Serodiagnostics of paratuberculosis Kari R. Lybeck
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1 Serodiagnostics of paratuberculosis Kari R. Lybeck Photo: Birte Graeber
2 Paratuberculosis Paratuberculosis Mycobacterium avium subsp. paratuberculosis (MAP) Chronic, granulomatous enteritis and lymphadenitis Production losses Threatens animal welfare MAP Highly resistant bacterium Survive for more than a year in the environment Difficult to control through preventive measures Crohn s disease in humans? Photo: Nils Leine
3 Hosts and transmission Hosts Domestic and wild ruminants plus camelids Young animals Other species may also be infected Transmission Faecal-oral Milk In utero
4 Development of the disease Long incubation (2-5 years or more) Stress factors->disease progression Some animals never develop clinical signs Animals may develop Reduced milk production Poor body condition ->emaciation Diarrhoea (cattle) Pathology Thickened intestinal wall with transverse folds Enlarged intestinal lymph nodes Photo: Mette Valheim
5 Immune responses to MAP Antibody production generally accredited little protective effect Cell-mediated immune responses important Interferon gamma (IFN-γ) essential Activation of macrophages MAP evades host defence Persistent infection-> continued recruitment of macrophages and lymphocytes->granulomatous inflammation Effective immune responses may control or clear infection Granulomatous inflammation in caprine intestine
6 Immune responses and faecal shedding I Cell-mediated immune response (IFN-γ) Humoral immune response (antibodies) Shedding of MAP in the faeces Time Infection Clinical signs Common belief: Cell-mediated immune responses during early stages Disease progression associated with increased faecal shedding, switch to humoral immune response, and waning cell-mediated immunity
7 References: Nielsen 10, Lybeck 11, Sweeney 12. Immune responses and faecal shedding II Early antibody production compared to IFN-γ responses in goat study 9% ELISA positive before IFN-γ positive 9% ELISA and IFN-γ positive simultaneously IgG2 antibodies-early stages May not be detected by standard ELISAs May indicate previous exposure IgG1 antibodies-progressive disease Plasma B-cell Shedding and antibodies detectable simultaneously Antibodies detectable before shedding or vice versa
8 Diagnosis of paratuberculosis Pathological examination Culture PCR Cell-mediated immunity (IFN-γ test) Antibody detection Combination of tests often used Low sensitivity of ante mortem tests in early stages Tests for cell-mediated immunity greatest potential Test strategy will depend on test purpose Disease control in infected herds Surveillance in presumed non-infected herds Photo: Berit Djønne
9 Antibody detection Antibodies often measurable from 2 to 6 years in cattle Testing before 2 years of age generally not recommended Influenced by dose, age at infection, MAP strain, species, natural versus experimental infection Goats: First antibody detection from 4-32 months (3 studies) Cattle study: Antibody positive 4.5 months after experimental infection at 12 months Younger cattle, longer time to become positive Reference : Mortier 14, Munjal 05, Stewart 06, Storset 01, Nielsen and Toft 08, OIE.
10 Lack of antibody response In the very late stages the antibody response may disappear Infected animals without antibodies in earlier stages Suggested these will not develop progressive disease Could still shed and transmit MAP 25% of culture positive goats negative on both IFN-γ and ELISA at all times Repeated MAP exposure or massive dose? In utero infection? References: Stabel 02, Stabel 09, Lybeck 11, Subharat 12.
11 Antibody tests Complement fixation (CF) and agar gel immunodiffusion (AGID) test Indirect ELISA most widely used today Absorbed ELISA better Se and Sp than CFT CFT still recommended by OIE Testing before export AGID lower Se and higher Sp compared with ELISA References: Bakker
12 Test evaluations Difficult to compare diagnostic test evaluations Variation in test antigens Animals in different disease stages A test should be evaluated in the specific population it is to be used Variation within and between laboratories Sample handling could influence result No optimal reference method for test evaluation Faecal culture often used Has itself unknown/low sensitivity in subclinical stages References: Adaska 02, Nielsen 02 and 10, Bakker 14, Alinovi 09, OIE.
13 ELISA test sensitivities I Reported accuracies of ELISAs have varied considerably Overall Se for MAP-infected animals 5-30% Se 50-90% in clinical stages Comparable accuracies in cattle, sheep, and goats Necessary to test sufficient number of adult animals (80?) to suggest low MAP prevalence References: Nielsen and Toft 08, Sergeant 08, Sweeney 12. Photo: Siri K. Sjurseth
14 ELISA test sensitivities II Organism detection claimed to have better sensitivity than ELISA Organism detection recommended in low prevalence herds Lower sensitively for ELISA not supported by a review of diagnostic tests accuracies ELISA preferred to Ziehl Neelsen staining on faeces for confirming suspected clinical cases References: Collins 06, Nielsen and Toft 08, Weber 09, Sweeney 12. Photo: Berit Djønne
15 ELISA values Serum antibodies more constantly present and titre higher as lesions become more extensive ELISA values are well correlated with likelihood and level of infection ELISA results often reported as positive or negative Information might be lost Considering the magnitude of the ELISA value recommended References: Collins 02, van Schaik 03, Lavers 15, Nilsen 07, Sweeney 12.
16 Test frequency Persistent or fluctuating antibody responses Frequent testing improves understanding of MAP status Important for identifying infected animals, possibly before shedding occurs A higher number of performed tests could lead to a higher number of false positive reactions Longer test interval, more animals likely to have seroconverted References: Hirst 02, Nielsen 10, Lu 08, Mortier 2014, Lavers 15.
17 False positive reactions I Specificity has generally been higher than 95% Confirmatory organism detecting test often used Antigens shared by MAP and other mycobacteria Cross-reacting antibodies to atypical mycobacteria reduced by preabsorption of sera with Mycobacterium phlei Other M. avium subspecies could give false positive reactions Absorption may negatively affect sensitivity The type of environmental mycobacteria could vary between geographic areas References: Bech-Neilson 92. Fredriksen 04, Nielsen 10,Nielsen and Toft 08.
18 False positive reactions II M. bovis infection can affect serological tests for MAP MAP ELISA values were significantly higher after skin testing for days (serum) MAP vaccination will result in positive antibody tests A positive ELISA not confirmed by culture or PCR need not be a false-positive Antibodies may be detectable before shedding Animals that control or have cleared the infection could be seropositive References: Nielsen 10, Bech-Neilson 92, Kennedy 14, Varges 09.
19 Milk ELISA Se and Sp regarded as comparable to serum Milk ELISA suggested most suitable in high prevalence herds Herds with < 2% prevalence considered likely false positives Serum ELISA: More likely positive during late lactation Milk ELISA: More likely positive during early and late lactation compared to mid-lactation Milk yield may significantly influence ELISA values Including milk yield in result interpretation suggested References: Nielsen 02, 10,13, Lavers 14, van Weering 07, Eisenberg 15.
20 Colostrum ELISA more likely positive when using colostrum Increased identification of infected animals? Binding of non-specific protein to ELISA wells suggested Non-specific bindings was low (0.8%) Indicate higher levels of MAP-specific antibodies in colostrum Further studies for determining Se and Sp suggested Decide on the use of colostrum batches to prevent transmission References: Nielsen 08, Nielsen 12, Zervens 13, Bakker 13.
21 Bulk tank milk Bulk tank milk ELISA performs relatively well when the withinherd prevalence is high Herds with a low prevalence may not be detected Few animals with antibodies, and variable levels in each individual Maximum S/P % for bulk milk much lower than for individual milk No clear separation of values to indicate likely infected and not likely infected References: Nielsen 00, 13 study, van Weering 07, Sweeney 12.
22 Antigens I The antigen influence test sensitivity and specificity Protoplasmatic antigens, lipid fractions, PPDj, extracts, and more Ongoing search for improved antigens MAP-specific and not require serum absorption Increase sensitivity in early stages Useful throughout the course of the disease Mix of antigens possible Could compromise test specificity References: Bakker 14. Li 2005
23 Antigens II Serological testing in early stages claimed successful using new antigens Antibody detection after 70 days with MAP-specific antigens 2 weeks with MAP stress-associated recombinant proteins ELISA using ethanol extract of MAP (EVELISA) claimed to reliably detect antibodies in low-shedding animals In general, new antigens have not lived up to their expectations References: Bakker 14, 06, Bannentine 08, Scott 10. Kawaji 12.
24 Thank you! Photo: Elisabeth Dahl Nybø
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