Method for the decontamination of biological tissue.

The present invention relates to methods for the decontamination of biological tissue, to compositions comprising antibodies against Campylobacter and complement, and to the use of such compositions as an antibacterial agent.

Bacteria of the genus Canxpylobacter are Gram-negative spiral shaped pathogenic bacteria, with a high motility and carrying a flagellum at one or both poles of the cell Several Campylobacter species have been found. Campylobacter jejuni is very often found in poultry. Frequently Campylobacter coli and (to a lesser extent) the recently found Campylobacter hyoilei are found, mostly in pigs.

Of these, Campylobacter jejuni is the most frequently isolated Campylobacter species in association with human diarrhoea. It is becoming more and more evident that the number of Campylobacter infections in humans exceeds the number of Salmonella infections. (Griffiths et al., Journ. of Applied Bacteriology 1990,69 : 281-301, Walker et al., Microbiological reviews 1986,50 : 81-94, Butler, J-P. , ISBN 0-8493-5446-3, RIVM Report No. 216852002, Bilthoven, the Netherlands).

It is difficult to avoid infection in humans with Campylobacter since, first of all, Campylobacter is a food borne zoonotic bacterium for which many animals, both wild '"and domestic, healthy or sick serve as a reservoir. In addition the bacterium has many different routes of transmission. Bacteria can survive in a dormant coccal form for several weeks on e. g. the surface of carcasses and in water. The bacterium can there- fore easily be transmitted to man through direct contact with animals or by means of contaminated water or food, e. g. milk or meat. C. jejuni is present in many healthy animals, e. g. avian species such as turkey and chicken, cattle, sheep, horses and ro- dents. Chicken meat, an important nutrient source in many countries world-wide is known to be very frequently contaminated with Campylobacter (Shane (1992), S. M., Avian Pathology 21: 189-213). This is not only the case in developing countries but also in e. g. Europe and the USA. Actually human campylobacteriosis currently is the most common cause of food poisoning in much of the industrialised world.

The important role of Campylobacter coli in campylobacteriosis has been described by Tam, C. C. et al. , in Journ. Infect. 47: 28-32 (2003).

Sources of Cantpylobacter (jejuni) colonisation in (broiler) chickens are extensively described in the sound review by Newell,. G. and Fearnley C. (Applied and Environ- mental Microbiology 69: 4343-4351 (2003) ) From this review, it becomes clear at the same time, that Campylobacter infection is practically ineradicably in poultry. The review concludes by saying that the lessons learned from Salmonella control in poul- try have been of little or no help in the control of Campylobacter in the same envi- ronment. Only the well-known methods for prevention of horizontal and vertical transmission of infection are applicable and should be carefully applied.

Campylobacter resides i. a. in the gut of poultry and pigs. Contamination of the meat frequently happens in the slaughterhouse when the intestinal tract, which is often heavily Canipylobacter-contaminated, is removed from the animal. Contamination during slaughter is very difficult to avoid. In the Netherlands, about 50 % of the chicken meat is contaminated, in spite of the high hygienic standards applied in meat industry. A recent overview of the epidemiology of Campylobacter in poultry is given in the Thesis of C. M. Karssen, (ISBN 90-71463-72-9). As a result of this high con- tamination pressure, about 300,000 persons annually in the Netherlands only (total population 15.000. 000) suffer from Campylobacter infection, caused by handling or eating undercooked poultry meat. These figures are not significantly different in other European countries. World-wide, annually more than 400.000. 000 cases are estimated to occur (Pace et al., Vaccine 1998,16 : 1563-1574). Campylobacter causes enteric infections in humans, and occasionally more severe diseases like abortion, meningitis, appendicitis, and urinary tract infection. (Blaser et al., New Engl. J. Med. 1981,305 : 1444-1452, Butzler et al., Clinics in Gastroenterol. 1979,8 : 737-765). Also, severe neurologic complications such as Guillain-Barré syndrome and Miller-Fisher syn- drome are sometimes seen (Schwerer et al. , 1995, J. Endotox. Res. 2: 395-403 and Salloway et al. , 1996, Infect. Immun. 64: 2945-2949). Diarrhoea due to Campylobac- ter jejuni is usually a self-limiting infection, lasting about 2-7 days. In young children, old people and immunocompromised patients, the disease is not self-limiting and re- quires antibiotic treatment. It is clear that, if a potential vaccine against Campylobac- ter for human use would be available, it could prevent humans from becoming in- fected. This would however require a standard vaccination comparable to vaccination

against e. g. mumps and measles. This is evidently not practical. A more logical ap- proach lies in avoiding the transmission from animal to man, specifically from poultry to man.

Such transmission could in principle be avoided through vaccination of animals, more specifically poultry and pigs, against Campylobacter at a moment in time shortly prior to slaughtering. This approach would however solve the problem only partially, be- cause in practise not all farmers are willing to vaccinate their animals prior to slaugh- tering.

The large scale use of antibiotics in animals in order to eradicate Campylobacter is for obvious reasons no option.

Treatment of livestock prior to slaughtering by feeding of antibodies against entero- toxigenic bacteria and even methods for cleaning of contaminated surfaces by spray- ing these surfaces with antibodies have been described for Escherichia coli and Sal- monella species in US-Patent Application US2003/0003104. The application uses an- tibodies derived from eggs, and thus free of complement. But, although claimed for other species as well, the method does not prevent or inhibit the replication of Campy- lobacter species. Campylobacter species simply continue to grow in the presence of antibodies.

Treatment of the meat shortly after slaughtering with disinfecting chemicals would of course be unacceptable, if only for the health safety risks connected to such an ap- proach.

It is clear from the data provided above, that there is an urgent need for novel ways of preventing transmission of Campylobacter from food and food animals to humans.

It is an objective of the present invention to provide such novel methods to avoid this transmission. To this end, a biological method is provided for the decontamination of biological tissue.

It was surprisingly found now, that Campylobacter is extremely sensitive to contact with antiserum against Capylobacter in the presence of complement. The combined action of antibodies against Campylobacter in the presence of complement not only binds to the bacterium, but kills the bacterium.

This finding is indeed unexpected: Campylobacter infection is restricted to the exter- nal cavities, specifically the gut, whereas the combination of antiserum and comple- ment, (if present at all) is restricted to the internal body, specifically to the blood and lymph in the body. Thus, even if an animal has anti-Cantpylobacter antibodies and complement in its blood (due to vaccination or infection), these will never come into- contact with the lumen of the gut, because external cavities and internal body are strictly separated by the epithelial surface lining.

An antibody response thus is very efficient to avoid or overcome the effects of sys- temic infection of the animal but it does not interfere with the presence of Campylo- bacter in the digestive tract of animals.

The surprising finding that Canapylobacter is extremely sensitive to contact with an- tiserum/complement forms now the basis to provide novel methods for the biological control of Campylobacter contamination of biological tissue.

Biological tissue is to be interpreted in a broad sense, relating to any tissue of biologi- cal origin. It can be vegetable tissue, such as leafs or roots, as well as tissue of animal origin such as meat or egg shells.- Examples of possibly contaminated vegetables are e. g. vegetables grown on mixed farms where chickens, pigs and cattle are kept. Meat, more specifically poultry and pig meat, is most frequently contaminated during the process of slaughter.

The most important application of methods according to the present invention will be the decontamination of meat, more specifically chicken, pig and cattle meat.

The method according to the invention for the biological control of contamination shortly relies on the contacting of vegetables or meat with antibodies against Cattipy- lobacter and complement.

The complement system plays an essential role in host defence against infectious agents and in the inflammatory process."Complement"and the"complement system" are known as terms of art from text books such as"Essential Immunology"by Ivan.

M. Roitt, ISBN 0-632-01994-8 (1988). The complement composition that will for rea-

sons of convenience be referred to further as"complement", consists of about twenty plasma proteins that function either as enzymes or as binding proteins. In addition to these plasma proteins, the complement system includes multiple distinct cell-surface receptors that exhibit specificity for the physiological fragments of complement pro- teins and that occur on inflammatory cells and cells of the immune system. There are also several regulatory membrane proteins that function to prevent autologous com- plement activation and protect host cells from accidental complement attack. The role of complement in host defence has been established through genetic deficiencies of certain complement components, which may result in life-threatening recurrent bacte- rial infections or immune complex diseases.

Complement thus is a complex mixture of proteins (see above). It is however present and readily available in serum. Therefore, serum is by far the most convenient source for complement. In the Examples provided below, a very simple, quick and reliable method for obtaining complement is presented.

Complement does not necessarily have to be derived from blood of the animal species from which the antibodies are derived. Complement from other animal species is usu- ally suitable as well. Complement from e. g. cows, horses and sheep is easily obtain- able in acceptable quantities. Guinea pig serum can even be commercially obtained.

In most cases however it would be most convenient to use antibodies and complement of the same species.

Antibodies or antiserum against Campylobacter can be obtained quickly and easily by vaccination of pigs, poultry or e. g. rabbits with inactivated Campylobacter cells in a water-in-oil suspension followed, after about four weeks, by bleeding, centrifugation of the coagulated blood and decanting of the sera.

Another source of antibodies is the blood or serum of e. g. poultry, pigs or cattle that have been naturally infected with Campylobacter. Antibody titters in infected animals will usually not be as high as titters found in deliberately vaccinated animals, but they are sufficiently high to be useful in the methods according to the invention.

Other methods for the preparation of antibodies, which may be polyclonal, mono- specific or monoclonal (or derivatives thereof) are well-known in the art. If polyclonal antibodies are desired, techniques for producing and processing polyclonal sera are

well-known in the art (e. g. Mayer and Walter, eds. Immunochemical Methods in Cell and Molecular Biology, Academic Press, London, 1987).

Monoclonal antibodies, reactive against Campylobacter can be prepared by immunis- ing inbred mice by techniques also known in the art (Kohler and Milstein, Nature, 256,495-497, 1975).

Methods for large-scale production of antibodies used in the invention are also known in the art. Such methods rely on the cloning of (fragments of) the genetic information encoding the protein according to the invention in a filamentous phage for phage dis- play. Such techniques are described i. a. at the"Antibody Engineering Page"under "filamentous phage display"at http ://aximtl. imt. uni-marburg. de/-reklaepphage. html. , and in review papers by Cortese, R. et al. , (1994) in Trends Biotechn. 12: 262-267., by Clackson, T. & Wells, J. A. (1994) in Trends Biotechn. 12: 173-183, by Marks, J. D. et al. , (1992) in J. Biol. Chem. 267: 16007-16010, by Winter, G. et al. , (1994) in Annu. Rev. Immunol. 12: 433-455, and by Little, M. et al. , (1994) Biotechn. Adv. 12: 539-555. The phages are subsequently used to screen camelid expression libraries ex- pressing camelid heavy chain antibodies. (Muyldermans, S. and Lauwereys, M., Journ. Molec. Recogn. 12: 131-140 (1999) and Ghahroudi, M. A. et al., FEBS Letters 414: 512-526 (1997) ). Cells from the library that express the desired antibodies can be replicated and subsequently be used for large scale expression of antibodies.

In the Example provided below, a simple method for inducing and subsequent har- vesting of antiserum is presented.

Thus, the invention relates to methods for the decontamination of biological tissue, preferably meat, which methods comprise the contacting of biological tissue, prefera- bly meat with antibodies against Campylobacter and complement.

The"contacting"should broadly be interpreted as bringing the tissue to be decon- taminated in contact with antibodies and complement. In principle, the contacting with the antibodies and the contacting with the complement need not necessarily take place at the same moment. In principle, the tissue could first be brought into contact with antibodies and subsequently with complement or vice versa. In practice the two

components, antibodies and complement, will mix anyway after the second of the two components is applied.

Preferably however, the contacting with the antibodies and the antiserum is done at the same time.

As can be seen from the Examples, this"contacting"can be done very quick and effi- ciently by adding an antibody/complement mixture to the biological tissue, preferably the meat to be decontaminated.

The duration of the contact is not very critical. A short"dip", in which the biological tissue is shortly dipped into the antibody/complement mixture and taken out again, is in principle already sufficient, due to the fact that the mixture will stick to the surface of the material and will remain there for minutes to hours, depending on possible fur- ther treatment of the biological tissue, c. q. the meat. Another efficient way of contact- ing the antibody/complement mixture with the biological tissue to be decontaminated is by spraying the antibody/complement mixture over the surface of the biological tis- sue.

The complement reaction is most efficient at 37 degrees Celsius. Therefore, for the most efficient decontamination, the temperature of the dipping fluid and preferably also that of the biological tissue should preferably be kept close to, or even better, at 37 degrees Celsius.

A preferred form of the invention relates to methods for the decontamination of bio- logical tissue wherein the biological tissue is meat.

An efficient moment for the method according to the invention to be applied, is the moment after removal of the intestines, or better the digestive tract from the slaugh- tered animal. The removal of the digestive tract is the moment at which the contai- nation of the meat usually takes place. Thereafter, the chances of contamination are low, because the source of the contamination is removed.

If the meat is sprayed or dipped shortly after removal of the digestive tract, there will be immediate decontamination, resulting in decontaminated meat, thereby avoiding further contamination of the eviscerating line. The step of dipping could easily be in- tegrated in the automated slaughter process. Merely as an example, a steel drum, somewhat larger than the animal to be dipped, could be filled with anti-

body/complement mixture and kept at 37 degrees Celsius. It then suffices to add to the slaughter line a facility that simply allows the hook to which the animal is at- tached, to descend until the animal remainders are fully immersed in the antiserum and complement, and to raise again thereafter.

It is clear that the method can be used not only during the process of slaughter, but equally well at any moment thereafter. Dipping or spraying could e. g. equally well be done shortly before the meat is packaged.

Due to the fact that poultry meat is the most likely source for human campylobacteri- osis, there clearly is a preference to use the method according to the invention for the prevention of contamination of poultry meat.

Therefore, in a more preferred embodiment, the method according to the invention is applied to poultry meat.

A quick, efficient and low-cost way of contacting biological tissue with antibodies in the method according to the invention is the use of antiserum from animals, preferably poultry, vaccinated against Canrzpylobacter, i. e. antiserum comprising antibodies against Campylobacter. Such antiserum comprises the antibodies needed for the method, and moreover, it comprises complement. In fact it suffices-for the method according to the invention to dip the biological tissue in the serum of animals having antibodies against Campylobacter. In this serum, both the antibodies and the comple- ment are present, and the biochemical conditions for the complement reaction are ex- cellent.

Thus, in an even more preferred embodiment of the method according to the inven- tion, the biological tissue is contacted with antiserum comprising (both complement and) antibodies against Campylobacter.

As can be seen from the Examples, the serum can be highly diluted. Use of a 3000 times dilution still provides significant killing of Campylobacter. The use of diluted serum is attractive, if only from a point of costs.

If however a more then 10 times diluted serum is used, additional complement should preferably be added, because complement should preferably not be diluted more than 10 times. More preferably, the complement is diluted 1: 1 (vol/vol) with serum.

The use of diluted antibodies, complement or antiserum can be advantageous, because these components usually have a relatively high protein content of proteins that are not relevant to the method as such. Dilution of the antibodies, complement or antise- rum can easily be done by adding e. g. physiological salt solution or another pharma- ceutically acceptable buffer. The advantage of a diluted components is that they carry less non-relevant proteins. As a result, a dilution is less sticky and can e. g. be sprayed more easily. A dilution of one or more of the components; antibodies, complement or antiserum with at least an equal volume of e. g. physiological salt (which thus leads to the antibodies, complement or antiserum to be diluted at least twice) is preferred. Of course a dilution should not exceed the level at which the antibodies, complement or antiserum are still exerting the decontaminating effect (see comments above).

In most cases, the cause of the contamination is a Campylobacter of the species Cam- pylobacter jejuni or Campylobacter coli.

Therefore, in a most preferred form of the method according to the invention, the an- tibodies or the antiserum used in the method according to the invention are raised against the Campylobacter species Campylobacter jejuni or Campylobacter coli.

Another embodiment of the present invention relates to the use of a composition com- prising antibodies against Campylobacter and complement as an antibacterial agent.

The use of this composition can be applied in all of the methods described herein.

Merely as an example; the composition an be used in the methods described accord- ing to the present invention, in which the composition is contacted with the biological tissue.

Finally, another embodiment of the present invention relates to a composition com- prising antibodies against Campylobacter and complement, wherein the composition is characterised by the fact that it does not comprise native antiserum, or wherein the composition is diluted compared to native antiserum.

This can be exemplified as follows: a composition which does not comprise native antiserum is a composition in which the antibody component or part of the antibody component is obtained from another source than the complement. Such a composition could e. g. be a composition comprising chicken complement and/or chicken serum, and additional rabbit antiserum against Campylobacter, or a composition comprising monoclonal mouse antibodies against Campylobacter and Guinee-pig-complement.

A composition which is diluted compared to native antiserum is a composition which comprises an antibody concentration and/or a complement concentration that is not found naturally in the serum of the animal from which the antiserum is taken. Merely as an example: an example of such a composition is an antiserum which has been di- luted with a buffer or physiological salt.

EXAMPLES Example 1 Used strain and growth conditions.

A standard well-defined virulent C. jejuni strain, strain 81116 (Nuijten et al., J. Biol.

Chem. , 265: 17798-17804 (1990) ) was grown on Campy Blood agar base (CBAB) solid media' (Becton Dickenson) under micro-aerophylic conditions, or in liquid Brucella broth (Difco) containing 1% yeast extract (Oxoid), abbreviated: BBYE.

Production of the vaccine.

Twenty colonies of strain 81116 were inoculated from a CBAB plate into 250 ml of BBYE and incubated for 21 hours at 42°C. The bacteria were inactivated by the addi- tion of a volume of 0.5 ml of a 37% formaldehyde solution (0.2% final concentration) and incubation for 16 hours at 4° while gently stirring. The culture was found to con- tain 6.9 x 10E8 cells/ml by microscopic count. The cells were spun down for 10 min- utes at 9000 x g and resuspended in BBYE to a concentration of 4 x 10E9 cells/ml.

The suspension was confirmed to be sterile by the absence of growth on CBAB plates. The vaccine was formulated in a water in oil suspension at 1.6 x 10E9 cells/ml.

Production of antiserum.

Ten four-week old SPF White leghorn chickens were held in an isolator under posi- tive pressure and were intramuscularly vaccinated with 1 ml of the vaccine and bled 4 weeks later from the jugular vein. Coagulated blood was centrifuged for 15 minutes at 3000 x g and the serum was pooled and designated serum A. The complement in se- rum A was inactivated by incubation for 30 minutes at 56°C.

Source of complement.

An SPF White leghorn chicken of approximately 8 weeks of age was exsanguinated from the jugular vein.

The blood was allowed to coagulate for 1 hour at approximately 25°C and then centri- fuged for 10 minutes at 3000 x g. The serum was decanted, and divided into 2 equal volumes. One volume was stored at 4°C and used as active complement. The other half was incubated at 56° for 30 minutes to inactivate the complement.

Example 2 In vitro antibody mediated killing of Campylobacter jejuni.

In a microtitre plate (NUNC), columns 2 to 12 were filled with 25 ul of PBS. Subse- quently, in column 1,50 ul of serum A was added. Duplicate dilutions were made by transferring 25 p1 from column 1 to column 2, from column 2 to column 3 etc. Then 25 gl of a suspension of C. jejuni strain 81116 containing approximately 470 cfu (as determined by viability counting on agar plates) was added. Then 25 ul of fresh com- plement (from an animal sacrificed on the same day) was added in all wells of row A.

In row B the same complement was added, but after heat inactivation (as described above). In row C, 25 ul of complement that had been stored for 5 days at 4°C was used and in row D, 25 ul of this 5 day-old complement was added after heat inactiva- tion.

The microtitre plate was mixed and incubated for 60 minutes at 37°C.

Then from each well, a 15 ut trop (comprising about 94 cfu) was placed on an agar plate, which was then incubated for 20 hours at 37°C under micro-aerophilic condi- tions. The number of cfu's were counted and are given in table 1.

Conclusion: The serum is still active at 3000-fold dilution.

The complement looses part of its activity upon storage for 5 days at 4°C, but is still active enough to support serum killing at 192-fold serum dilution. final dilution of serum A 3# 6# 12# 24# 48# 96# 192# 384# 768# 1536# 3072# 6144# 0 0 0 0 0 0 0 0 0 0 0 2 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 0 0 0 0 0 0 0 1 10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 Table 1: In vitro killing of C. jejuni by serum and complement. The number of cfu's are given for each well that was tested.

Example 3 Inactivation of C. jejuni on chicken meat.

The breast filet from the animal that was used as the source of complement was asepti- cally removed and placed into a large Petri-dish at 37°C. Droplets of 0.01 ml containing 20,200 or 2000 cfu of C. jejuni strain 81116 were placed in duplo on the surface of the muscle tissue. Then, three of the droplets (nr 1,3 and 5) were mixed on the surface of the chicken muscle with 20 pi of a 1 on 1 (vol/vol) mixture of serum A and fresh active complement. The other three droplets (nr 2,4, and 6) were mixed in a similar way with 20 pi of a 1 on 1 (vol/vol) mixture of serum A and inactivated complement (see table 2).

All samples were incubated for 60 minutes at 37°C. Then, a volume of 10 pi of PBS was added to the spot where the droplets had been placed on the meat and gently pipet- ted up and down 5 times. A volume of 10 pi was added to 40 pi of PBS and spread on a CBAB plate and incubated under micro-aerophylic conditions for 24 hours. The num- bers of colonies were counted (last column table 2). test nr of cfu serum A complement nr. of cfu added counted 1 20 es active 0 2 20 yes inactivated 26 3 200 yes active 0 4 200 es inactivated 57 5 2000 yes active 0 6 2000 yes inactivated >300 table 2: Inactivation of C. jejuni strain 81116 by serum and complement on chicken meat.

Conclusion: The combination of active complement and anti-Ca1npylobacter antiserum is able to completely inactivate C. jejuni cells on the surface of chicken meat.