Avian Pathology

ISSN: 0307-9457 (Print) 1465-3338 (Online) Journal homepage: http://www.tandfonline.com/loi/cavp20

Pathogenicity of fowl enteroviruses Mireille Decaesstecker , G. Charlier , J. Peeters & G. Meulemans To cite this article: Mireille Decaesstecker , G. Charlier , J. Peeters & G. Meulemans (1989) Pathogenicity of fowl enteroviruses, Avian Pathology, 18:4, 697-713 To link to this article: http://dx.doi.org/10.1080/03079458908418643

Published online: 12 Nov 2007.

Submit your article to this journal

Article views: 74

View related articles

Citing articles: 10 View citing articles

Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=cavp20 Download by: [37.44.207.181]

Date: 26 January 2017, At: 15:37

Avian Pathology, 18: 697-713, 1989

PATHOGENICITY OF FOWL ENTEROVIRUSES

Mireille DECAESSTECKER, G. CHARLIER, J. PEETERS and G. MEULEMANS National Institute for Veterinary Research, 99 Groeselenberg, 1180 Brussels, Belgium SUMMARY The pathogenicity of avian nephritis virus, entero-like particles described by McNulty et al. (Avian Pathology, 13: 429), the entero PV2 and entero 3 isolated in our laboratory, was studied by oral inoculation of one-day-old specific pathogen-free chickens. All viruses were shown by immunofluorescence and transmission electron microscopy to multiply in the cytoplasm of enterocytes but histological lesions of the intestine were only observed in chickens infected with McNulty's entero-like particles, entero PV2 and entero 3. Those lesions were present from 3 days post inoculation but were most prominent on the 7th day post inoculation. Variable histological lesions of the pancreas, proventriculus or kidneys were observed 14 days post inoculation with McNulty's entero-like particles, entero PV2 or entero 3. Avian nephritis virus principally induced kidney lesions. This demonstrates that members of a same species of fowl enteroviruses may have different tropisms and could induce different clinical signs and pathology as nephritis or malabsorption syndrome. INTRODUCTION In an accompanying paper (Decaesstecker and Meulemans, 1989) we have proposed the classification of fowl enteroviruses into two different species: the first, avian encephalomyelitis virus (AEV) (Butterfield et al., 1969) and the second, fowl enteroviruses including the avian nephritis virus (ANV) of Yamaguchi et al. (1979) and three different entero-like particles (ELPs) first isolated by McNulty et al. (1984) (ELP-1), Decaesstecker et al. (1986), -entero PV2 - and by Meulemans etal. (1986)-entero 3. Here we examine the pathogenicity of the viruses of the second species following experimental oral inoculation of one-day-old specific pathogen-free (SPF) chickens. MATERIALS AND METHODS Chickens and accommodation Chickens were hatched from SPF eggs (Valo Lohman) and reared from hatching Received 7 October 1988 Accepted 21 February 1989

698

Mireille Decaesstecker et al.

on wire floor in isolators. Experimental inoculation was performed by the oral route using 100 /jl virus preparation. The different groups of chickens were reared in separate isolators. Viruses Two entero-like viruses (entero PV2 and entero 3) isolated in our laboratory (Decaesstecker et al, 1986; Meulemans et al, 1986), the Japanese ANV of Yamaguchi et al (1979) and the ELP-1 described by McNulty et al. (1984) were used. The ANV and the ELP-1 were kindly supplied by Dr McFerran, Veterinary Research Laboratories, Stormont, Belfast, BT4 3SD, Northern Ireland. The ANV was multiplied by serial passages in chicken kidney cells (CKC) as described in Decaesstecker and Meulemans (1989). The final titre was 2.13 x 108TCID50/ml as determined by the tissue culture infectious dose 5O(TCID5O) method and calculated according to Reed and Muench (1938). The ELP-1, the entero PV2 and the entero 3 inocula were 20% (w/v) caecal and intestinal homogenates made in TNE (Tris 0.01 M, NaCl 0.1 M, EDTA 0.001 M pH 8.7) from 4-day-old SPF chickens inoculated at one day of age respectively with each of those viruses. Entero PV2 and entero 3 were shown to be free of other enteric pathogens as described in Decaesstecker et al. (1986). The ELP-1 strain was shown to contain reovirus contaminant particles (McNulty etal, 1984). Experimental design Experiment 1. Three groups of SPF chickens were inoculated respectively at 1, 3 or 7 days of age with the ELP-1. One to four chicks were killed from the first to the 7th or 8th day post inoculation (pi). Three intestinal areas were collected immediately after death. The first area was in the middle of the ascending loop of the duodenum (area 1), the second was lying 7 to 10 cm proximal to the yolk stalk remnant (area 2) and the third was the terminal ileum (area 3). Two SPF control birds were killed when at 5 days old and treated in the same way. Cryostat sections (5 /um) were immediately made from each intestinal sample and were examined by direct immunofluorescence for the presence of viral antigen. Experiment 2. Four groups of 1-day-old SPF chickens were inoculated orally respectively with the ELP-1, the entero PV2, the entero 3 or the ANV. A further group was the uninoculated control. Four or five birds from all the groups were killed on days 3, 7 and 14 pi. Three samples were collected from area 2 immediately after death: one was directly submitted to cryostat section for immunofluorescence staining, one was directly fixed for histological examination whilst the third was rinsed in PBS and fixed in buffered cacodylate glutaraldehyde for scanning electron microscopy (SEM) and transmission electron microscopy (TEM). TEM was only performed on 4day-old chickens. The caeca were recovered and frozen at -20°C for demonstration of the presence of viral particles by TEM. The kidneys, proventriculus and pancreas

Pathogenicity of fowl enteroviiuses

699

were recovered only from 15-day-old birds and immediately fixed for histological examination. Direct immunofluorescence test Cryostat sections were fixed in cold acetone for 10 min and stained for 1 h at 37°C with fluorescein conjugated hyperimmune serum against entero 3 produced as described by Decaesstecker and Meulemans (1989). The sections were then rinsed in water, mounted in buffered glycerol pH 8.5 and examined for the presence of viral antigen with a Leitz Wetzlar microscope using incident ultraviolet light. Virus demonstration in caecal contents In each group, the caecal contents were pooled and adjusted to 12 ml in TNE pH 8.7. Each sample was homogenised (v/v) in Arklone (ICI) and centrifuged 30 min at 12.000 g. The aqueous phase was recovered, treated with SDS 0.25% for 30 min at 37°C and spun at 90000 g in a SW40 rotor (Beckman) for 1 h. The pellet was resuspended in 0.5 ml H 2 O and examined by TEM after negative staining using 2% uranyl acetate for the demonstration of viral particles. Histological examination Samples fixed in 10% (v/v) formalin in PBS were dehydrated in ethanol and isopropanol, cleared in histosol, embedded in Paraplast-plus using the standard techniques and 5 pm sections were cut with a Reichert autocut. Sections were stained with haematoxylin-eosin and mounted using a synthetic resin medium: DPX (BDH). Scanning electron microscopy Tissues were fixed in 2.5% (w/v) cacodylate buffered glutaraldehyde pH 7.4 and postfixed in 1% (w/v) osmium tetroxide. The samples were then dehydrated in serial baths of ethanol and acetone and subjected to critical point drying in liquid carbon dioxide. They were then glued with carbon cement on an aluminium support and coated with a thin layer of gold (400 A) in a vacuum evaporator. Samples were examined in a Philips 501 microscope. Transmission electron microscopy The samples were fixed in 2.5% (w/v) cacodylate buffered glutaraldehyde pH 7.4, postfixed in 1% osmium tetroxide and stained with 2% uranyl acetate. They were dehydrated in ethanol and embedded in Taab 812 resin (Taab Laboratories, Reading, England). Villus/crypt ratio The villus crypt ratio was estimated after examination of four samples for each group. Five pieces of tissue were prepared for each bird for TEM. One piece of tissue was prepared for each bird for histology. RESULTS The first experiment was performed in order to determine the period and area of maximum ELP multiplication in the intestine and the effect of age at inoculation. Positive immunofluorescence reactions were observed in the cytoplasm of epithelial cells of all three intestinal areas as early as one day after inoculation. Most positive cells were found in area 2, followed by area 1 and then area 3. On day 1 pi, the

700

Mireille Decaesstecker et al.

positive cells were concentrated at the base of the intestinal villi whilst later on, they were scattered on the whole surface of the villi. One-day-old chickens were more susceptible to virus multiplication than older birds. As seen in Table 1, the number of positive cells and the duration of virus replication were a function of age at inoculation: we could detect the presence of ELP antigen in many cells for 8 days pi after inoculation at one day of age. This was reduced to 5 days after inoculation at 3 days of age and to 4 days when inoculation was performed at 7 days. However, individual variations among chicks were observed. No immunofluorescence was detected in 5 day old uninoculated chickens. On the basis of the above results, area 2 in birds inoculated at 1 day of age was used to compare the pathogenicity of ANV, ELP-1, entero PV2 and entero 3 after experimental inoculation. The results are presented in Table 2. The multiplication of the different viruses in area 2 of the intestine was demonstrated by positive immunofluorescence reactions. Fluorescence was observed in the cytoplasm of enterocytes 3 and 7 days after inoculation with ELP-1. entero PV2 or entero 3. A few cells with weak fluorescence were observed in some birds on day 14 pi. With ANV, some birds (3/5) were shown to be positive on day 3, and only one bird was found positive on day 7 pi. Moreover, the number of positive cells was smaller than in birds inoculated with the different ELP inocula. No immunofluorescence was found in intestinal sections of uninoculated control chickens. Intestinal virus replication was indicated by the presence of virus particles in the caecal contents of chickens 3 and 7 days after inoculation with ELP-1, entero PV2 and entero 3. Virus shedding lasted until 14 days pi with entero PV2. No viral particles could be observed in caecal preparations from the ANV group and from uninoculated chickens. Vacuoles containing crystalline arrays of virus particles located in the cytoplasm of enterocytes (Fig. 1) were observed by TEM of the villi, 3 days pi for each of the four inoculated viruses. However, they were more numerous for entero PV2 and relatively rare for ANV although present. Infected cells were mostly found at the base of the villi. The presence of crystalline arrays of virus was generally associated with vacuolar degeneration of enterocytes and with decreased length of the microvilli. These lesions lead to the complete desquamation of the epithelial cells in certain areas. Despite the limited observation of ANV crystals, enterocytes of the chickens belonging to this group were also sometimes vacuolated. Modifications of the intestinal villi were observed by SEM principally 3 and 7 days pi with ELP-1, entero PV2 and entero 3. Compared with the uninoculated chickens, the decrease of the villus/crypt length ratios was striking (Table 2, Figs 2a,b). This effect was most pronounced 7 days pi and disappeared on day 14 pi. Lesions of the enterocytes varying from decreased length of the microvillus border to the total desquamation of the intestinal epithelium (Fig. 2c) was observed from

Table 1. Direct immunofluorescent detection of the replication of an entero-like virus (McNulty et al., 1984) in three different intestinal areas after experimental oral inoculation ofSPF chickens at 1,3 or 7 days of age. Days post inoculation

Age at Intesinoculation tinal area (d) (18)* 3 (14)

1 2 3

ND ND**

1 2 3

7 (13)

1 2 3

Controls (2)

1 2 3

+ ND eg

ND

* = number of chickens analysed in this group; **:ND = not done. - = none; (+) = rare; + = occasional; ++ = often; +++ = widespread.

o

Table 2. Histopathological, immunofluorescent and ultra structural findings in the intestine after experimental oral inoculation of 1 -day-old SPF chickens with various fowl enteroviruses 3 days pi

o

14 days pi

7 days pi

Con- ELP-1 Ent- Ent- AN Con- ELP-1 Ent- Ent- AN Con- ELP-1 Ent- Ent- AN trols ero 3 ero trols ero 3 ero trols ero 3 ero PV2 PV2 PV2 (4)a (4) (4) (4) (4) (4) (4) (4) (4) (4) (4) (5) (5) (4) (5) 4 - 56 c 2 1-3 1-3 3 - 4 5 - 6 2 - 4 2 - 4 1-3 5 - 6 5 - 6 5 - 6 5 - 6 5 - 6 5 - 6 -(4) +(4) +(3) +(3) -(4) -(4) ++(4) +(1) +(1) -(4) -(4) -(5) -(5) -(4) -(5) -(4) +(4) +(1) +(2) -(4) -(4) +(1) +(2) -(4) -(4) -(4) -(5) -(5) -(4) -(5) -(4)

+(2) +(3) +(3) -(4)

-(4)

+(3) +(4) +(4) -(4)

-(4)

+(2) +(5) +(4) -(5)

3_5 -(4) -(4) -(4) -(4) -(4)

i_3 +(3) +(3) -(4) +(2) +(3)

5-6 -(4) -(4) -(4) -(4) -(4)

1-5 +(4) +(4) +(2) +(4) ++(4)

3-8 -(4) -(4) -(4) -(4) -(4)

4-8 -(5) -(5) ±(2) ±(2) ±(5)

-(4)

-(2) -(3) +(D +(2) ++(2)

-(3) +(D

-(5)

-

-

+

-

i_4 +(4) ++(3) -(4) +(D +(D

-(4)

3-6 -(4) -(4) -(4) +(D -(4)

1-5 +(4) +(3) +(D +(4) +(3)

0.5-5 3 - 6 +(4) ±(2) +(3) -(4) +(2) -(4) +(4) +(2) +(3) -(4)

-(2)e -(4) +(2) +(D ++(1) ++(2) )+++(l )

-(3) -d) +(2) +(D +(3) +d) ++(1) ++(2) ++(1) +++(1) +

d

++(2) +++(2 -

2-4 +(2) +(2) -(4) +(2) +(2)

+

+

+

-

+

+

+

+

-

+

+

-

8-106 -(5) ±(3) -(5) +(1) -(5) +(4) ±(2) -(4) ±(5) +(4)

-

8-10 -(5) -(5) -(5) -(5) -(5)

a: number of chickens examined; b: number of chickens presenting this lesion; c: inferior and superior limits observed; d: in this test 3 chickens were analysed; c: in this test 6 chickens were analysed. For light microscopy and SEM: - = absence of the lesion; ± = weak lesion; + = presence of the lesion; ++ = very conspicuous lesions.

5? sstecker et al.

Histology: intestine Villus/crypt ratio Lengthening of the crypts Degeneration of enterocytes Increased number of mononuclear cells in the lamina propria SEM: intestine Villus/crypt ratio Lengthening of the crypts Shortening of the villi Crypt swelling Cells swelling Degeneration of enterocytes Immunofluorescence: intestine Negative Few cells + Some cells ++ Numerous cells +++ TEM Presence of virus in the caeca Presence of virus in small intestinal sections

703

Pathogenicity of fowl enteroviruses

3 days pi with a peak 7 days pi. At 14 days pi, the general aspect of the intestinal villi was almost normal again. A swelling of the apical enterocytes of the villi was observed principally 7 days pi. The same lesions as those described above for SEM were established by light microscopy. Vacuolar degeneration of the enterocytes located at the top of the villi was observed 3 and 7 days pi. In addition, the lamina propria was infiltrated by mononuclear cells. This invasion persisted 14 days pi. In the group of chickens inoculated with the ANV, only a small increase of the crypt depth was observed 7 days pi. Swelling of the apical enterocytes was also observed by SEM at 3 days pi but mostly at 7 days pi. However,, this was never accompanied by cellular degeneration. No intestinal lesions were observed by histology. Histo-pathological changes of the pancreas, proventriculus and kidneys are summarised in Table 3. Localised desquamation of glandular epithelial cells of the proventriculus was observed 14 days pi with ELP-1 and with ANV. Infection by entero PV2 was associated with glandular necrosis and the same lesion was also observed in one chick inoculated with entero 3. In some cases these changes were associated with accumulation of necrotic material in the lumen of the glands and with mononuclear cell infiltration of the surrounding tissue (Figs 3d, e). There was also an increase and an hyperplasia of the lymphoid follicules in the lamina propria. Table 3. Histopathological changes of pancreas, proventriculus and kidney observed 14 days after experimental oral inoculation of 1-day-old. SPF chickens with various fowl enteroviruses 14 days post inoculation ELP-1 (5)

Entero 3 (5)

PV2 (4)

ANV (5)

1

0 0

0 1

3 2

0 1

0 0 0

0 4 3

1 0 1

3 0 0

0 2 1

0 0

1 1

0 1

0 2

1 1

0 0 0

0 0 0

3 0 0

0 0 0

1 1 2

Controls (4)* Pancreas

Areas of degeneration Lymphoid follicles

o**

Proventriculus

Glandular necrosis Epithelial desquamation Cellular debris in the lumen Lymphocytic reaction with cellular debris in the lumen Lymphoid follicles Kidney

Interstitial nephritis Tubulonephrosis Lymphoid follicles

* = number of chickens examined. ** = number of chickens presenting this lesion.

7 04

Mireille Decaesstecker et al.

In chickens inoculated with entero PV2, some areas of acinar degeneration with partial to complete disappearance of zymogen granules were observed in the exocrine glands of the pancreas (Figs 3b,c). The islets of Langerhans remained unaffected. Slight to moderate interstitial nephritis was found in chickens infected with entero 3 (Fig. 3a), whereas tubulonephrosis associated with interstitial nephritis was observed in one chick infected by ANV. In two other animals of the latter group some lymphoid follicles were also seen. These lesions were not observed in uninoculated control chicks. DISCUSSION Although all viruses used in this study belong to the same species (Decaesstecker and Meulemans, 1989), they showed a difference in tropism. ELP-1, entero PV2 and entero 3 were clearly shown to replicate intensively in enterocytes and to induce intestinal lesions after inoculation of one-day-old SPF chickens. In contrast, no important modification of the intestine was observed after infection with ANV, although some viral replication occurred in some enterocytes as demonstrated by TEM and immunofluorescence. This replication was however limited by comparison with the other viruses. Differences in location, severity and duration of the intestinal lesions (decrease of the villus/crypt ratio, lengthening of the crypts, shortening and stunting of the villi, cell swelling and degeneration) were recorded among ELP-1, entero PV2 and entero 3. Entero PV2 which produced the most important and long lasting lesions, was also shown to be excreted during the entire observation period in contrast with the other viruses tested. All enteroviruses tested induced proventricular lesions, but glandular necrosis and lymphoid cell infiltration were prominent in chickens infected with entero PV2. This virus caused also pancreatic degeneration. Enteric problems followed by growth retardation is a common feature of the malabsorption syndrome (McFerran et al., 1983); entero-like particles as ELP-1 (McNulty et al., 1984), entero PV2 and entero 3 (Decaesstecker et al., 1986, Meulemans et al., 1986) have already been incriminated in this pathology. Pancreatic and proventricular lesions are also reported in field cases of malabsorption syndrome. Pancreatic degeneration and fibrosis (Randall et ah, 1981; Reece et al., 1984; Riddell and Derow, 1984; Sinclair et al., 1984; Martland and Farmer, 1986) and hypertrophy of the glandular epithelium of the proventriculus with lymphoid cell infiltration (Kouwenhoven et al., 1978; Page et al., 1982) have been reported as features of the malabsorption/stunting syndrome. Entero PV2 which causes growth retardation (Decaesstecker et al., 1986; Meulemans et al., 1986), produced intestinal, proventricular and pancreatic lesions and is therefore a good candidate as the aetiological agent for malabsorption syndrome as already suggested (Decaesstecker et al., 1986;Meulemanse?a/., 1986). However, the pancreatic lesions observed after experimental inoculation of this virus were less severe as those described in the field cases. Extensive degeneration, atrophy and fibroplasia reported to occur in the exocrine pancreatic tissue (Randall et al., 1981) were not seen. This could be due to the early age at which our experimental

Pathogenicity of fowl enteroviruses

705

Fig. 1. Crystalline arrays of entero-like particles (the entero 3 strain) in a section of a villous enterocyte from area 2 of the intestine 3 days after oral inoculation of this virus in a one-day-old SPF chicken. Bar = 500 nm.

706

Miieille Decaesstecker et al.

Fig. 2. Scanning electron microscopy findings. a. General aspect of the intestinal villi and crypts of a 7-day-old SPF uninoculated control chicken. Bar = 100 pm. b. General aspect of the intestinal villi and crypts of a 7-day-old SPF chicken experimentally inoculated by the oral route at one-day-old with the entero PV2. Note the decrease of the villus length and the increase of the crypt length in comparison with the control chickens. Bar = 100 pm. c. Desquamated area on the top of the villi of a 7-day-old SPF chicken inoculated by the oral route at day-old with the entero PV2. Bar = 10 y.m.

Pathogenicity of fowl enterovituses

Fig. 2. Scanning electron microscopy findings.

707

708

Mireille Decaesstecker et at.

Fig. 3. Histological sections (haematoxylin-eosin staining) after oral infection of one-day-old chickens. a. Kidney section of an entero 3-infected chick: note interstitial infiltration of mononuclear cells. Bar = 50 nm. b. Pancreas section of an entero PV2-infected chick: note the degeneration of the acini (arrow heads) with the disparition of zymogen granules. Bar = 50 fim. c. Pancreas section of an entero PV2-infected chick: note the conservation of acini structure but with the dilatation of the lumen of some of them (arrow head). Bar = 50 (im. d. Proventriculus section of an entero 3-infected chick: note the presence of cellular debris in the lumen of a gland and the congested blood vessels (arrow head). Bar = 100 fim. e. Proventriculus section of an entero PV2-infected chick: note the important mononuclear cell infiltration of the submucosa. Bar = 100 \im.

Pathogenicity of fowl enteioviruses

ft

Fig. 3. Histological sections (haematoxylin-eosin staining) after oral infection of one-day-old chickens.

709

Pathogenicity of fowl enteroviruses

711

birds were killed. Indeed, the earliest changes observed in the field cases are also limited to the disappearance of zymogen granules (Randall et al., 1981). In this study, the entero 3 induced interstitial nephritis lesions which are specific of the ANV (Maeda et al, 1979). The clinical manifestations of ANV are quite different (Maeda et al, 1979) from the other enteroviruses and in contrast no important growth retardation was observed after experimental inoculation of ANV (McFerran and McNulty, 1986). Serological surveys for ANV in Japan (Imada et al., 1980) and in Northern Ireland (Connor et al., 1987) have shown the ubiquity of antibodies against this virus in broiler flocks and in some SPF flocks. However, as ANV is antigenically identical to other fowl enteroviruses (Decaesstecker and Meulemans, 1989) those studies may reflect the presence of other related enteroviruses as well as ANV. Therefore, interpretation of such serological studies is difficult as they indicate the presence of one or another fowl enterovirus having different tropisms and which could be involved in different clinical manifestations as interstitial nephritis or malabsorption syndrome. Acknowledgements This work was supported by a grant from l'Institut pour l'Encouragement de la Recherche Scientifique dans l'Industrie et l'Agriculture. The skilful technical assistance of Mrs Marina Ledecq and Mrs Riet Geeroms is gratefully acknowledged. REFERENCES Butterfîeld, W.K., Luginbuhl, R.E. and Helmboldt, CF. (1969). Characterisation of avian encephalomyelitis virus (an avian enterovirus). Avian Diseases, 13: 363-378. Connor, T.J., McNeilly, F., McFerran, J.B. and McNulty, M.S. (1987). A survey of avian sera from Northern Ireland for antibody to avian nephritis virus. Avian Pathology, 16: 15-20. Decaesstecker, M., Charlier, G. and Meulemans, G. (1986). Significance of parvoviruses, enterolike viruses and reoviruses in the aetiology of the chicken malabsorption syndrome. Avian Pathology, 15: 769-782. Decaesstecker, M., and Meulemans, G. (1989). Antigenic relationship between fowl enteroviruses. Avian Pathology, 18: xxxxxxxxxxxx. Imada, T., Yamaguchi, S., Miura, N. and Kawamura, H. (1980). Antibody survey against avian nephritis virus among chickens in Japan. National Institute of Animal Health Quarterly, 20: 79-80. Kouwenhoven, B., Davelaar, F.G. and Van Walsum, J. (1978). Infectious proventriculitis causing runting in broilers. Avian Pathology, 7:183-187. Maeda, M., Imada, T., Taniguchi, T. and Horiuuhi, T. (1979). Pathological changes in chicks inoculated with the picornavirus 'Avian Nephritis Virus'. Avian Diseases, 23: 589-596. Martland, M.F. and Farmer, H. (1986). Pancreatic duct obstruction in a stunting syndrome of broiler chickens. Veterinary Record, 118: 531-534. McFerran, J.B., McNulty, M.S., McCracken, R.M. and Greene, J. (1983). Enteritis and associated problems. In: Disease Prevention and Control in Poultry Production pp. 129-138. Edited by Hungerford, T.G. Sydney: University Press. McFerran, J.B. and McNulty, M.S. (1986). Recent advances in enterovirus infections of birds. In: Acute Virus Infections of Poultry, pp. 195-202. Edited by McFerran, J.B. and McNulty, M.S. Brussels: Martinus Nijhoff. McNulty, M.S., Allan, G.M., Connor, T.J., McFerran, J.B. and McCracken, R.M. (1984). An entero-like virus associated with the runting syndrome in broiler chickens. Avian Pathology, 13: 429-439.

712

Mireille Decaessteckei et al.

Meulemans, G.,Decaesstecker, M. and Charlier, G. (1986). Runting syndrome in broiler chickens. Experimental reproduction studies. In: Acute Virus Infections of Poultry, pp. 179-189. Edited by McFerran, J.B. and McNulty, M.S. Brussels: Martinus Nijhoff. Page, R.K., Fletcher, O.J., Rowland, G.N., Gaudry, J. and Villegas, P. (1982). Malabsorption syndrome in broiler chickens. Avian Diseases, 26: 618-624. Randall, C.J., Wyeth, P.J. and Higgins, R.J. (1981). Pancreatic lesions in stunted broilers. Veterinary Record, 109: 125-126. Reece, R.L., Hooger, P.T., Tate, S.H., Beddome, V.D., Forsyth, W.M., Scott, P.C. and Barr, D.A. (1984). Field, clinical and pthological observations of a runting and stunting syndrome in broilers. Veterinary Record, 115: 483-485. Reed, L.J. and Muench, J. (1938). A simple method of estimating fifty percent end point. American Journal of Hygiene, 27: 493-497. Riddell, C. and Derow, D. (1984). Infectious stunting and pancreatic fibrosis in broiler chickens in Saskatchewan. Avian Diseases, 29: 107-115. Sinclair, A.J., Embury, D.H., Smart, I.J., Barr, D.A., Reece, R.L., Hooper, P.T. and Gould, J.A. (1984). Pancreatic degeneration in broilers with runting and stunting syndrome. Veterinary Record, 115: 485-488. Yamaguchi, S., Imada, T. and Kawamura, H. (1979). Characterization of a picornavirus isolated from broiler chicks. Avian Diseases, 23: 571-581. RESUME Etude du pouvoir pathogéne de différents entérovirus des volailles

Le pouvoir pathogène de quatre entérovirus du poulet: virus de la néphrite aviaire, particules de pseudo-entérovirus décrits par McNulty et coll. (Avian Pathology, 13: 429), entérovirus PV2 et entéro 3 isolés dans notre laboratoire, a été étudié par inoculation orale de poulets âgés d'un jour exempts d'organismes pathogènes spécifiés. Il a été démontré par immunofluorescence et microscopie électronique que tous les virus se multipliaient dans le cytoplasme des entérocytes, mais des lésions histologiques n'ont été observées que chez les poulets infectés avec les particules de pseudo-entérovirus, l'entérovirus PV2 et l'entéro 3. Ces lésions étaient présentes dès le troisième jour après l'inoculation et étaient maximum le septième jour après l'inoculation. Des lésions histologiques variables du pancréas, du proventricule ou des reins ont été observées 14 jours après l'inoculation avec les particules de pseudoentérovirus de McNulty, l'entérovirus PV2 ou l'entéro 3. Le virus de la néphrite aviaire a induit principalement des lésions des reins. Ceci démontre que les membres d'une même espèce d'entérovirus des volailles peuvent avoir des tropismes différents et induire des signes cliniques et une pathologie aussi diverse qu'une néphrite ou un syndrome de malabsorption. Des études séro-épidémiologiques des entérovirus des volailles impliquent donc d'être corrélées avec les symptômes cliniques pour en interprêter correctement les résultats. ZUSAMMENFASSUNG Die Pathogenität von Geflügelviren

Die Pathogenität von vier Hühnerenteroviren: Aviären Nephritis Virus, durch McNulty et dl. (Avian Pathology, 13: 429) beschriebene Enteroähnliche Partikel und im eigenen Labor isolierten Viren Entero PV2 und Entero 3 wurde durch orale Inokulation von SPF-Eintagsküken untersucht. Durch Immunofluoreszenz und Transmissions-Elektronenmikroskopie wurde nachgewiesen, daß sich alle diese Viren im Zytoplasma von Enterozyten vermehren. Histologische Darmveränderungen wurden aber nur bei den mit den Enteroähnlichen

Pathogenicity of fowl enteroviruses

713

Partikeln von McNulty, mit Entero PV2 und Entero 3 infizierten Küken beobachtet. Die Veränderungen waren ab dem 3. Tag nach Inokulation vorhanden, aber am 7. Tag nach derselben am stärksten ausgeprägt. Variable histologische Veränderungen im Pankreas, Vormagen oder Nieren wurden 14 Tage nach Inokulation mit McNulty's Enteroähnlichen Partikeln, Entero PV2 oder Entero 3 beobachtet. Das Aviäre Nephritisvirus verursachte hauptsächlich Nierenveränderungen. Dies besagt, daj3 die Mitglieder der gleichen Geflügelenterovirusspecies einen verschiedenen Tropismus haben und verschiedene klinische Symptome und pathologische Veränderungen wie Nephritis oder Malabsorptionssyndrom verursachen können. Für eine korrekte Interpretation müssen daher konsequenterweise seroepidemiologische Untersuchungen der Geflügelenteroviren mit den klinischen Symptomen korreliert werden. RESUMEN Patógenicidad de los enterovirus aviares En pollitos de un día de edad libres de gérmenes patógenos específicos inoculados por vía oral, se estudió la patógenicidad de cuatro enterovirus: el virus de la nefritis aviar, las partículas similares a enterovirus descritas por McNulty et al. (Avian Pathology, 13: 429), el enterovirus PV2 y el enterovirus 3 aislados ambos en nuestro laboratorio. Mediante inmunofluorescencia y microscopía electrónica se comprobó que todos los virus citados se multiplicaban en el citoplasma de los enterocitos aunque sólamente se observaron lesiones histológicas intestinales en los pollitos infectados con las partículas de McNulty, el enterovirus PV2 y el 3. Dichas lesiones se detectaron a los 3 días post inoculación aunque fueron más evidentes el séptimo día postinoculación. El día 14 postinoculación se observaron lesiones histológicas en el páncreas, proventrículo o los ríñones de aves infectadas con las partículas de McNulty, el enterovirus PV2 o el 3. El virus de la nefritis aviar indujo fundamentalmente lesiones renales. Estos hallazgos demuestran que miembros de una misma especie de enterovirus aviares pueden mostrar tropismos diferentes e inducir distintos síntomas clínicos y anatomopatológicos como nefritis y el síndrome de malabsorción. Para la interpretación correcta de dichos hallazgos debieran hacerse estudios seroepidemiológicos de los enterovirus aviares correlacionándolos con los síntomas clínicos.