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Immunohistochemical detection of peste des petits ruminants viral antigens in tissues from cases of naturally occurring pneumonia in goats
Small Ruminant Research 51 (2004) 273–277
Technical note
Immunohistochemical detection of peste des petits ruminants viral antigens in tissues from cases of naturally occurring pneumonia in goats Z. Yener a,∗ , Y.S. Sa˘glam b , A. Temur c , H. Kele¸s d a
Department of Pathology, Faculty of Veterinary Medicine, Yuzuncu Yil University, Van 65080, Turkey b Department of Pathology, Faculty of Veterinary Medicine, Atatürk University, Erzurum, Turkey c Pathology Laboratory, Veterinary Control and Research Institute, Erzurum, Turkey d Department of Pathology, Faculty of Veterinary Medicine, Afyon Kocatepe University, Afyon, Turkey Accepted 4 April 2003
Abstract Peste des petits ruminants (PPR) is an important viral disease of goats and sheep characterized by erosive stomatitis, enteritis, and pneumonia. But the pneumonia is the most frequently recorded lesion in PPR infections. In this study, the presence of PPR viral antigens in goat’s pneumonia was investigated. For this purpose, a total of 1505 goat’s lungs slaughtered in Bitlis and Van slaughterhouses were grossly examined and pneumonia was detected in 74 cases (4.91%). Lesions were more frequently encountered in anteroventral lobes than caudal lobes. Immunohistochemical (IHC) examinations were performed on 42 pneumonic lungs (except the verminous pneumonia). IHC staining of formalin-fixed, paraffin-embedded tissues were carried out by the avidin–biotin–peroxidase complex (ABC) procedure. The presence of PPR viral antigens were detected in 17 (40%) out of 42 pneumonic lungs. Viral antigens were found most frequently in the cytoplasm and rarely in the nucleus, including bronchial glands, bronchial and bronchiolar epithelial cells, type II pneumocytes, syncytial cells, and alveolar macrophages. In conclusion, it was found that there was a close relationship between the pneumonia in goats and the presence of PPR viral antigens. The PPR incidence in pneumonic lungs of goats was detected to be very high in the present study performed in the region of Bitlis and Van. © 2003 Elsevier B.V. All rights reserved. Keywords: Peste des petits ruminants; Pneumonia; Immunohistochemistry; Goats
1. Introduction Peste des petits ruminants (PPR) is an acute or subacute contagious viral disease of goats and sheep characterized by fever, erosive stomatitis, conjunc∗ Corresponding author. Tel.: +90-432-225-11-28; fax: +90-432-225-11-27. E-mail addresses: [email protected], [email protected] (Z. Yener).
tivitis, gastroenteritis, and pneumonia (Bundza et al., 1988; Brown et al., 1991; Aruni et al., 1998). PPR is prevalent in most of Africa, the Middle East countries and in recent years, in Indian subcontinent (Shaila et al., 1996). The outbreaks of PPR are also recently reported in Turkey (Alcigir et al., 1996; Gul et al., 2001). The disease causes major health constraints in sheep and goats with considerable economic losses to the farmers (Ramadevi and Sreelatha, 1999). The causative agent, peste des petits ruminants virus
0921-4488/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0921-4488(03)00194-9
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Z. Yener et al. / Small Ruminant Research 51 (2004) 273–277
(PPRV), is closely related to rinderpest virus (RPV), which causes a similar but distinct disease in large ruminants (Sinnathamby et al., 2001). Rinderpest and PPR are especially distinguished between each other by their different range of pathogenicity and varied host range (Diallo et al., 1989). Unlike rinderpest, the lung lesions characterized by bronchitis, bronchiolitis, or bronchointerstitial pneumonia are the most consistent findings in PPR infections (Brown et al., 1991; Aruni et al., 1998). Immunohistochemical (IHC) studies have been performed on experimentally induced PPR and naturally occurring PPR infections of goats (Bundza et al., 1988; Brown et al., 1991; Saliki et al., 1994; Eligulashvili et al., 1999). However, IHC studies of the pneumonia caused by PPRV are scanty (Brown et al., 1991). The purpose of this investigation was to determine the role and presence, if any, of PPRV antigens using immunohistochemistry staining of formalin-fixed, paraffin-embedded tissues in pneumonia of slaughtered goats at abattoir.
2. Materials and methods An abattoir survey of goats was carried out between August and December 1999 in Bitlis and Van (neighboring Iran, provinces of East Anatolia) regions. The goats presented for slaughtering were predominantly young females. A total 1505 goats were grossly examined and pneumonia was detected in 74 cases (4, 91%). The distribution and gross appearance of the pneumonic areas were recorded and sampled at the slaughterhouse. Pneumonic lesions characterized by slightly or confluent consolidated purple-red or gray foci and irregular lobular foci of atelectasis were usually confined to the cranioventral regions of lungs. Selected tissues were preserved in 10% neutral buffered formalin. Pieces of pneumonic lungs were embedded in paraffin. Replicate sections were cut at 4–5 m for histopathologic examination. The prevalence of pneumonic lesions and their pathological characteristics in slaughtered goats were announced elsewhere (Yener et al., 2001). Sections repeated from these samples kept in paraffin blocks for 3 years were stained only for immunohistochemistry to demonstrate the presence of PPRV antigens on cases of 42 pneumonic lungs (except the verminous pneumonia). ICH staining was performed with the avidin–biotin–peroxidase complex
(ABC) procedure (Bundza et al., 1988; Brown et al., 1991), using commercially available immunoperoxidase kits (Shandon Inc., CadenzaTags peroxidase kit with AEC, No. 407300, PA, USA). The polyclonal rabbit anti-RPV serum was used for IHC staining (the anti-RPV serum was kindly supplied by Dr. E. Aksoy, Ankara Central Veterinary Control and Research Institute, Turkey). For tissue control, a non-pneumonic goat lung tissue was processed in an identical manner to the pneumonic lung tissues. For serum control, replicate sections of the pneumonic lung tissues were processed, substituting normal rabbit serum for rabbit anti-RPV serum.
3. IHC and histopathological findings In IHC staining, the presence of PPR viral antigens were detected in 17 (40%) out of 42 pneumonic lungs. No staining was observed in tissue from non-pneumonic lung or in pneumonic lung tissue on which the normal rabbit serum was used (serum control). Viral antigens were found most frequently in the cytoplasm and rarely in the nucleus of bronchial glands, bronchial and bronchiolar epithelial cells, type II pneumocytes, syncytial cells, and alveolar macrophages. Antigens were also found at the luminal surfaces of bronchial, bronchiolar and alveoli epithelial cells, within bronchial and bronchiolar exudates, and in the chondrocytes of cartilage around bronchi (Fig. 1a–d). Staining generally tended to be granular in the cytoplasm and linear in the luminal surfaces, on the other hand diffuse in the cytoplasm of the chondrocytes. The immunopositivity was strong in bronchi and bronchioles, especially around which had inflammatory foci, and which had also the greatest concentrations of viral antigens. In some cases, staining included the glandular epithelium and the chondrocytes of cartilage of bronchi. The immunoreaction in the type II pneumocytes, syncytial cells, and alveolar macrophages was weak. Of 42 pneumonic lung immunohistochemically examined in this study, 17 cases with immunopositivity showed multifocal necrotic bronchitis, bronchiolitis or bronchointerstitial pneumonia with varying degrees of severity. Inflammation in the bronchi and bronchioles was characterized with focal degeneration,
Z. Yener et al. / Small Ruminant Research 51 (2004) 273–277
275
Fig. 1. (a) Bronchial epithelial cells showing intracytoplasmic PPR antigens (320×); (b) viral antigens of PPR antigens are present in the cytoplasm of epithelial cells of bronchial glands (200×); (c) IHC staining demonstrating PPR antigens in the cytoplasm of chondrocytes around bronchi and within bronchiolar exudates (320×); (d) type II pneumocytes and syncytial cells displaying the viral antigens of PPR (320×) (avidin–biotin–peroxidase method, Mayer’s haematoxylin counterstain); (e) necrotizing bronchiolitis and a proliferative inflammatory reaction involving adjacent alveoli (200×) (haematoxylin-eosin).
necrosis, and ulceration. Alveoli next to affected bronchioles were altered. These alveoli consistently revealed marked hyperplasia of type II pneumocytes and increased numbers of alveolar macrophages, and mostly syncytial cell formations (Fig. 1e). But viral inclusion bodies were not seen in any of these cases. In some areas, the alveolar septa were irregularly thickened with lymphocytes and a lesser number of macrophages. There were lymphoid aggregates in some septa and also adjacent to small vessels and bronchioles. In many cases these proliferative lesions were detected together with the neutrophilic exudate, which suggested the secondary bacterial invaders.
4. Discussion PPR is caused by a paramyxovirus of the Morbillivirus genus. Other members of the genus include RPV, measles virus (MV), canine distemper virus (CDV), and phocid distemper virus (PDV) of sea mammals (seals). For many years, PPRV was considered a variant of RPV, specifically adapted for goats and sheep, which had lost its virulence for cattle. It is now known that the two viruses are distinct though closely related antigenically and morphologically (Bourdin et al., 1970; Gibbs et al., 1979; Osterhaus, 1992). The morbilliviral diseases of PPR in sheep and goats, and rinderpest in cattle, have similar clinical
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Z. Yener et al. / Small Ruminant Research 51 (2004) 273–277
signs, and similar inflammatory and necrotic lesions in the mouth and the gastrointestinal tract, but animals with rinderpest rarely develop significant pneumonia. In contrast, PPR have marked involvement of the respiratory tract characterized by the consolidating, interstitial pneumonia with necrotizing bronchiolitis (Conference, 2001). Rabbit anti-RPV serum have been successfully used for IHC detection of PPRV antigens in formalin-fixed, paraffin-embedded tissues of experimentally induced (Bundza et al., 1988; Brown et al., 1991) and naturally occurring PPR infections (Alcigir et al., 1996), because of their strong cross-reactivity. Even in this investigation, rabbit anti-RPV serum were used for IHC detection of PPRV antigens in cases of naturally occurring pneumonia in goats, and PPR viral antigens were detected in 17 (40%) out of 42 pneumonic lungs. Owing to the fact that PPRV has usually affecting the respiratory system, these results have been considered to be reproducible. The PPR incidence in pneumonic lungs of goats was found to be very high in the present study performed in the regions of Bitlis and Van, which suggested that the outbreaks of PPR might have occurred in these regions. Under light of literature information presented below, it can be considered that the present findings in investigation were in conformity with the literature. According to the reports of International Office of Epizootics (OIE); in 1998, after an outbreak of PPR in Iraq was reported, by its northern governorates to OIE; in 1999–2000 years, outbreaks of PPR in goats in Provinces in eastern Anatolia of Turkey were reported to OIE (Ozkul et al., 2002), which called to mind that these outbreaks to be related to movements of animals from the east. Furthermore, when it was surveyed the affected areas of Turkey regarding the presence of infection with PPRV, the regions of Van and Bitlis were found to indicate the highest percentage of seroprevalence (Ozkul et al., 2002). The PPRV infections have been characterized by the presence of viral antigens immunohistochemically in greatest concentrations in airway epithelium, including trachea, bronchi and bronchioles, and histopathologically the presence of bronchointerstitial pneumonia with necrotizing bronchiolitis, type II pneumocyte hyperplasia, syncytial cell formations, viral inclusion bodies in the syncytial cells and pneumocytes, and epithelial cells of bronchi and bronchioles
(Brown et al., 1991). In this study histopathologic and IHC findings were similar to those described above by Brown et al. (1991), but the viral inclusion bodies were not observed, differently viral antigens were also found in the chondrocytes of cartilage around bronchi and within bronchial and bronchiolar exudates. The absence of inclusion bodies could be explained by the fact that the animals examined in this study were surviving acute phase of PPR and recovering from PPR. Hence inclusion bodies or epithelial cells with inclusion may be depleted in these animals. Furthermore, Perl et al. (1994) stated that all morphological changes of PPR were seen in only the regions where endemic. Brown et al. (1991) suggested that PPR-initiated pneumonias may result in death due to bacterial pneumonia, and the primary viral etiology is overlooked. They also suggested that PPR virus had the potential to cause severe pneumonia. In this study, it was noted that there was usually mild involvement of respiratory system confined to the cranioventral regions of lungs, underscoring the primary viral lesion and secondary bacterial invaders were not considered will not enough to bring about a serious pneumonia, presumably due to environmental conditions in which the goats examined in the study survived, and their race features. As far as we could determine, this study most likely is the first report of demonstration of PPRV antigen in cases of naturally occurring pneumonia in goats slaughtered at abattoir. Moreover, in this study, the retrospective examination of preserved specimens of animals suspected from PPR has been shown to be possible immunohistochemically. This study was performed in the regions of Bitlis and Van, and a relationship was found between pneumonia and the presence of PPR viral antigens. The PPR incidence in pneumonic lungs was detected to be very high, probably because of the animals in the region surviving an outbreak of PPR. References Alcigir, G., Vural, S.A., Toplu, N., 1996. Turkiye’de kuzularda peste des petits ruminants virus enfeksiyonunun patomorfolojik ve immunohistolojik ilk tanimi (The first pathomorphological and immunohistological description of the peste petitis ruminants virus observed in lambs in Turkey). Ankara Univ. Vet. Fak. Derg. 43, 181–189. Aruni, A.W., Lalitha, P.S., Mohan, A.C., Chitravelu, P., Anbumani, S.P., 1998. Histopathological study of a natural outbreak of
Z. Yener et al. / Small Ruminant Research 51 (2004) 273–277 peste des petitis ruminants in goats of Tamilnadu. Small Rumin. Res. 28, 233–240. Bourdin, P., Rioche, M., Laurent, A., 1970. Emploi d’un vaccin antibovipestique produit sur cultures cellulaires dans la prophylaxie de la peste des petits ruminants au dahomey. Rev. Elev. Med. Vet. Pays Trop. 23, 295–300. Brown, C.C., Mariner, J.C., Olander, H.J., 1991. An immunohistochemical study of the pneumonia caused by peste des petits ruminants virus. Vet. Pathol. 28, 166–170. Bundza, A., Afshar, A., Dukes, T.W., Myers, D.J., Dulac, G.C., Becker, S.A.W.E., 1988. Experimental peste des petits ruminants (goat plague) in goats and sheep. Can. J. Vet. Res. 52, 46–52. Conference, 2001. In: Brown, C. (Coord.), Proceedings of the Wednesday Slide Conference 2000–2001, 14–22 March 2001, Department of Veterinary Pathology, The Armed Forces Institute of Pathology. Diallo, A., Taylor, W.P., Lefevre, P.C., Provost, A., 1989. Attenuation d’une souche du virus de la peste des petits ruminants: candidat pour un vaccin homologue vivant. Rev. Elev. Med. Vet. Pays Trop. 42, 311–319. Eligulashvili, R., Perl, S., Stram, Y., Friedgut, O., Scheichat, N., Samina, I., Trainin, Z., 1999. Immunohistochemical detection of peste des petits ruminants viral antigen in formalin-fixed, paraffin-embedded tissues from cases of naturally occurring infection. J. Vet. Diagn. Invest. 11, 286–288. Gibbs, E.P.J., Taylor, W.P., Lawman, M.J.P., Bryant, J., 1979. Classification of peste des petits ruminants virus as the fourth member of the genus Morbillivirus. Intervirology 11, 268–274. Gul, Y., Dabak, M., Issi, M., Ba¸sbug, O., 2001. Elazig’da 1999 yilinda koyun ve kecilerde gozlenen peste des petits ruminants olgulari (Peste des petits ruminants cases observed in sheep and goats at 1999 year in Elazıg province of Turkey). Fırat Üniv. Sag. Bil. Derg. 15, 35–38.
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Osterhaus, A.D.M.E., 1992. Studies on virus Infections of wild aquatic mammals in the ciba-geigy prize for research in animal health, 1991. Ciba-Geigy Ltd., Basel, Switzerland, p. 27. Ozkul, A., Akca, Y., Alkan, F., Barrett, T., Karaoglu, T., Dagalp, S.B., Anderson, J., Yesilbag, K., Cokcaliskan, C., Gencay, A., Burgu, I., 2002. Prevalence, distribution, and host range of peste des petits ruminants virus, Turkey. Research 8 (7). Perl, S., Alexander, A., Yakobson, B., Nyska, A., Harmelin, A., Sheikhal, N., Shimshony, A., Davidson, M., Abramson, M., Rapaport, E., 1994. Peste des petits ruminants (PPR) of sheep in Israel: case report. Isr. J. Vet. Med. 49, 59–62. Ramadevi, V., Sreelatha, C.H., 1999. Peste des petits ruminants (Ppr) in sheep and goats. http://www.intaspharma.com/neovet/ journal/page3637.htm. Saliki, J.T., Brown, C.C., House, J.A., Dubovi, E.J., 1994. Differential immunohistochemical staining of peste des petits ruminants and rinderpest antigens in formalin-fixed, paraffin-embedded tissues using monoclonal and polyclonal antibodies. J. Vet. Diagn. Invest. 6, 96–98. Shaila, M., Shamaki, D., Forsyth, M., Diallo, A., Goatley, L., Kitching, R., Barrett, T., 1996. Geographic distribution and epidemiology of peste des petits ruminants virus. Virus Res. 43, 149–153. Sinnathamby, G., Renukaradhya, G., Rajasekhar, M., Nayak, R., Shaila, M., 2001. Immune responses in goats to recombinant hemagglutinin–neuraminidase glycoprotein of peste des petits ruminants virus: identification of at cell determinant. Vaccine 19, 4816–4823. Yener, Z., Gurturk, K., Gulbahar, Y., Solmaz, H., 2001. Bitlis mezbahasinda kesilen kecilerde pnomoni olgulari uzerinde patolojik ve bakteriyolojik calismalar (Pathological and bacteriological studies on pneumonia in goats slaughtered at Bitlis slaughterhouse). Vet. Bil. Derg. 17, 13–20.
Technical note
Immunohistochemical detection of peste des petits ruminants viral antigens in tissues from cases of naturally occurring pneumonia in goats Z. Yener a,∗ , Y.S. Sa˘glam b , A. Temur c , H. Kele¸s d a
Department of Pathology, Faculty of Veterinary Medicine, Yuzuncu Yil University, Van 65080, Turkey b Department of Pathology, Faculty of Veterinary Medicine, Atatürk University, Erzurum, Turkey c Pathology Laboratory, Veterinary Control and Research Institute, Erzurum, Turkey d Department of Pathology, Faculty of Veterinary Medicine, Afyon Kocatepe University, Afyon, Turkey Accepted 4 April 2003
Abstract Peste des petits ruminants (PPR) is an important viral disease of goats and sheep characterized by erosive stomatitis, enteritis, and pneumonia. But the pneumonia is the most frequently recorded lesion in PPR infections. In this study, the presence of PPR viral antigens in goat’s pneumonia was investigated. For this purpose, a total of 1505 goat’s lungs slaughtered in Bitlis and Van slaughterhouses were grossly examined and pneumonia was detected in 74 cases (4.91%). Lesions were more frequently encountered in anteroventral lobes than caudal lobes. Immunohistochemical (IHC) examinations were performed on 42 pneumonic lungs (except the verminous pneumonia). IHC staining of formalin-fixed, paraffin-embedded tissues were carried out by the avidin–biotin–peroxidase complex (ABC) procedure. The presence of PPR viral antigens were detected in 17 (40%) out of 42 pneumonic lungs. Viral antigens were found most frequently in the cytoplasm and rarely in the nucleus, including bronchial glands, bronchial and bronchiolar epithelial cells, type II pneumocytes, syncytial cells, and alveolar macrophages. In conclusion, it was found that there was a close relationship between the pneumonia in goats and the presence of PPR viral antigens. The PPR incidence in pneumonic lungs of goats was detected to be very high in the present study performed in the region of Bitlis and Van. © 2003 Elsevier B.V. All rights reserved. Keywords: Peste des petits ruminants; Pneumonia; Immunohistochemistry; Goats
1. Introduction Peste des petits ruminants (PPR) is an acute or subacute contagious viral disease of goats and sheep characterized by fever, erosive stomatitis, conjunc∗ Corresponding author. Tel.: +90-432-225-11-28; fax: +90-432-225-11-27. E-mail addresses: [email protected], [email protected] (Z. Yener).
tivitis, gastroenteritis, and pneumonia (Bundza et al., 1988; Brown et al., 1991; Aruni et al., 1998). PPR is prevalent in most of Africa, the Middle East countries and in recent years, in Indian subcontinent (Shaila et al., 1996). The outbreaks of PPR are also recently reported in Turkey (Alcigir et al., 1996; Gul et al., 2001). The disease causes major health constraints in sheep and goats with considerable economic losses to the farmers (Ramadevi and Sreelatha, 1999). The causative agent, peste des petits ruminants virus
0921-4488/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0921-4488(03)00194-9
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Z. Yener et al. / Small Ruminant Research 51 (2004) 273–277
(PPRV), is closely related to rinderpest virus (RPV), which causes a similar but distinct disease in large ruminants (Sinnathamby et al., 2001). Rinderpest and PPR are especially distinguished between each other by their different range of pathogenicity and varied host range (Diallo et al., 1989). Unlike rinderpest, the lung lesions characterized by bronchitis, bronchiolitis, or bronchointerstitial pneumonia are the most consistent findings in PPR infections (Brown et al., 1991; Aruni et al., 1998). Immunohistochemical (IHC) studies have been performed on experimentally induced PPR and naturally occurring PPR infections of goats (Bundza et al., 1988; Brown et al., 1991; Saliki et al., 1994; Eligulashvili et al., 1999). However, IHC studies of the pneumonia caused by PPRV are scanty (Brown et al., 1991). The purpose of this investigation was to determine the role and presence, if any, of PPRV antigens using immunohistochemistry staining of formalin-fixed, paraffin-embedded tissues in pneumonia of slaughtered goats at abattoir.
2. Materials and methods An abattoir survey of goats was carried out between August and December 1999 in Bitlis and Van (neighboring Iran, provinces of East Anatolia) regions. The goats presented for slaughtering were predominantly young females. A total 1505 goats were grossly examined and pneumonia was detected in 74 cases (4, 91%). The distribution and gross appearance of the pneumonic areas were recorded and sampled at the slaughterhouse. Pneumonic lesions characterized by slightly or confluent consolidated purple-red or gray foci and irregular lobular foci of atelectasis were usually confined to the cranioventral regions of lungs. Selected tissues were preserved in 10% neutral buffered formalin. Pieces of pneumonic lungs were embedded in paraffin. Replicate sections were cut at 4–5 m for histopathologic examination. The prevalence of pneumonic lesions and their pathological characteristics in slaughtered goats were announced elsewhere (Yener et al., 2001). Sections repeated from these samples kept in paraffin blocks for 3 years were stained only for immunohistochemistry to demonstrate the presence of PPRV antigens on cases of 42 pneumonic lungs (except the verminous pneumonia). ICH staining was performed with the avidin–biotin–peroxidase complex
(ABC) procedure (Bundza et al., 1988; Brown et al., 1991), using commercially available immunoperoxidase kits (Shandon Inc., CadenzaTags peroxidase kit with AEC, No. 407300, PA, USA). The polyclonal rabbit anti-RPV serum was used for IHC staining (the anti-RPV serum was kindly supplied by Dr. E. Aksoy, Ankara Central Veterinary Control and Research Institute, Turkey). For tissue control, a non-pneumonic goat lung tissue was processed in an identical manner to the pneumonic lung tissues. For serum control, replicate sections of the pneumonic lung tissues were processed, substituting normal rabbit serum for rabbit anti-RPV serum.
3. IHC and histopathological findings In IHC staining, the presence of PPR viral antigens were detected in 17 (40%) out of 42 pneumonic lungs. No staining was observed in tissue from non-pneumonic lung or in pneumonic lung tissue on which the normal rabbit serum was used (serum control). Viral antigens were found most frequently in the cytoplasm and rarely in the nucleus of bronchial glands, bronchial and bronchiolar epithelial cells, type II pneumocytes, syncytial cells, and alveolar macrophages. Antigens were also found at the luminal surfaces of bronchial, bronchiolar and alveoli epithelial cells, within bronchial and bronchiolar exudates, and in the chondrocytes of cartilage around bronchi (Fig. 1a–d). Staining generally tended to be granular in the cytoplasm and linear in the luminal surfaces, on the other hand diffuse in the cytoplasm of the chondrocytes. The immunopositivity was strong in bronchi and bronchioles, especially around which had inflammatory foci, and which had also the greatest concentrations of viral antigens. In some cases, staining included the glandular epithelium and the chondrocytes of cartilage of bronchi. The immunoreaction in the type II pneumocytes, syncytial cells, and alveolar macrophages was weak. Of 42 pneumonic lung immunohistochemically examined in this study, 17 cases with immunopositivity showed multifocal necrotic bronchitis, bronchiolitis or bronchointerstitial pneumonia with varying degrees of severity. Inflammation in the bronchi and bronchioles was characterized with focal degeneration,
Z. Yener et al. / Small Ruminant Research 51 (2004) 273–277
275
Fig. 1. (a) Bronchial epithelial cells showing intracytoplasmic PPR antigens (320×); (b) viral antigens of PPR antigens are present in the cytoplasm of epithelial cells of bronchial glands (200×); (c) IHC staining demonstrating PPR antigens in the cytoplasm of chondrocytes around bronchi and within bronchiolar exudates (320×); (d) type II pneumocytes and syncytial cells displaying the viral antigens of PPR (320×) (avidin–biotin–peroxidase method, Mayer’s haematoxylin counterstain); (e) necrotizing bronchiolitis and a proliferative inflammatory reaction involving adjacent alveoli (200×) (haematoxylin-eosin).
necrosis, and ulceration. Alveoli next to affected bronchioles were altered. These alveoli consistently revealed marked hyperplasia of type II pneumocytes and increased numbers of alveolar macrophages, and mostly syncytial cell formations (Fig. 1e). But viral inclusion bodies were not seen in any of these cases. In some areas, the alveolar septa were irregularly thickened with lymphocytes and a lesser number of macrophages. There were lymphoid aggregates in some septa and also adjacent to small vessels and bronchioles. In many cases these proliferative lesions were detected together with the neutrophilic exudate, which suggested the secondary bacterial invaders.
4. Discussion PPR is caused by a paramyxovirus of the Morbillivirus genus. Other members of the genus include RPV, measles virus (MV), canine distemper virus (CDV), and phocid distemper virus (PDV) of sea mammals (seals). For many years, PPRV was considered a variant of RPV, specifically adapted for goats and sheep, which had lost its virulence for cattle. It is now known that the two viruses are distinct though closely related antigenically and morphologically (Bourdin et al., 1970; Gibbs et al., 1979; Osterhaus, 1992). The morbilliviral diseases of PPR in sheep and goats, and rinderpest in cattle, have similar clinical
276
Z. Yener et al. / Small Ruminant Research 51 (2004) 273–277
signs, and similar inflammatory and necrotic lesions in the mouth and the gastrointestinal tract, but animals with rinderpest rarely develop significant pneumonia. In contrast, PPR have marked involvement of the respiratory tract characterized by the consolidating, interstitial pneumonia with necrotizing bronchiolitis (Conference, 2001). Rabbit anti-RPV serum have been successfully used for IHC detection of PPRV antigens in formalin-fixed, paraffin-embedded tissues of experimentally induced (Bundza et al., 1988; Brown et al., 1991) and naturally occurring PPR infections (Alcigir et al., 1996), because of their strong cross-reactivity. Even in this investigation, rabbit anti-RPV serum were used for IHC detection of PPRV antigens in cases of naturally occurring pneumonia in goats, and PPR viral antigens were detected in 17 (40%) out of 42 pneumonic lungs. Owing to the fact that PPRV has usually affecting the respiratory system, these results have been considered to be reproducible. The PPR incidence in pneumonic lungs of goats was found to be very high in the present study performed in the regions of Bitlis and Van, which suggested that the outbreaks of PPR might have occurred in these regions. Under light of literature information presented below, it can be considered that the present findings in investigation were in conformity with the literature. According to the reports of International Office of Epizootics (OIE); in 1998, after an outbreak of PPR in Iraq was reported, by its northern governorates to OIE; in 1999–2000 years, outbreaks of PPR in goats in Provinces in eastern Anatolia of Turkey were reported to OIE (Ozkul et al., 2002), which called to mind that these outbreaks to be related to movements of animals from the east. Furthermore, when it was surveyed the affected areas of Turkey regarding the presence of infection with PPRV, the regions of Van and Bitlis were found to indicate the highest percentage of seroprevalence (Ozkul et al., 2002). The PPRV infections have been characterized by the presence of viral antigens immunohistochemically in greatest concentrations in airway epithelium, including trachea, bronchi and bronchioles, and histopathologically the presence of bronchointerstitial pneumonia with necrotizing bronchiolitis, type II pneumocyte hyperplasia, syncytial cell formations, viral inclusion bodies in the syncytial cells and pneumocytes, and epithelial cells of bronchi and bronchioles
(Brown et al., 1991). In this study histopathologic and IHC findings were similar to those described above by Brown et al. (1991), but the viral inclusion bodies were not observed, differently viral antigens were also found in the chondrocytes of cartilage around bronchi and within bronchial and bronchiolar exudates. The absence of inclusion bodies could be explained by the fact that the animals examined in this study were surviving acute phase of PPR and recovering from PPR. Hence inclusion bodies or epithelial cells with inclusion may be depleted in these animals. Furthermore, Perl et al. (1994) stated that all morphological changes of PPR were seen in only the regions where endemic. Brown et al. (1991) suggested that PPR-initiated pneumonias may result in death due to bacterial pneumonia, and the primary viral etiology is overlooked. They also suggested that PPR virus had the potential to cause severe pneumonia. In this study, it was noted that there was usually mild involvement of respiratory system confined to the cranioventral regions of lungs, underscoring the primary viral lesion and secondary bacterial invaders were not considered will not enough to bring about a serious pneumonia, presumably due to environmental conditions in which the goats examined in the study survived, and their race features. As far as we could determine, this study most likely is the first report of demonstration of PPRV antigen in cases of naturally occurring pneumonia in goats slaughtered at abattoir. Moreover, in this study, the retrospective examination of preserved specimens of animals suspected from PPR has been shown to be possible immunohistochemically. This study was performed in the regions of Bitlis and Van, and a relationship was found between pneumonia and the presence of PPR viral antigens. The PPR incidence in pneumonic lungs was detected to be very high, probably because of the animals in the region surviving an outbreak of PPR. References Alcigir, G., Vural, S.A., Toplu, N., 1996. Turkiye’de kuzularda peste des petits ruminants virus enfeksiyonunun patomorfolojik ve immunohistolojik ilk tanimi (The first pathomorphological and immunohistological description of the peste petitis ruminants virus observed in lambs in Turkey). Ankara Univ. Vet. Fak. Derg. 43, 181–189. Aruni, A.W., Lalitha, P.S., Mohan, A.C., Chitravelu, P., Anbumani, S.P., 1998. Histopathological study of a natural outbreak of
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