J. Vet. Med. B 39, 595-598 (1992) 0 1992 Paul Parey Scientific Publishers, Berlin and Hamburg ISSN 0931-1793
Instituto Nacional de Tecnologia Agropecuaria (INTA), Bariloche, Argentina
Bacillary Haemoglobinuria Diagnosis by the Peroxidase-Antiperoxidase (PAP) Technique F. A. UZAL~ ':., K. BELAK~, E. RIVERA*,C. A. ROBLES'and R. E. FEINSTEIN~ Address of authors: 1 Instituto Nacional de Tecnologia Agropecuaria (INTA), EEA Bariloche. C. C. 277-(8400) Bariloche, Argentina ZThe National Veterinary Institute (SVA). P. 0. Box 7073, S-750 07 Uppsala, Sweden
With one figure (Receivedfor publication October 24, 1991)
Summary A peroxidase-antiperoxidase (PAP) technique was used to diagnose bacillary haemoglobinuria in formalin-fixed, paraffin-embedded liver tissues of cattle. The PAP method revealed Clostridium haemolyticum in the zone of liver necrosis characteristic of the disease and also in culture smears of this microorganism, but C. novyi type B, C. chuuvoei, C. septicum and C.perfrngens types B and C remained unstained by the I'AP reaction. The PAP technique performed provides a specific, simple and rapid method to diagnose bacillary haemoglobinuria.
Introduction Bovine bacillary haemoglobinuria (BH) is characterized by an acute clinical course with fever, jaundice and haemoglobinuria. The causative agent Clostridium haernolyticum (C. novyi type D ) is found throughout the period of infection in a zone of liver necrosis (3, 5, 6). Ideally, a laboratory diagnosis of B H should be based on the cultivation of C. haemolyticum (3, 5). However, the fastidious cultural requirements of C. haemolyticum (3) and other factors, such as the early decomposition of tissues and contamination of other bacteria, may obscure the presence of C. haernolyticum (2) and hamper a laboratory diagnosis of BH. Immunohistochemical methods, such as the peroxidase-antiperoxidase (PAP) technique, have been used to demonstrate diverse microorganisms in tissue sections (4, 9). In the present study a PAP technique to diagnose bovine B H was performed using rabbit antibodies to vegetative forms of C.haemolyticum. Material and Methods Five Hereford cattle died from BH. Pure cultures of C. huemolyticum were isolated from the livers (data not shown), and the livers, with the zone of necrosis characteristic of BH, were examined microscopically. Samples of liver were fixed in 10% buffered formalin ( p H 7.4) for 24 hours, embedded in paraffin, sectioned at 4 pm and stained with haematoxylin and eosin ( H & E), and Giemsa and Gram stains. Liver samples, from a clinically healthy Hereford bull, that had been slaughtered, were processed as described above' and used as control. The time interval elapsed between death of the animals and the start of fixation ranged from 0 to 3 hours.
'k
Corresponding author.
U.S. Copyright Clearance Center Code Statement:
0931 - 1793/92/3908 -0595$02.50/0
596
UZAL,BELAK,RIVERA,ROBLESand FEINSTEIN
A C. haemolyticum strain isolated from one calf liver was cultured in chopped meat broth under anaerobic conditions for 48 hours at 37°C. To harvest the bacteria the growth medium was filtered, the liquid phase was centrifuged at 8 0 0 g~ for 60 minutes and the pellet obtained was further processed to obtain a vaccine, as described previously (9). T w o ml of that material was injected subcutaneously into two adult male New Zealand White rabbits, o n two occasions, with a 30-days interval. After further seven days, the rabbits were bled, the serum was separated, and stored at -20 " C until used as primary antibody (P-ah). Sera obtained prior to the immunization werc used as normal sera (NS) for control slides. To check the specificity of the primary antiserum produced, culture smears of C. haemolyricum, C. novyi type B, C. chuuvoei, C. septicum and C.perfringens types B and C, were air-dried, fixed by iminersion in pure acetone for 10 minutes at 4 ° C and incubated with P-ah o r NS respectively. For the PAP-technique, 4 pm thick sections were deparaffinized, rinsed in 0.01 M phosphate buffered saline (pH 7.2) and processed as described previously (4). Both culture smears and tissue sections were incubated with P-ah and NS diluted to 1:50, 1:100, 1:200, 1:400, 1:800 and 1:1,600.
Results Out of t h e six different P-ab dilutions tested, t h e 1:400 dilution resulted in t h e strongest staining of C. haemolyticum with t h e lowest background. T h e r e f o r e a 1:400 P-ab concentration w a s used thereafter. The PAP technique used here stained C. haemolyticum smears d a r k b r o w n . U s i n g NS, smears remained unstained. N e i t h e r P-ab n o r NS resulted in any PAP-staining of C. riowyi type B, C. chauvoei, C. septicum, C.perfringens type B a n d C.perfringens type C in c u l t u r e smears. Staining w a s n o t observed in sections w h e r e NS replaces P-ab, nor in liver sections f r o m t h e uninfected animal.
Fig. 1. PAP-stained C. haemolyticum (arrows) in a liver area of coagulation necrosis. Notice part of the rim of congestion and inflammatory infiltrate (i) that surrounds the necrotic area. Hematoxylin counterstaining. Bar: 10 pm
Peroxidase-Antiperoxidase Technique for Bacillary Haemoglobinuria
597
The five Hereford cattle examined displayed similar liver changes. The lesions noticed (using H & E, Giemsa and Gram stains) corresponded well with previous descriptions (5, 6). The PAP technique revealed numerous brown-stained rods, approximate size 3 x 1 pm; located singly or in clusters, in the sinusoids of the necrotic area and in the adjacent inner parts of the surrounding area of congestion, hemorrhage and infiltrating neutrophils (Fig. 1). In the inner margin of the rim of congestion and neutrophilic infiltration, granular or amorphous, PAP-stained material mixed with leukocytes, erythrocytes and unstained amorphous material was also found. Neither rods nor PAP-stained material were observed in the tissues surrounding the necrotic zone.
Discussion Bovine bacillary haemoglobinuria (BH) is routinely diagnosed by culturing the causal agent, C. huemolyticum. However, C. huemolyticum is very demanding in its cultural requirements and its cultivation is time-consuming (3, 6). For these reasons a simple and rapid method for BH diagnosis is needed. The PAP technique for C.huemolyticum described here is a simple and rapid method to perform, and it may also complement the cultural procedures to diagnose BH. C. huemolyticum and several other clostridial organisms in animal tissues and smears have been identified by immunofluorescence (1, 6, 10). However, a fluorescence microscope is required, the material must be fresh or specially prepared, sections can be stored for a few hours only and the tissue morphology is poor. All of these problems are solved with the PAP technique. Furthermore, the specificity of the immunofluorescent methods cited previously is not always uniform (1, 8, 10). The cultural examination of the BH livers investigated in this study confirmed that the rods shown with the PAP technique were, indeed, C. huemolyticum. The specificity of the PAP method was confirmed also by the lack of PAP-stain, as shown by the culture smears of other Clostridia and liver sections from control animal. The possibility to distinguish C. huemolyticum from C. novyi type B is noteworthy, since in cattle C. novyi type B, can cause lesions similar to BH (5, 6, 11). The PAP technique described here permits one to quickly and specifically stain C. huemolyticum in tissues processed for routine histology, thus providing a simple and rapid method to diagnose BH especially where laboratory facilities for anaerobic cultivation of the bacteria are not readily available. The PAP technique also enables retrospective studies. Some of the liver tissues currently examined were filed for long periods of time, of up to 10 years. Acknowledgements We wish to thank Ms C.NILSSON for technical assistance and Dr F.V. OLAECHEA for and P.L6PEZ for constructive criticism of the manuscript. We also thank Ms S.WESTERBERG preparing the manuscript.
References 1. BATTY, I., and P.W. WALKER, 1964: The identification of C.novyi (C.oedematzens) and C. tetuni by the use of fluorescent labelled antibodies. J. Pathol. Bacteriol. 88, 327-328. and 0.M. RADOSTITS, 1979: Veterinary Medicine. Bailliere 2. BLOOD, D. C., J. A. HENDERSON, Tindall, London. pp. 450-452. J. H., and J. F. TIMONEY, 1981: Hagan and Bruner’s infectious diseases of domestic 3. GILLESPIE, animals. Cornell University Press, Ithaca & London. pp. 851. K., A. UGGLA, T. SVENSSON, and L. SJOLAND,1988: Detection of Toxoplusma gondii 4. GUSTAFSSON, in liver tissue sections from Brown Hares (Lepus europaeus P.) and Mountain Hares (Lepus timidus L . ) using the Peroxidase Anti-Peroxidase (PAP) technique as a complement to conventional histopathology. J. Vet. Med. B 35, 402-407.
598
UZAL,BELAK,RIVERA,ROBLESand FEINSTEIN
5. KELLY,R. W., 1985: The liver and biliary system. In: K. V. F. JUBB,P. C. KENNEDY,and N. PALMER (eds.). Pathology of Domestic Animals. Academic Press, Inc., Orlando, FL. pp.240-312. 6. POWER,E. P., V. J. WHITE, E. J. MACKESSY, and J. WARD, 1987: Bacillary haemoglobinuria of cattle: confirmation of the disease in Ireland. Irish Vet. J. 41, 255-257. 7. SMITH,D. H., and J. F. BONE,1970: In: W. J. GIBBONS, E. J. CATCOTT, and J. F. SMITHCORS (eds.). Bovine Medicine and Surgery. Amer. Vet. Publ. Inc., Illinois. pp. 121-146. L., and A. J. BOOTH, 1984: Bacillary hemoglobinuria in cattle. Comp. Cont. Ed. Prac. 8. STOGDALE, Vet. 6, 284-290. 9. UZAL,F. A., E. RIVERA,and R. E. FEINSTEIN,1990: Indirect immunoperoxidase and immunofluorescence staining of Bordetella bronchiseptica in formalin-fixed, paraffin-embedded lung tissues of rabbits. J. Vet. Med. A 37, 1-7. 10. VAN KAMPEN,K. R., and P. C. KENNEDY,1968: Experimental Bacillary hemoglobinuria: intrahepatic detection of spores of Clostriditrm huemolyticum by immunofluorescense in the rabbit. Amer. J. Vet. Res. 29, 2173-2177. 1 1 . WILLIAMS,B. M., 1964: Clostrzdium oedematiens infections (Black disease and Bacillary haemoglobinuria) of cattle in Mid-Wales. Vet. Rec. 76, 591-596.
Instituto Nacional de Tecnologia Agropecuaria (INTA), Bariloche, Argentina
Bacillary Haemoglobinuria Diagnosis by the Peroxidase-Antiperoxidase (PAP) Technique F. A. UZAL~ ':., K. BELAK~, E. RIVERA*,C. A. ROBLES'and R. E. FEINSTEIN~ Address of authors: 1 Instituto Nacional de Tecnologia Agropecuaria (INTA), EEA Bariloche. C. C. 277-(8400) Bariloche, Argentina ZThe National Veterinary Institute (SVA). P. 0. Box 7073, S-750 07 Uppsala, Sweden
With one figure (Receivedfor publication October 24, 1991)
Summary A peroxidase-antiperoxidase (PAP) technique was used to diagnose bacillary haemoglobinuria in formalin-fixed, paraffin-embedded liver tissues of cattle. The PAP method revealed Clostridium haemolyticum in the zone of liver necrosis characteristic of the disease and also in culture smears of this microorganism, but C. novyi type B, C. chuuvoei, C. septicum and C.perfrngens types B and C remained unstained by the I'AP reaction. The PAP technique performed provides a specific, simple and rapid method to diagnose bacillary haemoglobinuria.
Introduction Bovine bacillary haemoglobinuria (BH) is characterized by an acute clinical course with fever, jaundice and haemoglobinuria. The causative agent Clostridium haernolyticum (C. novyi type D ) is found throughout the period of infection in a zone of liver necrosis (3, 5, 6). Ideally, a laboratory diagnosis of B H should be based on the cultivation of C. haemolyticum (3, 5). However, the fastidious cultural requirements of C. haemolyticum (3) and other factors, such as the early decomposition of tissues and contamination of other bacteria, may obscure the presence of C. haernolyticum (2) and hamper a laboratory diagnosis of BH. Immunohistochemical methods, such as the peroxidase-antiperoxidase (PAP) technique, have been used to demonstrate diverse microorganisms in tissue sections (4, 9). In the present study a PAP technique to diagnose bovine B H was performed using rabbit antibodies to vegetative forms of C.haemolyticum. Material and Methods Five Hereford cattle died from BH. Pure cultures of C. huemolyticum were isolated from the livers (data not shown), and the livers, with the zone of necrosis characteristic of BH, were examined microscopically. Samples of liver were fixed in 10% buffered formalin ( p H 7.4) for 24 hours, embedded in paraffin, sectioned at 4 pm and stained with haematoxylin and eosin ( H & E), and Giemsa and Gram stains. Liver samples, from a clinically healthy Hereford bull, that had been slaughtered, were processed as described above' and used as control. The time interval elapsed between death of the animals and the start of fixation ranged from 0 to 3 hours.
'k
Corresponding author.
U.S. Copyright Clearance Center Code Statement:
0931 - 1793/92/3908 -0595$02.50/0
596
UZAL,BELAK,RIVERA,ROBLESand FEINSTEIN
A C. haemolyticum strain isolated from one calf liver was cultured in chopped meat broth under anaerobic conditions for 48 hours at 37°C. To harvest the bacteria the growth medium was filtered, the liquid phase was centrifuged at 8 0 0 g~ for 60 minutes and the pellet obtained was further processed to obtain a vaccine, as described previously (9). T w o ml of that material was injected subcutaneously into two adult male New Zealand White rabbits, o n two occasions, with a 30-days interval. After further seven days, the rabbits were bled, the serum was separated, and stored at -20 " C until used as primary antibody (P-ah). Sera obtained prior to the immunization werc used as normal sera (NS) for control slides. To check the specificity of the primary antiserum produced, culture smears of C. haemolyricum, C. novyi type B, C. chuuvoei, C. septicum and C.perfringens types B and C, were air-dried, fixed by iminersion in pure acetone for 10 minutes at 4 ° C and incubated with P-ah o r NS respectively. For the PAP-technique, 4 pm thick sections were deparaffinized, rinsed in 0.01 M phosphate buffered saline (pH 7.2) and processed as described previously (4). Both culture smears and tissue sections were incubated with P-ah and NS diluted to 1:50, 1:100, 1:200, 1:400, 1:800 and 1:1,600.
Results Out of t h e six different P-ab dilutions tested, t h e 1:400 dilution resulted in t h e strongest staining of C. haemolyticum with t h e lowest background. T h e r e f o r e a 1:400 P-ab concentration w a s used thereafter. The PAP technique used here stained C. haemolyticum smears d a r k b r o w n . U s i n g NS, smears remained unstained. N e i t h e r P-ab n o r NS resulted in any PAP-staining of C. riowyi type B, C. chauvoei, C. septicum, C.perfringens type B a n d C.perfringens type C in c u l t u r e smears. Staining w a s n o t observed in sections w h e r e NS replaces P-ab, nor in liver sections f r o m t h e uninfected animal.
Fig. 1. PAP-stained C. haemolyticum (arrows) in a liver area of coagulation necrosis. Notice part of the rim of congestion and inflammatory infiltrate (i) that surrounds the necrotic area. Hematoxylin counterstaining. Bar: 10 pm
Peroxidase-Antiperoxidase Technique for Bacillary Haemoglobinuria
597
The five Hereford cattle examined displayed similar liver changes. The lesions noticed (using H & E, Giemsa and Gram stains) corresponded well with previous descriptions (5, 6). The PAP technique revealed numerous brown-stained rods, approximate size 3 x 1 pm; located singly or in clusters, in the sinusoids of the necrotic area and in the adjacent inner parts of the surrounding area of congestion, hemorrhage and infiltrating neutrophils (Fig. 1). In the inner margin of the rim of congestion and neutrophilic infiltration, granular or amorphous, PAP-stained material mixed with leukocytes, erythrocytes and unstained amorphous material was also found. Neither rods nor PAP-stained material were observed in the tissues surrounding the necrotic zone.
Discussion Bovine bacillary haemoglobinuria (BH) is routinely diagnosed by culturing the causal agent, C. huemolyticum. However, C. huemolyticum is very demanding in its cultural requirements and its cultivation is time-consuming (3, 6). For these reasons a simple and rapid method for BH diagnosis is needed. The PAP technique for C.huemolyticum described here is a simple and rapid method to perform, and it may also complement the cultural procedures to diagnose BH. C. huemolyticum and several other clostridial organisms in animal tissues and smears have been identified by immunofluorescence (1, 6, 10). However, a fluorescence microscope is required, the material must be fresh or specially prepared, sections can be stored for a few hours only and the tissue morphology is poor. All of these problems are solved with the PAP technique. Furthermore, the specificity of the immunofluorescent methods cited previously is not always uniform (1, 8, 10). The cultural examination of the BH livers investigated in this study confirmed that the rods shown with the PAP technique were, indeed, C. huemolyticum. The specificity of the PAP method was confirmed also by the lack of PAP-stain, as shown by the culture smears of other Clostridia and liver sections from control animal. The possibility to distinguish C. huemolyticum from C. novyi type B is noteworthy, since in cattle C. novyi type B, can cause lesions similar to BH (5, 6, 11). The PAP technique described here permits one to quickly and specifically stain C. huemolyticum in tissues processed for routine histology, thus providing a simple and rapid method to diagnose BH especially where laboratory facilities for anaerobic cultivation of the bacteria are not readily available. The PAP technique also enables retrospective studies. Some of the liver tissues currently examined were filed for long periods of time, of up to 10 years. Acknowledgements We wish to thank Ms C.NILSSON for technical assistance and Dr F.V. OLAECHEA for and P.L6PEZ for constructive criticism of the manuscript. We also thank Ms S.WESTERBERG preparing the manuscript.
References 1. BATTY, I., and P.W. WALKER, 1964: The identification of C.novyi (C.oedematzens) and C. tetuni by the use of fluorescent labelled antibodies. J. Pathol. Bacteriol. 88, 327-328. and 0.M. RADOSTITS, 1979: Veterinary Medicine. Bailliere 2. BLOOD, D. C., J. A. HENDERSON, Tindall, London. pp. 450-452. J. H., and J. F. TIMONEY, 1981: Hagan and Bruner’s infectious diseases of domestic 3. GILLESPIE, animals. Cornell University Press, Ithaca & London. pp. 851. K., A. UGGLA, T. SVENSSON, and L. SJOLAND,1988: Detection of Toxoplusma gondii 4. GUSTAFSSON, in liver tissue sections from Brown Hares (Lepus europaeus P.) and Mountain Hares (Lepus timidus L . ) using the Peroxidase Anti-Peroxidase (PAP) technique as a complement to conventional histopathology. J. Vet. Med. B 35, 402-407.
598
UZAL,BELAK,RIVERA,ROBLESand FEINSTEIN
5. KELLY,R. W., 1985: The liver and biliary system. In: K. V. F. JUBB,P. C. KENNEDY,and N. PALMER (eds.). Pathology of Domestic Animals. Academic Press, Inc., Orlando, FL. pp.240-312. 6. POWER,E. P., V. J. WHITE, E. J. MACKESSY, and J. WARD, 1987: Bacillary haemoglobinuria of cattle: confirmation of the disease in Ireland. Irish Vet. J. 41, 255-257. 7. SMITH,D. H., and J. F. BONE,1970: In: W. J. GIBBONS, E. J. CATCOTT, and J. F. SMITHCORS (eds.). Bovine Medicine and Surgery. Amer. Vet. Publ. Inc., Illinois. pp. 121-146. L., and A. J. BOOTH, 1984: Bacillary hemoglobinuria in cattle. Comp. Cont. Ed. Prac. 8. STOGDALE, Vet. 6, 284-290. 9. UZAL,F. A., E. RIVERA,and R. E. FEINSTEIN,1990: Indirect immunoperoxidase and immunofluorescence staining of Bordetella bronchiseptica in formalin-fixed, paraffin-embedded lung tissues of rabbits. J. Vet. Med. A 37, 1-7. 10. VAN KAMPEN,K. R., and P. C. KENNEDY,1968: Experimental Bacillary hemoglobinuria: intrahepatic detection of spores of Clostriditrm huemolyticum by immunofluorescense in the rabbit. Amer. J. Vet. Res. 29, 2173-2177. 1 1 . WILLIAMS,B. M., 1964: Clostrzdium oedematiens infections (Black disease and Bacillary haemoglobinuria) of cattle in Mid-Wales. Vet. Rec. 76, 591-596.
