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Distribution of Mycoplasma Gallisepticum in Selected Tissues and Organs of Artificially Infected Turkeys
Distribution of Mycoplasma Gallisepticum in Selected Tissues and Organs of Artificially Infected Turkeys R. L. PETERS 1 , J. E. FABEE2, W. D. KEENUM 3 AND H. M. DEVOLT 4 Department of Veterinary Science and Department of Microbiology, University of Maryland, College Park, Maryland (Received for publication January 14, 1966)
Veterinary Science.
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A IR-SAC infection (PPLO) in poultry is pneumonia-like organism is the cause of J~\ one of the most troublesome and per- chronic respiratory disease was advanced sistent ailments that prey upon the indus- by Markham and Wong (1952). Several try. This disease was the cause in 1963 and investigators, including Delaplane and 1964 for 38% and 40%, respectively, of Stuart (1943), Fahey and Crawley (1954), the total condemnations of poultry carcas- Gross (1956), Smibert et al. (1959a, b), ses under inspection regulations of the Poul- Fabricant and Levine (1962) have reporttry Inspection Service in the U.S.D.A. ed on several bacterial and viral agents in (McKee, 1965). An accurate and rapid the air-sac disease complex. Smibert et al. method of diagnosis is important to selec- (1959b) produced typical lesions by inocution of wholesome meat as well as for con- lating germ-free turkey poults with M. galtrol and eradication of disease in affected linarum {gallisepticum), an accomplishflocks. ment of special significance. He also reportIn a series of reports, Nelson (1935, ed the change from normally and predomi1936a, b) described the isolation of an orga- nantly gram-positive bacterial flora to prenism which he called "coccobacilliform dominantly gram-negative flora of the respibodies" from a fowl with coryza of slow ratory system following artificial inoculaonset and long duration. Van Herick and tion of conventional turkeys with M. gallEaton (1945), while passing a virus of isepticum. Mycoplasma have been routineatypical pneumonia in chicken embryos, ly isolated from respiratory organs of reported isolation of an organism which chickens and turkeys by Markham and was "pleuropneumonia-like." They con- Wong (1952), Adler et al. (1957), Grumcluded the eggs were the source of contami- bles et al. (1953), Taylor and Fabricant nation. Experimental proof that a pleuro- (1957), Taylor et al. (1957) and Smibert et al. (1959a). Other investigators have reported isolation of PPLO from organs not Condensation of a thesis submitted to the included in the respiratory system. Moulfaculty of the Graduate School, University of ton and Adler (1957) isolated the organism Maryland, as a partial fulfillment of the requirefrom synovial fluid. Yamamoto et al. ments for the degree Master of Science. Scientific Article No. A 1248. Contribution No. 3761 of the (1965) recovered the "N" type of MycoMaryland Agricultural Experiment Station. plasma from semen, vagina and bursa of 1 Graduate Assistant, Department of Veterinary Fabricius. The type strain, S6, was isolatScience and Department of Microbiology. 2 ed from brain tissue of a turkey by Zander Professor of Microbiology, Department of (1961). Cordy and Adler (1965) reported Microbiology. 3 Technician, Department of Veterinary Science. the presence of brain and muscle lesions in 1 Professor of Avian Pathology, Department of turkey poults after intravenous inoculation
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R. L. PETERS, J. E. FABER, W. D. KEENUM AND H. M.
DEVOLT
Artificial exposure to "air-sac" injection: Artificial exposure to "air-sac" infection in experimental turkeys was accomplished by injecting infected air-sac tissue. The infected air-sac tissue to be used for the experimental group was produced by preliminary injection of a few young poults with a broth culture of M. galUsepticum, strain S6*. After symptoms appeared, air-sac lesion tissue was harvested from freshly killed turkeys. The membranes were macerated in a Teflon grinder and suspended in proteose peptone # 3 (Difco) to make a 20% tissue suspension. A group of twenty, 6-week-old White Holland turkeys were each injected intratracheally with 0.5 ml. of the lesiontissue suspension and confined in wire cages in an isolated room. Bacteriological examination of the inoculum showed that it was heavily impregnated with M. galUsepticum and also contained 100 to 200 other organisms per ml. including Escherichia coli, Micrococcus, Streptococcus and diphtheroid sp.
MATERIALS AND METHODS
Selected organs and tissues from infected turkeys: At intervals of 3, 5, 7, 9, 11, 13, 15, 17, 21 and 30 days postinoculation, two turkeys were selected at random from the inoculated group and sacrificed. Immediately after being killed by breaking the neck, the carcass was immersed in Roccal, 1:1250, to destroy contaminating organisms on the feathers and skin. The skull was then opened to obtain brain tissue and skin carefully removed for the purpose of taking samples of muscle tissue. The body cavity of the carcass was then immediately opened and certain visceral organs removed with aseptic precautions. A list of all organs and tissues employed in the present study follows.
The general plan of investigation was to inoculate a group of young experimental turkeys with M. galUsepticum in air-sac lesion-tissue and to sacrifice birds at various intervals postinoculation to obtain certain tissues and organs for bacteriological examination. Aliquots from all tissue and organ suspensions were used to seed culture media and to prepare direct smears for immunofiuorescent tissue staining. Final identification of M. galUsepticum in both culture and direct smears was by immunofiuorescent staining. The object of the experiment was to determine the extent and frequency with which M. galUsepticum may be disseminated from the respiratory system to other tissues and organs. In addition, a comparison was made between results obtained by isolation in culture and identification in direct smears with immunofiuorescent tissue staining.
(a) Thoracic and abdominal air-sacs. (b) Brain (cerebral hemispheres). * M. galUsepticum, type strain Se, was obtained from H. E. Adler, Department of Avian Medicine, University of California, Davis, California.
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of M. galUsepticum following either intravenous or intramuscular inoculation with Newcastle virus. Jerstad (1964) isolated M. galUsepticum from fresh eggs and blood 4 to 126 days and at 7 days, respectively, after intravenous inoculation. Initial work on the demonstration of antigenic substances by immunofiuorescent tissue staining was carried out by Coons et al. (1942). Marshall et al. (1958) reported on the method of conjugating fluorescein isothiocyanate with whole serum and serum-globulin. Malizia et al. (1961) and Barille et al. (1962) published reports on identification of Mycoplasma in tissue cell culture by immunofluorescence. Corstvet and Sadler (1964) identified M. galUsepticum by staining colony imprints and impression smears from trachea and air-sac tissue with fluorescein-labeled immune serum. Noel et al. (1964) reported the identification of M. galUsepticum in lesion tissue by immunofluorescence.
DISTRIBUTION OF MYCOPLASMA GALLISEPTICUM
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isms were harvested from several liters of broth in a continuous-flow, Servall, RC-2, refrigerated centrifuge at 23,000 X g. Sedimented M. gallisepticum organisms were suspended in phosphate-buffered saline (pH 7.4) and washed 3 times by centrifugation. Antigen for the tube-agglutination test was suspended in a diluent of 0.85% saline with 0.3% formalin and adjusted to 76% light transmission (LT) in a Coleman # 9 nepho-colorimeter. For testing rabbit serum, two-fold dilutions were made with the same diluent. For testing turkey serum, Lung and kidney tissues were taken from two-fold dilutions were made with diluent the infected side when air-sac lesions were containing 8.5% saline and 0.3% formalin. unilateral. Kidney, heart, liver and spleen Antigen for labbit injection to produce imwere immersed in 70% alcohol for 60 sec. mune serum was adjusted in physiological to destroy surface contamination from con- saline to 68% LT in the nephelometer. Six, tiguous infected air-sac membranes. These 4- to 5-pound rabbits were given 6 intraorgans were then rinsed S times in sterile peritoneal injections of 5 ml. of the antigen saline to remove alcohol. Blood for culture at intervals of 3 to 4 days. Sixteen days and serological examination was drawn after the last injection, the rabbits were exwith a sterile syringe from a wing vein. The sanguinated and serum separated in the selected organs and tissues were stored in usual manner. Titers of pooled serums used sterile stoppered vials at — 60°C. until used for separation of immune globulin varied for bacteriological examination. The or- from 1:320 to 1:1280. gans, when used for examination in 1 to 6 Preparation of immunofluorescent stain: months, were thawed at room temperature Globulin (immune) was separated from for 2 hr. They were prepared for examina- pooled rabbit serums by precipitating overtion by mincing and macerating in a Teflon night at 4°C. with half-saturated ammongrinder with sterile saline. From 3 to S g. ium sulfate. Precipitate was concentrated of tissue were macerated with 7 ml. saline by centrifugation at 3,000 X g. for 20 min. after which aliquots of the tissue suspen- at 4°C, dissolved in a quantity of distilled sions were used to seed culture media and water equal to the original volume of pooled to prepare smears for direct microscopic serums, and precipitated 3 or more times examination with immunofluorescent stain- until no trace of hemoglobin remained. ing. Similar tissue and organ preparations After final dissolution in distilled water, were prepared from noninfected turkeys to crude globulin was dialyzed at 4°C. against be used as controls with the immu- 3 or more changes of physiological saline nofluorescent tissue staining technique. until the dialysate was free from ammonProduction of antigen and immune ium sulfate as determined by testing aliserum: Antigen for agglutination tests and quots with barium chloride. Total protein production of immune serum in rabbits was content of the globulin was then determined produced by growing M. gallisepticum by the biuret reaction using a Bausch and strain, S6, in phenol-red broth (described Lomb spectronic-20 spectrophotometer at below) but with penicillin omitted. Organ- 540 mjx. and a protein standard (Scientific (c) Biceps brachii, triceps and gastrocnemius muscles (2 g. of each pooled). (d) Heart. (e) Kidney (3 lobes). (f) Lung. (g) Liver (half of left lobe). (h) Spleen. (i) Thoracic-wall muscles (2 g. pooled pectoralis superficialis and external intercostals). (j) Blood (venipuncture).
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R. L. PETERS, J. E. FABER, W. D. KEENUM AND H. M.
Culturing blood and tissue suspensions for M. gallisepticum: The medium employed for production of antigen and for examination of tissue suspensions from infected turkeys was phenol-red broth (Difco) supplemented with 0.25% dextrose and thallium acetate (1:2,000). After autoclaving, the medium was further supplemented by addition of 2% of PPLO serum-fraction (Difco) and by 1,000 units penicillin per ml. One ml. of tissue suspension was added to each of 4 tubes of broth. Ten ml. of broth were added to a sterile tube containing 1 ml. of freshly drawn blood from infected turkeys. Cultures were incubated at 37°C. and held for at least 14 days. Those undergoing color change (red to orange) were pooled with aseptic precautions and centrifuged at 6,000 X g for 30 min. The resulting sediment was sus-
pended in 1 to 2 ml. of sterile saline and used to prepare slides for examination by immunofluorescent staining. In some instances, broth cultures were plated on semisolid media (without penicillin) to observe colony type. Semisolid media was prepared with heart-infusion agar (Bacto) diluted with heart-infusion broth (Bacto) to adjust agar to 0.75% and supplemented with 1% yeast extract, 1% proteose peptone #Z, and 4% PPLO serum fraction (all Difco) according to the method described by Kelton (1962). Both the broth and semisolid medium were adjusted to pH 7.8. Typical colonies were subjected to further examination by immunofluorescent staining according to the method of Clark et al. (1961). A block of agar was cut from a petri dish and placed, colony side down, on a carefully cleaned glass slide which was then immersed in distilled water preheated to 80° C. The temperature of the water was raised until the agar block dissolved. The slide was then rinsed, air-dried, and examined by immunofluorescent staining. Immunofluorescent staining technique: Three loopsful of culture sediment or tissue suspension were spread on the surface of each of 2 clean glass slides, permitted to air-dry, and fixed with low heat. Slides were then flooded with immunofluorescent stain and held in a moist chamber for 30 min. at room temperature. The slides were rinsed with carbonate-bicarbonate buffer (0.5 M, pH 9.0) and placed in a Coplin jar with excess buffer to permit diffusion of stain. The stained smears were next rinsed and drained of excess fluid by touching the edge to filter paper. The surface of the smear, however, was not allowed to dry. Flazo orange was next applied as a counter stain for 45 to 60 sec. except in the case of brain and kidney tissue when counter staining was limited to 30 sec. because of greater affinity of those tissues for flazo orange. For use as controls in immunofluorescent tissue staining smears were prepared by applying
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Products, Washington, D.C.). Globulin protein was adjusted to a concentration of 1% with saline and carbonate-bicarbonate buffer (O.SM, pH 9.0). Diluted globulin had a pH of 9.0 with a 10% change in volume. The globulin solution was labelled with crystalline fluorescein isothiocyanate (FITC, Baltimore Biol. Lb., Baltimore, Md.) according to the method of Marshall el al. (19S8) with the following modifications. FITC was first dissolved in a portion of carbonate-bicarbonate buffer to facilitate uniform coupling with protein. Fluorescein isothiocyanate was added to globulin solution in a ratio of 1 part dye to 40 parts protein (0.025 mg. FITC/mg. protein) and conjugation continued for 18 hr. at 4°C. The mixture was then passed through a dextran column (Sephadex G 25, Fine, Pharmacia Fine Chem., New York City,) to remove uncombined dye. Nonimmune globulin from normal rabbits was used in the same way to prepare fluorescein conjugate to be used as a control for immunofluorescent tissue staining technique. The stain not used immediately was stored at -60°C.
DEVOLT
DISTRIBUTION
O F MYCOPLASMA
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GALLISEPTJCVM
TABLE 1.—Results 0/ culture and immunoftuorescent staining on selected tissues from turkeys irtificially injected with Mycoplasma gallisepticum Days post-inoculation 3
|
5
7
9
1
11
Wing-band numbers 1 Tissue Air Sac
3
2
4
Kidney
Thoracic Wall
8
-
-
-
- - + + + - + + - - -
10
9
- + + - -
+ + + + + + - + -
F
C
F
+ + + - + + -
-
-
-
Days post-inoculation 13
IS
21
17
30
Wing-band numbers 11 Tissue Air Sac Brain Kidney Lung Spleen Trachea Thoracic Wall
13
12
c F c + - - - - - - - - + + + + + + + + F
-
15
14
i 16
17
18
19
20
c F c F c F c F c F c + + + + + + + + + + + ~ - + + + + _ ^ + + + + + - + + + + + + + + + + + + + + + + + + + + + - + + — + + + c F c + + - + - - + + - - + + + + F
F
Negative tests are indicated by (—). Positive tests are indicated by ( + ) . 1 F indicates immunofluorescent tissue stain. 2 C indicates culture.
the stain to similar tissue suspensions from organs of normal turkeys and also applying fluorescein-labeled nonimmune globulin to the tissues from infected turkeys. The criteria for identification of M. gallisepticum in tissue smears or in culture sediment was fluorescing coccoid bodies ranging from 0.2 p.. to 0.5 [x. in diameter. Fluorescene was considered to be positive at 2 + between the limits of slight fluorescence at 1 + and strong fluorescence at 4 + . Fluorescent microscopy: Microscopic examination of immunofluorescent stained tissue smears and culture sediment was made with a Carl Zeiss, Model W, microscope equipped with a cardioid darkfield condenser and nonfluorescing objectives. An Osram, HBO 200, high-pressure mercu-
ry vapor burner operating under a constant input of 120 volts was employed as a source of illumination. A three mm., Schott, BG-12 exciter filter was used in combination with a Kodak, G-1S barrier filter. Photographs were made with a Zeiss camera using Kodak, high-speed Ektachrome color films. RESULTS
Results of examinations of selected tissues and organs from artifically infected turkeys by culture and/or immunofluorescent tissue staining of tissue smears are given in Table 1. Culture sediment when stained with immunofluorescent technique resulted in fluorescing masses of M. gallisepticum (Fig. 1) Colonies re-
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- - + + -
Spleen Trachea
7
6
c F C + + + + + + + - - - + - - + + - - - + + + + + + + + + + — + +
Brain
Lung
5
c» F C F C F C F C F C F c + + ~ + + + + ~ + + + + + +
Fi
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R. L. PETERS, J. E. FABER, W. D. KEENUM AND H. M.
DEVOLT
FIG. 2. Colony of Mycoplasma gallisepticum fixed by "hot water'' method and stained with immunofluorescent technique (X600).
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FIG. 1. Culture sediment of Mycoplasma gallisepticum stained with immunofluorescent technique (X4500).
DISTRIBUTION OF MYCOPLASMA GALLISEPTICUM
919
moved from semisolid media and stained by immunofluorescent technique exhibited bright fluorescence (Fig. 2). Generally, in smears of macerated tissue suspensions, at least 3 fluorescing coccoid bodies in several microscope fields on duplicate slides were observed (Fig. 3). Organs and tissues found positive for M. gallisepticum included lung, air-sac membranes, kidney, trachea, thoracic-wall muscle, brain and spleen (one case). Tissue suspensions prepared from heart, liver and pooled gastrocnemius, biceps brachii and triceps muscles were negative in all infected turkeys. Tissue suspensions concurrently employed as controls for immunofluorescent staining technique were negative in all cases. M. gallisepticum injection in respiratory organs: Lung tissue suspensions were posi-
tive for M. gallisepticum in all infected turkeys. Air-sac tissue was positive in 19 out of 20 turkeys. In bird No. 12, sacrificed 13 days postinoculation, although exhibiting marked lesions, the organism was found neither by culture nor by immunofluorescent staining of air-sac membranes. Tissue suspensions prepared from macerated trachea were positive in 16 turkeys and negative both by culture and immunofluorescent staining in each of 2 turkeys sacrificed respectively on the 3rd and 11th day after inoculation. M. gallisepticum in nonrespiratory organs: M. galUsepticum was identified in the kidney of 6 turkeys being found in one bird sacrificed respectively on the 7th, 9th, 11th, 15th, 17th and 21st days postinoculation. Thoracic-wall muscle was positive in
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FIG. 3. Immunofluorescent stained smear of macerated lung from turkey infected with Mycoplasma gallisepticum (X7500).
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DEVOLT
4 turkeys including 2 sacrificed on the 7th and 1 each respectively on the 9th and 17th day postinoculation. M. gallisepticum was found also in brain tissue of 4 turkeys sacrificed respectively on the 9th, 15th, 17th, and 21st day after inoculation. In addition, the organism was found in the spleen of one turkey on the 17th day. M. gallisepticum infection thus was demonstrated in organs other than those comprising the respiratory system in 9 (45%) turkeys out of 20. Respiratory lesions, serum titers, cultures oj blood and nonrespiratory organs: The nature and extent of lesions in lungs and air-sacs, serum titers and cultures from blood and nonrespiratory organs are presented in Table 2. M. gallisepticum was found in freshly drawn blood from 5 turkeys including 1 each sacrificed respectively on the 11th, 13th, 15th, 17th and 21st days postinoculation. Considering a titer of 1:80 as positive to the tube-agglutination test, all turkeys examined after the 7th day had positive serum titers with the exception of 1 (No. 8) sacrificed on the 9th day. Positive titers ranged from 1:80 to 1:640 and in general were highest including and between the 11th and 17th days. By the 9th day after inoculation, frequent coughing was observed in the inoculated group. Generally both of the abdominal and thoracic air-sacs exhibited moderate to pronounced lesions after the 5th day. Macroscopic lesions were not observed in the trachea and air passages of the head. Air-sac lesions consisted of thickening of the membranes with purulent to fibrinous exudate and varied from moderate thickening with scant exudate to pronounced thickening with abundant exudate and congestion of one or both lungs. Less frequently, lesions were observed in cervical and intraclavicular airsacs. Nonrespiratory organs failed to exhibit macroscopic lesions.
^H « H *« T ( -*
Comparison oj results obtained by cul-
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r-
+ + +
Infecti Nonre Organ
O
D.
DISTRIBUTION OF MYCOPLASMA GALLISEPTICUM
SXX2 + SX22 - SD2 ri2 =
= —
2V(SX l 2 )(SX 2 *)
S = summation; Xx = result by immunofluorescence; X-2 = result by culture; D = deviation. DISCUSSION Isolation of M. gallisepticum from organs and tissues other than those included in the respiratory system have been reported by other investigators (noted above). In the present report, the relationship between infection in nonrespiratory organs and recovery of the organism from blood (culture) presents an interesting problem (Table 2). In 2 turkeys examined respectively on the 11th and 13th days, M. gallisepticum was isolated from freshly drawn blood but was not demonstrated in nonrespiratory organs while in 3 turkeys examined respectively on the 15th, 17th and 21st day, M. gallisepticum was recovered from blood and also demonstrated in nonrespiratory organs. Especially in the case of brain tissue, but also in the case of other nonrespiratory organs, metastasis by way of the blood stream may have occurred. On the other hand in the case of thoracic-wall muscle, although the sample examined did not include air-sac membranes, dissemina-
tion of infection from adjacent tissues was possible. Contiguous tissue was found, in preliminary experiments, to constitute a probable source of contamination, because M. gallisepticum was found on such organs as heart, kidney, spleen and liver. The organism was removed by dipping the organs momentarily in alcohol. These data, however, in the final analysis, fall short of explaining whether metastasis from respiratory to other organs in all instances proceeded by way of the blood stream or from infected contiguous tissue such as air-sac membranes. In support of the rapid method of diagnosis is the fact that in the present investigation, immunofluorescent tissue staining detected M. gallisepticum in 58/71 (81.7%) of the cases positive by either or both methods of examination. The results, however, strongly indicate the advisability of examining by culture suspected tissues negative to immunofluorescent staining technique since an additional 13 (18.3%) tissue suspensions were found positive solely by culture. SUMMARY AND CONCLUSIONS Twenty 6-week-old White Holland turkeys were inoculated intratracheally with Mycoplasma gallisepticum, strain S6, in air-sac lesion tissue. The birds were confined in wire cages in an isolated room. At various intervals between the 3rd and 30th days postinoculation, two were selected at random and sacrificed for autopsy and removal of selected tissues and organs for bacteriological examination. Macerates of tissues from infected birds were examined both by culture with phenol-red broth supplemented with 2% PPLO serum fraction and by direct examination of macerated tissues under the microscope with immunofluorescent staining. Two hundred tissues were examined including 10 selected organs from each turkey. The organism was demonstrated by culture and/or im-
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ture and immunofluorescent tissue staining: From a total of 200 tissue suspensions examined, 71 were positive by culture and/or immunofluorescent tissue staining. The two methods were in agreement in 48 (67.6%) tissues. In 23 cases in which the two methods were in disagreement, culture was positive and immunofluorescent tissue staining negative in 13/23 (56.5%) while culture was negative and immunofluorescent tissue staining positive in 10/23 (43.5%). The coefficient of correlation was calculated to be 0.823 according to the following formula (Snedecor, 1953).
921
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R. L. PETERS, J. E. FABEE, W. D. KEENUM AND H. M.
propagation of a virus in embryonated chicken eggs causing a chronic respiratory disease of chickens. Am. J. Vet. Res. 4 : 325-332. Fabricant, J., and P. P. Levine, 1962. Experimental production of complicated chronic respiratory disease infection ("air sac" disease). Avian Diseases, 6: 13-23. Fahey, J. E., and J. E. Crawley, 1954. Studies on chronic respiratory disease of chickens. II. Isolation of a virus. Canad. J. Comp. Med. 18: 13-21. Gross, W. B., 1956. Escherichia coli as a complicating factor in chronic respiratory disease of chickens and infectious sinusitis of turkeys. Poultry Sci. 35: 765-771. Grumbles, L. C , E. Phillips, W. A. Boney and J. P. Delaplane, 1953. Cultural and biochemical characteristics of the agent causing infectious sinusitis of turkeys and chronic respiratory disease of chickens. Southwestern Vet. 6: 166-168. Jerstad, A. C , 1964. Isolation of Mycoplasma galllisepticum from fresh eggs and blood. Avian Diseases, 8: 36-39. Kelton, W. H., 1962. Synchronized division of avian pleuropneumonia-like organisms. J. Bact. 83: 948-955. Malizia, W. F., M. F. Barille and D. B. Riggs, 1961. Immunofluorescence of pleuropneumonialike organisms isolated from tissue cell cultures. REFERENCES Nature (London), 191: 190-191. Adler, H. E., R. Yamamoto and J. Berg, 19S7. Markham, F. S., and S. C. Wong, 1952. PleuroStrain differences of pleuropneumonia-like orpneumonia-like organisms in the etiology of ganisms of avian origin. Avian Diseases, 1:19-27. turkey sinusitis and chronic respiratory disease Barille, M. F., W. F. Malizia and D. B. Riggs, of chickens. Poultry Sci. 3 1 : 902-904. 1962. Incidence and detection of pleuropneuMarshall, J. D., W. C. Eveland and C. W. Smith, monia-like organisms in cell cultures by fluores1958. Superiority of fluorescein isothiocyanate cent antibody and cultural procedures. J. Bact. (Riggs) for fluorescent-antibody technique with 84: 130-136. a modification of its application. Proc. Soc. Exp. Clark, H. W., R. C. Fowler and T. McP. Brown, Biol. Med. 98: 898-900. 1961. Preparation of Pleuropneumonia-like or- McKee, G. S., 1965. Poultry Inspection Service, ganisms for microscopic study. J. Bact. 8 1 : U.S.D.A., Washington, D.C. Personal communi500-502. cation. Coons, A. H., H. J. Creech, R. N. Jones and E. Moulton, J. E., and H. E. Adler, 1957. Pathogenesis Berliner, 1942. The demonstration of pneumoof arthritis in chicken embryos caused by a coccal antigen in tissues by the use of fluorespleuropneumonia-like organism. Am. J. Vet. cent antibody. J. Immunol. 45: 157-170. Res. 18: 731-734. Cordy, D. R., and H. E. Adler, 1965. Brain and Nelson, J. B., 1935. Coccobacilliform bodies assomuscle lesions caused by Mycoplasma gallisepticiated with an infectious fowl coryza. Science, cum in turkey poults. Am. J. Vet. Res. 26: 18682: 43. 190. Nelson, J. B., 1936a. Studies on an uncomplicated Corstvet, R. E., and W. W. Sadler, 1964. The coryza of the domestic fowl. V. A coryza of diagnosis of certain avian diseases with the slow onset. J. Exp. Med. 63 : 509-513. fluorescent antibody technique. Poultry Sci. 43: Nelson, J. B., 1936b. Studies on an uncomplicated 1280-1288. coryza of the domestic fowl. VI. CoccobacilliDelaplane, J. P., and H. O. Stuart, 1943. The form bodies in birds infected with the coryza
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munofluorescent tissue staining in 71 tissue macerates including lung, air-sac, trachea, thoracic-wall muscle, brain, kidney and spleen. Heart, liver and pooled gastrocnemius and biceps brachii and triceps muscles were negative in all turkeys. M. gallisepticum was found most frequently in lung and air-sac tissues. In 9 turkeys (45%), the organism was demonstrated in organs other than those included in the respiratory system. In 5 turkeys, isolation by culture was made from freshly drawn blood. By immunofiuorescent tissue staining, M. gallisepticum was demonstrated in 81.6% of the positive tissues. In an additional 18.4% of the tissue suspensions, the organism was isolated by culture alone. Examination of suspected tissue by culture is considered a necessary supplement to direct examination under the microscope with immunofluorescent tissue staining. The coefficient of correlation between the two methods of examination was 0.823.
DEVOLT
D I S T R I B U T I O N OF MYCOPLASMA
Taylor, J. R. E., and J. Fabricant, 1957. Studies on the isolation of the pleuropneumonia-like organism of chronic respiratory disease of fowls. Cornell Vet. 47 : 112-126. Taylor, J. R. E., J. Fabricant and P. P. Levine. 1957. A comparison of four in vitro methods for the isolation of the pleuropneumonia-like organism of chronic respiratory disease from tracheal exudate. Avian Diseases, 1: 101-104. Van Herick, W., and M. D. Eaton, 1945. An unidentified pleuropneumonia-like organism isolated during passages in chick embryos. J. Bact. 50:47-55. Yamamoto, R., H. B. Ortmayer, C. H. Bigland and M. L. Seely, 1965. Isolation of "N" Mycoplasma from different sites of the turkey. Poultry Sci. 44: 732-736. Zander, D. V., 1961. Origin of S« strain of Mycoplasma. Avian Diseases, 5: 154-156.
Selection for Body Weight at Eight Weeks of Age 3. R E A L I Z E D H E R I T A B I L I T I E S O F U N S E L E C T E D G.
TRAITS
I D E T A 1 AND P . B. S I E G E L
Virginia Polytechnic Institute, Blacksburg (Received for publication January 18, 1966) E R I T A B I L I T Y is a statistic which and downward directions and thereby describes a certain population in minimize influences of asymmetry in the terms of observed phenotypic variance response of the divergently selected lines. due to differences between individuals in Falconer (1953) has also shown t h a t a their heredity (Lush, 1948). Various reliable method for measuring additive methods for estimating heritability have gene action in two-way selection experibeen developed and their advantages and ments is b y the regression of the response disadvantages were discussed by Lerner on the cumulative selection differential. (1950, 1958) and Falconer (1960). H a r v e y and Bearden (1962) presented A reliable measure of additive gene ac- procedures for the calculation of expected tion m a y be obtained by dividing the genetic change for each t r a i t in s t a n d a r d difference between the means of two lines deviation units when selection was conselected in opposite directions by the ducted simultanously for two traits. cumulative selection differential. The use Magee (1965) investigated the same of random mating control populations problem associated with phenotypic differenables the refinement of being able to ences between the mean of the parents calculate the heritability in both upward (P) and the average of the population (P) in which they were hatched. He pointed 1 Present address: National Hyogo Poultry out the difference between the selection Breeding Station, Issai-cho, Tatsuno-si Hyogo-ken, differential for a directly selected single Japan.
H
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of slow onset. J. Exp. Med. 63: 515-522. Noel, J. K., H. M. De Volt and J. E. Faber, 1964. Identification of Mycoplasma galllisepticum in lesion tissue by immunofluorescence. Poultry Sci. 43 :145-149. Smibert, R. M., J. E. Faber and H. M. De Volt, 1959a. Studies on "air-sac" infection in poultry. 2. Bacterial flora of the respiratory system of turkeys associated with avian PPLO (pleuropneumonia-like organisms) in artificially induced aerosaccitis. Poultry Sci. 38: 1398-1404. Smibert, R. M., M. Forbes, J. E. Faber, A. R. Gabuten and H. M. De Volt, 1959b. Studies on "air-sac" infection in poultry. 1. Infection of germ-free turkeys with Mycoplasma gallinarum (avian PPLO) from sinal exudate and broth cultures. Poultry Sci. 38: 676-684. Snedecor, G. W., 1946. Statistical Methods (Fourth Edition) p-161.
923
GALLISEPTICUM
Veterinary Science.
913
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A IR-SAC infection (PPLO) in poultry is pneumonia-like organism is the cause of J~\ one of the most troublesome and per- chronic respiratory disease was advanced sistent ailments that prey upon the indus- by Markham and Wong (1952). Several try. This disease was the cause in 1963 and investigators, including Delaplane and 1964 for 38% and 40%, respectively, of Stuart (1943), Fahey and Crawley (1954), the total condemnations of poultry carcas- Gross (1956), Smibert et al. (1959a, b), ses under inspection regulations of the Poul- Fabricant and Levine (1962) have reporttry Inspection Service in the U.S.D.A. ed on several bacterial and viral agents in (McKee, 1965). An accurate and rapid the air-sac disease complex. Smibert et al. method of diagnosis is important to selec- (1959b) produced typical lesions by inocution of wholesome meat as well as for con- lating germ-free turkey poults with M. galtrol and eradication of disease in affected linarum {gallisepticum), an accomplishflocks. ment of special significance. He also reportIn a series of reports, Nelson (1935, ed the change from normally and predomi1936a, b) described the isolation of an orga- nantly gram-positive bacterial flora to prenism which he called "coccobacilliform dominantly gram-negative flora of the respibodies" from a fowl with coryza of slow ratory system following artificial inoculaonset and long duration. Van Herick and tion of conventional turkeys with M. gallEaton (1945), while passing a virus of isepticum. Mycoplasma have been routineatypical pneumonia in chicken embryos, ly isolated from respiratory organs of reported isolation of an organism which chickens and turkeys by Markham and was "pleuropneumonia-like." They con- Wong (1952), Adler et al. (1957), Grumcluded the eggs were the source of contami- bles et al. (1953), Taylor and Fabricant nation. Experimental proof that a pleuro- (1957), Taylor et al. (1957) and Smibert et al. (1959a). Other investigators have reported isolation of PPLO from organs not Condensation of a thesis submitted to the included in the respiratory system. Moulfaculty of the Graduate School, University of ton and Adler (1957) isolated the organism Maryland, as a partial fulfillment of the requirefrom synovial fluid. Yamamoto et al. ments for the degree Master of Science. Scientific Article No. A 1248. Contribution No. 3761 of the (1965) recovered the "N" type of MycoMaryland Agricultural Experiment Station. plasma from semen, vagina and bursa of 1 Graduate Assistant, Department of Veterinary Fabricius. The type strain, S6, was isolatScience and Department of Microbiology. 2 ed from brain tissue of a turkey by Zander Professor of Microbiology, Department of (1961). Cordy and Adler (1965) reported Microbiology. 3 Technician, Department of Veterinary Science. the presence of brain and muscle lesions in 1 Professor of Avian Pathology, Department of turkey poults after intravenous inoculation
914
R. L. PETERS, J. E. FABER, W. D. KEENUM AND H. M.
DEVOLT
Artificial exposure to "air-sac" injection: Artificial exposure to "air-sac" infection in experimental turkeys was accomplished by injecting infected air-sac tissue. The infected air-sac tissue to be used for the experimental group was produced by preliminary injection of a few young poults with a broth culture of M. galUsepticum, strain S6*. After symptoms appeared, air-sac lesion tissue was harvested from freshly killed turkeys. The membranes were macerated in a Teflon grinder and suspended in proteose peptone # 3 (Difco) to make a 20% tissue suspension. A group of twenty, 6-week-old White Holland turkeys were each injected intratracheally with 0.5 ml. of the lesiontissue suspension and confined in wire cages in an isolated room. Bacteriological examination of the inoculum showed that it was heavily impregnated with M. galUsepticum and also contained 100 to 200 other organisms per ml. including Escherichia coli, Micrococcus, Streptococcus and diphtheroid sp.
MATERIALS AND METHODS
Selected organs and tissues from infected turkeys: At intervals of 3, 5, 7, 9, 11, 13, 15, 17, 21 and 30 days postinoculation, two turkeys were selected at random from the inoculated group and sacrificed. Immediately after being killed by breaking the neck, the carcass was immersed in Roccal, 1:1250, to destroy contaminating organisms on the feathers and skin. The skull was then opened to obtain brain tissue and skin carefully removed for the purpose of taking samples of muscle tissue. The body cavity of the carcass was then immediately opened and certain visceral organs removed with aseptic precautions. A list of all organs and tissues employed in the present study follows.
The general plan of investigation was to inoculate a group of young experimental turkeys with M. galUsepticum in air-sac lesion-tissue and to sacrifice birds at various intervals postinoculation to obtain certain tissues and organs for bacteriological examination. Aliquots from all tissue and organ suspensions were used to seed culture media and to prepare direct smears for immunofiuorescent tissue staining. Final identification of M. galUsepticum in both culture and direct smears was by immunofiuorescent staining. The object of the experiment was to determine the extent and frequency with which M. galUsepticum may be disseminated from the respiratory system to other tissues and organs. In addition, a comparison was made between results obtained by isolation in culture and identification in direct smears with immunofiuorescent tissue staining.
(a) Thoracic and abdominal air-sacs. (b) Brain (cerebral hemispheres). * M. galUsepticum, type strain Se, was obtained from H. E. Adler, Department of Avian Medicine, University of California, Davis, California.
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of M. galUsepticum following either intravenous or intramuscular inoculation with Newcastle virus. Jerstad (1964) isolated M. galUsepticum from fresh eggs and blood 4 to 126 days and at 7 days, respectively, after intravenous inoculation. Initial work on the demonstration of antigenic substances by immunofiuorescent tissue staining was carried out by Coons et al. (1942). Marshall et al. (1958) reported on the method of conjugating fluorescein isothiocyanate with whole serum and serum-globulin. Malizia et al. (1961) and Barille et al. (1962) published reports on identification of Mycoplasma in tissue cell culture by immunofluorescence. Corstvet and Sadler (1964) identified M. galUsepticum by staining colony imprints and impression smears from trachea and air-sac tissue with fluorescein-labeled immune serum. Noel et al. (1964) reported the identification of M. galUsepticum in lesion tissue by immunofluorescence.
DISTRIBUTION OF MYCOPLASMA GALLISEPTICUM
915
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isms were harvested from several liters of broth in a continuous-flow, Servall, RC-2, refrigerated centrifuge at 23,000 X g. Sedimented M. gallisepticum organisms were suspended in phosphate-buffered saline (pH 7.4) and washed 3 times by centrifugation. Antigen for the tube-agglutination test was suspended in a diluent of 0.85% saline with 0.3% formalin and adjusted to 76% light transmission (LT) in a Coleman # 9 nepho-colorimeter. For testing rabbit serum, two-fold dilutions were made with the same diluent. For testing turkey serum, Lung and kidney tissues were taken from two-fold dilutions were made with diluent the infected side when air-sac lesions were containing 8.5% saline and 0.3% formalin. unilateral. Kidney, heart, liver and spleen Antigen for labbit injection to produce imwere immersed in 70% alcohol for 60 sec. mune serum was adjusted in physiological to destroy surface contamination from con- saline to 68% LT in the nephelometer. Six, tiguous infected air-sac membranes. These 4- to 5-pound rabbits were given 6 intraorgans were then rinsed S times in sterile peritoneal injections of 5 ml. of the antigen saline to remove alcohol. Blood for culture at intervals of 3 to 4 days. Sixteen days and serological examination was drawn after the last injection, the rabbits were exwith a sterile syringe from a wing vein. The sanguinated and serum separated in the selected organs and tissues were stored in usual manner. Titers of pooled serums used sterile stoppered vials at — 60°C. until used for separation of immune globulin varied for bacteriological examination. The or- from 1:320 to 1:1280. gans, when used for examination in 1 to 6 Preparation of immunofluorescent stain: months, were thawed at room temperature Globulin (immune) was separated from for 2 hr. They were prepared for examina- pooled rabbit serums by precipitating overtion by mincing and macerating in a Teflon night at 4°C. with half-saturated ammongrinder with sterile saline. From 3 to S g. ium sulfate. Precipitate was concentrated of tissue were macerated with 7 ml. saline by centrifugation at 3,000 X g. for 20 min. after which aliquots of the tissue suspen- at 4°C, dissolved in a quantity of distilled sions were used to seed culture media and water equal to the original volume of pooled to prepare smears for direct microscopic serums, and precipitated 3 or more times examination with immunofluorescent stain- until no trace of hemoglobin remained. ing. Similar tissue and organ preparations After final dissolution in distilled water, were prepared from noninfected turkeys to crude globulin was dialyzed at 4°C. against be used as controls with the immu- 3 or more changes of physiological saline nofluorescent tissue staining technique. until the dialysate was free from ammonProduction of antigen and immune ium sulfate as determined by testing aliserum: Antigen for agglutination tests and quots with barium chloride. Total protein production of immune serum in rabbits was content of the globulin was then determined produced by growing M. gallisepticum by the biuret reaction using a Bausch and strain, S6, in phenol-red broth (described Lomb spectronic-20 spectrophotometer at below) but with penicillin omitted. Organ- 540 mjx. and a protein standard (Scientific (c) Biceps brachii, triceps and gastrocnemius muscles (2 g. of each pooled). (d) Heart. (e) Kidney (3 lobes). (f) Lung. (g) Liver (half of left lobe). (h) Spleen. (i) Thoracic-wall muscles (2 g. pooled pectoralis superficialis and external intercostals). (j) Blood (venipuncture).
916
R. L. PETERS, J. E. FABER, W. D. KEENUM AND H. M.
Culturing blood and tissue suspensions for M. gallisepticum: The medium employed for production of antigen and for examination of tissue suspensions from infected turkeys was phenol-red broth (Difco) supplemented with 0.25% dextrose and thallium acetate (1:2,000). After autoclaving, the medium was further supplemented by addition of 2% of PPLO serum-fraction (Difco) and by 1,000 units penicillin per ml. One ml. of tissue suspension was added to each of 4 tubes of broth. Ten ml. of broth were added to a sterile tube containing 1 ml. of freshly drawn blood from infected turkeys. Cultures were incubated at 37°C. and held for at least 14 days. Those undergoing color change (red to orange) were pooled with aseptic precautions and centrifuged at 6,000 X g for 30 min. The resulting sediment was sus-
pended in 1 to 2 ml. of sterile saline and used to prepare slides for examination by immunofluorescent staining. In some instances, broth cultures were plated on semisolid media (without penicillin) to observe colony type. Semisolid media was prepared with heart-infusion agar (Bacto) diluted with heart-infusion broth (Bacto) to adjust agar to 0.75% and supplemented with 1% yeast extract, 1% proteose peptone #Z, and 4% PPLO serum fraction (all Difco) according to the method described by Kelton (1962). Both the broth and semisolid medium were adjusted to pH 7.8. Typical colonies were subjected to further examination by immunofluorescent staining according to the method of Clark et al. (1961). A block of agar was cut from a petri dish and placed, colony side down, on a carefully cleaned glass slide which was then immersed in distilled water preheated to 80° C. The temperature of the water was raised until the agar block dissolved. The slide was then rinsed, air-dried, and examined by immunofluorescent staining. Immunofluorescent staining technique: Three loopsful of culture sediment or tissue suspension were spread on the surface of each of 2 clean glass slides, permitted to air-dry, and fixed with low heat. Slides were then flooded with immunofluorescent stain and held in a moist chamber for 30 min. at room temperature. The slides were rinsed with carbonate-bicarbonate buffer (0.5 M, pH 9.0) and placed in a Coplin jar with excess buffer to permit diffusion of stain. The stained smears were next rinsed and drained of excess fluid by touching the edge to filter paper. The surface of the smear, however, was not allowed to dry. Flazo orange was next applied as a counter stain for 45 to 60 sec. except in the case of brain and kidney tissue when counter staining was limited to 30 sec. because of greater affinity of those tissues for flazo orange. For use as controls in immunofluorescent tissue staining smears were prepared by applying
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Products, Washington, D.C.). Globulin protein was adjusted to a concentration of 1% with saline and carbonate-bicarbonate buffer (O.SM, pH 9.0). Diluted globulin had a pH of 9.0 with a 10% change in volume. The globulin solution was labelled with crystalline fluorescein isothiocyanate (FITC, Baltimore Biol. Lb., Baltimore, Md.) according to the method of Marshall el al. (19S8) with the following modifications. FITC was first dissolved in a portion of carbonate-bicarbonate buffer to facilitate uniform coupling with protein. Fluorescein isothiocyanate was added to globulin solution in a ratio of 1 part dye to 40 parts protein (0.025 mg. FITC/mg. protein) and conjugation continued for 18 hr. at 4°C. The mixture was then passed through a dextran column (Sephadex G 25, Fine, Pharmacia Fine Chem., New York City,) to remove uncombined dye. Nonimmune globulin from normal rabbits was used in the same way to prepare fluorescein conjugate to be used as a control for immunofluorescent tissue staining technique. The stain not used immediately was stored at -60°C.
DEVOLT
DISTRIBUTION
O F MYCOPLASMA
917
GALLISEPTJCVM
TABLE 1.—Results 0/ culture and immunoftuorescent staining on selected tissues from turkeys irtificially injected with Mycoplasma gallisepticum Days post-inoculation 3
|
5
7
9
1
11
Wing-band numbers 1 Tissue Air Sac
3
2
4
Kidney
Thoracic Wall
8
-
-
-
- - + + + - + + - - -
10
9
- + + - -
+ + + + + + - + -
F
C
F
+ + + - + + -
-
-
-
Days post-inoculation 13
IS
21
17
30
Wing-band numbers 11 Tissue Air Sac Brain Kidney Lung Spleen Trachea Thoracic Wall
13
12
c F c + - - - - - - - - + + + + + + + + F
-
15
14
i 16
17
18
19
20
c F c F c F c F c F c + + + + + + + + + + + ~ - + + + + _ ^ + + + + + - + + + + + + + + + + + + + + + + + + + + + - + + — + + + c F c + + - + - - + + - - + + + + F
F
Negative tests are indicated by (—). Positive tests are indicated by ( + ) . 1 F indicates immunofluorescent tissue stain. 2 C indicates culture.
the stain to similar tissue suspensions from organs of normal turkeys and also applying fluorescein-labeled nonimmune globulin to the tissues from infected turkeys. The criteria for identification of M. gallisepticum in tissue smears or in culture sediment was fluorescing coccoid bodies ranging from 0.2 p.. to 0.5 [x. in diameter. Fluorescene was considered to be positive at 2 + between the limits of slight fluorescence at 1 + and strong fluorescence at 4 + . Fluorescent microscopy: Microscopic examination of immunofluorescent stained tissue smears and culture sediment was made with a Carl Zeiss, Model W, microscope equipped with a cardioid darkfield condenser and nonfluorescing objectives. An Osram, HBO 200, high-pressure mercu-
ry vapor burner operating under a constant input of 120 volts was employed as a source of illumination. A three mm., Schott, BG-12 exciter filter was used in combination with a Kodak, G-1S barrier filter. Photographs were made with a Zeiss camera using Kodak, high-speed Ektachrome color films. RESULTS
Results of examinations of selected tissues and organs from artifically infected turkeys by culture and/or immunofluorescent tissue staining of tissue smears are given in Table 1. Culture sediment when stained with immunofluorescent technique resulted in fluorescing masses of M. gallisepticum (Fig. 1) Colonies re-
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- - + + -
Spleen Trachea
7
6
c F C + + + + + + + - - - + - - + + - - - + + + + + + + + + + — + +
Brain
Lung
5
c» F C F C F C F C F C F c + + ~ + + + + ~ + + + + + +
Fi
918
R. L. PETERS, J. E. FABER, W. D. KEENUM AND H. M.
DEVOLT
FIG. 2. Colony of Mycoplasma gallisepticum fixed by "hot water'' method and stained with immunofluorescent technique (X600).
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FIG. 1. Culture sediment of Mycoplasma gallisepticum stained with immunofluorescent technique (X4500).
DISTRIBUTION OF MYCOPLASMA GALLISEPTICUM
919
moved from semisolid media and stained by immunofluorescent technique exhibited bright fluorescence (Fig. 2). Generally, in smears of macerated tissue suspensions, at least 3 fluorescing coccoid bodies in several microscope fields on duplicate slides were observed (Fig. 3). Organs and tissues found positive for M. gallisepticum included lung, air-sac membranes, kidney, trachea, thoracic-wall muscle, brain and spleen (one case). Tissue suspensions prepared from heart, liver and pooled gastrocnemius, biceps brachii and triceps muscles were negative in all infected turkeys. Tissue suspensions concurrently employed as controls for immunofluorescent staining technique were negative in all cases. M. gallisepticum injection in respiratory organs: Lung tissue suspensions were posi-
tive for M. gallisepticum in all infected turkeys. Air-sac tissue was positive in 19 out of 20 turkeys. In bird No. 12, sacrificed 13 days postinoculation, although exhibiting marked lesions, the organism was found neither by culture nor by immunofluorescent staining of air-sac membranes. Tissue suspensions prepared from macerated trachea were positive in 16 turkeys and negative both by culture and immunofluorescent staining in each of 2 turkeys sacrificed respectively on the 3rd and 11th day after inoculation. M. gallisepticum in nonrespiratory organs: M. galUsepticum was identified in the kidney of 6 turkeys being found in one bird sacrificed respectively on the 7th, 9th, 11th, 15th, 17th and 21st days postinoculation. Thoracic-wall muscle was positive in
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FIG. 3. Immunofluorescent stained smear of macerated lung from turkey infected with Mycoplasma gallisepticum (X7500).
920
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2
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KEENUM AND H.
M.
DEVOLT
4 turkeys including 2 sacrificed on the 7th and 1 each respectively on the 9th and 17th day postinoculation. M. gallisepticum was found also in brain tissue of 4 turkeys sacrificed respectively on the 9th, 15th, 17th, and 21st day after inoculation. In addition, the organism was found in the spleen of one turkey on the 17th day. M. gallisepticum infection thus was demonstrated in organs other than those comprising the respiratory system in 9 (45%) turkeys out of 20. Respiratory lesions, serum titers, cultures oj blood and nonrespiratory organs: The nature and extent of lesions in lungs and air-sacs, serum titers and cultures from blood and nonrespiratory organs are presented in Table 2. M. gallisepticum was found in freshly drawn blood from 5 turkeys including 1 each sacrificed respectively on the 11th, 13th, 15th, 17th and 21st days postinoculation. Considering a titer of 1:80 as positive to the tube-agglutination test, all turkeys examined after the 7th day had positive serum titers with the exception of 1 (No. 8) sacrificed on the 9th day. Positive titers ranged from 1:80 to 1:640 and in general were highest including and between the 11th and 17th days. By the 9th day after inoculation, frequent coughing was observed in the inoculated group. Generally both of the abdominal and thoracic air-sacs exhibited moderate to pronounced lesions after the 5th day. Macroscopic lesions were not observed in the trachea and air passages of the head. Air-sac lesions consisted of thickening of the membranes with purulent to fibrinous exudate and varied from moderate thickening with scant exudate to pronounced thickening with abundant exudate and congestion of one or both lungs. Less frequently, lesions were observed in cervical and intraclavicular airsacs. Nonrespiratory organs failed to exhibit macroscopic lesions.
^H « H *« T ( -*
Comparison oj results obtained by cul-
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r-
+ + +
Infecti Nonre Organ
O
D.
DISTRIBUTION OF MYCOPLASMA GALLISEPTICUM
SXX2 + SX22 - SD2 ri2 =
= —
2V(SX l 2 )(SX 2 *)
S = summation; Xx = result by immunofluorescence; X-2 = result by culture; D = deviation. DISCUSSION Isolation of M. gallisepticum from organs and tissues other than those included in the respiratory system have been reported by other investigators (noted above). In the present report, the relationship between infection in nonrespiratory organs and recovery of the organism from blood (culture) presents an interesting problem (Table 2). In 2 turkeys examined respectively on the 11th and 13th days, M. gallisepticum was isolated from freshly drawn blood but was not demonstrated in nonrespiratory organs while in 3 turkeys examined respectively on the 15th, 17th and 21st day, M. gallisepticum was recovered from blood and also demonstrated in nonrespiratory organs. Especially in the case of brain tissue, but also in the case of other nonrespiratory organs, metastasis by way of the blood stream may have occurred. On the other hand in the case of thoracic-wall muscle, although the sample examined did not include air-sac membranes, dissemina-
tion of infection from adjacent tissues was possible. Contiguous tissue was found, in preliminary experiments, to constitute a probable source of contamination, because M. gallisepticum was found on such organs as heart, kidney, spleen and liver. The organism was removed by dipping the organs momentarily in alcohol. These data, however, in the final analysis, fall short of explaining whether metastasis from respiratory to other organs in all instances proceeded by way of the blood stream or from infected contiguous tissue such as air-sac membranes. In support of the rapid method of diagnosis is the fact that in the present investigation, immunofluorescent tissue staining detected M. gallisepticum in 58/71 (81.7%) of the cases positive by either or both methods of examination. The results, however, strongly indicate the advisability of examining by culture suspected tissues negative to immunofluorescent staining technique since an additional 13 (18.3%) tissue suspensions were found positive solely by culture. SUMMARY AND CONCLUSIONS Twenty 6-week-old White Holland turkeys were inoculated intratracheally with Mycoplasma gallisepticum, strain S6, in air-sac lesion tissue. The birds were confined in wire cages in an isolated room. At various intervals between the 3rd and 30th days postinoculation, two were selected at random and sacrificed for autopsy and removal of selected tissues and organs for bacteriological examination. Macerates of tissues from infected birds were examined both by culture with phenol-red broth supplemented with 2% PPLO serum fraction and by direct examination of macerated tissues under the microscope with immunofluorescent staining. Two hundred tissues were examined including 10 selected organs from each turkey. The organism was demonstrated by culture and/or im-
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ture and immunofluorescent tissue staining: From a total of 200 tissue suspensions examined, 71 were positive by culture and/or immunofluorescent tissue staining. The two methods were in agreement in 48 (67.6%) tissues. In 23 cases in which the two methods were in disagreement, culture was positive and immunofluorescent tissue staining negative in 13/23 (56.5%) while culture was negative and immunofluorescent tissue staining positive in 10/23 (43.5%). The coefficient of correlation was calculated to be 0.823 according to the following formula (Snedecor, 1953).
921
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R. L. PETERS, J. E. FABEE, W. D. KEENUM AND H. M.
propagation of a virus in embryonated chicken eggs causing a chronic respiratory disease of chickens. Am. J. Vet. Res. 4 : 325-332. Fabricant, J., and P. P. Levine, 1962. Experimental production of complicated chronic respiratory disease infection ("air sac" disease). Avian Diseases, 6: 13-23. Fahey, J. E., and J. E. Crawley, 1954. Studies on chronic respiratory disease of chickens. II. Isolation of a virus. Canad. J. Comp. Med. 18: 13-21. Gross, W. B., 1956. Escherichia coli as a complicating factor in chronic respiratory disease of chickens and infectious sinusitis of turkeys. Poultry Sci. 35: 765-771. Grumbles, L. C , E. Phillips, W. A. Boney and J. P. Delaplane, 1953. Cultural and biochemical characteristics of the agent causing infectious sinusitis of turkeys and chronic respiratory disease of chickens. Southwestern Vet. 6: 166-168. Jerstad, A. C , 1964. Isolation of Mycoplasma galllisepticum from fresh eggs and blood. Avian Diseases, 8: 36-39. Kelton, W. H., 1962. Synchronized division of avian pleuropneumonia-like organisms. J. Bact. 83: 948-955. Malizia, W. F., M. F. Barille and D. B. Riggs, 1961. Immunofluorescence of pleuropneumonialike organisms isolated from tissue cell cultures. REFERENCES Nature (London), 191: 190-191. Adler, H. E., R. Yamamoto and J. Berg, 19S7. Markham, F. S., and S. C. Wong, 1952. PleuroStrain differences of pleuropneumonia-like orpneumonia-like organisms in the etiology of ganisms of avian origin. Avian Diseases, 1:19-27. turkey sinusitis and chronic respiratory disease Barille, M. F., W. F. Malizia and D. B. Riggs, of chickens. Poultry Sci. 3 1 : 902-904. 1962. Incidence and detection of pleuropneuMarshall, J. D., W. C. Eveland and C. W. Smith, monia-like organisms in cell cultures by fluores1958. Superiority of fluorescein isothiocyanate cent antibody and cultural procedures. J. Bact. (Riggs) for fluorescent-antibody technique with 84: 130-136. a modification of its application. Proc. Soc. Exp. Clark, H. W., R. C. Fowler and T. McP. Brown, Biol. Med. 98: 898-900. 1961. Preparation of Pleuropneumonia-like or- McKee, G. S., 1965. Poultry Inspection Service, ganisms for microscopic study. J. Bact. 8 1 : U.S.D.A., Washington, D.C. Personal communi500-502. cation. Coons, A. H., H. J. Creech, R. N. Jones and E. Moulton, J. E., and H. E. Adler, 1957. Pathogenesis Berliner, 1942. The demonstration of pneumoof arthritis in chicken embryos caused by a coccal antigen in tissues by the use of fluorespleuropneumonia-like organism. Am. J. Vet. cent antibody. J. Immunol. 45: 157-170. Res. 18: 731-734. Cordy, D. R., and H. E. Adler, 1965. Brain and Nelson, J. B., 1935. Coccobacilliform bodies assomuscle lesions caused by Mycoplasma gallisepticiated with an infectious fowl coryza. Science, cum in turkey poults. Am. J. Vet. Res. 26: 18682: 43. 190. Nelson, J. B., 1936a. Studies on an uncomplicated Corstvet, R. E., and W. W. Sadler, 1964. The coryza of the domestic fowl. V. A coryza of diagnosis of certain avian diseases with the slow onset. J. Exp. Med. 63 : 509-513. fluorescent antibody technique. Poultry Sci. 43: Nelson, J. B., 1936b. Studies on an uncomplicated 1280-1288. coryza of the domestic fowl. VI. CoccobacilliDelaplane, J. P., and H. O. Stuart, 1943. The form bodies in birds infected with the coryza
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munofluorescent tissue staining in 71 tissue macerates including lung, air-sac, trachea, thoracic-wall muscle, brain, kidney and spleen. Heart, liver and pooled gastrocnemius and biceps brachii and triceps muscles were negative in all turkeys. M. gallisepticum was found most frequently in lung and air-sac tissues. In 9 turkeys (45%), the organism was demonstrated in organs other than those included in the respiratory system. In 5 turkeys, isolation by culture was made from freshly drawn blood. By immunofiuorescent tissue staining, M. gallisepticum was demonstrated in 81.6% of the positive tissues. In an additional 18.4% of the tissue suspensions, the organism was isolated by culture alone. Examination of suspected tissue by culture is considered a necessary supplement to direct examination under the microscope with immunofluorescent tissue staining. The coefficient of correlation between the two methods of examination was 0.823.
DEVOLT
D I S T R I B U T I O N OF MYCOPLASMA
Taylor, J. R. E., and J. Fabricant, 1957. Studies on the isolation of the pleuropneumonia-like organism of chronic respiratory disease of fowls. Cornell Vet. 47 : 112-126. Taylor, J. R. E., J. Fabricant and P. P. Levine. 1957. A comparison of four in vitro methods for the isolation of the pleuropneumonia-like organism of chronic respiratory disease from tracheal exudate. Avian Diseases, 1: 101-104. Van Herick, W., and M. D. Eaton, 1945. An unidentified pleuropneumonia-like organism isolated during passages in chick embryos. J. Bact. 50:47-55. Yamamoto, R., H. B. Ortmayer, C. H. Bigland and M. L. Seely, 1965. Isolation of "N" Mycoplasma from different sites of the turkey. Poultry Sci. 44: 732-736. Zander, D. V., 1961. Origin of S« strain of Mycoplasma. Avian Diseases, 5: 154-156.
Selection for Body Weight at Eight Weeks of Age 3. R E A L I Z E D H E R I T A B I L I T I E S O F U N S E L E C T E D G.
TRAITS
I D E T A 1 AND P . B. S I E G E L
Virginia Polytechnic Institute, Blacksburg (Received for publication January 18, 1966) E R I T A B I L I T Y is a statistic which and downward directions and thereby describes a certain population in minimize influences of asymmetry in the terms of observed phenotypic variance response of the divergently selected lines. due to differences between individuals in Falconer (1953) has also shown t h a t a their heredity (Lush, 1948). Various reliable method for measuring additive methods for estimating heritability have gene action in two-way selection experibeen developed and their advantages and ments is b y the regression of the response disadvantages were discussed by Lerner on the cumulative selection differential. (1950, 1958) and Falconer (1960). H a r v e y and Bearden (1962) presented A reliable measure of additive gene ac- procedures for the calculation of expected tion m a y be obtained by dividing the genetic change for each t r a i t in s t a n d a r d difference between the means of two lines deviation units when selection was conselected in opposite directions by the ducted simultanously for two traits. cumulative selection differential. The use Magee (1965) investigated the same of random mating control populations problem associated with phenotypic differenables the refinement of being able to ences between the mean of the parents calculate the heritability in both upward (P) and the average of the population (P) in which they were hatched. He pointed 1 Present address: National Hyogo Poultry out the difference between the selection Breeding Station, Issai-cho, Tatsuno-si Hyogo-ken, differential for a directly selected single Japan.
H
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of slow onset. J. Exp. Med. 63: 515-522. Noel, J. K., H. M. De Volt and J. E. Faber, 1964. Identification of Mycoplasma galllisepticum in lesion tissue by immunofluorescence. Poultry Sci. 43 :145-149. Smibert, R. M., J. E. Faber and H. M. De Volt, 1959a. Studies on "air-sac" infection in poultry. 2. Bacterial flora of the respiratory system of turkeys associated with avian PPLO (pleuropneumonia-like organisms) in artificially induced aerosaccitis. Poultry Sci. 38: 1398-1404. Smibert, R. M., M. Forbes, J. E. Faber, A. R. Gabuten and H. M. De Volt, 1959b. Studies on "air-sac" infection in poultry. 1. Infection of germ-free turkeys with Mycoplasma gallinarum (avian PPLO) from sinal exudate and broth cultures. Poultry Sci. 38: 676-684. Snedecor, G. W., 1946. Statistical Methods (Fourth Edition) p-161.
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