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I sabelle Devaud

1
, J ean-Bernard Herin
1
, Stphane Lemire
2
, Leslie Maurin-Bernaud
3
,
Sylvain Goutebroze
3
, Franois-Xavier Le-Gros
4

1
Merial France, 106 rue Charles et Gabriel Voisin, 44154 Ancenis cedex 4-France

;
2
Merial S.A.S., 29 avenue Tony Garnier 69348 Lyon cedex 7-France;
3
Merial S.A.S., 01150 Saint Vulbas -France;

4
Merial S.A.S., 254 rue Marcel Mrieux 69007 Lyon- France

isabelle.devaud@merial.com



Key words: Muscovy duck, parvovirus, live vaccine, GM MDVP strain, protection, GPV challenge,
MDPV challenge, prevention of growth retardation & condemnation


Abstracts: A new generation live vaccine based on live MDV was recently developed; its under final
process of EU registration. It was shown to provide protection against both types of virus, GPV and
MDVP. Clinical studies managed by Merial R&D demonstrated the heterologous (GPV) and the
homologous protection (MDPV) in vaccinated populations of conventional ducklings in experimental
conditions. Both virus challenges were performed: protection against acute form of the disease was
highlighted, as well as protection against later chronic form. Recent parvovirus isolate genetic
characterization surveys in France, showed that both GPV and MDVP still represent a threat to the
production. MDPV has been showing since the first 1988 cases very low genetic variability.






INTRODUCTION

Muscovy duck species has been proven to be susceptible to parvovirus since the years 1970 (Palya
and al., 2013) . The goose parvovirus (GPV) or Derzsys disease virus was involved in clinical
cases associated with high levels of mortality at the origin. A live vaccine formulated with a GPV
viral strain was developed originally. A specific condition to the Muscovy duck virus was
characterized later in 1988 associated with high mortalities in vaccinated ducks in France, due to a
muscovy duck parvovirus (MDPV) (Jestin and al., 1991). Cross-protection with the available live
vaccine was not anymore sufficient, and a bivalent vaccine, live GPV combined to inactivated
adjuvanted MDPV vaccine was developed, as well as later on a bivalent vaccine for breeders in oil
emulsion. A new generation live vaccine based on live MDPV, GM strain grown on EB66 cell
culture, was recently developed and is under final process of EU registration.

MATERIAL AND METHODS

1/ Parvovirus epidemiological data in muscovy ducks

A diagnosis survey has been performed by Merial laboratory technical assistance (LTA) since 2008.
The LTA gives a yearly picture of the epidemiological situation following requests from the field:
diagnosis is performed, by testing samples from field suspicions, at once by histology on spleen,
heart, muscle, sciatic nerve, and by real-time quantitative polymerase chain reaction (qPCR) on
spleens. Various qPCR have been developed to detect both GPV and mDPV, either vaccine or wild
strain, by different primers on VP1.

P30 Protection of muscovy ducks against parvoviruses by vaccination:
An approach based on scientifical data
The benefit of a new generation live vaccine
Parvovirus disease routine diagnosis is established when sciatic neuritis, heart and muscle fiber
degeneration are found, then noted HISTO+, concomitant or not with a qPCR positive test, then
noted qPCR+.

Phylogenetic analysis is performed sequencing genes from GPV and MDPV isolates; a focus is
driven on 9 French MDPV field isolates: 3 from historical acute cases in 2005-2007 (references
508-169, 508-430 and 607-720A) and 6 from recent subacute cases in 2011. The French GenBank
reference strain (89384) is the tenth integrated in the analysis (Le Gall-Recule and al., 1994).

2/ Efficacy of a new generation live vaccine

Material and method
Several batches of 17 conventional Muscovy ducklings, with maternally derived antibodies, were
vaccinated at one day and 17 days of age with the attenuated Muscovy duck parvovirus GM strain.
(Maurin-Bernaud and al., 2013). Virulent independent challenges, both homologous and
heterologous, were performed on these ducklings aged 28 to 43 days. Depending on the trial, each
duck received a MDPV challenge (strain NX/1) or a Derzsys disease challenge (strain TJ), by
intramuscular injection in the thigh. Monitoring was performed for 2 weeks after the MDPV
challenge and 3 weeks after the GPV one. The ducks were checked on a daily basis. Morbidity rate
and growth following challenge were considered as primary endpoints for the analysis. The
frequency of affected ducks was compared between groups by using the Fisher-Exacts test. The
growth in both groups was compared by a Student t-test or Mann-Whitney test for MDPV
challenges (one weighing after challenge) and by a mixed linear model with repeated measures for
GPV challenges (2 weighings after challenge).


RESULTS

1/ Parvovirus epidemiological data in Muscovy ducks

Diagnosis survey highlights 52 (50%) parvovirus cases out of 104 clinical suspicions in 2012
confirmed by laboratory analysis : 5 occurrences of histological lesions HISTO+ and 47 cases
combining HISTO+ and qPCR+, including 42 qPCR+ for wild strain MDPV ( 45%) and 5 qPCR+
for wild strain GPV (5%).

Table 1: Experimental parvovirus disease diagnosis : 2012 survey
Data in cases ( in rate)






Sciatic neuritis, heart and muscle fiber degeneration are found, then noted HISTO+, parvovirus
qPCR positive test, then noted qPCR+.

Phylogenetic analysis shows very low genetic variability, since the first 1988 cases as previously
reported by similar studies (Chang and al., 2000). The phylogenetic tree summarizes the survey
(Figure 1).

Cases
MDPV
qPCR+
GPV
qPCR+
Parvovirus
qPCR-
HISTO + 42 (45 %) 5 (5%) 5 (5 %)
HISTO - 9 (10%) 2(2%) 41 (39%)


Figure 1: MDPV phylogenetic tree based on VP1 sequencing analysis.
Percentage of nucleotide difference is indicated by the scale and legend in italics





2/ Efficacy of a new generation live vaccine

Clinical protection
Post challenges at day 28, the morbidity rate observed in vaccinated groups was significantly lower
compared to the unvaccinated control group (p=0.00) : 47 vs 100 % in case of MDPV challenge, 24
vs 82 % in case of GPV challenge (Table 2) . A morbidity rate of 100% was observed at necropsy
in the unvaccinated birds post MDPV challenge and 82 % post GPV challenge, thus evidencing the
susceptibility of the birds and validating the high severity of the challenges. For the later challenge
at day 43, no morbidity was evidenced which was an expected finding considering the age-
dependent susceptibility of ducklings to the disease. However under these conditions, the protection
afforded by the vaccination can still be evaluated through growth performances.

Growth performance
The conditions of the study allowed a precise evaluation of the growth retardation of the animals,
which is a specific manifestation of the disease in the context of a 28 days old age challenge, and
the major manifestation of the disease in the context of a 43 days old age challenge. MDPV GM
strain vaccination by 2 injections at 1 and 17 days of age provided statistically significant protection
against bodyweight loss as compared to the unvaccinated birds.

Table 2: Protection rates and growth intakes in efficacy trials









DISCUSSION/CONCLUSION

Clinical studies managed by Merial R&D demonstrated the heterologous (GPV) and the
homologous (MDPV) protection in vaccinated conventional ducklings monitored in experimental
conditions. Diagnosis survey in France, one of the main Muscovy duck meat producers in the world,
2006c
Chine
2005b
Hongrie
89384-France
2011 isolates
(7 similar
isolates)


0.002
substitutions/site
Batches Year
2005a August 2005
2005b August 2005
2006c July 2006
6 field strains March, April,May 2011
89384-France
Genbank Z68272
1989



Challenge type Vaccinated vs controls Challenge D28 Challenge D43
Muscovy duck parvovirus
Morbidity rate % 47 vs 100 P=0,00 NA
Growth intake % +21 P=0,00 +11 P=0,01
Derzsys disease virus
Morbidity rate % 24 vs 82 P=0,00 NA
Growth intake % +10 P=0,00 +5 P=0,00

2005a


showed that both GPV and MDPV still represent a threat to the production. MDPV showed since
the first 1988 cases very low genetic variability. Vaccination represents the main tool for parvovirus
infection prevention in the context of Muscovy duck farming: this new monovalent vaccine allows
prevention of the mortality and lesions, classically associated with the acute form of the MDPV and
GPV diseases, as well as significantly reducting until 9 weeks of age growth retardation generally
associated with the chronic form of both diseases.


JESTIN, V., LE BRAS, M.O., CHERBONNEL, M., LE GALL, G. & BENNEJEAN, G. (1991)
Mise en vidence de parvovirus (virus de la maladie de Derzsy) trs pathognes dans les levages
de canards de Barbarie. Recueil de Mdecine Veterinaire 167, 849-857

CHANG, P.-C, SHIEN, J.-H., WANG, M.-S. & SHIEH, H.-K, Phylogenetic analysis of parvovirus
isolated in Taiwan from ducks and geese, Avian pathology (2000), 29, 45-49


LE GALL-RECUL, G. & JESTIN, V. (1994) Biochemical and genomic characterization of
muscovy duck parvovirus. Archives of Virology 139(1-2), 121-131

MAURIN-BERNAUD, L., MERDY, O., LE-GROS, F.X. & GOUTEBROZE, S. (2013) Efficacit
dun nouveau parvovirus attnu contre la parvovirose du canard de Barbarie et la maladie de
Derzsy. Actes des 10mes Journes de la recherche avicole et palmipdes foie gras du 26-28 mars
2013, La Rochelle, France, pp. 284-288

PALYA, V.J. (2013). Parvovirus Infections of Waterfowl. In: Diseases of Poultry, 13th Edition
(eds. Swayne D.E., Glissen J.R., McDougald L.R., Nolan L.K., Suarez D.L. Nair V.L.) Wiley-
Blackwell, pp 444-454