A Comparative Pathogenesis of Two Marek's Disease Virus Isolates JOSEPH J. GIAMBRONE,1 ROBERT J. ECKROADE2 and JOHN K. ROSENBERGER Department of Animal Science and Agricultural University of Delaware, Newark, Delaware 19711
Biochemistry,
(Received for publication December 16, 1977)
INTRODUCTION T h e in vivo sites of Marek's disease virus (MDV) replication are i m p o r t a n t in t h e p a t h o genesis of M D . T h e J M isolate of M D V has been f o u n d associated with virtually every organ of t h e chicken using i m m u n o t l u o r e s c e n t ( I F ) and agar gel precipitin (AGP) techniques (Adldinger and Calnek, 1 9 7 3 ; Calnek and Hitchner, 1 9 6 9 ; Calnek et al, 1 9 7 0 ; Purchase, 1 9 7 0 ; Spencer, 1 9 6 9 ; Spencer and Calnek, 1 9 7 0 ) . Research of this n a t u r e using t h e GA isolate has n o t been rep o r t e d . Since t h e G A isolate is c o m m o n l y used in m a n y laboratories, its pathogenesis, if different from isolate JM, should be u n d e r s t o o d . Presently, only Nazerian ( 1 9 7 1 ) has studied t h e in vivo sites of replication of t h e GA isolate. Using t h e electron microscope, M D V was observed in t h e kidney, spleen, gonad, feather follicles, and bursa of Fabricius of GA infected chickens. T h e objectives of this s t u d y were t o describe t h e early incidence, sequential spread, and character of t h e G A isolate and t o c o m p a r e its pathogenesis with t h e JM isolate. MATERIALS AND METHODS Experimental Animals. Three g r o u p s of t w e n t y , one-day-old specific p a t h o g e n free (SPF) Leghorn chicks were housed in separate
1 Present Address: Poultry Science Department, Auburn University, Auburn, Alabama 36830. 2 Present Address: Newbolten Center, University of Pennsylvania, Kennett Square, Pennsylvania.
1978 Poultry Sci 57:897-906
isolation r o o m s . Also k e p t in these facilities were t h r e e , four-month-old SPF roosters and s e v e r a l three-week-old SPF (MDV) d o n o r chickens. F o u r m a t u r e white domestic rabbits, o b t a i n e d from t h e West Jersey Biological Farms, Wenonah, N e w Jersey, were held t o g e t h e r in a separate isolated facility. Inoculation Procedures. T w o g r o u p s of t w e n t y , one-day-old chicks were injected intraabdominally with .2 ml per bird of a heparinized w h o l e b l o o d p o o l , o b t a i n e d from several J M - ( S e v o i a n a n d Chamberlain, 1 9 6 4 ) or GA-(Eidson a n d S c h m i t t l e , 1 9 6 8 ) inoculated t h r e e - w e e k - o l d S P F d o n o r birds displaying clinical signs of M D . Prior t o this s t u d y , each isolate had been serially p r o p a g a t e d in SPF chickens. T h e third group of one-day-old chicks were u n i n o c u l a t e d controls. Antiserum. T h r e e roosters and t h r e e rabbits were i m m u n i z e d t o t h e G A isolate of M D V , and serum was collected o n e week after t h e final injection. Each r o o s t e r was i n o c u l a t e d intraabdominally a t o n e d a y of age and again a t o n e m o n t h of age with .25 ml of a w h o l e b l o o d pool from several M D V infected chickens. Each rabbit was injected t h r e e times intramuscularly a t weekly intervals with a m i x t u r e containing 1 ml of feather follicle epithelium ( F F E ) e x t r a c t and 1 ml of F r e u n d s c o m p l e t e adjuvant. Following these inoculations, t w o intravenous injections were administered with 1 ml of F F E e x t r a c t o n l y . R a b b i t s were bled a n d sera collected o n e w e e k after t h e final injection. T h e F F E e x t r a c t was p r e p a r e d b y scraping t h e epithelium from feathers of chickens infected
897
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ABSTRACT The pathogenesis of the JM and GA isolates of Marek's disease virus (MDV) infection was compared by studying the sequential tissue distribution of MDV associated antigens with gross and microscopic lesions in infected chickens. With both MDV isolates the initial sites of viral replication were the lymphoid organs, skin, and feathers. Both the GA and JM isolates were viscerotropic as demonstrated by the presence of MDV antigens with gross and microscopic changes occurring in several visceral organs. The JM isolate was more neurotropic and had a greater affinity for the gonads than the GA isolates as evidenced by an early detection of MDV antigens and severe pathology in the peripheral nerves and gonads.
898
GIAMBRONE ET AL.
Indirect FA Test. Frozen tissues were sectioned at 6 (1, fixed in acetone, washed in PBS, and stained with either known positive or negative MDV chicken serum for thirty minutes as stated by Calnek and Hitchner (1969), except that the indirect test required an additional staining with FITCH serum. Sections were examined with a fluorescent microscope containing a halogen light source (Atlantic Instruments, Inc., Philadelphia, Pennsylvania). Pathological Observations. All tissues were examined for gross and microscopic lesions characteristic of MD according to Payne and Biggs (1967). Each suspension was fixed in 10% buffered formalin, sectioned at 6 ju and stained with H & E. The grading of microscopic changes was based on the presence of lymphoid cells, with the exception of the lymphoid
organs. The bursa of Fabricius was examined for atrophied follicles and cystic degenerations, while the spleen and thymus were examined for reticulum cell hyperplasia. Lesions were scored at grade I (+), II (++), and III (+++). Grade I was characterized by perivascular accumulations of small numbers of lymphocytes, grade II by a more severe perivascular lymphocytic accumulation that often formed focal aggregates, and grade III by massive lymphocytic aggregates throughout the tissue. Experimental Deisgn. The collection of tissues for all AGP tests, FA techniques, and tissue observations was identical. One uninoculated and two infected birds were sacrificed and tissues collected at three-day intervals until the twenty-first day postinoculation (PI). Tissues were then harvested at five-day intervals until the fortieth day PI. The following tissues were c o l l e c t e d : sciatic nerve, brachial plexus, thymus, spleen, bursa of Fabricius, duodenum, heart, trachea, lung, gonads, skin, and feathers. A section of each tissue, except the feathers, was fixed in formalin and the remaining portion frozen in petri dishes at —20 C for later use. All tissues were first screened for AGP antigen by the AGP test. Any sample not producing a precipitin line was examined by radial immunodiffusion. All negative tissues were then tested by immunoelectrophoresis, the more sensitive of the three methods. In addition, a section of each tissue, except the feathers, was processed for the FA test.
RESULTS Viral infection of selected tissues was based upon detection of MDV associated antigens using AGP and FA techniques. With either MDV isolate specific fluorescence occurred in two patterns: 1) In lymphoid organs granular nuclear and ring-shaped cytoplasmic fluorescence appeared in round lymphoid cells; 2) In non-lymphoid organs structural detail was less distinct and diffuse fluorescence filled the entire cell. Initially, MDV specific fluorescence in lymphoid tissues was localized in small groups of cells in the medullary portion of the follicles, while in more advanced cases, both the size of fluorescing granules and number of cells fluorescing increased, and occasionally cells of the follicular cortex fluoresced. In non-lymphoid tissues, IF antigens occurred in the epithelium lining the feather follicles of the skin, glandular
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with MDV. This material was homogenized and prepared as a 10% w/v extract in cold phosphate buffered (PBS) saline. Control serum was collected from uninoculated chickens and rabbits raised in isolation. Rabbit serum for immunoelectrophoresis was adsorbed with acetone-dried liver powder by the method of Lennette and Schmidt (1964). Rabbit antichicken gammaglobulin conjugated with fluorescein isothiocyanate (FITCH), obtained from Grand Island Biological Co., Inc., Grand Island, New York, was used in the indirect fluorescent antibody (FA) test. Immunodiffusion. All tissues except the feathers were prepared as 25% w/v extracts in PBS according to Calnek and Hitchner (1969). Five feathers per bird were collected and feather tips placed in agar according to Giambrone and Mathews (1973). The petri plates (60 X 15 mm) containing 10 ml of NaKPC^ buffer at 7.2 pH with 2% NaCl and .85% Noble agar were prepared by the method of Chubb and Churchill (1968) for all AGP tests and for radial immunodiffusion by Mancini (1965). All tissues were tested against known positive and negative MDV chicken serum. For immunoelectrophoresis, each tissue extract was electrophoresed using the LKB 68000A Immunoelectrophoresis Equipment (LKB Produkter AB, Stockholm Sweden) at 250 mv for 1 hr on microscope slides containing barbital buffer in 1% agar gel at 8.6 pH. After electrophoresis, either positive or negative MDV rabbit serum was added to each trough. All diffusion plates and slides were incubated at 37 C in a humidified chamber and examined for precipitin bands at 24 and 48 hr.
PATHOGENESIS OF MDV ISOLATES
Similar microscopic lesions occurred with both MDV isolates during the first three weeks PI (Table 1, 2, and 3). These changes were characterized by a perivascular accumulation of small numbers of lymphoid cells in the portal areas of the liver, submucosa of the proventriculus, bronchi and bronchioles of the lung, interstitial areas of the medullary portion of the kidneys, epi and myocardium, and epithelium lining the feather follicles. Also evident at this time in the bursa was a slight atrophy of the medullary portion of the follicles with cystic degenerations apparent. In the spleen and thymus, lymphoid depletions with concomitant r e t i c u l u m cell hyperplasia occurred. The atrophy in the bursa and lymphoid depletion in the spleen and thymus became more severe with time PI and involved both the medulla and cortex. Durng the forth and fifth weeks PI, the severity and extent of lymphocytic accumula-
tions increased in the liver, kidney, heart, and lung of both GA and JM infected birds. By the sixth week PI, the peripheral nerves and gonads of GA infected birds were severely affected. These severe lesions appeared as large lymphoid aggregates which displaced and compressed normal parenchymal cells, frequently resulting in gross changes and clinical signs. The major cellular composition of all lymphoid aggregates consisted of small, medium, and large lymphocytes with mediumsized predominating. Large immature lymphoblasts were more frequent in lesions of GA infected birds, while mitotic figures and MD cells were more prominent in JM lesions. MD cells and evidence of mitosis were infrequently seen with the GA isolate. Small compact encapsulated lymphoid foci were occasionally observed in the liver, lung, proventriculus, and duodenum of uninoculated control birds. These foci were also comprised of small, medium, and large lymphocytes. Gross lesions and clinical signs were absent from all non-infected birds. Clinical signs in GA infected birds were depression with ruffling of feathers, while a stilted gait and unilateral paralysis of the extremities occurred in JM inoculated chicks. With both MDV isolates, gross lesions appeared as small grayish white foci in the visceral organs, grayish solidification of the lung with unilateral enlargement, and discoloration of the peripheral nerves and gonads. By the sixth week PI, nodular tumors appeared in many of the visceral organs of GA infected birds, while bilateral nerve and gonad involvement occurred in JM challenged birds only. The major differences in the pathogenesis of the GA and JM isolates occurred in the peripheral nerves and gonads. The JM isolate was more neurotropic and had a greater affinity for the gonads. The JM isolate induced IF antigen ten days earlier and microscopic lesions seventeen days earlier in the peripheral nerves and gonads than the GA isolate. Also, only JM infected birds developed a stilted gait, crippling, and bilateral enlargement of the nerves and gonads. DISCUSSION
The early incidence and sequential distribution of MDV was similar for both the GA and JM isolates. With both isolates, the initial sites of MDV replication were the lymphoid organs,
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and mucosal epithelium of the proventriculus, bronchi lumen and alveolar parenchyma of the lung, interstitial areas of the gonads, epithelium lining both the trachea and kidney tubules, m y o c a r d i u m and epicardium between the peripheral nerve fibers, and surrounding the blood vessels of the liver. In the AGP tests, all tissue homogenates from GA or JM infected birds produced a reaction of identity with each organ extract, indicating that all AGP tissue antigens were serologically identical. Each feather yielded two precipitin lines. Corresponding tissue extracts from uninfected chickens did not produce precipitation. In radial immunodiffusion, a single circular precipitin line formed around each antigen well, and with Immunoelectrophoresis, one precipitin arc appeared a centimeter toward the anode side of each antigen well. With both MDV isolates the initial sites of viral replication were the bursa of Fabricius, thymus, and skin (IF antigen at nine days) and feathers (AGP antigen at twelve days) (Table 1 and 3). These tissues as well as the other lymphoid organs, spleen and cecal tonsil (IF antigen at twelve days), were constant sites for MDV replication with both isolates. During the forth and fifth weeks PI, both IF and AGP antigens occurred sporadically in the proventirculus, kidney, and liver of both GA and JM infected birds and in the gonads and peripheral nerves of JM infected birds. By the sixth week, the peripheral nerves and gonads of GA challenged birds displayed MDV antigens.
899
RE
RE
RE
RE(1)
Path AGP IF
Path AGP IF
Path AGP IF
Path AGP IF
18
21
25
30
RE(1) R
x
RE
RE
Path AGP IF
15
RE
RE I R
RE
x
x
RE
x
Thymus
RE
Spleen
Path AGP IF
Sciatic nerve
12
Brachial plexus
RE
Type of exam
Path AGP IF
Days PI
GA isolate
AF, C
X
AF, C R
AF
AF, C
AF, C
Bursa
Cecal tonsil
++(1)
X
++
+
Brachial plexus
++(1)
X
++
+
Sciatic nerve
RE A
X
RE
RE R
X
X
RE
RE
RE
RE
x
RE
+
Thymus
RE
RE
RE
RE
RE
Spleen
JM isolate
TABLE 1.—Sequential incidence of MDV antigens and severity of lesions in the tissues of chicks inoculated at one day of age with GA or JM isolate ofMDVa
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AF, C R
X
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AF, C
AF, C
AF, C
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Cecal tonsil
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PATHOGENESIS OF MDV ISOLATES
skin, and feathers. Using a JM isolate, Adldin-
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ger and Calnek (1973) and Purchase (1970) have found IF antigen as early as the first week PI in these tissues. Any differences in the chronological occurrence of viral antigens can result from variation in the genetic line, age, and sex of the bird; dose of viral inoculum (Adldinger and Calnek, 1973; Calnek and Hitchner, 1969; Calnek et al, 1970); or specificity of the conjugated antiserum. The early presence of MDV specific fluorescence in round cells of the lymphoid organs and lymphocytic infiltrations of the peripheral nerves observed with both MDV isolates indicates a productive nature of the infection and that these cells can spread MDV. Thus, it appears evident that the lymphoid organs are important to the early pathogenesis of both the GA and JM isolates. Although MDV dissemination probably resulted from these infected lymphoid cells, the appearance of MDV specific fluorescence
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non-
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lymphoid tissues of both GA and JM infected birds indicates that the infection actively involves those organs. Even though it was not possible to establish that IF and AGP antigens were similar, both antigens appear MDV induced since they were absent from control birds. MDV antigens produced by both the GA and JM isolates appear indistinguishable. Both the pattern of fluorescence and AGP antigens induced by either MDV isolate were serologically identical. The early microscopic changes observed with both the GA or JM isolates were identical and similar to those previously described (Eidson and Schmittle, 1968, Sevoian and Chamberlain, 1964). Initially, MD appeared as an accumulation of lymphoid cells, which were first perivascular, but later increased in number until the tissue was largely replaced by these cells. The presence of atrophied follicles and cystic degenerations in the bursa of Fabricius has also been previously reported for both the GA (Fletcher et al, 1972) and JM (Jakowski et al, 1969) isolates. Lymphoid depletion with concomitant reticulum cell hyperplasia in the spleen and thymus has been previously reported -for the JM isolate only . .(Sevoian . . . . and Chamber-
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Although the early tissue distribution of microscopic lesions were similar for both MDV isolates, differences between the GA and JM isolates were noted in the cellular composition of microscopic changes. These variations were
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Path AGP IF
Path AGP IF
Path AGP IF
Path AGP IF
Path AGP IF
15
18
21
25
Path AGP IF
Path AGP IF
Type of exam
12
PI
++(1)
Liver
Kidney
++ I
Proventriculus
GA isolate Duodenum Heart Liver
Kidney
Proventriculus
JM isolate
TABLE 2.—Sequential incidence of MDV antigens and severity of lesions in the tissues of chicks inoculated at one day of age with GA or JM isolate of MDV
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Heart
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PATHOGENESIS OF MDV ISOLATES
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similar to those described in which JM lesions frequnetly contained mitotic figures and MD cells (Nazerian, 1971; Sevoian and Chamberlain, 1964) which were infrequent in GA l e s i o n s , a l t h o u g h immature lymphoblasts (Nazerian, 1971) and lymphoreticular cells (Eidson and Schmittle, 1968) were prominent. The significance of this observation is unknown, but evidence of mitosis and abnormal cells in JM lesions indicates neoplasia, while their absence, together with frequent immature cells in GA lesions, indicates an inflammatory response. A problem arising in this and other studies (Payne and Biggs, 1967) was distinguishing between histopathological changes induced by MDV and non-specific lymphoid foci in several visceral organs or control birds. The presence of foci in apparently normal birds is well documented (Jordan, 1936; Lucas, 1949), and many researchers believe this to be a normal part of the lymphocytic system of the chicken. Nonspecific foci were usually small, compact, and encapsulated. The observation that the JM isolate is more neurotropic than the GA isolate agrees with other studies (Nazarian, 1971; Sevoian and Chamberlain, 1964), Characteristically, birds infected with the JM isolate develop predominately nerve lesions; however, some visceral lesions are present, especially in the gonads, while with the GA isolate, visceral lesions predominate with minimal nerve lesions occasionally evident (Nazerian, 1971). In this study, the GA isolate was slightly more viserotropic, inducing MDV antigens nine days earlier in the liver and four days earlier in the duodenum and lungs than the JM isolate. Also, only GA infected birds developed nodular tumors in the liver, spleen, and heart. However, variations of a few days in the appearance of MDV antigens were not highly significant and could result from differences in the initial titer of the GA and JM whole blood inoculum. With both MDV isolates, two different types of virus host cell interactions occurred in vivo. The tissues undergoing degeneration (thymus, bursa, and feather follicles) were a constant source of MDV antigens, while antigens occurred sporadically in tissues with lymphoid accumulations. It seems that viral replication results in degenerative changes in some tissues, while in others lymphocytic accumulations are not dependent on MDV replication. An incorporated viral genome found in other DNA
A
+
Path AGP IF
25
+ A x
x
A
+
Path AGP IF
21 A x
+ A x
Path AGP IF
Skin
18
Gonads
+ A x
Lung
Path AGP IF
Trachea
15
Feather tip
Path AGP IF
Skin
12
Gonads
Path AGP IF
Lung
9
Trachea
Path AGP IF
Type of exam
JM isolate
6
Days PI
GA isolate
TABLE 3.—Sequential incidence of MDV antigens and severity of lesions in the tissues of chicks inoculated at one day of age with GA or JM isolate of MDV
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PATHOGENESIS OF MDV ISOLATES
oncogenic viruses could induce l y m p h o p r o liferation w i t h o u t viral replication.
REFERENCES
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Adldinger, H. K., and B. W. Calnek, 1973. Pathogenesis of Marek's disease: Early distribution of virus and viral antigens in infected chickens. J. Nat. Cancer Inst. 50:1287-1298. Calnek, B. W., and S. B. Hi tenner, 1969. Localization of viral antigen in chickens infected with Marek's disease h e r p e s v i r u s . J. Nat. Cancer Inst. 43:935-949. Calnek, B. W., T. Ubertini, and H. K. Adldinger, 1970. Viral antigen virus particles, and infectivity of tissues from chickens with Marek's disease. J. Natl. Cancer Inst. 45:341—351. Chubb, R. C , and A. E. Churchill, 1968. Precipitating antibodies associated with Marek's disease. Vet. Rec. 8 3 : 4 - 7 . Eidson, C. S., and S. C. Schmittle, 1968. Studies on acute Marek's disease. I. Characteristics of isolate GA in chickens. Avian Dis. 12:467—475. Fletcher, O. J., C. S. Eidson, and S. H. Kleven, 1972. Bursal lesions in chickens inoculated with Marek's disease vaccines. Avian Dis. 16:153—162. Giambrone, J. J., and C. T. Mathews, 1973. The feather tip agar gel precipitin test as a diagnostic tool for Marek's disease. 45th Northeastern Conf. on Avian Dis. Morgantown, WV. Jakowski, K. M., T. W. Frederickson, R. E. Luginbuhl, and C. F. Helmboldt, 1969. Early changes in bursa of Fabricius from Marek's disease. Avian Dis. 13:215-222. Jordan, H. E., 1936. The relation of lymphoid tissue to the process of blood production in avian bone marrow. Amer. J. Anat. 59:249—296. Lennette, E. and N. J. Schmidt, 1964. Page 180 in Diagnostic procedures for viral and rickettsial diseases. Amer. Publ. Health Ass., Inc., New York. Lucas, A. M., 1949. Lymphoid tissue and its relation t o so-called normal lymphoid foci and to lymphomatosis. 1. Qualitative study of lymphoid areas in the pancreas of chickens. Amer. Path. 25:1197-1213. Mancini, G. A., 1965. Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry 2:235—254. Nazerian, K., 1971. Further studies on the replication of Marek's disease virus in the chicken and in cell culture. J. Nat. Cancer Inst. 4 7 : 2 0 7 - 2 1 7 ; Payne, L. N. and P. M. Biggs, 1967. Studies on Marek's disease. II. Pathogenesis. J.Nat. Cancer Inst. 39:281-302. Purchase, H. G., and P. M. Biggs, 1967. Characterization of five isolates of Marek's disease. Detection of antibody. Am. J. Vet. Res. 31:117-123. Purchase, H. G., 1970. Virus specific immunofluorescent and precipitin antigens and cell-free virus in tissues of birds infected with Marek's disease. Cancer Res. 30:1898-1908. Sevoian, M., and D. M. Chamberlain, 1964. Avian lymphomatosis. IV. Pathogenesis. Avian Dis. 8:281-310.
906
GIAMBRONE ET AL.
Spencer, J. L., 1969. Cultivation of Marek's disease virus in cell culture and application of immunofluorescence for virus detection. Ph.D. thesis, Cornell University.
Spencer, J. L., and B. W. Calnek, 1970. Marek's disease. Application of immunofluorescence for detection of antigen and antibody. Am. J. Vet. Res. 31:345-358.
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Biochemistry,
(Received for publication December 16, 1977)
INTRODUCTION T h e in vivo sites of Marek's disease virus (MDV) replication are i m p o r t a n t in t h e p a t h o genesis of M D . T h e J M isolate of M D V has been f o u n d associated with virtually every organ of t h e chicken using i m m u n o t l u o r e s c e n t ( I F ) and agar gel precipitin (AGP) techniques (Adldinger and Calnek, 1 9 7 3 ; Calnek and Hitchner, 1 9 6 9 ; Calnek et al, 1 9 7 0 ; Purchase, 1 9 7 0 ; Spencer, 1 9 6 9 ; Spencer and Calnek, 1 9 7 0 ) . Research of this n a t u r e using t h e GA isolate has n o t been rep o r t e d . Since t h e G A isolate is c o m m o n l y used in m a n y laboratories, its pathogenesis, if different from isolate JM, should be u n d e r s t o o d . Presently, only Nazerian ( 1 9 7 1 ) has studied t h e in vivo sites of replication of t h e GA isolate. Using t h e electron microscope, M D V was observed in t h e kidney, spleen, gonad, feather follicles, and bursa of Fabricius of GA infected chickens. T h e objectives of this s t u d y were t o describe t h e early incidence, sequential spread, and character of t h e G A isolate and t o c o m p a r e its pathogenesis with t h e JM isolate. MATERIALS AND METHODS Experimental Animals. Three g r o u p s of t w e n t y , one-day-old specific p a t h o g e n free (SPF) Leghorn chicks were housed in separate
1 Present Address: Poultry Science Department, Auburn University, Auburn, Alabama 36830. 2 Present Address: Newbolten Center, University of Pennsylvania, Kennett Square, Pennsylvania.
1978 Poultry Sci 57:897-906
isolation r o o m s . Also k e p t in these facilities were t h r e e , four-month-old SPF roosters and s e v e r a l three-week-old SPF (MDV) d o n o r chickens. F o u r m a t u r e white domestic rabbits, o b t a i n e d from t h e West Jersey Biological Farms, Wenonah, N e w Jersey, were held t o g e t h e r in a separate isolated facility. Inoculation Procedures. T w o g r o u p s of t w e n t y , one-day-old chicks were injected intraabdominally with .2 ml per bird of a heparinized w h o l e b l o o d p o o l , o b t a i n e d from several J M - ( S e v o i a n a n d Chamberlain, 1 9 6 4 ) or GA-(Eidson a n d S c h m i t t l e , 1 9 6 8 ) inoculated t h r e e - w e e k - o l d S P F d o n o r birds displaying clinical signs of M D . Prior t o this s t u d y , each isolate had been serially p r o p a g a t e d in SPF chickens. T h e third group of one-day-old chicks were u n i n o c u l a t e d controls. Antiserum. T h r e e roosters and t h r e e rabbits were i m m u n i z e d t o t h e G A isolate of M D V , and serum was collected o n e week after t h e final injection. Each r o o s t e r was i n o c u l a t e d intraabdominally a t o n e d a y of age and again a t o n e m o n t h of age with .25 ml of a w h o l e b l o o d pool from several M D V infected chickens. Each rabbit was injected t h r e e times intramuscularly a t weekly intervals with a m i x t u r e containing 1 ml of feather follicle epithelium ( F F E ) e x t r a c t and 1 ml of F r e u n d s c o m p l e t e adjuvant. Following these inoculations, t w o intravenous injections were administered with 1 ml of F F E e x t r a c t o n l y . R a b b i t s were bled a n d sera collected o n e w e e k after t h e final injection. T h e F F E e x t r a c t was p r e p a r e d b y scraping t h e epithelium from feathers of chickens infected
897
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ABSTRACT The pathogenesis of the JM and GA isolates of Marek's disease virus (MDV) infection was compared by studying the sequential tissue distribution of MDV associated antigens with gross and microscopic lesions in infected chickens. With both MDV isolates the initial sites of viral replication were the lymphoid organs, skin, and feathers. Both the GA and JM isolates were viscerotropic as demonstrated by the presence of MDV antigens with gross and microscopic changes occurring in several visceral organs. The JM isolate was more neurotropic and had a greater affinity for the gonads than the GA isolates as evidenced by an early detection of MDV antigens and severe pathology in the peripheral nerves and gonads.
898
GIAMBRONE ET AL.
Indirect FA Test. Frozen tissues were sectioned at 6 (1, fixed in acetone, washed in PBS, and stained with either known positive or negative MDV chicken serum for thirty minutes as stated by Calnek and Hitchner (1969), except that the indirect test required an additional staining with FITCH serum. Sections were examined with a fluorescent microscope containing a halogen light source (Atlantic Instruments, Inc., Philadelphia, Pennsylvania). Pathological Observations. All tissues were examined for gross and microscopic lesions characteristic of MD according to Payne and Biggs (1967). Each suspension was fixed in 10% buffered formalin, sectioned at 6 ju and stained with H & E. The grading of microscopic changes was based on the presence of lymphoid cells, with the exception of the lymphoid
organs. The bursa of Fabricius was examined for atrophied follicles and cystic degenerations, while the spleen and thymus were examined for reticulum cell hyperplasia. Lesions were scored at grade I (+), II (++), and III (+++). Grade I was characterized by perivascular accumulations of small numbers of lymphocytes, grade II by a more severe perivascular lymphocytic accumulation that often formed focal aggregates, and grade III by massive lymphocytic aggregates throughout the tissue. Experimental Deisgn. The collection of tissues for all AGP tests, FA techniques, and tissue observations was identical. One uninoculated and two infected birds were sacrificed and tissues collected at three-day intervals until the twenty-first day postinoculation (PI). Tissues were then harvested at five-day intervals until the fortieth day PI. The following tissues were c o l l e c t e d : sciatic nerve, brachial plexus, thymus, spleen, bursa of Fabricius, duodenum, heart, trachea, lung, gonads, skin, and feathers. A section of each tissue, except the feathers, was fixed in formalin and the remaining portion frozen in petri dishes at —20 C for later use. All tissues were first screened for AGP antigen by the AGP test. Any sample not producing a precipitin line was examined by radial immunodiffusion. All negative tissues were then tested by immunoelectrophoresis, the more sensitive of the three methods. In addition, a section of each tissue, except the feathers, was processed for the FA test.
RESULTS Viral infection of selected tissues was based upon detection of MDV associated antigens using AGP and FA techniques. With either MDV isolate specific fluorescence occurred in two patterns: 1) In lymphoid organs granular nuclear and ring-shaped cytoplasmic fluorescence appeared in round lymphoid cells; 2) In non-lymphoid organs structural detail was less distinct and diffuse fluorescence filled the entire cell. Initially, MDV specific fluorescence in lymphoid tissues was localized in small groups of cells in the medullary portion of the follicles, while in more advanced cases, both the size of fluorescing granules and number of cells fluorescing increased, and occasionally cells of the follicular cortex fluoresced. In non-lymphoid tissues, IF antigens occurred in the epithelium lining the feather follicles of the skin, glandular
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with MDV. This material was homogenized and prepared as a 10% w/v extract in cold phosphate buffered (PBS) saline. Control serum was collected from uninoculated chickens and rabbits raised in isolation. Rabbit serum for immunoelectrophoresis was adsorbed with acetone-dried liver powder by the method of Lennette and Schmidt (1964). Rabbit antichicken gammaglobulin conjugated with fluorescein isothiocyanate (FITCH), obtained from Grand Island Biological Co., Inc., Grand Island, New York, was used in the indirect fluorescent antibody (FA) test. Immunodiffusion. All tissues except the feathers were prepared as 25% w/v extracts in PBS according to Calnek and Hitchner (1969). Five feathers per bird were collected and feather tips placed in agar according to Giambrone and Mathews (1973). The petri plates (60 X 15 mm) containing 10 ml of NaKPC^ buffer at 7.2 pH with 2% NaCl and .85% Noble agar were prepared by the method of Chubb and Churchill (1968) for all AGP tests and for radial immunodiffusion by Mancini (1965). All tissues were tested against known positive and negative MDV chicken serum. For immunoelectrophoresis, each tissue extract was electrophoresed using the LKB 68000A Immunoelectrophoresis Equipment (LKB Produkter AB, Stockholm Sweden) at 250 mv for 1 hr on microscope slides containing barbital buffer in 1% agar gel at 8.6 pH. After electrophoresis, either positive or negative MDV rabbit serum was added to each trough. All diffusion plates and slides were incubated at 37 C in a humidified chamber and examined for precipitin bands at 24 and 48 hr.
PATHOGENESIS OF MDV ISOLATES
Similar microscopic lesions occurred with both MDV isolates during the first three weeks PI (Table 1, 2, and 3). These changes were characterized by a perivascular accumulation of small numbers of lymphoid cells in the portal areas of the liver, submucosa of the proventriculus, bronchi and bronchioles of the lung, interstitial areas of the medullary portion of the kidneys, epi and myocardium, and epithelium lining the feather follicles. Also evident at this time in the bursa was a slight atrophy of the medullary portion of the follicles with cystic degenerations apparent. In the spleen and thymus, lymphoid depletions with concomitant r e t i c u l u m cell hyperplasia occurred. The atrophy in the bursa and lymphoid depletion in the spleen and thymus became more severe with time PI and involved both the medulla and cortex. Durng the forth and fifth weeks PI, the severity and extent of lymphocytic accumula-
tions increased in the liver, kidney, heart, and lung of both GA and JM infected birds. By the sixth week PI, the peripheral nerves and gonads of GA infected birds were severely affected. These severe lesions appeared as large lymphoid aggregates which displaced and compressed normal parenchymal cells, frequently resulting in gross changes and clinical signs. The major cellular composition of all lymphoid aggregates consisted of small, medium, and large lymphocytes with mediumsized predominating. Large immature lymphoblasts were more frequent in lesions of GA infected birds, while mitotic figures and MD cells were more prominent in JM lesions. MD cells and evidence of mitosis were infrequently seen with the GA isolate. Small compact encapsulated lymphoid foci were occasionally observed in the liver, lung, proventriculus, and duodenum of uninoculated control birds. These foci were also comprised of small, medium, and large lymphocytes. Gross lesions and clinical signs were absent from all non-infected birds. Clinical signs in GA infected birds were depression with ruffling of feathers, while a stilted gait and unilateral paralysis of the extremities occurred in JM inoculated chicks. With both MDV isolates, gross lesions appeared as small grayish white foci in the visceral organs, grayish solidification of the lung with unilateral enlargement, and discoloration of the peripheral nerves and gonads. By the sixth week PI, nodular tumors appeared in many of the visceral organs of GA infected birds, while bilateral nerve and gonad involvement occurred in JM challenged birds only. The major differences in the pathogenesis of the GA and JM isolates occurred in the peripheral nerves and gonads. The JM isolate was more neurotropic and had a greater affinity for the gonads. The JM isolate induced IF antigen ten days earlier and microscopic lesions seventeen days earlier in the peripheral nerves and gonads than the GA isolate. Also, only JM infected birds developed a stilted gait, crippling, and bilateral enlargement of the nerves and gonads. DISCUSSION
The early incidence and sequential distribution of MDV was similar for both the GA and JM isolates. With both isolates, the initial sites of MDV replication were the lymphoid organs,
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and mucosal epithelium of the proventriculus, bronchi lumen and alveolar parenchyma of the lung, interstitial areas of the gonads, epithelium lining both the trachea and kidney tubules, m y o c a r d i u m and epicardium between the peripheral nerve fibers, and surrounding the blood vessels of the liver. In the AGP tests, all tissue homogenates from GA or JM infected birds produced a reaction of identity with each organ extract, indicating that all AGP tissue antigens were serologically identical. Each feather yielded two precipitin lines. Corresponding tissue extracts from uninfected chickens did not produce precipitation. In radial immunodiffusion, a single circular precipitin line formed around each antigen well, and with Immunoelectrophoresis, one precipitin arc appeared a centimeter toward the anode side of each antigen well. With both MDV isolates the initial sites of viral replication were the bursa of Fabricius, thymus, and skin (IF antigen at nine days) and feathers (AGP antigen at twelve days) (Table 1 and 3). These tissues as well as the other lymphoid organs, spleen and cecal tonsil (IF antigen at twelve days), were constant sites for MDV replication with both isolates. During the forth and fifth weeks PI, both IF and AGP antigens occurred sporadically in the proventirculus, kidney, and liver of both GA and JM infected birds and in the gonads and peripheral nerves of JM infected birds. By the sixth week, the peripheral nerves and gonads of GA challenged birds displayed MDV antigens.
899
RE
RE
RE
RE(1)
Path AGP IF
Path AGP IF
Path AGP IF
Path AGP IF
18
21
25
30
RE(1) R
x
RE
RE
Path AGP IF
15
RE
RE I R
RE
x
x
RE
x
Thymus
RE
Spleen
Path AGP IF
Sciatic nerve
12
Brachial plexus
RE
Type of exam
Path AGP IF
Days PI
GA isolate
AF, C
X
AF, C R
AF
AF, C
AF, C
Bursa
Cecal tonsil
++(1)
X
++
+
Brachial plexus
++(1)
X
++
+
Sciatic nerve
RE A
X
RE
RE R
X
X
RE
RE
RE
RE
x
RE
+
Thymus
RE
RE
RE
RE
RE
Spleen
JM isolate
TABLE 1.—Sequential incidence of MDV antigens and severity of lesions in the tissues of chicks inoculated at one day of age with GA or JM isolate ofMDVa
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X
AF, C R
X
AF, C
AF, C
AF, C
AF, C
Bursa
Cecal tonsil
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PATHOGENESIS OF MDV ISOLATES
skin, and feathers. Using a JM isolate, Adldin-
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ger and Calnek (1973) and Purchase (1970) have found IF antigen as early as the first week PI in these tissues. Any differences in the chronological occurrence of viral antigens can result from variation in the genetic line, age, and sex of the bird; dose of viral inoculum (Adldinger and Calnek, 1973; Calnek and Hitchner, 1969; Calnek et al, 1970); or specificity of the conjugated antiserum. The early presence of MDV specific fluorescence in round cells of the lymphoid organs and lymphocytic infiltrations of the peripheral nerves observed with both MDV isolates indicates a productive nature of the infection and that these cells can spread MDV. Thus, it appears evident that the lymphoid organs are important to the early pathogenesis of both the GA and JM isolates. Although MDV dissemination probably resulted from these infected lymphoid cells, the appearance of MDV specific fluorescence
in the resident
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non-
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lymphoid tissues of both GA and JM infected birds indicates that the infection actively involves those organs. Even though it was not possible to establish that IF and AGP antigens were similar, both antigens appear MDV induced since they were absent from control birds. MDV antigens produced by both the GA and JM isolates appear indistinguishable. Both the pattern of fluorescence and AGP antigens induced by either MDV isolate were serologically identical. The early microscopic changes observed with both the GA or JM isolates were identical and similar to those previously described (Eidson and Schmittle, 1968, Sevoian and Chamberlain, 1964). Initially, MD appeared as an accumulation of lymphoid cells, which were first perivascular, but later increased in number until the tissue was largely replaced by these cells. The presence of atrophied follicles and cystic degenerations in the bursa of Fabricius has also been previously reported for both the GA (Fletcher et al, 1972) and JM (Jakowski et al, 1969) isolates. Lymphoid depletion with concomitant reticulum cell hyperplasia in the spleen and thymus has been previously reported -for the JM isolate only . .(Sevoian . . . . and Chamber-
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lain, 1964).
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Although the early tissue distribution of microscopic lesions were similar for both MDV isolates, differences between the GA and JM isolates were noted in the cellular composition of microscopic changes. These variations were
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Path AGP IF
Path AGP IF
Path AGP IF
Path AGP IF
Path AGP IF
15
18
21
25
Path AGP IF
Path AGP IF
Type of exam
12
PI
++(1)
Liver
Kidney
++ I
Proventriculus
GA isolate Duodenum Heart Liver
Kidney
Proventriculus
JM isolate
TABLE 2.—Sequential incidence of MDV antigens and severity of lesions in the tissues of chicks inoculated at one day of age with GA or JM isolate of MDV
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Heart
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PATHOGENESIS OF MDV ISOLATES
903
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similar to those described in which JM lesions frequnetly contained mitotic figures and MD cells (Nazerian, 1971; Sevoian and Chamberlain, 1964) which were infrequent in GA l e s i o n s , a l t h o u g h immature lymphoblasts (Nazerian, 1971) and lymphoreticular cells (Eidson and Schmittle, 1968) were prominent. The significance of this observation is unknown, but evidence of mitosis and abnormal cells in JM lesions indicates neoplasia, while their absence, together with frequent immature cells in GA lesions, indicates an inflammatory response. A problem arising in this and other studies (Payne and Biggs, 1967) was distinguishing between histopathological changes induced by MDV and non-specific lymphoid foci in several visceral organs or control birds. The presence of foci in apparently normal birds is well documented (Jordan, 1936; Lucas, 1949), and many researchers believe this to be a normal part of the lymphocytic system of the chicken. Nonspecific foci were usually small, compact, and encapsulated. The observation that the JM isolate is more neurotropic than the GA isolate agrees with other studies (Nazarian, 1971; Sevoian and Chamberlain, 1964), Characteristically, birds infected with the JM isolate develop predominately nerve lesions; however, some visceral lesions are present, especially in the gonads, while with the GA isolate, visceral lesions predominate with minimal nerve lesions occasionally evident (Nazerian, 1971). In this study, the GA isolate was slightly more viserotropic, inducing MDV antigens nine days earlier in the liver and four days earlier in the duodenum and lungs than the JM isolate. Also, only GA infected birds developed nodular tumors in the liver, spleen, and heart. However, variations of a few days in the appearance of MDV antigens were not highly significant and could result from differences in the initial titer of the GA and JM whole blood inoculum. With both MDV isolates, two different types of virus host cell interactions occurred in vivo. The tissues undergoing degeneration (thymus, bursa, and feather follicles) were a constant source of MDV antigens, while antigens occurred sporadically in tissues with lymphoid accumulations. It seems that viral replication results in degenerative changes in some tissues, while in others lymphocytic accumulations are not dependent on MDV replication. An incorporated viral genome found in other DNA
A
+
Path AGP IF
25
+ A x
x
A
+
Path AGP IF
21 A x
+ A x
Path AGP IF
Skin
18
Gonads
+ A x
Lung
Path AGP IF
Trachea
15
Feather tip
Path AGP IF
Skin
12
Gonads
Path AGP IF
Lung
9
Trachea
Path AGP IF
Type of exam
JM isolate
6
Days PI
GA isolate
TABLE 3.—Sequential incidence of MDV antigens and severity of lesions in the tissues of chicks inoculated at one day of age with GA or JM isolate of MDV
ed from http://ps.oxfordjournals.org/ at University of New Orleans on June 4, 2015 Feather tip
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PATHOGENESIS OF MDV ISOLATES
oncogenic viruses could induce l y m p h o p r o liferation w i t h o u t viral replication.
REFERENCES
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Adldinger, H. K., and B. W. Calnek, 1973. Pathogenesis of Marek's disease: Early distribution of virus and viral antigens in infected chickens. J. Nat. Cancer Inst. 50:1287-1298. Calnek, B. W., and S. B. Hi tenner, 1969. Localization of viral antigen in chickens infected with Marek's disease h e r p e s v i r u s . J. Nat. Cancer Inst. 43:935-949. Calnek, B. W., T. Ubertini, and H. K. Adldinger, 1970. Viral antigen virus particles, and infectivity of tissues from chickens with Marek's disease. J. Natl. Cancer Inst. 45:341—351. Chubb, R. C , and A. E. Churchill, 1968. Precipitating antibodies associated with Marek's disease. Vet. Rec. 8 3 : 4 - 7 . Eidson, C. S., and S. C. Schmittle, 1968. Studies on acute Marek's disease. I. Characteristics of isolate GA in chickens. Avian Dis. 12:467—475. Fletcher, O. J., C. S. Eidson, and S. H. Kleven, 1972. Bursal lesions in chickens inoculated with Marek's disease vaccines. Avian Dis. 16:153—162. Giambrone, J. J., and C. T. Mathews, 1973. The feather tip agar gel precipitin test as a diagnostic tool for Marek's disease. 45th Northeastern Conf. on Avian Dis. Morgantown, WV. Jakowski, K. M., T. W. Frederickson, R. E. Luginbuhl, and C. F. Helmboldt, 1969. Early changes in bursa of Fabricius from Marek's disease. Avian Dis. 13:215-222. Jordan, H. E., 1936. The relation of lymphoid tissue to the process of blood production in avian bone marrow. Amer. J. Anat. 59:249—296. Lennette, E. and N. J. Schmidt, 1964. Page 180 in Diagnostic procedures for viral and rickettsial diseases. Amer. Publ. Health Ass., Inc., New York. Lucas, A. M., 1949. Lymphoid tissue and its relation t o so-called normal lymphoid foci and to lymphomatosis. 1. Qualitative study of lymphoid areas in the pancreas of chickens. Amer. Path. 25:1197-1213. Mancini, G. A., 1965. Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry 2:235—254. Nazerian, K., 1971. Further studies on the replication of Marek's disease virus in the chicken and in cell culture. J. Nat. Cancer Inst. 4 7 : 2 0 7 - 2 1 7 ; Payne, L. N. and P. M. Biggs, 1967. Studies on Marek's disease. II. Pathogenesis. J.Nat. Cancer Inst. 39:281-302. Purchase, H. G., and P. M. Biggs, 1967. Characterization of five isolates of Marek's disease. Detection of antibody. Am. J. Vet. Res. 31:117-123. Purchase, H. G., 1970. Virus specific immunofluorescent and precipitin antigens and cell-free virus in tissues of birds infected with Marek's disease. Cancer Res. 30:1898-1908. Sevoian, M., and D. M. Chamberlain, 1964. Avian lymphomatosis. IV. Pathogenesis. Avian Dis. 8:281-310.
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Spencer, J. L., 1969. Cultivation of Marek's disease virus in cell culture and application of immunofluorescence for virus detection. Ph.D. thesis, Cornell University.
Spencer, J. L., and B. W. Calnek, 1970. Marek's disease. Application of immunofluorescence for detection of antigen and antibody. Am. J. Vet. Res. 31:345-358.
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