adenovirus infection in lambs : ii. experimental infection of lambs

Zbl. Vet. Med. B, 24, 529-541 (1977) @ 1977 Verlag Paul Parey, Berlin und Hamburg ISSN 0514-7166/ASTM-Coden: ZVRBA2

From the Veterinary Institute, BCkCscsaba, Department of Epizootiology, University of Veterinary Sciences and Central Veterinary Institute,

Budapest, Hungary

Adenovirus Infection in Lambs 11. Experimental Infection of Lambs

BY

V. PALYA, S. BELAK and V. PALFI

With 10 figures

(Received f o r publication July 9, 1971)

Introduction In a previous paper (3) outbreaks of pneumoenteritis were reported in

two large-scale lamb fattening farms in the spring of 1974. The widespread infections were aetiologically related to adenoviruses belonging to different serotypes. Strain ORT/l 1 1 isolated in Farm “A” was serologically related to strain Het/3 isolated previously from sheep in Hungary (1, 2, 5). Strain GY/14 isolated in Farm “B” was serologically related to ovine adenovirus type 1. Since no data are available on the pathogenicity of ovine adenovirus type 1, isolated by MCFERRAN in Northern Ireland (4), experimental infection was carried out with the GY/14 strain.

Material and Methods Experimental animals

Eight colostrum-deprived Merino lambs were used for experimental infection. Lambs were aged 3 weeks and their sera contained no neutralizing antibodies to strain GY/14.

Virus Secondary lamb kidney cell cultures were used for the propagation of the

virus. Experimental infection was made with the 4th passage of the virus.

Experimental infection Five lambs were inoculated, each with 1 ml. of virus suspension

( los TCID,,) intratracheally and 1.5 ml. virus suspension intranasally (Nos. 1-5). Inoculation was repeated on the following day. One further animal (No. 6) was put in the inoculated group as contact control. Two lambs (Nos. 7 and 8 were inoculated with uninfected tissue culture supernatant in a

530 PALYA, BELAK and PALFI

similar way. Body temperature was recorded each day and from the 3rd pi. day onwards nasal and rectal swabs were collected daily. Urine was collected from four infected animals on the 6th and 7th days following infection and on the 8th day from a control animal. Inoculated animals were euthanized 2, 4, 6, 8 and 13 days pi. The contact control animal was killed on the 14th day pi. The uninfected control animals were killed on the 5th and 9th days pi. Sera collected a t the time of the inoculation and slaughtering were tested in neutralization tests. The killed animals were subjected to pathological and histopathological examinations.

Histological examinations Lungs, nasal- and tracheal mucous membranes, conjunctiva, retropharyn-

geal-, peribronchial- and mesenterial lymph nodes, spleen, liver, kidneys, small intestines and brain of three animals were examined (Nos. 4, 5 and 6). Tissue samples were fixed in 10 per cent formalin and embedded in paraffin. Sections were stained with haemalaun-eosin.

Bacteriological, virological and urine examinations Lungs, peribronchial lymph nodes, intestines, mesenteric lymph nodes,

spleens, livers and kidneys were cultured. Homogenates of the above mention- ed organs were prepared for the reisolation of the virus on secondary lamb kidney monolayers. Urine samples were tested with Hema-combistix reagent strips (Ames) to detect glucose, protein, blood and pH value.

Results Clinical observations

No animals died of the infection. Elevated temperature with 0.3 to 1.5 OC above the normal was observed in the infected animals between the 2nd and 6th days following infection. In animal No. 5 and in the contact control the primary rise was followed by a second one 5-6 days later.

Except for animal No. 1 all lambs sneezed and had nasal discharge on the 3rd and 4th days pi. During the temperature rise the animals showed anorexia and inappetence. In animal No. 1, which was killed 2 days following infection, no clinical sign except fever and anorexia was seen. Concurrently or one day later respiratory symptoms were observed in every animal. Respiratory symptoms prevailed for the complete observation period and animals Nos. 5 and 6 showed clinical signs of severe neumonia during the second febrile period. Along with these symptoms diarr K oea developed on the 4th day pi. and conjunctivitis occurred in animals Nos. 3 and 5.

All the four infected animals excreted protein in their urine on the 6th and 7th days pi. which was estimated at 30-100 mg./100 ml. urine based on the colour reaction. Glucose and blood could not be demonstrated, but a slight drop in p H value was observed as compared to the control,

Pathological examination Similar changes were seen in animals killed on the 2nd and 4th day pi.

In the lungs the ventral parts of the cardiac, apical and accessory lobes were spotted with dark-red consolidated areas. On dissecting these areas a small amount of exudate appeared on the surface.

Lungs of animals killed on days 6 and 8 pi. showed slight enlargement and consolidation. In the ventral part of the apical, cardiac, accessory and diaphragmatic lobes some lobules showed atelectasis. In the animal killed on

Adenovirus Infection in Lambs/II. 531

13th day pi. and in the contact control focal interstitial pneumonia as well as lesions indicating broncho-pneumonia in the apical and cardiac lobes were found.

Except for the animal killed on the 2nd pi. day serous, mucopurulent rhinitis was observed in all the lambs.

In the animal killed on the 2nd day pi. the retropharyngeal lymph node was slightly swollen, soft and contained an abundant amount of fluid. All the other animals had swollen retropharyngeal-, peribronchiolar and mesenteric lymph nodes. On incising, their surface appeared greyish-white, covered with excess amount of fluid and the normal structure would not be recognized.

In animals killed on days 6, 8, 13 and 14 pi. catarrhal enteritis was found. Lymphoid patches in animals killed on days 8 and 13 pi. were swollen. In animals necropsied on days 6 and 13 pi. conjunctivitis was found.

No gross pathological changes in the other organs of the experimentally infected animals were seen and every organ examined was apparently free of pathological changes in the controls.

Histological examination In the lungs of the lamb killed on the 2nd day pi. macroscopic lesions

proved to be areas with bronchioli filled with acidophil homogeneous exudate containing degenerated and detached epithelial cells, and inflammatory, cells including some neutrophil granulocytes (Fig. 1 and 2 ) . Homogeneous acidophil material was also seen in some of the alveoli. Blood vessels in the alveolar septa were congested and the nuclei of the alveolar epithelial cells were swollen. Changes described above were usually distinct and involved the whole lobule. In the adjacent lobuli only a few bronchioli contained acidophil

Fig. 1. Alveoli with atelectasis and exudate containing cellular elements. H-E 157 X


Fig. 2. Inflammatory cells and detached epithelial cells in the eosinophil exudate within the lumen of a bronchiole. H-E 780 X

Fig. 3. Bronchiolar epithelium proliferation and peribronchiolar inflammation. H-E 450 x

Adenovirus Infection in LambslII. 533

exudate, degenerated and detached epithelial cells and neutrophil granulocytes.

In animals killed on days 4, 6 and 8 pi. the lungs showed similar changes but the extent of involvement was different. Chan es also appeared

number of bronhioli contained degenerated epithelial cells, macrophages and neutrophil granulocytes embedded in a homogeneous exudate. Nuclei of the epithelial cells in the small bronchioli were swollen and granulated. In the animal killed on the 8th day pi. some bronchiolar epithelial cells showed proliferation and formed multiple layers, while peribronchiolar connective tissue was infiltrated by lymphocytes and histiocytes (Fig. 3). Peribronchiolar lymphoid follicles proliferated and hyperplasia could be observed. Both the atelectatic and adjacent parts had areas showing thickening of the alveolar septum due to the swelling of the alveolar epithelial cells and to the infiltration with macrophages. Neutrophil granulocytes were seldom found in these areas.

In animals killed on 13th and 14th day pi. the thickening of the alveolar septa was the most pronounced lesion. Alveolar septa contained a large number of lymphoid cells and histiocytes which resulted in a pronounced reduction of the alveolar lumen. Bronchiolar epithelial cells showed strong activity and formed multiple layers in some bronchioli. Hyperplasia and proliferation of peribronchiolar lymphoid follicles were observed in some areas and perivascular lympho-histiocyte proliferation occurred in the peribronchiolar connective tissue.

Except for the lamb killed on the 2nd day pi. the lambs showed degeneration and necrosis of the epithelial cells of the nasal mucous membrane. In one animal (No. 5) extensive necrosis of the epithelial cells was noticed. In all the three layers of the mucous membrane, mostly adjacent of the necrotic areas, infiltration with neutrophil granulocytes was observed. Hyperaemia

in the lobuli adjacent to the parts with macroscopically visib P e lesions. A large

Fig. 4. Infiltration of the nasal mucous membrane with histiocytes and lymphocytes. H-E 270 X

Zbl. Vet. Med., Reihe B, Bd. 24, Heft 7 37


and oedema were seen in the submucosa. In animals killed on days 8, 13 and 14 pi. well-defined areas in the propria showed perivascular lympho-histiocyte proliferation and hyperplasia of the lymphoid follicles (Fig. 4).

In lambs killed on the 13th and 14th day pi. the submucosa of the tracheal mucous membrane showed hyperaemia and oedema, and slight infiltration with neutrophil granulocytes was found in the epithelium and propria. In well-defined areas mononuclear infiltration was observed in the propria. The rest of the animals showed no histopathological changes in their trachea.

In the animal killed on the 2nd day pi. the retropharyngeal and peribronchial lymph nodes showed hyperaemia and in the large sinuses inflammatory exudate was observed regularly. Several neutrophil granulocytes were also seen among the inflammatory cells. All the other infected animals and the contact control showed similar changes in the retropharyngeal, peribronchiolar and mesenteric lymph nodes. Cortical follicles proliferated and became enlarged, and the medullary cords became thicker. The large sinuses were filled with proliferating endothelial and reticular cells, lymphocytes and a small number of neutrophil granulocytes (Fig. 5).

Fig. 5. Sinus endothelial proliferation and sinuses filled with reticular and lymphoid elements. H-E 270 X

Kidneys of every infected animal showed changes. The changes were usually confined to the tubuli and the glomeruli. Most pronounced chan es were observed in the animals killed 6, 8, 13 and 14 day pi. Glomeru K ar capillary endothelium and cells covering the parietal layer of the Bowman- capsule showed swollen nuclei, and at the same time, homogeneous exudate was seen in the lumen of the Bowman-capsule (Fig. 6) .

Similar exudate was also observed in some of the tubuli. Most pronounced changes were observed in the proximal convoluted part. Nuclei of the epithelial cells were swollen, roughly granulated and necrosis of the epithelial


Fig. 6 . Swelling of the nuclei in the glomerular cells, homogeneous exudate in the Bowman- capsule, tubular epithelium degeneration. H-E 700 X

Fig. 7. Necrotic tubular epithelial cells with thickening of the nuclear membrane and central basophil bodies. H-E 1,000 X

37'>


Fig. 8. Intertubular and perivascular proliferation of lyrnpho-histiocytes in the kidney. H-E 450 X

Fig. 9. Glornerulitis and periglomerulitis. H-E 450 X


cells in some of the tubuli was also found. A relatively large number of nuclei showed thidrening of the nuclear membrane and basophil inclusion bodies surrounded by a halo (Fig. 7). Marked proliferation of the epithelial cells was observed in the tubuli and irregular clusters of epithelial cells completely occluded the lumen. In animals killed on the 13th and 14th day pi. some focal intertubular and perivascular proliferation of lymphocytes and histiocytes was observed (Fig. 8). Inflammatory changes were also seen in some glomeruli.

In some glomeruli the parietal epithelial cells of the Bowman-capsule proliferated and periglomerular proliferation of lympho-histiocytes was seen (Fig. 9).

In the liver of the animals killed on the 8th, 13th and 14th day pi. proliferation of lympho-histiocytes and plasma cells appeared around the intralobular blood vessels, and epithelial proliferation of the bile capillaries was also found. Slight proliferation of the reticular cells characterized the changes in the spleens.

The intestinal mucous membrane was oedematous, hyperaemic and was infiltrated with inflammatory cells, while in animals killed on the 6th, 8th and 13th day pi. hyperplasia of the lymphoid follicles appeared.

The conjunctival submucosa showed hyperaemia and oedema, and the epithelial layer was infiltrated by neutrophil granulocytes in animals killed on the 6th and 14th day pi.

No pathological changes were found in the brain of the infected animals. Except for the lamb killed on the 2nd day pi. nuclear inclusions in

various cells were found in every animal. The incidence of inclusions was most common in cells of the animal killed on the 4th day pi. whereas only a few inclusions were found in the cells of lambs killed on the 13th and 14th day

Fig. 10. Intranuclear inclusions in the macrophage of a lymph node. H-E 1,200 X


pi. Nuclear inclusions could be observed primarily in the nasal and bronchial mucous membrane epithelium, in the alveolar epithelium and in the reticular cells of the peribronchial and retropharyngeal lymph nodes. In the animal killed on the 4th day pi. several cells listed above were found which contained slightly basophil or eosinophil granules in the swollen nuclei. These granules were arranged in a diffuse manner o r formed small clusters. Inclusions at their final stage of development stained basophil and were located at the center of the nucleus. Nuclear membrane thickening was also observed (Fig. 10).

Bacteriological and virological examinations No bacteria were isolated from the lungs, peribronchial and mesenteric

lymph nodes, spleens, livers and kidneys. A mixed flora of non-haemolytic E. coli and enterococci was found in the small intestines.

Virus could be reisolated from the nasal discharge of five lambs (Nos. 2, 3, 4, 5, 6 ) . Virus excretion was observed between the 3rd and 10th day of the infection. Rectal swabs yielded virus from four animals (Nos. 2, 4, 5, 6). Virus excretion by feces was confirmed between the 3rd and 5th day pi.

Virus isolation attempts failed to yield any virus from the homogenized respiratory and digestive tract and other parenchymal organ homogenates in spite of several passages.

Reisolated viruses proved to be identical with strain GY/14 on virus neutralization tests.

Neutralizing antibodies at a titre of 1 : 2 appeared first in the serum of the lamb (No. 3) killed on the 8th day pi. Serum of the animal killed on the 13th day pi. (No. 5) and of the contact control lamb showed neutralizing antibodies in a titre of 1 : 4.

Discussion Following experimental infection the most pronounced symptoms and

lesions developed in the respiratory tract. Respiratory symptoms lasted until the death of the animals and at even in two lambs killed at the end of the experiment this condition became more severe. These symptoms developed in the contact lamb, too.

The most characteristic pathological changes were found in the respiratory tract and also in the lymph nodes adjacent to the respiratory tract and the digestive tract and in the kidneys.

The primary atelectatic foci probably developed consequently to the exudative process affecting several bronchioli in the lobule and resulted in reduction or occlusion of the bronchiolar lumen. After the primary regressive changes of the alveolar epithelium proliferative response followed. As the inflammation proceeded intralobular interstitial pneumonia involved a greater part of the lungs with peribronchitis and proliferation of the peribronchial lymphoid follicles. Microscopic examinations revealed that larger parts of the lungs had become affected than was seen at the gross pathological examination.

Exudative changes in the nasal mucous membrane and regressive processes of the epithelium accompanied by inflammatory cell infiltration were found.

Acute and subacute inflammatory changes characterized the lesions found in the lymph nodes adjacent to the respiratory and digestive tracts. The most extensive changes, notably hypertrophy and hyperplasia, were found in the RHS and lymphoid elements.


Changes were later observed also in the kidneys. Glomerular lesions started with exudative processes, whereas lesions in the tubuli reflected degenerative processes. Glomerular damage was indicated also by the detec- tion of protein in the urine. Though in a slight form in later stages of the disease interstitial inflammation was also observed.

Findings of the experimental infection were similar to those observed in the natural cases; the difference appeared to be confined to the severity of the changes only. Proliferation of the peribronchial lymphoid follicles and of the bronchiolar epithelial cells was more pronounced in the natural cases.

In experimental infections the findings observed seemed to be similar to those observed in experimental infection with the virus strain Het/3 in colostrum-deprived lambs (2, 5). However, strain GY/14 caused more pronounced pathological changes in colostrum deprived lambs, because no peribronchitis, peribronchial lymphoid follicle hyperplasia and interstitial nephritis developed when animals had been infected with strain Het/3.

Intralobular interstitial pneumonia and the changes observed in the upper respiratory tract and adjacent lymphoid follicles were, in effect, identical in both cases.

In the natural cases and in the experimental infections demonstration of basophil nuclear inclusions in the nasal-, and bronchiolar epithelial cells, as well as in the cells lining the alveoli and in the reticular cells of the lymph nodes and lymphoid follicles seems to be characteristic and of diagnostic value. However, inclusions could only be demonstrated a t the beginning of the infection, more suitably between the 4th and 8th day following infection.

Based on the observations made during the natural outbreaks and the experimental infections we came to the conclusion that the adenovirus strains ORT/111 and GY/14 were pathogenic to lambs.

Summary Adenovirus strain GY/14 isolated during a natural outbreak was used in

experimental infection. Three weeks old lambs responded with temperature rise, respiratory symptoms and diarrhoea to the infection. Infection spread to a contact animal, too. Reisolation of the virus was successful from the nasal discharge and feces from the 3rd to loth, and the 3rd to 5th day following infection, respectively. In the killed experimental animals the pathological and histological changes observed were similar to those observed in natural cases. On comparing the natural outbreaks with the experimental infection the only difference appeared in the severity of the changes. Following the experimental infection characteristic nuclear inclusions appeared in the nasal and bronchiolar epithelium, in the alveolar septa1 cells and in the reticular cells of the lymph nodes. Epizootiologic observations and experimental results confirm the assumption that our adenovirus strains isolated from natural cases are pathogenic for lambs.

Zusammenf assung Adenovirusinfektionen bei Lfmmern

11. Experimentelle Infektion Nach experimenteller Infektion von 3 Wochen alten Lammern mit dem

ovinen Adenovirusstamm GY/14 erkrankten die Tiere fieberhaft unter respi- ratorischen Symptomen und an Diarrhoe.

540 PALYA, BELAK and PALPI

Die Reisolierung des Virus gelang aus Nasenschleim vom 3.--10. Tag und aus Kot vom 3.-5. Tag nach der Infektion. Der Versuch einer Kontakt- infektion verlief positiv.

Pathologisch-anatomische und histopathologische Veranderungen nach experimenteller Infektion entsprachen in ihre Art den bei naturlicher Infektion beobachteten, waren jedoch weniger stark ausgepragt. Auch nach experimenteller Infektion wurden in Epithelzellen der Nasen- und Bronchialschleim- haut, in den Alveolarse ten der Lunge und in den Retikulumzellen der Lymphknoten Kerneinschksse nachgewiesen.

Auf Grund ihrer Beobachtungen bei naturlicher und experimenteller Infektion wird geschlossen, dai3 der isolierte Adenovirusstamm fur Lammer pathogen ist.

R h m C Infections ?I Adenovirus h e z des agneaux

11. Infection expirimentale Des agneaux de trois semaines, infectks avec la souche d'adenovirus ovine

GY/14, prksentirent des symptbmes respiratoires et de la diarrhke avec fikvre. La rkisolation du virus fut possible du 3kme au lOkme jour A partir des. muqueuses nasales et du 3&me au 5&me jour 1 partir de fkces ap rb l'infection. L'essai d'une infection par contact fut positive.

Les lksions anatomo-pathologiques et histopathologiques ap rb l'infection expkrimentales furent semblables dans le fond A celles constatkes dans une infection naturelle, les lksions ktant toutefois moins ktendues. On a kgalement constatk la prksence d'inclusions nuclkaires aprks I'infection expkrimentale dans des cellules kpithkliales des muqueuses nasales et bronchiques, dans les septums alvkolaires des poumons et dans les cellules rkticulaires des gang- lions. On a dkduit des observations faites aprb des infections naturelles et expkrimentales que la souche d'Adenovirus isolke est pathogkne pour des agneaux.

Resumen Infecciones or adenovirus en corderos

11. In F ecci6n experimental Tras infecci6n experimental de corderos de 3 semanas de edad con la

estirpe ovina de adenovirus GY/14 enfermaban 10s animales de forma febril bajo sintomas respiratorios y diarreicos.

El reaislamiento del virus se logr6 a partir de moco nasal del 3er a1 10' dia y del estikrcol del 3"' a1 5' dia desde la infecci6n. El intento de una infecci6n por contact0 result6 positivo.

Las modificaciones anatomo e histo atol6gicas despuks de la infecci6n

natural, aunque eran menos evidentes. Tambikn tras infecci6n experimental se identificaron inclusiones nucleares en las cklulas epiteliales de las mucosas nasal y bronquial, en 10s septos alveolares del pulm6n y en las cklulas reti- culares de 10s ganglios linfiticos. A la vista de las observaciones en la infec- ci6n natural y en la experimental, se saca en conclusi6n qwe la estirpe aislada de adenovirus es pat6gena para corderos.

experimental correspondian en su indo P e a las observadas en la infecci6n

References 1. BELAK, S., and V. PALPI, 1974: An adenovirus isolated from sheep and its relationship

to type 2 bovine adenovirus. Arch. ges. Virusforsch. 46, 366-369.


2. BELAK, S., V. PALFI and E. TURY, 1975: Experimental infection of lambs with an adenovirus isolated from sheep and related to bovine adenovirus type 2. I. Virological studies. ActaVet. Hung. 21, 91-95.

3. BELAK, S., V. PALFI and V. PALYA, 1976: Adenovirus infection in lambs. I. Epizootiology of the disease. Zbl. Vet. Med. 23, 320-330.

4. MCFERRAN, J. B., R. NELSON and E. R. KNOX, 1971: Isolation and characterization of sheep adenoviruses. Arch. ges. Virusforsch. 3'1, 232-241.

5. TURY, E., S. BELAK and V. PALFI, 1975: Experimental infection of lambs with an adenovirus isolated from sheep and related to bovine adenovirus type 2. 11. Pathological and histopathological studies. Acta Vet. Hung. 21, 171-1 84.

Authors' address: Dr. V. PALYA, Veterinary Institute, H-5602 Bkkkscsaba, Pf. 2.; Dr. S. B E L ~ K , Dept. of Epizootiology, University of Vet. Sci., H-1581 Budapest 146, Pf. 22.; Dr. V. PALFI, Central Veterinary Institute, H-1581 Budapest 146, Pf. 2., Hungary.

adenovirus infection in lambs : ii. experimental infection of lambs

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