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Histopathological changes in the gills of Channa punctatus Bl. Infected with Henneguya waltairensis
Arch. Protistenk. 129 (1985): 199-202
Department of Zoology, Andhra University Waltair, India
Histopathological Changes in the Gills of Channapunctatus BL. Infected with Henneguya waltairensis1 ) By C. KALAVATI and C. C. NARASIMHAMURTI With 5 Figures Key words: Histopathological changes; Henneguya waltairensis
Summary The effect of the myxosporidian parasite, Henneguya waltairensis on the gills of Channa punctatus is studied. During the early stages of infection the parasite induces hypertrophy of the host tissue with associated vacuolization of the cell cytoplasm. In the later stages degenerative changes are more conspicuous and are associated with accumulation of macrophages. The rupture of the cyst is associated with the haemorrhage of capillaries. The parasite is pathogenic.
Introduction The effect of myxosporidian parasites on fishes and the degree of pathogenicity is a much debated question during the last quarter of the century. Various types of tissue reactions have been reported in fishes infected with myxosporidian parasites. NIGRELLI and SMITH (1940) reported an inflammatory response in the gills of Lcialuru» punctatus infected with Henneguya exilis. JAKOWSKA and NIGRELLI (1953) observed extensive damage associated with accumulation of lymphocytes and fibroblasts in hosts infected with H. electrieus. They also found degenerative changes in the liver, kidney and heart. Myxoboluslintoni caused marked changes in the epidermis and hypodermis leading to the formation of fibroblastic tumours in Cyprinoides variegatus (NIGRELLI and SMITH 1940). Hyperplasia of the epithelium, including squammous muscle cells was observed in Ameiurus nebulosus when infected with Henneguya ameiurensie (NIGRELLI and SMITH 1940). However, some of the myxosporidian parasites appear to be innocuous and may not induce any response in the host tissue (GREVEN 1956). AISA (1972) did not observe any histological changes in Tinea tinea infected with Myxobolus ellipsoides braemaeformis. DYKOVA and LOM (1978) studied in detail the histopathological changes caused by H. psorospermica and H. ereplini and observed various types of tissue reactions. They also found changes brought about by a change in the temperature. Obviously the histopathological changes produced by myxosporidian parasites vary and are of different types. In the present investigation the changes brought about in the gill lamellae of Channa punetatus BL. infected with Henneguya waltairensis have been studied using various histological and histochemical stains. 1) Abstract presented at the VI. International Congress of Protozoology held at Warsaw, Poland, in August 1982.
200
C.
KALAVATI
and C. C.
NARASIMHAMURTI
Materials and Methods Channa punctoius of different size groups are collected during different seasons of the year and the gills are examined for myxosporidian parasites. Infected gill filaments are isolated and cleaned free from adhering mucus using a fine camel hair brush without causing damage either to the lamellae or to the cysts. They are fixed either in 10 % neutral formalin, Carnoy's fluid or alcoholic Bonin's fluid and sectioned at 8 p.m thickness. They are stained either with Heidenhain's iron haematoxylin-Eosin, Azocarmine or according to the Feulgen's technique, periodic acid Schiff method, Alcian blue method, Dialysed iron technique, Malachite green, Mercuric Bromophenol blue, Van Gieson's technique or Verhoeff's technique to study the various histological and histochemical changes.
Observations Fingerlings of Channa punctata ranging in size from 2.5-4.5 cm collected during the months of July and August did not show any external indications of infection. Sections of the gills occasionally showed plasmodia in the blood capillaries, each with 2-8 nuclei. Young fish measuring 7.5-10.0 em in length collected during SeptemberOctober period sometimes showed opaque white cysts measuring 2.0-3.0/lm in diameter on the secondary gill lamellae or near the gill rachis. Infected gill lamellae showed conspicuous enlargement at the base. Sections of the cysts showed that they are surrounded by stratified epithelial cells and contained a large number of somatic nuclei along the periphery and a few deeply stained generative nuclei in the centre (Fig. 1). The cysts increased in size with growth and a mature cyst containing fully formed spores occupied the entire gill lamella (Fig. 2). The cells surrounding the growing cyst showed hypertrophy and vacoulization of the cytoplasm (Fig. 3). The hypertrophied host tissue surrounds the wall of the parasite and forms a layer of cells surrounding it. Sometimes the cell membrane becomes incipient and the layer appeared almost syncytial and showed cellular disintigration, A number of degenerating macrophages and eosinophiles are observed in the cytoplasm surrounding the cyst. These corpuscles may have been derived from the ruptured capillaries. Histochemical observations on the fully formed myxosporidian cysts showed a layer of stratified epithelium, a band of connective tissue and a layer of granulated cytoplasm beneath it. The granules in the cytoplasmic layer are positive to Alcian blue with 0.06 M MgCI2 , 0.2 M MgCl2 and 0.5 M MgCl2 suggesting the presence of Chondriotin mucins, a material generally present in the cartilage and skin of fishes. The connective tissue band is positive to PAS, Alcian blue, Aldehyde fuchsin after oxidation with Van Gieson's stain. It is stained deep blue with Azoearmine-aniline blue and deep red with Verhoeff's stain indicating that it is a connective tissue of collagen nature. The lumen of the cyst is filled with spores embedded in a matrix of mucus material. Each spore has a thin layer of mucus surrounding it and the two caudal processes lie parallel to each other. However, when the cysts burst and release the spores into the surrounding water the caudal processes assume a characteristic' 'U" shaped appearance with the mucus extending in the form of a fine film between them. Spores released by the rupture of the cyst are sometimes associated with the release of a number of blood cells into the surrounding mucus probably as a result of haemorrhage of the capillaries (Fig. 4). When the gill filaments are heavily infected they showed a frayed appearance (Fig. 5). There is no typical host response or regeneration oftissue at this stage. Heavily infected fish are generally lethargic and respired with a slow movement of the operculum. They did not survive for more than 1 h in the laboratory.
Hi stopathologi cal Ch anges
Fig. F ig. F ig. F ig. F ig.
1. 2. 3. 4. 5.
201
E arly cyst in the b a sal r egion of the gill lamella. Mature cyst occupyi ng the en ti re len gth of the gill lamella. Cyst (note the hypertrophy of the surro u n ding cells ). R uptured cyst showing the re lease of t he spo res. H eavily infected fish showing frayed gi ll filamen ts.
Discussion The histological cha nges broug ht about by H. waltairensis showed t hat in t he early cystic stages t he parasit e indu ces hypertrophy of the host t issue wit h ass ociated vac uoliza tion of t he cell cytoplasm. In the la t er st ages when t he eyst s are mature degenerat ive cha nges appea red more conspicuous and a re associated wit h t he accumulation of macrophages. It is obvious t hat t he parasit e is pa thogenic since t here is hypertrop hyand degenera ti on of t he tissue an d t he rupture of t he cyst is associated wit h haem-
202
C. KALAVATI and C. C. NARASIMHAMURTI, Histopathological Changes
orrhage. Similar observations are made by DYKOVA and LOM (1978) in H . psorospermica but they did not observe associated haemorrhage. However, GREVEN (1956) reported the occurrence of haemorrhage in the same parasite . ERGENS and LOM (1970) reported changes in ti ssues and formation of granulomatou s ti ssue as a host response in Henneguyapsoroepermica. Similar changes, particularly the appearance of granulomatous tissue has not been observed in H. uialtairensis. Th e occurrence of fra yed gill ti ssue, however, suggest s that the host respon se is poor. The rupture of the myxosporidian cyst probably leaves behind a scar which redu ces t he respiratory surface of the gill.
Acknowledgement Our thanks are due to the U nive rsit y Grants Commission, New D elhi, for the sanction of a D ep artmental support during which time this work has been carried out. We are also thankful to the au t h orit ies of the Andhra University for the excellent faciliti es provided to carry out this work.
Literature AISA, E.: Osservazioni sie una m issoboliasi delle Tinche. P arasit ologi a 14 (1972): 219-225. DYKOVA, 1., and LO;\I, J.: Histopathological ch a nges in the fi sh gills in fecte d with myxosporidian parasites of the genus H enn eguya. J . Fish. Biol. 12 (1978) : 197-202. EUGENS, R. , a nd LOM, J. : Caus a tive agents of the parasiti c di seases of fish . Praha 1970: 1-383. GREVEN, U. : E in Beitrag zum Verhalten von Myxosp oridien in den Kiemen einiger SiiJ3 wasser · fi sche, Z. Fisch er ei u . Hilfswiss. 5 (1956): 110-114. JAKOWSKA, S., and NIGRELLI, R . F .: The p athology of m y x osp oridioses in the elect ric eel, Electroph orus electricus (L.) cause d b y H enneguya »isceralis an d H. lectri ca spp . nov. Zoologica ' . N . Y. 38 (19 53): 183-191. NIGRELLI, R . F. , and SMITH, G. M. : A p ap illa ry cys t ic di sease a ffect ing barbe ls of Ameirus nebu · losus (L e Sue u r) ca use d by m yxosp oridian H enn egya ameiurensis sp . nov'. Zoologies N.Y. 25 (1940): 89-96. ; Author 's add ress: Dr. C. KALAVATI and Dr. C. C. NARASUIHAMURTI, D ep artment of Zoology , Andhra U nive rs ity, W altair 530 003, India .
Department of Zoology, Andhra University Waltair, India
Histopathological Changes in the Gills of Channapunctatus BL. Infected with Henneguya waltairensis1 ) By C. KALAVATI and C. C. NARASIMHAMURTI With 5 Figures Key words: Histopathological changes; Henneguya waltairensis
Summary The effect of the myxosporidian parasite, Henneguya waltairensis on the gills of Channa punctatus is studied. During the early stages of infection the parasite induces hypertrophy of the host tissue with associated vacuolization of the cell cytoplasm. In the later stages degenerative changes are more conspicuous and are associated with accumulation of macrophages. The rupture of the cyst is associated with the haemorrhage of capillaries. The parasite is pathogenic.
Introduction The effect of myxosporidian parasites on fishes and the degree of pathogenicity is a much debated question during the last quarter of the century. Various types of tissue reactions have been reported in fishes infected with myxosporidian parasites. NIGRELLI and SMITH (1940) reported an inflammatory response in the gills of Lcialuru» punctatus infected with Henneguya exilis. JAKOWSKA and NIGRELLI (1953) observed extensive damage associated with accumulation of lymphocytes and fibroblasts in hosts infected with H. electrieus. They also found degenerative changes in the liver, kidney and heart. Myxoboluslintoni caused marked changes in the epidermis and hypodermis leading to the formation of fibroblastic tumours in Cyprinoides variegatus (NIGRELLI and SMITH 1940). Hyperplasia of the epithelium, including squammous muscle cells was observed in Ameiurus nebulosus when infected with Henneguya ameiurensie (NIGRELLI and SMITH 1940). However, some of the myxosporidian parasites appear to be innocuous and may not induce any response in the host tissue (GREVEN 1956). AISA (1972) did not observe any histological changes in Tinea tinea infected with Myxobolus ellipsoides braemaeformis. DYKOVA and LOM (1978) studied in detail the histopathological changes caused by H. psorospermica and H. ereplini and observed various types of tissue reactions. They also found changes brought about by a change in the temperature. Obviously the histopathological changes produced by myxosporidian parasites vary and are of different types. In the present investigation the changes brought about in the gill lamellae of Channa punetatus BL. infected with Henneguya waltairensis have been studied using various histological and histochemical stains. 1) Abstract presented at the VI. International Congress of Protozoology held at Warsaw, Poland, in August 1982.
200
C.
KALAVATI
and C. C.
NARASIMHAMURTI
Materials and Methods Channa punctoius of different size groups are collected during different seasons of the year and the gills are examined for myxosporidian parasites. Infected gill filaments are isolated and cleaned free from adhering mucus using a fine camel hair brush without causing damage either to the lamellae or to the cysts. They are fixed either in 10 % neutral formalin, Carnoy's fluid or alcoholic Bonin's fluid and sectioned at 8 p.m thickness. They are stained either with Heidenhain's iron haematoxylin-Eosin, Azocarmine or according to the Feulgen's technique, periodic acid Schiff method, Alcian blue method, Dialysed iron technique, Malachite green, Mercuric Bromophenol blue, Van Gieson's technique or Verhoeff's technique to study the various histological and histochemical changes.
Observations Fingerlings of Channa punctata ranging in size from 2.5-4.5 cm collected during the months of July and August did not show any external indications of infection. Sections of the gills occasionally showed plasmodia in the blood capillaries, each with 2-8 nuclei. Young fish measuring 7.5-10.0 em in length collected during SeptemberOctober period sometimes showed opaque white cysts measuring 2.0-3.0/lm in diameter on the secondary gill lamellae or near the gill rachis. Infected gill lamellae showed conspicuous enlargement at the base. Sections of the cysts showed that they are surrounded by stratified epithelial cells and contained a large number of somatic nuclei along the periphery and a few deeply stained generative nuclei in the centre (Fig. 1). The cysts increased in size with growth and a mature cyst containing fully formed spores occupied the entire gill lamella (Fig. 2). The cells surrounding the growing cyst showed hypertrophy and vacoulization of the cytoplasm (Fig. 3). The hypertrophied host tissue surrounds the wall of the parasite and forms a layer of cells surrounding it. Sometimes the cell membrane becomes incipient and the layer appeared almost syncytial and showed cellular disintigration, A number of degenerating macrophages and eosinophiles are observed in the cytoplasm surrounding the cyst. These corpuscles may have been derived from the ruptured capillaries. Histochemical observations on the fully formed myxosporidian cysts showed a layer of stratified epithelium, a band of connective tissue and a layer of granulated cytoplasm beneath it. The granules in the cytoplasmic layer are positive to Alcian blue with 0.06 M MgCI2 , 0.2 M MgCl2 and 0.5 M MgCl2 suggesting the presence of Chondriotin mucins, a material generally present in the cartilage and skin of fishes. The connective tissue band is positive to PAS, Alcian blue, Aldehyde fuchsin after oxidation with Van Gieson's stain. It is stained deep blue with Azoearmine-aniline blue and deep red with Verhoeff's stain indicating that it is a connective tissue of collagen nature. The lumen of the cyst is filled with spores embedded in a matrix of mucus material. Each spore has a thin layer of mucus surrounding it and the two caudal processes lie parallel to each other. However, when the cysts burst and release the spores into the surrounding water the caudal processes assume a characteristic' 'U" shaped appearance with the mucus extending in the form of a fine film between them. Spores released by the rupture of the cyst are sometimes associated with the release of a number of blood cells into the surrounding mucus probably as a result of haemorrhage of the capillaries (Fig. 4). When the gill filaments are heavily infected they showed a frayed appearance (Fig. 5). There is no typical host response or regeneration oftissue at this stage. Heavily infected fish are generally lethargic and respired with a slow movement of the operculum. They did not survive for more than 1 h in the laboratory.
Hi stopathologi cal Ch anges
Fig. F ig. F ig. F ig. F ig.
1. 2. 3. 4. 5.
201
E arly cyst in the b a sal r egion of the gill lamella. Mature cyst occupyi ng the en ti re len gth of the gill lamella. Cyst (note the hypertrophy of the surro u n ding cells ). R uptured cyst showing the re lease of t he spo res. H eavily infected fish showing frayed gi ll filamen ts.
Discussion The histological cha nges broug ht about by H. waltairensis showed t hat in t he early cystic stages t he parasit e indu ces hypertrophy of the host t issue wit h ass ociated vac uoliza tion of t he cell cytoplasm. In the la t er st ages when t he eyst s are mature degenerat ive cha nges appea red more conspicuous and a re associated wit h t he accumulation of macrophages. It is obvious t hat t he parasit e is pa thogenic since t here is hypertrop hyand degenera ti on of t he tissue an d t he rupture of t he cyst is associated wit h haem-
202
C. KALAVATI and C. C. NARASIMHAMURTI, Histopathological Changes
orrhage. Similar observations are made by DYKOVA and LOM (1978) in H . psorospermica but they did not observe associated haemorrhage. However, GREVEN (1956) reported the occurrence of haemorrhage in the same parasite . ERGENS and LOM (1970) reported changes in ti ssues and formation of granulomatou s ti ssue as a host response in Henneguyapsoroepermica. Similar changes, particularly the appearance of granulomatous tissue has not been observed in H. uialtairensis. Th e occurrence of fra yed gill ti ssue, however, suggest s that the host respon se is poor. The rupture of the myxosporidian cyst probably leaves behind a scar which redu ces t he respiratory surface of the gill.
Acknowledgement Our thanks are due to the U nive rsit y Grants Commission, New D elhi, for the sanction of a D ep artmental support during which time this work has been carried out. We are also thankful to the au t h orit ies of the Andhra University for the excellent faciliti es provided to carry out this work.
Literature AISA, E.: Osservazioni sie una m issoboliasi delle Tinche. P arasit ologi a 14 (1972): 219-225. DYKOVA, 1., and LO;\I, J.: Histopathological ch a nges in the fi sh gills in fecte d with myxosporidian parasites of the genus H enn eguya. J . Fish. Biol. 12 (1978) : 197-202. EUGENS, R. , a nd LOM, J. : Caus a tive agents of the parasiti c di seases of fish . Praha 1970: 1-383. GREVEN, U. : E in Beitrag zum Verhalten von Myxosp oridien in den Kiemen einiger SiiJ3 wasser · fi sche, Z. Fisch er ei u . Hilfswiss. 5 (1956): 110-114. JAKOWSKA, S., and NIGRELLI, R . F .: The p athology of m y x osp oridioses in the elect ric eel, Electroph orus electricus (L.) cause d b y H enneguya »isceralis an d H. lectri ca spp . nov. Zoologica ' . N . Y. 38 (19 53): 183-191. NIGRELLI, R . F. , and SMITH, G. M. : A p ap illa ry cys t ic di sease a ffect ing barbe ls of Ameirus nebu · losus (L e Sue u r) ca use d by m yxosp oridian H enn egya ameiurensis sp . nov'. Zoologies N.Y. 25 (1940): 89-96. ; Author 's add ress: Dr. C. KALAVATI and Dr. C. C. NARASUIHAMURTI, D ep artment of Zoology , Andhra U nive rs ity, W altair 530 003, India .