Increased gene expression of a cytokine-related molecule and profilin after activation of Suberites domuncula cells with xenogeneic sponge molecule(s)

Porifera (sponges) constitute the lowest metazoan phylum. Experiments examined whether sponges can recognize self/nonself molecules. Cells from the marine sponge Suberites domuncula were incubated with membranes from either S. domuncula or another marine sponge, Geodia cydonium, as well as with recombinant alpha-integrin from G. cydonium. The cells responded immediately with a rise of intracellular Ca2+ ([Ca2+i]) if they were treated with membranes from G. cydonium but not after treatment by those from S. domuncula. This change of [Ca2+i] was also recorded with G. cydonium alpha-integrin. In parallel, the expression of two genes was strongly upregulated; one codes for a cytokine-related molecule, pre-B-cell colony-enhancing factor, and the other for profilin. These genes have previously been found to be highly expressed in human or echinoderm cells in the presence of xenogeneic proteins. Our data support the hypothesis that a primordial immune response system is present in sponges.     

Origin of the integrin-mediated signal transduction. Functional studies with cell cultures from the sponge Suberites domuncula.

Sponges (phylum Porifera) represent the phylogenetically oldest metazoan animals. Recently, from the marine sponge Geodia cydonium a first cDNA encoding a putative integrin receptor molecule was isolated. In the present study basic functional experiments have been conducted to test the hypothesis that in sponges integrin polypeptides also function as adhesion molecules and as outside-in signaling molecules. The sponge Suberites domuncula has been used for the experiments because from this sponge only has a cell culture been established. Here we report that aggregation factor (AF)-mediated cell-cell adhesion is blocked by the RGDS peptide which is known to interact with beta integrin. Both RGDS and AF were found to stimulate DNA synthesis within 24 h. The beta subunit of the integrin receptor was cloned from S. domuncula; the estimated 91-kDa molecule comprises the characteristic signatures. Evolutionary conservation of the beta integrin was assessed by comparison with corresponding beta integrin subunits from evolutionary higher metazoan taxa. Addition of RGDS or of AF to isolated cells of S. domuncula causes a rapid (within 1-2 min) increase in the intracellular Ca2+ concentration which is further augmented in the presence of Ca2+. Furthermore, incubation of the cells with RGDS or AF causes an activation of the GTP-binding protein Ras. In addition it is shown that after a prolonged incubation of the cells with RGDS and AF the expression of the genes coding for Ras and for calmodulin is upregulated. These results suggest that the integrin receptor functions in the sponge system not only as adhesion molecule but also as a molecule involved in outside-in signaling.     

The UNC-112 gene in Caenorhabditis elegans encodes a novel component of cell-matrix adhesion structures required for integrin localization in the muscle cell membrane

Embryos homozygous for mutations in the unc-52, pat-2, pat-3, and unc-112 genes of C. elegans exhibit a similar Pat phenotype. Myosin and actin are not organized into sarcomeres in the body wall muscle cells of these mutants, and dense body and M-line components fail to assemble. The unc-52 (perlecan), pat-2 (alpha-integrin), and pat-3 (beta-integrin) genes encode ECM or transmembrane proteins found at the cell-matrix adhesion sites of both dense bodies and M-lines. This study describes the identification of the unc-112 gene product, a novel, membrane-associated, intracellular protein that colocalizes with integrin at cell-matrix adhesion complexes. The 720-amino acid UNC-112 protein is homologous to Mig-2, a human protein of unknown function. These two proteins share a region of homology with talin and members of the FERM superfamily of proteins.We have determined that a functional UNC-112::GFP fusion protein colocalizes with PAT-3/beta-integrin in both adult and embryonic body wall muscle. We also have determined that UNC-112 is required to organize PAT-3/beta-integrin after it is integrated into the basal cell membrane, but is not required to organize UNC-52/perlecan in the basement membrane, nor for DEB-1/vinculin to localize with PAT-3/beta-integrin. Furthermore, UNC-112 requires the presence of UNC-52/perlecan and PAT-3/beta-integrin, but not DEB-1/vinculin to become localized to the muscle cell membrane.     

Receptor tyrosine kinase, an autapomorphic character of metazoa: identification in marine sponges

In the present review we summarize sequence data obtained from cloning of sponge receptor tyrosine kinases [RTK]. The cDNA sequences were mainly obtained from the marine sponge Geodia cydonium. RTKs (i) with immunoglobulin [Ig]-like domains in the extracellular region, (ii) of the type of insulin-like receptors, as well as (iii) RTKs with one extracellular speract domain, have been identified. The analyses revealed that the RTK genes are constructed in blocks [domains], suggesting a blockwise evolution. The phylogenetic relationships of the sequences obtained revealed that all sponge sequences fall into one branch of the evolutionary tree, while related sequences from higher Metazoa, human, mouse and rat, including also invertebrate sequences, together form a second branch. It is concluded that the RTK molecules have evolved in sponges prior to the "Cambrian Explosion" and have contributed to the rapid appearance of the higher metazoan phyla and that sponges are, as a taxon, also monophyletic. Due to the fact that protein tyrosine kinases in general and RTKs in particular have only been identified in Metazoa, they are, as a group qualified, to be considered as an autapomorphic character of all metazoan phyla.     

Gene structure and function of tyrosine kinases in the marine sponge Geodia cydonium: autapomorphic characters in Metazoa

Porifera (sponges) represent the most ancient, extant metazoan phylum. They existed already prior to the 'Cambrian Explosion'. Based on the analysis of aa sequences of informative proteins, it is highly likely that all metazoan phyla evolved from only one common ancestor (monophyletic origin). As 'autapomorphic' proteins which are restricted to Metazoa only, integrin receptors, receptors with scavenger receptor cysteine-rich repeats, neuronal-like receptors and protein-tyrosine kinases (PTKs) have been identified in Porifera. From the marine sponge Geodia cydonium, a receptor tyrosine kinase (RTK) has been cloned that comprises the characteristic structural topology known from other metazoan RTKs; an extracellular domain, the transmembrane region, the juxtamembrane region and the TK domain. Only two introns, within the coding region of the RTK gene, could be found, which separate the two highly polymorphic immunoglobulin-like domains, found in the extracellular region of the enzyme. The functional role of this sponge RTK could be demonstrated both in situ (grafting experiments) and in vitro (increase of intracellular Ca2+ level). Upstream of this RTK gene, two further genes coding for tyrosine kinases (TK) have been identified. Both are intron-free. The deduced aa sequence of the first gene shows no transmembrane segment; from the second gene--so far--only half of its catalytic domain is known. A phylogenetic analysis with the TK domains from these sequences and a fourth, from a novel scavenger RTK (all domains comprise the signature for the TK class II receptors), showed that they are distantly related to the insulin and insulin-like receptors. The presented findings support the 'introns-late' hypothesis for such genes that encode 'metazoan' proteins. It is proposed that the TKs evolved from protein-serine/threonine kinases through modularization and subsequent exon shuffling. After formation of the ancestral TKs, the modules lost the framing introns to protect the evolutionary novelty. Since cell culture systems of sponges are now available, it can be expected that soon also those mechanisms that control the developmental programs will be unravelled.     

Cloning and expression of new receptors belonging to the immunoglobulin superfamily from the marine sponge Geodia cydonium

 A cDNA encoding a receptor tyrosine kinase (RTK) was previously cloned and expressed from the marine sponge (Porifera) Geodia cydonium. In addition to the two intracellular regions characteristic for RTKs, two immunoglobulin (Ig)-like domains are found in the extracellular part of the sponge RTK. In the present study it is shown that no further Ig-like domain is present in the upstream region of the cDNA as well as of the gene hitherto known from the sponge RTK. Two different full-length cDNAs have been isolated and characterized in the present study, which possess two Ig-like domains, one transmembrane segment, and only a short intracellular part, without a TK domain. The two deduced polypeptides were preliminarily termed sponge adhesion molecules (SAM). The longer form of the SAM, GCSAML, encodes a deduced aa sequence, GCSAML, which comprises in the open reading frame 505 amino acids (aa) and has a calculated M _r of 53911. The short form, GCSAMS, has 313 aa residues and an M _r of 33987. The two Ig-like domains in GCSAML and GCSAMS are highly similar to the corresponding Ig-like domains in the RTKs from G. cydonium; the substitutions on both the aa and nt level are restricted to a few sites. Phylogenetic analyses revealed that the Ig-like domain 1 is similar to the human Ig lambda chain variable region, while the Ig-like domain 2 is related more closely to the human Ig heavy chain variable region. Transplantation experiments (autografting) were performed to demonstrate that the level of expression of the two new genes, GCSAML and GCSAMS, is upregulated during the self/self fusion process. Immunohistochemical analyses using antibodies raised against the two Ig-like domains demonstrate a strong expression in the fusion zone between graft and host. This finding has been supported by northern blotting experiments that revealed that especially GCSAML is strongly upregulated after autografting (up to 12-fold); the expression of GCSAMS reaches a value of 5-fold if compared with the controls. The results presented here demonstrate that the expression of the new molecules described, comprising two Ig-like domains, is upregulated during the process of autograft fusion. Received: 17 November 1998 / Revised: 15 March 1999     

Review: How was metazoan threshold crossed? The hypothetical Urmetazoa.

The origin of Metazoa remained--until recently--the most enigmatic of all phylogenetic problems. Sponges [Porifera] as "living fossils", positioned at the base of multicellular animals, have been used to answer basic questions in metazoan evolution by molecular biological techniques. During the last few years, cDNAs/genes coding for informative proteins have been isolated and characterized from sponges, especially from the marine demosponges Suberites domuncula and Geodia cydonium. The analyses of their deduced amino acid sequences allowed a molecular biological resolution of the monophyly of Metazoa. Molecules of the extracellular matrix/basal lamina, with the integrin receptor, fibronectin and galectin as prominent examples, cell-surface receptors (tyrosine kinase receptors), elements of nerve system/sensory cells (metabotropic glutamate receptor), homologs/modules of an immune system [immunoglobulin-like molecules, SRCR- and SCR-repeats, cytokines, (2-5)A synthetase], as well as morphogens (myotrophin) classify the Porifera as true Metazoa. As "living fossils", provided with simple, primordial molecules allowing cell-cell and cell-matrix adhesion, as well as processes of signal transduction as known in a more complex manner from higher Metazoa, sponges also show peculiarities. Tissues of sponges are rich in telomerase activity, suggesting a high plasticity in the determination of cell lineages. It is concluded that molecular biological studies with sponges as models will not only help to understand the evolution to the Metazoa but also the complex, hierarchical regulatory network of cells in higher Metazoa [reviewed in Progress in Molecular Subcellular Biology, vols. 19, 21 (1998) Springer Verlag]. The hypothetical ancestral animal, the Urmetazoa, from which the metazoan lineages diverged (more than 600 MYA), may have had the following characteristics: cell adhesion molecules with intracellular signal transduction pathways, morphogens/growth factors forming gradients, a functional immune system, and a primordial nerve cell/receptor system.     

Immunological and biological identification of tumour necrosis-like factor in sponges: endotoxin that mediates necrosis formation in xenografts.

Xenografts of the sponge Geodia cydonium in its closely related species G. rovinjensis resulted in a rapid rejection of the graft within a period of 5 days. We identified an immunoreactive tumour necrosis factor (TNF)-like activity in the xenograft (Mr of 30,000) two days after grafting. In-vivo injection of 5 micrograms human recombinant TNF-alpha induced cytotoxicity in sponge cells in the same pattern and time course as during natural xenograft rejection. Anti-TNF-alpha polyclonals were found to react with xenograft extracts, by Western blot analysis, as from day 2 after grafting. Using ELISA we detected the TNF-like activity from day 2 after grafting with peak levels at days 4 and 5, where the amount was 0.72 ng/micrograms tissue DNA. By day 1, gp27 (inhibitory aggregation factor) is already formed in the xenograft. In-vitro experiments on isolated G. cydonium cells showed that addition of purified gp27 induced the production of the TNF-like activity (up to 13.5 ng/ml). Evidence is presented that gp27 is a product of the gp180 lectin receptor. We conclude that gp27 induces TNF-like factor production, resulting in destruction and dissolution of the xenograft after 5 days.     

On the origin of Metazoan adhesion receptors: cloning of integrin alpha subunit from the sponge Geodia cydonium

Integrins are prominent receptors known from vertebrates and the higher phyla of invertebrates. Until now, no evidence has been provided for the existence of integrins in the lowest Metazoa, the sponges (Porifera). We have isolated and characterized a cDNA clone encoding the alpha subunit of integrin from the marine sponge Geodia cydonium (GCINTEG). The open reading frame encodes a polypeptide of 1,086 residues (118 kDa). The intracellular domain features the sequence Tyr- Phe-x-Gly-Phe-Phe-x-Arg, which is different in one residue from the characteristic consensus pattern for integrin alpha subunits. We conclude that sponges, the oldest multicellular animal phylum, already utilize the structural elements which are required for a tuned and controlled interaction among cells, and between cells and the extracellular matrix.      

Experimental Indication in Favor of the Introns-Late Theory: The Receptor Tyrosine Kinase Gene from the Sponge Geodia cydonium

We have analyzed the gene that encodes receptor tyrosine kinase (RTK) from the marine sponge Geodia cydonium, which belongs to the most ancient and simple metazoan groups, the Porifera. RTKs are enzymes found only in metazoa. The sponge gene contains two introns in the extracellular part of the protein. However, the rest of the protein (transmembrane and intracellular part), including the tyrosine kinase (TK)-domain, is encoded by a single exon. In contrast, all TK genes, so far known only from higher animals (vertebrates), contain several introns especially in the TK-domain. The TK-domain of G. cydonium shows similarity with numerous members of receptor as well as nonreceptor TKs. Phylogenetic analysis of the sponge TK-domain indicates that this enzyme branched off first from the common tree of metazoan TK proteins. Consequently, we assume that introns, found in the TK-domains of genes from higher animals, were inserted into these genes after splitting off the sponge taxa from other metazoan organisms (over 600 million years ago). Our results support the view that ancient genes were not ``in pieces.' Received: 8 August 1996 / Accepted: 4 November 1996     

Molecular Evolution of the Metazoan Extracellular Matrix: Cloning and Expression of Structural Proteins from the Demosponges Suberites domuncula and Geodia cydonium

One crucial event during evolution to multicellularity was the development of either direct cell–cell contact or indirect interaction via extracellular matrix (ECM) molecules. The identification of those polypeptides provides conclusive data on the phylogenetic relationship of metazoan phyla and helps us to understand the position of the Metazoa among the other kingdoms. Recently it became evident that the ECM of sponges is amazingly complex; it is composed of fibrous molecules, e.g., collagen, and their corresponding receptors, which are highly similar to those existing in other metazoan phyla. While these data already support the view of monophyly of Metazoa, additional studies are required to understand whether these molecules, which are similar in their primary sequence, also have the same function throughout the metazoan kingdom. In the present study we identified the ligand for one of the autopomorphic characters of Metazoa, the single-transmembrane receptor protein with the receptor tyrosine kinase (RTK) from G. cydonium, as an example: the putative mucus-like protein from G. cydonium. This protein was upregulated during autograft fusion in the homologous system with kinetics similar to those of the RTK. Additionally, a cDNA was isolated from S. domuncula whose deduced polypeptide displays a high sequence similarity to dermatopontin, an ECM molecule found exclusively in Metazoa. Furthermore, it is documented that expression of the fibrous ECM molecule collagen is regulated by the characteristic metazoan morphogens myotrophin and endothelial monocyte-activating polypeptide. These data indicate that the ECM of sponges is not an unstructured ground substance but provides the basis for integrated cell communication. Received: 26 October 2000 / Accepted: 1 February 2001     

Demonstration of an endocrine signaling circuit for insulin in the sponge Geodia cydonium.

The existence of an insulin-mediated cell-to-cell signaling in the sponge Geodia cydonium is demonstrated in this study by molecular biological and immunological techniques. The sequence of a sponge cDNA clone encoding preproinsulin was analyzed for the first time and determined to comprise a high homology to human preproinsulin (60-80% homology). The predicted polypeptide of preproinsulin from sponge contains two disulfide bridges which link the A- to the B-chain. The intra-A chain disulfide bridge is absent. Applying immunological and electron microscopical techniques it is shown that insulin is produced in specialized cells (spherulous cells). Experimental evidence is presented which indicates that the sponge preproinsulin (predicted Mr 11,850) is processed to insulin (Mr 5600; B-chain, Mr 3700 and A-chain, Mr 1900). Plasma membranes of sponge cells are shown to be provided with an insulin-binding receptor composed of two molecules (Mr 104,000 and Mr 98,000). Heterologous insulin (from bovine pancreas) was found to stimulate gene expression in G. cydonium cells. It is concluded that sponges are provided with an endocrine signaling circuit: signaling cells (spherulous cells), hormone (insulin), and hormone receptor bearing target cells which respond to the hormone stimulus.     

Integrin heterodimer and receptor complexity in avian and mammalian cells

We report data showing that the integrin receptor complex in chickens contains several discrete heterodimers all sharing the beta 1-integrin subunit combined separately with different alpha-subunits. Using antisera to synthetic peptides based on cDNA sequences of chicken and human alpha-integrin subunits to analyze the integrin complement of avian and mammalian cells, we show that band 2 of the chicken integrin complex contains alpha-subunits related to both alpha 3- and alpha 5-subunits of human integrins. alpha 3 beta 1 and alpha 5 beta 1 have both previously been shown in human cells to be fibronectin receptors and alpha 3 beta 1 can also act as a receptor for laminin and collagen. We also provide evidence for the presence, in band 1 of the chicken integrin complex, of a third integrin alpha-subunit which is also alpha 5 related. This integrin subunit exists in a separate heterodimer complex with beta 1 and binds to fibronectin-affinity columns. These results provide explanations for published data showing that the avian integrin complex contains receptor activity for a variety of extracellular matrix proteins. We conclude that the chicken integrin complex comprises a set of beta 1-integrin heterodimers equivalent to the human VLA antigens and includes at least two fibronectin receptors. Finally, we show that chicken embryo fibroblasts also contain a beta 3-class integrin related to the RGD receptors defined in various human cells.     

Analysis of the role of egg integrins in sperm-egg binding and fusion

Sperm-egg fusion is believed to be mediated via specific molecular interactions. Integrin alpha6beta1 is a strong candidate for a sperm receptor on the egg plasma membrane. However, the ability of the egg integrin alpha6beta1 to interact with molecules on intact sperm has not yet been proven. In this report, possible involvement of integrin alpha6beta1 in sperm-egg interactions was examined by biochemical and immunocytochemical analyses. To identify egg molecules that specifically interact with sperm, we first incubated sperm with biotin-labeled egg surface proteins. Under this condition, solubilized proteins from eggs inhibited sperm-egg fusion. Western blot analysis under reducing conditions indicated that a major-labeled band of 135 kDa bound to sperm. An immunodepletion experiment using the anti-integrin alpha6 antibody GoH3 indicated that the 135 kDa egg surface molecule that bound to sperm was the integrin alpha6 subunit. To investigate the potential involvement of integrin alpha6beta1 in sperm-egg fusion, we next examined the localization of integrin alpha6 and beta1 subunits before and after fertilization by confocal laser microscopy. At an early stage of sperm-egg fusion, the integrin alpha6 and beta1 subunits were accumulated at the sperm binding site. The frequency of cluster formation was closely related to that of sperm-egg fusion, indicating that integrin receptors are accumulated by sperm destined for fusion. Taken together, these results strongly suggest that the integrin alpha6beta1 is involved in sperm-egg binding leading to fusion via direct association of the integrin alpha6 with sperm.     

Expression of a soluble functional form of the integrin alpha4beta1 in mammalian cells

The integrin alpha4beta1(VLA4) has been expressed as a soluble, active, heterodimeric immunoglobulin fusion protein. cDNAs encoding the extracellular domains of the human alpha4 and beta1 subunits were fused to the genomic DNA encoding the human gamma1 immunoglobulin Fc domain and functional integrin fusion protein was expressed as a secreted, soluble molecule from a range of mammalian cell lines. Specific mutations were introduced into the Fc region of the molecules to promote alpha4beta1 heterodimer formation. The soluble alpha4beta1-Fc fusion protein exhibited divalent cation dependent binding to VCAM-1, which was blocked by the appropriate function blocking antibodies. The apparent Kd for VCAM-1 binding were similar for both the soluble and native forms of alpha4beta1. In addition, the integrin-Fc fusion was shown to stain cells expressing VCAM-1 on their surface by FACs analysis. This approach for expressing soluble alpha4beta1 should be generally applicable to a range of integrins.     

Caspase-mediated apoptosis in sponges: cloning and function of the phylogenetic oldest apoptotic proteases from Metazoa

Sponges (phylum Porifera) represent the phylogenetically oldest metazoan phylum. These animals have complex cell adhesion and powerful immune systems which allow the formation of a distinct body plan. Consequently, an apoptotic machinery has to be predicted that allows sponges to eliminate unwanted cells accumulating during development. With the marine sponge Geodia cydonium, it is shown that allografts of these animals undergo apoptosis as demonstrated by apoptotic DNA fragmentation. Extracts from allografts contain an enzymic activity characteristic for caspases; as substrate to determine the cleavage activity, Ac-DEVD-AMC was applied. cDNAs encoding predicted caspase-3-related proteins were isolated; they comprise the characteristic structure known from caspases of other metazoan phyla. The two cDNAs are assumed to originate from one gene by alternative splicing; the longer form comprises a caspase recruitment domain (CARD), whereas the shorter one is missing CARD. The expression of sponge caspase genes is up-regulated during allograft rejection. In vivo incubation experiments with Ac-DEVD-CHO (a caspase-3 inhibitor) showed a reduction of apoptotic DNA fragmentation, whereas Ac-LEHD-CHO (an inhibitor of caspase-9) caused no effect. It is concluded, that for the establishment of the metazoan body plan, both the adhesion molecules and the apoptotic molecules (described here) were essential prerequisites.     

Expression of recombinant platelet glycoprotein IIbIIIa results in a functional fibrinogen-binding complex

The ability of cDNAs encoding the human platelet glycoprotein IIbIIIa to be expressed and assembled into a functional integrin receptor was assessed by transient transfection into a human cell line. Transfection of full length cDNAs resulted in synthesis of high levels of integrin subunits which appear to be stable within the cell for several days. Coexpression of both subunits resulted in a proteolytically processed form of GPIIb that associated with GPIIIa as a heterodimeric complex as the cell surface. Transport to the cell surface required association of these subunits with each other or with endogenous integrin subunits. When expressed alone, the GPIIb subunit remained intracellular, while the GPIIIa subunit was found to complex with endogenous proteins and was mobilized to the cell surface. The GPIIbIIIa receptor complex facilitated attachment of cells to known ligands for GPIIbIIIa: fibrinogen, vitronectin, and von Willebrand factor. This adhesion was sensitive to inhibition by the peptide GRGDV and the monoclonal antibody AP2, known inhibitors of platelet aggregation     

High-efficiency utilization of the bovine integrin alpha(v)beta(3) as a receptor for foot-and-mouth disease virus is dependent on the bovine beta(3) subunit

We have previously reported that Foot-and-mouth disease virus (FMDV), which is virulent for cattle and swine, can utilize the integrin alpha(v)beta(3) as a receptor on cultured cells. Since those studies were performed with the human integrin, we have molecularly cloned the bovine homolog of the integrin alpha(v)beta(3) and have compared the two receptors for utilization by FMDV. Both the alpha(v) and beta(3) subunits of the bovine integrin have high degrees of amino acid sequence similarity to their corresponding human subunits in the ectodomains (96%) and essentially identical transmembrane and cytoplasmic domains. Within the putative ligand-binding domains, the bovine and human alpha(v) subunits have a 98.8% amino acid sequence similarity while there is only a 93% similarity between the beta(3) subunits of these two species. COS cell cultures, which are not susceptible to FMDV infection, become susceptible if cotransfected with alpha(v) and beta(3) subunit cDNAs from a bovine or human source. Cultures cotransfected with the bovine alpha(v)beta(3) subunit cDNAs and infected with FMDV synthesize greater amounts of viral proteins than do infected cultures cotransfected with the human integrin subunits. Cells cotransfected with a bovine alpha(v) subunit and a human beta(3) subunit synthesize viral proteins at levels equivalent to those in cells expressing both human subunits. However, cells cotransfected with the human alpha(v) and the bovine beta(3) subunits synthesize amounts of viral proteins equivalent to those in cells expressing both bovine subunits, indicating that the bovine beta(3) subunit is responsible for the increased effectiveness of this receptor. By engineering chimeric bovine-human beta(3) subunits, we have shown that this increase in receptor efficiency is due to sequences encoding the C-terminal one-third of the subunit ectodomain, which contains a highly structured cysteine-rich repeat region. We postulate that amino acid sequence differences within this region may be responsible for structural differences between the human and bovine beta(3) subunit, leading to more efficient utilization of the bovine receptor by this bovine pathogen.