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Occurrence of Ascocotyle (Phagicola) longa Ransom, 1920 (Digenea: Heterophyidae) in Mugil incilis from Cartagena Bay, Colombia
Veterinary Parasitology 168 (2010) 31–35
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Occurrence of Ascocotyle (Phagicola) longa Ransom, 1920 (Digenea: Heterophyidae) in Mugil incilis from Cartagena Bay, Colombia Dilia Galva´n-Borja, Jesu´s Olivero-Verbel *, Lı´a Barrios-Garcı´a Environmental and Computational Chemistry Group, Campus of Zaragocilla, Faculty of Pharmaceutical Sciences, University of Cartagena, Cartagena, Colombia
A R T I C L E I N F O
A B S T R A C T
Article history: Received 13 June 2009 Received in revised form 5 October 2009 Accepted 13 October 2009
Fishborne metacercariae have not been reported in Colombia. During an 8-month period, 250 mullet/lisa (Mugil incilis) were examined for trematode metacercariae in the liver. Average prevalence and intensity were 93.4 2.5% and 8476 1238 cysts per gram of liver, respectively. Monthly prevalence of the trematode, identified as Ascocotyle (Phagicola) longa Ransom (1920), was always high (>79%) and there was no difference between sampling periods (P > 0.05). Trematode intensity in lisa had a significant negative effect on host size (R = 0.325, P < 0.001), condition factor (R = 0.352, P < 0.001), and liver-somatic index (R = 0.372, P < 0.001); in contrast, a positive relationship was found with gill-somatic index (R = 0.446, P < 0.001). Histopathological analysis indicated the presence of abundant cysts together with inflammatory reactions involving macrophage aggregates and necrosis. These results suggest that trematodes infecting the liver of M. incilis affect the growth and well-being of these fish, and their consumption could represent a major risk for human infection. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Mugil incilis Ascocotyle (Phagicola) longa Liver Metacercaria Cartagena Bay Colombia
1. Introduction Colombia is one of the two countries in the world with greatest biodiversity. Despite this, very few surveys on fish have been reported, and the publications regarding parasites in these organisms are scarce. Parasites in fish can be determinant in the dynamics of the ecosystem, and because environmental degradation can affect fish-parasite ecology, those may function as biomarkers of environmental health (Hinck et al., 2008). In addition to the ecological impact, parasites in fish could cause economic losses to fisheries, and because some of them could potentially be transmitted to humans, they might represent an important hazard for human health, in particular when host fish are part of the diet for local communities.
* Corresponding author. Tel.: +57 5 6698180; fax: +57 5 6699771. E-mail addresses: [email protected], [email protected] (J. Olivero-Verbel). URL: http://www.reactivos.com 0304-4017/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2009.10.011
Among fish which are important for the diet in the north coast of Colombia is the Mugil incilis, commonly named mullet or lisa, one of the most abundant fish in the Caribean (Arrieta and De-La-Rosa, 2003; Rueda, 2001). This species is particularly valuable because it is present during the whole year and it is part of traditional dishes. Its importance as food contrasts with its capacity to serve as intermediate host for several parasites. We have previously reported that M. incilis is a host of nematodes of the Anisakidae family (Olivero et al., 2005). However, it is also parasited with trematodes belonging to the Heterophidae family, that commonly uses the Mugilidae as second intermediary host (Scholz et al., 2001). It is well known that metacercarial infections in fish could affect the prevalence of definitive hosts in local areas (Seo et al., 1982; Song et al., 1985; Chai and Lee, 2002). Therefore, studies about these infections in brackish water fish, could give useful information about the possible human health risks derived from fish consumption. The aim of this paper is to describe, for the first time, the current status of the presence of metacercariae infections in M. incilis from the Cartagena Bay, north of Colombia.
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2. Materials and methods A total of 250 specimens of M. incilis were collected between September 2007 and April 2008 in Cartagena Bay, at Castillo Grande beach (758320 O, 108230 N). Samples were placed in plastic bags and transported to the lab on ice. Before gill and liver isolation, size was determined by weighing and measuring the total length of the fish. The condition factor (CF) was calculated using the formula: CF = (Q/L3) 100, and the gill- and liver-somatic indices using the equation: tissue-somatic index = (tissue weight (g)/weight of fish (g)) 100. After isolation, livers were washed in saline solution and stored at 20 8C, until testing within the next 48 h. A liver subsample of 0.5 g was grounded on phosphate buffer saline (PBS) (1:10, w/v), and the metacercariae were separated by several washing and centrifugation cycles (9000 g, 3 min). The resultant pellet was washed five times with PBS and resuspended in 1 mL of the same buffer. Counts were performed in at least three subsamples of 10 mL each, using a Neubauer Camera under an Olympus BX41 optical microscope, and reported as the number of cysts per gram of liver. Cysts containing metacercaria were identified according to taxonomic keys of Scholz (1999). Infection parameters for the trematode, prevalence (% of infected fish) and intensity of infection (number of cysts per gram of liver) were estimated following Rozsa et al. (2000). 2.1. Histology Representative liver samples from fish collected at different months were trimmed, processed conventionally, embedded in paraffin, sectioned at about 4 mm, and stained with hematoxylin and eosin (H&E). Each slide was examined at low (40) and high magnification (400). 2.2. Data analysis Data are presented as the mean standard error of the mean. Data was log-transformed before analysis, but only parasite intensity reached normality after transformation. Thus, Kruskal–Wallis ANOVA was performed to evaluate differences between sampling months, and Spearman rank-
Table 1 Average morphometric and parasite indexes (trematodes in liver) found in Mugil incilis collected at Cartagena Bay. Variable
Mean standard error
Weight (g) Length (cm) Condition factor Liver-somatic index Gill-somatic index Parasite prevalence (%) Parasite intensity (parasites per gram of liver)
99.9 3.2 23.7 0.2 0.716 0.009 0.987 0.032 5.691 0.070 93.4 2.5 8476 1238
order correlation was used for detecting relationships between variables. Differences in prevalence of metacercariae between sampling months were determined using a Chisquare test with Yates’ correction. A P-value of 0.05 or less was considered to be significant. Statistical analysis was performed using Sigma Stat 3.0 (Chicago, IL, USA). 3. Results Average morphometric and parasitic indexes measured for M. incilis collected in Cartagena Bay are shown in Table 1. Morphological analysis of the larvae isolated from the cysts showed that the head crown of the metacercaria was armed with 16 collar spines, typical of Ascocotyle (Phagicola) longa Ransom, 1920. Cyst metacercarias (n = 60) isolated from the liver of M. incilis had in average 153.08 2.07 mm in diameter (Fig. 1). The prevalence and infection intensity of cysts (metacercaria) in liver of M. incilis from Cartagena Bay are shown in Fig. 2. Prevalences did not vary substantially between sampling periods, and with the exception of November, values were kept greater than 80%. Prevalences of 100% were found during September and April and lower values appeared on October and November (87.8 and 79.4%, respectively). Maximum values for parasitic intensity occurred on December and January. Correlation analysis between morphometric and parasitic intensity are shown in Fig. 3. Parasitic intensity showed a significant but negative correlation with weight (R = 0.325, P < 0.001), condition factor (R = 0.352,
Fig. 1. Metacercariae of Ascocotyle (Phagicola) longa from the liver of Mugil incilis collected at Cartagena Bay, Colombia. (A) Encysted and (B) excysted.
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P < 0.001) and liver-somatic index (R = 0.372, P < 0.001), whereas it was positive with respect to gill-somatic index (R = 0.446, P < 0.001). Photomicrographs of the liver tissue containing of trematode-infected fish are shown in Fig. 4. Histopathologic examination indicates that most metacercaria of A. (Phagicola) longa are found as single or multiple cysts, encircled by a capsule of fibrous tissue. However, it is also clear a host-dependent reaction, characterised by melanomacrophage cells trying to surround the large sized encysted metacercaria, and necrosis. 4. Discussion
Fig. 2. Monthly prevalence (A) and intensity (B) of cysts (metacercaria) counted in liver of M. incilis from Cartagena Bay.
The results of the present survey for metacercariae in M. incilis (Lisa) shows that this species is highly infected with A. (Phagicola) longa, and this report, the first in Colombian fish, represents a significant food safety concern, since Lisa is used in several traditional dishes in this country. Although digenean worms could be present in fish either as larval metacercariae or as adult worms (Bricknell et al., 2006), no adult forms of the trematode were detected. It has been found that cyst prevalences are basically constant along the year, with maximum values (100%) matching the starting of the rainy seasons (September and April), and being generally lower during the rainy months (October and November). Moreover, parasitic intensity was highest on January, during the peak of the dry season. It is clear that ecological studies are needed to determine the intermediate and definitive hosts for these parasites, the effects of seasonal changes in water quality
Fig. 3. Relationships between parasite intensity (A. (Phagicola) longa) and morphometric variables for M. incilis collected at Cartagena Bay, Colombia.
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Fig. 4. Liver sections of M. incilis stained with H&E showing typical cysts of A. (Phagicola) longa. C, cyst; MMC, melanomacrophage center; N, necrosis.
on parasitic prevalence, and the tendency for transmission, among other aspects (Skov et al., 2009). This information will provide insight on the possible approaches to control infection, and together with epidemiological data, it should be able to establish risk factors linked to human infection (Nguyen et al., 2007). Even more important is the fact that Colombia needs to increase its efforts to protect biodiversity, as it may halt adverse effects of trematode infections in vertebrate hosts (Schotthoefer et al., 2007). The negative but significant correlation between size (weight), condition factor and liver-somatic index with parasitic intensity, might suggest that parasites can be impacting fish growth and physiology, and in particular, liver homeostasis. Histological evaluation of liver tissues showed that most common lesions were parasite-related. Melanomacrophage centers were frequently observed around the encysted metacercariae, and those were usually accompanied by fibrosis, inflammation, and necrosis, caused by parasites migrating through the liver. The final outcome of this extensive parasitism is uncertain. However, for some fish such as the juvenile Mozambique tilapia (Tilapia mossambica), the monogenean Enterogyrus cichlidarum, a trematode which normally inhabits the stomach and anterior intestinal lumina, can invade the internal organs, and these infections have been associated with chronic morbidity and mortality of these fish (Noga and Flowers, 1995). The positive relationship between gill-somatic index and parasitic intensity (R = 0.446, P < 0.001) is an interesting finding. Although additional testing is necessary, at least two hypotheses can be proposed to explain this observation. First, metacercarias that have been detected in the gills have been also associated with cartilage proliferation leading to hyperplasia of gill epithelium (Olson and Pierce, 1997). Second, cercariae penetration into the fish leads to an increase in the frequency of respiratory-current reversals (Paller and Uga, 2008), which eventually could lead to an increase in gill size. In America, several species of fish have been reported to be infected with A. longa, i.e., Dormitator latifrons (Scholz et al., 2001), Dormitator maculates (Salgado-Maldonado et al., 2005) and Gobiesox fluviatilis (Scholz et al., 2001); and it is particularly abundant in mullets such as Mugil cephalus and Mugil curema from coastal lagoons such as El Jabalı´ and Salinas de Careyes (Scholz et al., 2001; Scholz, 1999). Although the infection of M. incilis with this parasite is not
surprising, the fact that it has such high parasite intensity deserves more scientific attention. The ecology of M. incilis might contribute to its capacity as an intermediate host for A. (Phagicola) longa, not only because this species is mainly detritivorous and sometimes omnivorous (Osorio, 1988), but also because the fries serve as an import source of food for other species. The results presented here, together with reports of nematodes in M. incilis from Colombia (Olivero et al., 2005), revealed that M. incilis is a second intermediate host and a likely source of human infections. Indeed, the capacity of A. longa as a potential zoonotic agent for human infection was initially reported in people from Brazil, after ingestion of raw Mugil sp. (Chieffi et al., 1990, 1992). Trematodes in fish have been used as valuable models to study animal invasions and environmental local and global changes (Taraschewski, 2006; Dzikowski et al., 2003). Investigations of the infection rates of metacercariae in second intermediate hosts, in combination with studies on adult worm infection rates in humans and larval infections in first intermediate hosts, can usefully describe the epidemiology of trematodes in a particular area. Trematode infection has been linked to fish mortality events in several fish species (Mohan et al., 1999), and this etiological factor should be considered when evaluating fish mortalities in Cartagena. In summary, livers of M. incilis from Cartagena Bay are highly parasitized with A. (Phagicola) longa. The presence of these parasites is correlated with smaller fish size, and also with a decrease in liver weight. The presence of inflammation and necrosis in fish livers infected with the parasite, may suggest a host reaction against it, and this effect impacts fish physiology by altering its normal development and health. Acknowledgements The authors thank Barbara Arroyo Salgado for her expertise, and the University of Cartagena. References Arrieta, L., De-La-Rosa, J., 2003. Estructura de la comunidad ı´ctica de la cie´naga de Mallorquı´n, Caribe Colombiano. Bol. Invest. Mar. Costeras 32, 231–242. Bricknell, I.R., Bron, J.E., Bowden, T.J., 2006. Diseases of gadoid fish in cultivation: a review. ICES J. Mar. Sci. 63, 253–266.
D. Galva´n-Borja et al. / Veterinary Parasitology 168 (2010) 31–35 Chai, J.Y., Lee, S.H., 2002. Food-borne intestinal trematode infections in the Republic of Korea. Parasitol. Int. 51, 129–154. Chieffi, P.P., Gorla, M.C., Torres, D.M., Dias, R.M., Mangini, A.C., Monteiro, A.V., Woiciechovski, E., 1992. Human infection by Phagicola sp. (Trematoda, Heterophyidae) in the municipality of Registro, Sa˜o Paulo State, Brazil. J. Trop. Med. Hyg. 95, 346–348. Chieffi, P.P., Leite, O.H., Dias, R.M., Torres, D.M., Mangini, A.C., 1990. Human parasitism by Phagicola sp. (Trematoda, Heterophyidae) in Canane´ia, Sa˜o Paulo state, Brazil. Rev. Inst. Med. Trop. Sao Paulo 32, 285–288. Dzikowski, R., Diamant, A., Paperna, I., 2003. Trematode metacercariae of fishes as sentinels for a changing limnological environment. Dis. Aquat. Org. 55, 145–150. Hinck, J.E., Blazer, V.S., Denslow, N.D., Echols, K.R., Gale, R.W., Wieser, C., May, T.W., Ellersieck, M., Coyle, J.J., Tillitt, D.E., 2008. Chemical contaminants, health indicators, and reproductive biomarker responses in fish from rivers in the Southeastern United States. Sci. Total Environ. 390, 538–557. Mohan, C.V., Shankar, K.M., Ramesh, K.S., 1999. Mortalities of juvenile common carp. A case study. Cyprinus carpio associated with larval trematodes infection. J. Aquacult. Trop. 14, 137–142. Nguyen, T.H., Nguyen, V.D., Murrell, D., Dalsgaard, A., 2007. Occurrence and species distribution of fishborne zoonotic trematodes in wastewaterfed aquaculture in northern Vietnam. Trop. Med. Int. Health 12, 66–72. Noga, E.J., Flowers, J.R., 1995. Invasion of Tilapia mossambica (cichlidae) viscera by the monogenean Enterogyrus cichlidarum. J. Parasitol. 81, 815–817. Olivero, J., Baldiris, R., Arroyo, B., 2005. Nematode infection in Mugil incilis (lisa) from Cartagena Bay and Totumo Marsh, north of Colombia. J. Parasitol. 91, 1109–1112. Olson, R.E., Pierce, J.R., 1997. A trematode metacercaria causing gill cartilage proliferation in steelhead trout from Oregon. J. Wildl. Dis. 33, 886–890. Osorio, D., 1988. Ecologı´a tro´fica de Mugil curema, M. incilis y M. liza (Pisces: Mugilidae) en la Cie´naga Grande de Santa Marta, Caribe colombiano: Ana´lisis cualitativo y cuantitativo. An. Inst. Invest. Mar. Punta Betı´n 18, 113–126.
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Paller, V.G., Uga, S., 2008. Attachment and penetration of Centrocestus armatus (Digenea: Heterophyidae) cercariae to gills of secondary intermediate fish hosts. J. Parasitol. 94, 578–583. Rozsa, L., Reiczigel, J., Majoros, G., 2000. Quantifying parasites in samples of hosts. J. Parasitol. 86, 228–232. Rueda, M., 2001. Spatial distribution of fish species in a tropical estuarine lagoon: a geostatistical appraisal. Mar. Ecol. Prog. Ser. 222, 217– 226. ˜ as-Carranza, G., SotoSalgado-Maldonado, G., Aguilar-Aguilar, R., Caban Galera, E., Mendoza-Palmero, C., 2005. Helminth parasites in freshwater fish from the Papaloapan river basin, Mexico. Parasitol. Res. 96, 69–89. Scholz, T., Aguirre-Macedo, M.L., Salgado-Maldonado, G., 2001. Trematodes of the family Heterophyidae (Digenea) in Mexico: a review of species and new host and geographical records. J. Nat. Hist. 35, 1733– 1772. Scholz, T., 1999. Taxonomic study of Phagicola longa (Ransom, 1920) (Digenea: Heterophyidae) and related taxa. Syst. Parasitol. 43, 147–158. Schotthoefer, A.M., Labak, K.M., Beasley, V.R., 2007. Ribeiroia ondatrae cercariae are consumed by aquatic invertebrate predators. J. Parasitol. 93, 1240–1243. Seo, B.S., Hong, S.T., Chai, J.Y., Lee, S.H., 1982. Study on Metagonimus yokogawai (Katsurada, 1912) in Korea. VI. The geographical distribution of metacercarial infection in sweetfish along the East and South Coast. Korean J. Parasitol. 20, 28–32. Skov, J., Kania, P.W., Dalsgaard, A., Jørgensen, T.R., Buchmann, K., 2009. Life cycle stages of heterophyid trematodes in Vietnamese freshwater fishes traced by molecular and morphometric methods. Vet. Parasitol. 160, 66–75. Song, C.Y., Lee, S.H., Jeon, S.R., 1985. Studies on the intestinal fluke. Metagonimus yokogawai Katsurada, 1912 in Korea Geographical distribution of sweetfish and infection status with Metagonimus metacercaria in south-eastern area of Korea. Korean J. Parasitol. 23, 123–138. Taraschewski, H., 2006. Hosts and parasites as aliens. J. Helminthol. 80, 99–128.
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Occurrence of Ascocotyle (Phagicola) longa Ransom, 1920 (Digenea: Heterophyidae) in Mugil incilis from Cartagena Bay, Colombia Dilia Galva´n-Borja, Jesu´s Olivero-Verbel *, Lı´a Barrios-Garcı´a Environmental and Computational Chemistry Group, Campus of Zaragocilla, Faculty of Pharmaceutical Sciences, University of Cartagena, Cartagena, Colombia
A R T I C L E I N F O
A B S T R A C T
Article history: Received 13 June 2009 Received in revised form 5 October 2009 Accepted 13 October 2009
Fishborne metacercariae have not been reported in Colombia. During an 8-month period, 250 mullet/lisa (Mugil incilis) were examined for trematode metacercariae in the liver. Average prevalence and intensity were 93.4 2.5% and 8476 1238 cysts per gram of liver, respectively. Monthly prevalence of the trematode, identified as Ascocotyle (Phagicola) longa Ransom (1920), was always high (>79%) and there was no difference between sampling periods (P > 0.05). Trematode intensity in lisa had a significant negative effect on host size (R = 0.325, P < 0.001), condition factor (R = 0.352, P < 0.001), and liver-somatic index (R = 0.372, P < 0.001); in contrast, a positive relationship was found with gill-somatic index (R = 0.446, P < 0.001). Histopathological analysis indicated the presence of abundant cysts together with inflammatory reactions involving macrophage aggregates and necrosis. These results suggest that trematodes infecting the liver of M. incilis affect the growth and well-being of these fish, and their consumption could represent a major risk for human infection. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Mugil incilis Ascocotyle (Phagicola) longa Liver Metacercaria Cartagena Bay Colombia
1. Introduction Colombia is one of the two countries in the world with greatest biodiversity. Despite this, very few surveys on fish have been reported, and the publications regarding parasites in these organisms are scarce. Parasites in fish can be determinant in the dynamics of the ecosystem, and because environmental degradation can affect fish-parasite ecology, those may function as biomarkers of environmental health (Hinck et al., 2008). In addition to the ecological impact, parasites in fish could cause economic losses to fisheries, and because some of them could potentially be transmitted to humans, they might represent an important hazard for human health, in particular when host fish are part of the diet for local communities.
* Corresponding author. Tel.: +57 5 6698180; fax: +57 5 6699771. E-mail addresses: [email protected], [email protected] (J. Olivero-Verbel). URL: http://www.reactivos.com 0304-4017/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2009.10.011
Among fish which are important for the diet in the north coast of Colombia is the Mugil incilis, commonly named mullet or lisa, one of the most abundant fish in the Caribean (Arrieta and De-La-Rosa, 2003; Rueda, 2001). This species is particularly valuable because it is present during the whole year and it is part of traditional dishes. Its importance as food contrasts with its capacity to serve as intermediate host for several parasites. We have previously reported that M. incilis is a host of nematodes of the Anisakidae family (Olivero et al., 2005). However, it is also parasited with trematodes belonging to the Heterophidae family, that commonly uses the Mugilidae as second intermediary host (Scholz et al., 2001). It is well known that metacercarial infections in fish could affect the prevalence of definitive hosts in local areas (Seo et al., 1982; Song et al., 1985; Chai and Lee, 2002). Therefore, studies about these infections in brackish water fish, could give useful information about the possible human health risks derived from fish consumption. The aim of this paper is to describe, for the first time, the current status of the presence of metacercariae infections in M. incilis from the Cartagena Bay, north of Colombia.
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2. Materials and methods A total of 250 specimens of M. incilis were collected between September 2007 and April 2008 in Cartagena Bay, at Castillo Grande beach (758320 O, 108230 N). Samples were placed in plastic bags and transported to the lab on ice. Before gill and liver isolation, size was determined by weighing and measuring the total length of the fish. The condition factor (CF) was calculated using the formula: CF = (Q/L3) 100, and the gill- and liver-somatic indices using the equation: tissue-somatic index = (tissue weight (g)/weight of fish (g)) 100. After isolation, livers were washed in saline solution and stored at 20 8C, until testing within the next 48 h. A liver subsample of 0.5 g was grounded on phosphate buffer saline (PBS) (1:10, w/v), and the metacercariae were separated by several washing and centrifugation cycles (9000 g, 3 min). The resultant pellet was washed five times with PBS and resuspended in 1 mL of the same buffer. Counts were performed in at least three subsamples of 10 mL each, using a Neubauer Camera under an Olympus BX41 optical microscope, and reported as the number of cysts per gram of liver. Cysts containing metacercaria were identified according to taxonomic keys of Scholz (1999). Infection parameters for the trematode, prevalence (% of infected fish) and intensity of infection (number of cysts per gram of liver) were estimated following Rozsa et al. (2000). 2.1. Histology Representative liver samples from fish collected at different months were trimmed, processed conventionally, embedded in paraffin, sectioned at about 4 mm, and stained with hematoxylin and eosin (H&E). Each slide was examined at low (40) and high magnification (400). 2.2. Data analysis Data are presented as the mean standard error of the mean. Data was log-transformed before analysis, but only parasite intensity reached normality after transformation. Thus, Kruskal–Wallis ANOVA was performed to evaluate differences between sampling months, and Spearman rank-
Table 1 Average morphometric and parasite indexes (trematodes in liver) found in Mugil incilis collected at Cartagena Bay. Variable
Mean standard error
Weight (g) Length (cm) Condition factor Liver-somatic index Gill-somatic index Parasite prevalence (%) Parasite intensity (parasites per gram of liver)
99.9 3.2 23.7 0.2 0.716 0.009 0.987 0.032 5.691 0.070 93.4 2.5 8476 1238
order correlation was used for detecting relationships between variables. Differences in prevalence of metacercariae between sampling months were determined using a Chisquare test with Yates’ correction. A P-value of 0.05 or less was considered to be significant. Statistical analysis was performed using Sigma Stat 3.0 (Chicago, IL, USA). 3. Results Average morphometric and parasitic indexes measured for M. incilis collected in Cartagena Bay are shown in Table 1. Morphological analysis of the larvae isolated from the cysts showed that the head crown of the metacercaria was armed with 16 collar spines, typical of Ascocotyle (Phagicola) longa Ransom, 1920. Cyst metacercarias (n = 60) isolated from the liver of M. incilis had in average 153.08 2.07 mm in diameter (Fig. 1). The prevalence and infection intensity of cysts (metacercaria) in liver of M. incilis from Cartagena Bay are shown in Fig. 2. Prevalences did not vary substantially between sampling periods, and with the exception of November, values were kept greater than 80%. Prevalences of 100% were found during September and April and lower values appeared on October and November (87.8 and 79.4%, respectively). Maximum values for parasitic intensity occurred on December and January. Correlation analysis between morphometric and parasitic intensity are shown in Fig. 3. Parasitic intensity showed a significant but negative correlation with weight (R = 0.325, P < 0.001), condition factor (R = 0.352,
Fig. 1. Metacercariae of Ascocotyle (Phagicola) longa from the liver of Mugil incilis collected at Cartagena Bay, Colombia. (A) Encysted and (B) excysted.
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P < 0.001) and liver-somatic index (R = 0.372, P < 0.001), whereas it was positive with respect to gill-somatic index (R = 0.446, P < 0.001). Photomicrographs of the liver tissue containing of trematode-infected fish are shown in Fig. 4. Histopathologic examination indicates that most metacercaria of A. (Phagicola) longa are found as single or multiple cysts, encircled by a capsule of fibrous tissue. However, it is also clear a host-dependent reaction, characterised by melanomacrophage cells trying to surround the large sized encysted metacercaria, and necrosis. 4. Discussion
Fig. 2. Monthly prevalence (A) and intensity (B) of cysts (metacercaria) counted in liver of M. incilis from Cartagena Bay.
The results of the present survey for metacercariae in M. incilis (Lisa) shows that this species is highly infected with A. (Phagicola) longa, and this report, the first in Colombian fish, represents a significant food safety concern, since Lisa is used in several traditional dishes in this country. Although digenean worms could be present in fish either as larval metacercariae or as adult worms (Bricknell et al., 2006), no adult forms of the trematode were detected. It has been found that cyst prevalences are basically constant along the year, with maximum values (100%) matching the starting of the rainy seasons (September and April), and being generally lower during the rainy months (October and November). Moreover, parasitic intensity was highest on January, during the peak of the dry season. It is clear that ecological studies are needed to determine the intermediate and definitive hosts for these parasites, the effects of seasonal changes in water quality
Fig. 3. Relationships between parasite intensity (A. (Phagicola) longa) and morphometric variables for M. incilis collected at Cartagena Bay, Colombia.
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Fig. 4. Liver sections of M. incilis stained with H&E showing typical cysts of A. (Phagicola) longa. C, cyst; MMC, melanomacrophage center; N, necrosis.
on parasitic prevalence, and the tendency for transmission, among other aspects (Skov et al., 2009). This information will provide insight on the possible approaches to control infection, and together with epidemiological data, it should be able to establish risk factors linked to human infection (Nguyen et al., 2007). Even more important is the fact that Colombia needs to increase its efforts to protect biodiversity, as it may halt adverse effects of trematode infections in vertebrate hosts (Schotthoefer et al., 2007). The negative but significant correlation between size (weight), condition factor and liver-somatic index with parasitic intensity, might suggest that parasites can be impacting fish growth and physiology, and in particular, liver homeostasis. Histological evaluation of liver tissues showed that most common lesions were parasite-related. Melanomacrophage centers were frequently observed around the encysted metacercariae, and those were usually accompanied by fibrosis, inflammation, and necrosis, caused by parasites migrating through the liver. The final outcome of this extensive parasitism is uncertain. However, for some fish such as the juvenile Mozambique tilapia (Tilapia mossambica), the monogenean Enterogyrus cichlidarum, a trematode which normally inhabits the stomach and anterior intestinal lumina, can invade the internal organs, and these infections have been associated with chronic morbidity and mortality of these fish (Noga and Flowers, 1995). The positive relationship between gill-somatic index and parasitic intensity (R = 0.446, P < 0.001) is an interesting finding. Although additional testing is necessary, at least two hypotheses can be proposed to explain this observation. First, metacercarias that have been detected in the gills have been also associated with cartilage proliferation leading to hyperplasia of gill epithelium (Olson and Pierce, 1997). Second, cercariae penetration into the fish leads to an increase in the frequency of respiratory-current reversals (Paller and Uga, 2008), which eventually could lead to an increase in gill size. In America, several species of fish have been reported to be infected with A. longa, i.e., Dormitator latifrons (Scholz et al., 2001), Dormitator maculates (Salgado-Maldonado et al., 2005) and Gobiesox fluviatilis (Scholz et al., 2001); and it is particularly abundant in mullets such as Mugil cephalus and Mugil curema from coastal lagoons such as El Jabalı´ and Salinas de Careyes (Scholz et al., 2001; Scholz, 1999). Although the infection of M. incilis with this parasite is not
surprising, the fact that it has such high parasite intensity deserves more scientific attention. The ecology of M. incilis might contribute to its capacity as an intermediate host for A. (Phagicola) longa, not only because this species is mainly detritivorous and sometimes omnivorous (Osorio, 1988), but also because the fries serve as an import source of food for other species. The results presented here, together with reports of nematodes in M. incilis from Colombia (Olivero et al., 2005), revealed that M. incilis is a second intermediate host and a likely source of human infections. Indeed, the capacity of A. longa as a potential zoonotic agent for human infection was initially reported in people from Brazil, after ingestion of raw Mugil sp. (Chieffi et al., 1990, 1992). Trematodes in fish have been used as valuable models to study animal invasions and environmental local and global changes (Taraschewski, 2006; Dzikowski et al., 2003). Investigations of the infection rates of metacercariae in second intermediate hosts, in combination with studies on adult worm infection rates in humans and larval infections in first intermediate hosts, can usefully describe the epidemiology of trematodes in a particular area. Trematode infection has been linked to fish mortality events in several fish species (Mohan et al., 1999), and this etiological factor should be considered when evaluating fish mortalities in Cartagena. In summary, livers of M. incilis from Cartagena Bay are highly parasitized with A. (Phagicola) longa. The presence of these parasites is correlated with smaller fish size, and also with a decrease in liver weight. The presence of inflammation and necrosis in fish livers infected with the parasite, may suggest a host reaction against it, and this effect impacts fish physiology by altering its normal development and health. Acknowledgements The authors thank Barbara Arroyo Salgado for her expertise, and the University of Cartagena. References Arrieta, L., De-La-Rosa, J., 2003. Estructura de la comunidad ı´ctica de la cie´naga de Mallorquı´n, Caribe Colombiano. Bol. Invest. Mar. Costeras 32, 231–242. Bricknell, I.R., Bron, J.E., Bowden, T.J., 2006. Diseases of gadoid fish in cultivation: a review. ICES J. Mar. Sci. 63, 253–266.
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