Parasites and hepatic histopathological lesions in lisa (Mugil incilis) from Totumo mash, North of Colombia

Objective. To assess the levels of parasitism by nematodes and trematodes, as well as the hepatic histopathological alterations present in Mugil incilis (Lisa) from Totumo marsh, North of Colombia. Materials and methods. Between July 2004 and June 2005, 500 fish were collected at Totumo Marsh (75°16’W and 10°44 ́N), North of Colombia. Morphometric and parasitic parameters were determined for each specimen, and the hepatic histopathological status of the liver was assessed by analyzing liver slides stained with hematoxylin and eosin. Results. Nematode larvae isolated from Mugil incilis corresponded to Contracaecum spp. Parasite prevalence was 60.49%. Parasitic mean abundance and mean intensity were 4.8±1.05 and 7.02±1.49, nematodes per fish, respectively. The correlation between nematode mean abundance and fish length was significant and positive (r=0.525, p<0.0001), but negative for condition factor (r=-0.109, p=0.014). Hepatic histopathological analysis revealed the presence of encapsulated trematode larvae as the main finding. However, the presence of inflammation, granulomas, steatosis and necrosis, were also registered as secondary alterations. Conclusions. Lisas collected at Totumo Marsh are parasitized with nematodes and trematodes. These fish have different histopathological lesions in the liver tissue, being the most important the presence of trematode encapsulated cyst that generate inflammatory reactions, and negatively correlate with morphometric markers of fish health.

The presence of larval stages of Anisakid nematodes in fish is a problem for commercial fishing industries due to the potential risk of transmission of zoonotic diseases such as anisakiasis (6)(7)(8), and foodborne allergenicity (9,10). Currently, fish diseases, in particular those caused by parasites, constitute one of the most important problems and challenges confronting fish farming (11).
This occurs because parasitism can be a critical factor contributing to fish mortalities (12), and in less worst case scenarios, it may induce severe histological changes in fish organs such as gills, liver and gonads. These lesions are useful as sensitive and reliable indicators of the health of wild fish populations (13).
In Northern Colombia, Lisa (Mugil incilis) is an economically important fish species, and it has been found to be parasitized with nematodes in Cartagena Bay, as well as in Totumo marsh (14). Recently, trematodes such as Ascocotyle longa, have also been found infecting the liver of this fish in Cartagena Bay (15). The goal of this research was to document the histopathological lesions found in Mugil incilis from Totumo marsh.

MATERIALS AND METHODS
Study area. Totumo marsh is a waterbody considered unpolluted by industrial sources. It is located approximately 60 km north from Cartagena, Colombia (75°16'-W and 10°44'-N). It has an average depth of seven inches and a drainage area of 2100 ha. Its climate is predominantly tropical sub-humid with an average temperature of 28°C, and a relative humidity of 75%. The average monthly precipitation ranges from 60 to 100 mm (14).

Fish collection.
A total of 500 specimens of Mugil incilis were captured with the aid of fishermen from July 2004 to June 2005, and transported on ice to the laboratory, where necropsies were immediately performed. The following physicochemical parameters for surface water were registered during sampling campaigns, using a pH-meter, conductimeter and a field thermometer, respectively: pH 7.8±0.2, conductivity 142.7±75.2 µS/cm and temperature 27.8±0.8ºC.

Morphometric
analysis and larvae identification. Morphometric and parasite infestation measurements were performed for each fish specimen. Total length and weight, organ weight, and number of nematode larvae were recorded. Fish condition factor (CF) was calculated using the formula CF=100×W/L3, where W = total weight (g), L = total length (cm). Liver-, gill-and spleen-somatic indexes (HSI), were obtained as (OW/W) × 100, where OW=organ weight (g), (16). These morphometric parameters are considered markers of general fish health (17,18).
Nematodes were recovered, fixed, clarified and preserved. Preliminary nematode identification was based on keys previously published, and on morphological features suggested in the literature (19,20). Prevalence (proportion infected), mean intensity (parasites per infected individual), and mean abundance (parasites per individuals examined) were determined according to methods used by Bush et al (21).

Histopathogical analysis.
Livers were stored in buffered formalin and processed for histopathological examination. From each block, paraffin sections (5 µm) were stained with hematoxylin-eosin. The lesions were recorded including those non-specific related to inflammatory changes (infiltration for inflammatory cells and granulomatosis), steatosis, necrosis and melanomacrophage centers (MMCs), among others (22). The histopathological study was carried out by individually examining each sample under the light microscope, evaluating the prevalence of finding lesions in each sample.
Statistical analysis. Data are presented as mean ± standard error. Data were evaluated for normality and variance homogeneity using Kolmogorov-Smirnov and Barlett tests, respectively. Spearman correlations between infection levels and biometric parameters were tested. Analysis of variance (ANOVA) was employed to evaluate differences among means for different months in a particular sampling station. When normality was not achieved, ANOVA was replaced by Kruskal-Wallis test. Differences between means were evaluated by Student's t-test. For all statistical analysis, the criterion for significance was p<0.05.

RESULTS
Morphometric parameters as well as nematode parasitic infection indices obtained for all sampled specimens are presented in table 1.  Correlation analysis for morphometric and parasitic parameters are shown in table 2. As expected, there was a strong and significant correlation between total host weight and length (r=0.935, p<0.001). A positive and significant correlation was detected between nematode mean abundance and host length (r=0.525, p<0.0001) and weight (r=0.547, p<0.0001). Inverse and significant correlations were observed for nematode mean abundance and both host condition factor (r=-0.109, p=0.014) and gill-somatic index (r=-0.123, p=0.014). However, correlations were absent with internal organ somatic factors.
Histopathological findings detected in the liver of Mugil incilis collected in Totumo marsh are presented in table 3 and figure 3. The most distinctive histopathological feature of the liver was the presence of encapsulated trematode cysts. Some of these formed granulome cysts
For Mugil incilis, the positive relationship between intensity of nematode and length and weight found might be the result of a time of exposuredependent process. This observation has also been detected for other parasites infecting different fish species such as Austrodiplostomum compactum (Digenea) in Plagioscion squamosissimus (24), Sphincterodiplostomum musculosum in Cyphocharax gilbert (25), and Spatulifer maringaensis in Sorubim lima (26), among others. Condition factor (CF) and hepato-somatic index are screening indices that provide information about the general condition of fish, and those are useful to identify exposure/effects of potential contaminants, or data on energy reserves. The low negative and significant correlation observed between condition factor and parasite mean abundance could indicate that these nematodes are impacting fish health; perhaps by affecting food metabolism or inducing stress factors on fish.
This work has also shown that the liver of Mugil incilis is parasitized with trematode encapsulated cysts. A previous report on this species from Cartagena bay has suggested that this encapsulated trematode larvae is Phagicola longa (15). Histopathological examination revealed that trematodes were encapsulated by connective tissue in association with accumulation of lymphocytes and melanomacrophages, as previously described by others authors (22), with a host reaction to the larval presence characterized by fibroblast proliferation, which forms a capsule of concentric layers of connective tissue. This phenomenum has also been observed for metacercariae of the digenean Posthodiplostomum minimum in bluegill, Lepomis macrochirus (27).
The deposition of collagen layers and fibroblasts in the liver of Mugil incilis, probably is the result of a host defense reaction. In this study, most granulomes were linked to the presence of melano-macrophages centers (MMCs). These histological effects have been suggested as biomarkers for water quality in terms of lack of oxygen and chemical pollution (28). However, this process may also be the result of different stages of chronic inflammatory response to severe tissue damage or to a variety of infections.
There are not reported data showing that nematodes, in particular Contracaecum spp., or trematodes, could be producing zoonoses in human beings that eats Mugil incilis, only from Totumo marsh, but also from urban areas such as Cartagena and Barranquilla, where these fish are sold. However, the lack of information regarding fish parasitism, and the health hazards linked with fish consumption, could help to increase the risk of parasitic zoonoses in the area, as has been reported for Peru (29).
In addition, although fish-borne parasitic zoonoses have been focused on people from low-and middle-income countries. This problem is rapidly expanding because of globalization and migration, among other factors (30). Therefore, taking into account that these fish are currently used as a protein source for many people, it is suggested to implement sanitary measurements to avoid zoonoses.