against MD tumours and persist together with virulent MDV1 in the host (Witter et al., 1971; Witter & Schat, 2003).
Whilst the primary target cell for MDV pathology is the lymphocyte, MDV1 is contagious and is readily transmitted by the airborne route (Biggs, 1985). This is due to replication of fully infectious virus in the epithelial cells of the keratinizing layer of the feather follicle epithelium, which then slough off and are shed as highly infective dander (Calnek et al., 1970). MDV2 is also contagious (Witter, 1987), but HVT does not spread readily between chickens infected early in life (Cho & Kenzy, 1975). Understanding the rate of shedding of the different MDVs, and factors influencing this, is a prerequisite to a detailed understanding of the epidemiology of MDV, but our current understanding of this is limited. Earlier reports suggested that shedding of MDV1 commences from 2 to 4 weeks after infection, well before the appearance of clinical signs, and may continue throughout the life of the chicken (Witter et al., 1971; Carrozza et al., 1973). Since then, studies using more sensitive molecular methods have found that MDV1 is shed as early as 7 days post-infection (Baigent et al., 2005; Islam et al., 2005b). In recent studies, it has been shown that all three serotypes of MDV are prevalent in dander collected from commercial broiler farms across Australia (Islam, 2006; Renz et al., 2006). These studies and others (e.g. Walkden-Brown et al., 2004, 2005) have also demonstrated that poultry dander is a suitable material for monitoring of MDV1, MDV2 and HVT on commercial poultry farms. However, the pattern of shedding of these viruses over time in chickens infected singly or co-infected with different serotypes of MDV under controlled conditions has yet to be reported.
In order to understand in more detail the dynamics of MDV shedding from commercial meat chickens, the current experiment was designed to: (i) measure the amount of MDV1, MDV2 and HVT present in dander shed from broiler chickens infected with one or more of these viruses; (ii) estimate the amount of dander shed by broiler chickens during their growth cycle; and (iii) calculate the profiles of total virus shed for each MDV serotype during the broiler chicken growth cycle.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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In this experiment, the individual isolator was the experimental unit. Each treatment combination was replicated in two separate purpose-built positive-pressure chicken isolators in a physical contamination level 2 animal house. The isolators had approximately 20 air changes per hour. The University of New England Animal Ethics Committee approved the experiment protocol, and chickens were maintained in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (NH & MRC, 2004). The experiment started on 28 February 2005 and terminated on 5 May 2005. All surviving chickens were euthanized at day 56 post-challenge (p.c.).
Experimental chickens.
The experiment commenced with 648 newly hatched chickens providing 27 per isolator and 54 per treatment combination initially. The chickens were commercial female Cobb broilers from parental stock vaccinated with the Rispens CVI988 strain of attenuated MDV1. They were hatched at a commercial hatchery and were unvaccinated.
Approximately five chickens per isolator were removed on each of days 7 and 14 p.c. to evaluate immunosuppression (data not presented), reducing the effective maximum chicken number to 17 per isolator.
Vaccination.
This was performed manually by subcutaneous injection of the recommended dose at hatch (day 0) on arrival of the chickens at the isolator laboratory at the University of New England (UNE).
Both the cell-associated (ca) HVT vaccine (caHVT strain FC-126, batch number HO2308) and the bivalent vaccine (caHVT strain FC-126 and caMDV2 strain SB1, batch number SBH 4101) were supplied by Bioproperties Pty Ltd. The dose rate used for both vaccines was 8000 p.f.u. per bird in 0.2 ml diluent supplied by the manufacturer. The dose for the bivalent vaccine was for both viruses combined, with an HVT : MDV2 ratio of 63 : 37.
MDV1 challenge.
Chickens were challenged with three different Australian isolates of MDV1 (MPF57, 02LAR and FT158) on day 5 p.v. For each isolate, a dose of 500 p.f.u. per chicken in 0.2 ml diluent was inoculated via the intra-abdominal route. Challenge virus was kindly provided by Professor Greg Tannock (RMIT, Melbourne, Australia) and virus titrations were performed on chick kidney cells in his laboratory. MPF57 is the standard Australian challenge MDV1 and was included as a reference strain. It was isolated in 1994 from a bivalent (HVT/MDV2)-vaccinated layer flock (De Laney et al., 1998). The isolate 02LAR was isolated from an unvaccinated broiler flock in 2002, whilst FT158 was isolated from a meat breeder flock vaccinated with Rispens CVI988 in 2002.
Collection of dander for MDV analysis.
For determination of MDV load in dander, samples were collected weekly from the dust deposits at the 9 ° bend in the exhaust air outlet duct of each isolator. For mock-challenged isolators (n=6), this commenced at 7 days p.v., and for those challenged with MDV1 (n=18), dust collection commenced at day 7 p.c. (=12 days p.v.). Using a disposable wooden spatula, approximately 1 g of dust was scraped into an Eppendorf tube following brief closure of the exhaust air outlet valve. After each collection, the outlet duct was thoroughly cleaned, so that the next collection represented the past 7 days of dust accumulation.
Estimation of daily dander production per chicken.
Six isolators were selected randomly to measure dander production, and dander production was measured weekly throughout the experiment from these isolators. Dander production was measured by capturing the total dander output in the exit air of each isolator in commercial vacuum cleaner filter bags over a period of 48 h for weeks 1–4 and over 24 h for weeks 5–8. Daily dander production per chicken was then calculated from the total dander collected, the duration of the collection period and the number of chickens in the isolator.
DNA extraction from dander.
DNA was extracted from 5 mg dander using a DNeasy tissue kit according to the manufacturer's instructions (Qiagen) and stored at –20 °C. Prior to use in the quantitative real-time PCR (qPCR) assay, extracted DNA was quantified by spectrophotometric analysis (SmartSpec TM 3000; Bio-Rad) for DNA yield and purity, and then diluted to a concentration of 5 ng µl–1 for use in the qPCR assay.
Assay of MDV genome copy number by qPCR.
The primers and probes used were specific for the MDV1 meq gene, MDV2 dnapol gene and HVT sorf1 gene as described previously by Islam et al. (2004). MDV1, MDV2 and HVT genome copy numbers were determined in the extracted DNA from dander samples according to the absolute quantification method described by Islam et al. (2006a, 2007) and Renz et al. (2006). Briefly, each reaction contained 0.3 µM each primer and 0.2 µM corresponding probe, 12.5 µl Platinum Quantitative PCR System-UDG (Invitrogen) and 5 µl DNA template (25 ng) in a total reaction volume of 25 µl. The cycling parameters consisted of 50 °C for 2 min and 95 °C for 2 min, followed by 40–45 cycles of denaturation at 94 °C for 15 s and annealing/extension at 60 °C for 45 s. Amplification and data acquisition were carried out using a Rotor Gene 3000 real-time PCR machine (Corbett Research). A standard curve for each primer set was generated in each assay and used to derive the copy number of target sequences in unknown samples. For each assay run, individual standard curves were generated using four to five 10-fold dilutions of MDV1, MDV2 and HVT standards of known concentration of virus. Samples that did not amplify or that had a threshold cycle (Ct) value below the lowest standard were coded as zero/negative. Viral loads in dander were expressed as viral copy number (VCN) (mg dander)–1, taking into account the total amount of DNA extracted from each sample. The lower limits of detection (mean±SEM) based on the mean calculated VCN of the lowest standard in each of the assays were 3.34±0.22, 11.73±5.02 and 60.80±30.91 VCN per reaction for the MDV1, MDV2 and HVT assays, respectively.
All samples were randomized across assays to minimize individual assay effects, and amplified in duplicate. Mean intra-assay co-efficients of variation for all qPCR runs were 0.68±0.11 % for MDV1, 1.67±1.09 % for MDV2 and 1.07±0.29 % for HVT based on Ct values. The co-efficients of variation based on VCN were 14.0±2.7 % for the MDV1, 30.3±18. 5 % for the MDV2 and 23.5±4.7 % for the HVT assays, respectively.
Statistical analysis.
Data were analysed using JMP version 5.1 statistical software (SAS Institute). MDV1, MDV2 and HVT viral load data obtained from qPCR assays of isolator dander were square-root-transformed to meet the assumptions of analysis of variance (Petrie & Watson, 2006). The transformed data were then analysed using a restricted maximum likelihood mixed model with each isolator fitted as a random effect and the relevant treatment effects and their significant interactions fitted as fixed effects. Differences between individual values were determined using Tukey's HSD test or specific contrasts within the model. Viral load data are presented in the text as back-transformed least-squares means with 95 % confidence intervals (CI). A significance level of P
0.05 was used throughout.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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MDV1 load in dander from chickens challenged with MDV1
MDV1 load varied significantly due to the effect of vaccination (P=0.002) and the number of days p.c. (P<0.0001), with significant interaction between these effects (P<0.0001, Fig. 1). However, the effect of challenge with three isolates of MDV1 (P=0.91) and its interaction with the number of days p.c. (P=0.083) were not significant (Fig. 2). The interaction between challenge virus and vaccination was also not significant (P=0.257).
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), with this effect being most marked between days 14 and 28 (Fig. 1
). MDV1 appeared in dander from day 7 p.c., increased markedly between days 14 and 28 and then plateaued until day 56.
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, Table 1). There were no significant differences due to the different challenge viruses used. HVT was present in dander from day 7 p.v. onwards. In the groups challenged with MDV1, HVT load increased sharply between days 12 and 26 p.v. and then decreased between days 26 and 40 p.v. before stabilizing. The pattern in unchallenged chickens was broadly similar but with a greatly reduced load (Fig. 3). The effect of vaccine type was significant (P=0.031), with slightly higher overall values in chickens vaccinated with HVT than in those vaccinated with the bivalent vaccine [160 221 vs 110 789 copies (mg dander)–1, respectively].
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, Table 1). MDV2 was not present in dander sampled at day 7 p.v., but was present from day 12 onwards. In MDV1-challenged groups, MDV2 load in dander increased sharply between days 12 and 19 p.v. and then slightly between 19 and 26 days p.v., after which little change occurred. There were no significant differences due to the different challenge viruses used. In mock-challenged chickens, a similar pattern was observed, but with greatly reduced levels. In mock-challenged groups, MDV2 loads in dander also increased sharply between days 12 and 19 p.v. and then slightly between days 19 and 26 p.v., before stabilizing.
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, with clear differences in the pattern of viral load over time for the different MDV serotypes.
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). There was no effect of isolator. Overall, dander production increased from 8.9±0.9 mg at day 7 to 232.4±22.5 mg at day 56.
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. Shedding of MDV1 increased sharply between days 7 and 28 p.c., with minor changes thereafter, for both vaccinated and unvaccinated groups (Fig. 7a). From day 35 onwards, both groups were shedding approximately 109 copies per chicken per day. Shedding of MDV2 and HVT also increased rapidly between days 12 and 28 p.v. and stabilized thereafter. However, shedding of the vaccinal viruses was much greater in MDV1-challenged than in mock-challenged chickens for both MDV2 and HVT (Fig. 7b). In challenged chickens vaccinated with the bivalent vaccine, shedding of MDV2 was always higher than that of MDV1, which in turn was always higher than that of HVT.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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There were no significant differences in MDV1 load in dander from birds challenged with three different virulent MDV1 isolates, but there were significant differences among different serotypes of MDV. Nazerian & Witter (1970), using feather follicle epithelium, found positive correlations between virulence, contagiousness and replication of virulent and attenuated MDV1 and HVT viruses. They found that virulent strains of MDV1 were more readily detected in the feather follicle epithelium and more easily transmitted to contact chickens than avirulent strains including attenuated MDV1 and HVT. However, in a recent study using qPCR, Baigent et al. (2005) reported that an avirulent vaccine strain of MDV1 (CVI988 Rispens) was readily detectable in the feather tips after only 7 days, and subsequently rose to levels in this tissue that were almost 103-fold greater than that in the lymphoid tissues (spleen, bursa of Fabricius and thymus). In the current study, shedding of avirulent HVT in dander was certainly lower than that of virulent MDV1, but shedding of the avirulent MDV2 was greater than that of MDV1, suggesting that there is not an overarching relationship between virulence and the rate of viral replication and shedding from the feather follicle epithelium.
Both HVT and a bivalent vaccine combining HVT and MDV2 significantly suppressed MDV1 shedding in dander between days 14 and 28 p.c., but did not prevent MDV1 shedding. Indeed, the effect of vaccination was modest, and older vaccinated chickens continued to shed large amounts of MDV1 (
109 copies day–1). The effects of HVT vaccination on MDV1 appear to be somewhat different in lymphoid tissues. In both splenic (Islam et al., 2005a) and circulating lymphocytes (Islam et al., 2006b), MDV1 load was unaffected by vaccination until days 21 and 28 p.c., respectively, with vaccinated chickens having significantly lower MDV1 loads thereafter. This differs from the pattern seen in dander in the present experiment in which the effects of HVT vaccination were greatest at days 14–28 and reduced thereafter, thus demonstrating early rather than late suppression of MDV1 replication in the feather follicle epithelium.
Shedding of HVT and MDV2 in dander was significantly higher in MDV1-challenged chickens than in mock-challenged chickens. This was an unexpected observation and clearly demonstrates that concurrent challenge with MDV1 enhanced both HVT and MDV2 replication and subsequent shedding in dander. The exact mechanism of such enhancement is unknown. It complicates the use of this technology for monitoring of vaccine efficiency, as higher vaccine virus loads in dander may not relate to vaccine quality or vaccination efficacy, but rather to co-infection with MDV1.
HVT was shed in dander throughout the experimental period, as has been reported previously (Islam et al., 2005b). It has also been shown that HVT is present in many dust samples from commercial broiler farms (Islam, 2006). Although HVT exhibits limited lateral transmission in older chickens (Cho & Kenzy, 1975), transmission between broiler-aged chickens infected early in life is not thought to occur (Cho & Kenzy, 1975). This was confirmed in a recent study using molecular methods (Tink et al., 2005). This indicates that the HVT shed by chickens is largely non-infective for other chickens. With the CVI988 strain of MDV1, the original uncloned isolate spread readily by contact (Rispens et al., 1972), but the clone CVI988/C showed limited transmission between in-contact birds (Witter et al., 1987). The ability to spread is affected by serial passage and attenuation of CVI988 and this characteristic may vary among different subclones of the virus (Baigent et al., 2005).
This study provides the first estimates of dander production by individual broiler chickens (based on capturing dander in isolator exhausts) during their life span. The pattern of dander production was a typical sigmoidal growth curve and appeared to be closely associated with body weight, which exhibited a similar-shaped curve over this period. The method of calculation of dander production assumed that all dander shed by chickens was expelled in the isolator exhausts. This assumption clearly underestimated dander production, as dander accumulated over time in the isolators, although it was clear that most of it was expelled. For this reason, the estimates provided are likely to have underestimated total dander production by a small factor.
The combination of quantitative evaluation of viral load in dust and quantitative estimation of dust production by individual chickens enabled the first estimates to be made of actual shedding rates of the different MDV serotypes in commercial chickens under typical vaccination conditions. The combination of rapid increases in viral load in dander over the first 4 weeks of life, and a concomitant increase in dander production by individual birds led to a very rapid rise in shedding of all of the MDV serotypes over this period, followed by a plateau of viral shedding rates. A key finding was that the MDV2 strain examined had very high shedding rates, several-fold higher than that of three MDV1 isolates, whilst shedding of HVT was between 10- and 100-fold lower than that of the other two MDV serotypes. More importantly, there was a major interaction between the viruses in their effects on shedding rate. Vaccination with HVT or MDV2 provided significant, but not complete, protection against Marek's disease, but had minor effects on viral shedding of MDV1 when birds were given a high challenge dose (500 p.f.u.) at day 5 of age. Indeed, vaccinated chickens continued to shed approximately 109 copies of MDV1 per day, approximately half the amount shed by unvaccinated chickens. On the other hand, infection with MDV1 caused marked increases (approx. 1–2 logs) in the shedding rate of HVT and MDV2.
The implications of these findings are numerous. Firstly, they provide data on which to base further studies into, or models of, the epidemiology of Marek's disease and the spread of both vaccinal and virulent viruses in chicken populations. From a diagnostic sense, the findings are mixed. The ability to quantify either vaccinal or MDV1 viral load in poultry dust has considerable diagnostic and monitoring potential for MD in industry (Walkden-Brown et al., 2004, 2005; Islam, 2006) and indeed currently is being used for this purpose in Australia. However, the potential application of qPCR for monitoring vaccinal virus to confirm efficacy of vaccination is limited by the finding in this experiment that high values for vaccinal virus may be less a reflection of successful vaccination than an indication of post-vaccinal challenge with MDV1. It is likely, therefore, that routine monitoring of MDV using shed dust samples would be best based on analysis of MDV1 load alone. The other area where the findings of the present experiment find application is that of evolutionary biology. Marek's disease is almost unique in that the combination of mass vaccination against this disease worldwide using vaccines that limit oncogenicity but do not preclude co-infection by virulent MDV1 appears to have resulted in a steady evolution in virulence of MDV1 such that MD vaccines fail every decade or so in the USA (Witter, 1998). Gandon et al. (2001) have proposed a theoretical framework by which imperfect vaccines such as the MD vaccines may contribute to the evolution of virulence. The present study provides objective and quantitative data to show that shedding of MDV1 in vaccinated chickens remains at high levels and thus may contribute to evolution in virulence of MDV1 to overcome the effects of vaccination.
In summary, this study has shown that: (i) all three serotypes of MDV are detectable in feather dander, even when all three are co-existing in the same population; (ii) there was no significant difference in viral shedding rates among three isolates of MDV1 of broadly similar virulence; (iii) vaccination with HVT alone or in combination with MDV2 had only limited effects on shedding of MDV1 by infected chickens; (iv) shedding of the vaccinal viruses MDV2 and HVT increases significantly with co-infection with virulent MDV1; (v) a commercial broiler chicken challenged with 500 p.f.u. MDV1 on day 5 of age sheds approximately 109 copies of the MDV1 genome per day from day 28 onwards, irrespective of vaccination status; and (vi) qPCR assays can play an important role in the monitoring of MD status in poultry flock by assaying MDV load in feather dander.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Renz, K. G., Islam, A., Cheetham, B. F. & Walkden-Brown, S. W. (2006). Absolute quantification using real-time polymerase chain reaction of Marek's disease virus serotype 2 in field dust samples, feather tips and spleens. J Virol Methods 135, 186–191.[CrossRef][Medline]
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Witter, R. L. (1987). New serotype 2 and attenuated serotype 1 Marek's disease vaccine viruses: comparative efficacy. Avian Dis 31, 752–765.[CrossRef][Medline]
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Witter, R. L. & Schat, K. A. (2003). Marek's disease. In Diseases of Poultry, 11th edn, pp. 407–465. Edited by Y. M. Saif, H. J. Barnes, J. R. Glisson, A. M. Fadly, L. R. McDougald & D. E. Swayne. Ames: Iowa State University Press.
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Received 5 March 2007;
accepted 23 April 2007.
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