Wildlife and paratuberculosis: a review.

Paratuberculosis (PTB) is an infectious granulomatous enteritis caused by Mycobacterium avium paratuberculosis (MAP) causing significant economic losses in livestock. However, PTB in free-living and captive wildlife has not been as extensively studied as in livestock. We reviewed the existing literature references on MAP to (i) determine the potential impact of MAP infection in wildlife species; (ii) analyze whether wildlife reservoirs are relevant regarding MAP control in domestic ruminants; (iii) assess the importance of MAP as the cause of potential interferences with tuberculosis diagnosis in wildlife. The mean MAP prevalence reported in wildlife was 2.41% (95% confidence interval 1.76-3.06). Although MAP should be considered an important disease in farmed cervids, its impact on free-ranging species is questionable. MAP reservoirs may exist locally but their significance for PTB control in livestock is quite limited. The most critical aspect derived of MAP infection in wildlife is the interference with tuberculosis diagnosis.

Subolesin/Akirin vaccines for the control of arthropod vectors and vectorborne pathogens.

Diseases transmitted by arthropod vectors such as mosquitoes, ticks and sand flies greatly impact human and animal health, and therefore, their control is important for the eradication of vectorborne diseases (VBD). Vaccination is an environmentally friendly alternative for vector control that allows control of several VBD by targeting their common vector. Recent results have suggested that subolesin (SUB) and its orthologue in insects, akirin (AKR) are good candidate antigens for the control of arthropod vector infestations and pathogen infection. SUB was discovered as a tick-protective antigen in Ixodes scapularis. Vaccination trials with recombinant SUB/AKR demonstrated effective control of arthropod vector infestations in various hard and soft tick species, mosquitoes, sand flies, poultry red mites and sea lice by reducing their numbers, weight, oviposition, fertility and/or moulting. SUB/AKR vaccination also reduced tick infection with tickborne pathogens, Anaplasma phagocytophilum, A. marginale, Babesia bigemina and Borrelia burgdorferi. The effect of vaccination on different hosts, vector species, developmental stages and vectorborne pathogen infections demonstrated the feasibility of SUB/AKR universal vaccines for the control of multiple vector infestations and for reduction in VBD.

No evidence that wild red deer (Cervus elaphus) on the Iberian Peninsula are a reservoir of Mycobacterium avium subspecies paratuberculosis infection.

The potential role of red deer (Cervus elaphus) as a reservoir of Mycobacterium avium subspecies paratuberculosis (MAP) infection is largely unknown. A total of 332 wild red deer were investigated using post-mortem examination, bacteriology and serology. Only three animals (1.12%) were found to have lesions on histopathological examination and no MAP bacteria were recovered on culture. The results suggest it is unlikely that wild red deer make a significant contribution to the maintenance of MAP infection in the region. The cross-reactivity of the ELISAs used indicates this diagnostic modality is ineffective in the detection of MAP infection in this species. The implications of these results for the control of this important pathogen in both livestock and wildlife are discussed.

Large-scale ELISA testing of Spanish red deer for paratuberculosis.

A role of wildlife species as paratuberculosis reservoirs is strongly suspected based on field and molecular epidemiologic evidence. This paper presents the first large-scale data on enzyme-linked immunosorbent assay (ELISA) against Mycobacterium avium subspecies paratuberculosis (MAP) antibodies in red deer from Spain, and tests the effect of host and environmental risk factors on antibody levels. A total of 257 out of 852 serum samples tested positive, yielding a total seroprevalence of 30.16% (95% CI 27.08-33.24). Sampling locality, presence of cattle and increasing age explained the variation in the individual ELISA optical density (OD) results. Data presented in this study strongly suggest that Spanish red deer are exposed to MAP. While contact with cattle was statistically significant, some wild populations showed the highest positivity to the ELISA. The results support the need of a careful study of MAP prevalence based on culture and molecular tools in order to clarify if deer play a significant role as paratuberculosis reservoirs for livestock, and if deer paratuberculosis is affecting hunting harvest, trophy quality, or wild animal welfare in Spain.

Prevalence of infection and 18S rRNA gene sequences of Cytauxzoon species in Iberian lynx (Lynx pardinus) in Spain.

The Iberian lynx (Lynx pardinus) is the most endangered felid in the world. Only about 160 individuals remain in 2 separate metapopulations in Southern Spain (Sierra Morena and Doñana). We obtained blood samples of 20 lynxes captured from 2004 to 2006, and determined the prevalence of infection and genetic diversity of Cytauxzoon spp. using 18S rRNA PCR and sequence analysis. Prevalence of infection was 15% (3 of 20). Cytauxzoon sp. was only detected in Sierra Morena. For phylogenetic analysis, we used the sequences reported in the present study and those characterized in different domestic and wild felids and ticks from North and South America, Asia and Europe. Three different Cytauxzoon sp. sequences were obtained. They were closely related to that obtained from a Spanish cat, but diverged in up to 1.0% with respect to the only previously reported sequence from an Iberian lynx. Conversely, the latter sequence clustered together with C. manul sequences obtained from Pallas cats (Otocolobus manul) in Mongolia. Our analysis yields a separate cluster of C. felis sequences from cats, wild felids and ticks in the United States and Brazil. These results suggest that at least 2 different Cytauxzoon spp. may be present in Iberian lynx. The apparent absence in one of the areas, together with the possibility of fatal cytauxzoonosis in lynxes makes necessary disease risks to be taken into account in management conservation strategies, such as translocations and re-introductions.

Immunohistochemical analysis of beta3 integrin (CD61): expression in pig tissues and human tumors.

CD61 is a membrane glycoprotein that associates with CD41 (alphaIIb) to form the heterodimeric complex gpIIb/IIIa (CD41/CD61), predominantly expressed in platelets and megakariocytes. CD61 or beta3 integrin also associates with alpha v (CD51) to form the vitronectin receptor, which is expressed in many tissues. We have used a monoclonal antibody against the porcine gpIIIa or CD61 (JM2E5) to study the distribution of this molecule in different normal pig tissues. As in humans, CD61 was broadly expressed in all tissues examined. In the kidney, strong expression of CD61 was observed in epithelial cells from renal tubules. In the testis, CD61 expression was detected in the Leydig cells. However, in liver, CD61 was weak or not detected. Many integrins are particularly involved in tumogenicity and in tumor progression mediating cell-cell interaction. Immunofluorescence experiments using cultured human tumor HeLa cells showed nuclear and cytoplasmic staining of mAb JM2E5. Immunohistochemical analysis of human tumor sections from several organs showed a heterogeneus distribution in metastatic cases from colon and breast carcinoma. However, no staining was found in metastasis from melanoma.

Overview of the Third International Workshop on Swine Leukocyte Differentiation Antigens.

The aim of the Third International Workshop on Swine Leukocyte Differentiation Antigens (CD workshop), supported by the Veterinary Immunology Committee (VIC) of the International Union of Immunological Societies (IUIS), was to standardize the assignment of monoclonal antibodies (mAb) reactive with porcine leukocyte differentiation antigens and to define new antibody clusters, using nomenclature in accordance with human and ruminant CD nomenclature, as agreed at the summary meeting of the Second International Swine CD Workshop in Davis, 1995: only mAb with proven reactivity for the orthologous porcine gene product or cross-reactivity for the human gene products, were given the full CD nomenclature, all other allocations were prefixed with "w". As in previous workshops, the overall organization was entrusted to the chair and first author, with support by the chair of the previous workshop and second author. In addition to the existing 26 pig leukocyte CD/SWC determinants established in previous workshops, this workshop established/confirmed another 11 CDs for pig leukocytes, identified by a total of 21 mAb: CD11R1 (2 mAb), CD11R2 (1 mAb), CD11R3 (4 mAb), wCD40 (1 mAb), wCD46 (4 mAb), wCD47 (3 mAb), wCD49d (1 mAb), CD61 (1 mAb), wCD92 (1 mAb), wCD93 (1 mAb) and CD163 (2 mAb).

Summary of the first round analyses of the Third International Workshop on Swine Leukocyte Differentiation Antigens.

The reactivity of 155 monoclonal antibodies submitted to the Third International Workshop on Swine Leukocyte Differentiation Antigens, together with 41 internal standards, was analysed by flow cytometry on 29 different pig cell targets as well as two human cell targets as a means of establishing suitable panels of monoclonal antibodies for more detailed clustering analyses by the various subsections of the workshop. Results were collected either without further gating, with gating based on FS/SS characteristics or with gating based on the co-expression of a reference antibody in two-colour flow cytometry. The CD or SWC reactivity of the internal standards had been established in previous workshops. Data sets were subsequently analysed by statistical clustering using the Leucocyte Typing Database IV software. The resulting 18 cluster groups were allocated to the appropriate second round sections of the workshop, after reviewing the overall cellular reactivity of each cluster as well as the specificity of known standards which clustered in a group.

Expression of CD61 (beta3 integrin subunit) on canine cells.

A monoclonal antibody (JM2E5) specific for the integrin beta3 chain, or CD61 or GPIIIa subunit, has been employed to determine the expression of the canine homologue CD41/CD61 or CD51/CD61 complex on different canine cells in peripheral blood lymphocytes, monocytes, granulocytes, platelets, erythrocytes, lymph-node cells, spleen cells and breast tumour cells). The canine homologue CD41/CD61 or CD51/61 was present on peripheral blood lymphocytes, monocytes, granulocytes, breast tumour cells and spleen cells as well as on platelets and it was absent from erythrocytes and lymph-node cells. An antigen with components of molecular masses of 25/100/120 kDa (under reducing conditions) was immunoprecipitated from canine peripheral lymphocytes and platelets, but not from granulocytes or monocytes. Expression on canine lymphocytes of the canine homologue of the human beta3 integrin chain was unexpected, based on the expression pattern of this molecule in human tissue.

Role and regulation of pig CD59 and membrane cofactor protein/CD46 expressed on pig aortic endothelial cells.

BACKGROUND: Hyperacute rejection in xenotransplantation is caused by activation of complement (C) on endothelium. We have previously shown that purified C-regulators of the pig (CD59 and membrane cofactor protein [MCP]) are efficient regulators of human C (HuC). The aim of this study was to clarify the role of endogenously expressed C-regulatory molecules on pig endothelium in the protection against hyperacute rejection. METHODS: Porcine aortic endothelial cells (PAEC) were harvested and cultured for various passages. PAEC were examined for the expression of endogenous pig CD59 and MCP by flow cytometry. PAEC were assessed for their susceptibility to lysis by HuC. The effect of phorbol 12-myristate 13-acetate and various cytokines on the expression of MCP and CD59 and C-susceptibility was assessed. RESULTS: Primary PAEC showed an initial high level of expression of pig CD59, however, upon culturing, CD59 levels decreased dramatically to about 20% after five passages. In contrast, levels of MCP doubled upon culturing of PAEC to confluency and remained stable during at least five passages. Primary cells and cells in the early passages were more resistant to HuC than cells that were cultured for longer. Blocking the function of CD59 but not of MCP using monoclonal antibody increased the susceptibility to HuC. Purified human CD59 incorporated to a level of expression similar to that of pig CD59 reversed the increased C-susceptibility, suggesting that pig and human CD59 are similarly protective against HuC. Increase of C-resistance and of expression of pig MCP, but not of CD59, was achieved upon incubation with phorbol 12-myristate 13-acetate. Tumor necrosis factor-alpha, interleukin-1beta, interleukin-4, or interferon-gamma had no effect on C-regulator expression or C-susceptibility. CONCLUSIONS: These data demonstrate the importance of using primary PAEC or cells in the first passages of culturing in in vitro models of xenotransplantation and show that pig MCP and, in particular, pig CD59 play an important role in protection of PAEC from HuC.

Pigs express multiple forms of decay-accelerating factor (CD55), all of which contain only three short consensus repeats.

We report the cloning of cDNAs encoding multiple isoforms of the pig analogue of human decay-accelerating factor (DAF; CD55). Screening of a pig muscle cDNA library using a human DAF probe identified a single clone that encoded a DAF-like molecule comprising three short consensus repeats (SCR) homologous with the amino-terminal three SCR in human DAF, a serine/threonine-rich (ST) region, and sequence compatible with a transmembrane domain and cytoplasmic tail. Northern blot and RT-PCR analysis showed that pig DAF was expressed in a wide range of tissues. Additional isoforms of DAF were sought using RT-PCR and 3'-rapid amplification of cDNA ends followed by sequencing. Isoforms containing a GPI anchor and with differing lengths of ST region were identified; no isoform containing a fourth SCR was found. Cloning of the GPI-anchored isoform from granulocytes confirmed that it was identical with the original transmembrane isoform through the three SCR and first portion of ST and was derived from a frame shift caused by splicing out 176 bp of sequence. A panel of mAbs was generated and used to analyze the distribution and anchoring of pig DAF in circulating cells. Pig DAF was expressed on all circulating cells and was transmembrane anchored on erythrocytes, but completely or partially GPI anchored on granulocytes and mononuclear cells. The transmembrane isoform of pig DAF was expressed on Chinese hamster ovary cells and was shown to affect regulatory activity for the classical pathway of human complement, but was only marginally active against pig serum.

Distribution of membrane cofactor protein (MCP/CD46) on pig tissues. Relevance To xenotransplantation.

Membrane cofactor protein (MCP; CD46) is a 50-60 000 MW glycoprotein, expressed on a wide variety of cells and tissues in man, which plays an important role in regulating complement activation. Human MCP has also been shown to be the receptor for measles virus. We have recently identified the pig analogue of MCP and demonstrated that pig MCP has cofactor activity for factor I-mediated cleavage of C3b when these components are derived either from pig or human. As a consequence, pig MCP is an efficient regulator of the classic and alternative pathways of human and pig complement. In order to define the potential importance of MCP in protecting against complement activation in the pig, we have conducted a comprehensive survey of its distribution in pig cells and organs. As in humans, MCP in the pig is broadly and abundantly distributed. Pig MCP is highly expressed on all circulating cells, including erythrocytes, in contrast to its absence on human erythrocytes. Multiple isoforms of MCP are found on cells and in tissues, probably representing products of alternative splicing analogous to those found in man. MCP is abundantly expressed throughout all tissues examined with particularly strong staining on the vascular endothelium. Connective tissue elements within liver and testis are also strongly stained by anti-pig MCP antibodies. Pig MCP is expressed only weakly on skeletal muscle cells and expression is absent from smooth muscle cells in the lung and vessel walls, sites at which human MCP is expressed. Of particular note, MCP is not expressed in B-cell areas of the germinal centres of lymph nodes.

Epitope mapping of 10 monoclonal antibodies against the pig analogue of human membrane cofactor protein (MCP).

Pig membrane cofactor protein (MCP; CD46) is a 50 000-60 000 MW glycoprotein that is expressed on a wide variety of cells, including erythrocytes. Pig MCP has cofactor activity for factor I-mediated cleavage of C3b and is an efficient regulator of the classical and alternative pathway of human and pig complement. A panel of 10 monoclonal antibodies (mAbs) was collected from two different laboratories; all of these mAbs were raised against pig leucocytes and all recognized the same complex banding pattern on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of erythrocyte membranes. All were shown to be reactive with pig MCP and were divided into four groups of mutually competitive antibodies based on competition studies for membrane-bound MCP and for soluble MCP, the latter by surface plasmon resonance (SPR) analysis. The antigenic properties of membrane-bound and soluble MCP were similar, although some interesting differences were revealed. None of the 10 mAbs were cross-reactive with human MCP and only one showed cross-reactivity with leucocytes from a panel of large mammals - a weak cross-reactivity with a subset of dog leucocytes. All antibodies in one of the epitope groups and some in a second epitope group were able to block the functional activity of pig MCP, as measured by inhibition of MCP-catalysed C3 degradation by factor I.

A monoclonal antibody raised against pig leukocytes and platelets recognizes fibrinogen from pig plasma.

Monoclonal antibody (mab) JM7E6 was produced through immunization of mice with porcine peripheral blood mononuclear cells and platelets. Biochemical characterization of the antigen showed three bands of 48, 55 and 60 kDa approximately, under reducing condition, and a single band greater than 200 kDa, under non-reducing condition. The antigen distribution among leukocyte subpopulations was reduced, but abundant in platelets, which suggests the recognition of a platelet antigen. However, immunohistochemical analysis of paraffin-embedded porcine tissues showed reactivity on blood vessel plasma, and indicates recognition of a plasma protein. ELISA and immunoblotting techniques, which were performed with commercially available porcine fibrinogen, not only confirmed the identification of this antigen, but also localized the epitope recognized by JM7E6 in the fibrinogen gamma light chain. JM7E6 failed to recognize human, ovine, bovine and dog fibrinogen molecules, thus showing species specificity of the epitope recognized by this antibody. Since JM7E6 is able to precipitate fibrinogen molecules from porcine leukocytes and platelets, it may be a valuable tool for some interesting clinical applications.

Characterization of the porcine homologue to human platelet glycoprotein IIb-IIIa (CD41/CD61) by a monoclonal antibody.

Human gpIIb-IIIa or CD41/CD61 is a Ca(2+)-complex dependent heterodimer, abundant on platelets, that plays a key role in hemostasis. This report describes a murine monoclonal antibody, JM2E5, able to recognize and immunoprecipitate the gpIIb/IIIa surface glycoprotein from porcine platelets. Immunoprecipitation analysis showed an antigen molecular weight of 115 and 85 kDa under nonreducing conditions, and of 110, 100 and 25 kDa under reducing conditions. Immunohistochemistry analyses of frozen sections from several porcine lymphoid organs gave specific staining on platelets. EDTA treated platelets were studied by flow cytometry indicating that the epitope recognized was Ca(2+)-complex independent. Western-blotting experiments with porcine platelet extracts gave an antigen molecular weight of 85 kDa under nonreducing conditions, thus localizing the epitope recognized by JM2E5 on the complex light chain gpIIIa or CD61. JM2E5 was also cross-reacting with human, bovine and horse platelets, as shown by flow cytometry. This mAb would allow further studies on this important adhesion molecule on horses, ruminants and pigs, and it should be especially useful as a general anti-porcine platelet reagent.

Purification and characterization of the pig analogue of human membrane cofactor protein (CD46/MCP).

A panel of mAbs were raised against pig lymphocytes. Seven mAbs immunoprecipitated a 50- to 60-kDa membrane-bound protein. This protein, termed JM4C8-Ag, was expressed on a wide variety of cells, including all circulating cells and cells of fibroblast, epithelial, and endothelial origin. The JM4C8-Ag was transmembrane-anchored and glycosylated. One of the Abs was used in immunoaffinity chromatography to isolate JM4C8-Ag from erythrocyte membranes. N-terminal amino acid analysis through the first 28 residues showed a 43% homology with the human complement regulatory molecule membrane cofactor protein (MCP; CD46). The purified protein had cofactor activity for factor I-mediated cleavage of human and pig C3b, confirming its identity as the pig analogue of human MCP. The purified protein also strongly inhibited lysis of rabbit erythrocytes by human and pig complement after activation of the classical or alternative pathway. This is the first report of a nonprimate analogue of MCP. The presence of a resident MCP on pig cells capable of acting as a cofactor in the control of human complement activation has consequences for the use of pig organs in xenotransplantation.

Platelet activation studies with anti-CD41/61 monoclonal antibodies.

The purpose of this study was to monitor platelet activation by following alpha IIb beta 3 integrin (GpIIb/IIIa complex or CD41/CD61) on the platelet surface by flow cytometry (FCM) analysis using workshop mAbs. The results obtained with the mAbs showed increased expression of the GpIIb/IIIa complex (about 40%) on the platelet membrane surface under thrombin stimulation.