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Turkey Leucocytozoon Infection
344
R. D. WYATT, P. B. HAMILTON AND R. E. WELTY total, lipid. Clin. Chem. Acta, 8: 376-381. Sunderman, F. W., R. P. MacFate, D. A. Mc'. Fayden, G. G. Stevenson and B. E^ Copeland, 1953. Symposium on clinical hemoglobinometry. Am. J. Clin. Path. 23: 519-598. Tatum, L. A. 1971. The Southern corn leaf blight epidemic. Science, 171: 1113-1116. Tucker, T. L., and P. B. Hamilton, 1971. The effect of ochratoxin in broilers. Poultry Sci. 50: 1637. Tung, H. T., W. E. Donaldson and P. B. Hamilton, 1971. Modification in lipid metabolism during aflatoxicosis. Poultry Sci. 50: 1637. Washburn, K. W., and W. M. Britton, 1971. Effects of diets containing Helminthosporiwm maydis blighted corn on growth rate, feed conversion, mortality and blood clotting time of broilers. Poultry Sci. 50: 1161-1164. Wooton, I. D. P., 1964. Micro-analysis in Medical Biochemistry. Grune and Stratton, Inc., New York. Wyatt, R. D., and P. B. Hamilton, 1971. The effect of rubratoxin in broiler chickens. Poultry Sci. 50: 1647. Zlatkis, A. B., B. Zak and A. J. Boyle, 1953. A new method for the direct determination of serum cholesterol. J. Lab. Clin. Med. 4 1 : . 486-492.
Turkey Leucocytozoon Infection 1. A RAPID DIAGNOSTIC STAINING METHOD 1 JUAN SOUS Department of Poultry Science, Clemson University, Clemson, South Carolina 29631 (Received for publication May 12, 1972)
ABSTRACT A staining method is described for the rapid detection and identification, even at low densities, of turkey leucocytozoon parasites in peripheral blood films. The method is based on the high staining contrast of leucocytozoon parasites in films stained with brilliant cresyl blue. This contrast allows the use of low magnification and permits rapid scanning of large areas. POULTRY SCIENCE 52:
INTRODUCTION
T
URKEY and other avian leucocytozoon infections are diagnosed by the direct microscopic observation and identifi1
Published with the approval of the Director' of the South Carolina AgficultViral Experiment Station as technical contribution no. 993.
344-346,
1973
cation of the gametocytes in stained blood smears. These blood films are usually stained by the Giemsa or Wright's stain methods. These methods, however, not only are time-consuming, which is a factor of primary practical importance in animal disease diagnostic laboratories, but also stain
Downloaded from http://ps.oxfordjournals.org/ at UPVA on May 21, 2015
Ciegler, A., E. B. Lillehoj, R. E. Peterson and H. H. Hall, 1966. Microbial detoxification of afiatoxin. Appl. Microbiol. 14: 934-939. Ciegler, A., and R. E. Peterson, 1968. Aflatoxin detoxification: hydroxydihydroaflatoxin Bi. Appl. Microbiol. 16: 665-666. Ciegler, A., J. L. Richard, J. J. Ellis and A. C. Pier, 1971. Mycotoxin production by Helminthosporium may dis. Bacteriol. Proc. 1971:6. Doupnik, B., O. H. Jones and J. C. Peckham, 1971. Lack of toxicity of Helminthosporium maydis—invaded corn and culture filtrates to chicks and mice. Appl. Microbiol. 22: 732733. Hamilton, P. B., R. R. Nelson and B. S. H. Harris, 1968. Murine toxicity of Cochliobolus carbonum. Appl. Microbiol. 16: 1719-1722. Hamilton, P. B., H. T. Tung and F. W. Cook, 1971a. The anemia of chickens caused by aflatoxin. Poultry Sci. SO: 1583. Hamilton, P. B., R. D. Wyatt and H. R. Burmeister, 1971b. Effect of fusariotoxin T-2 in chickens. Poultry Sci. 50: 1583. Smith, J. W., and P. B. Hamilton, 1970. Aflatoxicosis in the broiler chicken. Poultry Sci. 49: 207-215. Searcy, R. L., J. L. Korotzer and C. N. Bergquist, 1963. Micromeasurement of serum
TURKEY LEUCOCYTOZOON INFECTION
MATERIALS AND METHODS
The stain is prepared by dissolving 0.25 gm. of brilliant cresyl blue stain in 100 ml. of absolute methanol. The solution is filtered and stored in an air tight container to prevent evaporation of the methanol. The stain is stable for at least 4 months at room temperature. Thin or thick blood smears are prepared the conventional way and allowed to air dry. The slides are placed on a staining tray, and the blood films are covered with 8 to 10 drops of stain for 30 sec-
FIGS. 1 AND 2. Blood films showing Leu& blood of turkeys. Note their elongated, or slig ing cytoplasm (X625).
onds which serves to fix and stain the blood film. The stain is then diluted with an equal number of drops of phosphate buffer (pH 7.0) as had been used of dye and allowed to stand for 30 additional seconds. The smear is rinsed with tap water and examined immediately or allowed to air dry. If the latter course is taken, the blood film should be covered with a thin film of immersion oil which greatly facilitates the microscopic observation of all blood cellular elements. RESULTS AND DISCUSSION
Most blood cellular components are readily identifiable. The nuclei of erythrocytes and platelets stain dark blue and their cytoplasm is light bluish-green. The nuclei of basophils, neutrophils, and eosinophils stain very light blue and their cytoplasmic granules dark blue. Lymphocytes and monocytes show a very light blue nucleus and slightly darker cytoplasm. The morphology of leucocytozoon-parasitized leucocytes is similar to that seen in Giemsa and Wright stained preparations. They appear elongated, spindle-shaped, or slightly less elongated, 2 or 3 times longer than their normal size. As with other leucocytes, their large nuclei stain very light blue and so do their cytoplasm. The cytoplasm of
ozoon smithi macrogametocytes in the peripheral ' less elongated morphology, and their dark stain-
Downloaded from http://ps.oxfordjournals.org/ at UPVA on May 21, 2015
most blood cellular components to nearly the same color as the parasites. In an attempt to reduce labor costs and improve time efficiency without sacrificing the parastie's staining quality, a rapid and accurate staining method has been developed which should greatly increase the efficiency of screening blood films. In addition, the rapid and accurate detection of leucocytozoon parasites in thick blood smears from birds with low level parasitemia is made much easier, thus replacing the cumbersome buffy coat concentration method currently available for detecting leucocytozoon parasites at low levels. It is the purpose of this report to describe the preparation and staining procedure for this method.
345
346
J. SOLIS plasmic contrast are achieved consistently with this procedure. The stained blood films can be stored for months without significant stain fading. There is no need to remove the oil film. The advantages gained by this method in leucocytozoon case-detection lies in the user's ability to rapidly locate a leucocytozoon parasite even at low levels of parasitemia. Total time involved is about 1 minute from staining the air-dried slide to reading of the slide, as opposed to the conventional Wright's and Giemsa methods, or even the rapid Giemsa staining method, which take about 5, 45, and between 3 to 5 minutes, respectively. Such a method, included as a routine screening procedure, would allow a faster, yet accurate diagnosis, as compared to the presently used routine diagnostic staining methods. Within this framework, this technic should prove of value in the detection of avian leucocytozoon infections.
NEWS AND NOTES (Continued from page 340) development of the laboratory facilities of the Department of Avian Diseases at Cornell University, the research facilities on Hungerford Hill have been named the P. Philip Levine Research Laboratories for Avian Diseases. SASKATCHEWAN NOTES Dr. C. H. Bigland, Department of Veterinary Medicine, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, is on sabbatical leave for a year, conducting research on Mycoplasma meleagridis at the Field Station, Department of Veterinary Preventive Medicine, The University of Liverpool, 'Leahurst,' Neston, Wirral, Cheshire, England L64 7TE. TEXAS NOTES Dr. J. H. Quisenberry, Professor Emeritus of Poultry Science at Texas A and M University, has been selected by the Bangladesh Agricultural University to serve on a selection committee for ap-
pointment of full professors and associate professors in the University's Poultry Science Department. P.E.N.B.-U.E.P. On November 8, 1972, agreement was reached between the Poultry and Egg National Board and the United Egg Producers to join forces. The agreement commences January 1, 1973. The major function of P.E.N.B. will be in the determination of policy and in the creation of new and expanded programs for the encouragement of demand for eggs and egg products. In order to permit P.E.N.B. to expand its basic promotion activities, U.E.P. will provide management and housekeeping services for P.E.N.B. at cost. U.E.P. will use its best efforts to secure contributions to P.E.N.B. to be used exclusively by P.E.N.B. in its promotion program. All contributions and other funds received by P.E.N.B. are solely the funds of P.E.N.B. U.E.P. agrees,
(Continued on page 354)
Downloaded from http://ps.oxfordjournals.org/ at UPVA on May 21, 2015
the macrogametocyte stains characteristically dark blue and their nuclei light blue (Figs. 1, 2). Both cytoplasm and nuclei of the micorgametocytes stain pale blue. With this staining method, macrogametes are easily and rapidly localized and are distinguishable from normal blood constituents in a microscope with 10 X objective and 10 X eyepieces due to their greater staining contrast and larger size. Although microgametocytes are difficult to see, the female-male ratio appears to be about 8:1 and thus there is little difficulty in finding parasites. It is the greater intensity of the stain and the differential shape and size between parasitized host cells and normal blood cellular components that permits one to quickly detect leucocytozoon parasites while rapidly scanning large areas of blood films under low magnification; parasites are then examined under higher mangification and their morphology confirmed. Reproducibility of staining and sharp cyto-
R. D. WYATT, P. B. HAMILTON AND R. E. WELTY total, lipid. Clin. Chem. Acta, 8: 376-381. Sunderman, F. W., R. P. MacFate, D. A. Mc'. Fayden, G. G. Stevenson and B. E^ Copeland, 1953. Symposium on clinical hemoglobinometry. Am. J. Clin. Path. 23: 519-598. Tatum, L. A. 1971. The Southern corn leaf blight epidemic. Science, 171: 1113-1116. Tucker, T. L., and P. B. Hamilton, 1971. The effect of ochratoxin in broilers. Poultry Sci. 50: 1637. Tung, H. T., W. E. Donaldson and P. B. Hamilton, 1971. Modification in lipid metabolism during aflatoxicosis. Poultry Sci. 50: 1637. Washburn, K. W., and W. M. Britton, 1971. Effects of diets containing Helminthosporiwm maydis blighted corn on growth rate, feed conversion, mortality and blood clotting time of broilers. Poultry Sci. 50: 1161-1164. Wooton, I. D. P., 1964. Micro-analysis in Medical Biochemistry. Grune and Stratton, Inc., New York. Wyatt, R. D., and P. B. Hamilton, 1971. The effect of rubratoxin in broiler chickens. Poultry Sci. 50: 1647. Zlatkis, A. B., B. Zak and A. J. Boyle, 1953. A new method for the direct determination of serum cholesterol. J. Lab. Clin. Med. 4 1 : . 486-492.
Turkey Leucocytozoon Infection 1. A RAPID DIAGNOSTIC STAINING METHOD 1 JUAN SOUS Department of Poultry Science, Clemson University, Clemson, South Carolina 29631 (Received for publication May 12, 1972)
ABSTRACT A staining method is described for the rapid detection and identification, even at low densities, of turkey leucocytozoon parasites in peripheral blood films. The method is based on the high staining contrast of leucocytozoon parasites in films stained with brilliant cresyl blue. This contrast allows the use of low magnification and permits rapid scanning of large areas. POULTRY SCIENCE 52:
INTRODUCTION
T
URKEY and other avian leucocytozoon infections are diagnosed by the direct microscopic observation and identifi1
Published with the approval of the Director' of the South Carolina AgficultViral Experiment Station as technical contribution no. 993.
344-346,
1973
cation of the gametocytes in stained blood smears. These blood films are usually stained by the Giemsa or Wright's stain methods. These methods, however, not only are time-consuming, which is a factor of primary practical importance in animal disease diagnostic laboratories, but also stain
Downloaded from http://ps.oxfordjournals.org/ at UPVA on May 21, 2015
Ciegler, A., E. B. Lillehoj, R. E. Peterson and H. H. Hall, 1966. Microbial detoxification of afiatoxin. Appl. Microbiol. 14: 934-939. Ciegler, A., and R. E. Peterson, 1968. Aflatoxin detoxification: hydroxydihydroaflatoxin Bi. Appl. Microbiol. 16: 665-666. Ciegler, A., J. L. Richard, J. J. Ellis and A. C. Pier, 1971. Mycotoxin production by Helminthosporium may dis. Bacteriol. Proc. 1971:6. Doupnik, B., O. H. Jones and J. C. Peckham, 1971. Lack of toxicity of Helminthosporium maydis—invaded corn and culture filtrates to chicks and mice. Appl. Microbiol. 22: 732733. Hamilton, P. B., R. R. Nelson and B. S. H. Harris, 1968. Murine toxicity of Cochliobolus carbonum. Appl. Microbiol. 16: 1719-1722. Hamilton, P. B., H. T. Tung and F. W. Cook, 1971a. The anemia of chickens caused by aflatoxin. Poultry Sci. SO: 1583. Hamilton, P. B., R. D. Wyatt and H. R. Burmeister, 1971b. Effect of fusariotoxin T-2 in chickens. Poultry Sci. 50: 1583. Smith, J. W., and P. B. Hamilton, 1970. Aflatoxicosis in the broiler chicken. Poultry Sci. 49: 207-215. Searcy, R. L., J. L. Korotzer and C. N. Bergquist, 1963. Micromeasurement of serum
TURKEY LEUCOCYTOZOON INFECTION
MATERIALS AND METHODS
The stain is prepared by dissolving 0.25 gm. of brilliant cresyl blue stain in 100 ml. of absolute methanol. The solution is filtered and stored in an air tight container to prevent evaporation of the methanol. The stain is stable for at least 4 months at room temperature. Thin or thick blood smears are prepared the conventional way and allowed to air dry. The slides are placed on a staining tray, and the blood films are covered with 8 to 10 drops of stain for 30 sec-
FIGS. 1 AND 2. Blood films showing Leu& blood of turkeys. Note their elongated, or slig ing cytoplasm (X625).
onds which serves to fix and stain the blood film. The stain is then diluted with an equal number of drops of phosphate buffer (pH 7.0) as had been used of dye and allowed to stand for 30 additional seconds. The smear is rinsed with tap water and examined immediately or allowed to air dry. If the latter course is taken, the blood film should be covered with a thin film of immersion oil which greatly facilitates the microscopic observation of all blood cellular elements. RESULTS AND DISCUSSION
Most blood cellular components are readily identifiable. The nuclei of erythrocytes and platelets stain dark blue and their cytoplasm is light bluish-green. The nuclei of basophils, neutrophils, and eosinophils stain very light blue and their cytoplasmic granules dark blue. Lymphocytes and monocytes show a very light blue nucleus and slightly darker cytoplasm. The morphology of leucocytozoon-parasitized leucocytes is similar to that seen in Giemsa and Wright stained preparations. They appear elongated, spindle-shaped, or slightly less elongated, 2 or 3 times longer than their normal size. As with other leucocytes, their large nuclei stain very light blue and so do their cytoplasm. The cytoplasm of
ozoon smithi macrogametocytes in the peripheral ' less elongated morphology, and their dark stain-
Downloaded from http://ps.oxfordjournals.org/ at UPVA on May 21, 2015
most blood cellular components to nearly the same color as the parasites. In an attempt to reduce labor costs and improve time efficiency without sacrificing the parastie's staining quality, a rapid and accurate staining method has been developed which should greatly increase the efficiency of screening blood films. In addition, the rapid and accurate detection of leucocytozoon parasites in thick blood smears from birds with low level parasitemia is made much easier, thus replacing the cumbersome buffy coat concentration method currently available for detecting leucocytozoon parasites at low levels. It is the purpose of this report to describe the preparation and staining procedure for this method.
345
346
J. SOLIS plasmic contrast are achieved consistently with this procedure. The stained blood films can be stored for months without significant stain fading. There is no need to remove the oil film. The advantages gained by this method in leucocytozoon case-detection lies in the user's ability to rapidly locate a leucocytozoon parasite even at low levels of parasitemia. Total time involved is about 1 minute from staining the air-dried slide to reading of the slide, as opposed to the conventional Wright's and Giemsa methods, or even the rapid Giemsa staining method, which take about 5, 45, and between 3 to 5 minutes, respectively. Such a method, included as a routine screening procedure, would allow a faster, yet accurate diagnosis, as compared to the presently used routine diagnostic staining methods. Within this framework, this technic should prove of value in the detection of avian leucocytozoon infections.
NEWS AND NOTES (Continued from page 340) development of the laboratory facilities of the Department of Avian Diseases at Cornell University, the research facilities on Hungerford Hill have been named the P. Philip Levine Research Laboratories for Avian Diseases. SASKATCHEWAN NOTES Dr. C. H. Bigland, Department of Veterinary Medicine, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, is on sabbatical leave for a year, conducting research on Mycoplasma meleagridis at the Field Station, Department of Veterinary Preventive Medicine, The University of Liverpool, 'Leahurst,' Neston, Wirral, Cheshire, England L64 7TE. TEXAS NOTES Dr. J. H. Quisenberry, Professor Emeritus of Poultry Science at Texas A and M University, has been selected by the Bangladesh Agricultural University to serve on a selection committee for ap-
pointment of full professors and associate professors in the University's Poultry Science Department. P.E.N.B.-U.E.P. On November 8, 1972, agreement was reached between the Poultry and Egg National Board and the United Egg Producers to join forces. The agreement commences January 1, 1973. The major function of P.E.N.B. will be in the determination of policy and in the creation of new and expanded programs for the encouragement of demand for eggs and egg products. In order to permit P.E.N.B. to expand its basic promotion activities, U.E.P. will provide management and housekeeping services for P.E.N.B. at cost. U.E.P. will use its best efforts to secure contributions to P.E.N.B. to be used exclusively by P.E.N.B. in its promotion program. All contributions and other funds received by P.E.N.B. are solely the funds of P.E.N.B. U.E.P. agrees,
(Continued on page 354)
Downloaded from http://ps.oxfordjournals.org/ at UPVA on May 21, 2015
the macrogametocyte stains characteristically dark blue and their nuclei light blue (Figs. 1, 2). Both cytoplasm and nuclei of the micorgametocytes stain pale blue. With this staining method, macrogametes are easily and rapidly localized and are distinguishable from normal blood constituents in a microscope with 10 X objective and 10 X eyepieces due to their greater staining contrast and larger size. Although microgametocytes are difficult to see, the female-male ratio appears to be about 8:1 and thus there is little difficulty in finding parasites. It is the greater intensity of the stain and the differential shape and size between parasitized host cells and normal blood cellular components that permits one to quickly detect leucocytozoon parasites while rapidly scanning large areas of blood films under low magnification; parasites are then examined under higher mangification and their morphology confirmed. Reproducibility of staining and sharp cyto-