- Email: [email protected]
1959 viruses
J. COMP.
PATH.
1967.
VOL.
167
77.
EXPERIMENTAL
INFECTION
INFLUENZA
OF
A/TERN/SOUTH
CHICKENS
AFRICA/1961
CHICKEN/SCOTLAND/l959 II.
WITH AND
VIRUSES
PATHOLOGY BY
C. J. UYS Departmmt
of Pathology,
University
of Cape Town Medical
W. B.
School, Republic of South Africa
BECKER
Department of Bacteriology and U.C.T. and C.S.I.R. Virus Research Unit, University of Cape Town Medical School, Republic of South Africa INTRODUCTION
Tern virus (TV) was isolated from dead and dying Common Terns (Sterna hirundo) in which it caused an explosive epizootic in South Africa in 1961 and was characterized and classified as Influenza A/,Tern/South Africa/ 1961 (Becker, 196 1, 1966). As most of the dead terns were extensively putrified when recovered, the pathology of this infection could not be studied. However, the chicken proved to be a very susceptible experimental host in which the pathogenesis of Tern virus infection could be studied and compared with Chicken/Scotland virus* infection in chickens. The clinical picture and virology have been reported (Becker and Uys, 1967) and the pathological findings will be presented in this communication and compared with those reported in Fowl plague and Newcastle disease. MATERIALS
AND
METHODS
The exerimental data relevant to this investigation are recorded in detail in a previous communication (Becker and Uys, 1967). In brief, these experiments permitted histological examination of tissues : from 6-week old chickens l-6, 10, 12, 19 and 25 days after infection with TV; from ch;ckens 3, 4 and 14 days after infection with CV; and from control chickens at selected times. Formol-fixed and paraffinembedded tissues from pectoral muscle, liver, spleen, lung, kidneys and adnexae, heart, brain, comb, soft palate, skin, proventriculus and ventriculus, small intestine, and both eyeballs with their eyelids were examined by routine histological techniques. RESULTS
Tern Virus Infection (TVI) External naked-eye lesions were not obtrusive. Soon after the onset of symptoms the conjunctivae appeared congested and showed on the lower lid towards the inner canthus small, pale nodules of lymphoid tissue. The birds surviving 5 days or more showed necrotic foci on the comb and eyelids which healed in those that recovered. The spleen was invariably enlarged and pale, showing small pale flecks * Kindly Lasswade,
made Great
available Britain.
by
Dr.
J.
E. Wilson,
Veterinary
Laboratory,
Ministry
of Agriculture,
168
INFLUENZA
INFECTION
IN
CHICKENS
:
PATHOLOGY
beneath the capsular surface. The heart was enlarged, pale and flabby. The ventricles were dilated and the myocardium had a pale mottled appearance with small pale flecks beneath the endocardium. The liver was enlarged and mottled. The meninges occasionally showed small surface haemorrhages. Striking histological changes were observed in the spleen, myocardium, brain, eyelids and eyes, tissues of the comb and skeletal muscle. In contrast, the kidneys, lungs, liver, intestines, gonads and adrenals showed virtually no changes. At the sites affected, lymphoreticular tissues were the first to show changes, with subsequent extension to other tissue components. The first lesions were noted in the spleen and eyelid 2 days after inoculation and were followed by lesions in the comb and skin after 3 to 4 days, and in the eye, ocular muscles, heart and brain after 4 to 5 days (Table 1). TABLE PATHOGENESIS
OF THE
HISTOLOGICAL
1 LESIONS
IN TERN
VIRUS
INFECTION
Days after inoculation Tissue 1 Spleen Eyelid Comb, skin External ocular muscles Eye Myocardium Brain Gastro-intestinal tract
----
2 f-b ++ -
-
+,
3 +++ ++ + + -
4 +++ ++t +++ i- + + + + ++S-
5 -H ++ ++-i--t +++ +E+ +++-t +
6 .+ + ++ TT++’ +++\ ++++
10
12
19
25
-
+
--+
+
A-$+++ -t++
-+ +
-
+ + 1 -,. ++++ ++++
-
-
-= no histological lesions detected. + + + + = tissues severely affected. + +, + + + = intermediate grades of involvement.
Spleen. First, there was proliferation around the sinusoids of large pale cells with oval nuclei which resembled reticulum cells. This resulted in the formation of pale cellular areas which extended to replace most of the pulp, while the follicles remained non-reactive and inconspicuous. Many of these reticular cells became angular and resembled epithelioid cells; others showed phagocytic activity and appeared as macrophages. A general increase of heterophiles accompanied these changes in the pulp (Fig. 1). In most birds the lesion did not progress beyond this stage. However, in a small proportion these cellular foci showed central necrosis and fibrin exudation. The necrotic areas contained chromatin debris and were infiltrated by heterophiles (Fig. 2). The splenic changes reached a peak at about the 4th day, and thereafter the reticulum cells became noticeably fewer, while the necrotic areas showed signs of organisation. By the 6th day the appearances were near normal and by the 10th day no signs of the lesions existed. Eye and eyelid. Although all the chickens were inoculated into the right conjunctival sac, the lesions described below were bilateral. The lymphoid deposits
C.
J.
UYS
AND
W.
B.
BECKER
169
beneath the conjunctiva on the internal aspect of the eyelid showed the first changes soon after infection. Reticulum cell hyperplasia and concurrent heterophile infiltration occurred centrally after 2 days. Mononuclear cells and heterophiles extended from here widely into the loose connective tissue of the eyelid. Central necrosis of the hyperplastic lymphoid tissue occurred next and extended to involve the overlying conjunctiva, resulting in ulceration. By this time the mononuclear cell infiltration extended into the peri-orbital tissues, focally involving the ocular muscles, and was followed by necrosis of the muscle fibres. After the 4th day the subepithelial connective tissue of the external eyelid also showed an intense focal mononuclear infiltration. This focus soon showed central necrosis which was followed by heterophile infiltration and fibrin exudation. Extension of this lesion resulted in necrosis and ulceration of the surface squamous epithelium. Identical lesions, occurring concurrently, were observed in the comb (Fig. 3). Following the inflammatory changes in the eyelid, per-i-orbital tissues and ocular muscles, the mononuclear infiltration involved in turn the iris, ciliary body, choroid and ultimately the optic disc (Fig. 4). The foci of necrosis in the ocular muscles increased in size and the mononuclear infiltration became more intense (Fig. 5). By the 6th day the inflammatory reaction was most intense within the eye, while in the eyelid there were signs of regression as manifested by a rapid diminution and disappearance of inflammatory cells with complete return to normal of the tissues and lymphoid aggregates, except where there had been preceding necrosis. The mononuclear cells around the necrotic foci became distinctly epithelioid on character and were distributed in radial fashion.. Many had fused, resulting in the formation of multinucleate giant cells. These chronic lesions had a distinctly granulomatous appearance and were confined to comb and eyelid only (Figs. 6 and 7). In the ocular muscle the inflammatory cells disappeared and repair was seen as a marked sarcolemmal proliferation. Comb. The lesions occurred in two forms. First, after 3 days there was intense focal infiltration of mononuclears into the connective tissues of the dermis. This lesion was identical in appearance and behaviour to that in the external eyelid and similarly showed a predilection for pre-existing lymphoid nests. The focal necrosis, fibrin exudation and heterophile infiltration ensued, as above. The surface epithelium was involved and showed necrosis, crusting and ulceration, and ultimately these foci were transformed into giant cell granulomata. The second type of lesion was infrequent, became manifest only on the 5th day and was confined to the epidermis. Intracellular and extracellular oedema, followed by disintegration of the epithelial cells, led to intra-epidermal vesicle formation, Loss of the surface epithelium was followed by ulceration. Skin. This showed occasional focal lesions of the type seen in the dermis of the comb and external eyelid. In contrast to the other sites, the skin lesions did not undergo necrosis, ulceration and granuloma formation. Myocardium. Striking lesions were first observed after only 4 days. Initially, diffuse widespread interstitial oedema and hydropic degeneration of muscle fibres were noted. Here and there isolated groups of fibres were necrotic and there was a scanty infiltration of mononuclear cells. Overnight this picture changed to a florid myocarditis, involving both the right and left ventricles (Fig. 8). Few
170
INFLUENZA
INFECTION
IN
CHICKENS
:
PATHOLOGY
fibres appeased healthy, all showing hydropic degeneration and loss of staining reaction, and in addition there were numerous foci of frank necrosis associated with a florid mononuclear cell infiltration. In these foci, chromatin debris released by necrotic cells was scattered around and there was evidence of macrophage activity (Fig. 9). Usually, a scanty infiltration of heterophiles was noted in these foci and in one or two cases was intense. There was a general increased cellularity of the interstitial tissues away from the cellular necrotic foci due, in part, to proliferation of Anitsckow myocytes Up to the 10th day there was neither a diminution in the intensity of the inflammatory infiltration of the necrotic foci nor a change in its nature. In the later phases similar inflammatory cells extended into the subpericardial connective tissues and adjacent adipose tissues as well. By the 12th day, however, most of the inflammatory cells had disappeared and there was evidence of repair as illustrated by the predominence of interstitial cells in these foci. Brain. Lesions were noted on the 4th day. First, the meninges appeared slightly more cellular. In the brain substance there were small isolated foci of necrosis, seen as free-lying chromatin and proliferation of glial cells. These foci occurred close to small capillary-type blood vessels from which inflammatory cells were emigrating (Fig. 10). After 5 to 6 days, a striking picture of a more diffuse encephalitis had developed in both cerebrum and cerebellum. The meninges were obviously cellular due to a mononuclear mfiltrate. There was widespread and pronounced perivascular cuffing by similar cells (Figs. 11 and 12). The neuronal cells were hydropic and many were clearly necrotic. The brain substance showed oedema and a diffuse cellularity, possibly due to an increase of microglial and mononuclear cells; few heterophiles were noted. Engorged capillaries lined by prominent endothelial cells were apparent throughout the brain substance and in a. few birds there was evidence of recent capillary haemorrhage. Skeletal muscle. The florid necrotizing changes that occurred in the ocular muscles have already been described. The pectoral muscles, in contrast, were remarkably unaffected and at the most developed only occasional focal mononuclear infiltrations unassociated with necrosis. However, the pharyngeal and intercostal muscles late in the course of the disease showed necrosis and inflammatory changes which were similar to, but much less intense than, the ocular muscle lesions. Gastro-intestinal tract. Tissues from the proventriculus, ventriculus and intestine were free of lesions in nearly all cases. In one case, however, intestinal lymphoid tissue showed a reticular hyperplasia and heterophile infiltration similar to that encountered in lymphoid rests elsewhere. Chicken Virus Infection (CVZ) Externally, congestion and haemorrhages into the tissues were noteworthy, particularly in the skin around the joints and in the conjunctivae of the lower lids near the inner canthus. Birds surviving a few days had ulcers on the comb. The only other changes at autopsy were congestion of the proventriculus and pronounced injection of the cloaca1 mucous membrane. Histologically, the distribution of the lesions in the various organs and tissues and general tissue reactions resembled those of TVI. While CVI differed from
C.
J. UYS
AND
W.
B.
171
BECKER
TV1 mainly in that the intensity of the inflammatory reaction and the extent of tissue involvement in the affected organs were less pronounced, it showed additional features worthy of comment. Stress is laid on the differences rather than similarities. The changes in the spleen were indistinguishable from those of TVI. The two types of inflammatory lesion affected the eyelids as described under TVI, b’ut in contrast there was no evidence of extension to the eye or external ocular muscles. An added feature at this site was a necrotising vasculitis which resulted in thrombotic occlusion of the affected vessels and free haemorrhage into neighbouring tissues. The comb lesions were similar to those of the eyelids and included vascular necrosis, thrombosis and haemorrhage; in addition, however, subepithelial and
TABLE COMPARISON
OF THE DISEASE,
2
HISTOLOGICAL CHANGES PRODUCED AND TERN/SOUTH AFRICA/1961 AND
IN CHICKENS BY CHICKEN/SCOTLAND/l%9
Strains of Newcastle Tissue Heart Lung Spleen Gastro-intestinal Brain Eye External ocular
Fowlplague* virus
FOWL
PLAGUE, VIRUSES
disease virus* Tern vim
He@ordshire
NEWCASTLE
CalzjTomia
Chicken Gus
.New Jersq i
++++ : +++I-
tract
T ++++ ++++
Tmuscles
i,
+,
; +
I +
+++
+I+
++“++ +++ +++
+++I+ -
* Jungherr et al., 1946. - = no histological lesions detected. + + + + = tissues severely affected. + f, + + + = intermediate grades of involvement.
intra-epthelial vesicle formation were prominent with ultimate ulceration and crust formation, but no granulomatous lesions followed. The florid myocarditis of TV1 was absent, but patchy intra- and extracellular oedema existed and the interstitial cells were prominent. In the brain a meningeal reaction and perivascular cuffing were inconspicuous, but focal perivascular nodes of cellular necrosis and mononuclear infiltration were common. Reactive changes occurred in the lymphoid tissue of the proventricules, but necrosis was minimal. Other organs and skin showed no changes of note. DISCUSSION
The sequence of histopathological change in TV1 is based on the examination of chickens in experiments 2, 3 and 4 in the previous article (Becker and Uys, 1967). A comparison of the virological and histological findings can thus be made. Between the 6th and 10th day after inoculation the tissues had been cleared of TV and a good titre of serum antibodies was present. At this time the histological lesions were still active, but at 12 days healing had started and regression was
172
INFLUENZA
INFECTION
IN
CHICKENS
:
PATHOLOGY
virtually complete at approximately 3 weeks. Despite the large amount of virus regularly demonstrated in the lungs of chickens dying of TVI, there were no respiratory signs and no lesions were noted in the lungs either macroscopically or histologically. Earlier experiments (Becker and Uys, unpublished observations) had shown that the tissue tropism of TV did not vary according to the route of inoculation used, whether intramuscular, intraconjuntival, intranasal or oral. Jungherr, Tyzzer, Brandly and Moses (1946) found that the tropism of the strain of fowl plague virus they used was the same when inoculated intramuscularly or intratracheally, but by contrast the tropisms of some strains of Newcastle disease virus differed if the virus was inoculated by these routes (Brandly, 1959; Jungherr and Markham, 1962; Jungherr et al., 1946). The earliest change in TV1 occurred in the lymphoid tissue of the spleen, eyelid and comb as reactive hyperplasia, and was followed by striking inflammatory changes at these sites and in other organs such as the eye, ocular muscles, myocardium and brain. As most birds did not survive more than 6 days, the usual picture encountered was that of an acute focal inflammatory reaction characterized by a mononuclear infiltration associated with tissue necrosis which involved particularly the myocardium, brain, eye, eyelids and comb. In a few birds that recovered, the myocardium, brain, eye and comb were the last to return to normal. While signs of active inflammation persisted in the myocardium and brain longer than at other sites, these lesions, as at most other sites, appeared to undergo resolution, and despite their extremely florid appearance in the early stages they were not followed by any degree of reparatory fibrosis. This, however, did not apply to the inflammatory foci of the comb and eyelid which were replaced by characteristic giant cell granulomata; similarly the necrotic ocular muscles showed reparative sarcolemmal proliferation. Although it is known that there may be a certain degree of overlap in the histological changes in avian diseases produced by antigenically unrelated myxoviruses, as for instance fowl plague and Newcastle disease (Jungherr et al., 1946), it is in the pattern of organ involvement as a whole that significant differences may be demonstrated. The findings of Jungherr et al. (1946) on fowl plague and Newcastle disease, and ours relating to TV1 and CVI have been summarised in the present article. It seems clear that a distinctive pattern exists for each of these virus infections. The features which distinguish TV1 from most of the others are the degree of cardiac and brain involvement : these lesions do not occur in conjunction in such a florid form in any of the other infections. The comb and eyelid lesions seen in TV1 have not been stressed in reports on the other infections under consideration and particularly noteworthy are the distinctive granulomatous lesions which follow at these sites late in the course of TVI. While the pattern of pathological lesions of CVI most closely resembles that of TV1 it does not include the severe grade of myocarditis, the intra-ocular lesions or the late granulomata of the combs and lids. As an additional feature, CVI shows necrotising vascular lesions with rest&ant Iocal haemorrhage into the tissue. It would thus appear that the histopathological pattern of organ involvement in TVI, like the clinical picture, is sufficiently distinctive to be of use in the characterization of TV.
C.
J.
IJYS
APGD
W.
B.
BECKER
INFLUENZA
INFECTION
IN
CHICKENS
:
PATHOLOGY
C.
J.
UYS
AND
W.
B.
BECKER
C.
J.
UYS
AND
W.
B.
BECKER
173
SUMMARY
The viruses of influenza A/Tern/South Africa/l961 and Chicken/Scotland/ 1959 are antigenically closely related strains of avian influenza A, both of which produce fulminating infection in six-week-old chickens. The lesions produced experimentally in chickens by Tern virus are described and compared with those produced by Chicken virus under similar conditions, and with those reported in fowl plague and Newcastle disease virus infection. The findings indicate that the histopathological pattern of tissue involvement in Tern virus infection is distinctive and is therefore of use in the characterization of Tern virus. ACKNOWLEDGMENTS
We wish to record our thanks advice.
to Professor A. Kipps for his encouragement and
REFERENCES
Becker, W. B. (1961). S. Afr. med. J., 35, 1093; (1966). J. Hyg., Camb., 64, (in press). Becker, W. B., and Uys, C. J. (1967). f. camp; Path., 77, 159. Brandly, C. A. (1959). Diseases of Poultry, 4th Edit., H. E. Biester and L. H. Schwarts, Eds., p. 464. Iowa State University Press; Ames. Iowa. Jungherr, E. L., and Markham, F. S. (1962). Poultry Science, 41, 522. Jungherr, E. L., Tyzzer, E. E., Brandly, C. A., and Moses, H. E. (1946). Amer. J. vet. Res., 7, 250. [Received
for
LEGENDS Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig.
1.
publication,
TO
July
9th, 19661
ILLUSTRATIONS
Spleen showing pale areas of reticulum cell hyperplasia, one of the earliest reactions to infection. H. & E. x 110. 2. A focal area of necrosis in the spleen which sometimes occurs centrally in areas of reticulum cell hyperplasia. H. & E. x 110. 3. Intense focal infiltration occurring subepithelially in the comb and external eyelid 4 days after infection which is composed predominantly of mononuclear cells but includes a few heterophiles. Central necrosis commonly supervenes. H. & E. x 110. 4. Inflammatory infiltration involving choroid and retina. H. & E. x 110. infiltration of external ocular muscles and associated necrosis of muscle 5. Intense mononuclear fibres, with indications of reparative sarcolemmal proliferation. H. & E. x 270. 6. Focal granuloma which follows the inflammatory focus in comb and external eyelid of the types illustrated in Fig. 3. H. & E. x 110. showing giant cells and epithelioid cells arranged radially around central 7. Focal granuloma necrotic debris. H. & E. x 270. 8. Focal myocarditis. Early phases characterised by a focal mononuclear infiltration and incipient necrosis of cardiac muscle fibres. H. & E. x 110. 9. Focal myocarditis, showing intense inflammatory infiltration, and marked focal muscle fibre necrosis. Macrophage activity is also evident. H. & E. x 270. 10. Acute encephalitis showing perivascular mononuclear cell infiltration and glial reaction. H. & E. x270. 11. Acute encephalitis showing increased cellularity of meninges due to an inflammatory infiltrate. H. & E. x270. 12. Acute encephalitis showing the intensity of the inflammatory response in the brain substance, perivascular cuffing and general increased cellularity of brain and meninges. Increased vascularity is also present. H. & E. x 110.
PATH.
1967.
VOL.
167
77.
EXPERIMENTAL
INFECTION
INFLUENZA
OF
A/TERN/SOUTH
CHICKENS
AFRICA/1961
CHICKEN/SCOTLAND/l959 II.
WITH AND
VIRUSES
PATHOLOGY BY
C. J. UYS Departmmt
of Pathology,
University
of Cape Town Medical
W. B.
School, Republic of South Africa
BECKER
Department of Bacteriology and U.C.T. and C.S.I.R. Virus Research Unit, University of Cape Town Medical School, Republic of South Africa INTRODUCTION
Tern virus (TV) was isolated from dead and dying Common Terns (Sterna hirundo) in which it caused an explosive epizootic in South Africa in 1961 and was characterized and classified as Influenza A/,Tern/South Africa/ 1961 (Becker, 196 1, 1966). As most of the dead terns were extensively putrified when recovered, the pathology of this infection could not be studied. However, the chicken proved to be a very susceptible experimental host in which the pathogenesis of Tern virus infection could be studied and compared with Chicken/Scotland virus* infection in chickens. The clinical picture and virology have been reported (Becker and Uys, 1967) and the pathological findings will be presented in this communication and compared with those reported in Fowl plague and Newcastle disease. MATERIALS
AND
METHODS
The exerimental data relevant to this investigation are recorded in detail in a previous communication (Becker and Uys, 1967). In brief, these experiments permitted histological examination of tissues : from 6-week old chickens l-6, 10, 12, 19 and 25 days after infection with TV; from ch;ckens 3, 4 and 14 days after infection with CV; and from control chickens at selected times. Formol-fixed and paraffinembedded tissues from pectoral muscle, liver, spleen, lung, kidneys and adnexae, heart, brain, comb, soft palate, skin, proventriculus and ventriculus, small intestine, and both eyeballs with their eyelids were examined by routine histological techniques. RESULTS
Tern Virus Infection (TVI) External naked-eye lesions were not obtrusive. Soon after the onset of symptoms the conjunctivae appeared congested and showed on the lower lid towards the inner canthus small, pale nodules of lymphoid tissue. The birds surviving 5 days or more showed necrotic foci on the comb and eyelids which healed in those that recovered. The spleen was invariably enlarged and pale, showing small pale flecks * Kindly Lasswade,
made Great
available Britain.
by
Dr.
J.
E. Wilson,
Veterinary
Laboratory,
Ministry
of Agriculture,
168
INFLUENZA
INFECTION
IN
CHICKENS
:
PATHOLOGY
beneath the capsular surface. The heart was enlarged, pale and flabby. The ventricles were dilated and the myocardium had a pale mottled appearance with small pale flecks beneath the endocardium. The liver was enlarged and mottled. The meninges occasionally showed small surface haemorrhages. Striking histological changes were observed in the spleen, myocardium, brain, eyelids and eyes, tissues of the comb and skeletal muscle. In contrast, the kidneys, lungs, liver, intestines, gonads and adrenals showed virtually no changes. At the sites affected, lymphoreticular tissues were the first to show changes, with subsequent extension to other tissue components. The first lesions were noted in the spleen and eyelid 2 days after inoculation and were followed by lesions in the comb and skin after 3 to 4 days, and in the eye, ocular muscles, heart and brain after 4 to 5 days (Table 1). TABLE PATHOGENESIS
OF THE
HISTOLOGICAL
1 LESIONS
IN TERN
VIRUS
INFECTION
Days after inoculation Tissue 1 Spleen Eyelid Comb, skin External ocular muscles Eye Myocardium Brain Gastro-intestinal tract
----
2 f-b ++ -
-
+,
3 +++ ++ + + -
4 +++ ++t +++ i- + + + + ++S-
5 -H ++ ++-i--t +++ +E+ +++-t +
6 .+ + ++ TT++’ +++\ ++++
10
12
19
25
-
+
--+
+
A-$+++ -t++
-+ +
-
+ + 1 -,. ++++ ++++
-
-
-= no histological lesions detected. + + + + = tissues severely affected. + +, + + + = intermediate grades of involvement.
Spleen. First, there was proliferation around the sinusoids of large pale cells with oval nuclei which resembled reticulum cells. This resulted in the formation of pale cellular areas which extended to replace most of the pulp, while the follicles remained non-reactive and inconspicuous. Many of these reticular cells became angular and resembled epithelioid cells; others showed phagocytic activity and appeared as macrophages. A general increase of heterophiles accompanied these changes in the pulp (Fig. 1). In most birds the lesion did not progress beyond this stage. However, in a small proportion these cellular foci showed central necrosis and fibrin exudation. The necrotic areas contained chromatin debris and were infiltrated by heterophiles (Fig. 2). The splenic changes reached a peak at about the 4th day, and thereafter the reticulum cells became noticeably fewer, while the necrotic areas showed signs of organisation. By the 6th day the appearances were near normal and by the 10th day no signs of the lesions existed. Eye and eyelid. Although all the chickens were inoculated into the right conjunctival sac, the lesions described below were bilateral. The lymphoid deposits
C.
J.
UYS
AND
W.
B.
BECKER
169
beneath the conjunctiva on the internal aspect of the eyelid showed the first changes soon after infection. Reticulum cell hyperplasia and concurrent heterophile infiltration occurred centrally after 2 days. Mononuclear cells and heterophiles extended from here widely into the loose connective tissue of the eyelid. Central necrosis of the hyperplastic lymphoid tissue occurred next and extended to involve the overlying conjunctiva, resulting in ulceration. By this time the mononuclear cell infiltration extended into the peri-orbital tissues, focally involving the ocular muscles, and was followed by necrosis of the muscle fibres. After the 4th day the subepithelial connective tissue of the external eyelid also showed an intense focal mononuclear infiltration. This focus soon showed central necrosis which was followed by heterophile infiltration and fibrin exudation. Extension of this lesion resulted in necrosis and ulceration of the surface squamous epithelium. Identical lesions, occurring concurrently, were observed in the comb (Fig. 3). Following the inflammatory changes in the eyelid, per-i-orbital tissues and ocular muscles, the mononuclear infiltration involved in turn the iris, ciliary body, choroid and ultimately the optic disc (Fig. 4). The foci of necrosis in the ocular muscles increased in size and the mononuclear infiltration became more intense (Fig. 5). By the 6th day the inflammatory reaction was most intense within the eye, while in the eyelid there were signs of regression as manifested by a rapid diminution and disappearance of inflammatory cells with complete return to normal of the tissues and lymphoid aggregates, except where there had been preceding necrosis. The mononuclear cells around the necrotic foci became distinctly epithelioid on character and were distributed in radial fashion.. Many had fused, resulting in the formation of multinucleate giant cells. These chronic lesions had a distinctly granulomatous appearance and were confined to comb and eyelid only (Figs. 6 and 7). In the ocular muscle the inflammatory cells disappeared and repair was seen as a marked sarcolemmal proliferation. Comb. The lesions occurred in two forms. First, after 3 days there was intense focal infiltration of mononuclears into the connective tissues of the dermis. This lesion was identical in appearance and behaviour to that in the external eyelid and similarly showed a predilection for pre-existing lymphoid nests. The focal necrosis, fibrin exudation and heterophile infiltration ensued, as above. The surface epithelium was involved and showed necrosis, crusting and ulceration, and ultimately these foci were transformed into giant cell granulomata. The second type of lesion was infrequent, became manifest only on the 5th day and was confined to the epidermis. Intracellular and extracellular oedema, followed by disintegration of the epithelial cells, led to intra-epidermal vesicle formation, Loss of the surface epithelium was followed by ulceration. Skin. This showed occasional focal lesions of the type seen in the dermis of the comb and external eyelid. In contrast to the other sites, the skin lesions did not undergo necrosis, ulceration and granuloma formation. Myocardium. Striking lesions were first observed after only 4 days. Initially, diffuse widespread interstitial oedema and hydropic degeneration of muscle fibres were noted. Here and there isolated groups of fibres were necrotic and there was a scanty infiltration of mononuclear cells. Overnight this picture changed to a florid myocarditis, involving both the right and left ventricles (Fig. 8). Few
170
INFLUENZA
INFECTION
IN
CHICKENS
:
PATHOLOGY
fibres appeased healthy, all showing hydropic degeneration and loss of staining reaction, and in addition there were numerous foci of frank necrosis associated with a florid mononuclear cell infiltration. In these foci, chromatin debris released by necrotic cells was scattered around and there was evidence of macrophage activity (Fig. 9). Usually, a scanty infiltration of heterophiles was noted in these foci and in one or two cases was intense. There was a general increased cellularity of the interstitial tissues away from the cellular necrotic foci due, in part, to proliferation of Anitsckow myocytes Up to the 10th day there was neither a diminution in the intensity of the inflammatory infiltration of the necrotic foci nor a change in its nature. In the later phases similar inflammatory cells extended into the subpericardial connective tissues and adjacent adipose tissues as well. By the 12th day, however, most of the inflammatory cells had disappeared and there was evidence of repair as illustrated by the predominence of interstitial cells in these foci. Brain. Lesions were noted on the 4th day. First, the meninges appeared slightly more cellular. In the brain substance there were small isolated foci of necrosis, seen as free-lying chromatin and proliferation of glial cells. These foci occurred close to small capillary-type blood vessels from which inflammatory cells were emigrating (Fig. 10). After 5 to 6 days, a striking picture of a more diffuse encephalitis had developed in both cerebrum and cerebellum. The meninges were obviously cellular due to a mononuclear mfiltrate. There was widespread and pronounced perivascular cuffing by similar cells (Figs. 11 and 12). The neuronal cells were hydropic and many were clearly necrotic. The brain substance showed oedema and a diffuse cellularity, possibly due to an increase of microglial and mononuclear cells; few heterophiles were noted. Engorged capillaries lined by prominent endothelial cells were apparent throughout the brain substance and in a. few birds there was evidence of recent capillary haemorrhage. Skeletal muscle. The florid necrotizing changes that occurred in the ocular muscles have already been described. The pectoral muscles, in contrast, were remarkably unaffected and at the most developed only occasional focal mononuclear infiltrations unassociated with necrosis. However, the pharyngeal and intercostal muscles late in the course of the disease showed necrosis and inflammatory changes which were similar to, but much less intense than, the ocular muscle lesions. Gastro-intestinal tract. Tissues from the proventriculus, ventriculus and intestine were free of lesions in nearly all cases. In one case, however, intestinal lymphoid tissue showed a reticular hyperplasia and heterophile infiltration similar to that encountered in lymphoid rests elsewhere. Chicken Virus Infection (CVZ) Externally, congestion and haemorrhages into the tissues were noteworthy, particularly in the skin around the joints and in the conjunctivae of the lower lids near the inner canthus. Birds surviving a few days had ulcers on the comb. The only other changes at autopsy were congestion of the proventriculus and pronounced injection of the cloaca1 mucous membrane. Histologically, the distribution of the lesions in the various organs and tissues and general tissue reactions resembled those of TVI. While CVI differed from
C.
J. UYS
AND
W.
B.
171
BECKER
TV1 mainly in that the intensity of the inflammatory reaction and the extent of tissue involvement in the affected organs were less pronounced, it showed additional features worthy of comment. Stress is laid on the differences rather than similarities. The changes in the spleen were indistinguishable from those of TVI. The two types of inflammatory lesion affected the eyelids as described under TVI, b’ut in contrast there was no evidence of extension to the eye or external ocular muscles. An added feature at this site was a necrotising vasculitis which resulted in thrombotic occlusion of the affected vessels and free haemorrhage into neighbouring tissues. The comb lesions were similar to those of the eyelids and included vascular necrosis, thrombosis and haemorrhage; in addition, however, subepithelial and
TABLE COMPARISON
OF THE DISEASE,
2
HISTOLOGICAL CHANGES PRODUCED AND TERN/SOUTH AFRICA/1961 AND
IN CHICKENS BY CHICKEN/SCOTLAND/l%9
Strains of Newcastle Tissue Heart Lung Spleen Gastro-intestinal Brain Eye External ocular
Fowlplague* virus
FOWL
PLAGUE, VIRUSES
disease virus* Tern vim
He@ordshire
NEWCASTLE
CalzjTomia
Chicken Gus
.New Jersq i
++++ : +++I-
tract
T ++++ ++++
Tmuscles
i,
+,
; +
I +
+++
+I+
++“++ +++ +++
+++I+ -
* Jungherr et al., 1946. - = no histological lesions detected. + + + + = tissues severely affected. + f, + + + = intermediate grades of involvement.
intra-epthelial vesicle formation were prominent with ultimate ulceration and crust formation, but no granulomatous lesions followed. The florid myocarditis of TV1 was absent, but patchy intra- and extracellular oedema existed and the interstitial cells were prominent. In the brain a meningeal reaction and perivascular cuffing were inconspicuous, but focal perivascular nodes of cellular necrosis and mononuclear infiltration were common. Reactive changes occurred in the lymphoid tissue of the proventricules, but necrosis was minimal. Other organs and skin showed no changes of note. DISCUSSION
The sequence of histopathological change in TV1 is based on the examination of chickens in experiments 2, 3 and 4 in the previous article (Becker and Uys, 1967). A comparison of the virological and histological findings can thus be made. Between the 6th and 10th day after inoculation the tissues had been cleared of TV and a good titre of serum antibodies was present. At this time the histological lesions were still active, but at 12 days healing had started and regression was
172
INFLUENZA
INFECTION
IN
CHICKENS
:
PATHOLOGY
virtually complete at approximately 3 weeks. Despite the large amount of virus regularly demonstrated in the lungs of chickens dying of TVI, there were no respiratory signs and no lesions were noted in the lungs either macroscopically or histologically. Earlier experiments (Becker and Uys, unpublished observations) had shown that the tissue tropism of TV did not vary according to the route of inoculation used, whether intramuscular, intraconjuntival, intranasal or oral. Jungherr, Tyzzer, Brandly and Moses (1946) found that the tropism of the strain of fowl plague virus they used was the same when inoculated intramuscularly or intratracheally, but by contrast the tropisms of some strains of Newcastle disease virus differed if the virus was inoculated by these routes (Brandly, 1959; Jungherr and Markham, 1962; Jungherr et al., 1946). The earliest change in TV1 occurred in the lymphoid tissue of the spleen, eyelid and comb as reactive hyperplasia, and was followed by striking inflammatory changes at these sites and in other organs such as the eye, ocular muscles, myocardium and brain. As most birds did not survive more than 6 days, the usual picture encountered was that of an acute focal inflammatory reaction characterized by a mononuclear infiltration associated with tissue necrosis which involved particularly the myocardium, brain, eye, eyelids and comb. In a few birds that recovered, the myocardium, brain, eye and comb were the last to return to normal. While signs of active inflammation persisted in the myocardium and brain longer than at other sites, these lesions, as at most other sites, appeared to undergo resolution, and despite their extremely florid appearance in the early stages they were not followed by any degree of reparatory fibrosis. This, however, did not apply to the inflammatory foci of the comb and eyelid which were replaced by characteristic giant cell granulomata; similarly the necrotic ocular muscles showed reparative sarcolemmal proliferation. Although it is known that there may be a certain degree of overlap in the histological changes in avian diseases produced by antigenically unrelated myxoviruses, as for instance fowl plague and Newcastle disease (Jungherr et al., 1946), it is in the pattern of organ involvement as a whole that significant differences may be demonstrated. The findings of Jungherr et al. (1946) on fowl plague and Newcastle disease, and ours relating to TV1 and CVI have been summarised in the present article. It seems clear that a distinctive pattern exists for each of these virus infections. The features which distinguish TV1 from most of the others are the degree of cardiac and brain involvement : these lesions do not occur in conjunction in such a florid form in any of the other infections. The comb and eyelid lesions seen in TV1 have not been stressed in reports on the other infections under consideration and particularly noteworthy are the distinctive granulomatous lesions which follow at these sites late in the course of TVI. While the pattern of pathological lesions of CVI most closely resembles that of TV1 it does not include the severe grade of myocarditis, the intra-ocular lesions or the late granulomata of the combs and lids. As an additional feature, CVI shows necrotising vascular lesions with rest&ant Iocal haemorrhage into the tissue. It would thus appear that the histopathological pattern of organ involvement in TVI, like the clinical picture, is sufficiently distinctive to be of use in the characterization of TV.
C.
J.
IJYS
APGD
W.
B.
BECKER
INFLUENZA
INFECTION
IN
CHICKENS
:
PATHOLOGY
C.
J.
UYS
AND
W.
B.
BECKER
C.
J.
UYS
AND
W.
B.
BECKER
173
SUMMARY
The viruses of influenza A/Tern/South Africa/l961 and Chicken/Scotland/ 1959 are antigenically closely related strains of avian influenza A, both of which produce fulminating infection in six-week-old chickens. The lesions produced experimentally in chickens by Tern virus are described and compared with those produced by Chicken virus under similar conditions, and with those reported in fowl plague and Newcastle disease virus infection. The findings indicate that the histopathological pattern of tissue involvement in Tern virus infection is distinctive and is therefore of use in the characterization of Tern virus. ACKNOWLEDGMENTS
We wish to record our thanks advice.
to Professor A. Kipps for his encouragement and
REFERENCES
Becker, W. B. (1961). S. Afr. med. J., 35, 1093; (1966). J. Hyg., Camb., 64, (in press). Becker, W. B., and Uys, C. J. (1967). f. camp; Path., 77, 159. Brandly, C. A. (1959). Diseases of Poultry, 4th Edit., H. E. Biester and L. H. Schwarts, Eds., p. 464. Iowa State University Press; Ames. Iowa. Jungherr, E. L., and Markham, F. S. (1962). Poultry Science, 41, 522. Jungherr, E. L., Tyzzer, E. E., Brandly, C. A., and Moses, H. E. (1946). Amer. J. vet. Res., 7, 250. [Received
for
LEGENDS Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig.
1.
publication,
TO
July
9th, 19661
ILLUSTRATIONS
Spleen showing pale areas of reticulum cell hyperplasia, one of the earliest reactions to infection. H. & E. x 110. 2. A focal area of necrosis in the spleen which sometimes occurs centrally in areas of reticulum cell hyperplasia. H. & E. x 110. 3. Intense focal infiltration occurring subepithelially in the comb and external eyelid 4 days after infection which is composed predominantly of mononuclear cells but includes a few heterophiles. Central necrosis commonly supervenes. H. & E. x 110. 4. Inflammatory infiltration involving choroid and retina. H. & E. x 110. infiltration of external ocular muscles and associated necrosis of muscle 5. Intense mononuclear fibres, with indications of reparative sarcolemmal proliferation. H. & E. x 270. 6. Focal granuloma which follows the inflammatory focus in comb and external eyelid of the types illustrated in Fig. 3. H. & E. x 110. showing giant cells and epithelioid cells arranged radially around central 7. Focal granuloma necrotic debris. H. & E. x 270. 8. Focal myocarditis. Early phases characterised by a focal mononuclear infiltration and incipient necrosis of cardiac muscle fibres. H. & E. x 110. 9. Focal myocarditis, showing intense inflammatory infiltration, and marked focal muscle fibre necrosis. Macrophage activity is also evident. H. & E. x 270. 10. Acute encephalitis showing perivascular mononuclear cell infiltration and glial reaction. H. & E. x270. 11. Acute encephalitis showing increased cellularity of meninges due to an inflammatory infiltrate. H. & E. x270. 12. Acute encephalitis showing the intensity of the inflammatory response in the brain substance, perivascular cuffing and general increased cellularity of brain and meninges. Increased vascularity is also present. H. & E. x 110.