WO2017081232A1

WO2017081232A1 - Inactivated avian reovirus vaccine for use in a method to increase the hatchability of poultry eggs

Info

Publication number: WO2017081232A1
Application number: PCT/EP2016/077400
Authority: WO
Inventors: Peter DE HERDT; Hendrik Cornelius KOOPMAN
Original assignee: Intervet International B.V.; Intervet Inc.
Priority date: 2015-11-12
Filing date: 2016-11-11
Publication date: 2017-05-18
Also published as: CN108348597A; RU2018121296A; EP3373966A1; RU2018121296A3

Abstract

The present invention provides a way to increase the hatchability of poultry eggs, by applying a specific regime for the vaccination against avian Reovirus. It was found that the administration to poultry of an inactivated avian Reovirus vaccine, at least two times, prior to onset of lay, can lead to a production of 4 to 5 more chicks per breeder hen, over one egg-laying period. This advantageous effect can be obtained through the vaccinations, methods, and uses as defined herein.

Description

INACTIVATED AVIAN REOVIRUS VACCINE FOR USE IN A METHOD TO

INCREASE THE HATCHABILITY OF POULTRY EGGS

The present invention relates to the field of veterinary vaccinology, more specifically the invention relates to vaccination of poultry against avian Reovirus. In particular the invention relates to an inactivated avian Reovirus vaccine for use in the invention, and to methods and uses employing said vaccine.

The expected rise in the world's human population in the future will cause an increased demand for food such as animal protein. From the various possible sources, one of the preferred is protein from poultry meat and eggs, for being highly nutritious and relatively efficient to produce. The current world production of poultry protein is already at an impressive level, with 80 million tons of poultry meat, and 1.5 x 10^Λ12 eggs (67 million tonnes) being produced annually. While the vast majority applies to production of chickens, also turkeys, ducks and geese are of considerable economic relevance. To produce at such levels, modern methods of intensive poultry farming employ a combination of technological tools and optimised procedures to maintain the animals' health and performance at high level. Also the animal breeds used have been developed to be highly efficient in feed conversion. Next to high volumes however, this market also runs on low margins, and is therefore strongly cost aware. An improvement in the yield of meat, eggs, or number of offspring can therefore make a significant impact on a farmers' economy of operation, and assist in meeting increasing production goals.

Poultry eggs are produced by birds called layers, and meat is obtained from birds called broilers. In turn, these layers and broilers are derived from parent animals, called breeders. These are produced by specialised companies via an elaborate system of crossing and selecting that combines specific lines of animals to produce the male and female lines for the different generations. Such lines of parental birds are available commercially, for example layer breeds such as Hyline™ or ISA™, and broiler breeds such as Ross™ or Cobb™. The scale of operation increases exponentially for the different generations, going from production of (great-) grandparents, to parents, to production animals. Therefore the poultry breeder industry is also of considerable economic size, with the production of hatching eggs at about 5 % of the world's poultry egg production. Such hatching eggs differ from eggs for consumption (so-called table- eggs) in the fact that they (for the most part) are fertilised, and are intended for the generation of offspring.

Although there is considerable vertical integration in the poultry industry, typically separate operations are used to grow and keep breeder poultry. Breeders are raised on rearing farms, from day old chicks to about 18 weeks of age. Typically male and female birds are raised separately. At this type of farm the breeders will receive their initial series of vaccinations.

Chicken hens will come into lay around about 20 weeks of age, and the weekly number of eggs produced then rapidly increases and peaks between 28 and 32 weeks of age. Later egg production slowly decreases again, to below economic levels from about 60 weeks of age. At an age of 18-20 weeks, the breeders are transferred to breeder farms, where they stay to about 60 weeks of age. After an acclimatisation period, males are introduced at a male:female ratio of 1 :5 - 1 : 10. Commonly breeder farms will have a ground-plan that provides separate floor- and nesting areas, where the nests allow for the (automated) collection of eggs. Collected eggs are stored for a limited period (2 - 14 days) under controlled conditions, e.g. 16 °C and 80 % relative humidity (RH), in order to maintain the health of the fertilised eggs, but not yet stimulate embryo development.

Before placing in incubators, the hatching eggs undergo a selection, where eggs are removed that have a soiled, porous, cracked, or malformed shell, or eggs of aberrant size. This assures that only quality eggs will be incubated. Optionally this can be followed by an egg-surface decontamination, e.g. by fumigation.

At hatching farms the selected eggs are then placed in incubators, the so-called 'setting', which starts embryo development.

The conditions for egg incubation that allow growth of the embryo comprise for example incubation at about 37 °C and at about 90 % RH, and placing the eggs with the air-chamber upwards. Also the position-angle of the eggs is shifted regularly, the so-called turning.

In between setting and hatch the eggs will be checked for embryo development. Such inspection is typically done by candling: viewing an egg over a bright light, to inspect its quality and visualise the embryo. Eggs with dead embryos or empty, infertile eggs are removed, and eggs with healthy embryos are returned to incubation. This visualisation of an embryo can be done from about 1 -third of the incubation time; for chickens this correlates to 6-8 days of embryo development. However usually this selection is performed a few days before (expected) hatch and combined with a transfer to hatching trays to minimise the disturbance to the eggs, and to economise the handling time. For chicken eggs this is typically done at about 18 days of embryo development.

Chicken eggs will hatch at about 21 days of embryo development, for other poultry species this is commonly longer: turkey: 28 days; duck: 28 (Pekin) to 35 (Muscovy) days; Japanese quail: 17 days; Guinea fowl: 26 days; pheasant, partridge: 24 days; and goose: 28-32 days.

The day-old chicks are the final product of the hatching farm, and are sold on and transported to layer- or broiler rearing farms. Breeder birds are selected for the quality of their eggs, to give rise to large numbers of healthy progeny. Consequently one of the main criteria for assessing breeder bird quality is the hatchability of their eggs, i.e. the number of live chicks produced from the number of eggs that were incubated. Hatchability is different for each genus of poultry, and for the different breeds within one species. In addition, several other factors influence hatchability, such as: management conditions; general health; handling, storage and incubation of the eggs; and hen's age (see: Yassin et al., 2008, Poultry Sci., vol. 87, p. 2408-2417; Mauldin, J., Chapter 39, in: "Commercial chicken meat and egg production", 2002, Springer media, New York).

As an example: a modern chicken broiler breeder hen will lay about 180 eggs, which produce about 140 live chicks, over one laying period from about 26 - 60 weeks of age. For other poultry types and species this number is lower. Consequently there is a need in this field to improve upon this outcome, to optimise farming economy, and to accommodate future requirements for the production of animal protein.

It is therefore an object of the present invention to improve the hatchability of the eggs from breeder poultry.

There can be many reasons why the hatchability of breeder eggs is not higher. A simple cause may be that not every hen was fertilised. This can be managed by controlling the number and the quality of the male birds that are kept with the hens for fertilisation. More complex are causes for reduced fertility of the male or female birds themselves, which can be as variable as the quality of the feed, the management conditions in the poultry house, and the effects of various (infectious) diseases.

Commonly breeder birds are under strict veterinary care, and receive several vaccinations against various viral- and bacterial diseases. These vaccinations are intended to protect the vaccinated breeders themselves, but also to provide passive immune-protection to their progeny via maternally derived antibodies in the yolk of the egg. One of the vaccinations common for breeder birds is against avian Reovirus.

Avian Reoviruses are taxonomically classified in the species avian Orthoreovirus that is within the genus Orthoreovirus, and in the Reoviridae family. The virus particle is without envelope but has a double- shelled protein capsid. This capsid contains the double stranded RNA genome that exists of 10 segments. An overview of characterising features of an avian Reovirus is given in Benavente & Martinez- Costas (Virus Res., 2007, vol. 123, p. 105-1 19) and in the chapter Birnavirideae in Fields Virology (4th Edition 2001 , Lippincott Williams & Wilkins, ISBN-10: 0781718325). Avian Reoviruses are important pathogens causing severe disease in birds, and significant economic damage to commercial poultry farming operations; chickens, turkeys and other types of birds may be affected. Avian Reovirus is more pathogenic to young birds, because some age-related resistance to avian Reovirus infection develops in birds from about 4 weeks old. Symptoms and lesions include among others enteric- and respiratory disease, myocarditis and hepatitis. Most typical is viral arthritis (or:

Reoviral arthritis) and tenosynovitis, an inflammation of the tendon sheets. The resulting lameness can cause difficulties in walking to feed bins, resulting in starvation. Alternatively, viral enteric disease leading to malabsorbtion prevents an effective conversion of the feed. Also, in combination with several other viral and bacterial enteric disease agents, avian Reovirus is a factor in the so-called 'runting stunting syndrome', which has general symptoms of wasting and under-performance.

Vaccination against avian Reovirus can be applied both to older birds, and to chicks at very young age. However in practice only the parents are vaccinated, protecting both the parents themselves, as well as the progeny, both as embryo and as chicks.

Avian Reovirus vaccines are usually whole virus vaccines, that are of the attenuated live-, or of the inactivated type; both types are commonly administered by injection, either subcutaneous or intramuscular. Examples of commercial avian Reovirus vaccines (all from MSD AH, the Netherlands) are: attenuated live vaccines: Nobilis® Reo 1 133, or Nobilis® Reo 2177; or inactivated-adjuvanted vaccine such as Nobilis® Reo Inac (comprising strains 1733 and 2408). An inactivated Reovirus antigen can also be comprised in an inactivated combination vaccine, such as for example in Nobilis® Reo+IB+G+ND (MSD AH).

In the standard recommended vaccination regime, breeder birds are vaccinated against avian Reovirus two times: by a primary vaccination (priming) with live vaccine at young age, and by a secondary vaccination (booster) with inactivated vaccine several weeks later. For chickens this booster vaccination is usually given at about 16-18 weeks old, i.e. 2-6 weeks before the expected onset of lay (see:

Giambrone & Clay, 1986, Poultry Sci., vol. 65, p. 457-461 ; De Herdt et al., 1999, Avian Diseases, vol. 43, p. 271-278). In case of high field-infection pressure, one or more additional booster vaccinations may be given.

The vaccination schedule of applying a priming with a live vaccine, followed by a booster with an inactivated vaccine is the preferred schedule for a broad and lasting protection against avian Reovirus infection and disease. This is because this regime is known to induce high antibody titres in the target birds (see: (R.C. Jones, 2014: Reovirus infections, in: Swayne et al., eds.: Diseases of Poultry. 13th ed., Wiley-Blackwell, Ames, IA., USA, p. 351-374). However, some countries -e.g. Great Britain and Belgium-, have not allowed the introduction of live avian Reovirus vaccines. Consequently, in these countries the only alternative is to use a vaccination regime employing two or more vaccinations with inactivated avian Reovirus vaccine. Many farmers are not happy with this restriction of their choice for a vaccination regime; not only because inactivated Reovirus vaccine is more expensive than live vaccine, but also because it is a firmly established belief in this field that the Reovirus vaccination regime of live priming and inactivated boosting is preferable. For example, the Belgian department of the World Veterinary Poultry Association (WVPA) writes in its 2015 vaccination schedule advice for breeder chickens: "The application of a prior vaccination with a live Reovirus vaccine, 4 to 6 weeks before the final vaccination with inactivated vaccine, is recommended. At the moment there is no live Reovirus vaccine registered in Belgium." [translated from Dutch], see page 12, paragraph 5 of:

http://www.dgz.be/sites/default files WVPA_pluimvee_entschema_advies_2015.pdf.

As a result, it is common practice in Belgium to import live avian Reovirus vaccine from another country, and use that for live priming vaccination under an exemption rule.

Surprisingly it was found that the object of the present invention, the increase of the hatchability of breeder eggs, can be met by a specific regime of the vaccination of the breeder poultry against avian Reovirus: by changing from the preferred regime of a live priming followed by an inactivated boosting

('live+inac'), to a regime of administering at least two times an inactivated Reovirus vaccine ('2x inac') to a poultry animal. This way a significant increase of about 3.4 % in hatchability of breeder eggs has been found.

It is important to realise that at an average output of about 140 chicks per breeder hen, this invention allows an additional 4-5 chicks to be produced per breeder hen. This represents a considerable economic benefit for a poultry breeder farmer, which substantially surpasses improvements obtained by optimisation of the animal's genetics, or their maintenance conditions. Even more so: the economic advantage from this increased hatchability is so substantial, that it far outweighs the higher costs of the use of inactivated vaccine instead of live vaccine.

Consequently, through the present invention, a poultry breeder farmer now has the option to considerably improve the overall economic performance.

This invention is applicable to all production of poultry hatching eggs intended for the production of progeny, either for breeder-, broiler- or layer chicks.

This effect was totally unexpected, and in direct contrast to the strong general conviction in the field that a live+inac regime for Reovirus vaccination was preferable.

Also, even though a 2x inac regime has already been applied in the past, there has never been any realisation or description that this regime would have a positive effect on the hatchability of fertilised poultry breeder eggs. It is not known why the 2x inac vaccination regime for avian Reovirus has such a strong positive effect on hatchability as compared to the standard live+inac regime. Although the inventors do not want to be bound by any theory or model that might explain these observations, they speculate that this vaccination regime induces certain health effects in the breeder birds, which may lead to an increase of reproductive performance such as fertilisation efficiency. Alternatively, or in addition, these health effects may lead to stronger and healthier embryos, which is visible as a reduction of embryo mortality in ovo.

As discussed, vaccination with inactivated Reovirus vaccine is known: for instance Giambrone & Clay

(1986, Poultry Sci., vol. 65, p. 457-461 ) compare programs for the vaccination of broiler breeders "for the prevention of clinical Reovirus infection in broiler progeny". They conclude that the optimal regime consists of a live priming followed by two boosters with inactivated vaccine, all before onset of lay.

However they focus on serology data, and no reference or suggestion is made to any effect of such a regime on the hatchability of the breeder's eggs.

Several publications have considered the effect of avian Reovirus infection on hatchability. One possible mechanism for contamination and infection of the embryo could be through a vertical transmission of avian Reovirus via the egg. However unlike results obtained in vitro, under normal field conditions such transmission was found to be very low (Al-Muffarej, et al. 1996, Avian Pathol., vol. 25, p.

469-480).

One publication describes negative effects on hatchability from vaccination of breeders with a live vaccine against avian Reovirus: Giambrone et al., 1991 , Avian Diseases, vol. 35, p. 380-383. However in that study the live vaccine was applied during egg production, instead of before. Also the tested vaccine turned out to be unusually virulent; based on the pathology observed, it is likely that in that study a field- infection occurred with another virus, probably infectious bronchitis virus.

Therefore in one aspect the invention relates to an inactivated avian Reovirus vaccine for use in increasing the hatchability of poultry eggs, wherein the vaccine is administered at least two times to the poultry, prior to onset of lay. An "avian Reovirus" is well known in the art as a virus belonging to the species avian Orthoreovirus. These viruses and their induced diseases are e.g. described in well-known handbooks, like: "The Merck veterinary manual" (10th ed., 2010, CM. Kahn edt., ISBN: 091 191093X), and: "Diseases of Poultry" (13th ed., supra).

An avian Reovirus displays the characterising features of its taxonomic group-members such as the morphologic, genomic, and biochemical characteristics, as well as the biological characteristics such as physiologic, immunologic, or pathologic behaviour. As is known in the field, the classification of microorganisms is based on a combination of such features. The invention therefore also includes avian Reoviruses that are sub-classified therefrom in any way, for instance as a subspecies, strain, isolate, genotype, variant, subtype or subgroup and the like.

A "vaccine" is well known to be a composition which has an inherent medical effect. A vaccine comprises an immunologically active component, and a pharmaceutically acceptable carrier. The 'immunologically active component', is one or more antigenic molecule(s) that is recognised by the immune system of a target, and induces a protective immunological response. The response may originate from the targets' innate- and/or from the acquired immune system, and may be of the cellular- and/or of the humoral type.

A vaccine generally is efficacious in reducing the level or the extent of an infection, for example by reducing the viral load or shortening the duration of viral replication in a host animal. Here this refers to reducing the infection by avian Reovirus in its target organs such as tendon, joints, or intestines.

Also, or possibly as a results thereof, a vaccine generally is effective in reducing or ameliorating the symptoms of disease that may be caused by, or may the result of, such viral infection or replication.

Such a vaccine is colloquially referred to as a vaccine 'against' avian Reovirus, or as an 'avian Reovirus vaccine'.

The inactivated avian Reovirus vaccine as described herein, when used according to the invention, has the effect of increasing hatchability of poultry eggs. The inventors do not know whether this results from reducing infection by avian Reovirus, from a reduction of the lesions and clinical signs caused by such infection, or from both, or even from other effects, related or not. An "inactivated" vaccine is a vaccine comprising a micro-organism that has been rendered non-replicative by some method of inactivation. Common methods of inactivation are by applying e.g. heat, radiation, or chemicals such as formalin, beta-propiolactone, binary ethyleneimine, or beta-ethanolamine.

The avian Reovirus to be inactivated initially is the whole virus particle which can be derived from a viral culture, such as from the cell-pellet, the culture supernatant, or the whole culture. As the inactivation method affects the proteins, the lipids, and/or the nucleic acids of the virus particle, this may to some extend become damaged. Nevertheless this type of vaccine is commonly called a whole virus inactivated vaccine.

The selection of a particular viral substrate, and of a method of inactivation, is well within the routine capabilities of the person skilled in the art. An alternative for a whole virus inactivated vaccine, as described above, is a subunit vaccine. This can be prepared either from live- or from inactivated virus, by applying one or more (additional) steps for the fractionation or isolation of one or more parts of the viral particle. This comprises for instance preparing an extract, fraction, homogenate, or sonicate.

In a preferred embodiment the inactivated avian Reovirus vaccine for use in the invention is a whole virus inactivated vaccine.

The term inactivated is used as opposed to a "live" vaccine. Although the term live is biologically incorrect in respect of a viral agent, it is commonly used in this field. Consequently, for the invention the term 'live' refers to an avian Reovirus that is capable of replication, i.e. is replicative, non-inactivated.

The vaccine for use in the invention can in principle be any inactivated avian Reovirus vaccine. For the invention the term "hatchability" is defined as the number of chicks that hatched from a certain number of incubated eggs, expressed as a percentage.

Details of ways to determine hatchability are well known in the art. Consequently the determination of the effectiveness of an inactivated avian Reovirus vaccine on hatchability is well within the skills of the routine practitioner. In addition methods and preferences for determining and comparing hatchability are described in detail below.

Effectiveness of a vaccine against avian Reovirus can e.g. be done by monitoring the immunological response following vaccination or after a challenge infection, e.g. by monitoring the targets' signs of disease, clinical scores, serological parameters, or by re-isolation of the pathogen, and comparing these results to a vaccination-challenge response seen in mock vaccinated animals.

Various embodiments, preferences and examples of an inactivated avian Reovirus vaccine for use according to the invention will be outlined below. The "increasing" of the hatchability is relative to the hatchability as obtained by a vaccination regime of breeder poultry against avian Reovirus that does not apply the 2x inac regime of the invention; in particular as compared to the 'standard' vaccination regime against avian Reovirus of live+inac. The increase can be measured by methods well known in the art, and as described herein, and needs to have a statistical significance, in order to be credible. For example the p value should be 0.05 or less.

The inventors have analysed the effect of this vaccination regime, by monitoring of the production data of one large poultry breeder operation in Belgium over 2013 and 2014. Data from over 280.000 breeder birds, and their 4.7 million eggs were collected. Out of the many variables in play, a correlation analysis showed that next to the hen's age, also the type of avian Reovirus vaccination regime was strongly correlated to hatchability. Next by regression analysis using hen age and vaccination regime as independent variables, a positive effect for the dependent variable: hatchability was found from the 2x inac vaccination regime: an increase was found in hatchability about 3.4 %, at an adjusted R² value of 0.662, and a p value of < 0.001.

As the skilled person will appreciate, when applying the present invention the exact number of the increase in hatchability obtained for a certain experiment or for a series of experiments will depend on several factors. Amongst others these factors involve the general health status of the poultry used, their management conditions, the number of test animals, and the statistical analysis applied.

However, the inventors have found that the advantageous effect of the invention is readily apparent even when comparing groups of animals of different composition, and using different types of analysis. For the animals in the test groups described in the Examples section hereinafter, the calculated increase in hatchability resulting from the vaccination regime according to the invention, was well above any variability caused by management conditions or breed characteristics, which surmounts to an effect on hatchability of about ± 1 %. In fact the inventors typically observed increases in hatchability between about 2.5 and about 3.9 % depending on the selection of the groups considered, and the statistical analysis method applied. However in all cases this was highly significant and economically relevant.

The effect of the invention is preferably determined using relatively large groups of animals, and over a substantial period of time; preferably at least 10.000 breeders and 250.000 of their eggs. For less complicated analyses, for example when the age of the hens does not vary between test groups, statistical analysis may be done using an ANOVA. However for groups of animals of variable composition, a direct comparison is not suitable because of the age effect on hatchability, and therefore regression analysis of the data should be applied to calculate the increase in hatchability obtained, using age of the breeder hens and vaccination regime as independent variables. Details and preferences for the performance of the experiments and the calculations are described in the Examples section hereinafter. An example of such a regression analysis is also described in De Herdt et al., 2012 (Avian Dis., vol. 56, p. 365-368).

As the skilled person is well aware, such regression analysis requires the establishment of a regression formula, wherein the number and the impact of independent variables is adapted until the maximal R² value for this data set is obtained. This can conveniently be calculated using one of several available computer programs for statistical analysis, such as the programs: SPSS™ (IBM), SAS™ (SAS Institute), or R™ (R foundation).

The regression formula as a whole, and within the formula its constituting independent variables, should have a p value that indicates a significant effect. Therefore for the invention, the regression formula and the constituting independent variables should have a p value of 0.05 or less.

For the invention, "about" indicates that a number can vary between ± 25 % around its indicated value; preferably about means ± 20 % around its value, more preferably about means ± 15, 12, 10, 8, 6, 5, 4, 3, 2 % around its value, or even about means ± 1 % around its value, in that order of preference. "Poultry" refers to avians of agricultural relevance, such as: chicken, turkey, duck, goose, partridge, peacock, quail, pigeon, pheasant, guinea fowl, or ostrich.

For the invention, the terms "breeders", or "breeder poultry" refer to parental poultry animals, kept for producing fertilised eggs. The offspring chicks may be of broiler, layer, or of breeder type. Consequently, the breeders for the invention may be named broiler-breeders, layer-breeders or breeder (grand)parents.

The vaccine for use in the invention should be "administered" in the optimal way, which means that when the vaccine is a commercial vaccine, it is to be administered as prescribed on its product-leaflet.

When the vaccine is non-commercial, it must be applied in an optimal way in respect of its dose, volume, route, or formulation, as well in an optimal way with respect to the target animal's age, sex, or health status. The skilled person is perfectly capable of determining such optimal vaccine administration conditions. For an inactivated avian Reovirus vaccine that route will typically be by injection, specifically via i.m. or s.c.

For the invention, the administration "at least two times" refers to an administration for 2, 3, or more times, such as may be practically and economically feasible. Also, this refers to the administration to a particular animal; consequently when the advantageous effects of the invention are to be obtained from a group of poultry animals, then in principle every animal from that group needs to be administered the vaccine at least two times.

The "onset of lay" is the day a poultry hen lays her first egg. As this date is not exactly predictable for an individual bird, in practice this means the date of the expected onset of lay, as prediction for a group of birds considered. The age at which onset of lay occurs differs for the various kinds of poultry, and may differ for different breeds, and management conditions. This is well-known in the art, for example, for chickens onset of lay will commonly be at about 20-22 weeks of age, but is sensitive to management conditions such as the feeding- and lighting regime.

Expected dates for onset of lay for the different poultry species and -breeds, as well as optimal conditions for management and operation of breeders, are well-known in the art, and are for instance published in manuals and pocket guides from the main suppliers of breeders such as Aviagen, Cobb-

Vantress, or Hendrix Genetics.

For the invention, the administration "prior to onset of lay" means that the vaccination regime of the invention is to be completed before commencement of egg-laying. This in order to have established (a begin of) an effective immune response in the animals, before fertilised eggs will be produced. In practice this means that the booster vaccination needs to be administered before the onset of lay; in case more than one booster vaccination is administered, then the last of the booster vaccinations is to be administered before the onset of lay.

For the invention, "prior to" refers to a period of time between the last of the vaccine

administrations, and the onset of lay. This period should be at least 1 day. When the vaccination regime for the invention is applied to male poultry breeders, the period prior to onset of lay is calculated based on the expected onset of lay of the female birds of the group in which these males will be placed. The vaccination regime as described for the invention leads to an increase in hatchability of poultry eggs. One way in which this effect is reached, is by a reduction of the number of dead embryos in the incubated eggs. This is readily detectable during incubation, for example upon candling of the eggs that were incubated (after the quality selection), by counting live and dead embryos. When this number is compared to the number for embryo mortality in eggs that were incubated in the same way, but that were derived from breeders that did not receive a vaccination regime as described for the invention, the difference is evident.

Consequently, one of the further advantageous effects of the invention is the reduction of poultry embryo mortality. Therefore in an embodiment of the inactivated avian Reovirus vaccine for use in increasing the hatchability of poultry eggs according to the invention, the increase in hatchability results from a reduction in poultry embryo mortality.

Alternatively or additionally, the increase in hatchability obtained by the invention, can be accredited to an increased fertility of the breeder poultry (male and/or female), resulting in the production of a higher number of fertilised eggs, and consequently, more hatching chicks. This effect can be observed by comparing avian Reovirus vaccination regimes with respect to the number of chicks that hatch from the eggs that were incubated.

Consequently, one of the further advantageous effects of the invention is an increase in the fertility of breeder poultry.

Therefore in an embodiment of the inactivated avian Reovirus vaccine for use in increasing the hatchability of poultry eggs according to the invention, the increase in hatchability results from an increased fertility of the poultry.

As described, the cause for the advantageous effect of the invention on hatchability of poultry eggs is not clear, and may derive from an effect on the health and/or the fertility of the vaccinated parent poultry, next to, or in addition to, an effect on the health of the embryo. Consequently, it is advantageous to vaccinate either the female or the male birds of a group of breeder poultry. However preferably the vaccine is administered at least two times both to female and to male poultry.

Therefore in an embodiment, the inactivated avian Reovirus vaccine for use in increasing the hatchability of poultry eggs according to the invention, is administered both to female and to male poultry. As described above, the timing of the vaccination of the male poultry is derived from the expected onset of lay of the female birds of the group of breeders in which these males will be placed. In an embodiment, the inactivated avian Reovirus vaccine for use in the invention comprises an adjuvant. This applies in particular to inactivated vaccines, which are non-replicative, therefore require additional immune stimulation to be able to induce an effective, broad-spectrum, and lasting vaccination.

An "adjuvant" is a well-known vaccine ingredient, which in general is a substance that stimulates the immune response of a target in a non-specific manner. Many different adjuvants are known in the art. Examples of adjuvants are Freund's Complete or -Incomplete adjuvants, vitamin E, aluminium compositions such as Aluminium-phosphate or Aluminium-hydroxide, non-ionic block polymers and polyamines such as dextransulphate, Carbopol®, pyran, Saponin, such as : Quil A®, or Q-vac®. Saponin and vaccine components may be combined in an ISCOM® (EP 109.942, EP 180.564, EP 242.380).

Furthermore, peptides such as muramyldipeptides, dimethylglycine, tuftsin, are often used as adjuvant, and oil-emulsions, using mineral oil e.g. Bayol™ or Markol™, Montanide™ or light mineral (paraffin) oil; or non-mineral oil such as squalene, squalane, or vegetable oils, e.g. ethyl-oleate. Also combination products such as ISA™ (from Seppic) or DiluvacForte™ can advantageously be used.

An emulsion can be water-in-oil (w/o), oil-in-water (o/w), water-in-oil-in-water (w/o/w), or a double oil-emulsion (DOE), etc.

In a preferred embodiment the vaccine comprising an adjuvant, is a water-in-oil emulsion.

Such emulsions provide a depot effect in the vaccinated animal that slowly releases the antigen, thereby providing a prolonged stimulation of the target's immune system.

In a more preferred embodiment, the oily phase of the water-in-oil emulsion vaccine comprises a mineral oil, or an ethyl-oleate. In an embodiment, the inactivated avian Reovirus vaccine for use in the invention comprises inactivated avian Reovirus from a single biologic origin; e.g. from one subtype, serotype or genotype.

In a preferred embodiment the vaccine comprises inactivated avian Reoviruses from two or more sub-classifications as defined herein; for example being of different subtype, serotype, genotype, etc. For example the vaccine for use in the invention comprises one or more of the avian Reovirus strains: 1 133 (a.k.a. S1 133), 2177, ERS, 1733, 2408, the GEL series, and/or the ISR series. These are all well-known in the art, see e.g.: Kant et al. (2003, Vet. Res., vol. 34, p. 203-212), Liu et al. (2003, Virology, vol. 314, p. 336-349), and Lublin et al. (201 1 , vaccine, vol. 29, p. 8683-8688).

In an embodiment the inactivated avian Reovirus vaccine for use in the invention is Nobilis® Reo Inac.

In an embodiment, the inactivated avian Reovirus vaccine for use in the invention comprises inactivated avian Reovirus from different genotypes. This is for example described in European patent application number EP 14187650. Therefore, in an embodiment the inactivated avian Reovirus vaccine for use in the invention comprises inactivated avian Reoviruses from each one of two genotype groups: genotype group 1 and genotype group 4, as defined in European patent application number EP 14187650. The term "comprises" (as well as variations such as "comprise", "comprising", and "comprised") as used herein, refer(s) to all elements, and in any possible combination conceivable for the invention, that are covered by or included in the text section, paragraph, claim, etc., in which this term is used, even if such elements or combinations are not explicitly recited; and does not refer to the exclusion of any of such element(s) or combinations. Consequently, any such text section, paragraph, claim, etc., can also relate to one or more embodiment(s) wherein the term "comprises" (or its variations) is replaced by terms such as "consist of", "consisting of", or "consist essentially of.

In an embodiment the poultry for the invention is selected from the group consisting of chicken, turkey, duck, and geese. In a preferred embodiment the poultry is chicken, because for this poultry species the economic impact of the increase in hatchability is the largest for the poultry breeding sector.

In an embodiment of administration prior to onset of lay according to the invention, the vaccination regime of administering at least two times the inactivated avian Reovirus vaccine for use in the invention, is completed at least one week prior to onset of lay. Preferably the administration is completed at least two, three or even four weeks prior to onset of lay, in that order of preference.

In an embodiment, the regression formula used for the statistical assessment of the effect of the invention employs an 'adjusted' R² value. This compensates for possible interactions between the independent variables. For the invention, the regression formula preferably has an adjusted R² value of at least 0.200.

More preferably, the regression formula has an adjusted R² value of at least 0.300. More preferably the regression formula has an adjusted R² value of at least 0.350, 0.400, 0.450, 0.500, 0.550, 0.600, 0.650, 0.700, 0.750, or even of at least 0.800, in this order of preference.

In an embodiment, the regression formula as a whole, and the constituting independent variables have a p value of 0.05 or less. More preferably the regression formula and the constituting independent variables have a p value of 0.04, 0.03, 0.02, 0.01 , 0.005, 0.001 or less, or even of less than 0.001 , in that order of preference.

In an embodiment of the inactivated avian Reovirus vaccine for use in increasing the hatchability of poultry eggs according to the invention, one or more or all of the conditions apply selected from the group consisting of:

the vaccine is an adjuvated water-in-oil-emulsion,

the adjuvated water-in-oil-emulsion vaccine comprises a mineral oil or an ethyl-oleate, the vaccine is administered at least two times and at least one week prior to onset of lay, - the vaccine is administered both to female and to male poultry,

the poultry is chicken, the vaccine comprises inactivated avian Reoviruses from two or more sub-classifications as defined herein,

the regression formula used for the statistical assessment of the effect of the invention employs an adjusted R² value of at least 0.200,

- the regression formula used for the statistical assessment of the effect of the invention as a whole, and the constituting independent variables have a p value of 0.05 or less, and the inactivated avian Reovirus vaccine for use in the invention is Nobilis® Reo Inac.

Therefore, in an embodiment, the inactivated avian Reovirus vaccine for use in increasing the hatchability of poultry eggs according to the invention is an adjuvated water-in-oil-emulsion, is administered at least two times at least one week prior to onset of lay, is administered both to female and to male poultry, and the poultry is chicken.

The advantageous medical effects of the invention can also be described using different wording.

Therefore in a further aspect the invention relates to the use of an avian Reovirus for the manufacture of a vaccine for increasing the hatchability of poultry eggs, wherein the vaccine is an inactivated avian Reovirus vaccine, and the vaccine is administered at least two times to the poultry, prior to onset of lay. In an embodiment of the use of an avian Reovirus for the manufacture of a vaccine for increasing the hatchability of poultry eggs, one or more or all of the conditions apply selected from the group consisting of:

the increase in hatchability results from a reduction in poultry embryo mortality,

the increase in hatchability results from an increased fertility of the poultry,

- the vaccine is an adjuvated water-in-oil-emulsion,

the adjuvated water-in-oil-emulsion vaccine comprises a mineral oil or an ethyl-oleate, the vaccine is administered at least two times and at least one week prior to onset of lay, the vaccine is administered both to female and to male poultry,

the poultry is chicken,

- the vaccine comprises inactivated avian Reoviruses from two or more sub-classifications as defined herein,

the regression formula used for the statistical assessment of the effect of the invention as a whole, and the constituting independent variables have a p value of 0.05 or less, and the inactivated avian Reovirus vaccine for use in the invention is Nobilis® Reo Inac.

In an embodiment of the use for the manufacture according to the invention, the vaccine is an adjuvated water-in-oil-emulsion, is administered at least two times at least one week prior to onset of lay, is administered both to female and to male poultry, and the poultry is chicken. In a preferred embodiment of the use for the manufacture according to the invention, the vaccine is Nobilis® Reo Inac.

The manufacture of the different vaccines according to the invention is well-known in the art, and is within the routine capabilities of the skilled person. Such methods of manufacture will in general comprise steps for the propagation of avian Reovirus, e.g. in an in vitro cell-culture; inactivating the virus, e.g. with formalin; admixing and formulation of inactivated avian Reovirus antigenic material with pharmaceutically acceptable excipients; followed by apportionment into appropriate sized containers. The various stages of the manufacturing process will need to be monitored by adequate tests, for instance by immunological tests for the quality and quantity of the antigens; by micro-biological tests for inactivation, sterility and absence of extraneous agents; and ultimately by in vitro or in vivo experiments to determine vaccine efficacy and -safety. All these are well known to a skilled person, and are prescribed in Governmental regulations such as the Pharmacopoeia, and in handbooks such as "Remington: the science and practice of pharmacy" (2000, Lippincot, USA, ISBN: 683306472), and: "Veterinary vaccinology" (P. Pastoret et al. ed., 1997, Elsevier, Amsterdam, ISBN 0444819681 ).

A pharmaceutically acceptable carrier is for example a liquid such as water, physiological salt solution, or phosphate buffered saline solutions. In a more complex form the carrier can e.g. be a buffer comprising further additives, such as stabilisers or preservatives.

A vaccine for the invention is manufactured into a form that is suitable for administration to an avian target, and that matches with the desired route of application, and with the desired effect.

The vaccine is formulated as an injectable liquid, such as: a suspension, solution, dispersion, or emulsion. Commonly such vaccines are prepared sterile.

In an embodiment of a use for manufacture according to the invention, the vaccination regimen for the administration of an inactivated avian Reovirus vaccine for the invention is integrated into existing vaccination schedules of other vaccines that the target poultry may require, in order to reduce stress to the animals and to reduce labour costs. These other vaccines can be administered in a simultaneous, concurrent or sequential fashion, in a manner compatible with their registered use.

In a further embodiment the inactivated avian Reovirus vaccine for the invention can be comprised in physical combination with one or more other poultry vaccines, provided it does not significantly interfere with the advantageous effects of such Reovirus vaccination within the context of the present invention. For example the inactivated avian Reovirus vaccine for the invention can be added to the composition, dilution, or emulsion of another poultry vaccine. Alternatively, one or more other vaccine or -vaccine component(s) may be added to an inactivated avian Reovirus vaccine for the invention, to create such combination.

Therefore in an embodiment of a use for manufacture according to the invention, the vaccine is administered in a combination with another poultry vaccine. In a further aspect, the invention relates to a method for increasing the hatchability of poultry eggs, the method comprising the step of administering an inactivated avian Reovirus vaccine at least two times to the poultry, prior to onset of lay. In an embodiment of the method for increasing the hatchability of poultry eggs according to the invention, one or more or all of the conditions apply selected from the group consisting of:

the vaccine is an adjuvated water-in-oil-emulsion,

- the adjuvated water-in-oil-emulsion vaccine comprises a mineral oil or an ethyl-oleate,

the vaccine is administered at least two times and at least one week prior to onset of lay, the vaccine is administered both to female and to male poultry,

the poultry is chicken,

the vaccine comprises inactivated avian Reoviruses from two or more sub-classifications as defined herein,

the regression formula used for the statistical assessment of the effect of the invention as a whole, and the constituting independent variables have a p value of 0.05 or less, and

- the inactivated avian Reovirus vaccine for use in the invention is Nobilis® Reo Inac.

Therefore, in an embodiment of the method for increasing the hatchability of poultry eggs according to the invention , the vaccine is an adjuvated water-in-oil-emulsion, is administered at least two times at least one week prior to onset of lay, is administered both to female and to male poultry, and the poultry is chicken.

In a preferred embodiment of the method for increasing the hatchability of poultry eggs according to the invention, the vaccine is Nobilis® Reo Inac. In another aspect the invention relates to the use of an inactivated avian Reovirus vaccine for increasing the hatchability of poultry eggs, wherein the vaccine is administered at least two times to the poultry, prior to onset of lay.

In an embodiment of a use of an inactivated avian Reovirus vaccine for increasing the hatchability of poultry eggs according to the invention, one or more or all of the conditions apply selected from the group consisting of:

the vaccine is an adjuvated water-in-oil-emulsion,

the poultry is chicken,

- the regression formula used for the statistical assessment of the effect of the invention employs an adjusted R² value of at least 0.200,

Therefore, in an embodiment of a use of an inactivated avian Reovirus vaccine for increasing the hatchability of poultry eggs according to the invention, the vaccine is an adjuvated water-in-oil-emulsion, is administered at least two times at least one week prior to onset of lay, is administered both to female and to male poultry, and the poultry is chicken.

In a preferred embodiment of a use of an inactivated avian Reovirus vaccine for increasing the hatchability of poultry eggs according to the invention, the vaccine is Nobilis® Reo Inac.

The invention will now be further described by the following, non-limiting, examples.

Examples

Example 1 : Determination of hatchability The advantageous effect of the invention, the increase in hatchability of poultry breeder eggs, is preferably to be determined as follows:

1.1. Poultry breeder hens are assigned to different treatment groups

Poultry breeder hens are reared and kept under standard optimal management conditions in the business, and receive the recommended vaccinations, except for those for avian Reovirus. The hens in different treatment groups receive either 2 vaccinations with an inactivated avian Reovirus vaccine, or receive another way of Reovirus vaccination, such as a live Reovirus vaccine, given once or multiple times, or a combination of live and inactivated vaccine; provided the inactivated Reovirus vaccine is not given more than once.

Group sizes need to be adeguate to allow analyses and comparisons with sufficient statistical significance of p < 0.05.

1.2. The vaccination schedule is to be completed before the expected onset of lav

For chickens the onset of lay is typically at 20-22 weeks of age. Conseguently, for chickens the avian Reovirus vaccination regime is to be completed before the animals are 20 weeks old.

1.3. Before onset of lav, the hens are kept in the presence of fertile roosters, so they may be fertilised

Male poultry of adeguate number and guality need to be present in the same group, from about 20 weeks of age of the hens, to allow effective fertilisation.

1.4. Once the hens are in full lav, eggs are collected, and stored for a limited time

Eggs are collected and handled with care, and eggs from the different treatment groups are either marked in a non-invasive way, or kept separate, to be able to distinguish them. To maintain good viability, eggs are to be collected at least two times per week, and are stored prior to incubation not more than 14 days under appropriate conditions, preferably at about 16 - 18 °C, and about 75 % RH.

Full lay will be reached at about 4 weeks after onset of lay; for chickens this is at about 26 weeks of For the invention, eggs from hens between 26 and 59 weeks of age are to be used, as is described below.

1.5. Optionally the eggs can be decontaminated

Egg-shell decontamination can be performed at this stage by well known, but non-invasive technigues, such as fumigation. 1.6. Stored eggs are selected for quality and put into incubators to allow growth of the embryo

Quality selection of eggs at this stage incorporates the removal of eggs with soiled, porous, cracked, or malformed shell, or eggs with double yolk; such double yolk eggs are easily recognised for being overly large as compared to the other eggs from the same batch; for example having a more than 25 % greater height, measured from tip to tip.

The number of eggs that are set into incubators at this stage is the number to be used for the calculation of hatchability.

Conditions for incubation that allow growth of the embryo are well known in the art, and comprise for example incubation at about 37 °C and at about 90 % RH, and placing the eggs with the air-chamber upwards, and small-end down. Also the eggs are turned regularly; in practice this means that the tilt angle of the tray-plates holding the eggs is shifted once about every hour, by about 90°, by a mechanism built- into the incubator.

1.7. Incubated eggs may be candled, selected, placed in hatching trays and incubation is continued

Candling is a well-known method to assess incubated poultry eggs, whereby the eggs are inspected over a bright light. This allows selecting out eggs with healthy growing embryos from eggs with dead embryos or eggs which were not fertilised. Eggs with healthy embryos are returned to incubation.

Candling and selection for dead/infertile eggs can be done at any time after about the first 1 -third of the incubation has passed; for chicken eggs this would be at about 6-8 days of incubation. Candling can be done using a variety of eguipment, even fully automated candling- and selection machines are available.

The candled and selected eggs are placed in hatching trays to allow the chicks room to hatch. The trays are returned to incubators; no turning is reguired anymore.

For the invention, the steps of candling, selection and moving of the eggs to hatching trays is performed in one operation, at (for chicken eggs) about day 18 of embryo-development.

1.8. Hatched chicks are counted, and hatchability is calculated

The number of chicks that finally hatch (for chickens: at day 21 of e.d.) are counted on the day of hatch.

The number of chicks that hatched is then divided by the number of eggs that were initially set in the incubator (after the initial guality selection). The ratio yields the hatchability number, which is conveniently presented as a percentage. This percentage is calculated for each group of breeders tested. When the hatchability is compared between groups of breeders that differed in age, regression analysis is necessary to calculate the effect of the vaccination regime, over the effect of age.

1.9. Method of calculation

In order to observe the advantageous effect of the avian Reovirus vaccination regime according to the invention on the hatchability of poultry breeder eggs, the birds are preferably kept under field conditions. Also it is necessary to consider data from an entire laying period, as hatchability varies with the age of the hen. Therefore hatchability data from breeder hens with different ages cannot be directly compared, unless by setting a regression formula incorporating 'age of the breeder hens' as an independent variable. In order to be able to estimate the effect of the vaccination regime, 'vaccination regime' has to be used as an independent variable as well. Elements to be considered while developing an appropriate regression formula are:

To aim for the highest possible explanatory potential of the formula. This can be evaluated on the basis of the adjusted R² value, which can vary from 0 (no explanatory potential at all) tot 1 (explanatory potential of 100%), but values above about 0.2 already have credibility. In order to achieve this, it may be necessary to transform the independent variable 'age of the breeder hens in weeks' to an exponential function, e.g. such as 'age of the breeder hens in weeks' to the 6th power.

The p-value for the obtained regression formula has to be 0.05 or less, and

The p-value for each of the constituting independent variables within the formula has to be 0.05 or less.

The difference in hatchability between breeders vaccinated with the 2x inac regime versus alternative vaccination regimes, corrected for age of the birds, can be read from the regression formula, as the coefficient modifying the independent variable 'vaccination regime'.

Example 2: Testing the effect of avian Reovirus vaccination regime on hatchability of chicken breeder eggs.

Ross breeder flocks from one large Belgian breeder chicken operation that hatched in 2013 were reared for 18-20 weeks on 8 different farms in subseguent rounds and then divided over 14 production farms. The various farms were run using similar management conditions, and in all breeder flocks a standard vaccination schedule was used, except for the avian Reovirus vaccines. Breeder chickens on 7 of the 14 farms received one dose of live Nobilis® Reo 1 133 vaccine (MSD AH) at 8 weeks of age, and one dose of inactivated Nobilis® Reo Inac vaccine (MSD AH) at 18 weeks of age. The other 7 farms were vaccinated with 2 subseguent doses of the inactivated avian Reovirus vaccine only, at similar time points.

Eggs were collected, selected and incubated as described below. The day-old chicks that hatched from the eggs of these breeders were counted, and hatchability scores for each of the groups were determined. To compare them, and determine an overall effect of the avian Reovirus vaccination regime, a regression formula was built, that showed an adjusted R² value of 0.662 reflecting a high explanatory potential. All statistical analyses were performed using SPSS Statistics 23 (IBM) at a significance level of 0.05.

For the regression formula using 2x inac as the independent variable for 'vaccination regime', the result was:

% hatchability = 86.288 - 52.753 x 10 1 x (age of breeder hens in weeks)^A6 + 3.348 x (2x inac regime).

This indicated that -irrespective of age- breeders that received the 2x inac avian Reovirus vaccination regime had a 3.348 % higher hatchability, corresponding to an increased production of 4-5 chicks per breeder hen over a laying period.

Claims

1. Inactivated avian Reovirus vaccine for use in increasing the hatchability of poultry eggs, wherein the vaccine is administered at least two times to the poultry, prior to onset of lay.

2. The inactivated avian Reovirus vaccine for use according to claim 1 , wherein the increase in

hatchability results from a reduction in poultry embryo mortality.

3. The inactivated avian Reovirus vaccine for use according to claims 1 or 2, wherein the increase in hatchability results from an increased fertility of the poultry.

4. The inactivated avian Reovirus vaccine for use according to any one of claims 1 - 3, wherein the vaccine is administered both to female and to male poultry.

5. The inactivated avian Reovirus vaccine for use according to any one of claims 1 - 4, wherein the vaccine is an adjuvated water-in-oil-emulsion, and the vaccine is administered at least two times at least one week prior to onset of lay.

6. Use of an avian Reovirus for the manufacture of a vaccine for increasing the hatchability of poultry eggs, wherein the vaccine is an inactivated avian Reovirus vaccine, and the vaccine is administered at least two times to the poultry, prior to onset of lay.

7. Use according to claim 6, wherein the vaccine is an adjuvated water-in-oil-emulsion, the vaccine is administered at least two times at least one week prior to onset of lay, and the vaccine is administered both to female and to male poultry.

8. Method for increasing the hatchability of poultry eggs, the method comprising the step of

administering an inactivated avian Reovirus vaccine at least two times to the poultry, prior to onset of lay.

9. Method according to claim 8, wherein the vaccine is an adjuvated water-in-oil-emulsion, the vaccine is administered at least two times at least one week prior to onset of lay, and the vaccine is administered both to female and to male poultry.

10. Use of an inactivated avian Reovirus vaccine for increasing the hatchability of poultry eggs, wherein the vaccine is administered at least two times to the poultry, prior to onset of lay.

1 1. Use according to claim 10, wherein the vaccine is an adjuvated water-in-oil-emulsion, the vaccine is administered at least two times at least one week prior to onset of lay, and the vaccine is administered both to female and to male poultry.