Coccidiosis vaccine

Abstract

The present invention relates to an improved vaccine against Coccidiosis in poultry. The vaccine comprises live Eimeria oocysts in a composition with relatively high viscosity because of the inclusion of between 0.3 and 1.5% w/v Xanthan gum. Upon spray vaccination the new vaccine forms beads that are quickly ingested by the birds and which provide a rapid vaccine take, resulting in a quick and effective improvement of the signs of Coccidiosis upon challenge infection. Also the invention relates to a method for the heat sterilisation of a composition comprising Xanthan gum; by the inclusion of a concentration of a metal salt, an irreversible and dramatic reduction in viscosity can be largely prevented.

Claims

1. A method of vaccinating poultry against Coccidiosis, wherein the method comprises administering to poultry a Coccidiosis vaccine via a body spray; wherein the Coccidiosis vaccine comprises live oocysts of at least one species of Eimeria in a pharmaceutically acceptable carrier; wherein the vaccine comprises 0.3-1.5% w/v Xanthan gum that has been heat-sterilized in the presence of a concentration of a metal salt of at least about 0.1% w/v; wherein the Coccidiosis vaccine comprises less than about 0.4% w/v metal salt; and wherein the Coccidiosis vaccine has a viscosity of between 200 and 4000 mPa.Math.s.

2. The method of vaccinating poultry against Coccidiosis of claim 1, wherein the body spray is applied as a coarse spray.

3. The method of vaccinating poultry against Coccidiosis of claim 1, wherein the at least one species of Eimeria is selected from the group consisting of: E. acervulina, E. tenella, E. maxima, E. brunetti, E. mitis, E. praecox, E. mivati, E. hagani, E. necatrix, and any combination thereof.

4. The method of vaccinating poultry against Coccidiosis of claim 3, wherein the body spray is applied as a coarse spray.

Description

EXAMPLES

Example 1: Comparison of Viscosity of Xanthan Gum Compositions

(1) The viscosity of a solution of Xanthan gum in water was determined under different conditions, testing the effect of heat sterilisation with or without the inclusion of a concentration of metal salt.

(2) Some of the samples were autoclaved, by incubation for 30 minutes at 115° C.

(3) Viscosity measurements were performed with a Brookfield™ rotational viscometer according to the manufacturer's instructions. This equipment determines the viscosity of a liquid from the torque on a spindle, rotating at a defined number of rotations per minute, in a defined volume of that liquid. All samples were measured while equilibrated in a water bath at 25° C.

(4) TABLE-US-00001 TABLE 1 Viscosity of a solution of Xanthan gum in water % w/v Xanthan Pre- Viscosity gum treatment Additive (mPa .Math. s) 0.6 autoclaved none 25.5 0.6 autoclaved 0.15% w/v NaCl 593 0.6 autoclaved 0.15% w/v NaCl and 0.1% Carmine 586 0.6 none 0.15% w/v NaCl and 0.2% Carmine 660 2.4 autoclaved 0.15% w/v NaCl 8061

(5) These results demonstrate that the addition of a specific concentration of metal salt can almost completely prevent the otherwise dramatic decrease in viscosity that occurs upon heat sterilisation of a Xanthan gum dilution in water. The difference in viscosity between the samples autoclaved+salt and not-autoclaved+salt (here: about 11%), is considered to be non-severe.

Example 2: Effect of Xanthan Gum on Efficacy of a Multi-Species Live Coccidial Eimeria Vaccine

(6) 2.1. Introduction:

(7) To improve the delivery of a day-old coarse body spray administration of live Coccidiosis vaccines, the inventor tested a Xanthan gum vaccine formulation, next to a more traditional vaccine wherein the coccidia were diluted in water. It was tested if the vaccine would ‘bead’ on the birds, increasing the visibility of the vaccine, and leading to better vaccine ingestion and minimal wetting of the birds.

(8) 2.2. Experimental Design

(9) One hundred and twenty, one day-old chickens were divided into three groups of 40. All birds in group 1 were vaccinated with coccidia corresponding to those of the Paracox™ 8 vaccine, in water; the birds in group 2 were vaccinated with the same type of coccidia but in 0.6% w/v Xanthan Gum/0.2% w/v Nacl, and with 0.1% w/v Carmine (E120); both by coarse spray. Birds in group 3 were not vaccinated (see Table 2). All birds were kept in groups in separate floor pens for 21 days post vaccination. For the monitoring of (vaccinal) oocyst shedding, faecal samples were collected from the floor pens: for the vaccinated birds (groups 1 and 2) daily from day 4 to day 14 and on day 20 post vaccination (pv); for the control birds (group 3) on days 7, 14 and 20.

(10) On day 21 pv, thirty birds per group were uniquely identified by numbered tag, then each bird was challenged with 15,000 oocysts of a virulent E. tenella wild type strain. Next the challenged birds were transferred to collection cages (3 cages of 10 birds per group) in containment rooms. The remaining birds in groups 1 & 2 were euthanized and discarded. The remaining birds in group 3 were left in the floor pen to be weighed on days 21, 28 and 35. Two birds were not vaccinated and not challenged, to allow detection of any non-experimental infection.

(11) On day 5 post challenge (pc), in total 10 challenged birds (3 from each cage of the first two groups, and 4 from the third group) were euthanized and examined post mortem (without knowing the treatment group) for lesions associated with an E. tenella infection.

(12) On day 7 pc all remaining birds were weighed and a bulked faecal collection was taken from all cages, for enumeration of oocyst output. On day 14 pc all birds were weighed before being euthanized.

(13) Throughout the study all birds were observed daily for clinical signs related to an Eimeria infection, such as diarrhoea or bloody faeces, depression, or inappetance; no non-experimental infections were observed.

(14) 2.3. Materials and Methods

(15) 2.3.1. Test Animals

(16) Chickens used were SPF, of mixed sex, and one day old at the start of the experiment. All birds were checked for being of apparent good health and size before vaccination. The birds were allocated to the groups in a randomised way, on a first caught basis. Tap water and standard feed were available ad libitum. Challenge inoculation was by oral gavage.

(17) 2.3.2. Test Materials

(18) Vaccine coccidia (as in Paracox 8) and challenge materials (sterile, sporulated oocysts of E. tenella) were kept at 2-8° C. until use, and were well within their expiry date at the time of the experiment.

(19) The Paracox 8 vaccine coccidia were diluted to provide one animal dose in 0.21 ml; the vaccine for groups 1 and 2 were made up to contain 3,000 doses. The diluents were held in separate sterile glass Duran bottles either 315 ml of sterile water for group 1, or for group 2: 315 ml of (heat sterilised) 1.2% w/v Xanthan Gum (Xantural 11K, CP Kelco) and 0.4% w/v NaCl, both containing 0.2% w/v Carmine. Two, 5000 dose sachets were shaken and massaged vigorously for 1 minute to ensure re-suspension of the oocysts. The contents of these sachets were pooled. From the pooled vaccine 315 ml was taken and added to each of the diluents. Both formulations were then thoroughly mixed using a flea and magnetic stirrer for group 1, or shaken by hand for group 2, for at least 10 minutes prior to use.

(20) The concentration of the oocysts in the challenge material was determined using a modified Fuchs-Rosenthal counting chamber. From this a dilution was made to provide 15.000 oocysts per animal dose of 0.5 ml.

(21) 2.3.3. Spray Vaccination

(22) A Spraycox™ II machine was used following the manufacturer's instructions, to apply a coarse spray vaccine to the chicken hatchlings. As the machine is designed to vaccinate 100 chicks per tray, for the 40 chicks per groups used here, the tray area was reduced by 60%. A paper tray liner was used to provide a good surface which allowed the birds to move freely and preen themselves and each other.

(23) The target was to administer 0.21 ml of vaccine per bird, therefore 21 ml of vaccine was applied per tray. Prior to vaccination, the Spraycox machine's settings were calibrated by several mock runs to ensure that the correct dose (21 ml) was dispensed over the total internal surface area of the tray before the vaccination of the groups in the experiment. The prepared vaccines were shaken for 10 minutes prior to being transferred to the Spraycox machine, to ensure thorough mixing of the vaccine. Once vaccinated, birds were kept for at least 30 minutes in the trays in which they were vaccinated, in a well-lit and warm area before being transferred to the floor pens. The birds were monitored during this time to see how quickly the vaccine was taken up. Between each vaccination, the spray machine's settings were re-confirmed, and the machine was thoroughly rinsed out with warm water. The groups were vaccinated in order i.e. group 1 before 2.

(24) 2.3.4. Faecal Samples

(25) Post challenge, bulked faecal samples were collected from challenged cages between days 4 and 7 post challenge. The sample from each cage was separately collected on plastic sheets to determine the total oocyst output after challenge. Faeces were sprayed with water at least daily to ensure that they were kept moist. Each sample was double bagged, labelled to identify the contents, and stored at 2-8° C. until processing. The oocysts were recovered as per standard practices and the numbers calculated using the McMaster counting technique.

(26) 2.3.5. Data Analysis

(27) Data on the lesion scores, bodyweight gain, and oocyst output from pens of vaccinated challenged birds (groups 1 and 2) were compared with each other and with the control group (unvaccinated-challenged-group 3), using statistical tests. All analyses were performed by a statistician using SAS™ 9.3 with a significance level of 5%.

(28) 2.4. Results

(29) 2.4.1. Lesion Scores

(30) Lesions associated with E. tenella were determined by post mortem examination at day 5 pc. Results are summarised per group (n=10) in Table 2. Scoring was performed as described in the European Pharmacopoeia monograph 2326. In short: a score of 0 means no gross lesions, and scores of 1-4 mean increasing severity of thickening of the intestinal wall, increased amounts of blood, and decreased amounts of normal faeces. At a lesion score of 4 there can even be some mortality.

(31) The results show that the lesion scores in groups 1 and 3 were significantly higher than those in group 2 (P<0.0001), only overlapping at a score of 2. Although the birds in groups 1 and 3 only had high scores (2 or greater) the scores for the birds in group 1 were generally lower than the scores of the birds in group 3, however these are not significantly different to each other (P=0.6917).

(32) TABLE-US-00002 TABLE 2 Effect of vaccinations and challenge on lesion scores Lesion score (number of birds) Group - feature 0 1 2 3 4 1 - vaccine in water — — 1 6 3 2 - vaccine in Xanthan gum 3 5 2 — — 3 - unvaccinated — — 2 3 5

(33) 2.4.2. Bodyweight Gain

(34) Individual bodyweights were recorded on day 21 prior to challenge, and on days 7 and 14 pc, before euthanasia. A summary of weight measurement results per group (n=20) is presented in Table 3.

(35) The data shows that the birds in both of the vaccinated groups (1&2) gained significantly more weight than those in group 3, at 7 days pc (P<0.001—both groups) and at 14 days pc (P<0.001—both groups). With group 2 also gaining significantly more weight than group 1 at both time points (challenge+7 days: P=0.017; challenge+14 days: P=0.034).

(36) TABLE-US-00003 TABLE 3 Effect of vaccinations and challenge on bodyweight Average Average bodyweight (g) bodyweight gain (g) Group - feature d. 21 pv d. 7 pc d. 14 pc d. 0-7 pc d. 0-14 pc 1 - vaccine in water 191.23 274.43 375.45 83.20 184.22 2 - vaccine in 203.37 300.80 406.75 97.43 203.38 Xanthan gum 3 - unvaccinated 176.74 208.49 312.35 31.75 135.62

(37) 2.4.3. Oocyst Output

(38) The amounts of oocysts detected in the faecal collections are summarised in Tables 4 (taken post vaccination) and 5 (post challenge). Post vaccination there is good oocyst output showing that the vaccination was successful. Also the patterns of the oocyst output are as expected: an initial peak in oocyst output, directly resulting from the vaccination, followed by a drop in output, followed by a second peak, six days after the first, as the oocysts go through their second cycle. In group 2 the initial peak in oocyst output, although smaller than that in group 1, is earlier by three days and results in a larger second peak.

(39) Because immunity to coccidial Eimeria vaccines is strongly linked to oocyst cycling, therefore the birds in group 2 (vaccine in Xanthan gum) had a stronger immunity at the time of challenge infection, compared to group 1 (vaccine in water). The improved initial ingestion of the vaccine, and the reduced gut-transit time for the Xanthan gum containing vaccine, thus leads to a better and earlier development of immunity against Coccidiosis.

(40) TABLE-US-00004 TABLE 4 Effect of vaccination on oocyst output post vaccination. Number of oocysts/gram faeces (×10{circumflex over ( )}3) at X days pv Group - feature 4 5 6 7 8 9 10 11 12 13 14 20 1 - vaccine in water 0 0 0 2 5 0 1 0.5 1 1.5 12 5.5 2 - vaccine in Xanthan gum 0 3 0 0 0 4 5 33 8 15 1 3 3 - unvaccinated 0 0 0 0 0 0 0 0 0 0 0 0 Post challenge, there are clear numerical differences in the oocyst output between the groups, these differences are statistically significant with group 2 shedding significantly less oocysts than the other two groups (P < 0.0001). There is no statistically significant difference between the shedding by groups 1 and 3 (P = 0.0651).

(41) TABLE-US-00005 TABLE 5 Effect of vaccination and challenge on oocyst output post challenge. Group - feature Number of oocysts/gram faeces (×10{circumflex over ( )}6) 1 - vaccine in water 23.0 2 - vaccine in Xanthan gum 0.43 3 - unvaccinated 35.9
2.5. Conclusions

(42) The data support the conclusion that the use of Xanthan gum significantly improves the efficacy of an existing multi-species live coccidial Eimeria vaccine. The vaccine delivered in the Xanthan gum formulation provided greater immunity to the birds against challenge at 21 days post vaccination, as compared to the same vaccine but delivered in water. All of the three parameters used to assess vaccination efficacy: lesion scores, increase in bodyweight gain, and reduction of oocyst output, were significantly improved for the group receiving the vaccine in Xanthan gum, as compared to the traditional vaccine in water group and to the unvaccinated control group.

(43) While the vaccine had been prepared using heat-sterilised Xanthan gum, this had been provided with a concentration of metal salt so as to stabilise it during the heating.

(44) The strongly improved vaccine efficacy is due to an improved ingestion of vaccine, delivered in the Xanthan gum, demonstrated by the earlier output of vaccinal oocysts and improved re-cycling. This lead to an impressive reduction in lesion scores and oocyst output, as well as to an economically highly relevant increase in bodyweight gain post challenge.

Example 3: Improved Vaccine Take

(45) In an experiment of smaller scale, but otherwise highly similar in design and performance to the experiment described in Example 2, the efficacy of the vaccine take of a Coccidiosis vaccine according to the invention was compared to a standard vaccine without Xanthan gum.

(46) For such an experiment, ‘vaccine take’ is determined at 5-7 days post vaccination, at post mortem, by a microscopic examination of scrapings from the intestinal-wall. To detect the various species of Eimeria, different regions of the gut need to be monitored.

(47) When comparing a Coccidiosis vaccine according to the invention using 0.6% w/v Xanthan gum, and a vaccine based on Paracox 8 (i.e. coccidia in water), an improvement of the vaccine take was observed: from about 40-60% take for the coccidia in water, up to almost 80% take for the coccidia in Xanthan gum.

Example 4: Effect of Different Spray Formulations

(48) In early experiments, different gel-formulations for the delivery of coarse spray administration of live coccidiosis vaccines to day-old chicks were tested. The experiments compared solutions comprising either 3% polyvinylpyrrolidone (PVP) of type K-90, or 0.6% Xanthan gum in water. The negative control had the coccidia in plain water. All three formulations contained Carmine colorant (E120) at a final concentration of 0.1% w/v. The formulations were not heat-sterilised, consequently no salt was added.

(49) 4.1. Experimental Set-Up

(50) The experimental set-up was highly similar in design and performance to the experiment described in Example 2. In short: 75, day-old SPF chickens were randomly allocated to three groups of 25 birds. Each group was vaccinated with E. tenella by coarse spray, administered at a dose level of 0.21 ml, i.e about 500 coccidia per bird, using a commercial Spraycox II machine. The vaccine coccidia used were of the E. tenella strain as is present in the Paracox product.

(51) Post vaccination the birds were left to preen and take up the vaccine; they were then transferred to isolators at 1 group per isolator, and brooded for 7 days. Birds were observed daily. At 7 days p.v. the birds were euthanased and examined post mortem for the presence of oocysts or developing stages in their gut.

(52) Vaccine uptake was determined by microscopical examination of wet smears from both caecal pouches. Scores were either positive or negative depending on the presence or absence of parasitic stages, see Table 6.

(53) 4.2. Results

(54) The results obtained showed a clear advantage for the formulation with Xanthan gum over the two other formulations tested.

(55) TABLE-US-00006 TABLE 6 Effect of gel-formulation on vaccine uptake: Positive Negative % Vaccine Group birds birds uptake 1: Water 12 10 54.5 2: 0.6% Xanthan gum 17 7 70.8 3: 3% PVP K-90 10 13 43.5
4.3. Conclusions

(56) The uptake of the vaccine, as confirmed through the presence of parasitic stages seen in wet Cecal smears, is affected by the formulation in which it is applied. The use of 0.6% Xanthan gum (group 2) improved the uptake of the vaccine by 30%, namely from 54.5% with the traditional water medium (group 1) to 70.8%. Conversely the use of 3% PVP (group 3) actually reduced the uptake of vaccine by 20%, when compared with the water group, namely from 54.5 to 43.5%.

(57) Consequently, the use of the 0.6% Xanthan gum in the formulation of a spray vaccine of Eimeria oocysts has a clear positive effect on the uptake of oocysts, compared to the use of 3% PVP, or the use of conventional plain water as medium.

(58) Because all formulations tested contained Carmine, this experiment also demonstrated that the positive effect of Xanthan gum is distinctly separate from that of the colorant.

(59) Similarly, because no salt was added, this proves that presence of salt is not required when there is no heat-sterilisation.