GB1560934A

GB1560934A - Methods for the resistance of non-human mammals to gastro-intestinal disorders

Info

Publication number: GB1560934A
Application number: GB19161/75A
Authority: GB
Original assignee: Unilever PLC
Current assignee: Unilever PLC
Priority date: 1975-05-07
Filing date: 1975-05-07
Publication date: 1980-02-13
Also published as: FR2347932A1; IE43372B1; AU513712B2; BE841614A; ES447714A1; NL7604887A; DE2620287A1; ZA762727B; DE2620287C2; FR2347932B1; IE43372L; JPS51139618A; AU1375176A

Description

(54) METHODS FOR IMPROVING THE RESISTANCE OF NON-HUMAN MAMMALS TO GASTRO-INTESTINAL DISORDERS (71) We, UNILEVER LIMITED, a company organised under the laws of Great Britain, of Unilever House, Blackfriars, London E.C.4, England, do 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:- This invention relates to methods for improving the resistance of non-human mammals to gastro-intestinal disorders.

Mortality among mammals immediately after parturition and amongst the newborn is high: thus, for example, about 20% of pigs do not survive the first week of life, and about half of that number die because of gastro-intestinal disorders largely due to infection with pathogenic strains of one or more bacteria. Also, gastrointestinal disorders place a heavy strain on those that survive, and contribute to poor growth and to poor weight-gain. In stock-raising, a considerable loss of profit, for example in meat, ensues.

Such resistance as the newborn have to the bacterial infection to which they are exposed at birth, or immedately thereafter, is acquired from the mother. This protection (known as passive immune protection, because the anti-bodies conferring protection are received by the young animal, as distinct from being generated by it) is, in modern stock-raismg practice, put to severe test. To take pigs as an example, it is usual to bring sows into a common farrowing house a few days before parturition.

Because of the change of environment and the trauma of parturition, natural resistance of the sows (especially those having their first litter) to infection is much diminished. In consequence, the pathogenic strains of bacteria such as E. coli that are already present (but in small population) in the gut multiply. When these pathogens are excreted, the newborn young are exposed to infection by them, even before they take their first suck. Fresh arrivals of sows about to farrow, and the piglets they duly bear, are exposed to increasing possibility of infection from pathogens excreted by the dams and litters already in the farrowing house.

We have previously described, in British patent specifications Nos. 1,336,015, 1,401,280 and 1,462,384 the oral administration of endotoxins to domestic animals to improve their resistance to gastro-intestinal infection, and in particular to young at the weaning stage.

Such oral administration of endotoxins (whether neat, in the feed, or in the drinking water) to the pregnant animal stimulates the intestine of the animal to produce antibodies to the pathogens, and this results in a marked decrease in the amoum of thogens excreted, with consequent improvement in the environment with benefit to both mother and the young. With mammals, antibodies produced in the colostrum and milk by the oral administration to the mother give further benefit to the suckling young. Typical minimum dose rates are in the range 1 to 5 Haemagglutination Inhibition (HI) units (for measurement of HI units see for instance the Examples of British patent specification No. 1,336,015) of endotoxins of each pathogenic strain of bacterium per animal per day. A particularly suitable level for inclusion in a feed is 102 to 10 HI units of the endotoxins of each pathogen per kilogram Of feed.

In specifying as "endotoxins" the material to be introduced into the intestine, we do not mean to exclude from that material the presence of exotoxins or the presence of the cellular material within which the endotoxins are enclosed in the living bacterium; we mean merely that the endotoxins are of primary importance in obtaining the desired immunological effect while the exotoxins and cell debris are not. However, it will on occasion be convenient to leave either exotoxins or cell debris or both associated with the endotoxins; first, to save the trouble of separating them; and secondly, to enable such antigenic capacity as they possess to be utilised.

To act effectively the endotoxins are preferably freely available, for example by being water-soluble. The endotoxins should, of course, as is described in British patent specification No. 1,336,015, be substantially free from the living pathogenic organisms.

It is known that, by parenteral administration of appropriate antigenic material (essentially bacterial endotoxins) before parturition, there is produced in the mother's blood an increased level of antibodies against the appropriate pathogen.

These antibodies are transferred to the young, for example either by transfer across the placenta or by transfer to the colostrum and thence by absorption through the gastro-intesunal wall of the suckling young into its bloodstream. The pig is an example of an animal whose young acquire passive immunity atmost entirely via the colostrum and gastro-intestinal wall. Although without the passive immunity that is acquired the situation would be much worse, it is frequently found that the passive immunity is inadequate to cope with the highly infective environment described earlier.

We have now found that the effective antibody actively generated in a mammal by parenteral administration of endotoxins of pathogenic bacteria can be markedly improved by the oral administration of the endotoxins as described above to the mammal, whether pregnant or not. When the mammal is pregnant, there is consequent benefit both to the mother and to her young.

The invention provides a method for improving the resistance of a non-human mammal to gastro-intestinal disorders, in which method endotoxins of a pathogenic bacterium implicated in causing such gastroantestinal disorders in the mammal, substantially free from the living pathogenic bacterium, are administered to the mammal both orally and parenterally.

Particularly for mammals whose young acquire at least part of their passive immunity via the colostrum and gastro-intestinal wall, it has been found also that enhanced passive immunity is acquired by the young. This is especially important for those mammals, such as pigs, cows and sheep, where substantially all of the passive immunity of the young is acquired via the colostrum and gastro-intestinal wall.

Thus, according to a particularly important aspect of the present invention, endotoxins of a pathogenic bacterium against which protection is desired are repeatedly administered orally (i.e. are introduced into the intestine by way of the mouth) to a pregnant mammal whose young acquire at least part of their passive immunity via the colostrum and, towards the end of the gestation period, the endotoxins are also administered to the mammal parenterally.

If the pregnant mammal is a sow, notably enteropathogens against which protection is often desired are one or more of the E. coli serotypes which contain the endotoxins 08, 045, 0138, 0139, 0141, 0147, 0149 or 0157, or Closiridium weichit or Vibrio coli. If the pregnant mammal is a cow, notable enteropathogens are E. coli serotypes which contain the endotoxins 08, 09, 015, 026, 078, 086, 0114, 0115, 0137 or 0139, or Salmo7zella dublin or typhimurium. If the pregnant mammal is a ewe, notable enteropathogens are any of those already recited, or an E. coli serotype which contains the endotoxin 020. With pregnant cows or ewes, the antigenic material can if desired be protected from possible degradation in the rumen and yet still be available for absorption in the small intestine: see Phillipson, Proc, Nutr. Soc.

(1972) 31 159.

When both injection and oral administration are employed, it is strongly preferred that the same endotoxin should be injected as is orally administered.

It is a particular feature of the preferred form of the invention that only one injection is necessary. This is in sharp contrast to previous recommendations for parenteral treatment of dams to give passive immunity to their young. A further important feature is that the injected material need not contain an adjuvant, such as an emulsion. Such adjuvants have been recommended previously to ensure gradual release of the injected material. This is unnecessary, indeed disadvantageous, in the present invention.

The named effective endotoxins of the enteropathogens are stabIe to heating at 100oC, and are conveniently obtained by a bacteriumrsterilisation procedure which depends on heating, for example as described in our British patent specification No.

1,336,015. The preparation of feeds incorporating the endotoxins is also described there. A further technique is described hereinafter in Example 4. Pathogens other than those described above can be significant, in particular for other species, and here of course the appropriate endotoxins have to be isolated and used.

As explained above repeated oral administration of endotoxins to the pregnant animal stimulates the intestine of the animal to produce antibodies to the enteropathogens, and this results in a marked decrease in the amount of enteropathogens excreted, with consequent improvement in the environment and benefit to both mother and young. Additionally, the oral administration primes rhe blood circulatory, antibody system, so that antibody response to a parenteral administration of the endotixins is much enhanced. As already stated, parenteral administration of the endotoxins is carried out towards the end of the gestation period, and this ensures high antibody activity during the period of colostrum formation, which is roughly the lasr 10 days of gestation.

Preferably, parenteral administration of the endotoxins is carried out 15-30 days before the estimated date of parturition, following a course of oral administration in which, daily or at least on alternate days, the endotoxins have been orally administered for the preceding 3 weeks, preferably the preceding 4 or 5 weeks, at a dose rate of 10-100 HI units (heamagglutination inhibition units) of the endotoxins of each serotype per animal per day. It is preferable to prolong oral adminis talon until parturition, and even beyond, in order to minimise excretion of enteropathogens.

A convenient scheme is as follows (where S represents date of service by the sire): Pigs Cattle Sheep Begin repeated oral S to (S +30) S to (S+190) S to (S +70) administration in period Carry out parenteral (S +85) to (S+255) to (S +115) to administration in (5+100) (sol+270) (S+ 130) period Date of parturition (5+110) to (S +280) to (S+145) to (S +115) (S +290) (S+150) The colostrum antibody titres resulting from such a regime are unexpectedly high. The passive immune status of the offspring is accordingly much enhanced, and their suscepibility to infection correspondingly reduced.

With the combined parenteral and oral immunisation of the special aspect of the invention, there is the special advantage that, in particular, the class of antibody IgM is produced in the colostrum, at least in that of the sow, in unexpectedly high proportion, whereas a normal parenteral immunisation schedule produces predominantly the much less effective antibody class IgG.

The invention is further illustrated by the following Examples, in which the endotoxins employed were those of E. coli and had been obtained following the procedure described in Example 3A of our British patent specification No. 1,336,015.

EXAMPLE 1 This Example illustrates the increase in concentration of antibodies in the serum and in the colostrum of in-pig sows that is attainable by the administration of antigenic bacterial matter both orally and parenterally.

Two matched groups A and B, each of 8 sows, were taken and those in group A were fed on a pig feed that was of standard kind except for the inclusion in it of the endotoxins of each of seven pathogenic strains of the bacteriumi E. coiL, the strains having the O-antigen (endotoxins) serotype 08, 045, 0138, 0139, 0141, 0147 and 0149. Inclusion was at the level of 100 HI units (haemagglutination inhibition units; these are conveniently measured by the procedure set out in The Veterinary Record (1973) 92 630-636 or in Example 2c in our British patent specification No.

1,336,015) per kilogram of feed. The sows of Group B were fed on the standard feed not containing endotoxins.

Each group of sows was given its particular feed daily, from day S+30 (where S represents the date of service by the boar) to at least day S+115.

At day S+94 each sow in each group was given a parenteral injection of 3000 HI units of the endotoxins (O-antigen) of each of the above specified pathogenic E. coZi serotypes.

At days S+100 and S+107, and at day F+1 (where F represents date of farrowing) the serum of each sow was assayed for its content of antibodies. Imme diately after parturition, the colostrum was also sampled and assayed for antibody content; so, too, was milk sampled and assayed on days F+ 1, F+2, etc. Results are set out in the table below.

Sows Concentration of antibodies in PHA units Serum Colostrum or Milk Date Group A Group B Group A Group B S+100 640 80 S+107 1280 160 F 1280 160 2560 160 F+1 1280 80 640 80 F+2 1280 80 640 80 F+7 640 40 320 40 F+14 160 40 160 40 EXAMPLE 2 This Example compares the concentration of antibodies in the serum of piglets born to treated and untreated sows.

The piglets born to the sows of the groups A and B of Example 1 were suckled normally by their dams, and their blood was assayed for antibody concentration. Results were: Piglets Concentration of antibodies in PHA units in serum Date Group A Group B F 0 0 F+1 2560 80 F+2 2560 80 F+7 320 40 F+14 80 10 There is a clear increase in rhe antibody titre of the piglets suckled by sows treated according to the invention By the well known method of gel filtration, colostrum from both groups of sows and serum from the piglets suckled by them were examined to determine the class of antibody present. It was found that for Group A sows the ratio of immune globulin IgM to immunoglobulin IgG was 10: 1, whereas for Group B sows it was only 3:7. IgM is a far more potent antibacterial agent-by some 500 times-than IgG. Furthermore, IgM enhances the subsequent development of active immunity in the developing young, whereas IgG tends to suppress that development EXAMPLE 3 This Example illustrates the improved ability of piglets suckled by Group A sows to overcome bacterial challenge.

24 hours after birth, both groups of piglets (i.e. piglets from Group A and Group B sows) were challenged with an intravenous injection (1 ml) containing 5X109 live E. coli bacterial of serotype 0149: K91 K88a, c (L). Blood samples were taken during 1 hour immediately after injection, first at 5-minute intervals and then at 10-minute intervals, and the samples were assayed for the number of bacteria still living. Results were (in terms of organisms/ml of Blood): Piglets of Piglets of Time Group A sows Group B sows 0 108 10 5 106 108 10 5X104 5X101 15 104 2X107 20 103 101 25 < 103* 100 30 < 103 5X105 40 < 103 106 50 < 103 106 60 < 103 101 * < 103 not assayable In Group A, out of a group of 12 piglets there were no deaths and only three cases of mild hypothermia. In Group B (12 piglets), there was mild hypothermia in 4 pigs, and severe hypothermia and cyanosis in six. Of those six, four were dead within 3 hours of challenge.

EXAMPLE 4 Preparation of Endotoxin Material The procedure of Example 3A of British patent specification No. 1,336,015 was repeated except that after each final culture was produced it was heated to 600C for 30 minutes to release the endotoxins, and then cooled. 0.5 mls of formalin (40% formaldehyde) per 100 mls of culture was then added to each of the cooled cultures.

The cultures containing the free endotoxins were then combined. The combined cultures were not given a further heat treatment.

EXAMPLE 5 Groups of sows matched to those used in Example 1 were handled as follows.

The sows of Group C were handled exactly like those of Group A except that they were not injected.

The sows of Group D were handled exactly like those of Group B except that they were not injected.

At farrowing the concentrations of antibodies in PHA units in the colostrum were: Group A Group B Group C Group D 2560 160 80 20 10 WHAT WE CLAIM IS: 1. A method for improving the resistance of a non-human mammal to gastrointestinal disorders, in which method endotoxins of a pathogenic bacterium implicated in causing such gastro-intestinal disorders in the mammal, substantially free from the living pathogenic bacterium, are administered to the mammal both orally and parenterally.

2. A method according to Claim 1, wherein the mammal is pregnant.

3. A method according to Claim 2, wherein the mammal is one whose young acquire at least part of their passive immunity via the colostrum and gastro-intestinal wall.

4. A method according to Claim 3, wherein the mammal is one whose young acquire substantially all of their passive immunity via the colostrum and gastrointestinal wall.

5. A method according to Claim 4, wherein the mammal is a sow.

6. A method according to Claim 5, wherein the endotoxins are derived from one or more of the following: E. coli serotypes 08, 045, 0138, 0139, 0141, 0149 and 0157, Clostndium welchii and Vibrio coli.

7. A method according to Claim 4 wherein the mammal is a cow.

8. A method according to Claim 7, wherein the endotoxins are derived from one or more of the following: E. coli serotypes 08, 09, 015, 026, 078, 086, 0114, 0115, 0137 and 0139, Salmonella dublin and Salmonella typhimurium.

9. A method according to Claim 4, wherein the mammal is a ewe.

10. A method according to Claim 9, wherein the endotoxins are derived from one or more of the following: E. coli serotypes 08, 09, 015, 026, 045, 078, 086, 0114, 0115, 0137, 0138, 0139, 0141, 0149 and 0157, Glostridium zcelchii, Vibrw coli, Salmonella dublin and Salmonella tythimuriuF 11. A method according to any one of Claims 7 to 10, wherein the endotoxins are protected against possible degration in the rumen.

12. A method according to any one of Claim 2 to 11 wherein parenteral administration is effected 15-30 days before the estimated date of parturition.

13. A method according to any one of Claims 2 to 12, wherein parenteral administration follows a course of oral administration in which at least on alternate days the endotoxins have been orally administered for the preceding 3 weeks.

14. A method according to any one of the preceding claims, wherein at least 1 Haemagglutination Inhibition Unit of endotoxins of each pathogenic strain of bacterium is administered orally to each mammal each day.

**WARNING** end of DESC field may overlap start of CLMS **.

Claims

**WARNING** start of CLMS field may overlap end of DESC **.

In Group A, out of a group of 12 piglets there were no deaths and only three cases of mild hypothermia. In Group B (12 piglets), there was mild hypothermia in 4 pigs, and severe hypothermia and cyanosis in six. Of those six, four were dead within 3 hours of challenge.

EXAMPLE 4 Preparation of Endotoxin Material The procedure of Example 3A of British patent specification No. 1,336,015 was repeated except that after each final culture was produced it was heated to 600C for 30 minutes to release the endotoxins, and then cooled. 0.5 mls of formalin (40% formaldehyde) per 100 mls of culture was then added to each of the cooled cultures.

The cultures containing the free endotoxins were then combined. The combined cultures were not given a further heat treatment.

EXAMPLE 5 Groups of sows matched to those used in Example 1 were handled as follows.

The sows of Group C were handled exactly like those of Group A except that they were not injected.

The sows of Group D were handled exactly like those of Group B except that they were not injected.

At farrowing the concentrations of antibodies in PHA units in the colostrum were: Group A Group B Group C Group D

2560 160 80 20 10 WHAT WE CLAIM IS: 1. A method for improving the resistance of a non-human mammal to gastrointestinal disorders, in which method endotoxins of a pathogenic bacterium implicated in causing such gastro-intestinal disorders in the mammal, substantially free from the living pathogenic bacterium, are administered to the mammal both orally and parenterally.
2. A method according to Claim 1, wherein the mammal is pregnant.
3. A method according to Claim 2, wherein the mammal is one whose young acquire at least part of their passive immunity via the colostrum and gastro-intestinal wall.
4. A method according to Claim 3, wherein the mammal is one whose young acquire substantially all of their passive immunity via the colostrum and gastrointestinal wall.
5. A method according to Claim 4, wherein the mammal is a sow.
6. A method according to Claim 5, wherein the endotoxins are derived from one or more of the following: E. coli serotypes 08, 045, 0138, 0139, 0141, 0149 and 0157, Clostndium welchii and Vibrio coli.
7. A method according to Claim 4 wherein the mammal is a cow.
8. A method according to Claim 7, wherein the endotoxins are derived from one or more of the following: E. coli serotypes 08, 09, 015, 026, 078, 086, 0114, 0115, 0137 and 0139, Salmonella dublin and Salmonella typhimurium.
9. A method according to Claim 4, wherein the mammal is a ewe.
10. A method according to Claim 9, wherein the endotoxins are derived from one or more of the following: E. coli serotypes 08, 09, 015, 026, 045, 078, 086, 0114, 0115, 0137, 0138, 0139, 0141, 0149 and 0157, Glostridium zcelchii, Vibrw coli, Salmonella dublin and Salmonella tythimuriuF
11. A method according to any one of Claims 7 to 10, wherein the endotoxins are protected against possible degration in the rumen.
12. A method according to any one of Claim 2 to 11 wherein parenteral administration is effected 15-30 days before the estimated date of parturition.
13. A method according to any one of Claims 2 to 12, wherein parenteral administration follows a course of oral administration in which at least on alternate days the endotoxins have been orally administered for the preceding 3 weeks.
14. A method according to any one of the preceding claims, wherein at least 1 Haemagglutination Inhibition Unit of endotoxins of each pathogenic strain of bacterium is administered orally to each mammal each day.
15. A method according to Claim 14, wherein oral administration is by use of a

feed containing 102 to 105 Haemagglutination Inhibition Units of the endotoxins of each pathogen per kilogram of feed.
16. A method according to Claim 5 and substantially as hereinbefore described in Example 1.