Parasitol Res (2014) 113:451–455 DOI 10.1007/s00436-013-3674-y


Development of an indirect immunofluorescence technique for the diagnosis of toxoplasmosis in bottlenose dolphins María José Bernal-Guadarrama & Joan Salichs & Javier Almunia & Daniel García-Parraga & Nuhacet Fernández-Gallardo & María Ángeles Santana-Morales & Víctor Pacheco & Raquel N. Afonso-Lehmann & Daniel Déniz & Jacob Lorenzo-Morales & Basilio Valladares & Enrique Martínez-Carretero

Received: 10 October 2013 / Accepted: 25 October 2013 / Published online: 13 November 2013 # Springer-Verlag Berlin Heidelberg 2013

Abstract The diagnosis of toxoplasmosis is often complicated by the lack of specific clinical symptoms or postmortem features, in humans and other animals. The only diagnostic test described so far for the serological diagnosis of Toxoplasma gondii in marine mammals is the modified agglutination test (Dubey et al., Am J Vet Res 48(8):1239–1243, 1987). The development of more sensible and specific immunological techniques requires specific antibodies, which are currently unavailable in the scientific market. Indirect immunofluorescence (IIF) is one of the most widely used methods for the diagnosis of toxoplasmosis in humans (Auer et al., Parasitol Res 12:965–970, 2000). In order to develop and apply this technique to the bottlenose dolphin (Tursiops truncatus), immunoglobulins were firstly purified using ionexchange chromatography. The purified immunoglobulins were then injected in New Zealand rabbits in order to obtain polyclonal antibodies. These antisera were validated by the IIF technique, using as controls serum samples of dolphins infected by Toxoplasma. The results were visualized using antirabbit IgG labeled with fluorescein. This newly developed and specific serological assay was then tested with the dolphin M. J. Bernal-Guadarrama : M. Á. Santana-Morales : R. N. Afonso-Lehmann : D. Déniz : J. Lorenzo-Morales : B. Valladares : E. Martínez-Carretero University Institute of Tropical Diseases and Public Health of the Canary Islands, University of La Laguna, Av. Astrofísico F. Sánchez s/n, 38271 La Laguna, Tenerife, Spain M. J. Bernal-Guadarrama (*) : J. Salichs : J. Almunia : N. Fernández-Gallardo : V. Pacheco Loro Parque. Av. Loro Parque s/n, 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

collection of Loro Parque, Tenerife, Spain (group I), and L'Oceanogràfic of Valencia, Spain (group II). The obtained results in this study showed that none of the dolphins from group 1 were infected by T. gondii and two animals were positive in group 2. Furthermore, we conclude that this study has produced antibodies with high specificity against dolphin immunoglobulins and an IIF method which may be used as immunological diagnostic tools, especially for the serological diagnosis of toxoplasmosis.

Introduction Toxoplasma gondii is a parasitic protozoan from the phylum Apicomplexa, subclass coccidian, that has a wide distribution worldwide. Cats are the natural reservoirs of this parasite, which are also the definite host and the only animals in which the sexual cycle of the parasite is possible. The most frequent transmission route to humans is orally after the ingestion of cysts in raw or rare meats, through the ingestion of oocysts presented in contaminated water or vegetables, or through the environment in the case of individuals that are in close contact with cats. Toxoplasma gondii can infect marine mammals (Dubey and Beattie 1988) by either the ingestion of food or water contaminated with oocysts or, congenitally, by the transmission of tachyzoites from the mother to the fetus when the infection is acquired during pregnancy (Dubey and Beattie 1988). Toxoplasmosis has been reported in a wide variety of marine mammals both captive and wild (Yu et al. 2013). Cases of toxoplasmosis have been reported in cetaceans: estuarine dolphins (Sotalia guianensis) (Bandoli and Oliveira 1977), long-snouted spinner dolphins (Stenella longirostris) (Migaki et al. 1990), common bottlenose dolphins (Tursiops truncatus) (Cruickshank et al. 1990; Inskeep et al. 1990),


striped dolphins (Stenella coeruleoalba ) (Domingo et al. 1992), beluga whales (Delphinapterus leucas ) (Mikaelian et al. 2000), and Indo-Pacific bottlenose dolphins (Tursiops aduncus) (Jardine and Dubey 2002). In Spain, the disease was described from a Risso's dolphin (Grampus griseus), found stranded on a Mediterranean beach and which died a few hours later (Resendes et al. 2002). Analyses in other marine mammals have shown that muscle tissue infection has very little or no effect on the host, while infection of the nervous system can lead to acute encephalitis (Dailey 1985). The parasite has been isolated from common seals (Phoca vitulina) (Van Pelt and Dieterich 1973), and it has been also reported to cause disseminated organ infections in California sea lions (Zalophus californianus ) (Lauckner 1985) and has caused death from meningoencephalitis in manatees (Trichechus manatus) (Buergelt and Bonde 1983). Although the prevalence of the disease is relatively high in the population of Californian sea otters (Enhydra lutris nereis), the infection can be both asymptomatic and acute causing encephalitis (Thomas and Cole 1996; Cole et al. 2000; Lindsay et al. 2000). In veterinary medicine, toxoplasmosis is normally an asymptomatic process. However, nervous and respiratory problems have been associated with excess mucus secretion in dogs infected by this pathogen (Cordero del Campillo et al. 1999). In the case of cats, processes of enteritis, adenopathies, pneumonia, and encephalitis have been reported (Cordero del Campillo et al. 1999). Regarding cattle, goats and sheep present the most acute and constant pathologies. This is a consequence of the transplacental transmission mechanism that leads to the emergence of abortions and perinatal mortality. The only diagnostic test described so far for the serological diagnosis of T. gondii in marine mammals is the modified agglutination test (MAT) (Dubey et al. 1987). In light of the successful use of indirect immunofluorescence (IIF) methods for the diagnosis of toxoplasmosis in humans (Auer et al., 2000), we sought to develop this method for the diagnosis of this pathogen in bottlenose dolphins (T. truncatus). The specificity of the technique was successfully tested in various sera samples from the dolphin collection of Loro Parque, Tenerife, Spain, and L'Oceanogràfic of Valencia, Spain.

Material and methods Serum samples The serum samples used in this study were obtained from nine dolphins from Loro Parque, Tenerife, Spain, and 15 dolphins from L'Oceanogràfic of Valencia, Spain. Positive controls were kindly provided by Dr. Francisco Alonso de Vega (Department of Animal Health, Faculty of Veterinary,

Parasitol Res (2014) 113:451–455

University of Murcia, Spain (serum 1)) and Dr. J.P. Dubey (United States Department of Agriculture, Agricultural Research Service, Animal and Natural Resources Institute, Animal Parasitic Diseases Laboratory, Baltimore (serum 2)). These two positive controls obtained from stranded dolphins were previously validated for the presence of T. gondii, using the MAT (Dubey et al. 1987). Immunoglobulin purification 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. 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). Subsequently, 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 retaining limit of 12 kDa and treated as indicated by Sambrook et al. (1989). 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 process. Proteins thus obtained were lyophilized and kept at −20 °C until further use. As shown in Fig. 1, the first and second tubes collected during the purification process presented the highest concentration of immunoglobulins. Antidolphin IgG generation 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.

Parasitol Res (2014) 113:451–455 Fig. 1 Ten percent SDS-PAGE gel of the purified dolphins immunoglobulins. Two bands can be observed in every sample, the heaviest band (56 kDa) corresponds to the heavy-chain immunoglobulins while the smallest, with an apparent weight of approximately 31 kDa, corresponds to the light-chain immunoglobulins

453 M







Heavy chain

Light Chain

Lane m: High Molecular Weight Standard Mixture SDS 6H (Biorad) Lane M: Prestained SDS-PAGE standard (Biorad) Lane 1 : tube 1 Lane 2: tube 2 Lane 3: tube 3 Lane 4: tube 4 Lane 5: tube 5

Validation of the obtained antidolphin IgG specificity and serological study of dolphin collection from Loro Parque and L'Oceanogràfic of Valencia Slides containing inactivated parasites of the Sabin strain (T. gondii spot IIF, Bio-Mérieux Marcy L'Etoile, France) were used for the validation of the obtained antibody. Positive control 1 (kindly provided by Dr. Francisco Alonso de Vega, Department of Animal Health, Faculty of Veterinary, University of Murcia, Spain (serum 1)) was used for the validation tests and tittering of sera 1 and 2 (positives controls, described above).

Results The IIF assays demonstrated the high titre and specificity of the antibodies generated. Only the wells containing the

positive controls (sera 1 and 2) showed a positive reaction (Fig. 2a). Positive controls showed a titration level of 1:160 and 1:1280 for sera 1 and 2, respectively. As a negative control, phosphate-buffered saline (PBS) was used and no fluorescence was observed (Fig. 2b). A result was considered as being positive if fluorescence was observed from the parasites. Dolphins included in this study were divided into group 1 (Loro Parque Collection) and group 2 (L'Oceanographic of Valencia). In the case of group 1, three samples were collected for each three months (April–August–November 2004), and these serum samples were used at dilutions of 1/40 and 1/160. Analysis of these data allowed us to conclude that group 1 dolphins were negative for T. gondii in all cases and did not present any infection caused by this parasite. Regarding group 2 (L'Oceanogràfic of Valencia) (Table 1), a sample from each dolphin was collected and

Fig. 2 a IIF with serum positive control 2 at the dilution of 1/128 and b IIF negative control with PBS at the dilution of 1/1280




Parasitol Res (2014) 113:451–455

Table 1 Titering of dolphin sera included in group 2



Dilution 1/20

Dilution 1/40

Dilution 1/80

Dilution 1/160

Ref. Billy Ref. Grisel Ref. Laura Ref. Luiso

+ + + +

+ + + −

− − + −

− − − −

Ref. Loren Ref. Rocky Ref. Elly Ref. Lazo Ref. Josué Ref. Nika Ref. Jungla Ref. Kiara Ref. India Ref. Dam Ref. Lito

+ + + + + + + − + − −

+ − − − + + − − + − −

+ − − − + − − − − − −

+ − − − + − − − − − −

evaluated by IIF at dilutions of 1/20 and 1/20,480. The results showed a positive reaction in five serum samples (two individuals) from group 2 (see Table 2). The most widely used immunological techniques for the serological diagnosis of Toxoplasma are IIF and ELISA (Leslie et al. 2001). 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 et al. 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. Part of this problem is likely to be the lack of secondary antibodies. The antidolphin IgG generated in rabbits in this study did not show nonspecific or cross reactions with other pathogens, and it is the first disease in dolphins with a specific IIF test for its diagnosis. Moreover, the characteristics and working dilution of this second antibody indicates that it could be used for the serological diagnosis of any kind of infections in dolphins.

The results presented in this study showed that none of the dolphins from group 1 were infected by T. gondii, and two animals were positive in group 2. However, the presence of this parasite in wild animals has been previously reported (Leslie et al. 2001). If the transmission route of the parasite is investigated in the case of animals in captivity, it is usually found to be through feeding and the contamination of the pool water. Animals included in this study are fed with the following four fish species: Cuplea harengus, Scomber scombrus, Sprattus sprattus, and Mallotus villosus , which are kept at −20 °C until use. Toxoplasma cysts are inactivated by temperatures of −13 °C or lower (Dubey 1977), and thus, the risk of infection of these animals through feeding should be possible. Contamination of the pool water that comes straight from the sea has been previously been reported as a transmission route of this parasite even in humans (Conrad et al. 2005). The water that is used in the pools of both parks in this study is always pretreated by filtration and chlorination. However, it has been demonstrated that oocysts form Cryptosporidium and Toxoplasma sp. are able to resist detergents and disinfectants such as sodium hypochlorite (Rose et al. 2002; Dumètre and Dardé 2003), and thus, the elimination of Toxoplasma oocysts using this procedure is uncertain. As both parks use the same pretreatment procedure and feed the same fish from the same origin, it is possible that the positive animals suffered the infection before they arrived to the park. The half-life of antibodies in the peripheral blood of dolphin has not been established. In humans, generated anti-Toxoplasma IgGs remain for life in the infected individual and are responsible for the protection against this pathogen. Further studies in dolphins are required in order to conclude whether this process is similar to the one described in humans. In conclusion, this work has generated antidolphin IgGs for its use in different serological diagnostic techniques. In this study, it was used for the development of an IIF technique for the diagnosis of T. gondii in bottlenose dolphins (Tursiops truncatus ). The obtained results demonstrated that this technique is as fast, reliable, and specific a tool as that in humans for the diagnosis of this pathogen. Moreover, this technique could be used for the control of pregnant females in captivity in order to avoid undesired abortions.

Table 2 Tittering of dolphin sera from group 2 which were positive at the dilution of 1/160 Dolphin

Dilution 1/320

Dilution 1/640

Dilution 1/1,280

Dilution 1/2,560

Dilution 1/5,120

Dilution 1/10,240

Dilution 1/20,480

Ref. Loren Ref. Josué

+ +

+ +

+ +

+ +

− +

− +

− −

Parasitol Res (2014) 113:451–455 Acknowledgments The authors are grateful to the Loro Parque trainer team for their help in this work; thanks to them, the collection of samples 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. A special mention is given to Dr. J.P. Dubey (United States Department of Agriculture, Agricultural Research Service, Animal and Natural Resources Institute, Animal Parasitic Diseases Laboratory, Baltimore) and Dr. Francisco Alonso de Vega (Department of Animal Health, Faculty of Veterinary, University of Murcia, Spain). We are also grateful to Dr. Carlos Marcelino Rodríguez López (University of Wales) for his help in the discussion of this work. 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 diagnosis of toxoplasmosis in bottlenose dolphins.

The diagnosis of toxoplasmosis is often complicated by the lack of specific clinical symptoms or postmortem features, in humans and other animals. The...
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