"Detection of serum antibodies specific to Mycobacterium avium subsp. paratuberculosis in Rhea americana"
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1
Universidad de Buenos Aires, Cátedra de Inmunología, Facultad de Ciencias Veterinarias, Argentina
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2
Instituto Nacional de Tecnología Agropecuaria, EEA Balcarce, Argentina
INTRODUCTION
The health status is a determining factor in a herd, which in turn affects the quality of agro-food, as beef and milk. Paratuberculosis is an economically important disease affecting livestock and its productivity in our country. It is chronic, progressive infectious granulomatous enteritis in domestic ruminants and in wild mammals such as rabbits, rats, foxes, badgers and stoats. It has been associated with chronic inflammatory process of the human intestine known as Crohn's disease. The disease is caused by Mycobacterium avium subsp. paratuberculosis (Map), a ubiquitous microorganism with high resistance to adverse environments that facilitates its maintenance on the soil and the contamination of animal products and byproducts.
The most important mycobacteria affecting birds are grouped under the Mycobacterium avium Complex, which is comprised of M. avium subsp. avium, M. avium subsp. paratuberculosis (Map) and M. avium subsp. sylvaticum. Map has been isolated from healthy individuals of many bird species such as crows, sparrows and starlings, seagulls and other aquatic birds in Europe as well as in America, and a few cases of avian mycobacteriosis have been informed in farms and zoos involving ratites as rheas, emus and ostriches.
The objective of our work is to determine the presence of serum antibodies specific to Map in greater rhea (Rhea americana), a flightless bird native to South America. Our ultimate goal is to study the potential role of these birds as source of Map infection, due to the vicinity of their populations to livestock farms.
MATERIALS AND METHODS
A total of 55 birds belonging to a breeding farm located in Buenos Aires province were sampled: 19 chicks (younger than 6 month old); 30 juveniles (6-18 month old) and 6 adults (older than 18 month old). Blood was collected without anticoagulant by puncturing the brachial wing vein and serum was obtained and frozen at -20ºC until use.
All sera were pre-absorbed to minimize cross-reactions due to the immune response to saprophytic mycobacteria. To perform absorption, 25 μl of each serum to be tested were mixed with 100 μl of Mycobacterium phlei in a concentration adjusted to an optical density equal to 1, measured at 600 nm (OD 600 nm = 1), and incubated under constant stirring for 1 hour at 37 °C plus additional 16 hours at 4 ºC. The mixtures were centrifuged at 10,000 rpm for 30 min at 4 ºC; sera were then recovered from the supernatants and 20-fold diluted in 5% skim milk-PBS to achieve the working dilution of 1:100.
ELISA plates were coated with protoplasmic paratuberculosis antigen (PPA, Allied Monitor, Fayette, USA), and incubated for 16 hours at 4 °C. Then, plates were blocked with 10% skim milk in PBS during 1 hour at 37 ºC. Sample and control sera were added at a 1:100 dilution and incubated during 1 hour at 37 ºC, after which specific serum antibodies were identified through a goat serum specific to rhea-IgY (at a 1:500 dilution) that has been obtained in our laboratory, followed by a commercial peroxidase-labelled anti-goat IgG (Sigma-Aldrich, St. Louis, USA) at a 1:1000 dilution. Reference cattle sera that had been characterized as positive and negative to Map, were used as positive and negative controls. These sera were identified with a commercial peroxidase-labelled anti-bovine IgG (KPL, Gaithersburg, USA) diluted 1:1000. All dilutions were performed in 5% skim milk in PBS. The reactions were developed with ortho-phenylenediamine (OPD, Sigma-Aldrich), stopped with 0.5 M sulfuric acid, and read at 490 nm.
In addition, 12 stool samples were taken from recent depositions, as we were unable to collect feces directly from the cloaca. Samples were collected with clean disposable spoons, pooled and cultured in search for mycobacteria. Two grams of pooled feces were placed in a sterile tube and incubated with 40 ml of a sterile solution of 0.75% hexadecylpyridinium chloride (HPC, Sigma-Aldrich) for 30 minutes under constant stirring; the tube was incubated still for further 24 hours at room temperature to let the sediment to settle. The inoculum was aspirated from the interface between the supernatant and the sediment using a sterile Pasteur pipette, seeded onto slants containing Herrold’s medium supplemented with pyruvate (Sigma-Aldrich), mycobactin J (Allied Monitor) and amphotericin-B (Sigma), and incubated at 37 °C for a total of 20 weeks, during which tubes were observed every 15 days.
RESULTS
We could see that 10 (3 juveniles and 7 adults) out of the studied 55 rhea showed OD values > 0.450 with raw sera, while only 4 animals (1 juvenile and 3 adults) showed high OD values (0.412-0.833) in the absorbed PPA-ELISA. All the chicks gave OD values < 0.250 with raw as well as absorbed sera.
After nearly five months of fecal culture, colonies with typical Map morphology were observed on slants. Smears were performed, and acid-fast coccoid bacteria were identified by Ziehl Neelsen staining.
DISCUSSION
The serological tests commonly used for paratuberculosis in cattle, as indicated by the OIE terrestrial animal health code, are absorbed enzyme-linked immunosorbent assay (ELISA), together with agar gel immunodiffusion and complement fixation; the use of PPA as antigen to coat ELISA plates has been internationally endorsed. This absorbed PPA-ELISA is a technique carried out at our laboratory as a routine diagnostic practice; it has been demonstrated that it is useful to diagnose paratuberculosis in cattle, and that its sensitivity can vary according the antibody isotype that is identified. Indeed, it was found that the detection of specific IgG2 against PPA allowed improving the detection of infected cattle which underwent subclinical stage.
Reliable data on the Map-infection status of rheas are not available to date, so we could not get true positive and negative rhea control sera. To counteract this inconvenience, we have been using reference bovine sera as positive and negative controls in each PPA-ELISA test. We could not either perform statistical analyses; nevertheless, the results presented herein are preliminary but encouraging, as we have found differences in the OD values between animals, and all the results have been consistent among replicates in three independently performed experiments.
The gold standard for the diagnosis of paratuberculosis, as determined by the OIE, is the isolation of Map from fecal samples. In cultures performed from pooled feces samples obtained from a rhea farm, small colonies appeared by the 5th month of culture. This growth rate is slightly slower than that observed in colonies that come from cattle fecal samples. Ziehl Neelsen staining showed acid-fast cocci; it has been described that some species of nontuberculous mycobacteria, as those belonging to the avium complex, can be pleomorphic and show generally a coccoid appearance.
It will be necessary to study a larger number of both serum and fecal samples. In addition, we hope to obtain fecal samples from the cloaca of individuals that show high OD values in absorbed PPA-ELISA, in order to determine the biological significance of these results.
FINAL CONSIDERATIONS
The standardization of an indirect ELISA that allowed identifying the presence of specific Abs against Map in greater rheas is the first step in the development of a competitive PPA-ELISA that could be used as a serological diagnostic test in wild birds. The possibility of carrying out fecal cultures and characterization of growing mycobacteria, together with the identification of the humoral immune response to PPA, would allow us to assess the actual health status in wild populations in terms of infections with Mycobacterium avium subsp. paratuberculosis in our country.
Acknowledgements
This work was financially supported by Universidad de Buenos Aires (UBASeCyT 20020120200006BA and 20020130100607BA).
Keywords:
Paratuberculosis,
Diagnostic Tests, Routine,
reservoirs,
Rhea americana,
PPA-ELISA
Conference:
IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología, Medellin, Colombia, 13 Oct - 16 Oct, 2015.
Presentation Type:
Poster Presentation
Topic:
Veterinary and Comparative Immunology
Citation:
Jar
AM,
Redondo
F,
Ingratta
GG,
Maceira
NO and
Mundo
SL
(2015). "Detection of serum antibodies specific to Mycobacterium avium subsp. paratuberculosis in Rhea americana".
Front. Immunol.
Conference Abstract:
IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología.
doi: 10.3389/conf.fimmu.2015.05.00233
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Received:
29 May 2015;
Published Online:
14 Sep 2015.
*
Correspondence:
Dr. Ana M Jar, Universidad de Buenos Aires, Cátedra de Inmunología, Facultad de Ciencias Veterinarias, Buenos Aires, Argentina, anamjar2@yahoo.com