ISRAEL JOURNAL OF
VETERINARY MEDICINE
IMMUNOPEROXIDASE EXAMINATION OF
PNEUMONIC BOVINE LUNGS NATURALLY
INFECTED WITH PASTEURELLA HAEMOLYTICA
Vol. 56 (2) 2001
R. Haziroglu1, K. S. Diker2, M. Y. Gulbahar3 and O. Kul1
1 - Department of Pathology, 2 - Department Microbiology, Faculty of Veterinary
Medicine, University of Ankara, 06110 Ankara, Turkey
3 - Department of Pathology, Faculty of Veterinary Medicine, University of 100.
Yil
Summary
Ninety-three pneumonic lungs from cattle were examined by an immunoperoxidase
technique using avidin-biotin-peroxidase complex in formalin-fixed, paraffinembedded sections for Pasteurella haemolytica. In histological examination, strong
positive reactions against P. haemolytica were detected in 49 (52.68 %) lungs. This
agent was isolated bacteriologically in 42 (45.16 %) cases. Antigens were observed at
the surface and/or within the epithelial cells of bronchioles, macrophages, degenerated
leucocytes around the necrotic areas, necrotic alveolar walls, alveolar-bronchiolar
exudates, fibrins, distended interlobular septa.
Introduction
Pneumonia caused by Pasteurella haemolytica infection is common in cattle and is a
chief component of bovine respiratory tract disease (1). The disease is characterized
as a lobar exudative pneumonia in which fibrin is usually a prominent part of the
exudate, with fibrinous pleurisy a common sequela (1-3). By the immunoperoxidase
technique, coagulation necrosis is the most characteristic pneumonic lesions in calves
experimentally (4,5) and naturally (4,6,7) infected with P. haemolytica. Nevertheless,
there are a few reports of natural pneumonia associated with P. haemolytica using
immunoperoxidase technique (IP).
The present paper describes immunohistochemical and microbiological findings of
pneumonic bovine lungs associated with P. haemolytica. The distribution and
localization of the bacteria in pneumonic lung sections as detected by
immunoperoxidase are also described.
Materials and Methods
From March 1995 through June 1996, 93 pneumonic lungs from beef cattle, 12-16
months old were obtained from a local abattoir. Samples of lung tissue were taken
from pneumonic regions located usually in the right cranial lobes. The tissue sections
were fixed in 10 % buffered neutral formalin and processed by standard methods.
The avidin-biotin-peroxidase complex (ABPC) procedure was applied as
previously described (5,8). P. haemolytica type A1 strain kindly provided by
Dr.R.Sanchis (CNEVA, Laboratoire de Pathologie des Petits Ruminants et des
Abeilles, Biot, France) was used to produce primary antisera in rabbits. Primary
antisera against P. haemolytica had a titer of 1:2560 by ELISA.
The reagents used (except that the primary antiserum; whose dilution was twice the
ELISA concentration) were of commercial origin (Vectastain kit, Vector Laboratories
Inc, Burlingame, USA).
Results were recorded as strong if the antigens were widely distributed in the
section, and weak if the antigens were located infrequently in a limited number of
cells.
Lung sections from which P. haemolytica was or was not isolated were used as
positive and negative controls. To detect cross-reactions, lung sections naturally
infected with Haemophilus somnus were also examined using anti-P. haemolytica
antiserum. Control sections were also incubated with either nonimmune serum or
saline solution.
In bacteriological examination, the lung homogenates (1:10) were cultured on
blood Trypticase soy agar for 24-48 hours at 370C for isolation of P. haemolytica and
identified according to the criteria described previously (9).
Results
In the examination of sections by immunoperoxidase, a strong reaction specific to
P. haemolytica were detected in 49 (52.68 %) of 93 lungs. In bacteriological
examination, P. haemolytica were isolated from 42 (45.16 %) pneumonic lungs. A
weak reaction was observed in 18 of 93 cases. P. haemolytica was found in 49+18=67
(72.04 %) of 93 IP-positive lungs. Culture positive, IP negative cases were not
found.
Table 1: Histopathological, immunoperoxidase and bacteriological findings in 93 pneumonic cases
of calves
Histopathological
Findings
Number of
cases
Pasteurella haemolytica
IP
Isolation
strong
Proliferativeexudative
pneumonia
Purulent
Catarrhal
Haemorrhagic
Necrotic
Catarrhal-purulent
weak
14
6
5
6
11
4
1
1
28
5
2
10
3
8
5
2
10
Catarrhal-necrotic
Purulent-necrotic
Purulent-fibrinous
Fibrinous-necrotic
4
19
3
8
4
12
3
7
2
-
2
7
3
7
Total
Other lesions
93
49
18
42
Pleuritis
Fibrinous pleuritis
7
11
10
-
10
Antigens were present at the surface and/or within the epithelial cells of
bronchioles (Fig. 1), macrophages, necrotic alveolar walls distended interlobular septa
and serous exudates (Fig. 2). Antigen was also detected in the dense zones of
neutrophils, intraluminal leucocytes in bronchioles, and degenerated leucocytes
around the necrotic tissue. The distribution of antigens had a close association with
the presence of lesions. P. haemolytica was isolated from the lesions having strong
reaction in IP but was not generally isolated from the cases determined as weak
reaction in IP test. Specific staining was absent in the negative control tissue sections
and test sections in which the primary serum had been substituted by nonimmune
rabbit serum and saline solution. A cross-reaction was not observed between P.
haemolytica antiserum and H.somnus-infected lungs.
Fig. 1. P. haemolytica positive reaction in the epithelial cells of Fig. 2. Immunoreactive materials to P. haemolytica in the
bronchiole. ABPC method, Mayer's haemotoxylin counterstain necrotic alveolar walls and exudates. ABPC method, Mayer
X 240
haemotoxylin counterstain X 240
Discussion
In the present study, results did not always correlate between bacterial isolation and
immunoperoxidase detection of bacterial antigen from pneumonic lungs as reported
previously (5-8) and this contradiction might be attributable to the fact that the sample
taken for IP examination may have harboured none or a few organisms, and/or
antigenic differences may exist among strains. Moreover, the cases with positive IP
and negative culture may be interpreted by the presence of antigen from live and dead
bacteria whereas bacteriological cultures do not, obviously, detect dead organisms.
Negative culture results in the cases evaluated as "weak reaction" in IP test, may be
due to the low concentration of bacteria in the tissues examined.
The coagulation necrosis in pneumonic lungs is considered to be the most
characteristic lesion in P. haemolytica pneumonia (4-7). Also, coagulation necrosis
has been reported in the pneumonic lungs of cattle with several organisms (6,7,10,11).
Thus, not only P. haemolytica but other bacteria caused necrotic lesions in lungs of
calves with naturally acquired pneumonia. In the present study, necrotic lesions
(Table 1) were found in the 32 (34.40 %) of 93 pneumonic lungs. In these necrotic
lesions, P. haemolytica were detected bacteriologically and immunohistochemically
in 16 (50 %) and 23 strong + 2 weak = 25 (78.12 %) of 32 cases respectively.
However, in the 11 (11.8 %) fibrinous lesions (Table 1) of 93 pneumonic lungs, P.
haemolytica were determined using both tests in 10 (90.9 %) cases. This results may
show that fibrinous lesions are more encountered than those of necrotic lesions caused
by P. haemolytica pneumonia. Although fibrinous and necrotic lesions are also
observed in P. haemolytica pneumonia, it is suggested
that pneumonic cases should be teested by IP test.
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