Curr Microbiol (2015) 70:685–689 DOI 10.1007/s00284-014-0772-8
Evaluation of Two Commercially Available Immunological Kits for the Diagnosis of Helicobacter spp. in Bottlenose Dolphins (Tursiops truncatus) Marı´a Jose´ Bernal-Guadarrama • Nuhacet Ferna´ndez-Gallardo • Rafael Zamora-Padro´n • Vı´ctor Pacheco • Marı´a Reyes-Batlle • Basilio Valladares Jacob Lorenzo-Morales • Enrique Martı´nez-Carretero
•
Received: 29 April 2014 / Accepted: 4 December 2014 / Published online: 13 January 2015 Ó Springer Science+Business Media New York 2015
Abstract Helicobacter pylori is considered to be responsible for the most common gastric infections in humans worldwide. In animals, other Helicobacter species are linked to gastritis with and without the presence of ulcers in their respective hosts. Moreover, gastric ulcers have been reported for decades in wild and captive dolphins. Clinical signs include lack of appetite, anorexia, abdominal tenderness, depression, and occasional unresponsiveness. In this study, serum and stool of nine bottlenose dolphins from Loro Parque collection Tenerife, Spain were examined for the presence of Helicobacter spp. The aim of our study was to evaluate the use of two commercially available kits for the detection of H. pylori in humans: a stool antigen immunoassay (Letitest H. pylori CARD) and a Western blot assay (EUROLINE-WB H. pylori) that were adapted to identify specific Helicobacter spp. antibodies in the tested Loro Parque bottlenose dolphin collection. The utility of these diagnostic kits for their application in dolphins is demonstrated, and their use in the future for the diagnosis of Helicobacter spp. in both wild and captive dolphins is proposed in this study.
M. J. Bernal-Guadarrama (&) N. Ferna´ndez-Gallardo R. Zamora-Padro´n V. Pacheco Loro Parque, Avda. Loro Parque SN, 38400, Puerto de la Cruz, Tenerife, Spain e-mail:
[email protected] M. J. Bernal-Guadarrama M. Reyes-Batlle B. Valladares J. Lorenzo-Morales E. Martı´nez-Carretero University Institute of Tropical Diseases and Public Health of the Canary Islands, University of La Laguna, Avda. Astrofı´sico Fco. Sa´nchez S/N, 38203, La Laguna, Tenerife, Canary Islands, Spain
Introduction A variety of Helicobacter species have been identified from the gastrointestinal tract of many vertebrate species, including marine mammals. However, because of their fastidious conditions for growth, the isolation of these microorganisms continues to be challenging [8, 17]. Several species of Helicobacter have been reported to occur in association with conditions of gastrointestinal disease and may infect more than one host species [11]. Although most of Helicobacter species affecting land animals are widely known, a few species have also have been discovered in marine mammals. Of particular note is H. cetorum from marine mammals, defined to date primarily by its 16S rDNA sequences [6], which are more closely related to those of H. pylori and the big cat pathogen H. acinonychis [4] than to those of other known species. Moreover, PCR and 16S rDNA sequence data indicate that H. cetorum is present in oceans worldwide [6, 12]. Helicobacter cetorum and other Helicobacter species have been previously detected in stomach, stool, dental plaque, and gastric fluid of many marine mammals, including dolphins (Tursiops spp., Lagenorhynchus acutus, L. obliquidens, Delphinus delphis), beluga whales (Delphinapterus leucas), and seals (Phoca groenlandica, Arctocephalus spp.) [6, 9, 11, 19]. This is of particular importance in animals in captivity setting where the detection of such agents is essential for the diagnosis and treatment of disease [1]. Despite being highly prevalent in humans and other animals, the modes by which Helicobacter species are transmitted remains uncertain. Its isolation from dental plaque, saliva, and feces from humans suggests both oral– oral and fecal–oral transmission for H. pylori [15, 18]. Although the oral cavity could act as a primary bacterial
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reservoir [20], regurgitation of contaminated gastric juice also could be responsible for the local presence of Helicobacter [13]. In marine mammals, similar routes of transmission could be present for the Helicobacter spp. that have been isolated from feces [10] and detected in the dental plaque of captive dolphins [5]. Moreover, the detection of Helicobacter DNA has also been previously reported from regurgitated fish otoliths from marine mammals and also from water from pools inhabited by captive cetaceans and pinnipeds [7]. Among the reported clinical signs of Helicobacter spp. infection, the most common ones include lack of appetite, anorexia, abdominal tenderness, depression, and occasional unresponsiveness [10]. There are a variety of tests for the diagnosis of H. pylori infection, such as histological examination of gastric tissue samples, bacterial culture, rapid urease test, and PCR analysis of gastric tissue. However, these methods require the performance of invasive techniques such endoscopy [3, 14]. The dolphin stomach has three divisions: fore stomach, main stomach, and pylorus, which joins the duodenal ampulla. Lesions in the fore stomach and the cranial part of the main stomach can be observed by endoscopy, whereas the distal part of the main stomach and the pylorus are not accessible by endoscopy [9]. In contrary, there are other available tests that do not require endoscopy, such as the breath test, serological methods, PCR from the gastric juices, and ammonia detection [6, 10, 19]. In this study, the presence of Helicobacter spp. in nine bottlenose dolphins from Loro Parque collection, Tenerife, Spain was examined using two commercially available kits for the detection of H. pylori in humans which were adapted for their use in dolphins: a stool antigen immunoassay (Letitest H. pylori CARD) and a Western blot assay (EUROLINE-WB H. pylori) .
Materials and Methods Samples Stool and serum samples used in this study were obtained from nine dolphins (six females and three males) from Loro Parque collection, Tenerife, Spain (Table 1). Extractions were carried out after training of the animals by the staff from Loro Parque. Commercial Kits –
Letitest H. pylori CARD (Laboratorios Leti, Barcelona, Spain). The assay is an immunochromatographic test based on monoclonal antibodies specific to H. pylori
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antigens. The test was carried out as per manufacturer’s instructions. Briefly, the tube with the diluent and the stool sample were shaken to assure good sample dispersion. Five drops of sample were dispensed into the circular window marked with an arrow. The results were read after 10 min. If the result is negative, only one red band appears on the control line. If the sample is positive, a second red band also appears in the sample line. EUROLINE-WB H. pylori test (EUROIMMUN AG, Lu¨beck, Germany), a commercial anti-H. pylori Western blot kit was used as per manufacturer’s instructions with the exception of the application of an anti-dolphin IgG that was purified and generated by us in this study (described below). Briefly, the Western blot is based on a detergent extract of cultured H. pylori (strain ATCC 43504) separated according to molecular mass using discontinuous polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane. The blot strips contain a panel of antigens described as H. pylori-specific (p17, p19 (OMP), p26, p29 (UreA), p30 (OMP), p33, VacA (p95), and CagA (p120)) and also antigens that have been reported to be less specific owing to cross-reactivity [p41, p50, p54 (flagellin), p57 (HSp homolog), p66 (UreB), p67 (FSH), and p75] (Fig. 1). The positive criterion for this kit is the presence of a minimum of 2 bands corresponding to the listed proteins mentioned above.
Immunoglobulin Purification and Anti-dolphin IgG Generation In order to be able to apply the Western blot commercial kit (EUROLINE-WB H. pylori), antibody (dolphin’s immunoglobulins) purification was carried out by ionic interchange chromatography from a mix of 1 ml of serum from each dolphin of the Loro Parque collection as previously described by our laboratory [2]. Briefly, the mix was purified after washing it three times with Tris–HCl 0.1 M (pH 6.5) using a QAE Sephadex A-50 column (Pharmacia Biotech). A total of seven tubes were collected, each carrying a final volume of 500 ll. Protein quantification in each tube was carried out using the commercial kit Micro BCATM Protein Assay Reagent (Pierce Biotechnology, IL, USA). The obtained products from each wash were separated by electrophoresis in 10 % sodium dodecyl sulfatepolyacrylamide gel electrophoresis gels using the system Protean III (Bio-Rad) and stained for visualization using Coomassie blue (Merck). After that, tubes containing the higher concentrations of immunoglobulins were selected and were further purified by dialysis using a 16-mmdiameter cellulose membrane (dialysis tubing, cellulose membrane, Sigma, Tres Cantos, Madrid, Spain) with a
M. J. Bernal-Guadarrama et al.: Diagnosis of Helicobacter spp. in Bottlenose Dolphins
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Table 1 Obtained results with the EUROLINE-WB H. pylori test in the nine dolphins from Loro Parque collection included in this study Dolphin ref.
p120
p95
p75
p67
p66
p57
p54
P50
P41
P33
P30
P29
P26
P19
P17
Ruffles
?
?
-
-
?
?
?
?
?
-
?
?
-
?
?
Clara
?
?
-
-
?
-
-
-
-
-
?
-
-
-
-
Pacina
?
?
-
-
?
-
-
-
-
-
?
-
-
-
-
Taina
?
?
-
-
?
-
-
-
-
-
?
-
-
-
Luna Ce´sar
?
?
-
-
?
-
-
-
-
-
-
-
-
-
-
?
-
-
-
?
-
-
-
-
-
-
-
-
-
-
Sanibel
?
-
-
-
?
-
-
-
-
-
-
-
-
-
Paco
?
-
-
-
?
-
-
-
-
-
-
-
-
-
-
Joan
?
-
-
-
?
-
-
-
-
-
-
-
-
-
-
Band
Antigen
Specificity
120kDa
CagA,
CytotoxinA, associated protein, high specificity
p120 95kDa
VacA, p95
Vacuolizing cytotoxin A, high specificity
75kDa
p75
Unspecific
67kDa
FSH, p67
Flagellar Sheath Protein unspecific, because of cross reactivity to other bacteria
66kDa
UreB, p66
Heavy urease subunit, cross reactivity to other bacteria having urease
57kDa
HSP
Heat Shock Protein homolog, unspecific
homolog, p57 54kDa
Flagelin,
Flagellin, unspecific because of cross reactivity to other
p54
bacteria having flagella
50kDa
p50
Unspecific
41kDa
p41
Unspecific
33kDa
p33
Probably specific
30kDa
OMP, p30
Outer membrane Protein, species-specificity
29kDa
UreA, p29
Light urease subunit, high specificity
26kDa
p26
High specificity
19kDa
OMP, p19
Outer membrane Protein, species-specificity
17kDa
p17
Probably specific
Fig. 1 Results of EUROLINE-WB H. pylori test when dolphin reference Ruffles were tested for the time in this study (left). The table on the right describes the protein bands and their specificity to Helicobacter included in this test
retaining limit of 12 kDa and treated as indicated by Bernal-Guadarrama et al. [2]. The dialysis procedure continued with agitation for 24 h at 4 °C against a dialysis buffer of Tris–HCl 0.1 M (pH 6.5) 0.19, changing the
buffer three times during the process. Proteins thus obtained were lyophilized and kept at -20 °C until further use. After that two New Zealand rabbits, weighting around 3 kg, were used for immunization purposes. Vaccination
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M. J. Bernal-Guadarrama et al.: Diagnosis of Helicobacter spp. in Bottlenose Dolphins
Table 2 Obtained results with the EUROLINE-WB H. pylori test in the dolphin reference Ruffles after detecting the clinical symptoms of an Helicobacter infection for the second time in this animal (February 2008) Dolphin ref.
p120
p95
p75
p67
p66
p57
p54
p50
p41
p33
p30
p29
p26
p19
p17
Ruffles
?
?
-
-
?
?
?
?
-
?
?
-
?
?
?
was performed using three doses, leaving 2 weeks between each injection as follows: a first dose containing 500 lg of the emulsified protein with 500 ll of Freund’s complete adjuvant (CFA; Sigma) in a final total volume of 1 ml, while the subsequent two doses contained 500 lg of emulsified protein with 500 ll of Freund’s incomplete adjuvant (IFA; Sigma) in a final total volume of 1 ml. Injection was intramuscular, alternating legs between doses. A third rabbit, used as control, was inoculated once with CFA and twice with IFA in a final total volume of 1 ml. Rabbits were sacrificed and bled 45 days after the last injection of immunoglobulin as previously described [2].
Results and Discussion In order to validate the application of both commercial tests, the stool immunochromatographic test (Letitest H. pylori CARD) was firstly applied in the nine dolphins included in this study. It is important to mention that only two of the dolphins were showing any clinical signs of Helicobacter spp. infection (ref. Clara and Ruffles). These observed clinical signs included lack of appetite, abdominal tenderness intermittent regurgitation, and lethargy. Interestingly, only the dolphin reference Ruffles was positive for the Letitest H. pylori CARD test. After that serum samples from the dolphins with references Clara and Ruffles were tested by Western Blot using the EUROLINEWB H. pylori test. Regarding the dolphin with reference Clara, the Western blot kit was positive for the bands p120, p95 (human H. pylori highly specific bands), p66, and p30 (Table 1). In contrast, Ruffles reference was positive for the detection of the human highly specific bands p120, p95, and also the proteins p66, p33, p30, p29, p19, and p17 (Fig. 1; Table 1). Therefore, if proteins p120 and p95 are not considered, Ruffles was positive for more than two proteins reaching the positivity criterion of this kit (as described in the Materials and methods section). After that Ruffles was administered with 2 g of amoxicillin for 35 days and was tested again using both tests. Interestingly, the stool test was negative, whereas the Western blot kit was still positive and showing reaction with the same bands. In February 2008, Ruffles presented clinical signs of Helicobacter infection once more and thus both tests were
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performed again. The dolphin was positive for the Letitest H. pylori CARD test and the Western blot kit revealed the presence of the proteins p120, p95, p66, p57, p54, and p50 as well as p33, p30, p26, p19, and p17 (Table 2). Thus, the positivity criterion for the EUROLINE-WB H. pylori test in the tested dolphin was established (based on the obtained results) in the presence of more than two specific proteins of low molecular weight and the presence of proteins p57, p54, and p50. Regarding the rest of the dolphins included in this study, none of them were positive for the Letitest H. pylori CARD test and presented less than two specific proteins after performance of the EUROLINE-WB H. pylori test (Table 1). To the best of our knowledge, this is the first time that an approach as the one proposed in this study is used for the diagnosis of Helicobacter spp. infections in dolphins. Until now, only PCR-based approaches have been applied for the diagnosis in dolphins and other marine mammals. Both molecular and/or histological diagnosis of the pathogen require the performance of invasive techniques. Moreover, serology is also an invasive technique that requires drawing blood and also training of the animals to be able to perform it [9, 19]. Interestingly, many immunological-based approaches for the detection of this pathogen from stool or serum samples have been previously developed for the diagnosis of H. pylori in humans and are currently commercialized [16] and that should be evaluated and exploited for diagnostic purposes in other animals. Nevertheless, the evaluation of both commercial kits (Letitest H. pylori CARD and the EUROLINE-WB H. pylori test) in the tested dolphin collection has demonstrated that they could be used for the diagnosis and monitorization of Helicobacter spp. infections in dolphins. Furthermore, proteins p120 and p95 are not specific to Helicobacter spp. in dolphins. A positivity criterion in dolphins seems to be based in the presence of a minimum of two proteins from p33 to p17 based on the observed results with dolphin ref. Ruffles. Moreover, the presence of antibodies against p57, p54, and p50 could be related to the presence of Helicobacter spp. in dolphins. Nevertheless, further studies are needed including a higher number of dolphins in order to establish both commercial tests in a day-to-day basis for the detection of Helicobacter spp. at least in dolphins in captivity. However, the obtained results have validated the potential use of both kits and would be a base for the development and
M. J. Bernal-Guadarrama et al.: Diagnosis of Helicobacter spp. in Bottlenose Dolphins
application of further studies at least by Loro Parque and other similar zoological entities which would allow fast and non-aggressive detection of Helicobacter spp. infections in dolphins. Acknowledgments The authors are grateful to Loro Parque dolphin training team for their help in this work and also to Cristine Jo Dreiso¨rner and Andrea Ca´diz Ortiz from Loro Parque Veterinary Clinic; thanks to them, the collection of samples for the development of this work was possible. JLM was funded by a grant RYC-201108863 from the Ramo´n y Cajal Subprogramme of the Spanish Ministerio de Economı´a y Competitividad. MRB was funded by Becas de Investigacio´n Obra Social La Caixa-Fundacio´n Cajacanarias para Postgraduados 2014. The authors are also grateful to Loro Parque Fundacio´n for funding this research. Finally, we are grateful to the managing director of Loro Parque for allowing us to undertake this study.
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