Modified live vaccine strains of porcine reproductive and respiratory syndrome virus cause immune system dysregulation similar to wild strains.

Introduction: Porcine reproductive and respiratory syndrome virus (PRRSV) emerged about 30 years ago and continues to cause major economic losses in the pork industry. The lack of effective modified live vaccines (MLV) allows the pandemic to continue. Background and objective: We have previously shown that wild strains of PRRSV affect the nascent T cell repertoire in the thymus, deplete T cell clones recognizing viral epitopes essential for neutralization, while triggering a chronic, robust, but ineffective antibody response. Therefore, we hypothesized that the current MLV are inappropriate because they cause similar damage and fail to prevent viral-induced dysregulation of adaptive immunity. Methods: We tested three MLV strains to demonstrate that all have a comparable negative effect on thymocytes in vitro. Further in vivo studies compared the development of T cells in the thymus, peripheral lymphocytes, and antibody production in young piglets. These three MLV strains were used in a mixture to determine whether at least some of them behave similarly to the wild virus type 1 or type 2. Results: Both the wild and MLV strains cause the same immune dysregulations. These include depletion of T-cell precursors, alteration of the TCR repertoire, necrobiosis at corticomedullary junctions, low body weight gain, decreased thymic cellularity, lack of virus-neutralizing antibodies, and production of non-neutralizing anti-PRRSV antibodies of different isotypes. Discussion and conclusion: The results may explain why the use of current MLV in young animals may be ineffective and why their use may be potentially dangerous. Therefore, alternative vaccines, such as subunit or mRNA vaccines or improved MLV, are needed to control the PRRSV pandemic.

The order of immunoglobulin light chain κ and λ usage in primary and secondary lymphoid tissues of germ-free and conventional piglets.

In pigs (Sus scrofa), the initial immunoglobulin rearrangement of the κ light chain is replaced by λ before the heavy chains rearrange, and the light chains may rearrange even later. This study investigates whether these developmental differences are reflected in the usage of IGK and IGL genes. We found large differences between peripheral B cells and those developing in the bone marrow, and between B cells in germ-free piglets and conventional pigs. During early B cell development in the bone marrow, more 3' V and 5' J gene segments for both light chains are used. However, in the peripheral naive repertoire, more 5' IGLV and 3' IGLJ genes are used. A similar shift toward the use of more 5' IGKV and 3' IGKJ genes is observed later after antigen exposure in conventional pigs. The expression profile showed that most λ+ B cells are generated earlier, while κ+ B cells develop from late precursors that already contain the λ rearrangement. The initial λ rearrangement is retained in both λ+ and κ+ B lymphocytes, and multiple λ transcripts can be found in individual cells. The overall pool of the IGLV repertoire is therefore much larger and more diversified than for IGKV. The κ repertoire is further restricted to the preferential use of only two major IGKV genes, reflecting the limitation for only two consecutive rearrangements. Tracing of silenced λ transcripts in κ+ B cells further confirmed the unconventional mechanism of differential rearrangements in pigs. Our results underline the diversity of the immune system among mammals.

Comparative Aspects of Immunoglobulin Gene Rearrangement Arrays in Different Species.

Studies in humans and mice indicate the critical role of the surrogate light chain in the selection of the productive immunoglobulin repertoire during B cell development. However, subsequent studies using mutant mice have also demonstrated that alternative pathways are allowed. Our recent investigation has shown that some species, such as pig, physiologically use preferential rearrangement of authentic light chains, and become independent of surrogate light chains. Here we summarize the findings from swine and compare them with results in other species. In both groups, allelic and isotypic exclusions remain intact, so the different processes do not alter the paradigm of B-cell monospecificity. Both groups also retained some other essential processes, such as segregated and sequential rearrangement of heavy and light chain loci, preferential rearrangement of light chain kappa before lambda, and functional κ-deleting element recombination. On the other hand, the respective order of heavy and light chains rearrangement may vary, and rearrangement of the light chain kappa and lambda on different chromosomes may occur independently. Studies have also confirmed that the surrogate light chain is not required for the selection of the productive repertoire of heavy chains and can be substituted by authentic light chains. These findings are important for understanding evolutional approaches, redundancy and efficiency of B-cell generation, dependencies on other regulatory factors, and strategies for constructing therapeutic antibodies in unrelated species. The results may also be important for explaining interspecies differences in the proportional use of light chains and for the understanding of divergences in rearrangement processes. Therefore, the division into two groups may not be definitive and there may be more groups of intermediate species.

Revisiting the Pig IGHC Gene Locus in Different Breeds Uncovers Nine Distinct IGHG Genes.

IgG subclass diversification is common in placental mammals. It has been well documented in humans and mice that different IgG subclasses, with diversified functions, synergistically regulate humoral immunity. However, our knowledge on the genomic and functional diversification of IgG subclasses in the pig, a mammalian species with high agricultural and biomedical importance, is incomplete. Using bacterial artificial chromosome sequencing and newly assembled genomes generated by the PacBio sequencing approach, we characterized and mapped the IgH C region gene locus in three indigenous Chinese breeds (Erhualian, Xiang, and Luchuan) and compared them to that of Duroc. Our data revealed that IGHG genes in Chinese pigs differ from the Duroc, whereas the IGHM, IGHD, IGHA, and IGHE genes were all single copy and highly conserved in the pig breeds examined. Most striking were differences in numbers of IGHG genes: there are seven genes in Erhualian pigs, six in the Duroc, but only five in Xiang pigs. Phylogenetic analysis suggested that all reported porcine IGHG genes could be classified into nine subclasses: IGHG1, IGHG2a, IGHG2b, IGHG2c, IGHG3, IGHG4, IGHG5a, IGHG5b, and IGHG5c. Using sequence information, we developed a mouse mAb specific for IgG3. This study offers a starting point to investigate the structure-function relationship of IgG subclasses in pigs.

Perturbation of Thymocyte Development Underlies the PRRS Pandemic: A Testable Hypothesis.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes immune dysregulation during the Critical Window of Immunological Development. We hypothesize that thymocyte development is altered by infected thymic antigen presenting cells (TAPCs) in the fetal/neonatal thymus that interact with double-positive thymocytes causing an acute deficiency of T cells that produces "holes" in the T cell repertoire allowing for poor recognition of PRRSV and other neonatal pathogens. The deficiency may be the result of random elimination of PRRSV-specific T cells or the generation of T cells that accept PRRSV epitopes as self-antigens. Loss of helper T cells for virus neutralizing (VN) epitopes can result in the failure of selection for B cells in lymph node germinal centers capable of producing high affinity VN antibodies. Generation of cytotoxic and regulatory T cells may also be impaired. Similar to infections with LDV, LCMV, MCMV, HIV-1 and trypanosomes, the host responds to the deficiency of pathogen-specific T cells and perhaps regulatory T cells, by "last ditch" polyclonal B cell activation. In colostrum-deprived PRRSV-infected isolator piglets, this results in hypergammaglobulinemia, which we believe to be a "red herring" that detracts attention from the thymic atrophy story, but leads to our second independent hypothesis. Since hypergammaglobulinemia has not been reported in PRRSV-infected conventionally-reared piglets, we hypothesize that this is due to the down-regulatory effect of passive maternal IgG and cytokines in porcine colostrum, especially TGFß which stimulates development of regulatory T cells (Tregs).

T cells in swine completely rearrange immunoglobulin heavy chain genes.

Porcine thymus contains three independent populations of cells that have rearranged immunoglobulin heavy chain VDJH genes. The first population can be found exclusively in medulla and it consists of existing mature B cells and plasma cells. The second consists of developing B cells characterized by the presence of selected VDJH rearrangement, similar to B cell lymphogenesis in the bone marrow. The third population is entirely unaffected by selection mechanism for productive VDJH rearrangement and represents T lineage cells that rearrange immunoglobulin genes. Transcription of unselected VDJH repertoire is not allowed in T cells. Sequence analysis of unselected VDJH repertoire from T cells also revealed important consequences for B cell lymphogenesis and selection of B cell repertoire. As far as we know, this is the first evidence that some species completely rearrange VDJH genes in T cells. Our results also support the finding that B cells actively develop in the thymus.

Antibody repertoire development in fetal and neonatal piglets. XXIV. Hypothesis: The ileal Peyer patches (IPP) are the major source of primary, undiversified IgA antibodies in newborn piglets.

The ileal Peyers patches (IPP) of newborn germfree (GF) piglets were isolated into blind loops and the piglets colonized with a defined probiotic microflora. After 5 weeks, IgA levels in the intestinal lavage (IL) of loop piglets remained at GF levels and IgM comprised ∼70% while in controls, IgA levels were elevated 5-fold and comprised ∼70% of total Igs. Loop piglets also had reduced serum IgA levels suggesting the source of serum IgA had been interrupted. The isotype profile for loop contents was intermediate between that in the IL of GF and probiotic controls. Surprisingly, colonization alone did not result in repertoire diversification in the IPP. Rather, colonization promoted pronounced proliferation of fully switched IgA(+)IgM(-) B cells in the IPP that supply early, non-diversified "natural" SIgA antibodies to the gut lumen and a primary IgA response in serum.

Progress in the use of swine in developmental immunology of B and T lymphocytes.

The adaptive immune system of higher vertebrates is believed to have evolved to counter the ability of pathogens to avoid expulsion because their high rate of germline mutations. Vertebrates developed this adaptive immune response through the evolution of lymphocytes capable of somatic generation of a diverse repertoire of their antigenic receptors without the need to increase the frequency of germline mutation. The focus of our research and this article is on the ontogenetic development of the lymphocytes, and the repertoires they generate in swine. Several features are discussed including (a) the "closed" porcine placenta means that de novo fetal development can be studied for 114 days without passive influence from the mother, (b) newborn piglets are precocial permitting them to be reared without their mothers in germ-free isolators, (c) swine are members of the γδ-high group of mammals and thus provides a greater opportunity to characterize the role of γδ T cells and (d) because swine have a simplified variable heavy and light chain genome they offer a convenient system to study antibody repertoire development.

The comparative profile of lymphoid cells and the T and B cell spectratype of germ-free piglets infected with viruses SIV, PRRSV or PCV2.

Lymphocyte subsets isolated from germ-free piglets experimentally infected with swine influenza virus (SIV), porcine reproductive and respiratory syndrome virus (PRRSV) or porcine circovirus type 2 (PCV2) were studied and the profile of these subsets among these three infections was monitored. Germ-free piglets were used since their response could be directly correlated to the viral infection. Because SIV infections are resolved even by colostrum-deprived neonates whereas PRRSV and PCV2 infections are not, SIV was used as a benchmark for an effectively resolved viral infection. PRRSV caused a large increase in the proportion of lymphocytes at the site of infection and rapid differentiation of B cells leading to a high level of Ig-producing cells but a severe reduction in CD2-CD21+ primed B cells. Unlike SIV and PCV2, PRRSV also caused an increase in terminally differentiated subset of CD2+CD8α+ γδ cells and polyclonal expansion of major Vß families suggesting that non-specific helper T cells drive swift B cell activation. Distinct from infections with SIV and PRRSV, PCV2 infection led to the: (a) prevalence of MHC-II+ T cytotoxic cells, (b) restriction of the T helper compartment in the respiratory tract, (c) generation of a high proportion of FoxP3+ T cells in the blood and (d) selective expansion of IgA and IgE suggesting this virus elicits a mucosal immune response. Our findings suggest that PRRSV and PCV2 may negatively modulate the host immune system by different mechanisms which may explain their persistence.

The enigma of the lower gut-associated lymphoid tissue (GALT).

Artiodactyls possess GALT that appears in fetal life and is located at the extreme end of the ileum. These IPP contain mostly B cells and involute early in postnatal life. Rabbits have a similarly located lymphoid organ, called the sacculus rotundus. Studies in sheep and rabbits have led to the concept that the lower hindgut GALT represents primary lymphoid tissue for B cells and is necessary for normal B cell development, analogous to the bursa of Fabricius. This review traces the history of the observations and theories that have led to the existing concept concerning the role of lower GALT. We then review recent data from piglets with resected IPP that challenges the concept that the IPP is primary B cell lymphoid tissue and that artiodactyls and rabbits are members of the GALT group in the same context as gallinaceous birds. Eliminating the IPP as the primary lymphoid tissue for B cells leads to the hypothesis that the IPP acts as first-responder mucosal lymphoid tissue.

Antibody repertoire development in fetal and neonatal piglets. XII. Three IGLV genes comprise 70% of the pre-immune repertoire and there is little junctional diversity.

We characterized 239 lambda rearrangements from fetal and germfree (GF) piglets to: (1) determine if transcripts recovered from the earliest sites of B cell lymphogenesis were unique (2) determine what proportion of the genome is used to form the pre-immune repertoire (3) estimate the degree of somatic hypermutation and junctional diversity during ontogeny and (4) test whether piglets maintained germfree in isolators (GF piglets) have a more diversified repertoire than fetal piglets. We show that all expressed lambda genes belong to the IGLV3 and IGLV8 families and only IGLJ2 and IGLJ3 were expressed and used equally throughout fetal and neonatal life. Only genes of the IGLV8 family were used in yolk sac and fetal liver and in these tissues, IGLV8-10 comprised >50%. However, the IGLV8 genes recovered at these early sites of B cell lymphogenesis were recovered at all stages of development. Thus, no unique lambda rearrangement was recovered at the first sites of B cell development. The frequency of somatic hypermutation (SHM) in fetal piglets was ~5.9 per Kb equivalent, mutation were concentrated in CDR regions and did not increase in GF piglets. The average CDR3 length was 30 nt ± 2.7 and did not change in GF piglets. Similar to the heavy chain pre-immune repertoire in this species, three IGLV genes account for ~70% of the repertoire. Unlike the heavy chain repertoire, junctional diversity was very limited.

Antibody repertoire development in fetal and neonatal piglets. XVI. Influenza stimulates adaptive immunity, class switch and diversification of the IgG repertoire encoded by downstream Cγ genes.

Infection of germ-free isolator piglets with swine influenza (S-FLU) that generates dsRNA during replication causes elevation of immunoglobulins in serum and bronchoalveolar lavage, a very weak response to trinitrophenyl conjugates but an immune response to S-FLU. The increased immunoglobulin levels result mainly from the polyclonal activation of B cells during the infection, but model antigen exposure may contribute. The 10-fold increase in local and serum IgG accompanies a 10-fold decrease in the transcription of IgG3 in the tracheal-bronchial lymph nodes and in the ileal Peyer's patches. Infection results in class switch recombination to downstream Cγ genes, which diversify their repertoire; both features are diagnostic of adaptive immunity. Meanwhile the repertoires of IgM and IgG3 remain undiversified suggesting that they encode innate, natural antibodies. Whereas IgG3 may play an initial protective role, antibodies encoded by downstream Cγ genes with diversified repertoires are predicted to be most important in long-term protection against S-FLU.

Antibody repertoire development in fetal and neonatal piglets. XV. Porcine circovirus type 2 infection differentially affects serum IgG levels and antibodies to ORF2 in piglets free from other environmental factors.

Porcine circovirus type 2 (PCV2) is an important pathogen in the porcine respiratory disease complex (PRDC) and its persistence may be due to dysregulation of systemic immunity. We examined this contention using isolator piglets. We present data on Ig levels in serum and bronchio-alveolar lavage (BAL), on antibody response to PCV2 and to TNP conjugates used as model antigens in 48 PCV2-infected isolator piglets. We compared these to data from TNP-immunized isolator piglets colonized with a probiotic flora, those infected with swine influenza (S-FLU) and those infected with porcine respiratory and reproductive syndrome virus (PRRSV). We found that PCV2 infection does not cause generalized hypergammaglobulinemia that characterizes PRRSV infections, but causes an unexplained increase in serum IgA. All animals had serum IgG to the ORF2 gene product of PCR2, but neither IgA nor IgG anti-ORF2 responses in BAL. PCV2 infection is a poor adjuvant since only natural anti-TNP antibodies were found. Unexpectedly, immunization appeared to result in lower Ig levels and lower anti-ORF2 responses. There was extreme variation in serum Ig levels in response to infection that could in part be traced to genetic and gender differences. These data suggest that non-replicating vaccines are unlikely to result in a significant primary antibody response but may prime the system for a secondary antibody and cytotoxic response following actual infection. In any case, developers may have to contend with significant genetic differences in the response of piglets to PCV2.

The porcine antibody repertoire: variations on the textbook theme.

The genes encoding the heavy and light chains of swine antibodies are organized in the same manner as in other eutherian mammals. There are ∼30 VH genes, two functional DH genes and one functional JH gene, 14-60 Vκ genes, 5 Jκ segments, 12-13 functional Vλ genes, and two functional Jλ genes. The heavy chain constant regions encode the same repertoire of isotypes common to other eutherian mammals. The piglet models offers advantage over rodent models since the fetal repertoire develops without maternal influences and the precocial nature of their multiple offspring allows the experimenter to control the influences of environmental and maternal factors on repertoire development postnatally. B cell lymphogenesis in swine begins in the fetal yolk sac at 20 days of gestation (DG), moves to the fetal liver at 30 DG and eventually to the bone marrow which dominates until birth (114 DG) and to at least 5 weeks postpartum. There is no evidence that the ileal Peyers patches are a site of B cell lymphogenesis or are required for B cell maintenance. Unlike rodents and humans, light chain rearrangement begins first in the lambda locus; kappa rearrangements are not seen until late gestation. Dissimilar to lab rodents and more in the direction of the rabbit, swine utilize a small number of VH genes to form >90% of their pre-immune repertoire. Diversification in response to environmental antigen does not alter this pattern and is achieved by somatic hypermutation (SHM) of the same small number of VH genes. The situation for light chains is less well studied, but certain Vκ and Jκ and Vλ and Jλ are dominant in transcripts and in contrast to rearranged heavy chains, there is little junctional diversity, less SHM, and mutations are not concentrated in CDR regions. The transcribed and secreted pre-immune antibodies of the fetus include mainly IgM, IgA, and IgG3; this last isotype may provide a type of first responder mucosal immunity. Development of functional adaptive immunity is dependent on bacterial MAMPs or MAMPs provided by viral infections, indicating the importance of innate immunity for development of adaptive immunity. The structural analysis of Ig genes of this species indicate that especially the VH and Cγ gene are the result of tandem gene duplication in the context of genomic gene conversion. Since only a few of these duplicated VH genes substantially contribute to the antibody repertoire, polygeny may be a vestige from a time before somatic processes became prominently evolved to generate the antibody repertoire. In swine we believe such duplications within the genome have very limited functional significance and their occurrence is therefore overrated.

Antibody repertoire development in fetal and neonatal piglets. XXII. λ Rearrangement precedes κ rearrangement during B-cell lymphogenesis in swine.

VDJ and VJ rearrangements, expression of RAG-1, Tdt and VpreB, and the presence of signal joint circles (SJC) were used to identify sites of B-cell lymphogenesis. VDJ, VλJλ but not VκJκ rearrangements or SJC were recovered from yolk sac (YS) at 20 days of gestation (DG) along with strong expression of VpreB and RAG-1 but weak Tdt expression. VλJλ rearrangements but not VκJκ rearrangements were recovered from fetal liver at 30-50 DG. SJC were pronounced in bone marrow at 95 DG where VκJκ rearrangements were first recovered. The VλJλ rearrangements recovered at 20-50 DG used some of the same Vλ and Jλ segments seen in older fetuses and adult animals. Hence the textbook paradigm for the order of light-chain rearrangement does not apply to swine. Consistent with weak Tdt expression in early sites of lymphogenesis, N-region additions in VDJ rearrangements were more frequent at 95 DG. Junctional diversity in VλJλ rearrangement was limited at all stages of development. There was little evidence for B-cell lymphogenesis in the ileal Peyer's patches. The widespread recovery of VpreB transcripts in whole, non-lymphoid tissue was unexpected as was its recovery from bone marrow and peripheral blood monocytes. Based on recovery of SJC, B-cell lymphogenesis continues for at least 5 weeks postpartum.

Structure of the genomic sequence comprising the immunoglobulin heavy constant (IGHC) genes from Sus scrofa.

The number of immunoglobulin heavy chain (IGH) constant genes (IGHC) varies among mammals. To annotate the porcine IGHC genes, we sequenced the entire IGHC-containing genomic region from a single porcine haplotype. The resulting contiguous sequence included in 5' the IGH diversity (D) gene cluster and in 3' TMEM121, which flank the IGHC cluster in the human genome, suggesting that we had obtained the entire genomic region containing porcine IGHC. This region was about 190-kb long, in good agreement with those of other mammals. The porcine IGHC cluster contained 10 genes, IGHM, IGHD, six IGHG genes, IGHE and IGHA. The porcine IGHG genes formed a cluster between IGHD and IGHE, with IGHG3 considered as the most ancient IGHG gene, located at the beginning of the IGHG cluster. Furthermore, the porcine sequence contained two IGHG5 and two IGHG6 genes, but no IGHG genes for IgG2 and IgG4, suggesting flexibility within the IGHG cluster. We also recorded structural differences in the switch regions of the IGHC genes that may be important in their transcription. This haplotype can serve as a reference for future studies on other haplotypes and for functional analysis of porcine immunoglobulin (IG) isotypes.

Recent advances using FcRn overexpression in transgenic animals to overcome impediments of standard antibody technologies to improve the generation of specific antibodies.

This review illustrates the salutary effects of neonatal Fc receptor (FcRn) overexpression in significantly improving humoral immune responses in the generation of antibodies for immunotherapy and diagnostics. These include: (1) improved IgG protection; (2) augmented antigen-specific humoral immune response with larger numbers of antigen specific B cells, thus offering a wider spectrum of clones; (3) generation of antibodies against weakly immunogenic antigens; (4) significant improvements in the number and substantial developments in the diversity of hybridomas. FcRn transgenesis thus confers a number of practical benefits, including faster antibody production, higher antibody yields and improved generation of hybridomas for monoclonal antibody production. Notably, these efficiencies in polyclonal antibody production were also demonstrated in FcRn transgenic rabbits. Overall, FcRn transgenic animals yield more antibodies and provide a route to the generation of antibodies against antigens of low immunogenicity that are difficult to obtain using currently available methods.

Antibody repertoire development in fetal and neonatal piglets XXI. Usage of most VH genes remains constant during fetal and postnatal development.

Usage of variable region gene segments during development of the antibody repertoire in mammals is unresolved in part because of the complexity of the locus in mice and humans and the difficulty of distinguishing intrinsic from extrinsic influences in these species. We present the first vertical studies on VH usage that spans the fetal and neonatal period using the piglet model. We tracked VH usage in DNA rearrangements and in VDJ transcripts throughout 75 days of gestation (DG) in outbred fetuses, thereafter in outbred germfree and colonized isolator piglets, isolator piglets infected with swine influenza and in conventionally reared nematode-infected adults. Seven VH genes account for >90% of the pre-immune repertoire which is the same among tissues and in both transcripts and DNA rearrangements. Statistical modeling supports the view that proportional usage of the major genes remains constant during fetal life and that postnatal usage ranking is similar to that during fetal life. Changes in usage ranking are developmental not antigen dependent. In this species exposure to environmental antigens results in diversification of the repertoire by somatic hypermutation of the same small number of VH genes that comprise the pre-immune repertoire, not by using other VH gene available in the germline. Therefore in swine a small number of VH genes shape the antibody repertoire throughout life questioning the need for extensive VH polygeny.

Ileal Peyer's patches are not necessary for systemic B cell development and maintenance and do not contribute significantly to the overall B cell pool in swine.

Based on studies of sheep, ileal Peyer's patches (IPP) have been regarded as a type of primary lymphoid tissue similar to the bursa of Fabricius in chicken. Because bursectomy results in B cell deficiency, we wondered whether resection of the IPP of piglets would have a similar effect. Comparison of IPP-resected, surgical shams and untreated germ-free piglets, all of which were later colonized with a defined commensal flora, demonstrated that resection of the IPP did not alter the level and phenotype of B and T cells in lymphoid tissues and the blood 10 wk after surgery. Additionally, colonization of IPP caused a shift from the fetal type of lymphocyte distribution to the adult type that is characterized by prevalence of B cells, with many of them representing IgA(+) switched B cells or displaying a more mature CD2(-)CD21(+) and CD2(-)CD21(-) phenotype. Moreover, colonization leads to appearance of effector CD4(+)CD8(+) αß T helper and CD2(+)CD8(-) γδ T cells. Comparison of germ-free with colonized pigs and experiments utilizing surgical transposition of jejunal Peyer's patch into terminal ileum or construction of isolated ileal loops indicated that lymphocyte development in IPP is dependent on colonization. Although our studies confirmed higher mitotic and apoptotic rates in IPP, they failed to identify any cell populations that resemble developing B lineage cells in the bone marrow. These results indicate that porcine IPP are not required for systemic B cell generation or maintenance, but they are secondary lymphoid tissue that appears important in immune responses to colonizing bacteria.