Arch Microbiol (2014) 196:785–790 DOI 10.1007/s00203-014-1020-0

ORIGINAL PAPER

Development of an indirect immunofluorescence technique for the evaluation of generated antibody titers against Erysipelothrix rhusiopathiae in captive bottlenose dolphins (Tursiops truncatus) María José Bernal‑Guadarrama · Daniel García‑Parraga · Nuhacet Fernández‑Gallardo · Rafael Zamora‑Padrón · Víctor Pacheco · María Reyes‑Batlle · Basilio Valladares · Jacob Lorenzo‑Morales · Enrique Martínez‑Carretero 

Received: 22 May 2014 / Revised: 30 June 2014 / Accepted: 17 July 2014 / Published online: 27 July 2014 © Springer-Verlag Berlin Heidelberg 2014

Abstract  Erysipelothrix rhusiopathiae is the causative agent of erysipelas, a disease of many mammalian and avian species, mainly swine and turkeys. In cetaceans, erysipelas is considered to be the most common infection in juvenile individuals, which have not been vaccinated. Moreover, the disease manifest in both forms, the dermatologic and the acute septicemic forms, has been reported in various species of dolphins and whales. It is difficult to diagnose erysipelas by currently available approaches. Moreover, it is mainly based on culture methods and also PCR methods, which are currently being developed. At the present stage, prophylactic approaches are based on antibiotic therapy and vaccination mostly with porcine erysipelas vaccines. In the present study, an Indirect Immuno Fluorescence method for the detection of dolphin antibodies levels against E. rhusiopathiae was developed and applied in two different groups of captive bottlenose dolphins (Tursiops truncatus) from Loro Parque (Tenerife, Canary Islands, Spain) and L’Oceanogràfic de Valencia (Valencia, Spain) Communicated by Erko Stackebrandt. M. J. Bernal‑Guadarrama · N. Fernández‑Gallardo · R. Zamora‑Padrón · V. Pacheco  Loro Parque, Avda. Loro Parque SN, 38400 Puerto de la Cruz, Tenerife, Spain e-mail: [email protected] D. García‑Parraga  L’Oceanogràfic de Valencia, Junta de Murs s/n, 46013 Valencia, Spain M. Reyes‑Batlle · B. Valladares · J. Lorenzo‑Morales (*) · E. Martínez‑Carretero  University Institute of Tropical Diseases and Public Health of the Canary Islands, University of La Laguna, Avda. Astrofísico Fco. Sánchez, S/N, 38203 La Laguna, Tenerife, Canary Islands, Spain e-mail: [email protected]

in order to check the tittering levels of antibodies after application of porcine erysipelas vaccines in the studied dolphins. Keywords  Erysipelothrix rhusiopathiae · Bottlenose dolphin · Diagnosis · Vaccination · IIF

Introduction Erysipelothrix rhusiopathiae is the causative agent of erysipelas, a disease of many mammalian and avian species, mainly swine and turkeys (Dunn et al. 2001; Wang et al. 2010; Melero et al. 2011). This ubiquitous microorganism is a gram-positive, nonspore-forming, nonacidfast, rod-shaped bacterium that can survive for long periods of time in the environment, including marine locations (Wang et al. 2010). Moreover, it has been isolated from the cutaneous slime of both fresh and salt water fish, insects, mollusks and crustaceans among others (Seibold and Neal 1956; Lauckner 1985; Kinsel et al. 1997; Wang et al. 2010; Opriessnig et al. 2013). In humans, the disease (known as erysipeloid) is considered a zoonosis, which is caused by contact of the individuals with contaminated animals, their products, wastes or soil (Hunt et al. 2008; Wang et al. 2010; Melero et al. 2011). The disease can manifest in three forms, which have been described in humans: a localized cutaneous form, a generalized cutaneous form and a septicemic form, which is associated with endocarditis and if untreated often results in death (Wang et al. 2010). In cetaceans, erysipelas is considered to be the most common infection in juvenile individuals, which have not been vaccinated. Interestingly, the dermatologic and acute septicemic forms have been reported in various species of dolphins including Tursiops truncatus, Stenella plagiodon,

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Grampus griseus and T. aduncus and also in pilot, beluga and killer whales among others (Seibold and Neal 1956; Chastel et al. 1975; Thurman et al. 1983; Kinsel et al. 1997; Melero et al. 2011; Opriessnig et al. 2013). Among the clinical signs of this disease in cetaceans, nonspecific symptoms such as anorexia and weakness should be considered. Nevertheless, the pathognomonic sign of erysipelas in many species is the presence of diamond-shaped skin lesions (Wang et al. 2010). In cetaceans, the lesions appear as gray, rhomboid plaques with well-defined edges that occur over the entire body (Kinsel et al. 1997; Dunn et al. 2001; Melero et al. 2011). Regarding captive cetaceans, it has been previously reported that the main sources of infection could be mainly due to dead fish that have not been well preserved but other likely sources are humans with erysipeloid, food preparation sites, opportunistic colonization of wounds, and flying insects serving as vectors between contaminated areas (Wood and Shuman 1981; Higgins 2000; Melero et al. 2011). Therefore, most cases of erysipelas have been reported in captive animals although this pathogen has also been informed in stranded animals (Melero et al. 2011). Diagnosis of erysipelas can be difficult to reach if not recognized clinically. Moreover, culture methods for the isolation of E. rhusiopathiae involve the use of selective and enrichment media as well as commercially available blood culture media, which are satisfactory for primary isolation from blood since E. rhusiopathiae is not particularly fastidious. A number of selective media for the isolation of Erysipelothrix have been also described being the Erysipelothrix selective broth (ESB) the most commonly used medium. Nevertheless, all these media present good aspects, but none of them are ideal (Shimoji et al. 1998a, b; Wang et al. 2010). The API Coryne System 2.0 (Biomerieux), a commercial strip system, is considered as a rapid and reliable system for the identification of E. rhusiopathiae (Soto et al. 1994). However, it is based on a number of biochemical reactions for the identification of coryneform bacteria and related genera (including E. rhusiopathiae), and currently, molecular identification mainly based on PCR is quickly gaining favor as a tool for the identification of this pathogen (Yamazaki 2006; Wang et al. 2010). Current prophylactic approaches are based on antibiotic therapy penicillin being the usual drug of choice. Moreover, vaccination is considered to be a useful procedure to control the problem in farms (Wang et al. 2010). Most of the available vaccines in the market are composed of attenuated E. rhusiopathiae strains or bacterins and are effective in pigs and turkeys between 6 and 12 months (Groschup and Timoney 1990; Wood 1992; Swan and Lindsey 1998; Wang et al. 2010). Regarding vaccination in dolphins, porcine erysipelas vaccines have been tested with successful results (Lacave et al. 2001; Sitt et al. 2010).

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Arch Microbiol (2014) 196:785–790

In the present study, an Indirect Immuno fluorescence (IIF) method for the detection of dolphin antibodies against E. rhusiopathiae was developed and applied in two different groups of captive bottlenose dolphins (Tursiops truncatus) in order to check the tittering levels of anti-erysipelas antibodies after application of porcine erysipelas vaccines in the studied dolphins.

Materials and methods Serum samples The serum samples used in this study were obtained from nine dolphins (6 female and 3 male individuals) from Loro Parque, Tenerife, Spain (group 1, Table 1), and 11 dolphins (7 male and 4 female individuals) from L’Oceanogràfic of Valencia, Spain (group 2 Table 2). Samples were extracted before and after vaccination of the some of the individuals included in this study (Tables 1 and 2). ER Bac Plus® (Pfizer) was used for the vaccination of the dolphins included in group 1, whereas ERYSORB PLUS (Vetoquinol Especialidades Veterinarias, Madrid, Spain) was used for group 2. Vaccines were chosen by the veterinarians of each park independently. Immunoglobulin purification and antidolphin IgG generation Antibody (dolphin’s immunoglobulins) purification was carried out by ion-exchange chromatography from a mix of 1 ml of serum from each dolphin of the Loro Parque collection as previously described by our laboratory (BernalGuadarrama et al. 2014). Briefly, the mix was purified after three washes 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 μl. Protein quantification in each tube was carried out using the commercial kit Micro BCA™ Protein Assay Reagent (Pierce Biotechnology, IL, USA). The obtained products from each wash were separated by electrophoresis in 10 % polyacrylamide sodium dodecyl sulfate (SDS)polyacrylamide 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-mm-diameter cellulose membrane (dialysis tubing, cellulose membrane, Sigma, Tres Cantos, Madrid, Spain) with a retaining limit of 12 kDa and treated as indicated by Bernal-Guadarrama et al. (2014). Dialysis continued with agitation for 24 h at 4 °C against a dialysis buffer of Tris–HCl 0.1 M (pH 6.5) 0.1 × , changing the buffer three times during the

Arch Microbiol (2014) 196:785–790 Table 1  Titering of dolphin sera included in group one (Loro Parque) before and after vaccination

787 Dolphin reference and date of sample collection

Jan-40

Jan-80

1/160

1/320

1/640

Vaccination

Pacina (08/07/09) Pacina (08/09/09) Ruffles (08/07/09) Ruffles (08/09/09) Joan (08/07/09) Joan (08/09/09) Sanibel (08/07/09) Sanibel (08/09/09) Luna (08/07/09) Luna (08/09/09) Paco (08/07/09) Paco (08/09/09) Taina (08/07/09) Taina (08/09/09) Clara (08/07/09)

+ + − + + + − + − + − + − + −

− + − + − + − + − + − + − + −

− + − + − + − + − + − + − + −

− + − + − + − + − + − + − + −

− − − − − − − − − − − − − − −

08-07-2009

Clara (08/09/09)

process. Proteins thus obtained were lyophilized and kept at −20 °C until further use. Two New Zealand rabbits, weighting around 3 kg, were used for immunization purposes. Vaccination was performed using three doses, leaving 2 weeks between each injection as follows: a first dose containing 500 μg of the emulsified protein with 500 μl of Freund’s complete adjuvant (CFA; Sigma) in a final total volume of 1 ml, while the subsequent two doses contained 500 μg of emulsified protein with 500 μl 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 (Bernal-Guadarrama et al. 2014). Preparation of the Indirect Immunofluorescence (IIF) slides and serological study of dolphin collection from Loro Parque and L’Oceanogràfic of Valencia The Erysipelothrix rhusiopathiae strain 19194™ from the American Type Culture Collection (ATCC) (Microbiologics, Scharlab, Barcelona, Spain) was cultured following manufacturer´s instructions. This strain is provided in Lyfo Disk ® vials, and thus, strain was grown in blood agar (Biomerieux) following the procedure provided by the manufacturer. Briefly, 1 vial of Erysipelothrix rhusiopathiae Lyfo Disk ® was dissolved in 500 µl of sterile Phosphate Buffer Saline (PBS). Blood agar plates were seeded with this bacterial solution and incubated at 35 °C for 48 h. Bacterial colonies were then picked up and transferred to an

+

+

+

+

08-07-2009 08-07-2009 08-07-2009 08-07-2009 08-07-2009 08-07-2009 08-07-2009



Eppendorf tube containing 500 µl of PBS. These tubes were centrifuged at 500 g for 10 min, and the supernatant was discharged. Then, 80 µl of Periodate-Lysine-Paraformaldehyde fixative (PLP) was added to the pellets, and 6 µl of the mixture was added to the wells of IF slides (Teflon-coated 6-mm-well IF slides, Biomerieux), incubated 30 min at 37 °C and washed twice with PBS and left to dry at 37 °C. Dolphin serum samples were used at the dilutions of 1/20, 1/40, 1/80 and 1/160. The same serum samples were considered as positive controls since the animals were vaccinated. The previously generated antidolphin IgG was used as the secondary antibody at a dilution of 1/100, and an antirabbit IgG labeled with FITC (Sigma, Madrid, Spain) was used as the tertiary antibody at a dilution of 1/60. For the experiments, 30 µl of serum sample was placed in the wells and PBS was used as a negative control. Samples were incubated in the slides at 37 °C, 30 min and after incubation were washed three times with PBS/Tween 20. After that, 20 µl of the generated antidolphin IgG was added and incubated at 37 °C for 30 min. Then, slides were washed three times with PBS/Tween 20, and 20 µl of the third antibody (anti-rabbit IgG labeled with FITC) was added and incubated at 37 °C, 30 min, and the slide was three times as mentioned above. The slides were then observed using a Leitz Dialux 20 fluorescence microscope. A result was considered as being positive if fluorescence was observed.

Results and discussion Dolphins included in this study were divided into group 1 (Loro Parque Collection) and group 2 (L’Oceanographic of

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Arch Microbiol (2014) 196:785–790

Table 2  Titering of dolphin sera included in group two (L’Oceanogràfic) before and after vaccination Dolphin reference and date of sample collection

Jan-40

Jan-80

1/160

1/320

1/640

1/1,280

Vaccinated

Dam (03/01/07) Dam (22/03/07) Josué (03/01/07) Josué (26/03/07) Josué (11/09/08) Kiara (03/01/07) Kiara (26/02/07) Kiara (06/06/07) Laura (03/01/07)

− + − + + − − − −

− + − + + − − − −

− + − + + − − − −

− − − + − − − − −

− − − + − − − − −

− − − + − − − − −

03-01-2007

Laura (26/02/07) Laura (12/10/08) Lazo (03/01/07) Lazo (20/02/07) Lazo (08/12/08) Lito (03/01/07) Lito (08/03/07) Lito (06/11/08) Loreto (03/01/07) Loreto (20/02/07) Loreto (13/09/08) Nala (08/06/06) Nala (16/02/07) Nala (03/01/09) Neptuno (08/06/06) Neptuno (06/02/07) Neptuno (07/02/09) Nika (05/11/06) Nika (07/11/08) Rocky (03/01/07) Rocky (26/02/07) Rocky (03/11/08)

+ + − − − − − − − + − − + + − − + − + + +

+

+ − − − − − − − − − − − + − − − + − + + +



+ − − − − − − − − − − − − − − − − − + − −



Valencia). All evaluated serum samples from the individuals included in group 1 showed a titration level of 1:320 after vaccination (Table 1). This patter was characterized by a titration level of 1:40 (dolphin reference Pacina and Joan, Table 1) or null the same day after vaccination and increased to 1:320 when serum samples were checked again 2 months after vaccination (Table 1). Therefore, it seemed that vaccination with the ER Bac Plus® (Pfizer) induced the same levels of antibodies against E. rhusiopathiae in all the animals included in this group. Regarding the animals included in group 2, vaccination only induced production of antibody levels against E. rhusiopathiae in 4 dolphins. Moreover, no similar patterns were observed in any of these four animals: Two of them showed a titration level of 1:320 (ref. Laura and Nika), 1:160 in the dolphin with reference Dam and reference Josué showed

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+ − − − − − − − − − − − − − − − − − + − −



− − − − − − − − − − − − − − − − − − − − −



− − − − − − − − − − − − − − − − − − − − −

03-01-2007

03-01-2007

03-01-2007

03-01-2007

03-01-2007

03-01-2007

Before vacc. After vacc. Before vacc. After vacc. Before vacc. After vacc. 03-01-2007



a titration level of 1:1,280, which even kept to 1:160, 18 months after vaccination (Table 2). Four dolphins did not show any sign of anti-erysipelas antibodies titer after vaccination, and other three dolphins only showed titration levels of 1:80. Therefore, it could be stated that even when the vaccine ERYSORB PLUS (Vetoquinol Especialidades Veterinarias, Madrid, Spain) used in this group induced high titration levels in three individuals, most of the dolphins of group 2 did not generate any antibodies against the pathogen after vaccination. Erysipelas has been described in cetaceans in two forms: a septicemic form and a dermatologic form, and both are similar to those observed in infected swine (Dierauf and Gulland 2001: Wang et al. 2010). Signs of septicemic disease in infected cetaceans are usually of nonspecific, and death within a few hours of clinical presentation is not

Arch Microbiol (2014) 196:785–790

unusual (Dierauf and Gulland 2001; Sitt et al. 2010). The dermatologic form of the disease occurs periodically and is typically accompanied by a leukocytosis and anorexia with subsequent development of skin lesions, the latter being most commonly visible on the trunk (Sitt et al. 2010). After that, septicemia and death could follow within days or weeks if the animal is not treated (Wang et al. 2010). Regarding the value of implementing a vaccination program for erysipelas, it is important to mention that this fact became apparent after observing that the majority of reported clinical cases were related to animals that had not been vaccinated (Dierauf and Gulland 2001; Sitt et al. 2010). Even though there is a wide variety of porcine erysipelas vaccines, only around 1/3 of them have been demonstrated to be effective in cetaceans (Dierauf and Gulland, 2001). Moreover, most of the tested vaccines have shown adverse clinical reactions and even death of individuals (Howard et al. 1983; Dierauf and Gulland 2001; Sitt et al. 2010). In a recent study, it was shown that ER Bac® Plus (Pfizer Animal Health, Exton, PA) porcine erysipelas vaccine was able to induce a vaccine-induced memory in bottlenose dolphins in captivity (Sitt et al. 2010). These observations are also supported in the present study, as it was observed that all animals from group 1 (Loro Parque collection) showed titration levels of 1:320 after vaccination with ER Bac® Plus. Another commercial porcine erysipelas vaccine that has been evaluated for protection in dolphins is Eurovac Ery (Eurovet, Belgium). However, it was evaluated in a mouse model, and no positive correlation between the amount of antibodies at the moment of challenge and the induced protection was established (Lacave et al. 2001). Among the most widely used immunological techniques for the serological diagnosis of any pathogen are IIF and ELISA. However, both techniques require the use of a second antibody against the immunoglobulins of the mammal’s sample to be analyzed. Dolphins present an immunological system that resembles that described for terrestrial mammals, including humans (Leslie and Gulland 2001). Despite the fact that diagnosis of infectious diseases in marine mammals is of high importance from a conservational point of view, there are no commercial kits available for the immunological diagnosis of diseases in these animals. The same problem is raised if evaluation of vaccination procedures is considered as not simple and/or noninvasive methods are currently available for dolphins or other marine mammals. Part of this problem is likely to be the lack of secondary antibodies. The antidolphin IgG generated in rabbits (Bernal-Guadarrama et al. 2014) and tested in this study did not show nonspecific or cross-reactions with other pathogens, and erysipelas is the second disease in dolphins with a specific IIF test for its diagnosis.

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Moreover, the results obtained in this study indicated that it could be used for the evaluation of generated antibody titers after vaccine application in dolphins, at least in the case of erysipelas. Acknowledgments  The authors are grateful to Loro Parque and L’Oceanogràfic de Valencia dolphin training team for their help in this work and also to Cristine Jo Dreisörner and Andrea Cádiz Ortiz from Loro Parque Veterinary Clinic and the staff from L’Oceanogràfic de Valencia Veterinary Clinic; thanks to them, the collection of samples for the development of this work was possible. JLM was funded by a Grant RYC-2011-08863 from the Ramón y Cajal Subprogramme of the Spanish Ministerio de Economía y Competitividad. MRB was funded by Becas Fundación Cajacanarias para Postgraduados 2014. The authors are also grateful to Loro Parque Fundación for funding this research. Finally, we are grateful to the managing direction of Loro Parque for allowing us to undertake this study.

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Development of an indirect immunofluorescence technique for the evaluation of generated antibody titers against Erysipelothrix rhusiopathiae in captive bottlenose dolphins (Tursiops truncatus).

Erysipelothrix rhusiopathiae is the causative agent of erysipelas, a disease of many mammalian and avian species, mainly swine and turkeys. In cetacea...
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