Resumen de Epidemiología molecular y desarrollo de anisákidos. Expresión diferencial de la actividad proteolítica en especies gemelas de anisakis simplex S.L
1. Introduction and justification It is known as anisakidosis the pathology produced by anisakid larvae, which are parasitic nematodes of the Anisakidae family. Larval third stage (L3) of this family can be found in fish and squid, which act as intermediate/paratenic hosts, and larval fourth stage (L4) and adults in marine mammals and piscivorous birds, which are definitive hosts of the parasites of the subfamily Anisakinae. Human can suffer anisakidosis when consuming raw or undercooked fish that is infected by the L3 of any of the four genera that have been related to anisakidosis: Anisakis, Pseudoterranova, Contracaecum or Hysterothylacium (Myers 1975; Ishikura et al. 1993; Fernández-Caldas et al. 1998; Takahashi et al. 1998).
This pathology usually occurs with digestive and/or allergic symptoms, and despite being an underdiagnosed infection (Navarro Suárez et al. 2014), it is estimated that there have been more than 30,000 cases worldwide, most of which have been described in Japan (Takahashi et al. 1998; Audicana et al. 2003). In addition, it is estimated that in this country between 2,000 and 3,000 new cases are described every year (Umehara et al. 2007). In Spain, a large number of cases have also been described (Arenal Vera et al. 1991; Repiso Ortega et al. 2003; del Rey-Moreno et al. 2008), being the European country where most cases have been diagnosed, most of them due to the consumption of anchovies in vinegar (Bao et al. 2017). The larvae of the genus Anisakis are the most important because of its clinical importance: it is estimated that around 97% of all the cases described worldwide are caused by this type of larvae (Audicana et al. 2003). The pathology produced larvae of the genus Anisakis is known specifically as anisakiasis/anisakiosis, and there are several species involved: A. simplex sensu stricto, A. pegreffii and, rarely, A. physeteris s.l. The first two belong to a complex of sibling species morphologically indistinguishable in L3 stage, and although it has been demonstrated by studies both in experimental animals and in vitro, that A. simplex s.s. is more pathogenic than A. pegreffii, both are capable to produce anisakiasis (Quiazon et al. 2011; Arizono et al. 2012; Romero et al. 2013).
A possible way to address this difference in pathogenicity would be through the study of the proteolytic activity. Proteases are a type of protein of great importance in parasitic nematodes, where they have been related to their pathogenicity and they have a crucial role in the invasion of host tissues (McKerrow et al. 2006), as well as participating in various biological functions, such as nutrition, moulting or embryonic development (Malagón et al. 2013). The study of the proteolytic activity of the previously described species could help to identify the possible differences among them, justifying this difference in pathogenicity. In addition, changes in proteolytic activity that occur during the development process could be related to some biological function. Also of interest are cathepsins, which are a type of proteases of special importance in parasitic nematodes. These molecules have been postulated as possible therapeutic targets, being of great importance in the development of vaccines (Dalton et al. 1996; Renard et al. 2000; Robinson et al. 2008; Knox 2012).
On the other hand, it is interesting to analyze the presence of anisakid larvae in fish of commercial and culinary interest, which allows to take preventive measures to avoid anisakidosis. There are several species that can affect humans, so it is also relevant to know which species are parasitizing the fish we consume. It is also important to deep the knowledge into the biology and development of these parasites, and also to identify the characteristics that allow to distinguish between the different species with clinical importance. In addition, the life cycle of several species of anisakids is not elucidated and there are different opinions about the embryonic development that occurs inside the egg. Knowing thoroughly the life cycle of a parasite allows to design strategies to control it, which is fundamental in any parasitosis. In the case of anisakids, which have complex life cycles and with many species not yet well differentiated involved, much research is still needed.
2. Materials and Methods 2.1. Sample collection for epidemiological and life cycle studies Sardines (Sardina pilchardus) and blue whiting (Micromesistius poutassou) landed in several harbors of Spain were transported in ice flakes to lab and then immediately measured and weighed, after which they were dissected. Larvae removed from the abdominal cavity were maintained on ice bath until their morphological identification (Berland 1961). The viscera and the musculature of the fish were subjected to a pepsin digestion for 2-8 hours separately. All the extracted larvae were morphologically identified and then frozen at -20 °C until molecular identification using PCR-RFLP technique, for which NC2 and NC5 primers described by Zhu et al. (1998) and the restriction enzymes Taq1 and Hinf1 Fast Digest, following the method used by other authors (Martín-Sánchez et al. 2005), were employed.
For life cycle studies, female adults from Contracaecum multipapillatum s.l. obtained from brown pelican (Pelecanus occidentalis) were dissected and eggs were taken from the most external part of the uteri. Then, they were axeniced and placed in saline solution at different concentrations. The development of the eggs was monitored to distinguish the different stages and hatching and survival of the larvae in the saline solutions, which were renewed weekly, was recorded. After 1 month under these conditions, eggs and larvae were moved from the saline solution to a Grace’s insect medium solution supplemented with 2% v/v basal medium Eagle’s vitamins (100×) solution, 1 mM L-cysteine, 1 g/L glucose, 20% v/v heat-inactivated foetal bovine serum and 1% v/v RPMI-1640 amino acid solution (50×), adjusting pH to 7.2.
The C. multipapillatum s.l. larvae obtained inmediately after egg hatching and others at different development stages were processed for scanning electron microscopy (SEM) without any further preparation. Likewise, larvae of A. physeteris collected from blue whiting from Mediterranean Sea and L4 obtained from in vitro culture (Iglesias et al. 1997, 2001) were fixed in hot 70% (v/v) ethanol and preserved for preparation for examination by SEM. The fixed larvae underwent critical point drying and cut into 3 sections, to avoid the distortion of the larvae which occurs when fresh or only fixed are cut. The anterior and posterior sections were separated for SEM preparation while a small cylindrical part of the central section was used for molecular identification.
2.2. In vitro culture for protease and cathepsin assays and preparation of Anisakis extracts The following larval stages of Anisakis type I from blue whiting from Cantabrian Sea and Mediterranean Sea, were taken to study: L3 freshly collected from fish (L3-0h), L3 after 24 hours in culture (L3-24h), larvae from fourth larval stage (L4) 24 hours after moulting (L4-24h), and L4 after 14 days in culture (L4-14d). For the L3-0h stage, the larvae immediately after its extraction from the fish and washing in sterile cold saline solution were frozen. For the rest of the stages, the larvae were axenized and individually placed in culture as indicated in other works (Iglesias et al. 1997, 2001). Once the desired stage was obtained, the larvae were washed and frozen at -20 °C until use.
For extract preparation, larvae were separately homogenized in a small volume of 50 mM Tris/HCl buffer with 20% glycerin w/v at pH 7.8, in order to stabilize the proteins and avoid its rapid degradation (Gianfreda and Scarfi 1991; Iyer and Ananthanarayan 2008), finally completing upto 500 μl. They were subsequently centrifuged at 19,000 x g for 20 minutes at 4 ºC, and the supernatant was used for enzymatic assays.
2.3. Proteolytic and cathepsin assays To determine the proteolytic activity, soluble extract of L3-0h of A. simplex s.s. and discontinuous system of buffers of pH 2 to 11 were used. The proteolytic activity was determined by measuring the fluorescence emitted after the degradation of the fluorogenic substrate bodipy FL-casein with excitation λ 490 nm and excitation λ 510 nm. The final concentration into well was: 50 mM of buffer, 50 μl of extract/ml (equivalent to 25 μg of protein), 1 mM of CaCl2 and 5 μg of substrate/ml, for a final volume of 200 μl (Malagón et al. 2010).
Inhibition tests were carried out at the two pHs in which maximum proteolytic activity was detected (Malagón et al. 2011). The following control enzymes and inhibitors specific to each group of proteases were used: pepsin (64 U/ml) and pepstatin A (0.02mM), respectively, for aspartic proteases; thermolysin (0.2 U/ml) and 1,10-phenanthroline (2 mM), for metalloproteases; papain (0.24 U/ml) and E64 [trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane] (0.05 mM), for the cysteine proteases; chymotrypsin (0.1 U/ml) and AEBSF [4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride] (2 mM), for serine proteases. The final concentration into well was: 50 mM buffer, 1 mM CaCl2, enzymes and inhibitors as described above, 50 μl of extract/ml (equivalent to 25 μg of protein), 5 μg of substrate/ml. The substrate was the same, i.e. bodipy FL casein.
For the determination of cathepsin L and B activities, procedures of Malagón et al. (2010) were employed. Ionic strength was maintained constant at 0.6 M by addition of NaCl as necessary. Dithiothreitol (DTT) was added to buffers for 1 mM final concentration into well. Two fluorogenic substrates were used: Z-FR-AMC (N-α-benciloxicarbonil-L-fenilalanil-L-arginina-7-amido-4-metil-cumarina) to determine cathepsins B and L, and Z-RR-AMC (N-α-benciloxicarbonil-L-arginil-L-arginina-7-amido-4-metil-cumarina) to determine cathepsin B. Fluorescence was detected using a fluorometer with excitation at λ 355 nm and emission at 460 nm. The final concentration in well was: 100 mM of tris-maleic buffer with DTT at 1mM, 50 μl of extract/ml (equivalent to 10 μg of protein), 150 μl of substrate (100 μM)/ml (final concentration 15 μM), and bidistilled water to complete the final volume upto 200 μl.
2.4. Statistical study SPSS 20.0 was used for the analysis of epidemiological risk factors in fish. The following variables were used: size, weight, sex, condition factor, origin of the fish and month of capture. Univariate model was designed using Anisakis infection as a dependent variable, and those variables that had shown a statistically significant association were selected to perform a multivariate model.
For the study of proteases and cathepsins in A. simplex s.s. and A. pegreffii, SPSS 22.0 was also used. An analysis of the variance (ANOVA) was carried out, after checking that the residuals of the variables followed a normal distribution by the Shapiro-Wilk test (p>0.05 in all cases) and with the support of Q-Q graphics. After ANOVA, a post hoc study was carried out using the Bonferroni test for the variables that had shown significant differences. The level of significance was set at p<0.05. When the residuals of the variables did not follow a normal distribution, Kruskal-Wallis test was performed and a post hoc study using the Mann-Whitney U test in the variables that had shown statistically significant differences was carried out.
3. Results 3.1. Epidemiological and life cycle studies Three Anisakis genotypes were identified in the Atlantic Ocean: A. simplex sensu stricto, A. pegreffii and a hybrid genotype between these two species; and four genotypes were found in the Mediterranean Sea: the same three previously mentioned and A. physeteris. Sardine and blue withing infection was associated with fish length/weight, which agree with the results of other authors (Adroher et al. 1996; Valero et al. 2000; Rello et al. 2008). The public health authorities must continue to emphasize the need for suitable thermal treatment (freezing or cooking) of the fish prior to consumption.
In Contracaecum multipapillatum s.l., hatching and survival of the larvae were greater at 15 °C than at 24 °C, and increased salinity resulted in a slight increase in hatching but seemed to reduce survival at 24 °C, but not at 15 °C. The newly hatched larvae were ensheathed and highly motile. When placed in culture medium, the hatched larvae grew within their sheath, and a small percentage exsheathed completely 2 weeks later. Although they did not moult during culture, SEM and optical microscopy revealed a morphology typical of third-stage larvae. Thus, we suggest that newly hatched larvae from eggs of C. multipapillatum s.l. are third larval stage but with sheath of the second larval stage, as occuring in other anisakids. We suggest a life cycle of C. multipapillatum in which L3 hatch from the egg, copepods are involved as first intermediate hosts, mullets as intermediate/paratenic hosts and brown pelicans as final hosts in the geographical area of Bahía de La Paz, Mexico In A. physeteris the development of a row of denticles on each of the three prominent lips, almost reaching the buccal commisures, was observed in the L4. Pores of unknown function were found in the upper external part of each lip. Clearly developed cephalic papillae, amphids, and deirids were also observed in L4, while, although present in L3, they were beneath the cuticle. Phasmids were detected in L4 but not in L3. In females the vulva could be seen by light microscopy, apparently still covered by the cuticle.
3.2. Proteolytic assays: proteases and cathepsins Two peaks of maximum proteolytic activity were detected at pH 6.0 and 8.5. Activity was detected in all developmental stages in A. simplex s.s. y A. pegreffii. At pH 6.0, L4 larvae showed higher proteolytic activity than L3 larvae in both species (p<0.001), the majority of which was due to metalloproteases and aspartic proteases, that could be related to nutrition (Dziekońska-Rynko and Rokicki 2005). At pH 8.5, proteolytic activity was higher in A. simplex s.s. than in A. pegreffii (p<0.01). At this pH, most of the activity was due to metalloproteases in all developmental phases of both species, although in L3-0h, the activity of these proteases was significantly higher (p<0.03) in A. simplex s.s. than in A. pegreffii. This could be related to the greater invasive capacity of the former: some authors relate metalloproteases with pathogenicity of some anisakids (Malagón et al. 2011). Serine proteases, which have also been implicated in the pathogenicity of A. simplex (Sakanari and McKerrow 1990; Morris and Sakanari 1994), show a higher activity (p<0.05) in A. simplex s.s. than in A. pegreffii. These differences in metalloproteases at L3-0h and in serine proteases could be contributing to the previously reported differences in pathogenicity between these two Anisakis species.
The cathepsin-like activities in A. simplex s.s. and A. pegreffii, were also studied. With Z-FR-AMC, substrate for cathepsin L, activity at acid pH, with a peak at pH 5.0, was detected. This activity was fully inhibited with E64, a cysteine proteases specific inhibitor, and might be involved in digestive processes, among others. The activity of A. simplex s.s. is higher than the one shown by A. pegreffii (p=0.06). With Z-RR-AMC, substrate from cathepsin B, a peak at pH 8.0 in L4-stages of A. simplex s.s. is detected. This activity was not inhibited with E64 but with AEBSF, showing to be composed by 90% with serine proteases and with some contribution of metalloproteases. These characteristics suggest that the detected activity might be produced by a trypsin-like serine protease, previously described in the scientific literature (Sakanari and McKerrow 1990; Morris and Sakanari 1994), although further research is needed to confirm this hypothesis. In any case, it is noticeable that the activity is concentrated within phase L4 of A. simplex s.s. and that it is very low in A. pegreffii (p=0.001).
4. Conclusions 1. There is a correlation between both sardines and blue whiting length/weight and the Anisakis infection. Risk of infection increases with fish size. The greater prevalence in sardines were from La Coruña, NW Spain (28,3%) followed by Ondarroa, northern Spain (5%). The species found in this study were A. simplex s.s., A. pegreffii, hybrids genotypes between them, and A. physeteris (this only in the Mediterranean blue whiting).
2. Third larval stage (L3) of Contracaecum multipapillatum s.l. hatches from the egg ensheathed with L2 cuticule and become infective without any further moulting, being the morphology of newly hatched larvae identifiable to the L3 found in fish. The SEM studies of the L3 and L4 stages of Anisakis physeteris have allowed the identification of diverse structures in them, highlighting the sensory ones. 3. 3. Aspartic- and metalloprotease activities predominate at pH 6.0 while, at pH 8.5, the activity of metalloproteases in A. simplex s.s. and A. pegreffii. Serine proteases show higher activity in the former than in A. pegreffii (p<0.05) and they might be contributing to the differential pathogenicity showed by these two species. Metalloproteases activity is greater in A. simplex s.s. than in A. pegreffii in the L3 just obtained from fish (L3-0h), that could be related to their different tissue penetration capacity. No cathepsin B-like have been detected under our experimental conditions in any of the studies species. Cathepsin L-like shows a peak at pH 5.0 and decreasing with development and it could be related to nutrition.
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