Method to sporulate coccidial oocysts purified from animal faeces, sporulated oocysts obtained with this method and a vaccine containing these sporulated oocysts

Abstract

The invention pertains to a method to sporulate coccidial oocysts purified from animal faeces (5), the method comprising providing the purified oocysts as a layer (40) on a supporting surface (22), maintaining the layer at least intermittently in an oxygen containing gaseous environment (60) having a relative humidity of at least 15% and maintaining the temperature of the oocysts between 19 C. and 37 C. The invention also pertains to sporulated oocysts obtained with this method and to a vaccine containing such sporulated oocysts.

Claims

1. A method to sporulate coccidial oocysts purified from animal faeces, the method comprising providing the purified oocysts as a layer on a supporting surface, maintaining the layer at least intermittently in an oxygen containing gaseous environment having a relative humidity of at least 15% and maintaining the temperature of the oocysts between 19 C. and 37 C.

2. The method of claim 1, wherein the thickness of the layer is below 10 mm.

3. The method of claim 1, wherein the supporting surface is a perforated deck that is able to let water pass, but does not let the oocysts pass.

4. The method of claim 3, wherein the supporting surface has mesh openings around a minimum dimension of the oocysts.

5. The method of claim 3, wherein the supporting surface has mesh openings around 10 m.

6. The method of claim 1, wherein the supporting surface is in the form of a perforated deck that is drum shaped, and wherein the layer of oocysts is provided on the internal surface of the drum-shaped deck.

7. The method of claim 6, wherein the drum-shaped deck is placed partly in a volume of water and partly in the gaseous environment, mounted with its longitudinal axis extending in parallel with a surface of this volume of water, wherein the deck is rotated for maintaining the layer at least intermittently in the oxygen containing gaseous environment, and wherein the drum-shaped deck is rotated around its longitudinal axis.

8. The method of claim 7, wherein the drum-shaped deck is rotated at a circumferential speed of 5-20 rpms.

9. The method of claim 7, wherein the volume of water is kept at a temperature between 19 C. and 37 C.

10. The method of claim 7, wherein the volume of water is kept at a temperature of about 28 C.

11. The method of claim 1, wherein after sporulation of the oocysts, a further aqueous medium containing an anti-infective is provided to the layer to remove contamination by a sieving step, whereafter the layer is washed to remove the anti-infective.

12. The method of claim 1, wherein the supporting surface is a sieve deck having an effective mesh opening around the minimum dimension of the oocysts, wherein a thin layer is provided by collecting faeces of animals containing the coccidial oocysts, diluting the faeces in an aqueous medium wherein separating a coarse fraction comprising macroscopic particulate matter from the diluted faeces is optional, and collecting an aqueous fraction containing the oocysts, sieving the aqueous fraction over a first sieve deck other than the said supporting surface, the first sieve deck having mesh openings to let the oocysts pass in order to obtain an aqueous filtrate comprising the oocysts and a residue on the first sieve deck comprising particles larger than the oocysts, and then sieving the aqueous filtrate over the said supporting surface.

Description

EXAMPLES

(1) FIG. 1 diagrammatically shows a system for collecting oocysts for sporulation.

(2) FIG. 2 schematically shows an embodiment of a system according to the invention.

(3) FIG. 3 schematically shows an embodiment of a sieve deck for use in a method or system according to the invention.

(4) FIG. 4 schematically shows another embodiment of a system according to the invention.

(5) FIG. 5 schematically shows a sieve deck for use as a support to let purified oocysts sporulate.

(6) Example 1 describes process data regarding a method according to the invention.

(7) FIG. 1

(8) FIG. 1 diagrammatically shows a system for collecting animal faeces containing coccidial oocysts from host animals, and separating a coarse fraction comprising macroscopic particulate matter from the faeces and collecting a fraction containing the oocysts for further purification. In general, a number of different methods of preparing oocysts for further purification are known in the art. Any one or combination of such methods may be used prior to further purification. A preferred method is set out below.

(9) To begin, once host animals (typically chickens) begin shedding the organism, the oocysts can be collected. Most commonly, the chickens are kept in cages (1), and are fed solid food (2) and water (3). Faeces 5 are collected from the cages, and a waste stream containing other material (feathers, straw etc) is discarded. Once collected, the faeces are brought over to a slurry tank 6 and mixed with added water (7). The resulting diluted fecal material is provided to a sieve 9 for removal of the coarse material in the faeces such as stones, remains of shavings, grid, remains of animal feed etc. For this, the sieve comprises two consecutive plate sieves, the upstream sieve having mesh openings of 2 mm, and the downstream sieve having mesh openings of 125 m. The resulting residues (11) are discarded, and the filtrate is collected as an aqueous fraction 10 containing the oocysts.

(10) FIG. 2

(11) FIG. 2 schematically shows an embodiment of a system 20 according to the invention. In this embodiment the system comprises a longitudinal, tube-like housing 23 having two internal sieve decks, viz. an upstream sieve deck 21 and a downstream sieve deck 22. In this embodiment the sieve decks are made of woven stainless steel wires, according to a plain weaving pattern. The aqueous fraction 10 (see FIG. 1) is provided to the top of sieve deck 21 in order to sieve this fraction. This deck has mesh openings such that the oocysts pass to obtain an aqueous filtrate 32 comprising the oocysts, and a first residue 31 comprising particles larger than the oocysts. The aqueous filtrate 32 is provided to the top of second sieve deck 22, which sieve deck has mesh openings to obstruct passing of the oocysts through this sieve deck. This way a second residue 40 comprising the purified oocysts and a waist filtrate 42 comprising particles smaller than the oocysts is obtained.

(12) The size of the mesh openings should be chosen to effective collect oocysts of the desired shape. For example, to collect oocysts of a size range between 15 and 25 m, the first sieve deck may have mesh openings of 25 m, and the second sieve deck may have mesh openings of about 14 m. In this case, since the mesh openings correspond almost exactly with the size of oocysts, a lot of additional water may be needed (provided as a separate feed to the top of sieve deck 21) to actually have the oocysts pass the first sieve deck. In another set-up, for example to collect oocysts of a size range between 20 and 30 m, the first sieve deck may have mesh openings of 40 m, and the second sieve deck may have mesh openings of about 15 m. In yet another set-up, for example to collect oocysts of a size range between 10 and 40 m, the first sieve deck may have mesh openings of 42 m, and the second sieve deck may have mesh openings of about 10 m. In still another embodiment, for example to collect oocysts of a size range between 12 and 48 m, the first sieve deck may have mesh openings of 50 m, and the second sieve deck may have mesh openings of about 10 m.

(13) FIG. 3

(14) FIG. 3 schematically shows an embodiment of a sieve deck 21 (mesh openings 50 m) for use in a method or system according to the invention. In this embodiment, the sieve deck is an endless deck in the form of a drum. The drum is rotatably supported on axis 25 and is internally provided with a stationary container 26. In use, the aqueous fraction is loaded on the inside of this drum, below the container 26, and the drum is rotated while sieving the aqueous fraction. Also, during this sieving action additional aqueous medium having a temperature of about 28 C. is added to the inside of the drum 21. The aqueous filtrate is collected in container 27. Above the drum is situated a row of spray heads 28. These heads can be used to add water to the drum, either to serve as a lubricant whilst sieving, or, after the sieving has ended, to release the residue from the drum and collect it in the container 26. This container can be removed by sliding it over axis 25.

(15) FIG. 4

(16) FIG. 4 schematically shows another embodiment of a system 20 according to the invention, in which embodiment a semi continuous process for applying the method according to the invention can be run. This system comprises a rotating sieve 91 (having mesh openings of 150 m), which is surrounded by housing 9. This sieve and housing correspond to item 9 in FIG. 1. To this sieve 91 an aqueous fraction 8, comprising diluted chicken faeces (see FIG. 1, albeit that this fraction is optionally pre-sieved over a 2 mm sieve), is fed for separating the coarse fraction comprising macroscopic particulate matter from the diluted faeces. The aqueous fraction 10 containing the oocysts is collected and fed to drum-shaped rotated sieve 21 (see also FIG. 3) as present in housing 23. As described in conjunction with FIG. 3, this sieve deck has mesh openings of 50 m and separates the aqueous fraction in an aqueous filtrate 11 comprising the oocysts and a first residue (not shown), comprising particles larger than the oocysts. This residue can be removed as described here above. The aqueous filtrate 11 is fed to rotating sieve 22, which sieve is housed in housing 23. This sieve 22 has mesh openings of 10 m to obstruct passing of the oocysts through this sieve deck, such that a residue comprising the purified oocysts is build up on the internal side of this drum-shaped sieve deck 22. The waist filtrate 12 comprises particles smaller than the oocysts, such as any bacteria.

(17) FIG. 5

(18) FIG. 5 schematically shows a sieve deck for use as a support to let purified oocysts sporulate. In this embodiment, the residue 40 is build up as a 2.5-3.5 mm layer on the inside of sieve deck 22 (having mesh openings of 10 m). The drum-shaped sieve deck 22 is placed partly in a volume of water (50; kept at a temperature of 28 C.) and partly in the gaseous environment 60, such that about 20% of the total circumference of the drum is below the level of the volume of water. The drum is mounted with its longitudinal axis 25 extending in parallel with the surface of this volume of water. During sporulating the drum is rotated for maintaining the layer 40 intermittently in the oxygen containing gaseous environment 60. The drum is revolved at 10-12 rpm through the water 50. The relative humidity of the gaseous environment in the drum is 100%. It was found that this way the oocysts can sporulate (almost all) within 48 hours.

Example 1

(19) Example 1 describes process data regarding a method according to the invention using the system of FIG. 4. In this system small drum shaped decks are used having a length of 40 cm and a drum diameter of 80 cm (drums having a length of up to 2.80 meters and a diameter of up to 2.0 meters can be advantageously used in the set-up of FIG. 4). The decks have meshes of stainless steel wires woven according to a plain weave, with mesh openings as described in conjunction with FIG. 4. The drums rotate at 10-12 revelations per minute.

(20) The faeces of 60 white leghorn chickens (infected with Eimeria) aged 26-31 days was collected (approximately 25 grams of faeces per chicken per day), mixed with 200 liters of water, and the coarse fraction was separated using a 2 mm sieve. Approximately 50 liters of this mixture (containing about 2.25 kg of faeces) was loaded into the system, wherein during sieving about 5-10 liters of water per minute was added to sieve decks 21 and 22. This resulted in about 120 grams of purified oocysts (a composition containing an estimated amount of about 85 grams of non oocysts faecal particles, typically fine sand grains, silt and clay particles, and about 35 grams of oocysts) on sieve deck 22 after 35 minutes of sieving, at a calculated yield of approximately 81% for Eimeria acervulina and approximately 100% for Eimeria maxima. Using the traditional method of flotation and centrifugation, this takes about 6 hours, with typical yields of about 50-60% for both species.

(21) Optionally, depending on the amount of contamination still present an additional washing step may be performed by mixing the residue in a 6% hypochlorite (anti-infective) solution and load it into drum 22. Water is continuously added at about 5-10 liters per minute to remove the hypochlorite, and after 15 minutes the residue is ready for further processing.

(22) After sporulating as described in conjunction with FIG. 5, typical sporulating rates are 85% for Eimeria acervulina and 90% for Eimeria maxima (cf typical values of 40% to a maximum of 80% for the traditional process using potassium dichromate). These sporulated oocysts can serve as antigen in a coccidiosis vaccine as known in the prior art.