Pathogenesis of lethal influenza virus infection in turkeys

J.

COMP. PATH. 1972. VOL. 82.

129

PATHOGENESIS

OF LETHAL INFECTION

I. EXTRANEURAL

IN PHASE

INFLUENZA

VIRUS

TURKEYS OF

INFECTION

BY

0. NARAYAN,+ J. T HORSEN, T. J. HULLAND, G. ANKELI and P.G. JOSEPH Defiartment

of Vekrinay Ontario

Microbiology

Veterinary

and Immunology and Department of PathoIopy College, University of Guelph, Ontario, Canada

(Hulland),

INTRODUCTION

Unlike influenza A virus infections in man, horsesand swine, where the respiratory system is the chief target of infection, avian influenza is a diseasecomplex which may vary from mild respiratory infection to fatal generalized disease. Pereira (1969) has classifiedthese avian viruses into six serological families of which two, the fowl plague group and the more recently discovered family comprising A/Turkey/Ontario/7732-66 A/Tern/S.Africa/ 196l., A/Chicken/Scotland/1959, and A/Turkey/Ontario/6213-66, are pathogenic for chickens and turkeys. The virulent members of these two groups produce a highly contagious and rapidly fatal diseasein chickens. The clinicopathological signs of these infections have been described by several authors; those on fowl plague by Rohrer (1947) and Jungherr, Tyzzer, Brandly and Moses (1946), and those causedby strains of the new group by Becker and Uys (1967) and Narayan, Lang and Rouse (1969a, b). A common feature of the pathological lesionscaused by all these strains is multiple foci of necrosis in nearly all the organs of the bird. Despite the easeof transmission of the infections in birds and the ready means of cultivation and quantitation of the agents in embryonated eggs, only a limited number of pathogenesisstudies of these infections have been reported. Firstly, Uys and Becker (1967), using pathological criteria to compare the effects of A/Chicken/ Scotland/l959 and A/Tern/S. Africa/61 in chickens, pointed out subtle differences between the two strains. They showed that the tern virus infection was distinctive in the degree of cardiac and brain involvement, whereasthe Scottish fowl virus infection resulted in a high degree of necrotic vasculitis and haemorrhages into the superficial tissues.Furthermore, viraemia was only transitory in the tern virus infection, but was present until death in the other. Many of the pathological features present in both infections were reproduced in chickens and turkeys infected with A/Turkey/ Ontario/66 (V7732). Indeed, the lesions caused by V7732 in chickens were very similar to those causedby the Scottish virus, suggestinga closer antigenic relationship between these two viruses than between the Scottish and tern viruses. However, antigenic relationships of pathogenic avian influenza viruses based on pathological criteria should be made with caution for two reasons. (a) The same strain of virus may have different mechanismsof pathogenesis in different speciesof birds. For example, although V7732 killed chickens and turkeys, it caused haemorrhagic lesions in the former speciesonly, suggesting viral infection of the vascular endothelium in chickens but not in turkeys. (b) Lang (personal communication) isolated field strains * Presentaddress: Department of Neurology, Traylor Building 709, The Johns Hopkins University School

of Medicine,

Baltimore,

Maryland,

U.S.A.

130

INFLUENZA

VIRUS

IN

TURKEYS:

EXTRANEURAL

PHASE

OF

INFECTION

of avian influenza virus which, under experimental conditions, killed chickens not turkeys and vice versa. As there are no reports in the literature concerning virulence of tern or the Scottish virus for turkeys, further comparisons between Scottish virus and V7732 must remain speculative.

but the the

Since V7732 was isolated from turkeys during an epizootic, a pathogenesis study was undertaken using this species. It is based on a sequential examination of tissues by virus quantitation, histopathology and immunofluorescence. A subsequent paper deals with the central nervous system phase of the infection.

MATERIALS

AND

METHODS

Experimental turkeys. Female turkeys were reared in isolation quarters and for III antibody to virus 7732 and virus 6213 prior to infection. They were at approximately 12 weeks of age. Virus strain. Influenza A/Turkey/Ontario/773266 (V7732), of the 8th embryo passage, was used throughout the study. Inocula were prepared by allantoic fluid in tryptose phosphate broth (TPB).

screened infected chicken diluting

Sampling Procedures Viraemia studies. Four birds were infected intratracheally with 200 EID,, and 1 ml. of blood was taken from each bird 6 hours after infection and at 6 hour intervals up to the time of death. These samples were snap frozen and stored at -2lOC. They were assayed for virus by the technique described below. After 4 days several duplicate samples of blood were collected in anticoagulant and the plasma fractions kept for virus assay as described below. The relationship of virus in plasma to the evolution of lesions in the brain is reported in a subsequent paper dealing with the CNS phase of the infection. Pathogenesis studies. Preliminary experiments on the dose response of turkeys to V7732 established that 200 EID,, of virus deposited in the tracheas of 20 birds resulted in death of the birds within 10 days. Twenty-five birds were infected intratracheally with 200 EID,, of V7732 suspended in 0.4 ml. of tryptose phosphate broth (TPB) and placed in individual numbered cages. Birds were selected in ascending numerical order at 1, 2, 4, 6, 9 and 12 hours and at 12 hour intervals thereafter, up to 8 days after infection (PI). Two control birds, given 0.4 ml. TPB at the start of the experiment, were killed 8 days later. Each bird was anaesthetized with pentobarbitol sodium and blood samples were collected for virus titration and serology. The jugular and femoral veins were cut and the bird then perfused with cold saline. Several tissues were examined and 3 samples of each were usually taken; one for virus assay, one for immunofluorescence and one for histological study. Those samples intended to be examined by the first 2 tests were frozen in a dry-ice-alcohol mixture and stored at -2lOC. Samples for histological study were fixed in formalin and 5 ,.L thick paraffin sections were stained with haematoxylin and eosin. Tissues for virus titration were ground in pre-chilled Ten Broeck grinders and 10 per cent. extracts (wt./vol.) prepared in TPB. The extracts were centrifuged at 3000 g for 15 min. at 4OC. and the supernatant fluids titrated in 9 or lo-day-old embryonated hen eggs by allantoic inoculations. For fluorescent antibody studies, frozen sections of various tissues were stained directly with fluorescein*-labelled turkey antibody to V7732. Sometimes, a counterstain of rhodamine red conjugated bovine serum (Difco) was added to the conjugate at a concentration of 1:40. * Fluorescein isothiocyanate, Sigma

Chemicals,

St.

Louis, Missollri.

0. NARAYAN

f?t d.

131

RESULTS

Clinical

Signs

No signs of infection were seen in the birds killed at the intervals from 2 to 24 hours after infection. Those killed between 1.5 and 4 days showed ruffled feathers, diarrhoea, anorexia and various degrees of listlessness. One bird killed at 4 days, was incoordinated between 3 and 3.5 days, and by the 4th day was unable to stand; opisthotonos and paddling movements of the legs were also evident. The birds sampled at 5, 6, 6.5 and 7 days also had nervous signs which varied from incordination and spastic paralysis to somnolence. In all cases, these signs appeared within 12 to 24 hours of the time the birds were last examined. For example, the one killed at 6 days was merely listless at 5 days, but had become completely incoordinated at 5.5 days. Some died at varying intervals after the onset of nervous signs and were discarded.

I

Fig.

1.

Viraemia birds.

Viraemia

patterns

Patterns

in turkeys

2

3 4 5 Days after infection

infected

with

V7732.

“D”

6

7

indicates

a

time

of death

of individual

(Fig. I)

The time of first appearance of viraemia varied widely among the birds. Virus was first detectable in bird 1 at 12 hours and in bird 4 at 2 days after infection. Regardless of the time of appearance, the first 12 hours of viraemia were characterized by a rapid increase in titre. The first bird died during the exponential phase of viraemia. In birds surviving for some days after the peak of viraemia, there was a gradual levelling off and decline in blood titres. The second and third birds died during this stage. Viraemia in bird 4 declined and was negative by day 7 : at this time, the bird was comatose and died within the next 24 hours. Examination of a serum sample shortly before death revealed a low titre of serum neutralizing antibody. Virus titres on whole blood samples taken during the more extensive pathogenesis study are shown in Figs. 2 and 3. Such plasma samples as were tested for virus were positive, but this aspect of the study was incomplete and the titres are not reported here. B

ISFLUESZ.4

VIRUS

IN TURKEYS:

EXTRANEURAL

OF INFECTION

PHASE

,I2345870

+*+ ++ +

; I

I

Fig. 2.

234567 Days after

A comparison of virus titres in the spleen and blood with a histogram showing the degree of immunofluorescence and histological changes at various intervals. Signs + , + + and + -1-t indicate slight, moderate and severe reactions.

C?

Heart ----Blood

7

2 Days

Fig. 3.

’ infection

3 4 567 after infection

‘-\ comparison of virus titres in the heart and blood with a histogram showing the degrees of immunof?uorescence and histological changes at various intervals after infection. Signs +, + ?~ and + +~ ;- indicate slight, moderate and severe reactions.

et

0. NARAYAN

Virus Assay, ZmmunofEuorescence

133

al.

and Histological

Changes

No virus was detected in any of the tissues of birds killed at 9 hour and 2 hours after infection. At 4 hours, low titres were observed in lung and kidney SW pensions and by 12 hours, virus was isolated from the lung, kidney, ovary and spleen (Table 1). Fluorescence was noted for the first time at 12 hours in a few cells of these affected organs. Higher infectivity titres were noted in the 24 hour samples and fluorescence was noted both in parenchymal cells and in cells of the diffuse lymphocytic tissue within the visceral organs. The lung, gonad, kidney, bursa and thymus maintained high titres of virus, well above the declining blood values, over the entire period, but wide fluctuations were observed in the spleen over the test period. The trend, however, was a declining titre, parallel to, but well above that of the blood. The spleen extracts of the 6 and 6.5 day birds, which did not show viraemia, were qualitatively assayed for antibody by antibodyvirus dissociation tests and found to be positive. Fluctuations in titres of liver extracts were similar to those found in the spleen : they remained at a high level, with the exception of the 6 day sample. Pancreatic titrcs, like those of cardiac muscle, were detected after the viraemia had attained its maximum, reaching a peak between 3 to 4 days: with the exception of 2 samples, they remained at these levels until the end of the period.

SEQUBNTIAL

Hours after infection Tissues Trachea Lung Blood g;:.“d’ Adrenal Bursa F. Thymus Spleen Liver Brain Pancreas Heart Marrow Leg muscle * Virus

OF

1

V7732

IN VARIOUS

36

OF TURKEYS

48

60

144

Neg. 1*5* Neg. 1.2 Neg. NlTt Neg. Neg. Neg. Neg. N/T N/T N/T N/T N/T

2.2 1.0 Neg.

N/T l-8 Neg.

f :;

N/T 7-5

1.2

Neg.

;:: N/T 1 .il Neg. Neg. Neg. N/T N/T N/T N/T N/T

2”:; Neg. Neg. Neg. 2.8 Neg. Neg. Neg. Neg. Neg. N/T

;:; Neg. 1.2 PjJ~~.

;:;

Neg. Neg. Neg. Neg. 1.5 Neg.

35:; 3.2 5.5

expressed

as log,,

EID,,

24

TISSUES

6

I

12

TABLE

4

I

titre

DEVELOPMENT

per 0.02 g. tissue.

156

2:;

27:; Neg. N/T t N/T

= not tested

The sequential development of fluorescence and morphological changes in individual organs are described below. Generally, degenerative lesions occurred in areas in which intense fluorescence was noted earlier. Trachea. No fluorescence or pathological changes were observed in tracheal epithelium in any of the sections. Occasionally, a few fluorescent mononuclear cells were observed in the lamina propria. Lung.

Specific

fluorescence

was recognized

in mononuclear

leucocytes

among

134

INFLUENZA

VIRUS

IN TURKEYS:

EXTRANEURAL

PHASE

OF INFECTION

the air capillaries and in parabronchial (bronchiolar) epithelial cells of some lobules at 1.5 days. This focal and lobular distribution of fluorescence was maintained throughout, but varied in intensity from bird to bird. The most intense infection was noted at 4 days; large foci of fluorescence were seen in the air capillaries in the mononuclear leucocytes which had infiltrated these areas. Fluorescence in parenchymal cells were confined to the epithelial cells of the parabronchi (bronchiole) and the parabronchial atria (Fig. 4). The epithelium of the secondary and primary bronchi was not affected. Morphological alterations were lobular in distribution and, with the exception of necrosis of lymph nodules, which could be found in any area of the lung parenchyma, most of the pathological changes were located in the region of the air capillaries and parabronchi. Intense and diffuse heterophilic infiltration and necrosis of lymph nodules were noted in the sections examined at 1.5 and 2 days. The degenerate nodules were also infiltrated with heterophils. Pathological changes were most severe between 2.5 and 4 days. Dense mononuclear leucocyte infiltration and degenerative changes were noted in some lobules, varying in intensity from lobule to lobule. The lumens of the parabronchi in these lobules were filled with cellular debris and the cuboidal epithelial cells of these air tubes as well as those of the parabronchial atria were swollen and and many had sloughed (Fig. 5). Many other lobules were completely consolidated. The epithelium of the larger bronchi was little affected at any stage of the study. Spleen (Fig. 2). Fluorescence in this organ increased from that seen in small groups of lymphocytes at 1 day and involved almost the entire parenchyma, except the vascular endothelium, at 1.5 days (Fig. 6). Widespread fluorescence persisted up to 2.5 days after which it gradually decreased. By 4 days, fluorescence was restricted to individual cells in sinusoids and foci which were infiltrated with fibrinoid material. The first morphological alteration noted in the splenic parenchyma was a diffuse infiltration of heterophils in the sinusoids at 1 day. By 1.5 days, the heterophil population had increased considerably and most of the lymphocytes had disappeared. Lymph nodules were in various stages of necrosis, and many were infiltrated by heterophils. The sinusoidal area was infiltrated by a proteinaceous “fibrinoid” material in which were suspended degenerate lymphocytes, reticulum cells and heterophils (Fig. 7). Many of the large, pale-staining cells, concentric around arterioles and comprising the adenoid sheaths, also underwent degeneration. The vascular endothelium of the sheaths was unaffected. These changes were accentuated on day 2, and by 2.5 days the organ had undergone subtotal necrosis. Reparative changes were noted in subsequent sections and included mobilization of fibrinoid debris by macrophages and repopulation of the sinusoids with large lymphocytes. By day 7, the only evidence of infection in the section was an occasional encapsulated focus of necrosis. Large numbers of lymph nodules were also present.

Thymus. This organ, which contained no fluorescing cells at day 1, had between 50 and 75 per cent. of its cells infected by 1.5 days. The fluorescence was evenly distributed between the small, densely packed lyphocytes in the cortical area and the more sparsely populated medullary zone. The fluorescence in the thymus

0. NARAYAN

etal.

135

was most extensive at 1.5, 2 and 2.5 days. Widespread necrosis, accompanied by intense heterophil infiltration, was noted throughout the thymus from l-5 days onwards. Kidney. Fluorescence was only recognized in cells of the renal tubules. The reaction was first recognized in a few cells at day 1 and in subsequent sections, had spread down the tubules (Fig. 8). Lesions, likewise, were confined to renal tubules, in which cellular changes were first detectable at 2-5 days. Some cells within a few tubules appeared swollen and vacuolated and others had undergone fragmentation. Nuclei of the latter had either undergone pyknosis or karyorrhexis (Fig. 9). The most severe changes were noted in the day 4 section. Ovary. Although small groups of cells of the ovarian stroma revealed fluorescence, the follicles were not affected. Pathological changes were seen as diffuse heterophilic infiltration and necrosis of sporadic lymph nodules in the early stages, and vacuolation and lysis of individual parenchymal cells of the stroma at later intervals. Heart (Fig. 3). Fluorescence was found consistently in myocardial fibres after 1*5 days. It occurred as granular specks in the perinuclear area of one or two fibres at l-5 days and spread contiguously to involve larger numbers of fibres at subsequent intervals (Fig. 10). N umerous foci were recognized in the sections taken at 6.5 and 7 days. Pathological lesions were not identified until 5 days. Initially, fibres lost their striations and the sarcoplasm assumed a homogeneous appearance. This was accomplished or followed by a gradual loss of sarcoplasm. The nuclei seemed unaffected. Multiple foci of myocardial degeneration were noted at 6.5 and 7 days. In these affected areas, many nuclei were attached to remnants of degenerated sarcomeres, and others were clumped together in a syncytial arrangement (Fig. 11). Liver. Viral antigen was sporadically recognized in Kupffer cells in the liver sinusoids and occasionally in small groups of hepatocytes. The largest number of Kupffer cells was seen at days 2, 3 and 4. The only morphological alteration noted was a diffuse heterophilic infiltration. Gastro-intestinal tract. Starting at day 1, virus spread rapidly to involve most of the lymphatic elements in the proventriculus and intestinal tract. In the caecal tonsils, maximum fluorescence was noted at 2 and 2-5 days. The epithelium of the intestinal mucosa was not involved in any of the sections examined at later intervals. Pancreas. The appearance of small amounts of fluorescence in acinar cells at day 1 was followed by the spread of fluorescence to other cells of the acinus in subsequent sections, and then from acinus to acinus. At 2.5 days, several small groups of affected acini could be identified with fluorescence both in the nuclei and cytoplasm of affected cells (Fig. 12). This was followed by the development of almost confluent fluorescence in sections taken at later intervals. The first evidence of morphological changes was observed at day 3, when several small foci of acinar degeneration were seen. The cells were either swollen or ruptured and nuclei either lysed or condensed. The architecture of the gland in these areas was completely disrupted. Large numbers of heterophils were

136

INFLUENZA

VIRUS

IN TURKEYS:

EXTRANEURAL

PHASE

OF INFECTION

present in these lesions, but not in the rest of the apparently normal parenchyma. Confluent necrosis was seen at 25 days and in most of the subsequent sections. Pancreatic ducts and blood vessels were not affected. Bursa of Fabricius. Specific fluorescence was recognized in this organ in the 1.5 day samples.Appearing initially in large lymphoid cells of the medullary zone, fluorescence gradually spread to other lymphocytes of both the cortex and medulla of individual lobules. The row of epithelial cells separating the medulla and cortex of each follicle was also involved. Infection was restricted to several isolated follicles. The acute signs of degeneration, preceded by large numbers of heterophils observed in the spleen and thymus were absent. Four of the 7 samples examined between l-5 and 4.5 days had signs of lymphocyte depletion and necrosis in the medulla. More extensive involvement of bursal follicles was noted in sections taken between 6 and 7 days. Depletian and widespread necrosis of both cortical and medullary cells was noted. Striated muscle. No fluorescence or lesions were seen in muscles of the leg or breast. DISCUSSION

Although the infection in turkeys was caused by an influenza A virus, the evolution of the diseaseand distribution of the agent resembled more closely that described for certain paramyxoviruses than infections produced by the mammalian influenza A group. Initial spread of the agent by the blood, followed by multiplication in lympho-epithelial tissues and development of viraemia are typical of canine distemper (Appel, 1969)., rinderpest (Plowright, 1964) and NewcastIe disease(Karzon and Bang, 1951). Certain unique features were evident, however, in this infection. Among these was the limited virus involvement of the respiratory tract. Replication and pathological changes were noted only in the capillary region and the epithelial cells lining the parabronchi and parabronchial atria. The ciliated columnar epithelium of the larger bronchi and trachea was not involved. Bronchiolar and alveolar changes have been reported for human influenza (Hers, Mazurel and Mulder, 1958), but infection and lysis of mucosal cells of the upper respiratory tract are by far the commonest manifestation of influenza in many laboratory animals. Another unique feature was the mechanism of spread of the virus to all the tissues of the body. Plasma viraemia has not previously been reported for avian influenza. The influenza A virus of fowl plague has been reported to be associated with leucocytes of chickens during the viraemic phase of the disease (Todd, 1928). The source of the plasma viraemia in this study could not be determined accurately, because viral replication occurred in a large number of various cell types, including pancreatic acini, myocardium, renal tubules and the primary lymphocytic organs (spleen, thymus, caecal tonsils and bursa of Fabricius). Significantly, the vascular endothelium within affected organs did not support replication. However, because of similar day to day fluctuations of virus titres in the blood and spleen, it is probable the latter organ may have played a significant role in maintaining viraemia. Although the sequential infection of various organs may have depended on

0.

Fig. 4. Fig. 5. Fig.

6.

NARAYAN

et

d.

Fluorescence in cuboidal epithelial cells of a parabronchus and in cells of the parabronchial atria at 4 days. 400 x Cellular exudate in the lumens of a parabronchus and parabronchial atria of the lung at 4 days. H.E. 400 x Widespread fluorescence in the splenic parenchyma at 36 hours. 400 x

INFLUENZA

Fig.

7.

Fig. 8. Fig. 9.

VIRUS

IN

TURKEYS:

EXTRANEURAL

PHASE

OF INFECTION

Diffuse necrosis of the spleen with fibrinoid material deposited in sinusoidal area around adenoid sheaths. Capillaries of the sheaths are unaffected. The necrotic remains of a lymph nodule can be seen in the bottom left hand corner of the photograph. H.E. 400 x Cytoplasmic and nuclear fluorescence in renal tubular cells at 4 days. 1000 x Renal tubular nephrosis at 4 days. H.E. 800 x

0.

Fig. Fig. Fig.

NARAYAN

et d.

10. A focus of fluorescence in a few myocardial fibres at 2 days. 400 11. Focal myocardial degeneration at 7 days. H.E. 300 x 12. Nuclear and cytoplasmic fluorescence in acinar cells of the pancreas

x

at 2.5 days.

600

x

0.

NARAYAN

137

et d.

the route of inoculation, the dynamic changes occurring in each organ as the disease progressed were of considerable interest. The two extremes in this case were the spleen and the heart. The former organ was diffusely affected by 1.5 days and had undergone subtotal necrosis by 2.5 days. Repair and lymphocyte repopulation were evident by 4 days. Myocardial involvement occurred only after viraemia became established. Although there was a progressive increase in fluorescence in myocardial fibres from day 2 onwards, severe histological lesions were not observed until 7 days, at which time splenic lesions were almost completely resolved. SUMMARY

This report describes the pathogenesis of infection following intratracheal infection of 12-week-old turkeys with 200 EIDao of A/Turkey/Ontario/66 (V7732) influenza virus, which killed all birds in about 10 days. The incubation phase of the infection lasts from 2 to 24 hours, during which time the virus was disseminated from the lungs to other visceral organs by the blood. No clinical signs or pathological alterations were noted. Diarrhoea and listlessness were typical signs of the second or visceral phase, which involved the spread of the agent to the lymphoepithelial tissues. Rapid viral replication caused widespread lysis of cells and resulted in a pronounced plasma viraemia. The degenerative, inflammatory and reparative changes noted varied according to the time of infection and individual susceptibilities of various organs. The spleen was the first organ to undergo both necrosis and repair. The heart was the last to undergo degeneration and birds died before cardiac lesions could be resolved. ACKNOWLEDGMENTS

Financial assistance from the Ontario Department of Agriculture and Food, the Ontario Turkey Hatchery Association and the Ontario Veterinary College Research Committee are gratefully acknowledged. REFERENCES

Appel, M. J. G. (1969). A mer. J. vet. Res., 30, 1167. Becker, W. B., and Uys, C. J. (1967). J. camp. Path., 77, 159. Hers, J. F. P., Mazurel, N., and Mulder, J. (1958). Lance& ii, 1141. Jungherr, E., Tyzzer, E. E., Brandly, C. A., and Moses H. E. (1946). Amer.

J. vet.

Res. 7,250.

Karzon, D. T., and Bang, F. B. (1951). J. exp. Med., 93, 267. Narayan, O., Lang, G., and Rouse, B. T. (1969a). Arch ges. Virusforsch., (1969b). Ibid., 166. Pereira, H. G. (1969). Proc. Royal Sot. Med., 62, 43. Plowright, W. (1964). J. Hyg. Camb., 62, 257. Rohrer, H. (1947). Mh. vet. Med., 2, 33. Todd, C. (1928). &it. 1. exp. Path., 9, 101. Uys, C. J., and Becker, W. B. (1967). J. camp. Path., 77, 167. [Received

for publication,

April

15th, 19711

26, 149