Journal of Fisheries and Aquatic Science
ISSN 1816-4927
www.academicjournals.com OPEN ACCESS Journal of Fisheries and Aquatic Science
ISSN 1816-4927 DOI: 10.3923/jfas.2016.304.310
Research Article Huffmanela spp. (Nematoda, Trichosomoididae) from Microchirus azevia: Tissue Location and Correspondence of Host Muscle Discoloration with Parasite Burden
1Alexandra Esteves, 2Irene Oliveira, 3Paula Ramos, 4Ana Carvalho, 5Nuno Nazário and 1Fernanda Seixas
1Department of Veterinary Sciences, Animal and Veterinary Research Centre (CECAV), Universidade de Trás-os-Montes e Alto Douro (UTAD), 1013 Apartado, 5001-801, Vila-Real, Portugal 2Centre for the Research and Technology of Agro-Environmental and Biological Sciences (CITAB), Universidade de Trás-os-Montes e Alto Douro (UTAD), 1013 Apartado, 5001-801, Vila-Real, Portugal 3Laboratory for Pathology of Aquatic Animals, Portuguese Institute of the Sea and Atmosphere (IPMA), I.P. Avenida de Brasília, 1449-006 Lisboa, Portugal 4Veterinary Surgeon, Universidade de Trás-os-Montes e Alto Douro (UTAD), 1013 Apartado, 5001-801, Vila-Real, Portugal 5General Directorate for Veterinary and Food, Ministry of Agriculture, Rural Development and Fishery (DGAV), Ministério de Agricultura, do Desenvolvimen to Rural e Pescas, Largo da Academia Nacional das Belas Artes, nº2, 1249-105, Lisboa, Portugal
Abstract Forty four samples of Microchirus azevia (de Brito Capello) were analyzed, 75% of which presented black discoloration of musculature. Usually, the specimens presenting this black discoloration are rejected as unfit for consumption. Bipolar eggs somewhat resembling those of the nematode genus Huffmanela (Nematode, Trichosomoididae) were identified as the cause of the colour changes on the fish muscle. From this study it can be concluded that, although the skin can work as a camouflage for the observed color changes, there is a statistically significant relationship between the degree of color change and the number of eggs microscopically observed per field. The existence of a positive association (using SCA) between the classification of color changes before and after skin removal is a positive aspect regarding the validation of the veterinary inspector’s work when evaluating fish. It is important to highlight that some of the samples without skin color changes at microscopic examination presented trichinelloid eggs. The identification of Huffmanela sp. in a new host (Microchirus azevia) and the evidence that the presence of this parasite is not always accompanied by macroscopically evident changes, strengthens the belief that this infection may have been often underdiagnosed.
Key words: Huffmanela, Microchirus azevia, discoloration grade, eggs location
Received: February 04, 2016 Accepted: March 22, 2016 Published: June 15, 2016
Citation: Alexandra Esteves, Irene Oliveira, Paula Ramos, Ana Carvalho, Nuno Nazário and Fernanda Seixas, 2016. Huffmanela spp. (Nematoda, Trichosomoididae) from Microchirus azevia: Tissue location and correspondence of host muscle discoloration with parasite burden. J. Fish. Aquat. Sci., 11: 304-310.
Corresponding Author: Alexandra Esteves, Department of Veterinary Sciences, Animal and Veterinary Research Centre (CECAV), University of Trás-os-Montes e Alto Douro (UTAD), Quinta de Prados, Bloco Laboratorial, B1.03 5001-801 Vila Real, Portugal Tel: 351 259 350 659/351 259 350 480
Copyright: © 2016 Alexandra Esteves et al. This is an open access article distributed under the terms of the creative commons attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.
Competing Interest: The authors have declared that no competing interest exists.
Data Availability: All relevant data are within the paper and its supporting information files. J. Fish. Aquat. Sci., 11 (4): 304-310, 2016
INTRODUCTION associated and (3) To provide a microscopic description of the affected tissues and eventually search for new locations on Microchirus (Zevaia) azevia Capello, 1867 is present on fish organism with the aim of contributing to a better a demersal habitat on mud and sand of continental shelf from understanding of the Huffmanela sp., biology. the shore down to 250 m. Its food consists of a wide range of small benthic invertebrates, mainly amphipods and MATERIALS AND METHODS polychaetes (Algeria). Its distribution includes the Eastern Atlantic (Portugal Southward; a single record from South-West The analyzed sample consisted of 44 Microchiru sazevia of the British Isles) and the Mediterranean (Western portion, obtained on Atlantic coast from 12 catches in different days. Spain and Algeria) (http://species-identification.org/species. From the analyzed fish specimens, 33 (75%) exhibited php?species_group=fnam&id=1995). unusual black discoloration on the dorsal and ventral Veterinaries working at auction centers on the Portuguese musculature and 11 (25%) didn’t exhibit any discoloration coast have described the presence of unusual black change. discoloration on musculature of the pleuronecti form fish All specimens were measured, weighed and classified in Microchirus azevia (de Brito Capello). These color changes are a three grades system, based on the degree of discoloration of evident on the ventral (blind) face and also on dorsal faces of the ventral surface as observed through the skin, as grade (0) theses specimens. Usually, the specimens presenting this Specimens presented no skin color change; grade (1) black discoloration are rejected by fishermen or classified as Specimens presented a slight black discoloration of the skin unfit for consumption by veterinarian services due to their and grade (2) Specimens showed pronounced black repulsive appearance. discoloration. At a preliminary stage, it is verified that these unusual After this preliminary classification, ventral skin was color anomalies (black discolorations) of the muscle could removed from fish specimens, exposing the musculature. be explained by the presence of dark brown nematode Specimens were then subject to a reclassification and eggs compatible with genera Huffmanela. Huffmanela considering the degree of muscle discoloration as it was Moravec et al.1 comprises 20 valid species2-7 of extra-intestinal previously described. This classification was adapted from the nematodes, whose adults deposit eggs in the skin, gill, somatic one proposed by Mendes et al.12 for Trisopterus lucus. musculature, swimbladder, bone and mesenteries5,6. The fish were then subject to post mortem examination Advanced dark-shelled eggs of this parasite occur at the histopathology laboratory of the Universidade de frequently in masses in the host fish’s tissues, appearing as Trás-os-Montes e Alto Douro and fixed in 10% formalin. Tissue black spots. The eggs are lead with thin shells and then in the samples were processed for light microscopy in accordance host tissue, they increase in size and change in colour. The with standard methods, sectioned at 3 :m and stained with chitinous layer of the egg shell becomes thicker and gradually Haematoxylin and Eosin (H and E). turns from colourless to brown or even black8. To confirm whether the macroscopic discoloration Taxonomy of Huffmanela spp. has largely been based grading was in accordance with the parasitism intensity, the on egg morphology with considerations of host affiliation, egg number of eggs were evaluated microscopically on infection site and host geographic location1-4,9-11. Huffmanela longitudinal sections of the muscle. Ten microscopic sp. had already been described in the gadid Trisopterus luscus consecutive fields of muscle tissue were evaluated, using a 20x Linnaeus caught off the Portuguese coast by Mendes et al.12 power magnification objective in a blind trial and the eggs and redescribed by Esteves et al.10. average was calculated. To allow a better characterization of In the present study, it is evaluated that 44 Microchiru the eggs, in the absence of fresh samples, the fixed muscle sazevia, 75% of which presented black discoloration in the was submitted to muscle scraping and then the eggs were muscle. The aims of the present study were (1) to confirm the wet-mounted in lactophenol on cover slipped glass slides and presumptive diagnosis that the black discoloration observed photographed using a light microscope (Nikon Eclipse E600) on Microchirus azevia specimens were due to the presence equipped with a camera (Nikon DXM 1200) and the Image J of nematode eggs of the genus Huffmanela, (2) To confirm version 1.44o software. Fifty six eggs were measured in whether the degree of discoloration of fish and the observed micrometers and data were expressed as Mean±Standard number of eggs, counted in histological sections were Deviation (SD).
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Statistical analysis: Statistical analysis was performed using Kruskal-Wallis test was used to compare fish size, fish SPSS software (version 20.0 SPSS Inc., Chicago). To study the weight and egg counts among the 3 discoloration grades and statistical analysis associations between the discoloration the Dunn test, as post hoc test for multi comparison between degree on samples before and after removal of the ventral groups, when applicable. Values of p<0.05 were considered as skin, a chi-square test with residual adjusted values was used. statistically significant. If p was lower than 0.05 cells containing adjusted residuals above |2.0| were analyzed. Due to small sample size, a Monte RESULTS Carlo correction was applied. Kendall’s Tau-b measure of association was calculated. Forty four Microchirus azevia specimens were analyzed. Nevertheless, a Simple Correspondence Analysis (SCA) Macroscopically all, but 11 fish exhibited unusual black spots was performed. As the sanitary decision made on fish auction on the musculature, easily observed through the skin. Twenty and markets is based on fish without previously manipulation nine fishes showed slight dark muscle discoloration (grade 1) (with the skin), all remaining statistical analysis were made and 4 fishes showed severe dark discoloration of the dorsal based on the discoloration grading before removal of fish skin. and abdominal musculature (grade 2) (Fig. 1). The gill, skin and internal organs showed no macroscopic abnormalities. Table 1: Color grading classification before and after removal of ventral skin The reclassification after the removal of the fish’s ventral Grade of discoloration With the skin n (%) Without the skin n (%) skin resulted in some changes on classification (Table 1). Five 0 11 (25%) 6 (14%) 1 29 (66%) 28 (64%) (45.45%) of the fish specimens initially classified as grade 2 4 (9%) 10 (23%) 0 in the first evaluation were reclassified as grade 1 after
(a)
(b)
(c)
Fig. 1(a-c): Black discoloration classification on ventral musculature (after removal of ventral skin), (a) Grade 0, (b) Grade 1 and (c) Grade 2
306 J. Fish. Aquat. Sci., 11 (4): 304-310, 2016 the skin removal. Similarly, 7 (24.13%) of the samples initially some presented vitelline material, but lacked a recognizable classified as grade 1 were reclassified as grade 2. Only one larva. These eggs showing microscopic features compatible sample (7.14%) showed a decrease in grade after skin with Huffmanela spp. were located in the connective tissue removal, fromgrade 2 to grade 1 (Table 1). between muscle cells and were surrounded by inflammatory A significant association between the observed cells within granulomas. discoloration degree before and after skin removal was The granulomas presented isolated or multiple oval or found (c2 = 26.72, p<0.001, Tau-b = 0.688). Nevertheless, the deformed eggs in the centre; frequently there were phagocyte application of Simple Correspondence Analysis (SCA) based on cells inside the shrunken and destroyed eggs. Some fish also a contingency table, demonstrated an association between showed laminated concentric layers of proteinaceous material the observed discoloration degree before and after skin surrounding the eggs (Fig. 3b). The muscle cells exhibited removal (Fig. 2), which is reflected by the small graph distance myodegeneration, necrosis, atrophy and hypertrophy. between dots (with and without skin), representing grades of Occasionally, nematode specimens of an uncertain discoloration and the similar plan position on the graph. It is developmental stage (larvae or adults) were observed inside important to highlight this association by the small graph and or between the muscle cells (Fig. 3c). distance between the grade 0 of each measurement (with and Eggs were also detected in the skeletal muscle of without skin) in opposition to grades 1 and 2. oropharynx (n = 22) (Fig. 3b), in the skeletal muscle of the gill After the discoloration grading analysis, all specimens arch (n = 16) and operculum (n = 7), in the subcutaneous were submitted to a necropsy and histopathological exam. connective tissue (n = 7), in the skin (n = 2) (Fig. 3d), in the Microscopically the skeletal muscle showed ovoid dark shelled connective tissue surrounding the kidney (n = 4) and the trichinelloid embryonated eggs dispersed throughout the ovary (n = 1) in the subperitoneal connective tissue (n = 1) or muscle tissue, forming a linear or stripped pattern or in the kidney stroma (n = 1). The fish’s intestinal serosa or the occasionally small aggregates or spots of eggs in a variable digestive smooth muscle exhibited no abnormalities. No other number (Fig. 3a) in the endomysium or occasionally inside adult nematode specimens were observed in the various muscle cells or forming granulomas. Most eggs had a distinct analysed organs. plug and contained an in-folded larva, however, occasionally The muscle scraping revealed the presence of dark-brown
Degree before withdrawn the ventral skin eggs with larvae, seldom brown eggs containing vitelline Degree after withdrawn the ventral skin material and rare clear embryonated eggs or free larvae 1 (Fig. 4). 1 1 Brown eggs showed light-colored protruding plugs. The 0 0 size of dark-brown eggs with larvae (n = 50), including polar 0 plugs were 74.77-86.26 µm (80.42±2.88) long, 36.86-42.27 µm 2 -1 (39.08±1.36) wide, polar plugs were 4.82-6.87 µm
Dim. 2 (15.3%) 2 Dim. (5.86±0.54) long and 14.27- 19.82 µm (16.67±1.36) wide. Size 2 of eggs containing vitelline material, but no recognizable larva -2 (n = 4) including polar plugs were 80.45‒88.11 µm (84.48±3.87), diameter 37.65-38.97 µm (38.21±0.55), -3 polar plugs were 5.02-6.74 µm (5.95±0.36) long and -3 -2 -10 1 14.93-17.31 µm (16.72±0.87) wide. Clear non-operculate eggs Dim. 1 (84.7%) (n = 2) measured 47.03-51.52 µm (49.85±2.45) long and Fig. 2: Simple correspondence analysis of the color change 26.82-32.23 µm (28.97±2.87) wide. classification before (black dot) and after (white dot) Some brown bi-operculated eggs and clear eggs showed removal of the skin evidences that they were entirely surrounded by a thin
Table 2: Weight, length and eggs counts in the analyzed samples according to the skin grading discoloration Grade 0 X±SD (Median) Grade 1 X±SD (Median) Grade 2 X±SD (Median) p Fish weight (g) 47.59±11.52 (49.01) 48.88±11.27 (46.89) 55.08±10.98 (53.41) 0.41ns Fish length (cm) 15.090±0.96 (15.200) 15.49±1.20 (15.500) 16.10±1.11 (16.000) 0.19ns Eggs number/10 fields 3.39±4.81a (0.360) 11.60±7.06b (9.40) 11.83±7.76b (9.92) 0.002** a,b: Average values with unlike superscript letters within one row differ significantly at p<0.05, X±SD: Mean+/-Standard Deviation, ns: Not significant, **Highly significant
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(a) (b)
(c) (d)
Fig. 3(a-d): (a) Brown and clear embryonated eggs in a stripped pattern or small aggregates, (b) Oropharynx showing brown eggs surrounded by laminated proteinaceous material in the muscle, (c) Eggs and nematode specimens of uncertain developmental stage and (d) Skin showing brown trichinelloid embryonated eggs (a, b, c, d Bar = 100 µm)
Fig. 4: Muscle scraping showing dark-brown eggs with larvae and vitelline material and a clear egg (Bar = 50 µm)
308 J. Fish. Aquat. Sci., 11 (4): 304-310, 2016 transparent envelope, which in plugged eggs surrounded or the digestive smooth muscle exhibited no abnormalities, inclusively the polar plug. It is only observed 2 free larvae, but showing a clear tropism of this parasite to the skeletal we measured the diameter of 53 larvae, mostly inside the eggs muscle. Although apparently there is a high grade of that varied from 7.4-10.04 µm (8.79±0.59). microenvironment specificity, occasionally eggs were The fish analyzed were 27.21-75.07 g and the length observed in the skin (in the dermis), connective tissue and in ranged from 13.50-18.21 cm (data not shown). Simple data the kidney. observation showed that apparently there was no relationship The usual dark skin coloration of the dorsal face of azevias between fishes weight, length and the discoloration grading masks the black spots observed macroscopically on the observed. Table 2 shows the specimens’ data (weight, length) muscle, turning diagnosis of this parasitosis difficult in fish and the egg count, according to the grading classification inspection and auction. As aforementioned, fishes with based on the discoloration degree before skin removal. abnormal muscle discoloration are considered as unfit for A significant difference in the egg counts were observed human consumption and clearly the usual color of the dorsal between specimens from skin discoloration grades 1 and 2 face of the fishes bring a diagnostic problem and infection and fish with no skin color change. It was possible to confirm may be under diagnosed. Notwithstanding, even in specimens that there was no statistical association between weight or with no clear muscle color abnormality, classified as fit for lengths of the fishes and the discoloration grading. human consumption, it could find some residual number of It is notable that 7 (63.63%) out of 11 fishes classified as dark shelled trichinelloid embryonated eggs throughout the grade 0 before skin removal showed trichinelloid eggs in muscle tissue. muscles in the histological examination. Five of these cases It seems clear that skin can work as a camouflage for the were reclassified as grade 1 after skin removal: The mean egg color changes on fish muscle. This situation appears to be counts varied from 3.07-14.81 per 10 fields in these cases. more serious considering that on some of the samples without However, the 2 fishes that maintained a grade 0 classification skin color changes, the discoloration change stands out after after skin removal also showed a residual number of eggs in skin removal and at microscopic examination, trichinelloid the muscle (respectively 0.30 and 0.36). eggs were counted. All these fishes were approved for consumption on sanitary inspection. DISCUSSION However, it was proven the existence of a statistically significant relationship between the degree of color change Huffmanela Moravec et al.1 comprises 20 valid species2-7 and the number of eggs per field. and some of them in various teleost families, including The existence of a positive association (using SCA) Gadidae. The Huffmanela species briefly described from between the classification of color changes before and after Trisopterus luscus off Portugal by Mendes et al.12 has been skin removal is a positive aspect regarding the validation of 10 redescribed but still innominated. the veterinary inspector’s work when evaluating fish. However, Taxonomy of Huffmanela spp. has largely been based on one cannot ignore than more than 45% of the fish classified morphological structure and measurements of trichinelloid with 0 on color change before removal of ventral skin were eggs, as well as the host type, infection’s location and host afterwards reclassified as 1. 1-4,9-11 geographic location . According to what was said before, it isnot surprising that In the present study, it is observed trichinelloid eggs in the appearance of Huffmanela eggs in human stools has the muscle of various specimens of the pleuronectiform fish already been reported1,13,14, apparently as a result of prior Microchiru sazevia captured in the Atlantic sea. The eggs consumption of infected fish. However, these parasites seem features are in accordance with microscopic characteristics to be unimportant from a public health point of view. Until previously described by Moravec et al.1 as the genus know, there is no evidence of any consequence of this parasite Huffmanela. To our knowledge, this is the first description of on human health. Huffmanela eggs in Microchiru sazevia (de Brito Capello). The identification of Huffmanela spp. in a new host and Bi-operculated embryonated eggs were located in the the evidence that the presence of this parasite is not always muscle and connective tissue between muscle cells and accompanied by macroscopically evident changes, were surrounded by inflammatory cells forming granulomas, strengthens the belief that this infection may have been often suggesting that this parasite is not the fish’s intestinal serosa under diagnosed.
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ACKNOWLEDGMENT 7. Justine, J.L. and T. Iwaki, 2014. Huffmanela hamo sp. n. (Nematoda: Trichosomoididae: Huffmanelinae) from the The authors would like to thank CECAV-UTAD, dagger-tooth pike conger Muraenesox cinereus off Japan. CITAB-UTAD and the research is supported by national funds Folia Parasitol., 61: 267-271. 8. Zdarska, Z., D.G. Huffman, F. Moravec and J. Nebesarova, by FCT-Portuguese Foundation for Science and Technology, 2001. Egg shell ultrastructure of the fish nematode under the UID/CVT/00772/2013, UID/AGR/04033 projects Huffmanela huffmani (Trichosomoididae). Folia. Parasitol., and by FEDER/COMPETE/POCI-Operational Competitiveness 48: 231-234. and Internationalization Programme, under Progect 9. Justine, J.L., 2004. Three new species of Huffmanela POCI-01-0145-FEDER-006958. The authors wish to thank A. Moravec, 1987 (Nematoda: Trichosomoididae) from the gills Leite and L. Bento for their expert technical assistance. of marine fish off New Caledonia. Syst. Parasitol., 59: 29-37. 10. Esteves, A., F. Seixas, S. Carvalho, N. Nazario, M. Mendes REFERENCES and C. Martins, 2009. Huffmanela sp. (Nematoda: Trichosomoididae) muscular parasite from Trisopterus 1. Moravec, F., B. Koudela, K. Ogawa and K. Nagasawa, luscus captured off the Portuguese coast. Dis. Aquat. Org., 1998. Two new Huffmanela species, H. japonica n. sp. and 84: 251-255. H. shikokuensis n. sp. (Nematoda: Trichosomoididae) from 11. Bullard, S.A., C.F. Ruiz, A. McElwain, M.J. Murray, marine fishes in Japan. J. Parasitol., 84: 589-593. J.D. Borucinska and G.W. Benz, 2012. Huffmanela cf. 2. Moravec, F., 2001. Trichinelloid Nematodes Parasitic Carcharhini (Nematoda: Trichosomoididae: Huffmanelinae) In Cold-Blooded Vertebrates. Academia, Praha, from skin of a sandbar shark, Carcharhinus plumbeus, in the ISBN-13: 9788020008053, pp: 429. Pacific Ocean. J. Parasitol., 98: 333-340. 3. Justine, J.L., 2007. Huffmanela spp. (Nematoda, 12. Mendes, M., L.M.M. de Carvalho, P. Ramos, I. Fazendeiro Trichosomoididae) parasites in coral reef fishes off New and M.M. Afonso-Roque, 2005. Presence of Huffmanela Caledonia, with descriptions of H. balista n. sp. and H. longa eggs (Nematoda: Trichosomoididae), in the faneca n. sp. Zootaxa, 1628: 23-41. muscle-Trisopterus luscus (Linnaeus, 1758) of the coast of 4. Justine, J.L., 2011. Huffmanela plectropomi n. sp. Portugal. Proceedings of the 9th Congresso Iberico de (Nematoda: Trichosomoididae: Huffmanelinae) from the Parasitologia, October 25-28, 2005, Coimbra, Portugal. coralgrouper Plectropomus leopardus (Lacepede) off New 13. Schouten, H., R.M. Suriel-Smeets and M.A. Kibbelaar, 1968. Caledonia. Syst. Parasitol., 79: 139-143. The simultaneous occurrence of ova resembling Dicrocoelium 5. Ruiz, C.F. and S.A. Bullard, 2013. Huffmanela markgracei sp. dentriticum or Capillaria hepatica in the stools of n. (Nematoda: Trichosomoididae) from buccal cavity of inhabitants of Curaco. Trop. Geogr. Med., 20: 271-275. Atlantic sharpnose shark, Rhizoprionodon terraenovae 14. Gallego, J., C. Riera and M. Portus, 1993. Huffmanela sp. eggs (Carcharhiniformes: Carcharhinidae), in the Northwestern Gulf (Nematoda: Trichosomoididae), as a human spurious parasite of Mexico off Texas. Folia Parasitol., 60: 353-358. in a child from Barcelona (Spain). Folia Parasitol., 40: 208-210. 6. Ruiz, C.F., C.L. Ray, M. Cook, M.A. Grace and S.A. Bullard, 2013. A new species of Trichosomoididae (Nematoda) from skin of red snapper, Lutjanus campechanus (Perciformes: Lutjanidae), on the Texas-Louisiana Shelf, Northern Gulf of Mexico. J. Parasitol., 99: 318-326.
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