Vet Pathol 28166-170 (1991)
An Immunohistochemical Study of the Pneumonia Caused by Peste des Petits Ruminants Virus C. C. BROWN,J. C. MARINER, AND H. J. OLANDER Foreign Animal Disease Diagnostic Laboratory, National Veterinary Services Laboratory-Science and Technology- Animal and Plant Health Inspection Services, US Department of Agriculture, Greenport NY; and Department of Veterinary Pathology, University of California, Davis, CA Abstract. Ten goats were inoculated with peste des petits ruminants virus, a paramyxovirus closely related to rinderpest virus. All goats developed severe clinical disease, 81 10 having coughing or dyspnea as prominent clinical signs. In addition, all of the goats had stomatitis and diarrhea. Histopathologic and immunohistochemical studies were done only on the respiratory tracts. Pathologic changes ranged from mild multifocal bronchiolitis and bronchitis to severe bronchointerstitial pneumonia. Lesions were more severe in anteroventral than caudal lobes. The histologic nature of the viral process in the goat lungs had many features in common with the processes of pneumonia in dogs, due to canine distemper, or pneumonia in human beings, due to measles virus. Immunohistochemical staining of formalin-fixed, paraffin-embedded respiratory tract tissue was performed using an indirect system with rabbit anti-rinderpest virus serum, biotinylated anti-rabbit antibody, streptavidinalkaline phosphatase, and nitroblue tetrazolium chromogen. Staining was sensitive, highlighting the presence of viral antigen in both lung and trachea of all goats. Viral antigen was found in both cytoplasm and nucleus of tracheal, bronchial, and bronchiolar epithelial cells, type I1 pneumocytes, syncytial cells, and alveolar macrophages. In general, the amount of staining correlated directly with the severity of the inflammatory process.
Key words: Goat; peste des petits ruminants virus; pneumonia; immunohistochemistry.
Peste des petits ruminants (PPR) is a viral disease Materials and Methods of goats and sheep characterized principally by sto- Animals matitis, diarrhea, oculonasal discharge, and pneumoTen, mixed-breed goats (four females, five males, and one nia. Originally described in West Africa, the disease is now known to be responsible for morbidity and mor- castrated male), 4 to 7 months of age, became clinically ill after experimental inoculation with peste des petits rumitality throughout much of subSaharan Africa north of nants (PPR) virus. All goats were part of a larger study dethe equator, the Arabian peninsula, and, recently, In- signed to evaluate the efficacy of a Vero cell adapted rinderdia.6.9The causative agent is closely related to rinder- pest vaccine for protection against challenge with PPR virus. pest virus, and these two viruses are classified along Those results are reported elsewhere (Mariner, manuscript with the viruses of measles and canine distemper in in preparation). Five of the goats (Nos. 5-9) were vaccinated the genus Morbillivirus of the Family Paramyxoviri- with a very low dose of rinderpest vaccine and failed to seroconvert. The other five (Nos. 1 4 , 10) were control andae. Pathologic changes associated with PPR have been imals for the vaccine study and received no vaccination. All described. There is generally severe erosive stomatitis ten animals developed severe clinical disease after being inand an enterocolitis that shows many similarities to oculated with PPR virus. During the experimental period, that caused by rinderpest Pulmonary changes the goats were housed in a high-containment facility and given water and pelleted hay ad libitum. Animals were exthat occur have been less well characterized and de- amined daily, and their rectal temperatures and clinical signs scribed as a necrotizing bronchitis/bronchiolitisSand were recorded. an interstitial pneumonia.' It was the aim ofthis study to examine by histopathology and immunohistochem- Inoculation of virus istry the respiratory tracts of goats infected with PPR Ten goats were inoculated with 1.0 ml of goat blood stavirus to determine the relationship of the virus to the bilate (Meilig-Sudan, also called Sudan 72-2 strain) that had lesions and the importance of the pneumonia in the been stored at -70 C for 5 years. Inocula were given subcutaneously just anterior to the prescapular lymph node. disease complex. 166
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Table 1. Clinical signs and extent of histopathologic and immunochemical staining of the lungs of ten goats infected with peste des petits ruminants virus.
No.
'Oat
Age (months)
Type
Of
Death*
Time of Death (days after inoculation)
Lungs Histologic ImmunoChanges* chemistry§
Clinical Signst
3 C 4 E 8 Stomatitis, diarrhea, coughing A 1 Diarrhea, stomatitis 4 E 10 A 1 11 Stomatitis, diarrhea, dyspnea 4 D A 2 4 Dyspnea, diarrhea, stomatitis E 8 C 3 Diarrhea, stomatitis 7 D 10 B 3 Dyspnea, diarrhea 7 E 10 A 2 Coughing, stomatitis 7 E 10 B 3 Coughing, diarrhea 7 E 10 A 3 12 Coughing, dyspnea, stomatitis 7 D A 3 Dyspnea, stomatitis D 7 13 * D = died. E = euthanatized. t Listed in decreasing order of seventy. $ Extent of histopathologic changes: 1 = one or more microscopic foci of bronchiolar or limited bronchiolar/alveolar reaction; 2 = multiple, sometimes coalescing foci of bronchointerstitial inflammation; and 3 = severe inflammation with extensive consolidation +/- supenmposed neutrophilic exudate. 9 Extent of immunohistochemical staining: A = distinct focal staining of selected airway epithelialcells; B = distinct staining of numerous airways; C = heavy staining involving most airways and alveoli in foci of consolidation. I
2 3 4 5 6 7 8 9 10
Pathology
Six of the ten animals (Nos. 1,2,4,6-8) were euthanatized with Pentothal (Abbott Laboratories, North Chicago, IL, USA) or T6 1 (Hoescht-Roussel, Somerville, NJ, USA). The other four (Nos. 3,5,9, 10) were found dead. Selected tissues were preserved in 10% neutral buffered formalin. The right lung was fixed by intrabronchial infusion with formalin. Pieces ofcranial and caudal right lung and trachea were embedded in paraffin. Replicate sections were cut at 4-5 pm for histopathologic and immunohistochemical examination. For histopathologic study, tissues were stained with hematoxylin and eosin. Immunohistochemistry
For immunohistochemistry, sections were placed on polyL-lysine coated glass microscope slides. After 2 hours incubation at 48 C, sections were deparaffinized in xylene and washed briefly in phosphate-buffered saline (PBS). All washes and dilutions were done in PBS containing 0.005% polyoxyethylene sorbitan monolaurate. Tissues were digested with 0.1% trypsin in Tris (pH 7.6) with 0.1% CaCI, and 0.1% NaCl for 30 minutes at 37 C. Enzyme activity was stopped with 2% glycine in PBS. Blocking of nonspecific staining was achieved with 2% normal goat serum for 30 minutes at 37 C. The blocking serum was drained and replaced by rabbit anti-rinderpest serum at a 1500 dilution and incubated overnight at 4 C. After a 15-minute wash, sections were flooded with biotinylated goat anti-rabbit antibody (Vector Laboratories, Burlingame, CA, USA) for 1.0 hour at 37 C. After another 15-minute wash, sections were covered with streptavidin-alkaline phosphatase (Enzo Biochem, New York, NY, USA) diluted 1 5 0 in PBS with 5 mM EDTA (ethylenediaminetetraacetate), 0.1% bovine serum albumin, and 0.5% Triton X-100. The final step was the addition ofthe substrate and chromogen mixture of 10 x phosphate and nitroblue
tetrazolium (Boehringer Mannheim, Indianapolis, IN, USA) in 100 mM Tris-HCI, 100 mM NaCI, and 50 mM MgCI, (pH 9.5). After 45 minutes, color development was stopped by rinsing the sections in distilled water. Sections were counterstained lightly with aqueous eosin. For tissue control, a noninfected goat lung was processed in an identical manner to the infected tissue. For serum control, replicate sections of selected infected lungs and trachea were processed, substituting normal rabbit serum for rabbit anti-rinderpest serum. Preparation of primary antiserum
dilution of a 10% suspension of spleen One ml of a taken from a rabbit infected with the Nakamura (lapinized) strain of rinderpest virus was inoculated intravenously into an adult New Zealand white rabbit. The rabbit was given 1.O ml of the same suspension intravenously decreasing by a tenfold dilution weekly for 5 consecutive weeks. Four weeks after the last inoculation, the rabbit was again given 1.0 ml of a 10-1dilution. Blood was drawn 3 weeks later. The resulting serum had a serum neutralization index of 4.66 log,,.
Results Clinical disease
All animals became febrile (greater than 40.5 C) and depressed at 4 to 7 days after inoculation. Prominent clinical signs during the period of fever are presented in Table 1. Oral erosions, diarrhea, and dyspnea were the prominent signs and clinically visible lesions noted. In addition, two animals (Nos. 2, 4) had crusty exudates over the conjunctiva, and three (Nos. 1,4,8) had dried mucous exudate partially occluding the external nares. Animals were euthanatized when severely clin-
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Fig. 1. Lung; goat No. 1 . This lung from a goat infected with peste des petits ruminants virus demonstrates cranioventral consolidation and atelectasis.
ically ill at 8 to 10 days after inoculation. Four goats (Nos. 3, 5, 9, 10) were found dead 10 to 13 days after inoculation. Gross pathologic findings
Euthanatized animals were necropsied within 1 hour postmortem. Of the four animals (Nos. 3,5,9, 10) that were found dead, three (Nos. 3, 5, 9) were still warm at the time of necropsy. The tissues of the fourth animal, (No. 10)were moderately to markedly autolyzed. All but one of the animals (No. 2) had gross evidence of pneumonia at necropsy. In most, lesions were confined to anteroventral areas and consisted of consolidation and atelectasis of the most dependent portions (Fig. 1). In two animals (Nos. 9, lo), pleuritis was present, with multiple tenuous adhesions between parietal and visceral pleura. In addition, goat No. 10 had hydrothorax and multiple nodules of consolidation scattered throughout the caudal lobes. Histopathologic findings
Only the lungs and tracheas were processed for histopathology. All sections of lung had similar lesions with varying degrees of severity. Inflammation was centered on small airways, with focal degeneration, necrosis, and ulceration of bronchiolar epithelium. Recovering epithelium was often proliferative and irregular, with normal cuboidal lining cells being replaced by poorly-organized stratified squamous epithelium. Alveoli adjacent to affected bronchioles were also altered, with marked hyperplasia of type I1 pneumocytes, increased numbers of alveolar macrophages, and frequent formation of syncytial cells (Fig. 2). These syncytial cells varied in size but could be quite large with up to 50 nuclei. In the more severely affected lungs, foci of bronchiolo-alveolar reaction coalesced to form areas of consolidated pulmonary parenchyma. The epithelium of larger airways, i.e., bron-
Fig. 2. Lung; goat No. 6. There is a proliferative inflammatory reaction involving bronchioles and adjacent alveoli. Note syncytial cells. HE. Bar = 50 pm.
chi and trachea, had multiple foci of degeneration and necrosis, often extending into glands. The epithelium was proliferative and disorganized, with marked squamous metaplasia in some sections of trachea. In four of the animals (Nos. 5, 7, 8,9) a neutrophilic exudate was superimposed on the proliferative bronchiolo-alveolar lesion. In one of these (No. 9), bacterial colonies were present. Viral inclusion bodies were seen in tissue from every animal and occurred in tracheal, bronchial, and bronchiolar epithelium, type I1 pneumocytes, syncytial cells, and occasionally, alveolar macrophages. Inclusions occurred both in the nucleus and the cytoplasm. The nuclear inclusions were eosinophilic and usually did not fill the nucleus. Cytoplasmic inclusions were also eosinophilic but often multiple within a single cell and generally smaller than the nuclear inclusion and more difficult to visualize against the eosinophilic cytoplasm. Immunohistochemical findings
Immunochemical staining of antigen in the lungs was sensitive. Viral antigen was detected in every piece of lung and trachea from every infected goat. There was no staining in tissue from a noninfected animal or
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Fig. 3. Lung; goat No. 1. Note immunohistochemical staining demonstrating peste des petits ruminants antigen within epithelial cells, syncytial cells, and alveolar macrophages. Immunohistochemical staining using biotinylated antibody, streptavidin alkaline phosphatase and nitroblue tetrazolium chromogen. Eosin counterstain. Bar = 100 pm. Fig. 4. Trachea; goat No. 5. Note immunohistochemical staining of trachea demonstrating rinderpest antigen within both surface and glandular epithelium. Immunohistochemical staining using biotinylated antibody, streptavidin alkaline phosphatase and nitroblue tetrazolium chromogen. Eosin counterstain. Bar = 50 pm.
visible grossly. The changes ranged from mild multifocal bronchiolitis and bronchitis to severe bronchointerstitial pneumonia complicated by secondary bacteria. Lesions were more severe in anteroventral than caudal lobes. The histologic nature of the viral process in goats had many features similar to the process of pneumonia due to canine distemper virus in dogs or measles virus in human beings. As in the latter two viruses, the exudative component is most prominent at the terminal bronchioles, often spilling over into adjacent alveoli, making the process “bronchointerstitial” in character.2 Also, in both canine distemper and human measles, multinucleate giant cells are characteristic, and secondary bacterial infections are very c ~ m m o n .A~ possible .~ feature distinguishing PPR virus-induced lesions from canine distemper virus-induced lesions is that PPR virus tends to cause more severe damage in bronchi and trachea than what is reported for canine distemper. Coughing or dyspnea were prominent clinical signs in 8/10 goats. This was especially true in the six older goats (Nos. 5-10), five of which (Nos. 6-10) had dyspnea and coughing as their most severe signs. In these five, the histologic lung lesions were more severe than Discussion in the younger goats. In contrast, in 314 younger aniAll of the goats infected with peste des petits ru- mals, stomatitis and diarrhea were clinically more seminants (PPR) virus developed pulmonary changes, vere than respiratory problems. Perhaps the younger although in one of these (goat No. 2), changes were not animals succumbed to dehydration caused by diarrhea
in infected tissue on which the normal rabbit serum was used (serum control). Staining was most definitive when tissues were sectioned and stained the day following overnight embedding. Antigen was present in greatest concentrations in airway epithelium, including trachea, bronchi, and bronchioles. In general, staining tended to be diffuse in the cytoplasm and discretely globular in intranuclear locations. Within the lungs, antigen was evident both in the bronchi and bronchioles. Inflammatory foci around bronchioles stained heavily for antigen and also had staining of selected pneumocytes and alveolar macrophages. Syncytial cells were invariably positive by immunohistochemistry, with diffuse slightly granular staining of the cytoplasm and distinct globular intranuclear inclusions (Fig. 3). In the trachea, the staining was most concentrated in the apical portions of the proliferating surface epithelium. In the more severely affected tracheas, staining included the glandular epithelium as well (Fig. 4). In the less severely affected animals, tracheal staining was focal, with groups of cells containing antigen alternating with others that were free of staining.
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before the lesions in the lungs could become fully developed. In 4/6 older animals, the primary viral lesion in the lung was complicated by a pyogenic component, presumably due to secondary bacterial invaders. It may be that PPR-initiated pneumonias result in death due to bacterial pneumonia, and the primary viral etiology is overlooked. In any case, in this study, there was considerable primary viral damage in the lungs of many goats, underscoring the potential of PPR virus to cause a serious pneumonia. Immunochemical staining using primary rabbit antirinderpest virus serum was sensitive, highlighting the presence of PPR virus antigen in airway epithelium at all levels of the respiratory tract. Serologic cross-reactivity between PPR virus and rinderpest virus is well doc~rnented.'.~.~.~ In general, the amount of staining correlated directly with the severity of the inflammatory process; however, even in some sections of lung where there were very minimal pathologic changes, the presence of PPR virus could still be detected by distinct blue-black epithelial staining of selected areas of bronchioles, bronchi, or trachea. In areas where there was more inflammatory change, with consolidation of pulmonary parenchyma, PPR virus was demonstrated in alveolar lining and inflammatory cells as well. Syncytial cells, when present, consistently demonstrated the presence of PPR virus. Peste des petits ruminants virus is difficult to isolate from clinical cases of the disease. In this study, the presence of the virus was demonstrated consistently using a simple immunohistochemical technique on formalin-fixed, paraffin-embedded tissue. Even in a goat in which no lung lesions were detectable grossly (No. 2), PPR virus antigen was easily detected in the lungs and trachea. In the animal that was quite autolyzed at the time of necropsy (No. lo), PPR virus an-
tigen was still detectable in the lung. It should be possible to use this immunohistochemical technique to diagnose naturally-occumng cases of PPR. Acknowledgements The authors thank Dr. A. Dardiri for the peste des petits ruminants virus blood stabilate and Dr. C. Mebus for the splenic suspension of lapinized rinderpest virus.
References 1 Bundza A, Afshar A, Dukes TW, Myers DJ, Dulac GC, Becker SAWE Experimental peste des petits ruminants (goat plague) in goats and sheep. Can J Vet Res 5246-52, 1988 2 Dungworth DL: The Respiratory System. In: Pathology of Domestic Animals, ed. Jubb KVF, Kennedy PC, Palmer N, 3rd ed., pp. 413-556. Academic Press, Orlando, FL, 1985 3 Gibbs EPJ, Taylor WP, Lawman MJP, Bryant J: Classification of peste des petits ruminants virus as the fourth member of the genus Morbillivirus. Intervirology 22268274, 1979 4 Robbins SL, Cotran RS: Pathologic Basis of Disease, 2nd ed., pp. 440442. WB Saunders, Philadelphia, PA, 1979 5 Rowland AC, Scott GR, Hill DH: The pathology of an erosive stomatitis and enteritis in West African dwarfgoats. J Pathol9883-87, 1969 6 Shaila MS, Purushothaman V, Bhavasar D, Venugopal K, Venkatesan RA: Peste des petits ruminants of sheep in India. Vet Rec 125602, 1989 7 Taylor W P Protection of goats against peste des petits ruminants with attenuated rinderpest virus. Res Vet Sci 27:32 1-324, 1979 8 Taylor W P Serological studies with the virus of peste des petits ruminants in Nigeria. Res Vet Sci 26236-242, 1979 9 Taylor W P The distribution and epidemiology of peste des petits ruminants. Prev Med 2: 157-1 66, 1984
Request reprints from Dr. C. C. Brown, National Veterinary Services Laboratories, Foreign Animal Disease Diagnostic Laboratory, PO Box 848, Greenport, NY 1 1944 (USA).
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An Immunohistochemical Study of the Pneumonia Caused by Peste des Petits Ruminants Virus C. C. BROWN,J. C. MARINER, AND H. J. OLANDER Foreign Animal Disease Diagnostic Laboratory, National Veterinary Services Laboratory-Science and Technology- Animal and Plant Health Inspection Services, US Department of Agriculture, Greenport NY; and Department of Veterinary Pathology, University of California, Davis, CA Abstract. Ten goats were inoculated with peste des petits ruminants virus, a paramyxovirus closely related to rinderpest virus. All goats developed severe clinical disease, 81 10 having coughing or dyspnea as prominent clinical signs. In addition, all of the goats had stomatitis and diarrhea. Histopathologic and immunohistochemical studies were done only on the respiratory tracts. Pathologic changes ranged from mild multifocal bronchiolitis and bronchitis to severe bronchointerstitial pneumonia. Lesions were more severe in anteroventral than caudal lobes. The histologic nature of the viral process in the goat lungs had many features in common with the processes of pneumonia in dogs, due to canine distemper, or pneumonia in human beings, due to measles virus. Immunohistochemical staining of formalin-fixed, paraffin-embedded respiratory tract tissue was performed using an indirect system with rabbit anti-rinderpest virus serum, biotinylated anti-rabbit antibody, streptavidinalkaline phosphatase, and nitroblue tetrazolium chromogen. Staining was sensitive, highlighting the presence of viral antigen in both lung and trachea of all goats. Viral antigen was found in both cytoplasm and nucleus of tracheal, bronchial, and bronchiolar epithelial cells, type I1 pneumocytes, syncytial cells, and alveolar macrophages. In general, the amount of staining correlated directly with the severity of the inflammatory process.
Key words: Goat; peste des petits ruminants virus; pneumonia; immunohistochemistry.
Peste des petits ruminants (PPR) is a viral disease Materials and Methods of goats and sheep characterized principally by sto- Animals matitis, diarrhea, oculonasal discharge, and pneumoTen, mixed-breed goats (four females, five males, and one nia. Originally described in West Africa, the disease is now known to be responsible for morbidity and mor- castrated male), 4 to 7 months of age, became clinically ill after experimental inoculation with peste des petits rumitality throughout much of subSaharan Africa north of nants (PPR) virus. All goats were part of a larger study dethe equator, the Arabian peninsula, and, recently, In- signed to evaluate the efficacy of a Vero cell adapted rinderdia.6.9The causative agent is closely related to rinder- pest vaccine for protection against challenge with PPR virus. pest virus, and these two viruses are classified along Those results are reported elsewhere (Mariner, manuscript with the viruses of measles and canine distemper in in preparation). Five of the goats (Nos. 5-9) were vaccinated the genus Morbillivirus of the Family Paramyxoviri- with a very low dose of rinderpest vaccine and failed to seroconvert. The other five (Nos. 1 4 , 10) were control andae. Pathologic changes associated with PPR have been imals for the vaccine study and received no vaccination. All described. There is generally severe erosive stomatitis ten animals developed severe clinical disease after being inand an enterocolitis that shows many similarities to oculated with PPR virus. During the experimental period, that caused by rinderpest Pulmonary changes the goats were housed in a high-containment facility and given water and pelleted hay ad libitum. Animals were exthat occur have been less well characterized and de- amined daily, and their rectal temperatures and clinical signs scribed as a necrotizing bronchitis/bronchiolitisSand were recorded. an interstitial pneumonia.' It was the aim ofthis study to examine by histopathology and immunohistochem- Inoculation of virus istry the respiratory tracts of goats infected with PPR Ten goats were inoculated with 1.0 ml of goat blood stavirus to determine the relationship of the virus to the bilate (Meilig-Sudan, also called Sudan 72-2 strain) that had lesions and the importance of the pneumonia in the been stored at -70 C for 5 years. Inocula were given subcutaneously just anterior to the prescapular lymph node. disease complex. 166
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Table 1. Clinical signs and extent of histopathologic and immunochemical staining of the lungs of ten goats infected with peste des petits ruminants virus.
No.
'Oat
Age (months)
Type
Of
Death*
Time of Death (days after inoculation)
Lungs Histologic ImmunoChanges* chemistry§
Clinical Signst
3 C 4 E 8 Stomatitis, diarrhea, coughing A 1 Diarrhea, stomatitis 4 E 10 A 1 11 Stomatitis, diarrhea, dyspnea 4 D A 2 4 Dyspnea, diarrhea, stomatitis E 8 C 3 Diarrhea, stomatitis 7 D 10 B 3 Dyspnea, diarrhea 7 E 10 A 2 Coughing, stomatitis 7 E 10 B 3 Coughing, diarrhea 7 E 10 A 3 12 Coughing, dyspnea, stomatitis 7 D A 3 Dyspnea, stomatitis D 7 13 * D = died. E = euthanatized. t Listed in decreasing order of seventy. $ Extent of histopathologic changes: 1 = one or more microscopic foci of bronchiolar or limited bronchiolar/alveolar reaction; 2 = multiple, sometimes coalescing foci of bronchointerstitial inflammation; and 3 = severe inflammation with extensive consolidation +/- supenmposed neutrophilic exudate. 9 Extent of immunohistochemical staining: A = distinct focal staining of selected airway epithelialcells; B = distinct staining of numerous airways; C = heavy staining involving most airways and alveoli in foci of consolidation. I
2 3 4 5 6 7 8 9 10
Pathology
Six of the ten animals (Nos. 1,2,4,6-8) were euthanatized with Pentothal (Abbott Laboratories, North Chicago, IL, USA) or T6 1 (Hoescht-Roussel, Somerville, NJ, USA). The other four (Nos. 3,5,9, 10) were found dead. Selected tissues were preserved in 10% neutral buffered formalin. The right lung was fixed by intrabronchial infusion with formalin. Pieces ofcranial and caudal right lung and trachea were embedded in paraffin. Replicate sections were cut at 4-5 pm for histopathologic and immunohistochemical examination. For histopathologic study, tissues were stained with hematoxylin and eosin. Immunohistochemistry
For immunohistochemistry, sections were placed on polyL-lysine coated glass microscope slides. After 2 hours incubation at 48 C, sections were deparaffinized in xylene and washed briefly in phosphate-buffered saline (PBS). All washes and dilutions were done in PBS containing 0.005% polyoxyethylene sorbitan monolaurate. Tissues were digested with 0.1% trypsin in Tris (pH 7.6) with 0.1% CaCI, and 0.1% NaCl for 30 minutes at 37 C. Enzyme activity was stopped with 2% glycine in PBS. Blocking of nonspecific staining was achieved with 2% normal goat serum for 30 minutes at 37 C. The blocking serum was drained and replaced by rabbit anti-rinderpest serum at a 1500 dilution and incubated overnight at 4 C. After a 15-minute wash, sections were flooded with biotinylated goat anti-rabbit antibody (Vector Laboratories, Burlingame, CA, USA) for 1.0 hour at 37 C. After another 15-minute wash, sections were covered with streptavidin-alkaline phosphatase (Enzo Biochem, New York, NY, USA) diluted 1 5 0 in PBS with 5 mM EDTA (ethylenediaminetetraacetate), 0.1% bovine serum albumin, and 0.5% Triton X-100. The final step was the addition ofthe substrate and chromogen mixture of 10 x phosphate and nitroblue
tetrazolium (Boehringer Mannheim, Indianapolis, IN, USA) in 100 mM Tris-HCI, 100 mM NaCI, and 50 mM MgCI, (pH 9.5). After 45 minutes, color development was stopped by rinsing the sections in distilled water. Sections were counterstained lightly with aqueous eosin. For tissue control, a noninfected goat lung was processed in an identical manner to the infected tissue. For serum control, replicate sections of selected infected lungs and trachea were processed, substituting normal rabbit serum for rabbit anti-rinderpest serum. Preparation of primary antiserum
dilution of a 10% suspension of spleen One ml of a taken from a rabbit infected with the Nakamura (lapinized) strain of rinderpest virus was inoculated intravenously into an adult New Zealand white rabbit. The rabbit was given 1.O ml of the same suspension intravenously decreasing by a tenfold dilution weekly for 5 consecutive weeks. Four weeks after the last inoculation, the rabbit was again given 1.0 ml of a 10-1dilution. Blood was drawn 3 weeks later. The resulting serum had a serum neutralization index of 4.66 log,,.
Results Clinical disease
All animals became febrile (greater than 40.5 C) and depressed at 4 to 7 days after inoculation. Prominent clinical signs during the period of fever are presented in Table 1. Oral erosions, diarrhea, and dyspnea were the prominent signs and clinically visible lesions noted. In addition, two animals (Nos. 2, 4) had crusty exudates over the conjunctiva, and three (Nos. 1,4,8) had dried mucous exudate partially occluding the external nares. Animals were euthanatized when severely clin-
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Fig. 1. Lung; goat No. 1 . This lung from a goat infected with peste des petits ruminants virus demonstrates cranioventral consolidation and atelectasis.
ically ill at 8 to 10 days after inoculation. Four goats (Nos. 3, 5, 9, 10) were found dead 10 to 13 days after inoculation. Gross pathologic findings
Euthanatized animals were necropsied within 1 hour postmortem. Of the four animals (Nos. 3,5,9, 10) that were found dead, three (Nos. 3, 5, 9) were still warm at the time of necropsy. The tissues of the fourth animal, (No. 10)were moderately to markedly autolyzed. All but one of the animals (No. 2) had gross evidence of pneumonia at necropsy. In most, lesions were confined to anteroventral areas and consisted of consolidation and atelectasis of the most dependent portions (Fig. 1). In two animals (Nos. 9, lo), pleuritis was present, with multiple tenuous adhesions between parietal and visceral pleura. In addition, goat No. 10 had hydrothorax and multiple nodules of consolidation scattered throughout the caudal lobes. Histopathologic findings
Only the lungs and tracheas were processed for histopathology. All sections of lung had similar lesions with varying degrees of severity. Inflammation was centered on small airways, with focal degeneration, necrosis, and ulceration of bronchiolar epithelium. Recovering epithelium was often proliferative and irregular, with normal cuboidal lining cells being replaced by poorly-organized stratified squamous epithelium. Alveoli adjacent to affected bronchioles were also altered, with marked hyperplasia of type I1 pneumocytes, increased numbers of alveolar macrophages, and frequent formation of syncytial cells (Fig. 2). These syncytial cells varied in size but could be quite large with up to 50 nuclei. In the more severely affected lungs, foci of bronchiolo-alveolar reaction coalesced to form areas of consolidated pulmonary parenchyma. The epithelium of larger airways, i.e., bron-
Fig. 2. Lung; goat No. 6. There is a proliferative inflammatory reaction involving bronchioles and adjacent alveoli. Note syncytial cells. HE. Bar = 50 pm.
chi and trachea, had multiple foci of degeneration and necrosis, often extending into glands. The epithelium was proliferative and disorganized, with marked squamous metaplasia in some sections of trachea. In four of the animals (Nos. 5, 7, 8,9) a neutrophilic exudate was superimposed on the proliferative bronchiolo-alveolar lesion. In one of these (No. 9), bacterial colonies were present. Viral inclusion bodies were seen in tissue from every animal and occurred in tracheal, bronchial, and bronchiolar epithelium, type I1 pneumocytes, syncytial cells, and occasionally, alveolar macrophages. Inclusions occurred both in the nucleus and the cytoplasm. The nuclear inclusions were eosinophilic and usually did not fill the nucleus. Cytoplasmic inclusions were also eosinophilic but often multiple within a single cell and generally smaller than the nuclear inclusion and more difficult to visualize against the eosinophilic cytoplasm. Immunohistochemical findings
Immunochemical staining of antigen in the lungs was sensitive. Viral antigen was detected in every piece of lung and trachea from every infected goat. There was no staining in tissue from a noninfected animal or
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Fig. 3. Lung; goat No. 1. Note immunohistochemical staining demonstrating peste des petits ruminants antigen within epithelial cells, syncytial cells, and alveolar macrophages. Immunohistochemical staining using biotinylated antibody, streptavidin alkaline phosphatase and nitroblue tetrazolium chromogen. Eosin counterstain. Bar = 100 pm. Fig. 4. Trachea; goat No. 5. Note immunohistochemical staining of trachea demonstrating rinderpest antigen within both surface and glandular epithelium. Immunohistochemical staining using biotinylated antibody, streptavidin alkaline phosphatase and nitroblue tetrazolium chromogen. Eosin counterstain. Bar = 50 pm.
visible grossly. The changes ranged from mild multifocal bronchiolitis and bronchitis to severe bronchointerstitial pneumonia complicated by secondary bacteria. Lesions were more severe in anteroventral than caudal lobes. The histologic nature of the viral process in goats had many features similar to the process of pneumonia due to canine distemper virus in dogs or measles virus in human beings. As in the latter two viruses, the exudative component is most prominent at the terminal bronchioles, often spilling over into adjacent alveoli, making the process “bronchointerstitial” in character.2 Also, in both canine distemper and human measles, multinucleate giant cells are characteristic, and secondary bacterial infections are very c ~ m m o n .A~ possible .~ feature distinguishing PPR virus-induced lesions from canine distemper virus-induced lesions is that PPR virus tends to cause more severe damage in bronchi and trachea than what is reported for canine distemper. Coughing or dyspnea were prominent clinical signs in 8/10 goats. This was especially true in the six older goats (Nos. 5-10), five of which (Nos. 6-10) had dyspnea and coughing as their most severe signs. In these five, the histologic lung lesions were more severe than Discussion in the younger goats. In contrast, in 314 younger aniAll of the goats infected with peste des petits ru- mals, stomatitis and diarrhea were clinically more seminants (PPR) virus developed pulmonary changes, vere than respiratory problems. Perhaps the younger although in one of these (goat No. 2), changes were not animals succumbed to dehydration caused by diarrhea
in infected tissue on which the normal rabbit serum was used (serum control). Staining was most definitive when tissues were sectioned and stained the day following overnight embedding. Antigen was present in greatest concentrations in airway epithelium, including trachea, bronchi, and bronchioles. In general, staining tended to be diffuse in the cytoplasm and discretely globular in intranuclear locations. Within the lungs, antigen was evident both in the bronchi and bronchioles. Inflammatory foci around bronchioles stained heavily for antigen and also had staining of selected pneumocytes and alveolar macrophages. Syncytial cells were invariably positive by immunohistochemistry, with diffuse slightly granular staining of the cytoplasm and distinct globular intranuclear inclusions (Fig. 3). In the trachea, the staining was most concentrated in the apical portions of the proliferating surface epithelium. In the more severely affected tracheas, staining included the glandular epithelium as well (Fig. 4). In the less severely affected animals, tracheal staining was focal, with groups of cells containing antigen alternating with others that were free of staining.
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before the lesions in the lungs could become fully developed. In 4/6 older animals, the primary viral lesion in the lung was complicated by a pyogenic component, presumably due to secondary bacterial invaders. It may be that PPR-initiated pneumonias result in death due to bacterial pneumonia, and the primary viral etiology is overlooked. In any case, in this study, there was considerable primary viral damage in the lungs of many goats, underscoring the potential of PPR virus to cause a serious pneumonia. Immunochemical staining using primary rabbit antirinderpest virus serum was sensitive, highlighting the presence of PPR virus antigen in airway epithelium at all levels of the respiratory tract. Serologic cross-reactivity between PPR virus and rinderpest virus is well doc~rnented.'.~.~.~ In general, the amount of staining correlated directly with the severity of the inflammatory process; however, even in some sections of lung where there were very minimal pathologic changes, the presence of PPR virus could still be detected by distinct blue-black epithelial staining of selected areas of bronchioles, bronchi, or trachea. In areas where there was more inflammatory change, with consolidation of pulmonary parenchyma, PPR virus was demonstrated in alveolar lining and inflammatory cells as well. Syncytial cells, when present, consistently demonstrated the presence of PPR virus. Peste des petits ruminants virus is difficult to isolate from clinical cases of the disease. In this study, the presence of the virus was demonstrated consistently using a simple immunohistochemical technique on formalin-fixed, paraffin-embedded tissue. Even in a goat in which no lung lesions were detectable grossly (No. 2), PPR virus antigen was easily detected in the lungs and trachea. In the animal that was quite autolyzed at the time of necropsy (No. lo), PPR virus an-
tigen was still detectable in the lung. It should be possible to use this immunohistochemical technique to diagnose naturally-occumng cases of PPR. Acknowledgements The authors thank Dr. A. Dardiri for the peste des petits ruminants virus blood stabilate and Dr. C. Mebus for the splenic suspension of lapinized rinderpest virus.
References 1 Bundza A, Afshar A, Dukes TW, Myers DJ, Dulac GC, Becker SAWE Experimental peste des petits ruminants (goat plague) in goats and sheep. Can J Vet Res 5246-52, 1988 2 Dungworth DL: The Respiratory System. In: Pathology of Domestic Animals, ed. Jubb KVF, Kennedy PC, Palmer N, 3rd ed., pp. 413-556. Academic Press, Orlando, FL, 1985 3 Gibbs EPJ, Taylor WP, Lawman MJP, Bryant J: Classification of peste des petits ruminants virus as the fourth member of the genus Morbillivirus. Intervirology 22268274, 1979 4 Robbins SL, Cotran RS: Pathologic Basis of Disease, 2nd ed., pp. 440442. WB Saunders, Philadelphia, PA, 1979 5 Rowland AC, Scott GR, Hill DH: The pathology of an erosive stomatitis and enteritis in West African dwarfgoats. J Pathol9883-87, 1969 6 Shaila MS, Purushothaman V, Bhavasar D, Venugopal K, Venkatesan RA: Peste des petits ruminants of sheep in India. Vet Rec 125602, 1989 7 Taylor W P Protection of goats against peste des petits ruminants with attenuated rinderpest virus. Res Vet Sci 27:32 1-324, 1979 8 Taylor W P Serological studies with the virus of peste des petits ruminants in Nigeria. Res Vet Sci 26236-242, 1979 9 Taylor W P The distribution and epidemiology of peste des petits ruminants. Prev Med 2: 157-1 66, 1984
Request reprints from Dr. C. C. Brown, National Veterinary Services Laboratories, Foreign Animal Disease Diagnostic Laboratory, PO Box 848, Greenport, NY 1 1944 (USA).
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