- Email: [email protected]
Electron Microscopy Studies of Four Strains of Chronic Respiratory Agent
960
R. L. REAGAN, W. C. DAY AND A. L. BRUECKNER
respectively for the 24 and 30 percent protein lots. In case of phosphorus more was retained by the poults reported on in this paper. SUMMARY
REFERENCES Ackerson, C. W., M. J. Blish and F. E. Mussehl' 1938. The utilization of food elements by growing chicks. V. A comparison of cottonseed meal and linseed oil meal as portions of the protein concentrate. Univ. of Nebraska Agr. Expt. Sta. Res. Bull. 100. Ackerson, C. W., and F. E. Mussehl, 1947. The utilization of food elements by growing poults. Univ. of Nebraska Agr. Expt. Sta. Res. Bull. 151.
Electron Microscopy Studies of Four Strains of Chronic Respiratory Agent R. L. REAGAN, W. C. D A Y AND A. L. BRUECKNER Live Stock Sanitary Service Laboratory, University of Maryland, College Park, Maryland (Received for publication February 23. 1953)
I
N PREVIOUS electron micrographs of the virus of chronic respiratory disease in chickens (Tucker strain) it was demonstrated by Reagan et al. (1951) that this strain contained virus-like filaments which had an average width of 53 millimicrons. The morphological characteristics of this strain of chronic respiratory disease (CRD) of chickens appeared to be similar in size and shape to the causative agent of infectious sinusitis of turkeys. The present studies were undertaken to determine whether the causative agents of different strains of chronic respiratory disease differ morphologically and whether more than one agent could be detected by electron microscopy. METHODS AND MATERIALS
The four strains of CRD were obtained from the following men: Dr. Henry Van Roekel of the Agricultural Experiment Station, Amherst, Massachusetts, (strains A and B); Dr. Harold E. Moses of the
Agricultural Experiment Station, Lafayette, Indiana, (strain No. B706)*; and Dr. James A. Bivins of the New Jersey Agricultural Experiment Station, New Brunswick, New Jersey, (Kassenoff strain). These strains were proven by the above-mentioned investigators to cause CRD in fowl. All strains were stored in the — 40°C. dry ice cabinet upon arrival. Each strain was subjected to two additional egg passages at this laboratory. Five- to sevenday embryonating White Leghorn chicken eggs taken from an unvaccinated, Newcastle disease and chronic respiratory disease free flocks were employed. In all cases * Strain B706 was obtained from Dr. Harold E. Moses of the Agricultural Experimental Station, Lafayette, Indiana. This strain was designated as strain B706. This agent was shown to produce sinusitis in turkeys by Dr. Moses, sinusitis and aerosacculitis in chickens, and death of chicken embryos; it was quite resistant to penicillin (1,000 O. U. per ml) and sensitive to streptomycin (0.781 mg. per ml).
Downloaded from http://ps.oxfordjournals.org/ at Serials Section, Dixson Library on June 13, 2015
1. Two lots of day-old "Beltsville White" poults were individually fed equal amounts of rations containing 24 and 30 percent of protein. 2. The lot fed the diet with 30 percent protein showed a greater gain in a shorter period while consuming equal amounts of feed. 3. The percentage utilization of nitrogen, calcium and phosphorus was higher
in the poults fed the 24 percent protein ration. 4. The average nitrogen, calcium and phosphorus content of five-week-old poults was 3.30, 0.96 and 0.63 percent respectively.
E L E C T R O N MICROSCOPY OF C H R O N I C R E S P I R A T O R Y A G E N T
961
eggs were inoculated with 0.1 cc. of infected material and the route of inoculation was via the yolk sac. Allanto-amdiotic fluids from those eggs succumbing between the sixth and tenth days post inoculation were harvested and a separate pool prepared for each strain. This fluid was then centrifuged for three minutes a t
1000 r.p.m. in an angle centrifuge in order to remove any large particles. T h e supern a t a n t fluid from each strain was then centrifuged for one hour a t 30,000 r.p.m. in a Spinco ultracentrifuge. There was less t h a n a 1°C. change in the temperature of the refrigerated outer jacket. Each sediment was then resuspended in distilled
FIG. 2. Electron micrograph of chronic respiratory agent (strain A from Dr. Van Roekel) showing virus-likefilamentsshadowed with chromium at tangent 2/10. X 72,000.
Downloaded from http://ps.oxfordjournals.org/ at Serials Section, Dixson Library on June 13, 2015
FIG. 1. Electron micrograph of chronic respiratory agent (strain B from Dr. Van Roekel) showing x-bodies, shadowed with chromium at tangent 2/10. X20,000.
962
R. L. REAGAN, W. C. D A Y AND A. L. BRUECKNER
water, allowed to stand for five minutes and placed on parlodion screens which had been prepared forty-eight hours previously. The screens were then shadowed with chromium at arc tangent 2/10 (Williams and Wyckoff, 1946). Normal controls were made from allanto-amniotic fluid harvested from fourteen-day embryonating eggs. This fluid was subjected to the same treatment described previously for the infected m a t e rial. T h e C R D and normal control screens were then examined under an R C A electron microscope, type E M U . Several hundred fields were examined for each strain
and likewise several hundred fields for the normal controls. RESULTS AND DISCUSSION Figure 1 shows spherical particles which we designate as x-bodies and which have an average diameter of 200 to 250 millimicrons. Figure 2 shows virus-like filaments which measure 50 to 60 millimicrons in width and are stringy in appearance. Figure 3 shows x-bodies measuring 400 millimicrons in diameter (average). Figure 4 shows virus-like filaments which measure 50 to 60 millimicrons in width. Figure 5 shows a plaque containing x-
Downloaded from http://ps.oxfordjournals.org/ at Serials Section, Dixson Library on June 13, 2015
FIG. 3. Electron micrograph of chronic respiratory agent (strain No. B706 from Dr. Moses) showing x-bodies, shadowed with chromium at tangent 2/10. X91,200.
ELECTRON MICROSCOPY OF CHRONIC RESPIRATORY AGENT
963
FIG. 4. Electron micrograph of chronic respiratory agent (strain No. B706 from Dr. Moses) showing virus-like filaments, shadowed with chromium at tangent 2/10. X 72,000.
bodies which have an average diameter of 400 millimicrons. Figure 6 shows xbodies measuring 400 millimicrons in diameter and also contains virus-like filaments measuring 50 to 60 millimicrons in width. Observations made from electron micrographs of the previously-mentioned strains indicate that 3 of the strains (strains A, No. B706, and Kassenoff) contain virus-like filaments which are approximately the same in width (50 to 60 mil-
limicrons). Two of these same strains (No. B706 and Kassenoff strain) also contain x-bodies which measure 400 millimicrons in diameter. However in another strain (strain B) x-bodies were demonstrated which measured 250 millimicrons in diameter. This strain did not contain any virus-like filaments. A previously-mentioned strain (strain A) which was obtained from the same investigator (Van Roekel), did not contain any x-bodies. Xbodies or virus-like filaments such as de-
Downloaded from http://ps.oxfordjournals.org/ at Serials Section, Dixson Library on June 13, 2015
i 3 « l
964
R. L. REAGAN, W. C. Day AND A. L. BREUCKNER
FIG. 6. Electron micrograph of chronic respiratory agent (Kassenoff strain from Dr. Bivins) showing x-bodies and virus-like filaments, shadowed with chromium at tangent 2/10. X 72,000.
Downloaded from http://ps.oxfordjournals.org/ at Serials Section, Dixson Library on June 13, 2015
FIG. 5. Electron micrograph of chronic respiratory agent (Kassenoff strain from Dr. Bivins) showing x-bodies, shadowed with chromium at tangent 2/10. X72,000.
ELECTRON MICROSCOPY OF CHRONIC RESPIRATORY AGENT
electron micrographs of the Tucker strain. This was possibly due to the fact that the Tucker virus material was filtered through a type ST, size L3 Seitz filter prior to examination under electron microscope. It is unlikely that an x-body measuring 400 millimicrons in diameter could pass through the above-mentioned filter. SUMMARY
Electron micrographs of 4 chronic respiratory disease agents are presented. Virus-like filaments measuring in width between 50-60 millimcrons were present in 3 strains. Bodies designated as x-bodies varying somewhat in diameter were also demonstrated in 3 of the strains. In one strain the x-bodies were 250 millimicrons in diameter but in two other strains they measured 400 millimicrons in diameter. Normal allanto-amniotic fluid was treated in the same manner as were the infected fluids. No virus-like filaments or x-bodies could be seen upon examination of the normal fluid. REFERENCES Delaplane, J. P., 1948. Some recent observations of lesions in chick embryos induced by the virus of a chronic respiratory disease of chickens. Cornell Vet. 38: 192-194. Johnson, C. P., 1951. Now we have "air sac colds." Broiler Growing, 2: 12, 26. Murphy, J. S., D. T. Karzon and F. B. Bang, 19S0. Studies of influenza A (PR8) infected tissue cultures by electron microscopy. Proc. Soc. Expt. Biol. Med. 73: 596-599. Reagan, R. L., A. L. Brueckner and J. P. Delaplane, 1951. Morphological observation by electron microscopy of the virus (Tucker strain) of a chronic respiratory disease of chickens. Poultry Sci. 15: 113-115. Van Roekel, H., O. M. Olesiuk and H. A. Peck, 1952. Chronic respiratory disease in chickens. Amer. J. Vet. Res. 13:252-259. Williams, R. C , and R. W. G. Wyckoff, 1946. Application of metallic shadow-casting to microscopy. J. Applied Physics 17: 23-33.
Downloaded from http://ps.oxfordjournals.org/ at Serials Section, Dixson Library on June 13, 2015
scribed above could not be found in the control specimens upon examination under the electron microscope. There is no explanation at this time why two of the strains of chronic respiratory disease contained what appeared to be two different agents and the other two strains contained only a single agent each, even though all four strains apparently produced the same symptoms in fowl. It is however, theoretically possible that with different strains of the chronic respiratory disease, two agents must be present in order to produce typical symptoms of the disease. The .exact nature of the etiological agent of CRD has been to date very difficult to define. Several investigators (Van Roekel et al., 1952; Delaplane, 1948) have indicated that it is virus-like in appearance. Another investigator (Johnson, 1951) has stated that the agent is a rickettsial-like organism. In reference to the x-bodies demonstrated in these studies, these bodies resemble in size the psittacosis-lymphogranuloma venereum group of viruses because this group of viruses measures approximately 450 millimicrons in diameter and the x-bodies except for those found in strain B (250 millimicrons) measure approximately 400 millimicrons in diameter. In regards to the virus-like filaments found in three of the strains, it is impossible to determine at this time the exact nature of this material. However they do resemble to some degree the virus filaments of influenza A as demonstrated in electron micrographs by Murphy et al. (1950) In a previous electron microscope study of the Tucker strain (Reagan et al., 1951) of CRD, virus-like filaments were found which resembled those of the present investigation in that they measured 53 millimicrons in width. However, no x-bodies were seen in the
965
R. L. REAGAN, W. C. DAY AND A. L. BRUECKNER
respectively for the 24 and 30 percent protein lots. In case of phosphorus more was retained by the poults reported on in this paper. SUMMARY
REFERENCES Ackerson, C. W., M. J. Blish and F. E. Mussehl' 1938. The utilization of food elements by growing chicks. V. A comparison of cottonseed meal and linseed oil meal as portions of the protein concentrate. Univ. of Nebraska Agr. Expt. Sta. Res. Bull. 100. Ackerson, C. W., and F. E. Mussehl, 1947. The utilization of food elements by growing poults. Univ. of Nebraska Agr. Expt. Sta. Res. Bull. 151.
Electron Microscopy Studies of Four Strains of Chronic Respiratory Agent R. L. REAGAN, W. C. D A Y AND A. L. BRUECKNER Live Stock Sanitary Service Laboratory, University of Maryland, College Park, Maryland (Received for publication February 23. 1953)
I
N PREVIOUS electron micrographs of the virus of chronic respiratory disease in chickens (Tucker strain) it was demonstrated by Reagan et al. (1951) that this strain contained virus-like filaments which had an average width of 53 millimicrons. The morphological characteristics of this strain of chronic respiratory disease (CRD) of chickens appeared to be similar in size and shape to the causative agent of infectious sinusitis of turkeys. The present studies were undertaken to determine whether the causative agents of different strains of chronic respiratory disease differ morphologically and whether more than one agent could be detected by electron microscopy. METHODS AND MATERIALS
The four strains of CRD were obtained from the following men: Dr. Henry Van Roekel of the Agricultural Experiment Station, Amherst, Massachusetts, (strains A and B); Dr. Harold E. Moses of the
Agricultural Experiment Station, Lafayette, Indiana, (strain No. B706)*; and Dr. James A. Bivins of the New Jersey Agricultural Experiment Station, New Brunswick, New Jersey, (Kassenoff strain). These strains were proven by the above-mentioned investigators to cause CRD in fowl. All strains were stored in the — 40°C. dry ice cabinet upon arrival. Each strain was subjected to two additional egg passages at this laboratory. Five- to sevenday embryonating White Leghorn chicken eggs taken from an unvaccinated, Newcastle disease and chronic respiratory disease free flocks were employed. In all cases * Strain B706 was obtained from Dr. Harold E. Moses of the Agricultural Experimental Station, Lafayette, Indiana. This strain was designated as strain B706. This agent was shown to produce sinusitis in turkeys by Dr. Moses, sinusitis and aerosacculitis in chickens, and death of chicken embryos; it was quite resistant to penicillin (1,000 O. U. per ml) and sensitive to streptomycin (0.781 mg. per ml).
Downloaded from http://ps.oxfordjournals.org/ at Serials Section, Dixson Library on June 13, 2015
1. Two lots of day-old "Beltsville White" poults were individually fed equal amounts of rations containing 24 and 30 percent of protein. 2. The lot fed the diet with 30 percent protein showed a greater gain in a shorter period while consuming equal amounts of feed. 3. The percentage utilization of nitrogen, calcium and phosphorus was higher
in the poults fed the 24 percent protein ration. 4. The average nitrogen, calcium and phosphorus content of five-week-old poults was 3.30, 0.96 and 0.63 percent respectively.
E L E C T R O N MICROSCOPY OF C H R O N I C R E S P I R A T O R Y A G E N T
961
eggs were inoculated with 0.1 cc. of infected material and the route of inoculation was via the yolk sac. Allanto-amdiotic fluids from those eggs succumbing between the sixth and tenth days post inoculation were harvested and a separate pool prepared for each strain. This fluid was then centrifuged for three minutes a t
1000 r.p.m. in an angle centrifuge in order to remove any large particles. T h e supern a t a n t fluid from each strain was then centrifuged for one hour a t 30,000 r.p.m. in a Spinco ultracentrifuge. There was less t h a n a 1°C. change in the temperature of the refrigerated outer jacket. Each sediment was then resuspended in distilled
FIG. 2. Electron micrograph of chronic respiratory agent (strain A from Dr. Van Roekel) showing virus-likefilamentsshadowed with chromium at tangent 2/10. X 72,000.
Downloaded from http://ps.oxfordjournals.org/ at Serials Section, Dixson Library on June 13, 2015
FIG. 1. Electron micrograph of chronic respiratory agent (strain B from Dr. Van Roekel) showing x-bodies, shadowed with chromium at tangent 2/10. X20,000.
962
R. L. REAGAN, W. C. D A Y AND A. L. BRUECKNER
water, allowed to stand for five minutes and placed on parlodion screens which had been prepared forty-eight hours previously. The screens were then shadowed with chromium at arc tangent 2/10 (Williams and Wyckoff, 1946). Normal controls were made from allanto-amniotic fluid harvested from fourteen-day embryonating eggs. This fluid was subjected to the same treatment described previously for the infected m a t e rial. T h e C R D and normal control screens were then examined under an R C A electron microscope, type E M U . Several hundred fields were examined for each strain
and likewise several hundred fields for the normal controls. RESULTS AND DISCUSSION Figure 1 shows spherical particles which we designate as x-bodies and which have an average diameter of 200 to 250 millimicrons. Figure 2 shows virus-like filaments which measure 50 to 60 millimicrons in width and are stringy in appearance. Figure 3 shows x-bodies measuring 400 millimicrons in diameter (average). Figure 4 shows virus-like filaments which measure 50 to 60 millimicrons in width. Figure 5 shows a plaque containing x-
Downloaded from http://ps.oxfordjournals.org/ at Serials Section, Dixson Library on June 13, 2015
FIG. 3. Electron micrograph of chronic respiratory agent (strain No. B706 from Dr. Moses) showing x-bodies, shadowed with chromium at tangent 2/10. X91,200.
ELECTRON MICROSCOPY OF CHRONIC RESPIRATORY AGENT
963
FIG. 4. Electron micrograph of chronic respiratory agent (strain No. B706 from Dr. Moses) showing virus-like filaments, shadowed with chromium at tangent 2/10. X 72,000.
bodies which have an average diameter of 400 millimicrons. Figure 6 shows xbodies measuring 400 millimicrons in diameter and also contains virus-like filaments measuring 50 to 60 millimicrons in width. Observations made from electron micrographs of the previously-mentioned strains indicate that 3 of the strains (strains A, No. B706, and Kassenoff) contain virus-like filaments which are approximately the same in width (50 to 60 mil-
limicrons). Two of these same strains (No. B706 and Kassenoff strain) also contain x-bodies which measure 400 millimicrons in diameter. However in another strain (strain B) x-bodies were demonstrated which measured 250 millimicrons in diameter. This strain did not contain any virus-like filaments. A previously-mentioned strain (strain A) which was obtained from the same investigator (Van Roekel), did not contain any x-bodies. Xbodies or virus-like filaments such as de-
Downloaded from http://ps.oxfordjournals.org/ at Serials Section, Dixson Library on June 13, 2015
i 3 « l
964
R. L. REAGAN, W. C. Day AND A. L. BREUCKNER
FIG. 6. Electron micrograph of chronic respiratory agent (Kassenoff strain from Dr. Bivins) showing x-bodies and virus-like filaments, shadowed with chromium at tangent 2/10. X 72,000.
Downloaded from http://ps.oxfordjournals.org/ at Serials Section, Dixson Library on June 13, 2015
FIG. 5. Electron micrograph of chronic respiratory agent (Kassenoff strain from Dr. Bivins) showing x-bodies, shadowed with chromium at tangent 2/10. X72,000.
ELECTRON MICROSCOPY OF CHRONIC RESPIRATORY AGENT
electron micrographs of the Tucker strain. This was possibly due to the fact that the Tucker virus material was filtered through a type ST, size L3 Seitz filter prior to examination under electron microscope. It is unlikely that an x-body measuring 400 millimicrons in diameter could pass through the above-mentioned filter. SUMMARY
Electron micrographs of 4 chronic respiratory disease agents are presented. Virus-like filaments measuring in width between 50-60 millimcrons were present in 3 strains. Bodies designated as x-bodies varying somewhat in diameter were also demonstrated in 3 of the strains. In one strain the x-bodies were 250 millimicrons in diameter but in two other strains they measured 400 millimicrons in diameter. Normal allanto-amniotic fluid was treated in the same manner as were the infected fluids. No virus-like filaments or x-bodies could be seen upon examination of the normal fluid. REFERENCES Delaplane, J. P., 1948. Some recent observations of lesions in chick embryos induced by the virus of a chronic respiratory disease of chickens. Cornell Vet. 38: 192-194. Johnson, C. P., 1951. Now we have "air sac colds." Broiler Growing, 2: 12, 26. Murphy, J. S., D. T. Karzon and F. B. Bang, 19S0. Studies of influenza A (PR8) infected tissue cultures by electron microscopy. Proc. Soc. Expt. Biol. Med. 73: 596-599. Reagan, R. L., A. L. Brueckner and J. P. Delaplane, 1951. Morphological observation by electron microscopy of the virus (Tucker strain) of a chronic respiratory disease of chickens. Poultry Sci. 15: 113-115. Van Roekel, H., O. M. Olesiuk and H. A. Peck, 1952. Chronic respiratory disease in chickens. Amer. J. Vet. Res. 13:252-259. Williams, R. C , and R. W. G. Wyckoff, 1946. Application of metallic shadow-casting to microscopy. J. Applied Physics 17: 23-33.
Downloaded from http://ps.oxfordjournals.org/ at Serials Section, Dixson Library on June 13, 2015
scribed above could not be found in the control specimens upon examination under the electron microscope. There is no explanation at this time why two of the strains of chronic respiratory disease contained what appeared to be two different agents and the other two strains contained only a single agent each, even though all four strains apparently produced the same symptoms in fowl. It is however, theoretically possible that with different strains of the chronic respiratory disease, two agents must be present in order to produce typical symptoms of the disease. The .exact nature of the etiological agent of CRD has been to date very difficult to define. Several investigators (Van Roekel et al., 1952; Delaplane, 1948) have indicated that it is virus-like in appearance. Another investigator (Johnson, 1951) has stated that the agent is a rickettsial-like organism. In reference to the x-bodies demonstrated in these studies, these bodies resemble in size the psittacosis-lymphogranuloma venereum group of viruses because this group of viruses measures approximately 450 millimicrons in diameter and the x-bodies except for those found in strain B (250 millimicrons) measure approximately 400 millimicrons in diameter. In regards to the virus-like filaments found in three of the strains, it is impossible to determine at this time the exact nature of this material. However they do resemble to some degree the virus filaments of influenza A as demonstrated in electron micrographs by Murphy et al. (1950) In a previous electron microscope study of the Tucker strain (Reagan et al., 1951) of CRD, virus-like filaments were found which resembled those of the present investigation in that they measured 53 millimicrons in width. However, no x-bodies were seen in the
965