PARATUBERCULOSIS: AN UPDATE

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1 This manuscript has been published in the IVIS website with the permission of the congress organizers. To return to the Table of Content click here or go to PARATUBERCULOSIS: AN UPDATE J.R. Stabel USDA-ARS-National Animal Disease Center, Ames, USA 1. INTRODUCTION The disorder known as paratuberculosis was first described in 1895 by Johne and Frothingham (Johne & Frothingham, 1895). They identified acid-fast staining organisms in granulomatous lesions in the intestines of affected cattle, indicating some type of mycobacterial organism. In 1910, the organism was cultured from cattle and classified as a mycobacterium by Twort (Twort, 1910; Twort & Ingram, 1912). The organism was fully characterized several years later and named Mycobacterium paratuberculosis (later changed to Mycobacterium avium subsp. paratuberculosis). Paratuberculosis is widely distributed both nationally and internationally in domestic ruminants such as cattle, sheep, and goats, as well as wildlife, such as deer, antelope, and bison. The prevalence of the disease in the US is unknown because comprehensive studies have not been conducted to date. In 1996, the National Animal Health Monitoring System conducted a survey of dairy farms in 20 states in the US using serologic analysis and results yielded estimates of 20-40% of dairy herds had some level of infection within the herd (Wells et al. 1998). At the time of that study, it was estimated that annual losses in the US from paratuberculosis in dairy herds would exceed $200 million. Economic losses due to paratuberculosis result from early culling or death from clinical disease, reduced reproductive efficiency and feed efficiency, and decreased milk production (Johnson-Ifearulundu et al. 1999; Ott et al. 1999). The rising incidence of paratuberculosis in the past 10 years suggests that this figure sorely underestimates the current financial burden of this disease. The primary route of transmission of infection is through ingestion of fecal matter, milk or colostrum containing M. paratuberculosis microorganisms, although in utero transmission can occur as well. It is generally thought that neonates are the most susceptible to infection due their undeveloped immune system and the degree of exposure to M. paratuberculosis if their dams are clinically infected (Larsen et al. 1975). However, it has also been demonstrated that adult animals can become infected after exposure to large doses of the bacteria (Rankin, 1962). After infection, animals can remain asymptomatic for long periods of time, up to 2 to 5 years post-infection. During this subclinical phase of infection, cattle will shed minimal amounts of M. paratuberculosis in their feces yet can still be a detriment to the herd due to stealthy contamination of the environment, possibly resulting in further spread of infection throughout the herd. During the clinical phas of

2 infection, fecal shedding of the microorganism is quite high and can exceed cfu/g of feces (Chiodini et al. 1984). The terminal clinical stage of disease is characterized by chronic diarrhea, rapid weight loss, diffuse edema, decreased milk production, and infertility. 2. DIAGNOSIS Diagnosis of paratuberculosis is difficult due to the fastidious growth pattern of the bacterium and the long periods of incubation required for microbiologic culture. Solid medium methods of culture generally require 8 to 12 weeks for growth, whereas incubation periods are reduced to 2 to 8 weeks using the more recent liquid medium methods of culture (Bactec, MGIT, Trek ESP). Since infected animals may shed the organisms intermittently in their feces, use of fecal culture alone as a diagnostic test may result in a misrepresentation of infection within the herd; between 25 to 50% of infected animals are detected by fecal culture (Whitlock et al. 2000; Nielsen et al. 2002). Serologic tests for diagnosis of paratuberculosis such as agar gel immunodiffusion (AGID), enzyme-linked immunosorbent assay (ELISA) and complement-fixation (CF) are relatively easy to perform but suffer from a lack of sensitivity (Colgrove et al. 1989; Clarke et al. 1996). As technology advances, this situation should improve. A recent report describing a flow cytometric assay to detect M. paratuberculosis demonstrated an increase in sensitivity for serologic detection (Eda et al. 2005). Still, antibody-based serologic tests may fall short of detecting animals in the early stages of infection due to a negligible humoral immune response (Stabel, 2000). Assays that measure cell-mediated immune function may be more appropriate detection tools in early infection. Stabel (1996) observed significantly higher secretion of IFN-χ by PBMC from naturally infected cows with subclinical disease compared to control noninfected cows after stimulation of cells with either concanavalin A, a T cell mitogen, or a whole cell sonicate of M. paratuberculosis. Studies to evaluate the sensitivity and specificity of two cell-mediated immune function tests, the IFN-χ assay and the intradermal skin test, in cattle and sheep have demonstrated promise for early detection (Robbe-Austerman, personal communication). Using tissue culture as a reference standard in known infected and negative sheep flocks, preliminary results suggest that the IFN-χ test has a sensitivity of 41% and a specificity of 98% using a M. paratuberculosis whole-cell sonicate as the antigen preparation. Results using a johnin purified protein derivative (JPPD) demonstrated wide variability, dependent upon which lot of JPPD was used. The IFN-χ test can be used for the diagnosis of paratuberculosis, but the specificity is hindered due to extensive cross-reactions with closely related mycobacteria ubiquitously present in the environment. Furthermore, it has been demonstrated that young calves and uninfected cattle often respond to mycobacterial whole-cell antigen mixtures or secreted proteins in the IFN-χ test without having any evidence of infection (McDonald et al. 1999). It is intuitive that the specificity could be improved by using specific antigens.however, few M. paratuberculosis-specific antigens have been described to date. The close genetic relationship between M. paratuberculosis and M. avium has hindered progress in the identification of specific antigens for M. paratuberculosis. In spite of this obstacle, the power of comparative genomic approaches has enabled the characterization of a few M. paratuberculosis-specific antigens as published by our laboratory within the past two years (Bannantine et al. 2004a; Bannantine et al. 2004b; Paustian et al. 2004). Finally, novel cell surface proteins on M. paratuberculosis, if antigenic, will also enable a more thorough characterization of the antigenic repertoire produced by this pathogen and provide targets to exploit, not only for diagnosis, but for vaccine development as well. Diagnosis of M. paratuberculosis infections is confounded by the genetic similarity between M. avium subsp. paratuberculosis and nonpathogenic environmental mycobacteria, especially other members of the Mycobacterium avium complex (MAC). MAC bacteria, comprising M. avium and M. intracellulare isolates, possess a high degree of genetic similarity, but are capable of infecting a

3 diverse range of host species. M. avium subsp. paratuberculosis is traditionally distinguished from other mycobacteria by its dependence on exogenous mycobactin, although intermittent mycobactin dependence has been reported among M. avium subsp. paratuberculosis isolates and within other species (Matthews & McDiarmid, 1979; Barclay and Ratledge, 1983). Sequencing of the genomes of isolates of M. avium subsp. avium and M. paratuberculosis has been completed (Li et al. 2005; Wu et al. 2006) and comparative analysis has demonstrated a homology of greater than 97% at the nucleotide level (Bannantine et al. 2002; Paustian et al. 2005). This has prompted work to define novel and specific antigens of M. paratuberculosis that can be utilized as diagnostic tools (Stabel & Bannanatine, 2005). Other novel coding sequences specific to M. paratuberculosis have demonstrated potential as serologic diagnostic tools in experimental format but will have to be field testing in valid formats to determine true value (Paustian et al. 2004). 3. HOST IMMUNITY Understanding the host response to infection with M. paratuberculosis is central to the development of better diagnostic tests, for the characterization of protective immunogens for use as vaccine candidates, and in the identification of potential therapeutic agents. A reciprocal relationship has been documented between the host T cell responses and the extent of disease during mycobacterial infections (Orme, 1993; Koets et al. 2002; Welsh et al. 2005). A temporal shift in Th1 to Th2-dominated immune responses has been demonstrated in both experimentally and naturally infected sheep and cattle with paratuberculosis (Thorel et al. 1992; Stabel, 2000). Th1-mediated immunity appears to be essential to keep infection from progressing from subclinical to clinical disease. Expresssion of cytokines by various Th cell populations is uniquely associated with the polarity of disease states in mycobacterial infections. In early studies in our laboratory, we found that activated peripheral blood mononuclear cells isolated from subclinically infected cows secrete higher levels of IFN-χ after stimulation with mitogens or M. paratuberculosis antigens compared to healthy control cows or clinically infected cows (Stabel, 1996; Stabel & Whitlock, 2001). Further, cells from clinically infected cows secreted lower levels of IFN-χ, TNF-α, and IL-2, cytokines associated with Th1-mediated responses, after stimulation with either concanavalin A or M. paratuberculosis antigen (Stabel, 2000). More recently, we demonstrated that cows in the latter stages of infection have upregulated gene expression of IL-10 and TGF-β as compared to healthy control cows or cows with subclinical disease (Khalifeh & Stabel, 2004). Further studies conducted with PBMC obtained from naturally infected cows, demonstrated a specific upregulation of IL-10 and TGF-β production in culture supernatants in response to stimulation with live M. paratuberculosis (Khalifeh & Stabel, 2004). Almost complete abrogration of IFN-χ secretion was noted after the addition of TGF-β or TGF-β plus IL-10 to cell cultures from healthy and subclinically infected cows, yet only a moderate reduction in IFN-χ was noted for clinically infected cows. Although infection of unfractionated PBMC with live M. paratuberculosis upregulated IFN-χ secretion in all animal groups, particularly healthy controls, the inhibitory effects of TGF-β and IL- 10 were most predominant in the cell cultures from healthy controls and subclinically infected cows. Compared to healthy cows, naturally infected animals had higher numbers of viable M. paratuberculosis recovered from their cultures after in vitro infection. The addition of exogenous IL-10 and TGF-β to PBMC isolated from healthy controls inhibited the bactericidal activity of these cells as evidenced by the increased number of viable M. paratuberculosis recovered from those cultures compared to cell cultures containing medium alone. These studies are the first to report significant effects of M. paratuberculosis on the production of the Treg cell cytokines, IL-10 and TGF-β. Previous reports demonstrated an upregulation of IL-10 gene expression in peripheral blood mononuclear cells from clinically infected cows compared to healthy control cows (Coussens et al. 2002) and increased expression of IL-10 was also noted in bovine monocyte-derived macrophages stimulated with live M. paratuberculosis (Weiss et al. 2002). Results from our studies are similar to those demonstrated with other mycobacterial diseases,

4 such as tuberculosis (Toosi et al. 1995; Toosi & Ellner, 1998). Cells from patients with advanced tuberculosis produced higher levels of TGF-β as compared to cells from patients in early stages of infection (Toosi et al. 1995; Othieno et al. 1999). Further, in vitro infection of cells from patients with tuberculosis with live M. tuberculosis stimulated production of IL-10 and TGF-β (Othieno et al. 1999). These data suggest an important immune regulatory role of IL-10 and TGF- β in mycobacterial infections, and that the effects observed with M. paratuberculosis may be directly related to their effects on macrophage activation via IFN-χ and killing of M. paratuberculosis. 4. VACCINATION Studies evaluating protective responses to vaccination for paratuberculosis have desmonstrated that both cellular and humoral immune responses are induced (Kohler et al. 2001; Muskens et al. 2002), yet vaccination does not prevent infection. Rather, vaccination for paratuberculosis effectively reduces fecal shedding of the bacterium and allays the onset of clinical signs (Kormendy, 1994; Wentink et al. 1994; Fridriksdottir et al. 2000; Kalis et al. 2001). In the US, only one commercial vaccine preparation is available for paratuberculosis and that consists of a heat-killed whole-cell suspension in oil. Although effective in some management systems as a control measure for paratuberculosis, it does induce unfavorable complications such as severe granulomatous lesions at the site of injection and an interference with skin testing and other serologic analyses. Internationally, commercial vaccines have fared much better and countries such as Spain, Australia, New Zealand, and The Netherlands promote the use of vaccination to control the spread of paratuberculosis. More recently, DNA vaccines have been proposed as an alternative approach to the whole-cell preparations. DNA vaccines offer the advantage of being inexpensive and simple to produce, are stable at a variety of temperatures, and do not contain elements that induce allergic or hyposensitivity responses, which are often associated with conventional vaccines (Babiuk et al. 2000). One study describing immunization of mice with an expression library demonstrated protection against colonization of M. paratuberculosis in tissues upon challenge (Huntley et al. 2005). Immunization of lambs with singular mycobacterial genes (p85a-mav, p85a-bcg; phsp65) cloned into a mammalian expression vector induced protective responses upon oral challenge with M. paratuberculosis (Sechi et al. 2006). 5. CONCLUSIONS Significant progress has been made in the field of paratuberculosis research in recent years. Sequencing of the M. paratuberculosis genome has generated information that can be utilized to select antigens for improved diagnostics and immunogens for vaccines. Further research involving new technologies such as DNA microarrays and proteomics will provide insights into hostpathogen interactions, and this along with a more thorough understanding of host immune responses to the pathogen will allow development of intervention strategies to control the spread of this disease. 6. SUMMARY Paratuberculosis (Johne s disease) is a chronic, progressive enteric disease of ruminants caused by infection with Mycobacterium avium subsp. paratuberculosis. Economic losses from this disease are estimated to be $200US/infected cow/year and are the result of animal culling, reduced milk production, poor reproductive performance, and reduced carcass value. Johne s disease has become a high priority disease in the cattle industry. The latest estimates of herd prevalence in the US range from 20 to 40% of dairy herds having at least one serologically positive animal. There are no adequate estimates of herd prevalence in beef cattle in the US. The economic impact of this disease on the dairy industry was estimated to be over $200 million per year in 1996 and is growing each year with the continued spread of this disease. In addition, M. avium subsp. paratuberculosis has

5 been implicated as a causative factor in Crohn s disease, a chronic inflammatory bowel disease of human beings, which has served as a further impetus to control this disease in our national cattle industry. One of the major objectives of research in paratuberculosis is to improve diagnostic tests for the detection of the disease. Improved detection will provide tools to reduced the environmental load of the bacterium and allay the spread of the disease within herds. Research on host immunology and pathogenesis to infections with M. avium subsp. paratuberculosis will allow design of more rational diagnostic and control procedures. 7. KEY WORDS Paratuberculosis, cattle, diagnostics, immunology. 8. RESUME La paratuberculose (Maladie de Johne) est une entérite chronique et progressive des ruminants due à une infection par Mycobacterium avium subsp. paratuberculosis. Les pertes économiques dues à cette maladie sont estimées à $200US/vache infectée/an et proviennent de la réforme des animaux, de la baisse de la production laitière, de la diminution de la fertilité et de la dépréciation des carcasses. La paratuberculose est devenue une maladie à haute priorité dans la filière bovine. Les dernières estimations de sa prévalence aux Etats-Unis sont de 20 à 40 % des troupeaux laitiers ayant au moins un animal séropositif. Mais il n existe aucune donnée concernant sa prévalence dans les élevages allaitants. L impact économique de cette maladie sur la filière laitière a été estimé supérieur à $200 millions par année en 1996 et augmente tous les ans du fait de l extension continue de la maladie. De plus, M. avium subsp. paratuberculosis a été reconnu comme agent causal dans la maladie de Crohn, une inflammation intestinale chronique de l homme, ceci ayant donné un nouvel élan pour la prévention de cette maladie dans la filière bovine. L un des objectifs majeurs de la recherche sur la paratuberculose est d améliorer les tests de diagnostic pour la détection de la maladie. Ceci permettrait de réduire la pression environnementale des bactéries et de modérer la dissémination de la maladie au sein des troupeaux. La recherche sur l immunologie de l hôte et sur la pathogenèse des infections à M. avium subsp. paratuberculosis permettra de concevoir des procédures de diagnostic et de prévention plus rationnelles. 9. MOTS CLES Paratuberculose, bovin, diagnostics, immunologie. 10. REFERENCES Babiuk LA, Babiuk S L et al. - Nucleic acid vaccines: research tool or commercial reality. Vet Immunol Immunopathol, 2000; 76:1-23. Bannantine JP, Baechler E et al. Genome scale comparison of Mycobacterium avium subsp. paratuberculosis with Mycobacterium avium subsp. avium reveals potential diagnostic sequences. J Clin Microbiol, 2002; 40: Bannantine JP, Barletta RG et al. Application of the genome sequence to address concerns that Mycobacterium avium subspecies paratuberculosis might be a foodborne pathogen. Foodborne Pathog Dis, 2004a; 1:3-15. Bannantine JP, Hansen JK et al. Expression and immunogenicity of proteins encoded by sequences specific to Mycobacterium avium subsp. paratuberculosis. J Clin Microbiol, 2004b; 42: Barclay R, Ratledge C. Iron-binding compounds of Mycobacterium avium, M. intracellulare, M. scrofulaceum, and mycobactin-dependent M. paratuberculosis and M. avium. J Bacteriol, 1983; 153: Chiodini RJ, Van Kruiningen HJ et al. Ruminant paratuberculosis (Johne s disease): the current status and future prospects. Cornell Vet, 1984; 74:

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