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Liver Histology in Virus Hepatitis of Ducklings
Liver Histology in Virus Hepatitis of Ducklings W. M. CORREA Departmento de Histologia e Embriologia da Faculdade de Medicina Veterindria da Universidade de Sao Paulo, Brasil (Received for publication August 25, 1958)
work reported herein, probably because this work was done with field cases, in which the livers always showed connective tissue growth when the ducklings resisted the disease. Since histological aspects of field cases have not been completely studied previously (c/. Hagan and Bruner, 1952) the hepatic histological findings are given in this paper. MATERIAL AND METHODS
At first, sections of liver, spleen, kidneys, air sacs and then later brain, were collected for study, but it was soon established that with the exception of the liver, the alterations in the other tissues were variable. Subsequent histological observations were therefore made on liver only. From among a total of 107 sick or dead ducklings originating in several flocks and which were brought to the "Instituto Biologico de Sao Paulo" for examination, 50 livers were selected for study. Organ specimens were cut about 0.5 cm. thick and fixed in 10 percent formalin and imbedded in paraffin. Subsequently, they were cut at 5 p, and routinely stained with hematoxilineosin. Other stains employed were: Giemsa's azur II, van Gieson's picrofuschsin, UnaTaenzer-Livini's acid orcein and Bielchow-
FIG. 1. Liver showing dilated sinusoids with blood and initial connective growth. H.E.—200X. FIG. 2. Detail of Plate 1, showing, at the right down corner, hepatic cells vacuolated. H.E.—400XFIG. 3. Severe lymphoplasmocytic infiltration. H.E.—100 X. FIG. 4. Detail of Plate 3. H.E— 1000XFIG. 5. Growth of biliary ducts. H.E. 200X. FIG. 6. Hepatic tubules with cells detached, from the normal reticular membrane. Note the sinusoids with blood cells just at the periphery of the tubules (arrows). H.E. Bielchowsky. 400X. 516
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T EVINE and Fabricant (1950) described •*-' a new disease, occurring amongst ducklings three days to four weeks of age, and were able to isolate a virus responsible for the illness. Asplin and McLauchlan (19S4) reported on work with the disease and developed a vaccine for it (Asplin, (19S6). The disease has been reported in Illinois and British Columbia, by Hanson and Alberts (1956) and MacPherson and Avery (1957), respectively. The two groups isolated and studied the virus. Correa (1957) obtained similar results after experimental inoculations of embryonating eggs with virual material of ducklings having a clinical picture similar to that described by previous investigations. Macro- and microscopic studies of organs, injured by the disease, were presented. Since experimental inoculation and virus isolation in ducklings are difficult, studies were directed toward establishing information for a diagnosis based on histological alterations. The results of the studies are reported herein. Hanson (1958) described the histological aspects of the disease in susceptible young ducks, infected by a virus, either adapted in embryonated eggs, or not. The lesions differ slightly from those observed in the
VIRUS HEPATITIS OF DUCKLINGS
517
Downloaded from http://ps.oxfordjournals.org/ at UNIVERSITY OF VIRGINIA on April 12, 2015
FIG. 7. Abnormal number of reticular fibers amongst hepatic tubules; compare with 6. Bielchowsky—100X. Fie. 8. Case of advanced cirrhosis with abundant growth of the reticular fibers. Bielchowsky—25 X-
518
W. M. CORREA
sky's silver impregnation. All sections were examined with the usual optical microscope. Some were also observed with the phasecontrast microscope, but nothing special was seen. RESULTS AND OBSERVATIONS
DISCUSSION
It is of interest to note that the primary action of the virus is the vascular disturbance, dilating the sinusoids and separating the tubules one from another (Elias, 1957). Then, the hepatic cells begin to show degenerative changes and a lymphoplasmocytic infiltration starts, like a response to the antigenic stimulation of the lymphoid tissue (Ehrich and Harris, 1942; Ehrich, 1953; Roberts, 1954; Yoffey and Courtice, 1956). The degenerative changes of hepatic cells are probably a result of the synergic action of the virus and the vascular abnormality. When the primary phase of inflammation has passed, first the biliary ducts and later the connective tissue grow abundantly, replacing the degenerated tubules and lobules. The anatomic picture is closely related to the histologic lesions, showing at first a congestion (dilation of many blood sinusoids), afterwards the organ volume increase (inflammation phase) and finally the liver hardens owing to the cirrhotic process (connective and biliary growth). The retention of biliary pigments, the related nuclear degenerative changes of hepatic cells, and the heterophil infiltration, are not as frequent as the other changes. With the exception of brain, which showed a lymphocytic meningitis, the irregular damages in the spleen, kidney and air
Downloaded from http://ps.oxfordjournals.org/ at UNIVERSITY OF VIRGINIA on April 12, 2015
Gross examination of the liver revealed congestion, subcapsular focal hemorrhages, and some focal necrosis and cirrhosis. Microscopically the hepatic cords appeared separated with apparent dilation of many blood sinusoids. Some cells were isolated as a result of cord degeneration with consequent dissociation. The hepatic cells were larger and the cytoplasm more homogeneous and acidophilic than in the normal cell. Small irregular cytoplasmic vacuoles were observed after careful examination. In some cases there was a lysis of the cytoplasm, but the ectoplasmic zone was less rarefied. The nuclei were slightly enlarged showing a vesicular-like structure with one or more areas of condensed chromatin. Pyknosis and karyorrhexis were seen only in cases of great biliary retention. In these cases there were granules and conglomerates of biliary pigment in the Kupffer cells and also inside and outside the spongy hepatic cells. Generally, phagocytosis was associated with nuclear remnants of liver cells. Throughout the organ, but especially in degenerated areas, there was a diffuse infiltration of lymphoplasmocytes which was more or less evident according to the case; likewise, an inconstant and less evident heterophil infiltration was seen near portal areas. Cellular connective tissue appeared to invade areas that showed advanced degeneration. This tended to form cirrhotic lodges. In the connective tissue of the intercord spaces (chiefly in that surrounding portal
spaces) there was discreet to massive proliferation of biliary capillaries. The amount varied in relation to the length of sickness. This proliferation was always disorientated and of variable cronology showing solid and tunnelized cords. Silver impregnations showed that the number of the reticular fibers had increased, resulting in a large amount of cirrhotic stroma arising among the hepatic cords and even between the cells.
VIRUS HEPATITIS OF DUCKLINGS
sacs were reported in a previous paper (Correa, 1957). By use of the methods described, viral inclusion bodies were not seen, thus indicating that they are not formed in organs affected with this disease. SUMMARY AND
CONCLUSIONS
REFERENCES Asplin, F. D., and J. D. McLauchlan, 19S4. Duck virus hepatitis. Vet. Record, 66: 456^458. Asplin, F. D., 1956. The production of ducklings resistant to virus hepatitis. Vet. Record, 68: 412-413.
Correa, W. M., 1957. Notas preliminares sobre a hepatite a virus dos marrecos no Brasil. Rev. Fac. Med. Vet. S. Paulo, 6 (1). Ehrich, W. E., 19S3. Adaptation phase in inflammation. The Mechanism of Inflammation. Acta Inc. Medical Publishers, Montreal, Canada, p. 25. Ehrich, W. E., and T. N. Harris, 1942. The formation of antibodies, in the popliteal lymph node in rabbits. J. Exp. Med. 76: 335-348. Elias, H., 1957. Evolution and the liver. Chicago Med. School Quarterly, 18: 49. Hagan, W. A., and D. W. Bruner, 1952. Las Enfermedades Infecciosas de los Animates Domesticos. 2nd ed. La Presensa M6dica Mexicana, Mexico, p. 745. Hanson, L. E., 1958. Histological lesions in ducks with virus hepatitis. Amer. J. Vet. Res. 19: 712718. Hanson, L. E., and J. O. Alberts, 1956. Virus hepatitis in ducklings. J. Amer. Vet. Med. Assoc. 128: 37-38. Levine, P. P., and J. Fabricant, 1950. A hithertoundescribed virus disease of ducks in North America. Cornell Vet. 40: 71-86. MacPherson, L. W., and R. J. Avery, 1957. Duck virus hepatitis in Canada. Canad. J. Comp. Med. Vet. Sci. 2 1 : 26-31. Roberts, K. B., 1954. The localizations of antigens and the sites of antibody formation. Lectures on General Pathology, W. B. Saunders Co., London, p. 328. Yoffey, J. M., and F. C. Courtice, 1956. Lymphatics, Lymph and Lymphoid Tissue. Edward Arnold Ltd., London, p. 276 and 399.
The Use of High Quality Fat and the Effect of Protein Level in Broiler Diets R. G. BEILHARZ AND M. W. MCDONALD Poultry Experiment Station, Seven Hills, New South Wales, Australia (Received for publication September 2, 1958)
INTRODUCTION
R
ECENT surpluses of animal fats in the U.S.A. have stimulated interest in its wide incorporation into broiler diets. The resulting high energy diets have been shown to improve the efficiency of feed utilization
when measured as pounds of feed consumed per pound of gain (Sunde, 1956; Donaldson et al., 1957). This improvement may possibly be due partly to lowered feed wastage than occurs with light, bulky mashes. It has also been shown that high energy
Downloaded from http://ps.oxfordjournals.org/ at UNIVERSITY OF VIRGINIA on April 12, 2015
Various methods were utilized to study the histologic alterations in the liver of ducklings affected with virus hepatitis. The histological features were described, discussed and compared with the anatomopathological picture. Ducklings' virus hepatitis may be diagnosed with the aid of the following data: (1) Ducklings between 3 days and 4 weeks of age. (2) Histologic liver damage: (a) Sinusoidal dilation with hepatic tubules separated and damaged, (b) Lymphoplasmocytic infiltration, (c) Growth of biliary capillaries followed by (d) Growth of connective tissue leading to a cirrhosis.
519
work reported herein, probably because this work was done with field cases, in which the livers always showed connective tissue growth when the ducklings resisted the disease. Since histological aspects of field cases have not been completely studied previously (c/. Hagan and Bruner, 1952) the hepatic histological findings are given in this paper. MATERIAL AND METHODS
At first, sections of liver, spleen, kidneys, air sacs and then later brain, were collected for study, but it was soon established that with the exception of the liver, the alterations in the other tissues were variable. Subsequent histological observations were therefore made on liver only. From among a total of 107 sick or dead ducklings originating in several flocks and which were brought to the "Instituto Biologico de Sao Paulo" for examination, 50 livers were selected for study. Organ specimens were cut about 0.5 cm. thick and fixed in 10 percent formalin and imbedded in paraffin. Subsequently, they were cut at 5 p, and routinely stained with hematoxilineosin. Other stains employed were: Giemsa's azur II, van Gieson's picrofuschsin, UnaTaenzer-Livini's acid orcein and Bielchow-
FIG. 1. Liver showing dilated sinusoids with blood and initial connective growth. H.E.—200X. FIG. 2. Detail of Plate 1, showing, at the right down corner, hepatic cells vacuolated. H.E.—400XFIG. 3. Severe lymphoplasmocytic infiltration. H.E.—100 X. FIG. 4. Detail of Plate 3. H.E— 1000XFIG. 5. Growth of biliary ducts. H.E. 200X. FIG. 6. Hepatic tubules with cells detached, from the normal reticular membrane. Note the sinusoids with blood cells just at the periphery of the tubules (arrows). H.E. Bielchowsky. 400X. 516
Downloaded from http://ps.oxfordjournals.org/ at UNIVERSITY OF VIRGINIA on April 12, 2015
T EVINE and Fabricant (1950) described •*-' a new disease, occurring amongst ducklings three days to four weeks of age, and were able to isolate a virus responsible for the illness. Asplin and McLauchlan (19S4) reported on work with the disease and developed a vaccine for it (Asplin, (19S6). The disease has been reported in Illinois and British Columbia, by Hanson and Alberts (1956) and MacPherson and Avery (1957), respectively. The two groups isolated and studied the virus. Correa (1957) obtained similar results after experimental inoculations of embryonating eggs with virual material of ducklings having a clinical picture similar to that described by previous investigations. Macro- and microscopic studies of organs, injured by the disease, were presented. Since experimental inoculation and virus isolation in ducklings are difficult, studies were directed toward establishing information for a diagnosis based on histological alterations. The results of the studies are reported herein. Hanson (1958) described the histological aspects of the disease in susceptible young ducks, infected by a virus, either adapted in embryonated eggs, or not. The lesions differ slightly from those observed in the
VIRUS HEPATITIS OF DUCKLINGS
517
Downloaded from http://ps.oxfordjournals.org/ at UNIVERSITY OF VIRGINIA on April 12, 2015
FIG. 7. Abnormal number of reticular fibers amongst hepatic tubules; compare with 6. Bielchowsky—100X. Fie. 8. Case of advanced cirrhosis with abundant growth of the reticular fibers. Bielchowsky—25 X-
518
W. M. CORREA
sky's silver impregnation. All sections were examined with the usual optical microscope. Some were also observed with the phasecontrast microscope, but nothing special was seen. RESULTS AND OBSERVATIONS
DISCUSSION
It is of interest to note that the primary action of the virus is the vascular disturbance, dilating the sinusoids and separating the tubules one from another (Elias, 1957). Then, the hepatic cells begin to show degenerative changes and a lymphoplasmocytic infiltration starts, like a response to the antigenic stimulation of the lymphoid tissue (Ehrich and Harris, 1942; Ehrich, 1953; Roberts, 1954; Yoffey and Courtice, 1956). The degenerative changes of hepatic cells are probably a result of the synergic action of the virus and the vascular abnormality. When the primary phase of inflammation has passed, first the biliary ducts and later the connective tissue grow abundantly, replacing the degenerated tubules and lobules. The anatomic picture is closely related to the histologic lesions, showing at first a congestion (dilation of many blood sinusoids), afterwards the organ volume increase (inflammation phase) and finally the liver hardens owing to the cirrhotic process (connective and biliary growth). The retention of biliary pigments, the related nuclear degenerative changes of hepatic cells, and the heterophil infiltration, are not as frequent as the other changes. With the exception of brain, which showed a lymphocytic meningitis, the irregular damages in the spleen, kidney and air
Downloaded from http://ps.oxfordjournals.org/ at UNIVERSITY OF VIRGINIA on April 12, 2015
Gross examination of the liver revealed congestion, subcapsular focal hemorrhages, and some focal necrosis and cirrhosis. Microscopically the hepatic cords appeared separated with apparent dilation of many blood sinusoids. Some cells were isolated as a result of cord degeneration with consequent dissociation. The hepatic cells were larger and the cytoplasm more homogeneous and acidophilic than in the normal cell. Small irregular cytoplasmic vacuoles were observed after careful examination. In some cases there was a lysis of the cytoplasm, but the ectoplasmic zone was less rarefied. The nuclei were slightly enlarged showing a vesicular-like structure with one or more areas of condensed chromatin. Pyknosis and karyorrhexis were seen only in cases of great biliary retention. In these cases there were granules and conglomerates of biliary pigment in the Kupffer cells and also inside and outside the spongy hepatic cells. Generally, phagocytosis was associated with nuclear remnants of liver cells. Throughout the organ, but especially in degenerated areas, there was a diffuse infiltration of lymphoplasmocytes which was more or less evident according to the case; likewise, an inconstant and less evident heterophil infiltration was seen near portal areas. Cellular connective tissue appeared to invade areas that showed advanced degeneration. This tended to form cirrhotic lodges. In the connective tissue of the intercord spaces (chiefly in that surrounding portal
spaces) there was discreet to massive proliferation of biliary capillaries. The amount varied in relation to the length of sickness. This proliferation was always disorientated and of variable cronology showing solid and tunnelized cords. Silver impregnations showed that the number of the reticular fibers had increased, resulting in a large amount of cirrhotic stroma arising among the hepatic cords and even between the cells.
VIRUS HEPATITIS OF DUCKLINGS
sacs were reported in a previous paper (Correa, 1957). By use of the methods described, viral inclusion bodies were not seen, thus indicating that they are not formed in organs affected with this disease. SUMMARY AND
CONCLUSIONS
REFERENCES Asplin, F. D., and J. D. McLauchlan, 19S4. Duck virus hepatitis. Vet. Record, 66: 456^458. Asplin, F. D., 1956. The production of ducklings resistant to virus hepatitis. Vet. Record, 68: 412-413.
Correa, W. M., 1957. Notas preliminares sobre a hepatite a virus dos marrecos no Brasil. Rev. Fac. Med. Vet. S. Paulo, 6 (1). Ehrich, W. E., 19S3. Adaptation phase in inflammation. The Mechanism of Inflammation. Acta Inc. Medical Publishers, Montreal, Canada, p. 25. Ehrich, W. E., and T. N. Harris, 1942. The formation of antibodies, in the popliteal lymph node in rabbits. J. Exp. Med. 76: 335-348. Elias, H., 1957. Evolution and the liver. Chicago Med. School Quarterly, 18: 49. Hagan, W. A., and D. W. Bruner, 1952. Las Enfermedades Infecciosas de los Animates Domesticos. 2nd ed. La Presensa M6dica Mexicana, Mexico, p. 745. Hanson, L. E., 1958. Histological lesions in ducks with virus hepatitis. Amer. J. Vet. Res. 19: 712718. Hanson, L. E., and J. O. Alberts, 1956. Virus hepatitis in ducklings. J. Amer. Vet. Med. Assoc. 128: 37-38. Levine, P. P., and J. Fabricant, 1950. A hithertoundescribed virus disease of ducks in North America. Cornell Vet. 40: 71-86. MacPherson, L. W., and R. J. Avery, 1957. Duck virus hepatitis in Canada. Canad. J. Comp. Med. Vet. Sci. 2 1 : 26-31. Roberts, K. B., 1954. The localizations of antigens and the sites of antibody formation. Lectures on General Pathology, W. B. Saunders Co., London, p. 328. Yoffey, J. M., and F. C. Courtice, 1956. Lymphatics, Lymph and Lymphoid Tissue. Edward Arnold Ltd., London, p. 276 and 399.
The Use of High Quality Fat and the Effect of Protein Level in Broiler Diets R. G. BEILHARZ AND M. W. MCDONALD Poultry Experiment Station, Seven Hills, New South Wales, Australia (Received for publication September 2, 1958)
INTRODUCTION
R
ECENT surpluses of animal fats in the U.S.A. have stimulated interest in its wide incorporation into broiler diets. The resulting high energy diets have been shown to improve the efficiency of feed utilization
when measured as pounds of feed consumed per pound of gain (Sunde, 1956; Donaldson et al., 1957). This improvement may possibly be due partly to lowered feed wastage than occurs with light, bulky mashes. It has also been shown that high energy
Downloaded from http://ps.oxfordjournals.org/ at UNIVERSITY OF VIRGINIA on April 12, 2015
Various methods were utilized to study the histologic alterations in the liver of ducklings affected with virus hepatitis. The histological features were described, discussed and compared with the anatomopathological picture. Ducklings' virus hepatitis may be diagnosed with the aid of the following data: (1) Ducklings between 3 days and 4 weeks of age. (2) Histologic liver damage: (a) Sinusoidal dilation with hepatic tubules separated and damaged, (b) Lymphoplasmocytic infiltration, (c) Growth of biliary capillaries followed by (d) Growth of connective tissue leading to a cirrhosis.
519