Volume 45 Supplement 1

Proceedings of the 16th Internordic Symposium of the Nordic Committee for Veterinary Scientific Cooperation (NKVet)

Open Access

Rearing and Caring for a Future Xenograft Donor Pig

  • Axel Kornerup Hansen1,
  • Kirsten Dahl1 and
  • Dorte Bratbo Sørensen1
Acta Veterinaria Scandinavica200445(Suppl 1):S45

https://doi.org/10.1186/1751-0147-45-S1-S45

Published: 31 March 2004

Introduction

Due to the shortage of human organs for allotransplantation, essential efforts aimed at developing a transgenic source animal (donor) for animal-human xenotransplantation have been performed over the last decade. Although man is concordant with old world primates, and the choice of this species would be an obvious way to prevent hyperacute rejection, there are several good reasons for preferring pigs to primates (Table 1). Therefore, concerns for future maintenance problems should be related to the pig. It can be questioned whether maintenance conditions of the transgenic xenograft donor pigs will differ significantly from non-transgenic pigs. Due to the fact that a xenograft donor pig is not yet fully available, little factual knowledge about this issue is available. However, in relation to a future xenograft donor pig basic and applied research has already created knowledge usable for the prediction of its health and welfare. Transgenic knock out mice partly equivalent to a future donor pig have existed for years, and basic knowledge on the function of the strategic transgenes is also available. Therefore, essential information on the impact of these modifications may be achieved by combining this information. Basically, these will be reared and cared for as for other pigs, but two considerations may make the maintenance principles different. Firstly, transgenesis may have induced new health and welfare problems in these pigs, and secondly, the specialized use of the pig may induce new hazards to the xenograft recipients and even to mankind, as such.
Table 1

Advantages and disadvantages choosing between pig and primates as xenograft source animals.

Characteristic

Primates

Pigs

Hyperacute rejection

No

Yes

Breeding performance offspring/year/mother (app)

1

20

Knowledge on immunology and disease

Moderate

High

Risk of xenozoonotic infections

High

Moderate

Physiological compatibility

High

Moderate

Life span (years)

10 – 15

20 – 60

Size (kg)

5 – 15

10 – 300

Potential health and welfare problems of a future xeno-source pig

Several types of gene-modified pigs aimed at avoiding complement activation against the alpha-gal epitope have been produced, but so far, only three types seem to be have a realistic future as xenograft source animals:
  1. 1.

    Pigs with complement regulatory factors (CRF) inserted,

     
  2. 2.

    Pigs with a targeted mutation of the galactosyltransferase gene, so-called alpha-gal knock out pigs, and

     
  3. 3.

    The combination of 1 and 2.

     

Both mice [7, 12, 18, 33] and pigs [12, 24] transgenic for CRF's have been produced, and within both species multiple transfection or crossbreeding have been used to produce double transgenic animals, either with two CRF's [4, 6] or one CRF and one alpha-gal related gene [5, 33]. None of the papers report on any pathological, behavioural, clinical or otherwise welfare-related problems related to the pig carrying a human CRF. Animals transgenic for such genes will still express their own species specific CRF's, which will identify their organs as species-homologue, while the organ is placed in situ in the live xenograft source animal. Therefore, these transgenes are indeed less likely to cause problems for the xenograft donor, but closer examination of CRF transgenic animals are needed to state, whether, and to which extent, they are affected by the transgenesis.

Also alpha-gal knock out animals are available as both mice [28, 32] and pigs [10, 19]. Dahl et al. [9] have listed four possible health and welfare consequences of this targeted mutation:

  • Reduced sperm adhesion to zona pellucida.

  • Increased sensibility to sepsis

  • Increased sensibility to autoimmune diseases

  • Cataract formation

Apart from these points, alpha-gal knock out animals seem to have the same size, appearance and clinical chemistry as their wild type litter mates, and their organs do not seem to differ macro- or microscopically [28, 31]. After two years of observation of alpha-gal knock out mice in of one of the colonies the life span and health status seem to be normal [22]. So, only little knowledge is available on health and welfare of alpha-gal knock out pigs, apart from the fact that they are viable.

Reduced sperm adhesion to zona pellucida

Gal transferases play an important role for the attachment of spermatozoa to zona pellucida [26, 27] by binding to N-acetylglucosamine residues in the zona pellucida, while releasing alternative substrates that prevent the binding of other capacitated spermatozoa [25]. It might, therefore, be expected that it would be difficult to produce homozygotic alpha-gal knock out animals, because the alpha-gal depleted sperm may not be able to bind to the zona pellucida and thus prevent further entry of other spermatozoa. Since two groups have reported the successful production of alpha-gal knock out mice [28, 32] these problems do not fully impair fertilisation, but one group reports that matings between heterozygous alpha-gal knock out carrier mice do not result in the expected 1:2:1 ratio [28]. At least, it is, therefore, reasonable to assume that alpha-gal depleted spermatozoa are less competitive due to the lack of the gal transferase, although not fully incapable of fertilisation.

Increased sensibility to sepsis

Alpha-gal antibodies more frequently bind to Enterobacteriaceae isolates from patients with sepsis than to isolates from the stool of healthy humans [14]. In these patients these antibodies do not seem to initiate complement-mediated lysis of alpha-gal epitope containing bacteria. Neither do they provide a protected site for C3 deposition. In the same way, bacteria isolated from the gallstones of patients with sepsis, have been shown to always express the a-gal epitope [34]. These findings seem to indicate, that the alpha-gal antibodies are capable of protecting certain bacteria from complement-mediated lysis, and pigs with circulating alpha-gal antibodies may therefore express a higher degree of susceptibility to septicaemias caused by e.g. Escherichia coli.

Increased sensibility to autoimmune diseases

In human patients elevated titres of alpha-gal antibodies have been found in patients suffering from diseases with autoimmune elements, such as thyreoiditis. This could be due to the exposure of cryptic alpha-gal epitopes present in cells of certain tissues, e.g. thyroidea [11], or rearrangement and subsequent increased expression caused by binding of alpha-gal antibodies to the epitope on the cell surface [29], but it may also simply be an artefact caused by alpha-gal epitopes present on thyroid tissues used for bioassays [30]. Elevated titers of alpha-gal antibodies have also been found in rheumatoid arthritis patients with kidney damage after treatment with gold and/or D-penicillamine [20].

Cataract formation

One of the two groups producing alpha-gal knock out mice reports that these animals develop cortical cataracts at the age of 4–6 weeks [28]. Cataract formation is associated with significant membrane leakage, suggesting that the mouse lens is very sensitive to biochemical disturbance [22]. No such changes are reported by the other group [32].

Potential health and welfare problems of xenograft recipients

Most concern about xenozoonotic infections is obviously related to the risk of activating porcine endogenous retroviruses (PERV) in the recipients, but it is unlikely that this risk can be dealt with in the production and maintenance process. However, it is clear that ordinary zoonotic infections known to be present in pigs, such as Salmonella sp., influenzaviruses, encephalymyocarditis virus and rotaviruses may compose a risk for the recipients. Furthermore, as pig production is of major economic impact in a number of countries, it is unacceptable to have human xenograft recipients as carriers of unwanted porcine infections. These are both problems to be solved in the production phase.

Health protection in xenograft donor pigs

The main problem in the production of xenograft source pigs to be dealt with in the production phase seems to be protection against infections. Protective systems with terms, such as Specific Pathogen Free (SPF) or Minimal Disease (MD), exist for pig production in many Western countries. Most of such regulations are aimed at improving agricultural economy rather than the more sophisticated use of the porcine organs for xenotransplantation. E.g. Danish SPF regulations ensure protection against Mycoplasma hyopneumoniae, Actinobacillus pleuropneumonia Type 2, Sarcoptes scabiei, Serpulina hyodysenteriae and toxin-producing Pasteurella multocida [1]. Agents of interest in xenotransplantation, such as influenzaviruses and Erysipelothrix rhusiopathiae are common in these pig herds [2]. Therefore, commercial laboratory pig producers as well as xenotransplantation research facilities generally follow even stricter regimes [16].

Pigs for upstart of breeding are produced by caesarian section or embryo transfer. Breeding and rearing is performed behind barriers that protect against unwanted infections. The staff needs to shower into the facility, redress in decontaminated clothes, and they are under strict quarantine if they have been in contact with other pigs. Diet, equipment and materials are decontaminated through autoclaves or chemical locks, and the herd is currently screened for a high number of agents by a range of methods [17], e.g. as proposed for sentinel animals by the Federation of European Laboratory Animal Science Associations [23]. Such regimes are under current development, as it may still prove difficult to remain free of any kind of porcine infection in countries where there is major production of pigs for agricultural purposes, and consequently inhabitants have massive contact with porcine products.

If alpha-gal antibodies are capable of protecting Enterobacteriaceae spp. from complement-mediated lysis [14], this is very likely to become a problem in colonies of alpha-gal knock out pigs. Septicaemias caused by Enterobacteriaceae spp. are one of the most common causes of death in pig herds. It may occur in both piglets and young pigs [3] and as many as 2% of the population may die from this [8]. E. coli is the most common cause, but also another alpha-gal epitope expressing bacterium, Klebsiella pneumoniae, is a common cause [13, 21]. In the pig, all components of the native and the acquired immune systems develop in utero and are functional at birth, although less efficient [15]. Therefore, lacking the ability to lyse septicaemic bacteria by complement may increase the incidence of septicaemias. If this is the case, this problem has to be dealt with in two ways. Either, the production systems must be changed to reduce factors, such as stocking densities and post-weaning stress compared to ordinary pig herds, or the pigs have to be bred in isolators, i.e. systems with no contact with the external environment or with humans which ensures full control of all infections. The latter will be time consuming and expensive.

Authors’ Affiliations

(1)
Centre for Bioethics and Risk Assessment, Division of Laboratory Animal Science and Welfare, Department of Pharmacology and Pathobiology, Royal Veterinary and Agricultural University

References

  1. Anonymous: Betingelser for deklareret kontrol med svinedysenteri; Betingelser for deklareret kontrol med skab; Betingelser for deklareret kontrol med ondartet lungesyge (Hp2); Betingelser for deklareret kontrol med smitsom nysesyge [Conditions for declared monitoring of porcine dysenteria; Conditions for declared monitoring of scabies; Conditions for declared monitoring of evil lung disease (Hp2); Conditions for declared monitoring of infectious cough]. 1987, Copenhagen, Danske Slagterier [Danish Meathouse Organisation]Google Scholar
  2. Anonymous: Annual Report. 2001, Frederiksberg, National Veterinary Laboratory & State Institute for Virus ResearchGoogle Scholar
  3. Bertschinger HU, Fairborther JM: Eschericia coli infections. Diseases of swine. Edited by: Straw B, D'Allaire S, Mengeling WL, Taylor DJ. 1999, Oxford: Blackwell Science, 8: 431-468.Google Scholar
  4. Byrne GW, McCurry KR, Martin MJ, McClellan SM, Platt JL, Logan JS: Transgenic pigs expressing human CD59 and decay-accelerating factor produce an intrinsic barrier to complement-mediated damage. Transplantation. 1997, 63: 149-155. 10.1097/00007890-199701150-00027.PubMedView ArticleGoogle Scholar
  5. Costa C, Zhao L, Burton WV, Rosas C, Bondioli KR, Williams BL, Hoagland TA, Dalmasso AP, Fodor WL: Transgenic pigs designed to express human CD59 and H-transferase to avoid humoral xenograft rejection. Xenotransplantation. 2002, 9: 45-57. 10.1034/j.1399-3089.2002.0o142.x.PubMedView ArticleGoogle Scholar
  6. Cowan PJ, Shinkel TA, Aminian A, Romanella M, Wigley PL, Lonie AJ, Nottle MB, Pearse MJ, d'Apice AJF: High-level co-expression of complement regulators on vascular endothelium in transgenic mice: CD55 and CD59 provide greater protection from human complement-mediated injury than CD59 alone. Xenotransplantation. 1998, 5: 184-190. 10.1111/j.1399-3089.1998.tb00026.x.PubMedView ArticleGoogle Scholar
  7. Cowan PJ, Shinkel TA, Witort EJ, Barlow H, Pearse MJ, d'Apice AJ: Targeting gene expression to endothelial cells in transgenic mice using the human intercellular adhesion molecule 2 promoter. Transplantation. 1996, 62: 155-160. 10.1097/00007890-199607270-00002.PubMedView ArticleGoogle Scholar
  8. Cutler RS, Fahy VA, Spicer EM, Cronin GM: Preweaning Mortality. Diseases of swine. Edited by: Straw B, D'Allaire S, Mengeling WL, Taylor DJ. 1999, Oxford: Blackwell Science, 8: 985-1001.Google Scholar
  9. Dahl K, Sandøe P, Johnsen PF, Lassen J, Hansen AK: Outline of a risk assessment: Welfare of a future xenotransplantation donor pig. Anim Welf. 2003,Google Scholar
  10. Dai YF, Vaught TD, Boone J, Chen SH, Phelps CJ, Ball S, Monahan JA, Jobst PM, McCreath KJ, Lamborn AE, Cowell-Lucero JL, Wells KD, Colman A, Polejaeva IA, Ayares DL: Targeted disruption of the alpha 1,3-galactosyltransferase gene in cloned pigs. Nature Biotechn. 2002, 20: 251-255. 10.1038/nbt0302-251.View ArticleGoogle Scholar
  11. Etienne-Decerf J, Malaise M, Mahieu P, Winand R: Elevated anti-alpha-galactosyl antibody titres. A marker of progression in autoimmune thyroid disorders and in endocrine ophthalmopathy?. Acta Endocrinol (Copenh). 1987, 115: 67-74.Google Scholar
  12. Fodor WL, Williams BL, Matis LA, Madri JA, Rollins SA, Knight JW, Velander W, Squinto SP: Expression of a functional human complement inhibitor in a transgenic pig as a model for the prevention of xenogeneic hyperacute organ rejection. Proc Natl Acad Sci USA. 1994, 91: 11153-11157. 10.1073/pnas.91.23.11153.PubMedPubMed CentralView ArticleGoogle Scholar
  13. Glastonbury JRW: A survey of preweaning mortality in the pig. Austral Vet Jl. 1976, 52: 272-276. 10.1111/j.1751-0813.1976.tb00105.x.View ArticleGoogle Scholar
  14. Hamadeh RM, Jarvis GA, Galili U, Mandrell RE, Zhou P, Griffiss JM: Human natural anti-Gal IgG regulates alternative complement pathway activation on bacterial surfaces. J Clin Invest. 1992, 89: 1223-1235. 10.1172/JCI115706.PubMedPubMed CentralView ArticleGoogle Scholar
  15. Hammerberg C, Schurig GG, Ochs DL: Immunodeficiency in young pigs. Am J Vet Res. 1989, 50: 868-874.PubMedGoogle Scholar
  16. Hansen AK: Microbiological quality of laboratory pigs. Scand J Lab Anim Sci. 1998, 25: 145-152.Google Scholar
  17. Hansen AK, Farlov H, Bollen P: Microbiological monitoring of laboratory pigs. Lab Anim. 1997, 31: 193-200. 10.1258/002367797780596248.PubMedView ArticleGoogle Scholar
  18. Ijzermans J, Schraa EO, Bonthuis F, Yannnoutsos N, Marquet RL: In vivo evaluation of human membrane cofactor protein in transgenic mice. Transplant Proc. 1996, 28: 671-672.PubMedGoogle Scholar
  19. Lai LX, Kolber-Simonds D, Park KW, Cheong HT, Greenstein JL, Im GS, Samuel M, Bonk A, Rieke A, Day BN, Murphy CN, Carter DB, Hawley RJ, Prather RS: Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer coning. Science. 2002, 295: 1089-1092. 10.1126/science.1068228.PubMedView ArticleGoogle Scholar
  20. Malaise MG, Davin JC, Mahieu PR, Franchimont P: Elevated antigalactosyl antibody titers reflect renal injury after gold or D-penicillamine in rheumatoid arthritis [published erratum appears in Clin Immunol Immunopathol 1986 Nov; 41(2):303]. Clin Immunol Immunopathol. 1986, 40: 356-364. 10.1016/0090-1229(86)90040-1.PubMedView ArticleGoogle Scholar
  21. Nielsen NC, Riising HJ, Larsen JL, Bille N, Svendsen J: Preweaning mortality in pigs: Acute septicaemias. Nordisk Veterinærmedicin. 1975, 27: 129-139.PubMedGoogle Scholar
  22. Pearse MJ, Cowan PJ, Shinkel TA, Chen CG, d'Apice AJF: Anti-Xenograft Immune Responses in alpha-1,3-Galactosyltransferase KnockOut Mice. alpha-Gal and Anti-Gal. Edited by: Galili U, Avila JL. 1999, New York: Kluwer Academic/Plenum Publishers, 281-310.View ArticleGoogle Scholar
  23. Rehbinder C, Baneux P, Forbes D, van Herck H, Nicklas W, Rugaya Z, Winkler G: FELASA recommendations for the health monitoring of breeding colonies and experimental units of cats, dogs and pigs – Report of the Federation of European Laboratory Animal Science Associations (FELASA) Working Group on Animal Health. Lab Anim. 1998, 32: 1-17. 10.1258/002367798780559428.PubMedView ArticleGoogle Scholar
  24. Rosengard AM, Cary NRB, Langford GA, Tucker AW, Wallwork J, White DJG: Tissue Expression of Human-Complement Inhibitor, Decay-Accelerating Factor, in Transgenic Pigs – A Potential Approach for Preventing Xenograft Rejection. Transplantation. 1995, 59: 1325-1333.PubMedView ArticleGoogle Scholar
  25. Shapiro BM, Eddy EM: When sperm meets egg: biochemical mechanisms of gamete interaction. Int Rev Cytol. 1980, 66: 257-302.PubMedView ArticleGoogle Scholar
  26. Shur BD, Hall NG: A role for mouse sperm surface galactosyltransferase in sperm binding to the egg zona pellucida. J Cell Biol. 1982, 95: 574-579. 10.1083/jcb.95.2.574.PubMedView ArticleGoogle Scholar
  27. Shur BD, Hall NG: Sperm surface galactosyltransferase activities during in vitro capacitation. J Cell Biol. 1982, 95: 567-573. 10.1083/jcb.95.2.567.PubMedPubMed CentralView ArticleGoogle Scholar
  28. Tearle RG, Tange MJ, Zannettino ZL, Katerelos M, Shinkel TA, vanDenderen BJW, Lonie AJ, Lyons I, Nottle MB, Cox T, Becker C, Peura AM, Wigley PL, Crawford RJ, Robins AJ, Pearse MJ, DApice AJF: The alpha-1,3-galactosyltransferase knockout mouse – Implications for xenotransplantation. Transplantation. 1996, 61: 13-19. 10.1097/00007890-199601150-00004.PubMedView ArticleGoogle Scholar
  29. Thall A, Etienne-Decerf J, Winand RJ, Galili U: The alpha-galactosyl epitope on human normal and autoimmune thyroid cells. Autoimmunity. 1991, 10: 81-87. 10.3109/08916939109004811.PubMedView ArticleGoogle Scholar
  30. Thall A, Etienne-Decerf J, Winand RJ, Galili U: The alpha-galactosyl epitope on mammalian thyroid cells. Acta Endocrinol (Copenh). 1991, 124: 692-699.Google Scholar
  31. Thall A, Etiennedecerf J, Winand RJ, Galili U: The Alpha-Galactosyl Epitope on Mammalian Thyroid-Cells. Acta Endocrinol. 1991, 124: 692-699.PubMedGoogle Scholar
  32. Thall AD, Maly P, Lowe JB: Oocyte Gal-Alpha-1,3gal Epitopes Implicated In Sperm Adhesion to The Zona-Pellucida Glycoprotein Zp3 Are Not Required for Fertilization in the Mouse. J Biol Chem. 1995, 270: 21437-21440. 10.1074/jbc.270.37.21437.PubMedView ArticleGoogle Scholar
  33. van Denderen BJW, Salvaris E, Romanella M, Aminian A, Katerelos M, Tange MJ, Pearse MJ, DApice AJF: Combination of decay-accelerating factor expression and alpha 1,3-galactosyltransferase knockout affords added protection from human complement-mediated injury. Transplantation. 1997, 64: 882-888. 10.1097/00007890-199709270-00016.PubMedView ArticleGoogle Scholar
  34. Wetter LA, Hamadeh RM, Griffiss JM, Oesterle A, Aagaard B, Way LW: Differences in outer membrane characteristics between gallstone-associated bacteria and normal bacterial flora. Lancet. 1994, 343: 444-448. 10.1016/S0140-6736(94)92691-3.PubMedView ArticleGoogle Scholar

Copyright

© The Author(s); licensee BioMed Central Ltd. 2004

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.