NZ530709A - Improved IGA production method - Google Patents

Abstract

Disclosed herein is a method for increasing the production of immunoglobulin A (IgA) in the milk of a ruminant mammal that comprises the following steps: (a) immunising the mammal with an antigen to stimulate secretion of IgA in milk and (b) collecting milk from the mammal on a once-daily basis.

Description

53070 Inteitafttual Property Office of N.Z. 2 \ JAN 2005 j : -r- Jj \f ^ j j PATENTS FORM NO. 5 ^ Fee No. 4: $250.00 PATENTS ACT 1953 COMPLETE SPECIFICATION After Provisional No: 530709 Dated: 21 January 2004 IMPROVED IGA PRODUCTION METHOD We AgResearch Limited, a New Zealand company of East Street, Ruakura Campus, Hamilton, New Zealand, hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed to be particularly described in and by the following statement: 1 IMPROVED IGA PRODUCTION METHOD TECHNICAL FIELD The invention relates to an improved method for producing immunoglobulin A (IgA) in ruminant mammals and associated milk product. More specifically the invention 5 relates to a method for producing milk containing increased amounts of immunoglobulin A (IgA) using a combination of an immunisation protocol and once-daily milking.

BACKGROUND ART Immunoglobulin A (IgA) is a well documented immunoglobulin present in almost all body fluids. It is understood to play a major role in the protection of the host from infection by pathogenic organisms invading via the mucosal surfaces of the respiratory, gastrointestinal and urogenital tracts. IgA participates in the clearance of pathogenic bacterial, viral or parasitical organisms and a variety of ingested or 15 inhaled antigens from the mucosal surfaces by neutralising toxins and viral particles, inhibiting adherence of bacterial pathogens and preventing colonisation and penetration of mucosal surfaces by pathogenic microorganisms. The key role of immunoglobulins, including IgA, in milk therefore is to provide local protective immunity in the gastrointestinal tract of the offspring during the suckling period.

Immunoglobulins have come to be recognised as useful in the pharmaceutical and veterinary fields for treating bacterial or viral infections of the gut, and more generally in the treatment of disease and inflammation. Over the years various techniques for producing immunoglobulins have been proposed. A particularly 25 popular method is for the induction and production of immunoglobulins from 2 ruminant milk. This approach has particular advantages in that the immunoglobulin produced in the milk is in a form suitable for immediate consumption, or may be processed into appropriate formulae or products. It is safe to use and the industry infrastructure for producing milk containing antibodies 5 is already in place.

The immune system of a ruminant appears to differ from its human counterpart in that the immunoglobulin dominant in bovine mammary secretions is immunoglobulin G (IgG). Accordingly, the main focus of antibody production in milk by active immunisation has been on IgG, although theoretically, the preferred 10 immunoglobulin would be IgA for the reasons outlined above.

Some attempts have been made to produce increased levels of IgA in ruminant milk. Proposals for vaccination by a single administration route such as parenteral, subcutaneous, intravenous, systemic, oral, intraperitoneal, intramuscular, intramammary and the like have been suggested. Generally, these routes of 15 administration have resulted in the predominant production of IgG. Systemic immunisation produced both IgA and IgM in milk, but only at low concentrations.

The response was enhanced when intramuscular/sub-cutaneous (IM) and intramammary (IMM) immunisation processes were combined (Am. J. Vet. Res3).

Combinations of intraperitoneal (IP) and intramammary (IMM) infusions have also 20 been shown to produce IgA and IgG, (Immunology5; Res. in Vet.Sci2, Res. in Vet.Sci7, The Ruminant Immune System in Health and Disease6). It is noted that this route leads to limited enhancement of IgA production (The Ruminant Immune System in Health and Disease6). A combination of IM and IMM immunisation gave rise to a predominance of IgG in the milk (Aus. J. Dairy Technology4), as well as 25 increasing generally the levels of IgG, IgA, and immunoglobulin M (IgM) (Am. J. Vet. Res3). Significant variability in the antibody titres produced between animals 3 was also noted.

The predominance of the production of lgG1 is consistent with the findings that IgG's produced are the major immunoglobulins in ruminant mammary secretions. Intramammary immunisation techniques have generally not been preferred as a 5 route for vaccination under field conditions due to the high chance of mammary infection (Aus. J. Dairy Technology4). However, other work suggests that this may not be the case (Am. J. Vet. Res3).

It should be noted that much of the published literature concerning immunoglobulin production in mammary gland secretions is directed to disease prevention in animals or their offspring. Few are directed to the production of immunoglobulin enriched milk for the purposes of obtaining the immunoglobulins themselves.

An exception to this is a process for the production of a protein concentrate containing immunological factors of lactic origin in Swiss Patent No. 1,573,995. Nearly 20 years ago, this patent disclosed a process for producing milk with a high antibody titre, by intracisternal instillation into the mammary gland, parental injection (subcutaneous, intravenous), injection into the retromammary ganglionic system by scarification, by oral ingestion or by a combination of several of these modes. The only specific immunisation protocol for obtaining colostrum and transition milk disclosed involved some 11 immunisation steps over a period of 8 weeks prior to calving. This protocol comprises multiple parental (including intravenous) administration steps, with several IMM administration steps interspersed and requires two oral administration steps in the week prior to calving.

This protocol is not in widespread use today. The immunisation plan is onerous in the number of steps involved and is not in fact optimised for immunoglobulin A 25 production. Indeed, the patent is misleading in suggesting that IgA's are preponderant in ruminant maternal milk; a misconception that may have resulted from the knowledge that IgA is predominant in human milk. As established in other 4 teachings (see for example, Aus. J. Dairy Technology4), IgG is the predominant immunoglobulin produced in the maternal milk in cows.

It has also been shown in the intervening years that oral delivery of antigens results in little or no increase of IgA titres in mammary secretions when compared 5 with non-inoculated controls (Am. J. Vet. R3). It is presumed that the presence of the rumen may preclude the antigen reaching the small intestine. Accordingly, the oral administration step called for by Hilpert is now contraindicated.

Similarly, intravenous injection would not generally be recommended for immunisation purposes because of the possible adverse effects such as 10 anaphylactic shock (ILAR Journal1).

More recently, in the applicant's related patent, US 6,616,927 (the '927 patent), a set of protocols are described that increase the production of IgA in the milk of a ruminant. Whilst the concentration of IgA produced increases using the '927 patent protocols, the absolute yield of IgA produced is still low as the IgA is diluted 15 by normal milk production volumes.

In a further patent application by the applicant, NZ 521969, a process is described for determining the extent of production change in animals milked at a particular frequency, in particular by measuring lactoferrin content. Once-daily milking is considered.

In general, most New Zealand dairy herds are milked twice-daily, routinely early in the morning and again late in the afternoon, primarily as this routine maximises total milk yields. Overseas, milking may occur more frequently still.

Multiple daily milking is however, both labour and resource intensive.

Studies have been conducted into once-daily milking to take advantage of the 25 potential savings benefits in both labour and associated shed costs for milk production. Once-daily milking has not however been considered in terms of alterations in IgA production.

These studies have shown that one universal characteristic of once-daily milking is a reduced milk yield. Another characteristic of once-daily milking is the accelerated 5 regression of the udder, leading to an increased rate of decline of milk yield as lactation progresses. As such, the declines in milk yields from once-daily milking means these practices are not commonly employed in New Zealand for milk operations.

There is currently a need for a process for inducing production of IgA in milk at 10 higher levels and/or yields than have previously been obtained by known antigen administration processes. A process which additionally increases consistency between animals in the production of IgA is also desirable. A commercial process which optimises production of IgA while simplifying the immunisation protocol is also sought. A process that increases the concentration and/or yield of IgA 15 produced is likely to also be of value.

It is an object of the present invention to address the foregoing problems or at least to provide the public with a useful choice.

All references, including any patents or patent applications cited in this specification are hereby incorporated by reference. No admission is made that any 20 reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents form part of the common 25 general knowledge in the art, in New Zealand or in any other country. 6 It is acknowledged that the term 'comprise' may, under varying jurisdictions, be attributed with either an exclusive or an inclusive meaning. For the purpose of this specification, and unless otherwise noted, the term 'comprise' shall have an inclusive meaning - i.e. that it will be taken to mean an inclusion of not only the 5 listed components it directly references, but also other non-specified components or elements. This rationale will also be used when the term 'comprised' or 'comprising' is used in relation to one or more steps in a method or process.

Further aspects and advantages of the present invention will become apparent from the ensuing description which is given by way of example only.

DISCLOSURE OF INVENTION According to one aspect of the present invention there is provided a method for increasing the production of immunoglobulin A (IgA) in the milk of a ruminant mammal including the steps of: (a) immunising the mammal with an antigen to stimulate secretion of IgA in milk; and (b) collecting milk from the mammal on a once-daily basis.

The advantage found by the applicant in completing the above steps is that unexpectedly, the concentration and/or yield or titre of IgA in the collected milk is 20 greater than that obtained from either step (a) or (b) alone.

In one embodiment, the method is non-therapeutic i.e. only used to collect IgA in milk. In an alternative embodiment, the method is used to include IgA production as well as assist in treating and/or preventing disease.

Daily IgA concentration and/or yield may be determined based on the level of total 7 IgA by weight produced in the milk; or alternatively by the total IgA by weight recovered in the milk collected.

In preferred embodiments of the present invention, the method used to determine the extent of production change in ruminant animals prior to administration of the 5 above method may be that described in New Zealand Patent No. 521969.

However, it should be appreciated that this should not be seen as limiting. Other methods may be used to determine the production change for a ruminant animal.

In preferred embodiments of the present invention, the ruminant mammals of interest are dairy cows. However, this should not be seen as limiting as it is the 10 applicant's understanding that this method may also be applicable to other milk producing ruminant animals such as goats and sheep.

Preferably, step (a) immunisation is completed using the protocols described in US 6,616,927. However this should not be seen as limiting as other protocols in the art may also be used.

Most preferably, the immunisation protocol may involve immunisation at the following sites and times: Week 0: intra muscular in the neck (IM) and intra peritoneal (IP); Week 4: IM, IP and intra mammary in all four teats (IMM); Week 6: IMM; Week 7: IM and IP.

Preferably, week 0 is a date approximately 10-15 weeks prior to the earliest expected birth date of progeny from the mammal e.g. the calving date of the cow.

The term 'antigen' as used herein refers to any material capable of inducing an 8 antigenic response in the treated mammal. Antigens may be selected according to the ultimate utility of the IgA formulation. That is, if the formulation is to be used for generating passive immunity, the antigen against which such immunity is sought should be used. Antigenic substances which may be employed in the 5 method of the invention may be selected from the group consisting of: bacteria, viruses, yeasts, mycoplasms, proteins, haptens, animal tissue extracts, plant tissue extracts, spermatozoa, fungi, pollens, dust, chemical antigens, mammalian cells, and combinations thereof.

Where haptens are to be used as antigens, these should first be conjugated to 10 carrier substances such as proteins using chemistry known in the art.

Useful bacterial antigens may be selected from the group consisting of species selected from: Escherichia, Staphlococcus, Salmonella, Pneumococcus, and combinations thereof. Particularly preferred bacterial antigens may be selected from the group consisting of: Escherichia coli, Staphlococcus, Clostridium difficile, 15 Vibriocholerae, Heliobacter pylori, and combinations thereof.

Preferred yeast antigens include species of Candida. A particularly preferred yeast antigen is Candida albicans.

Useful viral antigens may be selected from the group consisting of: rotavirus, herpes, fowlpox, rhinopneumonitis, coronavirus, parvovirus, influenza, and 20 combinations thereof.

Protein antigens may be selected from the group consisting of: tumour necrosis factor, insulin-like growth factors, somatostatin, viral cell surface proteins, bacterial cell surface proteins, conjugated protein antigens, and combinations thereof.

Chemical antigens may be selected from the group consisting of: pollens, 25 pesticides, insecticides, fungicides, toxins, and combinations thereof. 9 Complex antigens comprising a combination of two or more antigens of the types identified are also envisaged. One such preferred complex antigen is 3K Scourguard (SmithKllne Beecham, Royal Oak, Auckland, New Zealand) or Rotavec K99 (Schering-Plough Animal Health Ltd, Upper Hutt, New Zealand). 5 These vaccines contain pathogenic E. coli, bovine rotavirus, and coronavirus.

Useful Mycoplasma antigens may be selected from the group consisting of: Mycoplasma pneumoniae, Cryptosporidium parvum, and combinations thereof.

Generally, the antigenic substances are suspended in liquid medium for infusion or injection according to known protocols. Any appropriate carriers, diluents, buffers, and adjuvants known in the art may be used. Suitable suspension liquids include saline solution, water, and physiologic buffers.

Preferably the antigen is administered together with at least one adjuvant. Suitable adjuvants for use with the antigens of the invention may be selected from the group consisting of: Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIC) 65, cholera toxin B subunit, aluminium hydroxide AI(OH)3, Bordetella pertussis, muramyl dipeptide, cytokines, saponin, and combinations thereof. Oil based adjuvants and in particular FCA and FIC are preferred.

Preferably, the antigen is included in an immunogen formulation including: an antigen being heat-killed Candida albicans cells at a concentration of approximately 1g dry weight/litre; Freund's incomplete adjuvant 750ml/litre; and sterile saline 249 ml/l. However, it should be appreciated by those skilled in the art that the immunogen formulation may vary according to various factors, for example, the ruminant mammal to which the immunogen formulation is administered.

Preferably, the method of collection of the milk may be by standard milk collection methods such as by hand or by automated devices used on existing dairy farms.

In preferred embodiments the term 'once-daily milking' refers to milking of the ruminant approximately once every 24 hours. It should be appreciated however that the term substantially is used as the time period may vary, for example to meet herding requirements and time associated with milking of the ruminant 5 mammal i.e. the first and last ruminant mammal to be milked.

A feature of once-daily milking is the accelerated regression of the udder as lactation progresses, leading to an increased rate of decline of milk yield.

In this regard, it has been shown that changing animals to a once-daily milking regime is a useful means of differentially increasing the yield of lactoferrin from 1 o milk (Proceedings of the New Zealand Society of Animal Production8). The increase in milk lactoferrin concentration triggered by once-daily milking is understood by the applicant as being due to the induction and expression of the lactoferrin gene within the milk secretory cells of the udder.

It has further been shown that changing animals to a once-daily milking regime 15 increases the concentration and yield of IgG in milk (J. Dairy Sci.9). The increase in milk IgG concentration triggered by once daily milking is understood by the applicant to be due to the increased leakage of serum based IgG into milk.

It should be appreciated by those skilled in the art that the IgA secreted and collected in the milk of the ruminant during and following the immunisation protocol 20 of step (a) is secretory IgA produced by plasma cells within the udder and not serum based IgA. The mechanisms underlying the once daily milking effect on IgA are therefore different to lactoferrin or IgG which are serum based.

Preferably, the concentration of IgA produced using steps (a) and (b) ranges from approximately 0.5 to 1.2 mg/ml.

Preferably, the yield of IgA produced using steps (a) and (b) ranges from 11 approximately 6 to 14 grams per mammal per day.

Most preferably, the concentration and yield are measured via an assay where the term 'assay' may be any experiment or diagnostic test conducted to determine the concentration or activity of a milk component. This can include tests such as 5 ELISA, Bradford or Lowry protein assays and so forth. Such assays are well known and it is anticipated that a skilled addressee would easily be able to match a given assay to IgA measurement.

In a further embodiment of the invention, IgA may be isolated from the milk collected. The isolated IgA may be further purified if desired using known 10 techniques.

In a further option, steps (a) and/or (b) are repeated on a desired frequency e.g. over a period of days, weeks, months or by lactation period of the ruminant.

According to a further aspect of the present invention there is provided ruminant milk containing IgA produced in accordance with the method substantially as 15 described above.

According to a further aspect of the present invention there is provided isolated and/or purified IgA produced in accordance with the method substantially as described above.

According to a further aspect of the present invention there is provided milk for 20 treatment of a disease including elevated levels of IgA produced by the method of: (a) immunising a ruminant mammal with an antigen to stimulate secretion of IgA in the milk; and; (b) collecting the milk from the ruminant mammal on a once-daily basis; characterised in that the titre of IgA in the collected milk is greater than that 12 obtained from either step (a) or (b) alone.

Preferably, the milk described above has a concentration of IgA ranging from approximately 0.5 to 1.2 mg/ml.

It should be appreciated from the above description that there is provided a 5 method for producing a milk with an increased concentration and/or yield or titre of IgA that unexpectedly improves on concentrations and/or yields found in prior art methods.

In addition, as the method utilises once-daily milking, considerable time and labour savings for the farmer result by not having to milk animals twice-daily (or more often). There is also a considerable saving in associated shed costs, equipment and so forth from less frequent milking.

A further advantage of once-daily milking is that herds do not need to be brought in from the pasture as frequently, allowing more grazing time for the animals.

As IgA is a high value item, this method may be commercially important to farmers by increasing the amount of value added product (IgA) produced.

BRIEF DESCRIPTION OF DRAWINGS Further aspects of the present invention will become apparent from the following description which is given by way of example only and with reference to the 20 accompanying drawings in which: Figure 1 shows the total IgA concentration before and after once-daily milking in immunised cows changed from twice-daily to once-daily milking in a first embodiment of the present invention; and, 13 Figure 2 shows the volume of IgA per day before and after once-daily milking in immunised cows changed from twice-daily to once-daily milking in a first embodiment of the present invention; Figure 3 shows the total IgA concentration before and after once-daily milking 5 in immunised cows changed from twice-daily to once-daily milking in a second embodiment of the present invention; Figure 4 shows the volume of IgA per day before and after once-daily milking in immunised cows changed from twice-daily to once-daily milking in a second embodiment of the present invention; Figure 5 shows the total IgA concentration for immunised cows with once-daily milking in one udder half and twice-daily milking in a second udder half; Figure 6 shows the total IgA concentration for cows before and during once-daily milking; Figure 7 shows the total IgA yield for cows before and during once-daily milking.

BEST MODES FOR CARRYING OUT THE INVENTION With reference to the Figures, experiments are described below showing the 20 correlation between IgA concentration and IgA yield for cows milked on a once-daily basis and immunised in accordance with the methods described above. 14 Experiment 1 Eight lactating cows were immunised for the first time with heat killed Candida albicans cells at a concentration of approximately 1 gram dry weight per litre in solution using an IgA immunisation protocol involving immunisation at the following 5 sites and times: Week 0: intra muscular in the neck (IM) and intra peritoneal (IP); Week 4: IM, IP and intra mammary in all four teats (IMM); Week 6: IMM; Week 7: IM and IP.

The cows were milked on a once-daily basis for 3 days at approximately day 130 of lactation (day 124 ± 5).

As shown below in Table 1, during the three days of once-daily milking the concentration of total IgA and Candida albicans specific IgA in milk produced by the cows increased by 26% and 63% by weight, respectively. Milk yield decreased 15 by 13% (wt), so overall yield of IgA was not significantly altered.

Pre-Once-daily milking Once-daily milking Increase Total IgA concentration mg/ml 0.30 0.38 26% Total IgA yield (g/d) 3.35 3.80 13% AnW-Candida albicans IgA titre concentration (ktU) 1.63 2.66 63% Anti-Candida albicans IgA titre yield (kktU) .0 28.9 44% Table 1 The results above are further described in Figure 1, which shows the change in concentration of total IgA from pre-once-daily milking levels. The solid bar on the graph indicates the period of once-daily milking.

Figure 2 shows the change in yield of IgA per day before, during and after the 5 period of once-daily milking where the solid bar indicates the period of once-daily milking.

Experiment 2 Eleven lactating cows which had been immunised with Candida albicans were 10 tested using the IgA immunisation protocol described in Experiment 1 in the previous lactation season. The cows were re-immunised for the experiment with Candida albicans using the IgA immunisation protocol of Experiment 1.

The cows were put onto once-daily milking for 7 days at approximately day 90 of lactation (day 91 ± 3).

As shown in Figures 3 and 4, during the once-daily milking period of 7 days, both the concentration and yield of total IgA increased.

Figure 3 shows the concentration of IgA in milk collected before, during and after the 7 day period of once-daily milking. Note the solid bar indicates the period of once-daily milking. IgA concentration was significantly higher than average pre 20 once-daily milking on days 2 to 7 of once-daily milking.

Figure 4 shows the yield of IgA before, during and after a period of once-daily milking. Like Figure 3, the solid bar indicates the period of once-daily milking. IgA yield was significantly higher on average than pre once-daily milking on days 2,4,5 and 6 of once-daily milking. 16 Experiment 3 Ten of the cows from Experiment 2 were put onto partial once-daily milking at approximately day 190 (day 191 ±8) of lactation for 3 days. One udder half was 5 milked twice-daily and the other udder half was milked once-daily. Samples were collected separately from each udder half before and after the period of once-daily milking.

Figure 5 shows the IgA concentration in udder halves before and after a period of 3 days unilateral once-daily milking. IgA concentrations in twice-daily (morning and 10 afternoon) milked udder halves were normalised to give a single concentration for the day using the formula: [(morning milk yield) * (morning IgA concentration) + (afternoon milk yield) * (afternoon IgA concentration)] / (daily milk yield) It was found that IgA concentration from the once-daily milked udder half was 15 higher than that from twice-daily milked half. These results indicate production of a local factor within the immunised udder is necessary for the increase in milk IgA due to once daily milking.

Experiment 4 To compare the effect of once-daily milking and immunisation together against either method alone, a further experiment was undertaken. 21 cows were immunised using the standard IgA immunisation protocol as described in Experiment 1 but with cows remaining on twice-daily milking as per standard New Zealand dairy practice. 17 11 out of 21 of the cows were responders to the immunisation treatment and twice-daily milking, showing an increased milk IgA concentration (above 0.35 mg/ml). of the 21 cows were non-responders to the protocol treatment with milk IgA concentrations of less than 0.2 mg/ml.

All cows were then milked on a once-daily basis for seven days at approximately day 90 of lactation (d93 ± 4).

Milk yield and concentrations of IgA in the milk on three days of twice-daily milking and the last four days of once-daily milking were averaged to smooth any point variations.

As shown in Figures 6 and 7, the combination of once-daily milking and the immunisation protocol resulted in an unexpected further increase in both IgA concentration and yield. No change was noted for non-responders.

The above results indicate that the effect of once-daily milking on milk IgA may be dependent on successful IgA response to immunisation protocol.

By way of reference, concentrations of IgA in milk of non-immunised animals are on average 0.1 mg/ml.

Experiment 5 Further experiments were completed to determine the effect if any that the stage of 20 lactation of the animal has on IgA concentration and yield.

As can be seen from the results described in Experiments 1 to 4 and based on further experiments undertaken by the applicant at various stages of lactation, i.e. at least for days 90, 124 and 191 of lactation, an increase in milk IgA 18 concentrations was obtained for all times suggesting that stage of lactation may not be an important factor.

Experiment 6 Another experiment investigated the influence of a different antigen to stimulate secretion of IgA into milk and also a longer period of once daily milking. Twenty-four cows were immunised with a mixture of heat killed Escherichia coli strains, inactivated bovine rotavirus and E. coli K99 antigen (sourced from Rotavec K99 vaccine from Schering-Plough Animal Health, Upper Hutt, New Zealand) using the 10 IgA immunisation protocol involving immunisation at the following sites and times: Week 0: intra muscular in the neck (IM) and intra peritoneal (IP); Week 4: IM, IP and intra mammary in all four teats (IMM); Week 6: IMM; Week 7: IM and IP.

The cows were put onto once-daily milking for 3-4 weeks at approximately 4 months of lactation. Milk was pooled from each animal and sampled two times whilst cows were milked twice a day and two times during once daily milking.

As shown below in Table 2, once-daily milking resulted in an unexpected further increase in IgA concentration by 18% and 20%, respectively, compared with twice 20 daily milking values in immunised animals. 19 Date of sampling Milking frequency (per day) IgA concentration (mg/ml) Nov 2 0.40 Nov 2 0.43 Nov 1 0.49 7 Nov 1 0.50 Table 2 The above results indicate that the influence of once daily milking on the increase in IgA is common to different antigens used to stimulate secretion of IgA into milk 5 and also that the further increase in concentration of IgA is maintained for several weeks following once daily milking.

It should be appreciated by those skilled in the art that alternative immunisation protocols may be used to achieve the improved concentration and/or yields.

Aspects of the present invention have been described by way of example only and 10 it should be appreciated that modifications and additions may be made thereto without departing from the scope thereof as defined in the appended claims.

REFERENCES: 1. Lynn R. Jackson and James G. Fox, Institutional Policies and Guidelines on 15 Adjuvants and Antibody Production, ILAR Journal, Vol 37, No.3, 1995. 2. R. F. Sheldrake, A. J. Husband, Specific antibody-containing cells in the mammary gland of non-lactating sheep after intraperitoneal and intramammary immunisation, Research in Veterinary Science 1985, 38, 312-316. 3. Linda J. Saif, PhD; K. Larry Smith, PhD; Bonnie J. Landmeier, MS; Edward H. 20 Bohl, DVM, PhD; Kenneth W. Theil, PhD; Debra A. Todhunter, PhD, Immune response of pregnant cows to bovine rotavirus immunization, Am J Vet Res, Vol 45, No.1. 4. R. F. Sheldrake, Immune Mechanisms of the Ruminant Mammary Gland, Australian Journal of Dairy Technology- March/June, 1987.

. R. F. Sheldrake, A. J. Husband, D. L. Watson & W. Cripps, The effect of intraperitoneal and intramammary immunization of sheep on the numbers of antibody-containing cells in the mammary gland, and antibody titres in blood serum and mammary secretions, Immunology 1985 56 605. 6. A. K. Lascelles, K. J. Beh, T. K. Mukkur and D. L. Watson, The mucosal immune system with particular reference to ruminant animals, The Ruminant Immune System in Health and Disease. 7. R.F. Sheldrake, A.J.Husband, Origin of antibody-containing cells in the ovine mammary gland following intraperitoneal and intramammary immunisation, Research in Veterinary Science 1988, 45, 156-159. 8. Farr et al, Proceedings of the New Zealand Society of Animal Production 62\ 15 pp 225-236 9. Lacy-Hulbert SJ, Woolford MW, Nicholas GD, Prosser CG & Stelwagen K, Effect of milking frequency and pasture intake on milk yield and composition of late lactation cows. Journal of Dairy Science 1999, 82, 1232-1239. 21

Claims (25)

WHAT WE CLAIM IS:

1. A method for increasing the production of immunoglobulin A (IgA) in the milk of a ruminant mammal including the steps of: (a) immunising the mammal with an antigen to stimulate secretion of IgA in milk; (b) collecting milk from the mammal on a once-daily basis; and characterised in that the concentration and titre of IgA in the collected milk is greater than that obtained from either step (a) or (b) alone.

2. The method of claim 1 wherein the mammal is a dairy cow.

3. The method as claimed in any preceding claim wherein the mammal is immunised at the following sites and times: (a) week 0: intra muscular in the neck (IM) and intra peritoneal (IP); (b) week 4: IM, IP and intra mammary in all four teats (IMM); (c) week 6: IMM; (d) week 7: IM and IP.

4. The method as claimed in claim 3 wherein, week 0 is a date approximately 10-15 weeks prior to the earliest expected birth date of progeny from the mammal.

5. The method as claimed in any preceding claim wherein the antigen substance or substances are selected from the group consisting of: bacteria, viruses, yeasts, mycoplasms, proteins, haptens, animal tissue extracts, plant tissue extracts, spermatozoa, fungi, pollens, dust, chemical antigens, mammalian cells, and combinations thereof. j Inte.^cfuiTp^ 22 Olfina of" m I RPflCn/r-

6. The method as claimed in claim 5 wherein the haptens are conjugated to carrier proteins.

7. The method as claimed in claim 5 wherein the bacterial antigen species are selected from the group consisting of: Escherichia, Staphlococcus, Salmonella, Pneumococcus, and combinations thereof.

8. The method as claimed in claim 5 wherein the bacterial antigens are selected from the group consisting of: Escherichia coli, Staphlococcus species, Clostridium difficile, Vibriocholerae species, Hetiobacter pylori, and combinations thereof.

9. The method as claimed in claim 5 wherein the yeast antigen is from the species Candida.

10. The method as claimed in claim 5 wherein the yeast antigen is Candida albicans.

11. The method as claimed in claim 5 wherein the viral antigen is selected from the group consisting of: rotavirus, herpes, fowlpox, rhinopneumonitis, coronavirus, parvovirus, influenza, and combinations thereof.

12. The method as claimed in claim 5 wherein the protein antigens are selected from the group consisting of: tumour necrosis factor, insulin-like growth factors, somatostatin, viral cell surface proteins, bacterial cell surface proteins, conjugated protein antigens, and combinations thereof.

13. The method as claimed in claim 5 wherein the chemical antigens are selected from the group consisting of: pollens, pesticides, insecticides, fungicides, toxins, and combinations thereof.

14. The method as claimed in claim 5 wherein the Mycoplasma antigens are 23 selected from: Mycoplasma pneumoniae, Cryptosporidium parvum, and combinations thereof.

15. The method as claimed in any of the above claims wherein the antigen is administered with at least one adjuvant.

16. The method as claimed in claim 15 wherein the adjuvants are selected from the group consisting of: Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIC) 65, cholera toxin B subunit, aluminium hydroxide AI(OH)3; BordeteJIa pertussis, muramyl dipeptide, cytokines, saponin, and combinations thereof.

17. The method as claimed in any preceding claim wherein the antigen is included in an immunogen formulation including: an antigen being heat-killed Candida albicans cells at a concentration of substantially 1g dry weight/litre; Freund's incomplete adjuvant 750ml/litre; and sterile saline 249 ml/I.

18. The method as claimed in any of the above claims wherein the IgA produced in the collected milk is substantially secretory IgA produced by plasma cells within the udder.

19. The method as claimed in any of the above claims wherein the concentration of IgA produced using steps (a) and (b) ranges from approximately 0.5 to 1.2 mg/ml.

20. The method as claimed in any of claims 1 to 19 wherein the yield of IgA produced using steps (a) and (b) ranges from approximately 6 to 14 grams per mammal per day.

21. The method as claimed in any of the above claims wherein steps (a) and/or (b) are repeated. 24

22. Ruminant milk for treatment of a disease including elevated levels of IgA produced by the method as claimed in any of claims 1 to 21.

23. The milk as claimed in claim 22 wherein the milk has a concentration of IgA ranging from approximately 0.5 to 1.2 mg/ml.

24. A method for increasing the production of immunoglobulin A (IgA) in the milk of a ruminant mammal substantially as hereinbefore described with reference to the examples and figures.

25. Ruminant milk for treatment of a disease including elevated levels of IgA produced by the method substantially as hereinbefore described with reference to the examples and figures. AgResearch Limited by their authorised agents JAMES & WELLS per: Office of '\i 'z \ 4 FEB 2005 25