J. Cornp. Path. 1994 Vol.

110, 329-339

Pneumonia in Pigs Infected with Pseudorabies Virus

and Haemophilus parasuis Serovar 4
M. Narita, K. Kawashima, S. Matsuura*, A. Uchimura and
Y. Miura
National Institute of Animal Health, Ministry of Agriculture, Forestry and Fisheries,
Kannondai, Tsukuba, Ibaraki, 305 Japan and *Matsumoto Animal Hygiene Center,
Matsumoto, Nagano, 390 Japan

Summary
Six HPCD (hysterectomy-produced, colostrum-deprived) pigs were inocu-
lated endobronchially with pseudorabies virus (PRV) in the right caudal lobe
by means of a bronchoscope, Two pigs, killed on days 5 and 7, had severe
purulent pneumonia in the right caudal lobe, associated with an accidental
Haemophilus parasuis serovar 4. infection. The three surviving animals were
treated with antibiotics. The pigs infected with PRV had neerotizing
bronchiolitis and alveolitis. PRY antigen was closely associated with necrotic
foci, and was sometimes surrounded by profuse H. parasuis antigen. PRV
antigen and IgO- and IgA-containing cells were also detected in
bronchioalveolar lavage fluid. These results suggested that the PRV infection
destroyed respiratory epithelial cells and allowed H, parasuis to proliferate in
the lungs.

Introduction
Pulmonary alveolar macrophages provide primary protection in the lower
region of the respiratory tract and can ingest viruses, bacteria and mycoplas-
mas. Some viruses are known to affect these defence mechanisms and thus
enable bacteria to proliferate (Kasza et al., 1969; Jericho and Langford, 1978;
Pijoan and Ochoa, 1978; Charloy, 1983, 1986; Yates et al., 1983; Fuentes and
Pijoan, 1987; Iglesia and Trujano, 1992).
Pseudorabies is an acute and often fatal disease in pigs caused by a
herpesvirus. Some strains of pseudorabies virus (PRV) produce pneumonia in
addition to lesions in the central nervous system (Baskerville, 1972, 1973;
Narita et al., 1989, 1990). However, there is little information on the inflamma-
tory and i m m u n e effector cells in bronchioalveolar lavage (BAL) fluid i~ PRV
infected pigs (Narita et al., 1993).
The purpose of the present study was to describe pneumonic lesions resulting
from endobronchial inoculation of P R V into the right caudal lobe of pigs by
means of a bronchoscope. Accidentally, however, the P R V infected pigs were
also infected with Haemophilus parasuis serovar 4. Therefore, the effect of co-
infection with P R V and H. parasuis is reported.
0021-9975/94/040329 + 11 $08,00/0 © 1994 Academic Press Limited
330 M. Narita et al.
M a t e r i a l s and M e t h o d s
Virus
PRV, strain Yamagata S-81 (ys-81), isolated in Japan (Fukusho et al., 1981), was
used. The virus was passaged s'erially three times in pig kidney-cell culture and its
infectivity was 107 TCIDs0/ml.

Animals
Eight 50-day-old hysterectomy-produced and colostrum-deprived (HPCD) pigs (six
experimental, two control) were used. The animals were housed separately in isolated
experimental units.

Experimental Procedures
Before inoculation with PRV, the pigs were sedated with azaperone (5 mg/kg of body
weight, intramuscularly), and anaesthetized with pentobarbital sodium (10 mg/kg of
body weight, intravenously). Then, six pigs (nos 1-6) were inoculated endobronchially
with 2 ml of a 1 in 1000 dilution of the virus suspension (104 TCIDs0/ml ) by means of
an Olympus Type B3 bronchoscope. The latter was inserted into the right main stem
bronchus. Two pigs (nos 7 and 8; controls) were inoculated endobronchially with 2 ml
of growth medium.
The initial plan was to kill the infected pigs singly on post-inoculation days (PID) 3,
5, 7, 10 and 14, and the non-infected controls on PID 7 and 14. However, pigs 2 and 3,
killed on PID 5 and 7, had severe purulent pneumonia, and a bacterium was isolated in
pure culture from the pneumonic lesions. The cultural and biochemical characteristics
of the isolate were those ofH. parasuis. The organism was ;serotyped by gel diffusion as
described previously (Morozumi and Nicolet, 1986), and was identified as H. parasuis
serovar 4. The experiment was stopped, and benzylpenicillin (300 000 units/head,
intramuscularly) was injected on PID 6 and 7 into the three surviving PRV-infected
pigs (nos 4, 5 and 6). These three treated animals were killed on PID 10. The two
non-infected control pigs (nos 7 and 8) were killed on PID 7 and 14, respectively.

Viral Examination
Nasal secretions from each surviving pig were collected daily on cotton swabs
throughout the experiment. The swabs were immersed in 5 ml of growth culture
medium and stored at - 80"C until used. Tissues from the middle part of the .right
caudal lung lobe, bronchial lymph node, tonsil, trigeminal ganglion and frontal part of
the brain were collected for virus isolation. Each sample was cultivated in a PK-15 cell
culture, which was then observed for cytopathic effect.

Cytological Evaluation of Bronchioalveolar Lavage (BAL) Fluid

At necropsy, a 4 mm-diameter cannula was inserted into the right main stem bronchus,
and 30 ml of sterile phosphate buffered saline (PBS) were introduced and recovered by
suction. The BAL fluid was placed in a sterile bottle. Counts of nucleated cells/ml of
BAL fluid were made with a Coulter counter, the centrifuged cells being stained with
Diff-Quik stain (International Reagent Corporation, Koube, Japan). Evaluation
included a 200-cell differential count and a morphological description of the cells. The
cells in BAL fluid were fixed with cold acetone; PRV antigen and porcine IgG-, IgM-
and IgA-containing ceils were demonstrated by the avidin-biotin-complex (ABC)
(Veetastain, Vector Laboratories, Burlingame, CA, U.S.A.) immunoperoxidase (IP)
method, as described by Narita et al. (1989). Anti-PRV rabbit serum was used as the
primary antibody at a dilution of 1 in 2048. Anti-porcine IgG and IgM rabbit sera
(Miles Laboratories, Naperville, IL, U.S.A.), and anti-porcine IgA goat serum (Bethyl
 P s e u d o r a b i e s V i r u s a n d H. p a r a s u i s i n P i g s 331
Table 1
Detection of inflammatory and i m m u n e effector cells in BAL fluid from the right caudal lobe

Percentage of total cells that cor~sistedof PR V antigen detected in

Pig PID Total number macro- lpmpho- neutro. BAL cells containing
no. of cells phages cytes phils cells IgG IgM IgA

1 3 1.02 x 108 75 24 1 + + + - - -
2 5 2.03 × 10~ 2 1 97 + - - -
3 7 3.40 x 109 1 1 98 + - - -
1.10x 106* 83 16 I . . . .
4 10 1.06 × 106 64 34 2 + + - - +
5 10 0.59 x 106 75 20 5 + + + - +
6 10 1.09 x 106 55 42 3 - + - +
7 7 0.89 × 106 81 18 1 . . . .
8 14 1.10× 104 82 17 1 . . . .

* = Cells in left c a u d a l lobe.

PID = Post-inoculatlon day.
A n t i g e n d e m o n s t r a t i o n : - , n e g a t i v e ; + , slight; + + , m o d e r a t e ; + + + , striking.

Laboratories Inc, Montgomery, TX, U.S.A.) were used at a dilution of 1 in 2560, 1 in

160 and 1 in 160, respectively. Cells from pigs 7 and 8 and sera from non-immunized
rabbits and goats were used for control studies.

Pathological Examination
Specimens from each pig were fixed in buffered 10% formalin and embedded in
paraffin wax, and sections were cut and stained with haematoxylin and eosin (HE).
Immunohistologically, PRV and H. parasuis serovar 4 antigens were demonstrated
by the ABC method. Anti-H. parasuis serovar 4 rabbit serum was kindly provided by Dr
T. Morozumi, National Institute of Animal Health, Japan, and used as the primary
antibody at a dilution of 1 in 8192.

Results
Clinical Observations
The first clinical sign of infection was an increase in body temperature (up to
40°C). The animals were depressed and refused food. On PID 6, severe
respiratory signs with sneezing, coughing and fever (up to 41°C) were evident
in the four surviving infected pigs. Three animals (nos 4, 5 and 6) treated with
antibiotics on P I D 6 and 7 had apparently recovered by PID 9-10. None of the
pigs showed nervous signs. The two non-infected control pigs (nos 7 and 8)
were free of clinical abnormalities.

BAL Fluid Examination

The results are shown in Table 1. The total cell number was 0"6-1"1 million
cells/ml, and there was little difference between that of infected pigs 1, 4, 5 and
6 and of the non-infected controls. The proportion oflymphocytes was slightly
higher than that in control BAL fluid. The total cell number in pigs 2 and 3,
which had purulent pneumonic lesions, was much higher and was almost
332 M. N a r i t a et at.

completely composed of neutrophils. However, the total cell n u m b e r in BAL

fluid from the left caudal lobe (in which there was no detectable pneumonia)
of pig 3 was the same as that in the non-infected controls.
P R V antigen was detected in BAL fluid of pigs 1, 2, 3, 4 and 5 (Fig. 1), in
IgG-containing cells of pigs 5 and 6 (Fig. 2) and in IgA-containing cells of pigs
4, 5 and 6 (Fig. 3). No IgM-containing cells were detected in any of the pigs.

Virus Isolation
Virus was first detected in nasal secretion on PID 3 in four infected pigs (nos 1,
2, 3 and 6). After that, three pigs (nos 4, 5 and 6) excreted the virus up to PID
10. T h e highest virus titres were 101"75TCIDs0/ml on PID 3 in pig 1, 104.25 on
PID 3 in pig 2, 105'7~ on PID 5 in pigs 3, 4 and 6, and l0 Gon PID 9 in pig 5.
Virus was isolated from a bronchial lymph node (1 04'25/g) in pig 1, lungs
(10 a's) in pig 2, trigeminal ganglion (1015 to 10 3"75) in pigs 1, 2 and 5, and tonsils
(101'25 to 103) in pigs 1, 2, 4 and 5.

Pathology
The pneumonic lesions in pigs 2 and 3, killed on P I D 5 and 7, were localized in
the middle to caudal parts of the right caudal lobe and in the accessory lobe.
The pneumonic areas were dark and solid, and showed haemorrhages (Fig. 4).
No fibrinous pleurisy was observed. The lymph nodes associated with the
airways were enlarged. Of the three pigs treated with antibiotics and killed on
PID 10, two (nos 4 and 5) had no pneumonic lesions, but one (no. 6) had a
small, focal, well demarcated lesion in the right middle lobe.
Microscopically, the findings in pig 1, killed on PID 3, were focal necrotizing
bronchitis, bronchiolitis, alveolitis and intranuclear inclusion bodies. The latter
were scattered throughout the right caudal lobe but not seen elsewhere. Pigs 2
and 3, killed on PID 5 and 7, had severe purulent pneumonia and alveoli filled
with neutrophils. T h e interlobular septa and peribronchial regions showed
serofibrinous or fibrinous exudate accompanied by focal necrotizing bronchiol-
itis in the centre of the purulent pneumonic lesion (Figs 5 and 6). Pigs 4, 5 and
6, killed on PID 10, had slight purulent pneumonia with necrotic foci
surrounded by fibroblasts, macrophages and lymphocytes, which resulted in a
small nodule.
Severe lymphadenitis was observed in the mandibular, bronchial and
mediastinal lymph nodes of pigs 2, 3, 4 and 6, tonsillitis in pig 5, trigeminal
ganglionitis in pigs 2, 3, 5 and 6, and encephalomyelitis in pigs 2, 3, 5 and 6.
There was no evidence ofpolyserositis or arthritis. No lesions were observed in
the two non-infected controls (Table 2).
Immunohistologically, PRV antigen was detected first in the epithelial cells
of the bronchioles and alveoli; subsequently, it was observed in neighbouring
alveoli. In two pigs killed on PID 10, P R V antigen was confined to the
necrotizing nodule, and closely associated with the necrotic foci. I-Iaemophilus
parasuis serovar 4 antigen was observed in pigs 2, 3, 4 and 6, associated with the
purulent pneumonic lesions. In the alveoli and interlobular septa, bacterial
 Pseudorabies V i r u s a n d H. parasuis i n P i g s 333

Fig, 1. PRV antigen-positive cells in BAL fluid from pig 1 killed on day 3, ABC/IP. x 400.
Fig. 2. lgG-containing cells in BAL fluid from pig 6 killed on day 10. ABC/IP, × 400.
Fig, 3, IgA-containlng celts in BAL fluid fi'om pig 4 killed on day 10. ABC/IP. x 400.
Fig, 4. The pneumonic lesion in pig 2 was localized in the right caudal lobe and was dark, solid and showed
haemorrhage.
334 M. Narita e t al.

Fig. 5. Severe purulent pneumonitis containing focal necrotic loci (N) in pig 3 killed on day 7. HE. x 50.
Fig. 6, Severe purulent pneumonia containing focal necrotic foci (N) surrounded with neutrophils in pig 3
killed on day 7, HE. x 50.
Fig. 7, PRV antigen in necrotic loci (N) from the same section as Fig. 5. ABC/IP. x 50.
Fig. 8. PRV antigen in the necrotic loci (N) from the same section as Fig, 6, ABC]IP~ x 50.
 Pseudorabies Virus and H. parasuls in Pigs 335

I I I L I ~ I 1 I I I l 1
1 J I I I I ~ L J I I 1 l
I I I I I I I I I I l I I I

I I I I I I I I II ~ l l I
I I I I I I I I I I I I I
I I I I I I I I I I I I 1 I

I I I I I I I I I I~ I I
III I++IIII + + +

+II l l + l l l l 1 l 1
~+I 1 77777777 ~ ~+ 7
°~

1 l l + I I l l l 1 1
L~
'~ ''7''''' .+ -+ 7
~+
m
0 ,++++<<< , ,
777 - - ~
i + + + + + + + + +

o +

,m
~<< '++''''< < <
o~
-++ 7777777+ ~ + ?
+~

,,+ ,,+,,,,, < <

777 777F7777 + , 7

.-~ ~.¢:: o o
336 M. Narita e t al.

Fig. 9. H. parasuis antigens in infiltrated neutrophils and macrophages surrounding the proliferated PRV
antigens in the section shown in Fig. 5. ABC/IP. x 50.
Fig. 10. H. parasuis antigens adjacent to the necrotic loci of P R V in the section shown in Fig. 6. ABC/IP.
x 50.

antigen was detected in the cytoplasm of infiltrating neutrophils and macro-

phages. A small amount of antigen was scattered in alveolar macrophages in
pigs 4 and 6. In pigs 2 and 3, the bacterial antigen was widely distributed and
surrounded the PRV-rich areas (Figs 7 and 8). Some foci contained both
antigens (Figs 9 and 10).
In addition to its presence in the lungs, P R V antigen was detected in the
tonsils, trachea and trigeminal ganglia, and H. parasuis antigen was seen in the
tonsils, lymph nodes and trachea (Table 2).
No histological lesions or viral or bacterial antigens were found in the two
control pigs.

Discussion
There are many reports of swine pneumonia induced by viruses and bacteria,
alone or together (Kasza et al., 1969; Baskerville, 1972; Pijoan and Ochoa,
1978; Charley, 1983; Yates et al., 1983; Fuentes and Pijoan, 1987; Iglesia and
Trujano, 1992). Primary virus infection of the respiratory tract is thought to
assist bacterial proliferation. In the present study, two experimentally PRV-
infected pigs (nos 2 and 3) showed severe clinical signs and had purulent
pneumonitis, localized to the right caudal lobe. H. parasuis serovar 4, together
with PRV, was isolated in pure culture from the pneumonic lesion. However,
 P s e u d o r a b i e s Virus and H. parasuis in Pigs 337
there was no polyserositis or arthritis. Three PRV-infected pigs, treated with
antibiotics, had only a small consolidation in the right caudal lobe. These
results suggested that PRV infection in the respiratory epithelial cells assisted
infection by H. parasuis.
Endobronchial inoculation is an efficient method of producing pulmonary
infections (Van Leengoed and Kamp, 1989; Narita et al., 1993). Microscopi-
cally, the PRV-infected pigs showed focal necrotizing bronchitis, bronchiolids,
alveolitis, and intranuclear inclusion bodies scattered throughout the right
caudal lobe. The two pigs infected with both PRV and H. parasuis had severe
acute purulent pneumonia. Alveoli were filled with numerous neutrophils and
interlobular septa were distended with serofibrinous exudate. The three pigs
treated with antibiotics had no suppurative pneumonic changes, only a small
necrotizing nodule in the right caudal lobe. Except for the purulent pneumo-
nia, the histopathological lesions resembled those already described in PRV
infection of plgs (Baskerville, 1972; 1973; Narita et al., 1989, 1993).
Several investigators have demonstrated differences in virulence between
different H. parasuis strains by experimental inoculation of pigs. Nielsen (1990)
examined the virulence of the reference strains of serovars 1 to 7 by exposing
specific-pathogen-free (SPF) pigs to an aerosol containing 10~ cells; only
serovars 1 and 4 caused clinical signs and lesions. Kielstein (1991) also
inoculated SPF pigs, intraperitoneally and intratracheally, with these refer-
ence strains; only serovars 1 and 5 were virulent by intratracheal inoculation.
In the present study, the contaminating H. parasuis serovar 4 produced severe
purulent pneumonia in the group of PRV infected pigs despite its medium
virulence for pigs. This suggested that PRV affected the defence mechanism
and allowed the proliferation ofH. parasuis in the lung.
Recently, many infectious agents have been identified by the immunoperox-
idase method. In the present study, PRV antigen was localized in the
degenerating epithelial cells and in the centre of the necrotic foci and nodules.
H. parasuis serovar 4 antigen was detected first in the purulent pneumonic
lesion of a pig killed on PID 5, and was later found to be distributed widely in
the alveoli and interlobular septa. PRV antigen in the lesion was surrounded
by deposits of H. parasuis antigen. Some loci contained both antigens. These
findings suggested that PRV infection destroyed respiratory epithelial cells,
thus opening the door to infection with H. parasuis.
It is known that the normal ratio of macrophages to neutrophils in BAL
fluid in domestic animals is approximately 10:1; however, in calves with
respiratory disease, the neutrophils increase and this ratio may approach 1:10
(Allen et al., 1992; Crisman et al., 1992; Traub-Dargatz et al., 1992). After PRV
infection, the total cell number in BAL fluid increases slightly and the ratio of
lymphocytes also increases (Narita et al., 1993). In the present study, the, two
pigs with purulent pneumonitis showed an increase in the total number of cells
in BAL fluid; these cells consisted almost completely of neutrophils. The total
cell number in BAL fluid from the left caudal lobe of one pig did not differ
significantly from that of the right caudal lobe of another PRV-infected pig
and of non-infected control pigs. However, the proportion of lymphocytes was
slightly greater than in non-infected control pigs. Moreover, PRV antigen and
338 M. Narita et al.
IgG- and IgA-containing cells were detected in the cells of BAL fluid from the
PRV-infected pigs. These cells would stimulate the immune responses in the
lungs and result in an increase of lymphocytes and IgG- and IgA-containing
cells. The findings support t h e view that the cells of BAL fluid m a y be
important indicators of respiratory disease and function as stimulants of the
local immune response against invading pathogens.

Acknowledgments
The authors thank Dr T. Morozumi for isolation and serological identification of H.
parasuis, and Mr Y. Ando and T. Fujisawa for preparing the photographs.

References
Allen, J. W., Viel, L., Batema~, K. G. and Rosendal, S. (1992). Changes in the
bacterial flora of the upper and lower respiratory tracts and bronchoalveolar
lavage differential cell counts in feedlot calves treated for respiratory disease.
Canadian Journal of VeterinaryResearch, 56, 177-183.
Baskerville, A. (1972). Ultrastructural changes in the pulmonary airways of pigs
infected with a strain of Aujeszky's disease virus. Research in Veterinary Science, 13,
127-132.
Baskerville, A. (1973). The histopathology of experimental pneumonia in pigs
produced by Aujeszky's disease virus. Research in Veterinary Science, 14, 223-228.
Charley, B. (1983). Interaction of influenza virus with swine alveolar maerophages:
influence of anti-virus antibodies and cytochalasin B. Annales de Virologic, 134,
51-5.9.
Charley B. (1986). Effects of immunopotentiating agents on alveolar macrophage
properties. Comparative Immunology, Microbiology and Infectious Disease, 9, 155-159.
Cris~r~, M. V., Hodgson, D. R. and Bayly, W. M. (1992). Effects of transport on
constituents of bronchc~alveolar lavage fluid from horses. Cornell Veterinarian, 82,
233-246. .~ ~ i
Fuentes, M. C. and Pijoan, C. (1987). Pne~amonia'in pigs induced by intranasal
challenge exposure with pseudorabies x*irusa n d PasteureUa multocida. American
Journal of Veterinary Research, 48, 1446-1448. , ;
Fukusho, A., Shimizu, M , Kubo, M , Nanba, K., Shimizu~ Y., Konno, S., Suzuki, K.
and Otakl, T. (1981). The first outbreak of Aujeszky s disease m swine m Japan.
Bulletin of the National Institute of Animal Health, 82, 5-11. '
Iglesia, J. G. and Trujano, M. (1992). Inoculation of pigs with Streptococcussuis type 2
alone or in combination with pseudorabies virus. American Journal;of Veterinary
Research, 53, 364-367. ~.
Jericho, K. W. F. and Langford, E. V. (1978). Pneumonia in calves produced with
aerosols of bovine herpesvirus 1 and Pasteurella haemolytica. Canadian Journal of
Comparative Medicine, 42, 269-277.
Kasza, L., Hodges, R. T., Betts, A. O. and Trexler, P. C. (1969). Pneumonia in
gnotobiotic pigs produced by simultaneous inoculation of a swine adenovirus and
Mycoplasma hyopneumoniae. Veterinary Record, 84, 262-267.
Kielstein, P. (1991). Zur Glasserschen Krankheit des Schweines-Untersuchungen tiber
Zusammenhange zwischen serologischen Haemophilus-parasuis-Stammen. Monats-
heftefffr Veteriniirmedizin, 46, 137-142.
Morozumi, T. and Nicolet, J. (1986). Some antigenic properties of Haemophilusparasuis
and a proposal for serological classification. Journal of Clinical Microbiology, 23,
138-142.
Narita, M., Imada, T. and Haritani, M. (1990). Comparative pathology of HPCD
pigs infected with wild-type and arc-T-resistant strains of Aujeszky's disease virus.
Journal of Comparative Pathology, 102, 63-69.
 P s e u d o r a b i e s Virus and H. parasuis in Pigs 339
Narita, M., Imada, T., Haritani, M. and Kawamura, H. (1989).
Immunohistopathologic study of pulmonary and lymphatic tissues from
gnotobiotic pigs infected with ara-T-resistant strain of pseudorabies virus.
American Journal of VeterinaryResearch, 50, 1940-1945.
Narita, M., Kawashima, K.~ Matsuura, S., Uchimura, A. and Miura, Y. (1993).
Pneumonia in pigs inoculated with pseudorabies virus by means of a
bronchoscope. Journal of ComparativePathology, 109, 335-344.
Nielsen, R. (1990). New diagnostic techniques: a review of the HAP group of bacteria.
Canadian Journal of VeterinaryResearch, 54, $68-$72.
Pijoan, C. and Ochoa, G. (1978). Interaction between a hog cholera vaccine strain and
Pasteurella multocidain the production of porcine pneumonia. Journal of Comparative
Pathology, 88, 167-170.
Traub-Dargatz, J. L., McKinnon, A. O., Thrall, M. A.,Jones, R. L., Bruyninckx, W.,
Blancquaert, A. M. B. and Dargatz, D. A. (1992). Evaluation of clinical signs of
disease, bronchoalveolar and tracheal wash analysis, and arterial blood gas
tensions in 13 horses with chronic obstructive pulmonary disease treated with
prednisone, methyl sulfonmethane, and clenbuterol hydrochloride. American
Journal of VeterinaryResearch, 53, 1908-1916.
Yates, W. D. G., Jericho, K. W. F. and Doige, C. E. (1983). Effect of viral dose on
experimental pneumonia caused by aerosol exposure of calves to bovine
herpesvirus 1 and Pasteurella haemolytica. CanadianJournal of ComparativeMedicine,
47, 57-63.
Van Leengoed, L. A. M. G. and Kamp, E. M. (1989). Endobronchial inoculation of
various doses of Haemophilus (Actinobacillus) pleuropneumonia in pigs. American
Journal of VeterinaryResearch, 50, 2054-2059.
I Received,October4th, 1993 ]
Accepted,January 4th, 1994]