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The Diagnosis of Certain Avian Diseases with the Fluorescent Antibody Technique
The Diagnosis of Certain Avian Diseases With the Fluorescent Antibody Technique R . E . CORSTVET AND W . W . SADLER Department of Public Health, University of California, Davis, California (Received for publication April 28, 1964)
A
Since the present work was begun, there have been reports describing successful use of the fluorescent antibody technique in demonstrating M. gallisepticum (Noel et al., 1964) and IBV (Mohanty et al., 1964) in infected tissues.
Since preliminary investigation showed that the indirect fluorescent method did not serve well for identifying colonies of avian mycoplasma, H. gallinarum, or E. coli a simpler method of preparing conjugated antiserum was sought. Published methods for the preparation of fluorescein-labeled antibody require that the serum or conjugate be subjected to time-consuming manipulations such as fractionation, dialysis, and concentration. In addition, various procedures have been used to eliminate or reduce nonspecific staining. Thus, the present study was undertaken to develop a rapid identifaction test for certain avian pathogens in infected tissues as well as in artificial media. MATERIALS AND METHODS
Antigen propagation and preparation of immune sera. The NDV and IBV were propagated in the allantoic fluids of embryonated chicken eggs 8-11 days old. The NDV immune serum was prepared by immunizing four week old Cornish cross chickens with NDV strain Bi and challenging them with NDV strain GB eight weeks later. Hyperimmune serum to IBV was prepared by infecting chickens with the Massachusetts strain of IBV and challenging them eight weeks later with this same strain. Multiple doses of a dense suspension of washed, broth-grown cells of Mycoplasma gallisepticum (S 6 ), Mycoplasma sp. of N characteristic ( # 5 2 9 ) , Haemophilus gallinarum (0083), or Escherichia coli (strain d) were given intravenously to rabbits and fryer stock chickens for the production of
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NUMBER of Mycoplasma species are associated with diseases of poultry. Some are common in mixed poultry infections associated with other pathogens such as Newcastle disease virus (NDV), infectious bronchitis virus (IBV), Haemophilus gallinarum, and Escherichia coli (Osteen, 1962). All of these agents have been differentiated by morphological, biochemical, serological, bird infectivity, and/ or lesion characteristics. These manipulations are time-consuming, however, and may still leave doubt (especially with the Mycoplasma spp.) as to the identity and purity of the agent(s) in question. Furthermore, attempts often fail to isolate these agents from infected tissues; where isolation is accomplished there is often doubt as to whether the tissue was contaminated from some other tissue or an external source. It is clear that a rapid test is needed to differentiate these pathogenic agents, especially the Mycoplasma spp., in artificial media and infected tissues. The fluorescent antibody technique was thought of as a possible means of accomplishing this since it has been used to identify various species and serotypes of human mycoplasma as well as various bacteria and viruses, including NDV (Prince and Ginsberg, 1957; Burnstein and Bang, 1958; Maestrone and Coffin, 1961).
FLUORESCENT ANTIBODY TECHNIQUE
were made with fluorescein isothiocyanate (Baltimore Biological Laboratories), and the various viral or bacterial antisera prepared in chickens, turkeys or rabbits. The protein content of the serum which varied between 50-60 mg./ml. was estimated from the ratio of optical densities at 260 and 280 mjji. (mg. protein per mil. = 1.55 D28o — 0.76 D26o; optical path = 1 cm.) (Colwick and Kaplan, 1957). Three to 8 ml. of the undiluted serum at 0°C. was adjusted to pH 8.8 by adding 0.5 M sodium bicarbonate buffer at pH 8.8 (15% of the serum volume). The fluorescein isothiocyanate crystals (1 mg. per 40 mg. protein) were then floated on the liquid surface and allowed to stand for approximately 10 minutes. The mixture was then stirred with a magnetic stirrer for 16 to 20 hours at 4°C, after which the conjugated serum was placed on the column without prior dialysis. Then 0.025 M phosphate buffer, pH 7.2, was percolated through the column. Collection of eluate was begun as soon as a yellow color appeared, and ended as soon as it disappeared. The eluted conjugated proteins were stored at 4°C. and used over a period of several months without noticeable loss of staining ability. Preparation and staining of tissue sections, tissue impressions, and bacterial colony imprints. The staining efficiency of the various conjugated antisera was determined by staining cryostat sections and impressions of tissues from normal chickens or turkeys and from chickens or turkeys infected with either NDV, IBV, Haemophilus gallinarum, Mycoplasma gallisepticum, Mycoplasma of N characteristic or Mycoplasma synoviae; and by staining various bacterial colony imprints, smears of chicken red blood cells, and coverslip preparation of HeLa cells both infected and unifected with NDV strain GB. Cryostat sections of infected and normal chickens and turkeys were cut at 6-10
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immune serum. Sera from turkeys artificially infected with M. gallisepticum (S6) and chickens artificially infected with M. synoviae (NTF) were also used for conjugation with fluorescein isothiocyanate. The propagation of all of the Mycoplasma spp. except M. synoviae for colony imprints was done with PPLO agar (Bacto PPLO broth Difco), 1% yeast autolysate (Albimi Lab. Inc.), 1.55% Bacto agar (Difco), 15% horse serum (Adler et al., 1962). M. synoviae was grown on the agar medium described by Chalquest (1962). Haemophilus gallinarum strains and Escherichia coli were grown on Bacto blood agar base (Difco) plus 5% ox serum; the H. gallinarum was grown in association with a Micrococcus sp. feeder in an atmosphere of 10% carbon dioxide. Preparation of the cellulose column. Diethylaminoethylcellulose (DEAE-cellulose, Bio-Rad Laboratories) was washed according to the method of Levy and Sober (1960), with some modification. The DEAE-cellulose was washed twice with 0.5 N NaOH, then with distilled water until the pH of the supernate was 7.0-7.2, and then with 0.025 M phosphate buffer, pH 7.2, until the pH of the supernate was that of the buffer. All washings were done for 15-30 minutes, with ten times the starting amount of DEAE-cellulose, and the latter was recovered on a Buchner filter. A magnetic stirrer made all washings much more efficient. When the DEAE-cellulose was equilibrated with the 0.025 M phosphate buffer, pH 7.2, the coarse particles were allowed to settle out for 10 to 15 minutes. The coarse particles were packed into glass columns, 12 mm. outside diameter, to a height of 3.5 cm. for every 1 ml. of the sample to be fractionated. The flow rate of the column was between 0.7 and 1 ml. per minute. The column was kept at 0°C. Conjugation of the serum. Conjugations
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R. E . CORSTVET AND W . W . SADLER
line, pH 7.2. When colony imprints were to be stained, the conjugates were not absorbed. The dilution of the conjugated anti-NDV serum to be used for staining tissue sections and impressions was determined by the use of the red blood cell smears. The highest dilution of conjugates which still gave maximum brilliancy of fluorescence was then used to stain the tissue preparations. The dilutions of conjugate used for staining the NDV were generally 1:2-1:10; the dilution varied slightly with the particular conjugate preparation. The dilution of conjugated anti-bacterial serum used to stain colony imprints was the highest dilution which gave the maximum brilliancy of fluorescence, generally 1:10 to 1:30. The dilution of conjugate used to stain bacterial antigens in chicken or turkey tissues was one doubling dilution less than that which gave maximum brilliancy of fluorescence with the colony imprint system. Fluorescent staining of the preparations was begun by placing one drop of conjugate on each tissue section or impression or colony imprint; the slides or coverslips were then placed in a moist chamber at 37°C. for 25 to 30 minutes. The preparations were then washed twice in two changes of 0.01 M phosphate-buffered saline, pH 7.2 (5 and 20 minutes, respectively), rinsed once with distilled water, and allowed to air-dry partially before being mounted. The mounting fluid was 0.01 M phosphate-buffered saline, pH 7.2, with 10 percent glycerol. These preparations were examined by Zeiss microscope with on Osram HBO 200-W maximal-pressure mercury-vapor arc. The ultraviolet excitation filters used were UG 5 plus UG 2 or UG 5, and the barrier was the combination 0/41. RESULTS AND DISCUSSION
The method of preparing fluorescein antibody conjugates was satisfactory for rabbit,
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microns and stored at — 6°C, either without chemical fixation or with fixation in acetone for ten minutes at room temperature. Airdried tissue impressions and HeLa cell preparations were fixed overnight in cold acetone at — 6°C. and stored at that temperature until stained. Bacterial colony imprints were prepared by touching a coverslip to colonies which had grown on the surface of agar medium for 1 to 13 days at 37°C. The imprints were then air-dried, passed twice through a cool flame from a Bunsen burner, and stained immediately or stored at — 6°C. for periods up to six months. Colony imprints were also prepared according to the method of Clark et al. (1961). The RBC smears were prepared by mixing 1 ml. of a 0.5 percent suspension of chick RBC's with 1 ml. of unifected chicken embryo allantoic fluid or allantoic fluid containing either NDV strain GB or IBV strain Massachusetts and allowing it to stand one hour at 4°C. Both unifected and IBV-infected allantoic fluids were used as controls. The mixture was then centrifuged ten minutes at 160-170 X G, and the supernate was decanted. A loopful of the packed cells was smeared on a glass slide, allowed to air-dry, fixed for ten minutes in acetone at room temperature, and stained the same day. HeLa cells grown in coverslips were inoculated with allantoic fluid containing NDV strain GB and incubated at 37°C. Forty-eight to 72 hours after inoculation, these coverslips were washed once in 0.01 M phosphate-buffered saline pH 7.2, and stained. The nonspecific staining components were absorbed from the serum conjugate by mixing the conjugate 1:1 with a 20 percent suspension of tissues of a normal four month old chicken. A homogenized suspension of trachea, spleen, liver and brain was prepared in 0.01 M phosphate buffered sa-
FLUORESCENT ANTIBODY TECHNIQUE
avian mycoplasma or for H. gattinarutn. In all instances, fixation of the colony was excellent; however, the mycoplasma antigen was evidently destroyed completely by heat, since no fluorescence was obtained with the homologous conjugated chicken antiserum. With H. gallinarum, fluorescence was obtained with the homologous conjugated antiserum but the brilliancy of the stain was greatly diminished. This would indicate a partial destruction or alteration of its antigen. Very gentle heat fixation of the colony imprints gave excellent preparations. The small amount of agar picked up on the coverslip took up some of the conjugate, but this was not detrimental, since negative and positive colonies were easily discernible. Although Clark et al. (1963) reported that the indirect fluorescent test could be used to stain colonies of the human strains of mycoplasma, attempts failed to use the indirect test on the bacterial colonies represented in Table 1. Addition of complement to the staining reagents had no beneficial effect, nor did variations in staining time up to 60 minutes at 37°C. Various preparations of chicken or rabbit antiglobulin conjugates and immune serum which functioned well in the indirect test with tissues did not give a positive test with the same antigen in the form of colony imprints. Table 1 clearly illustrates the specificity of the fluorescent antibody technique for differentiation and indentification of various avian pathogens in artificial media. There was no significant immunological cross reaction between the various Mycoplasma spp. or between them and the other bacterial antigens. The two field isolates of M. gallisepticum reacted only with the anti-Af. gallisepticum conjugate. The five mycoplasma isolates which were characterized as "N" type by morphological, biochemical, and serological procedures reacted only with the anti-Mycoplasma sp. "N"
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chicken, and turkey antisera containing either viral or bacterial antibodies. The antibody titer of the conjugated fractionated serum was generally one to two doubling dilutions less than that of the unconjugated unfractionated serum. Absorption of the conjugates to be used for staining colony imprints or the RBC smears was not necessary. With avian tissues and HeLa cells, however, there was a difference between unabsorbed and absorbed conjugates in the amount of nonspecific staining. With the unabsorbed conjugates the cells of all infected and normal chicken tissues took a diffuse, dull, nonspecific stain that increased the brightness of the background. However, this was not a problem in observing the specific fluorescence. This lighter background was completely eliminated by absorbing the conjugate. In the brain there was brilliant nonspecific staining of a very few small clusters of nonparenchymal cells associated with vascular areas in some of the infected and unlfected sections. However, specific staining of NDV antigen was excellent and easily discernible in the cytoplasm of the Purkinje cells and cells in the perivascular and vascular areas. In some of the infected and uninfected tracheal and air sac sections and impressions there were a few small nonparenchymal cells which stained with one brilliant granule in the center. Absorption of the conjugate eliminated this nonspecific staining in the brain, but not in the trachea and air sac. With the HeLa cells infected with NDV strain GB, nonspecific staining of the undiluted conjugate and a 1:5 unabsorbed dilution was 2.5+, based on a 4+ maximum. Even after absorption with normal HeLa cells this conjugate was not considered to be very satisfactory because of the nonspecific staining that was still present. The hot-water colony fixation method of Clark et al. (1961) for human strains of mycoplasma was not satisfactory for the
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R . E . CORSTVET AND W . W . SADLER
conjugate. M. synoviae, M. sp. #695, H. gallinarum, and E. coli antigens also reacted only with their respective homologous antiserum conjugates. The anti-NDV conjugate served as a control to show that hyperimmune anti-viral serum did not contain a nonspecific serum component which would react with the bacterial antigens. In addition to the antigens in Table 1, the conjugated anti-Af. gallisepticum (S6) serum
and H. gallinarum (0083) serum did not give positive staining with four Kidd strains of Mycoplasma (goat pathogen) Cordy et al., 1955) or with Mycoplasmas LA and LB (isolated from sewage by Dr. Edwards, London, England; subculture obtained from Dr. H. E. Adler), H3-10 (saprophyte of chickens) (Adler et al., 1961) and three strains of Haemophilus parahemolyticus (isolated from the stifle of a sheep, brain
Chicken immune serum conjugate Colony imprint 1
MG—l 2 MG—23 N—l 4 N—2" N—3 4 N
_44
N—5 6 MS—l 6 MS—2" MS—3 6 1' 28 38 48
59 Oio
11
HG—l HG—212 HG—3 12 Esch. coli strain "d" 13 Micrococcus sp. (feeder) 1
M. gallisepticum Se
Mycoplasma sp. " N " 529
Pos14 Pos Neg15 Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg
Neg Neg Pos Pos Pos Pos Pos Neg Neg
MycoMycoplasma sp. Synovitis plasma sp. 695 NTF Neg Neg Neg
— —
H. gallinarum 0083
Esch. coli Strain "d"
NDV Strain GB
Neg Neg Neg Neg Neg
Neg Neg Neg
Neg Neg
Neg
— —
Neg
— —
— — — —
— — — —
—
Neg
—
—
Neg Neg Pos Pos Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg
Neg
—
—
—
Neg
Pos
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
16
—
Neg Neg Neg Neg Neg Neg Neg Neg
Neg Neg
—
Neg
Neg Neg Neg Neg Neg Neg Neg Neg
Neg Neg Neg Neg Neg Neg Neg Neg
—
—
—
—
Pos Neg
— —
Neg
Neg Neg Neg Neg Neg Neg Neg
— —
Pos Pos Pos
Neg
Neg
— —
— —
— —
1- to 13-day agar plate culture. M. gallisepticum isolated by Dr. D. V. Zander (1961). M. gallisepticum isolate (Richey) obtained from Dr. S. Schmittle, Georgia. 4 Mycoplasma sp. (#1 = 529; #2 = 400; #3 = 2-5; #4=Byl4) of N characteristic isolated by Dr. R. Yamamoto from cases of turkey poult air-sacculitis. 5 Mycoplasma sp. of N characteristic isolated in our laboratory from a case of turkey poult arrsacculitis. 6 Mycoplasma synoviae (#1 = N T F ; #2 = 1853; #3 = Maine) obtained from Dr. J. Fabricant, (1960). 7 Mycoplasma sp. (#695) isolated by Drs. H. W. Yoder and M. S. Hofstad (1962). 8 Mycoplasma sp. (#2 = #8; 3 = #23; 4 = #24) other than M. gallisepticum or N isolated by Dr. R. Yamamoto from cases of turkey poult airsacculitis (1964). 9 Mycoplasma sp. (Tu) obtained from Dr. H. E. Adler (1957). 10 Human strain of Mycoplasma (#880) isolated from a tumor by Dr. Horosowitz, Memorial Hospital, Albany, N. Y. (subculture obtained from Dr. H. E. Adler). 11 H. gallinarum (0083) isolated by Dr. L. A. Page (1962). 12 H. gallinarum (#2 = 17756; #3=17764) isolated by Dr. R. Yamamoto from chicken field cases. 13 Esch. coli, strain " D , " isolated by Dr. W. B. Gross (1956). 14 Pos=specific fluorescence. 16 Neg=no specific fluorescence. 16 — = n o t done. 2
3
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TABLE 1.—Identification of microorganisms with the direct fluorescent antibody technique
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FLUORESCENT ANTIBODY TECHNIQUE TABLE 2.—Direct fluorescent staining of infected avian tissues Infected avian. tissue1 Absorbed conjugated chicken Antiserum
1 2 3 4
Chicken abdom airsac
MGS, H . G . 0 0 8 3 s ™ V 2
Neg
Neg 4
Neg
Pos
3
MGS,
Chicken foot pad
NDV Str GB
M.S. N T F
Turkey abdom air sac " N " 529
Neg
Pos
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Pos
Neg Pos
— —
Neg Neg
Pos Neg
Neg
Neg
Pos
Neg
Pos
Neg
Cryostat section 6-10 microns thick. Pos = specific fluorescence. Neg=no specific fluorescence. —=not done.
abscess in a steer and a swine isolate). The colony imprint system further confirmed the identification and grouping of the various avian mycoplasma, which to date has been done by more tedious serological, biochemical, and pathological tests. Identification and differentiation by the fluorescent antibody technique was possible not only with antigens propagated on artificial media but also with antigens propagated in avian tissues (Table 2). The pathogens in avian tissues, as was the case with these antigens in artificial media, stained only with the homologous antiserum conjugate. M. gallisepticum (S6) in the trachea or abdominal air sac was identified and differentiated from Mycoplasma sp. "N", Mycoplasma synoviae, H. galUnarum, and NDV. The fact that S6 and N may be differentiated in tissues is important because both of these agents can be associated with airsacculitis in turkeys (Adler et al., 1958). M. synoviae is not known to cause air sac disease in poultry, but both this mycoplasma and S6 produce joint lesions— so their differentiation in tissues is important. H. galUnarum and NDV were clearly separated by the fluorescent antibody technique, not only from each other but from
the Mycoplasma spp. as well, an important fact since combinations of these agents are often associated with poultry disease syndromes. The data in Table 3 demonstrate the applicability of these techniques to both the diagnosis of disease and the study of pathogenesis of selected diseases. This table summarizes the results of the direct fluorescent staining by absorbed conjugate of chicken tissues infected with NDV strain GB and a correlation with clinical signs. As one might expect, the antigen was demonstrable in the digestive and upper respiratory tissues before clinical signs were observable. Both the numbers of epithelial cells infected and the concentration of antigen per cell were greatest on the fourth day postinoculation. The proventriculus tissue smears were negative on that day, possibly because the infected parenchymal cells were so few that they were not picked up by the tissue impression. The fifth, sixth, and seventh-day impressions of the proventriculus contained only 2-3 positive epithelial cells, with few intracytoplasmic fluorescent particles. On these days there were a number of phagocytic cells in the tissue without NDV antigen. None of the air sac epithelial
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M. gallisepticum {§$) Mycoplasma sp. " N " 529 Mycoplasma synoviae (Synovitis NTF) H. galUnarum (0083) Newcastle Disease Virus strain G.B. (NDV)
Chicken trachea
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TABLE C5.—Correlation
of clinical signs with detection of Newcastle disease virus strain GB in tissues of 10-week-old intratracheally infected chickens by the direct fluorescent antibody technique Tissue impressions8
Sacrificed—day post inoculation 02
None
Proventriculus
Abdominal Air sac
Trachea
_ 4
—
—
—
2+
6
1
None
2+
2
Slightly depressed; slight hoarseness
3+
—
3+
3
Moderately depressed; rales; slight leg weakness; sneezing
4+
2+
3+
Slight rales; partial leg paralysis; clonic spasms; depressed
—
4+
4+
Paralysis; clonic spasms, depressed; no rales
1+
3 +
1+
Paralysis; clonic spasms; depressed; no rales
1+
—
1+
Slight rales; Hyperexcitable; clonic spasms head; marked leg weakness; ataxia.
1+
—
—
4 5 6 7
1
Clinical signs at time of autopsy
1
One bird sacrificed at daily intervals through 8 days. Sacrificed immediately prior to inoculation. Impressions of liver, brain, spleen, cervical glands, and lung were negative for each of the above postinoculation days. Heart blood was negative for days 1 through 4; S through 7, not done. 4 No fluorescence in epithelial cells. 6 1 through 4 + ; progression of not only more epithelial cells with viral antigen but more particles and aggregates of antigen per cell. 2
3
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cells showed intracytoplasmic fluorescence sue sections of the spleen, liver and bursa on the sixth and seventh days postinocula- from the bird on the fourth postinoculation tion. However, on the sixth day there were day, stained with fluorescent antibody a few phagocytic cells which contained showed that NDV antigen was present in NDV antigen in their cytoplasm. From a the cytoplasm of the cells lining the liver diagnostic point of view these results would sinusoids and bursal parenchymal cells, and indicate that proventriculus and trachea in the vascular endothelial cells of all three would be the tissue of choice, since the NDV tissues. The reason the spleen and liver was present in one or both of these tissues sections were positive and the impressions earlier and later than in the abdominal were not, is doubtless due to the fact that a air sac. tissue impression will usually not pick up The liver, spleen, cervical nodes, and the vascular architecture. Frozen tissue secheart blood impressions from the chickens tions of the brain on the fourth day postillustrated in Table 3 were all negative for inoculation displayed NDV antigen in the NDV antigen; these were good impressions cytoplasm of the Purkinje cells and in the and there was no nonspecific tissue fluores- endothelial cells of the blood vessels. Occacence with the absorbed conjugate. The sionally vascular areas would be seen with fact that the heart blood was negative is at fluorescent particles in the cells immediately variance with a previously published report surrounding these areas which gave the im(Maestrone and Coffin, 1961). Frozen tis- pression of fluorescent perivascular cuffing.
FLUORESCENT ANTIBODY TECHNIQUE
SUMMARY
A modified method of conjugate preparation produced conjugates that were satisfactory for differentiating various avian pathogens in artificial media and tissues. This simplified method saved considerable time over previously published methods by eliminating fractionation of the unconjugated serum, dialysis, and concentration. This was desirable since the indirect fluorescent test did not function with colony imprints and also, after conjugation and fractionation, the majority of the antibody protein was recovered; a distinct advantage in the case of the often-lo-titer avian mycoplasma antiserum. The results indicate that the fluorescent test can be very useful in evaluating mycoplasma cultures as to purity and greatly simplifies the identification of Mycoplasma spp. or serological types as well as other avain pathogens. The results also showed that tissue imprints of trachea, air sac, and proventriculus could be used in identifying NDV by the fluorescent antibody technique.
REFERENCES Adler, H. E., M. Shifrine and H. Ortmayer, 1962. Interpretation of Mycoplasma gallisepticum serologic tests for turkeys. X I I World's Poultry Congress: 322-324. Adler, H. E., M. Shifrine and H. B. Ortmayer, 1961. M. inoccuum sp. n., a saprophyte from chickens. J. Bacteriol. 82: 239-240. Adler, H. E., J. Fabricant, R. Yamamoto and J. Berg. 1958. Isolation and identification of pleuropneumonia-like organisms of avian origin. Am. J. Vet. Res. 19: 440-447. Adler, H. E., R. Yamamoto and J. Berg, 1957. Strain differences of pleuropneumonia-like organisms of avian origin. Avian Dis. 1: 19-26. Burnstein, T., and F. B. Bang, 1958. Infection of the upper respiratory tract of the chick with a mild (vaccine) strain of Newcastle disease virus. II. Studies on the pathogenesis of the infection. Bull. Johns Hopkins Hosp. 102: 135157. Chalquest, R. R., 1962. Cultivation of the infectious-synovitis-type pleuropneumonia-like organisms. Avian Dis. 6 : 36-43. Chalquest, R. R., and J. Fabricant, 1960. Pleuropneumonia-like organisms associated with synovitis in fowls. Avian Dis. 4 : 515-539. Clark, H. W., J. S. Bailey, R. C. Fowler and T. Mc P. Brown, 1963. Identification of Mycoplasmataceae by the fluorescent antibody method. J. Bacteriol. 85: 111-118. Clark, H. W., R. C. Fowler and T. McP. Brown, 1961. Preparation of pleuropneumonia-like organisms for microscopic study. J. Bacteriol. 81: 500-502. Colwick, S. P., and N. O. Kaplan, 1957. Methods in Enzymology, 1st ed. 3 : 451-454. Cordy, D. R., H. E. Adler and R. Yamamoto, 1955. A pathogenic PPLO from goats. Cornell Vet. 45: 50-68. Gross, W. B., 1956. Escherichia coli as a complicating factor in chronic respiratory disease of chickens and infectious sinusitis of turkeys. Poultry Sci. 3 5 : 765-771. Levy, H. G., and H. A. Sober, 1960. A simple chromatographic method for preparation of gamma globulin. Proc. Soc. 103 : 250-252. Maestrone, G., and D. L. Coffin, 1961. Studio della malattia di Newcastle a mezzo della tecnica degli anticorpi fluorescenti. Nota II. Osservazioni sulla infezione seperimentals nel pulcino. Arch. Vet. Ital. 12: 193-199. Mohanty, S. B., H. M. De Volt and J. E. Faber, 1964. A fluorescent antibody study of infectious bronchitis virus. Poultry Sci. 4 3 : 179182.
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With the Bj strain of NDV given intratracheally to chickens, one can see specific fluorescent particles in the tracheal epithelial cells 24 through at least 120 hours postinoculation. The air sac has not yet been followed in this sequence, but the epithelial cells of the abdominal air sac show large aggregates of intracytoplasmic fluorescent NDV antigen at four days postinoculation. Work with IBV-infected chicken tissues was limited. Chickens infected intratracheally with the Massachusetts strain of IBV and sacrificed at the time rales were observed (72-96 hours postinoculation) contained the antigen in tracheal, thoracic, and abdominal air sac epithelial cells. The specific fluorescence was seen as a diffuse area around the nucleus, and was mostly associated with cells which appeared to be degenerating.
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Noel, J. K., H. M. De Volt and J. E. Faber, 1964. Identification of Mycoplasma gallisepticum in lesion tissue by immunofluorescence. Poultry Sci. 43 : 145-149. Osteen, O. L., 1962. Avian pleuropneumonia-like organisms: Research basis for a control and eradication program for the poultry industry. Proc. U. S. Livestock Sanitary Assoc. 66th annual meeting: 240-245. Page, L. A., 1962. Haemophilus infections in chickens. I. Characteristics of 12 Haemophilus isolates recovered from diseased chickens. Am. J. Vet. Res. 23 : 85-95.
Prince, A. M., and H. S. Ginsberg, 1957. Immunohistochemical studies on the interaction between Erhlich ascites tumor cells and Newcastle disease virus. J. Exptl. Med.: 105: 177188. Yamamoto, R., and C. H. Bigland, 1964. Pathogenicity to chicks of Mycoplasma associated with turkey airsacculitis. Avian Dis. In press. Yoder, H. W., Jr., and M. S. Hofstad, 1962. A previously unreported serotype of avian Mycoplasma. Avian Dis. 6: 147-160. Zander, D. V. 1961. Origin of S« strain Mycoplasma. Avian Dis. 5: 154-156.
and Development Department staff of Dawe's Laboratories, Inc., Chicago, Illinois. He will work with other Dawe's nutritionists in providing nutritional counselling and other technical services to feed manufacturers. He received a B.S. degree, majoring in poultry husbandry, and a M.S. degree in poultry and animal nutrition at the University of Minnesota. He is a member of the Poultry Science Association and the Association of Vitamin Chemists. ARBOR ACRES NOTES Dr. John F. Grimes has been named Director of Leghorn Research for Arbor Acres Farm, Inc., Glastonbury, Connecticut. HY-LINE NOTES Dr. John Van Zandt, and Donald D. Jackson have joined the staff of Hy-Line Poultry Farms, Des Moines, Iowa, as Service Specialists. Dr. Van Zandt will assist Dr. Dean Young, Hy-Line Service Department Head, in handling emergency service calls in the United States. Jackson will plan and carry out field management experiments. SALSBURY FELLOWSHIP Dwight S. Bond, Auburn University, has been awarded the 1964 Dr. Salsbury Fellowship. Bond was awarded the Fellowship in 1963 and was the unanimous choice of the National Turkey Federation Awards Committee for continuance for a second year. The Fellowship, consisting of $1,500 is awarded annually to the most outstanding graduate student in the field of study relating to turkey disease problems.
HEISDORF AND NELSON NOTES Dr. Kenneth Goodwin has joined the Genetic Department of Heisdorf and Nelson Farms, Inc., Kirkland, Washington. He obtained a B.S. degree in 1948, a M.S. in 1950, and a Ph.D. in 1952 at Cornell University, specializing in genetics and physiology. P.E.N.B. NOTES At the annual meeting of the Poultry and Egg National Board, held in Philadelphia, April 27-29, the following officers were elected: President— Blanton Smith, Nashville, Tenn.; First VicePresident—John Salsbury, Charles City, Iowa; Second Vice-President—Earl Hess, Ephrata, Pa.; Secretary—L. A. Wilhelm, Chicago, 111.; Treasurer •—Ralph Treat, Wooster, Ohio; Assistant Treasurer—T. R. Greenlee, Chicago, 111.; Executive Committee Members—Edward Covell, Easton, Md.; Chester B. Franz, St. Louis, Mo.; John Holton, Indianapolis, Ind.; Russell Rathbone, Seattle, Wash.; Morris Smith, Ellicott City, Md.; and General Manager—Lloyd Geil, Chicago, 111. Ted W. Elliott, formerly with the American Dairy Association, has been named Director of Marketing and Merchandising. He will be responsible for developing egg merchandising programs acceptable and usable by major food outlets and egg distributors to stimulate sales in major population centers. Dr. Richard C. Eaton, Director of Public Relations for the Ralston Purina Company, has been named Chairman of the Feed Manufacturers Committee. He received a B.S. and a M.S. degree from Kansas State College, and a Ph.D. degree
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NEWS AND NOTES (Continued from page 1278)
A
Since the present work was begun, there have been reports describing successful use of the fluorescent antibody technique in demonstrating M. gallisepticum (Noel et al., 1964) and IBV (Mohanty et al., 1964) in infected tissues.
Since preliminary investigation showed that the indirect fluorescent method did not serve well for identifying colonies of avian mycoplasma, H. gallinarum, or E. coli a simpler method of preparing conjugated antiserum was sought. Published methods for the preparation of fluorescein-labeled antibody require that the serum or conjugate be subjected to time-consuming manipulations such as fractionation, dialysis, and concentration. In addition, various procedures have been used to eliminate or reduce nonspecific staining. Thus, the present study was undertaken to develop a rapid identifaction test for certain avian pathogens in infected tissues as well as in artificial media. MATERIALS AND METHODS
Antigen propagation and preparation of immune sera. The NDV and IBV were propagated in the allantoic fluids of embryonated chicken eggs 8-11 days old. The NDV immune serum was prepared by immunizing four week old Cornish cross chickens with NDV strain Bi and challenging them with NDV strain GB eight weeks later. Hyperimmune serum to IBV was prepared by infecting chickens with the Massachusetts strain of IBV and challenging them eight weeks later with this same strain. Multiple doses of a dense suspension of washed, broth-grown cells of Mycoplasma gallisepticum (S 6 ), Mycoplasma sp. of N characteristic ( # 5 2 9 ) , Haemophilus gallinarum (0083), or Escherichia coli (strain d) were given intravenously to rabbits and fryer stock chickens for the production of
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NUMBER of Mycoplasma species are associated with diseases of poultry. Some are common in mixed poultry infections associated with other pathogens such as Newcastle disease virus (NDV), infectious bronchitis virus (IBV), Haemophilus gallinarum, and Escherichia coli (Osteen, 1962). All of these agents have been differentiated by morphological, biochemical, serological, bird infectivity, and/ or lesion characteristics. These manipulations are time-consuming, however, and may still leave doubt (especially with the Mycoplasma spp.) as to the identity and purity of the agent(s) in question. Furthermore, attempts often fail to isolate these agents from infected tissues; where isolation is accomplished there is often doubt as to whether the tissue was contaminated from some other tissue or an external source. It is clear that a rapid test is needed to differentiate these pathogenic agents, especially the Mycoplasma spp., in artificial media and infected tissues. The fluorescent antibody technique was thought of as a possible means of accomplishing this since it has been used to identify various species and serotypes of human mycoplasma as well as various bacteria and viruses, including NDV (Prince and Ginsberg, 1957; Burnstein and Bang, 1958; Maestrone and Coffin, 1961).
FLUORESCENT ANTIBODY TECHNIQUE
were made with fluorescein isothiocyanate (Baltimore Biological Laboratories), and the various viral or bacterial antisera prepared in chickens, turkeys or rabbits. The protein content of the serum which varied between 50-60 mg./ml. was estimated from the ratio of optical densities at 260 and 280 mjji. (mg. protein per mil. = 1.55 D28o — 0.76 D26o; optical path = 1 cm.) (Colwick and Kaplan, 1957). Three to 8 ml. of the undiluted serum at 0°C. was adjusted to pH 8.8 by adding 0.5 M sodium bicarbonate buffer at pH 8.8 (15% of the serum volume). The fluorescein isothiocyanate crystals (1 mg. per 40 mg. protein) were then floated on the liquid surface and allowed to stand for approximately 10 minutes. The mixture was then stirred with a magnetic stirrer for 16 to 20 hours at 4°C, after which the conjugated serum was placed on the column without prior dialysis. Then 0.025 M phosphate buffer, pH 7.2, was percolated through the column. Collection of eluate was begun as soon as a yellow color appeared, and ended as soon as it disappeared. The eluted conjugated proteins were stored at 4°C. and used over a period of several months without noticeable loss of staining ability. Preparation and staining of tissue sections, tissue impressions, and bacterial colony imprints. The staining efficiency of the various conjugated antisera was determined by staining cryostat sections and impressions of tissues from normal chickens or turkeys and from chickens or turkeys infected with either NDV, IBV, Haemophilus gallinarum, Mycoplasma gallisepticum, Mycoplasma of N characteristic or Mycoplasma synoviae; and by staining various bacterial colony imprints, smears of chicken red blood cells, and coverslip preparation of HeLa cells both infected and unifected with NDV strain GB. Cryostat sections of infected and normal chickens and turkeys were cut at 6-10
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immune serum. Sera from turkeys artificially infected with M. gallisepticum (S6) and chickens artificially infected with M. synoviae (NTF) were also used for conjugation with fluorescein isothiocyanate. The propagation of all of the Mycoplasma spp. except M. synoviae for colony imprints was done with PPLO agar (Bacto PPLO broth Difco), 1% yeast autolysate (Albimi Lab. Inc.), 1.55% Bacto agar (Difco), 15% horse serum (Adler et al., 1962). M. synoviae was grown on the agar medium described by Chalquest (1962). Haemophilus gallinarum strains and Escherichia coli were grown on Bacto blood agar base (Difco) plus 5% ox serum; the H. gallinarum was grown in association with a Micrococcus sp. feeder in an atmosphere of 10% carbon dioxide. Preparation of the cellulose column. Diethylaminoethylcellulose (DEAE-cellulose, Bio-Rad Laboratories) was washed according to the method of Levy and Sober (1960), with some modification. The DEAE-cellulose was washed twice with 0.5 N NaOH, then with distilled water until the pH of the supernate was 7.0-7.2, and then with 0.025 M phosphate buffer, pH 7.2, until the pH of the supernate was that of the buffer. All washings were done for 15-30 minutes, with ten times the starting amount of DEAE-cellulose, and the latter was recovered on a Buchner filter. A magnetic stirrer made all washings much more efficient. When the DEAE-cellulose was equilibrated with the 0.025 M phosphate buffer, pH 7.2, the coarse particles were allowed to settle out for 10 to 15 minutes. The coarse particles were packed into glass columns, 12 mm. outside diameter, to a height of 3.5 cm. for every 1 ml. of the sample to be fractionated. The flow rate of the column was between 0.7 and 1 ml. per minute. The column was kept at 0°C. Conjugation of the serum. Conjugations
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R. E . CORSTVET AND W . W . SADLER
line, pH 7.2. When colony imprints were to be stained, the conjugates were not absorbed. The dilution of the conjugated anti-NDV serum to be used for staining tissue sections and impressions was determined by the use of the red blood cell smears. The highest dilution of conjugates which still gave maximum brilliancy of fluorescence was then used to stain the tissue preparations. The dilutions of conjugate used for staining the NDV were generally 1:2-1:10; the dilution varied slightly with the particular conjugate preparation. The dilution of conjugated anti-bacterial serum used to stain colony imprints was the highest dilution which gave the maximum brilliancy of fluorescence, generally 1:10 to 1:30. The dilution of conjugate used to stain bacterial antigens in chicken or turkey tissues was one doubling dilution less than that which gave maximum brilliancy of fluorescence with the colony imprint system. Fluorescent staining of the preparations was begun by placing one drop of conjugate on each tissue section or impression or colony imprint; the slides or coverslips were then placed in a moist chamber at 37°C. for 25 to 30 minutes. The preparations were then washed twice in two changes of 0.01 M phosphate-buffered saline, pH 7.2 (5 and 20 minutes, respectively), rinsed once with distilled water, and allowed to air-dry partially before being mounted. The mounting fluid was 0.01 M phosphate-buffered saline, pH 7.2, with 10 percent glycerol. These preparations were examined by Zeiss microscope with on Osram HBO 200-W maximal-pressure mercury-vapor arc. The ultraviolet excitation filters used were UG 5 plus UG 2 or UG 5, and the barrier was the combination 0/41. RESULTS AND DISCUSSION
The method of preparing fluorescein antibody conjugates was satisfactory for rabbit,
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microns and stored at — 6°C, either without chemical fixation or with fixation in acetone for ten minutes at room temperature. Airdried tissue impressions and HeLa cell preparations were fixed overnight in cold acetone at — 6°C. and stored at that temperature until stained. Bacterial colony imprints were prepared by touching a coverslip to colonies which had grown on the surface of agar medium for 1 to 13 days at 37°C. The imprints were then air-dried, passed twice through a cool flame from a Bunsen burner, and stained immediately or stored at — 6°C. for periods up to six months. Colony imprints were also prepared according to the method of Clark et al. (1961). The RBC smears were prepared by mixing 1 ml. of a 0.5 percent suspension of chick RBC's with 1 ml. of unifected chicken embryo allantoic fluid or allantoic fluid containing either NDV strain GB or IBV strain Massachusetts and allowing it to stand one hour at 4°C. Both unifected and IBV-infected allantoic fluids were used as controls. The mixture was then centrifuged ten minutes at 160-170 X G, and the supernate was decanted. A loopful of the packed cells was smeared on a glass slide, allowed to air-dry, fixed for ten minutes in acetone at room temperature, and stained the same day. HeLa cells grown in coverslips were inoculated with allantoic fluid containing NDV strain GB and incubated at 37°C. Forty-eight to 72 hours after inoculation, these coverslips were washed once in 0.01 M phosphate-buffered saline pH 7.2, and stained. The nonspecific staining components were absorbed from the serum conjugate by mixing the conjugate 1:1 with a 20 percent suspension of tissues of a normal four month old chicken. A homogenized suspension of trachea, spleen, liver and brain was prepared in 0.01 M phosphate buffered sa-
FLUORESCENT ANTIBODY TECHNIQUE
avian mycoplasma or for H. gattinarutn. In all instances, fixation of the colony was excellent; however, the mycoplasma antigen was evidently destroyed completely by heat, since no fluorescence was obtained with the homologous conjugated chicken antiserum. With H. gallinarum, fluorescence was obtained with the homologous conjugated antiserum but the brilliancy of the stain was greatly diminished. This would indicate a partial destruction or alteration of its antigen. Very gentle heat fixation of the colony imprints gave excellent preparations. The small amount of agar picked up on the coverslip took up some of the conjugate, but this was not detrimental, since negative and positive colonies were easily discernible. Although Clark et al. (1963) reported that the indirect fluorescent test could be used to stain colonies of the human strains of mycoplasma, attempts failed to use the indirect test on the bacterial colonies represented in Table 1. Addition of complement to the staining reagents had no beneficial effect, nor did variations in staining time up to 60 minutes at 37°C. Various preparations of chicken or rabbit antiglobulin conjugates and immune serum which functioned well in the indirect test with tissues did not give a positive test with the same antigen in the form of colony imprints. Table 1 clearly illustrates the specificity of the fluorescent antibody technique for differentiation and indentification of various avian pathogens in artificial media. There was no significant immunological cross reaction between the various Mycoplasma spp. or between them and the other bacterial antigens. The two field isolates of M. gallisepticum reacted only with the anti-Af. gallisepticum conjugate. The five mycoplasma isolates which were characterized as "N" type by morphological, biochemical, and serological procedures reacted only with the anti-Mycoplasma sp. "N"
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chicken, and turkey antisera containing either viral or bacterial antibodies. The antibody titer of the conjugated fractionated serum was generally one to two doubling dilutions less than that of the unconjugated unfractionated serum. Absorption of the conjugates to be used for staining colony imprints or the RBC smears was not necessary. With avian tissues and HeLa cells, however, there was a difference between unabsorbed and absorbed conjugates in the amount of nonspecific staining. With the unabsorbed conjugates the cells of all infected and normal chicken tissues took a diffuse, dull, nonspecific stain that increased the brightness of the background. However, this was not a problem in observing the specific fluorescence. This lighter background was completely eliminated by absorbing the conjugate. In the brain there was brilliant nonspecific staining of a very few small clusters of nonparenchymal cells associated with vascular areas in some of the infected and unlfected sections. However, specific staining of NDV antigen was excellent and easily discernible in the cytoplasm of the Purkinje cells and cells in the perivascular and vascular areas. In some of the infected and uninfected tracheal and air sac sections and impressions there were a few small nonparenchymal cells which stained with one brilliant granule in the center. Absorption of the conjugate eliminated this nonspecific staining in the brain, but not in the trachea and air sac. With the HeLa cells infected with NDV strain GB, nonspecific staining of the undiluted conjugate and a 1:5 unabsorbed dilution was 2.5+, based on a 4+ maximum. Even after absorption with normal HeLa cells this conjugate was not considered to be very satisfactory because of the nonspecific staining that was still present. The hot-water colony fixation method of Clark et al. (1961) for human strains of mycoplasma was not satisfactory for the
1283
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R . E . CORSTVET AND W . W . SADLER
conjugate. M. synoviae, M. sp. #695, H. gallinarum, and E. coli antigens also reacted only with their respective homologous antiserum conjugates. The anti-NDV conjugate served as a control to show that hyperimmune anti-viral serum did not contain a nonspecific serum component which would react with the bacterial antigens. In addition to the antigens in Table 1, the conjugated anti-Af. gallisepticum (S6) serum
and H. gallinarum (0083) serum did not give positive staining with four Kidd strains of Mycoplasma (goat pathogen) Cordy et al., 1955) or with Mycoplasmas LA and LB (isolated from sewage by Dr. Edwards, London, England; subculture obtained from Dr. H. E. Adler), H3-10 (saprophyte of chickens) (Adler et al., 1961) and three strains of Haemophilus parahemolyticus (isolated from the stifle of a sheep, brain
Chicken immune serum conjugate Colony imprint 1
MG—l 2 MG—23 N—l 4 N—2" N—3 4 N
_44
N—5 6 MS—l 6 MS—2" MS—3 6 1' 28 38 48
59 Oio
11
HG—l HG—212 HG—3 12 Esch. coli strain "d" 13 Micrococcus sp. (feeder) 1
M. gallisepticum Se
Mycoplasma sp. " N " 529
Pos14 Pos Neg15 Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg
Neg Neg Pos Pos Pos Pos Pos Neg Neg
MycoMycoplasma sp. Synovitis plasma sp. 695 NTF Neg Neg Neg
— —
H. gallinarum 0083
Esch. coli Strain "d"
NDV Strain GB
Neg Neg Neg Neg Neg
Neg Neg Neg
Neg Neg
Neg
— —
Neg
— —
— — — —
— — — —
—
Neg
—
—
Neg Neg Pos Pos Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg
Neg
—
—
—
Neg
Pos
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
16
—
Neg Neg Neg Neg Neg Neg Neg Neg
Neg Neg
—
Neg
Neg Neg Neg Neg Neg Neg Neg Neg
Neg Neg Neg Neg Neg Neg Neg Neg
—
—
—
—
Pos Neg
— —
Neg
Neg Neg Neg Neg Neg Neg Neg
— —
Pos Pos Pos
Neg
Neg
— —
— —
— —
1- to 13-day agar plate culture. M. gallisepticum isolated by Dr. D. V. Zander (1961). M. gallisepticum isolate (Richey) obtained from Dr. S. Schmittle, Georgia. 4 Mycoplasma sp. (#1 = 529; #2 = 400; #3 = 2-5; #4=Byl4) of N characteristic isolated by Dr. R. Yamamoto from cases of turkey poult air-sacculitis. 5 Mycoplasma sp. of N characteristic isolated in our laboratory from a case of turkey poult arrsacculitis. 6 Mycoplasma synoviae (#1 = N T F ; #2 = 1853; #3 = Maine) obtained from Dr. J. Fabricant, (1960). 7 Mycoplasma sp. (#695) isolated by Drs. H. W. Yoder and M. S. Hofstad (1962). 8 Mycoplasma sp. (#2 = #8; 3 = #23; 4 = #24) other than M. gallisepticum or N isolated by Dr. R. Yamamoto from cases of turkey poult airsacculitis (1964). 9 Mycoplasma sp. (Tu) obtained from Dr. H. E. Adler (1957). 10 Human strain of Mycoplasma (#880) isolated from a tumor by Dr. Horosowitz, Memorial Hospital, Albany, N. Y. (subculture obtained from Dr. H. E. Adler). 11 H. gallinarum (0083) isolated by Dr. L. A. Page (1962). 12 H. gallinarum (#2 = 17756; #3=17764) isolated by Dr. R. Yamamoto from chicken field cases. 13 Esch. coli, strain " D , " isolated by Dr. W. B. Gross (1956). 14 Pos=specific fluorescence. 16 Neg=no specific fluorescence. 16 — = n o t done. 2
3
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TABLE 1.—Identification of microorganisms with the direct fluorescent antibody technique
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FLUORESCENT ANTIBODY TECHNIQUE TABLE 2.—Direct fluorescent staining of infected avian tissues Infected avian. tissue1 Absorbed conjugated chicken Antiserum
1 2 3 4
Chicken abdom airsac
MGS, H . G . 0 0 8 3 s ™ V 2
Neg
Neg 4
Neg
Pos
3
MGS,
Chicken foot pad
NDV Str GB
M.S. N T F
Turkey abdom air sac " N " 529
Neg
Pos
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Pos
Neg Pos
— —
Neg Neg
Pos Neg
Neg
Neg
Pos
Neg
Pos
Neg
Cryostat section 6-10 microns thick. Pos = specific fluorescence. Neg=no specific fluorescence. —=not done.
abscess in a steer and a swine isolate). The colony imprint system further confirmed the identification and grouping of the various avian mycoplasma, which to date has been done by more tedious serological, biochemical, and pathological tests. Identification and differentiation by the fluorescent antibody technique was possible not only with antigens propagated on artificial media but also with antigens propagated in avian tissues (Table 2). The pathogens in avian tissues, as was the case with these antigens in artificial media, stained only with the homologous antiserum conjugate. M. gallisepticum (S6) in the trachea or abdominal air sac was identified and differentiated from Mycoplasma sp. "N", Mycoplasma synoviae, H. galUnarum, and NDV. The fact that S6 and N may be differentiated in tissues is important because both of these agents can be associated with airsacculitis in turkeys (Adler et al., 1958). M. synoviae is not known to cause air sac disease in poultry, but both this mycoplasma and S6 produce joint lesions— so their differentiation in tissues is important. H. galUnarum and NDV were clearly separated by the fluorescent antibody technique, not only from each other but from
the Mycoplasma spp. as well, an important fact since combinations of these agents are often associated with poultry disease syndromes. The data in Table 3 demonstrate the applicability of these techniques to both the diagnosis of disease and the study of pathogenesis of selected diseases. This table summarizes the results of the direct fluorescent staining by absorbed conjugate of chicken tissues infected with NDV strain GB and a correlation with clinical signs. As one might expect, the antigen was demonstrable in the digestive and upper respiratory tissues before clinical signs were observable. Both the numbers of epithelial cells infected and the concentration of antigen per cell were greatest on the fourth day postinoculation. The proventriculus tissue smears were negative on that day, possibly because the infected parenchymal cells were so few that they were not picked up by the tissue impression. The fifth, sixth, and seventh-day impressions of the proventriculus contained only 2-3 positive epithelial cells, with few intracytoplasmic fluorescent particles. On these days there were a number of phagocytic cells in the tissue without NDV antigen. None of the air sac epithelial
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M. gallisepticum {§$) Mycoplasma sp. " N " 529 Mycoplasma synoviae (Synovitis NTF) H. galUnarum (0083) Newcastle Disease Virus strain G.B. (NDV)
Chicken trachea
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TABLE C5.—Correlation
of clinical signs with detection of Newcastle disease virus strain GB in tissues of 10-week-old intratracheally infected chickens by the direct fluorescent antibody technique Tissue impressions8
Sacrificed—day post inoculation 02
None
Proventriculus
Abdominal Air sac
Trachea
_ 4
—
—
—
2+
6
1
None
2+
2
Slightly depressed; slight hoarseness
3+
—
3+
3
Moderately depressed; rales; slight leg weakness; sneezing
4+
2+
3+
Slight rales; partial leg paralysis; clonic spasms; depressed
—
4+
4+
Paralysis; clonic spasms, depressed; no rales
1+
3 +
1+
Paralysis; clonic spasms; depressed; no rales
1+
—
1+
Slight rales; Hyperexcitable; clonic spasms head; marked leg weakness; ataxia.
1+
—
—
4 5 6 7
1
Clinical signs at time of autopsy
1
One bird sacrificed at daily intervals through 8 days. Sacrificed immediately prior to inoculation. Impressions of liver, brain, spleen, cervical glands, and lung were negative for each of the above postinoculation days. Heart blood was negative for days 1 through 4; S through 7, not done. 4 No fluorescence in epithelial cells. 6 1 through 4 + ; progression of not only more epithelial cells with viral antigen but more particles and aggregates of antigen per cell. 2
3
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cells showed intracytoplasmic fluorescence sue sections of the spleen, liver and bursa on the sixth and seventh days postinocula- from the bird on the fourth postinoculation tion. However, on the sixth day there were day, stained with fluorescent antibody a few phagocytic cells which contained showed that NDV antigen was present in NDV antigen in their cytoplasm. From a the cytoplasm of the cells lining the liver diagnostic point of view these results would sinusoids and bursal parenchymal cells, and indicate that proventriculus and trachea in the vascular endothelial cells of all three would be the tissue of choice, since the NDV tissues. The reason the spleen and liver was present in one or both of these tissues sections were positive and the impressions earlier and later than in the abdominal were not, is doubtless due to the fact that a air sac. tissue impression will usually not pick up The liver, spleen, cervical nodes, and the vascular architecture. Frozen tissue secheart blood impressions from the chickens tions of the brain on the fourth day postillustrated in Table 3 were all negative for inoculation displayed NDV antigen in the NDV antigen; these were good impressions cytoplasm of the Purkinje cells and in the and there was no nonspecific tissue fluores- endothelial cells of the blood vessels. Occacence with the absorbed conjugate. The sionally vascular areas would be seen with fact that the heart blood was negative is at fluorescent particles in the cells immediately variance with a previously published report surrounding these areas which gave the im(Maestrone and Coffin, 1961). Frozen tis- pression of fluorescent perivascular cuffing.
FLUORESCENT ANTIBODY TECHNIQUE
SUMMARY
A modified method of conjugate preparation produced conjugates that were satisfactory for differentiating various avian pathogens in artificial media and tissues. This simplified method saved considerable time over previously published methods by eliminating fractionation of the unconjugated serum, dialysis, and concentration. This was desirable since the indirect fluorescent test did not function with colony imprints and also, after conjugation and fractionation, the majority of the antibody protein was recovered; a distinct advantage in the case of the often-lo-titer avian mycoplasma antiserum. The results indicate that the fluorescent test can be very useful in evaluating mycoplasma cultures as to purity and greatly simplifies the identification of Mycoplasma spp. or serological types as well as other avain pathogens. The results also showed that tissue imprints of trachea, air sac, and proventriculus could be used in identifying NDV by the fluorescent antibody technique.
REFERENCES Adler, H. E., M. Shifrine and H. Ortmayer, 1962. Interpretation of Mycoplasma gallisepticum serologic tests for turkeys. X I I World's Poultry Congress: 322-324. Adler, H. E., M. Shifrine and H. B. Ortmayer, 1961. M. inoccuum sp. n., a saprophyte from chickens. J. Bacteriol. 82: 239-240. Adler, H. E., J. Fabricant, R. Yamamoto and J. Berg. 1958. Isolation and identification of pleuropneumonia-like organisms of avian origin. Am. J. Vet. Res. 19: 440-447. Adler, H. E., R. Yamamoto and J. Berg, 1957. Strain differences of pleuropneumonia-like organisms of avian origin. Avian Dis. 1: 19-26. Burnstein, T., and F. B. Bang, 1958. Infection of the upper respiratory tract of the chick with a mild (vaccine) strain of Newcastle disease virus. II. Studies on the pathogenesis of the infection. Bull. Johns Hopkins Hosp. 102: 135157. Chalquest, R. R., 1962. Cultivation of the infectious-synovitis-type pleuropneumonia-like organisms. Avian Dis. 6 : 36-43. Chalquest, R. R., and J. Fabricant, 1960. Pleuropneumonia-like organisms associated with synovitis in fowls. Avian Dis. 4 : 515-539. Clark, H. W., J. S. Bailey, R. C. Fowler and T. Mc P. Brown, 1963. Identification of Mycoplasmataceae by the fluorescent antibody method. J. Bacteriol. 85: 111-118. Clark, H. W., R. C. Fowler and T. McP. Brown, 1961. Preparation of pleuropneumonia-like organisms for microscopic study. J. Bacteriol. 81: 500-502. Colwick, S. P., and N. O. Kaplan, 1957. Methods in Enzymology, 1st ed. 3 : 451-454. Cordy, D. R., H. E. Adler and R. Yamamoto, 1955. A pathogenic PPLO from goats. Cornell Vet. 45: 50-68. Gross, W. B., 1956. Escherichia coli as a complicating factor in chronic respiratory disease of chickens and infectious sinusitis of turkeys. Poultry Sci. 3 5 : 765-771. Levy, H. G., and H. A. Sober, 1960. A simple chromatographic method for preparation of gamma globulin. Proc. Soc. 103 : 250-252. Maestrone, G., and D. L. Coffin, 1961. Studio della malattia di Newcastle a mezzo della tecnica degli anticorpi fluorescenti. Nota II. Osservazioni sulla infezione seperimentals nel pulcino. Arch. Vet. Ital. 12: 193-199. Mohanty, S. B., H. M. De Volt and J. E. Faber, 1964. A fluorescent antibody study of infectious bronchitis virus. Poultry Sci. 4 3 : 179182.
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With the Bj strain of NDV given intratracheally to chickens, one can see specific fluorescent particles in the tracheal epithelial cells 24 through at least 120 hours postinoculation. The air sac has not yet been followed in this sequence, but the epithelial cells of the abdominal air sac show large aggregates of intracytoplasmic fluorescent NDV antigen at four days postinoculation. Work with IBV-infected chicken tissues was limited. Chickens infected intratracheally with the Massachusetts strain of IBV and sacrificed at the time rales were observed (72-96 hours postinoculation) contained the antigen in tracheal, thoracic, and abdominal air sac epithelial cells. The specific fluorescence was seen as a diffuse area around the nucleus, and was mostly associated with cells which appeared to be degenerating.
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Noel, J. K., H. M. De Volt and J. E. Faber, 1964. Identification of Mycoplasma gallisepticum in lesion tissue by immunofluorescence. Poultry Sci. 43 : 145-149. Osteen, O. L., 1962. Avian pleuropneumonia-like organisms: Research basis for a control and eradication program for the poultry industry. Proc. U. S. Livestock Sanitary Assoc. 66th annual meeting: 240-245. Page, L. A., 1962. Haemophilus infections in chickens. I. Characteristics of 12 Haemophilus isolates recovered from diseased chickens. Am. J. Vet. Res. 23 : 85-95.
Prince, A. M., and H. S. Ginsberg, 1957. Immunohistochemical studies on the interaction between Erhlich ascites tumor cells and Newcastle disease virus. J. Exptl. Med.: 105: 177188. Yamamoto, R., and C. H. Bigland, 1964. Pathogenicity to chicks of Mycoplasma associated with turkey airsacculitis. Avian Dis. In press. Yoder, H. W., Jr., and M. S. Hofstad, 1962. A previously unreported serotype of avian Mycoplasma. Avian Dis. 6: 147-160. Zander, D. V. 1961. Origin of S« strain Mycoplasma. Avian Dis. 5: 154-156.
and Development Department staff of Dawe's Laboratories, Inc., Chicago, Illinois. He will work with other Dawe's nutritionists in providing nutritional counselling and other technical services to feed manufacturers. He received a B.S. degree, majoring in poultry husbandry, and a M.S. degree in poultry and animal nutrition at the University of Minnesota. He is a member of the Poultry Science Association and the Association of Vitamin Chemists. ARBOR ACRES NOTES Dr. John F. Grimes has been named Director of Leghorn Research for Arbor Acres Farm, Inc., Glastonbury, Connecticut. HY-LINE NOTES Dr. John Van Zandt, and Donald D. Jackson have joined the staff of Hy-Line Poultry Farms, Des Moines, Iowa, as Service Specialists. Dr. Van Zandt will assist Dr. Dean Young, Hy-Line Service Department Head, in handling emergency service calls in the United States. Jackson will plan and carry out field management experiments. SALSBURY FELLOWSHIP Dwight S. Bond, Auburn University, has been awarded the 1964 Dr. Salsbury Fellowship. Bond was awarded the Fellowship in 1963 and was the unanimous choice of the National Turkey Federation Awards Committee for continuance for a second year. The Fellowship, consisting of $1,500 is awarded annually to the most outstanding graduate student in the field of study relating to turkey disease problems.
HEISDORF AND NELSON NOTES Dr. Kenneth Goodwin has joined the Genetic Department of Heisdorf and Nelson Farms, Inc., Kirkland, Washington. He obtained a B.S. degree in 1948, a M.S. in 1950, and a Ph.D. in 1952 at Cornell University, specializing in genetics and physiology. P.E.N.B. NOTES At the annual meeting of the Poultry and Egg National Board, held in Philadelphia, April 27-29, the following officers were elected: President— Blanton Smith, Nashville, Tenn.; First VicePresident—John Salsbury, Charles City, Iowa; Second Vice-President—Earl Hess, Ephrata, Pa.; Secretary—L. A. Wilhelm, Chicago, 111.; Treasurer •—Ralph Treat, Wooster, Ohio; Assistant Treasurer—T. R. Greenlee, Chicago, 111.; Executive Committee Members—Edward Covell, Easton, Md.; Chester B. Franz, St. Louis, Mo.; John Holton, Indianapolis, Ind.; Russell Rathbone, Seattle, Wash.; Morris Smith, Ellicott City, Md.; and General Manager—Lloyd Geil, Chicago, 111. Ted W. Elliott, formerly with the American Dairy Association, has been named Director of Marketing and Merchandising. He will be responsible for developing egg merchandising programs acceptable and usable by major food outlets and egg distributors to stimulate sales in major population centers. Dr. Richard C. Eaton, Director of Public Relations for the Ralston Purina Company, has been named Chairman of the Feed Manufacturers Committee. He received a B.S. and a M.S. degree from Kansas State College, and a Ph.D. degree
(Continued on page 1296)
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NEWS AND NOTES (Continued from page 1278)