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Avian Pathology

ISSN: 0307-9457 (Print) 1465-3338 (Online) Journal homepage: http://www.tandfonline.com/loi/cavp20

Big liver and spleen disease of broiler breeders

J.K. Clarke , G.M. Allan , D.G. Bryson , W. Williams , D. Todd , D.P. Mackie & J.B.
McFerran

To cite this article: J.K. Clarke , G.M. Allan , D.G. Bryson , W. Williams , D. Todd , D.P. Mackie
& J.B. McFerran (1990) Big liver and spleen disease of broiler breeders, Avian Pathology, 19:1,
41-50, DOI: 10.1080/03079459008418654

To link to this article: http://dx.doi.org/10.1080/03079459008418654

Published online: 12 Nov 2007.

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Avian Pathology, 19: 41-50, 1990

BIG LIVER AND SPLEEN DISEASE


OF BROILER BREEDERS

J.K. CLARKE 1 , G.M. ALLAN2, D.G. BRYSON 2 , W. WILLIAMS 3 ,


D. TODD 2 , D.P. MACKIE 2 and J.B. McFERRAN 2
1
Department of Microbiology and Genetics,
Massey University, Palmerston North, New Zealand
2
Veterinary Research Laboratories,
Stormont, Belfast BT4 3SD, Northern Ireland
3
Golden Coast Poultry Ltd,
PO Box 301, Levin, North Island, New Zealand

SUMMARY
Five adult broiler breeders were inoculated with faeces and tissue extracts
from birds with big liver and spleen (BLS) disease. Five birds were placed in
contact with the inoculated birds. After 8 weeks all inoculated and four of the
five in-contact birds were shown to be infected as evidenced by the detection of
an immune response and/or the demonstration of BLS antigen in spleen and
tissue smears, by an immunofluorescence test and by agar gel immunodiffusion.
Using appropriate antisera BLS antigen was found mainly in phagocytic liver
cells and in splenic macrophages and dendritic cells. Fewer cells containing
antigen were found in the kidney.
Double staining of liver and spleen tissue for BLS antigen and chicken
immunoglobulin indicated that many cells were positive for both. It is
possible that BLS antigen in most cells may represent phagocytosed material
rather than a replicating agent. If so, while this could explain the failure to
detect virus-like particles, it implies that the primary site of replication of the
BLS agent and its nature remain to be established.

INTRODUCTION
Big liver and spleen (BLS) disease of commercial broiler breeder hens is a condition
which has been recognised in Australia by one of us (WW). It is characterised by
enlargement of the liver and spleen, increased mortality and a drop in egg production.
The disease is infectious and the organism involved is presumed to be a virus
(Handlinger and Williams, 1989). Clearly, in order to characterise the causal agent, it

Received 1 December 1988


Accepted 7 April 1989
Address for correspondence: G.M. Allan, Virology Department, Veterinary Research Laboratories,
Stormont, BELFAST BT4 3SD
42 J.K. Clarke et at.

is desirable to propagate it in vitro and to observe it by electron microscopy. This might


be facilitated if the distribution and quantity of BLS agent antigens in the tissues of
infected birds were established.
We have undertaken an examination for BLS agent antigens in the liver, spleen and
kidney of birds which were infected experimentally by oral and intraperitoneal
inoculation. To confirm the infectious nature of the disease, serum and tissues from
five birds placed in contact with the inoculated birds were also examined. Antigen was
detected in tissue smears using a direct immunofluorescent test (IFT). The quantity of
antigen present was assayed by an agar gel immunodiffusion (AGID) test and the
distribution of antigen and immunoglobulin in liver, spleen and kidney tissue was
assessed both by a direct IFT and by immunoperoxidase staining of cryostat and
paraffin sections.

MATERIALS AND METHODS


BLS agent
Liver, spleen, serum and faeces from infected birds were obtained from P. Curtin,
Inghams Enterprises NSW, Australia.
Infection of birds
Five 30-week-old male broiler breeder birds were inoculated with BLS material. Each
bird was inoculated orally with 1 ml of a mixture of equal parts of 20% liver and spleen
suspension in phosphate buffered saline pH 7.2 (PBS). A further 1 ml of a mixed liver
and spleen suspension was inoculated intraperitoneally. These birds were placed in
contact with another five uninoculated birds. All birds were killed after 8 weeks and
liver, spleen, kidneys and blood were collected from each.
Agar gel immunodiffusion (AGID) tests
One percent agar in 8% NaCl adjusted to pH 7.4 with HC1 was used. Antigen consisted
of a liver and spleen tissue from infected birds homogenised in PBS to give a 20%
suspension. This tissue and a positive BLS antiserum derived from infected birds was
obtained from Mr P. Curtin, Inghams Enterprises NSW, Australia. The test was read
after overnight incubation at room temperature.
The specificity of all AGID reactions was ensured by including positive sera in each
test. A reaction was recorded only if a line of identity between the test material and the
standard sera was observed.
Production of antisera to BLS antigen
Birds inoculated orally and intraperitoneally with BLS material were bled at weekly
intervals and tested for antibody by AGID. Sera which showed a strong precipitin line
of identity, when tested in parallel with standard BLS antibody obtained from
Australia, were pooled. This antiserum showed no reaction with a control antigen
prepared from liver and spleen tissue of uninoculated birds.
Production of BLS antigen for rabbit immunisation
A partially purified BLS antigen was prepared as follows. Liver tissue obtained from
broiler breeders infected with BLS agent in Australia was homogenised in a blender to
give a 50% suspension in PBS. The suspension was clarified by low speed
centrifugation and then at 100,000g for 3 h. The supernatant showed a strong reaction
by AGID and was stored at -70C in 2 ml aliquots.
Big liver and spleen disease of broilers 43

Aliquots were fractionated by gel filtration using a column (1.0 x 80 cm) of Sephadex
G200 (Pharmacia) equilibrated with 0.1 M TRIS-HCL pH 8.0 (elution buffer). Five 2-ml
fractions which eluted close to the total volume of the column were positive by AGID.
Those fractions were pooled and subjected to ion exchange chromatography using a
column (0.9 x 10 cm) of DE52 (Whatman) equilibrated with elution buffer. Under
these conditions BLS antigen did not bind to the column. Those 'flow through'
fractions which contained BLS antigen when tested by AGID were pooled. This
preparation contained about 0.2 mg/ml of protein and was used to immunise
rabbits.
Rabbit antisera to BLS antigen
Partially purified BLS antigen (2 ml) was emulsified in an equal volume of Freund's
complete adjuvant. One millilitre was inoculated intramuscularly into each hind leg
and 2 ml was inoculated subcutaneously at the base of the neck. This procedure was
repeated after 3 weeks and blood was collected 4 weeks after the second inoculation.
The antiserum gave a single line of identity when tested with BLS antigen in parallel
with an avian antiserum to this antigen. It gave no reaction with control tissue in the
AGID test.
Immunofluorescence techniques
For direct staining of BLS antigen in cryostat sections or tissue smears, rabbit and
avian antisera were labelled with fluorescein isothiocyanate (FITC), purified by
Sephadex and DEAE cellulose chromatography and absorbed with tissue homogenates.
Acetone-fixed smears and frozen sections of liver, spleen and kidney were stained and
examined as described previously (McNulty and Allan, 1983).
Immunoglobulin present in tissue was stained using a mouse monoclonal anti-
chicken immunoglobulin followed by a fluorescein-labelled rabbit anti-mouse
immunoglobulin. Double immunofluorescence staining to determine if antigen and
antibody were located in the same cells was also carried out. Cryostat sections were
initially incubated for 60 min at 37C with equal volumes of rabbit antiserum to BLS
and mouse monoclonal to chicken immunoglobulin. The sections were washed in
three changes of PBS and stained with goat/A/rabbit FITC for a further 60 min. The
sections were washed again in PBS and restained with goat/A/mouse rhodamine
conjugate. After a further 60 min incubation the sections were washed again, mounted
in buffered glycerol and examined.
Histopathological and immunoperoxidase examination
Blocks of liver, spleen and kidney from two experimentally infected birds (5287 and
5296) and from two serologically positive in-contact birds (5289 and 5298) were taken
into neutral buffered formalin, processed by standard paraffin methods and stained
by haematoxylin and eosin. Paraffin sections of these organs were also stained using
an avidin-biotin-peroxidase complex technique (Bryson et al. 1988) using primary
rabbit antiserum to BLS at a dilution of 1:500.
Propagation of BLS agent in vitro
Suspensions of liver and spleen material were inoculated into the following cell
cultures: primary chick kidney, primary chick embryo liver, primary chick embryo
fibroblasts, MDCC-MSB1 cells, primary duck kidney and primary duck embryo liver. The
inoculated cultures were examined daily for evidence of cytopathic effect for at least
two weeks and examined for the presence of BLS antigen by immunofluorescence.
44 J-K- Clarke et al.

Electron microscopy
Specimens of liver, spleen and kidney shown by immunofluorescence to have many
cells positive for BLS antigen were processed by standard techniques and ultra-thin
sections were examined in the electron microscope.
Following centrifugation at 3,000 g for 30 min supernatant fluids from liver, spleen and
kidney homogenates were ultracentrifuged and the deposits applied to carbon coated
grids, stained with 4% sodium phosphotungstate pH 7.2 and examined in the electron
microscope. Gel precipitin lines from AGID tests carried out using known BLS
positive antigen and antisera, were cut from the agar, washed extensively in 0.01 M
phosphate buffered saline pH 7.2 and prepared for electron microscopy as above.

RESULTS
Attempted characterisation of the BLS agent
Examination of negatively stained material and ultrathin sections of liver, spleen and
kidney from infected birds failed to reveal either virus-like particles or any other
micro-organism.
Attempts to propagate the agent in cell cultures were not successful.
Antibody response
Initially, all birds were serologically negative. However, evidence that all five
inoculated birds were infected by the BLS agent was provided by the development of
antibody detectable by AGID. By this criterion three of the five in-contact birds were
also infected.
Antigen in tissues
Smears of liver, spleen and kidney tissue were stained directly with FITC labelled BLS
antiserum prepared in rabbits in an attempt to demonstrate BLS antigen. The results
are recorded in Table 1. With one exception (bird 3), antigen was detected in all three
tissues of those birds which had serological evidence of infection. Two in-contact birds
remained serologically negative. However, antigen was detected in smears of spleen
and kidney tissue from one of these birds.
The concentration of BLS antigen was similar in liver and spleen. Antigen was not
detected in the kidney by AGID (Table 1).
Distribution of antigen
Cryostat sections of liver, spleen and kidney from three inoculated birds (1, 2 and 3)
and one in-contact serologically positive bird (6) were examined by direct immuno-
fluorescence using avian and rabbit antisera. The results obtained with both antisera
were similar.
Liver tissue typically contained fluorescing cells or groups of fluorescing cells
scattered throughout the tissue. Most positive cells were lining sinusoids.
Spleen tissue also contained many fluorescing cells (Fig. 1) and in some birds over 25%
of cells were positive. Most positive cells exhibited a relatively dull fluorescence but
patches of such cells were often punctuated with occasional brightly fluorescing cells.
Kidney tissue also contained groups of positive cells although the cell type involved
was not established. In all cases the fluorescence was cytoplasmic.
Big liver and spleen disease of broilers 45

Table 1. Antigen distribution and antibody titres in five birds inoculated orally with
BLS material and five in contact birds

Antigen
Sero-
Bird Tissue AGID conversion
IFT titre

Inoculated
1 Liver
Spleen
Kidney
2 Liver
Spleen
Kidney
3 Liver
Spleen
Kidney
4 Liver 4
Spleen 4
Kidney
5 Liver 16
Spleen 4
Kidney
In contact
6 Liver
Spleen
Kidney
7 Liver 8
Spleen 2
Kidney
8 Liver 16
Spleen 16
Kidney
9 Liver
Spleen
Kidney
10 Liver
Spleen
Kidney

IFT = Immunofluorescence test on tissue: + = few positive cells;


+ + + + = many positive cells.
AGID = Agar gel immunodiffusion.
46 J-K. Clarke et al.

Staining of BLS
In the course of attempts to detect BLS antigen by indirect immunofluorescence,
cryostat sections of BLS infected liver and spleen were, as control material, stained
using FITC labelled anti-chick globulin. The pattern of staining resembled that
obtained when tissue was stained with BLS antisera. As might be expected however,
staining with anti-chick globulin, but not with BLS antiserum was prevented when the
antisera were diluted in normal chicken serum. In further investigations, liver and
spleen material from BLS infected birds and control liver and spleen were stained
using a mouse monoclonal anti-chick globulin produced in this laboratory and a
FITC labelled rabbit anti-mouse immunoglobulin antisera. The staining observed in
BLS infected material was much more extensive than that seen in control material
from uninfected birds and the pattern obtained was similar to that seen when sections
were stained directly with BLS antisera (Fig. 2).
Histopathological and immunoperoxidase examination
The major histopathological features noted in liver sections were marked vacuolation
of hepatocyte cytoplasm and perivascular accumulations of lymphoid cells.
Immunoperoxidase examination revealed widespread staining of BLS antigen in the
Kupffer cells of the hepatic sinusoids (Fig. 3). Staining was intracytoplasmic in
distribution and the pattern was of a diffuse granular staining of the cytoplasm,
superimposed on which was a more intense staining of larger irregular deposits
resembling inclusions or phagosomes. Staining was not observed in hepatocyte
cytoplasm, but was noted in scattered cells at the peripheries of perivascular lymphoid
aggregates.
In the splenic sections hyperplasia of white pulp with formation of lymphoid follicles
was noted. There was necrosis of scattered individual cells, but extensive necrosis of
the splenic parenchyma was not a feature. Immunoperoxidase examination revealed
widespread staining of BLS antigen in macrophages and dendritic cells both in white
and red pulp (Fig. 4). Histopathological changes in kidneys were minimal and
consisted mainly of small interstitial accumulations of lymphocytes. In three birds
(nos 2,5 and 6) BLS antigen staining was largely restricted to scattered cells within the
renal interstitium and to interstitial lymphoid aggregates. However in bird 8
particulate antigen staining was also observed in epithelial cells of renal tubules.
Antigen staining was infrequently observed in glomeruli.

DISCUSSION
Field observations in Australia and laboratory transmission of infection indicate that
BLS is an infectious disease (W. Williams, unpublished). The present investigation
confirmed the transmissibility of infection both by the inoculation of infected
material and by a natural, but unknown, route to in-contact birds. It also
demonstrated that susceptibility to infection is not confined to those strains of broiler
breeder birds present in Australia.
Evidence for infection was provided by the detection of antigen using the AGID test
on tissue homogenates and direct immunofluorescence on smears of liver, spleen and
kidney. Antibody development was also demonstrated using AGID. Immuno-
fluorescence was more sensitive than AGID for the detection of antigen and provides
an alternative rapid and sensitive method for the detection of BLS antigen in tissue
samples from infected birds.
Big liver and spleen disease of broilers 47

Fig. 1. Cryostat section of spleen from a broiler breeder infected with BLS agent and stained with a
FITC conjugated BLS antiserum. Note specific intracyloplasmic staining.

Fig. ,2. Cryostat section of spleen from the same broiler breeder as shown in Fig. 1. Stained with a
mouse monoclonal anti-chick immunoglobulin and counterstained with a FFTC conjugated
rabbit anti-mouse immunoglobulin. Note that the numbers, distribution and pattern offluorescing
cells are'very similar to those shown in Fig. 1.
48 J.K. Clarke et al

bill 'fr.L'Jt^C'-vbL -il. ;j:;r--:i>:-. ^/ji'l.A-I'^V^ -'-..""w


Fig 5. Formalin fixed section of liverfrom a broiler breeder infected with BLS agent and
stained with a BLS antiserum using an ABC peroxidase method. Note immunoperoxidase
positive staining ofKupffer cells and vacuolation of hepatocytes.

Fig. 4. Formalin fixed section of spleen from a broiler breeder infected with BLS agent
and stained with a BLS antiserum using an ABC peroxidase method. Note
immunoperoxidase staining of splenic macrophages.
Big liver and spleen disease of broilers 49

The failure to observe the aetiological agent by electron microscopy or to propagate it


in vitro was not unexpected. Initially, it seemed likely that our failure was a reflection of
the relatively small number of cells which can be examined by electron microscopy.
However, in liver and especially spleen, immunofluorescence showed that a high
proportion of cells contained BLS antigen. It is probable that many such cells were
observed in the electron microscope, but no virus-like particles were observed, so it
might be premature to equate the presence of antigen in a cell with the presence of a
replicating agent.
Antibody as well as antigen was present in most birds. Antigen was concentrated in
phagocytic cells in spleen and liver and many cells positive for BLS antigen by
immunofluorescence also appeared to contain immunoglobulin (Fig. 2). This raises
the possibility that the antigen observed in the cytoplasm of cells may, at least in part,
have been phagocytosed. The widespread non-focal, distribution of antigen in liver
(Fig. 3) is consistent with this possibility. The histopathological lesions observed in
our experiments were similar to those described in the early stages of the naturally
occurring disease (W. Williams, unpublished).
If a significant proportion of the antigen present in tissues represents phagocytosed
antigen and antigen-antibody complexes rather than replicating virus, this might
explain the apparent absence of virus-like particles. However, other explanations are
possible especially since the causal agent of BLS has not yet been proven to be a
virus.
Further work is required to elucidate the aetiology of BLS. Such work is of practical
importance outside Australia since we have serological evidence that some broiler
breeders in the UK are infected with the agent (Connor, unpublished).
Acknowledgements
We thank D. Pollock, J. McNair and M. Platten for excellent technical assistance.

REFERENCES
Bryson, D.G., Cush, P.F., McNulty, M.S.. Platten, M. and Allan, G. (1988). The demonstration of respiratory
syncytial virus antigen in pneumonic bovine lung by an avidin-biotin-peroxidase (A.B.C.)
staining method. In: Proceedings of 4th International Symposium of Veterinary Laboratory
Diagnosticians, Amsterdam, 504-509.
Handlinger, J.H. and Williams, W. (1989). An egg drop associated with splenomegaly in broiler breeders.
Avian Diseases, 32: 773-778.
McNulty, M.S. and Allan, G.M. (1983). Applications of immunofluorescence in veterinary viral diagnosis.
In: Recent Advances in Virus Diagnosis, pp. 15-26. Edited by McNulty, M.S. and McFerran, J.B.
The Hague: Martinus Nijhoff.

RESUME
Maladie du foie et de la rate hypertrophiques chez
des reproducteurs de type chair
Cinq reproducteurs de type chair ont t inoculs avec des fcs et des extraits tissulaires
d'oiseaux atteints de la maladie du foie et de la rate hypertrophiques (BLS). Cinq oiseaux
ont t placs au contact de ceux inoculs. Aprs huit semaines tous les oiseaux inoculs et
quatre des cinq oiseaux contact ont rvl tre infects comme le montrent la prsence
d'une rponse immunitaire et/ou celle de l'antigne BLS dans la rate et les empreintes de
tissus par le test d'immunofluorescence et d'immunodiffusion en glose.
50 J-K- Clarke et al.

En utilisant des antisrums appropris, l'antigne BLS a t trouv principalement dans les
cellules phagocytaires du foie et les macrophages de la rate ainsi que les cellules
dendritiques. Quelques cellules contenant l'antigne ont t trouves galement dans le
rein.
La double coloration des tissus hpatiques et splniques par l'antigne BLS et
l'immunoglobuline des poulets ont indiqu que de nombreuses cellules taient positives
pour les deux. Il est possible que l'antigne BLS dans la plupart des cellules, puisse
reprsenter du matriel phagocyt plutt qu'un agent se rpliquant. S'il en tait ainsi, ce qui
expliquerait la non dtection des particules virales, il serait ncessaire d'tablir le site
primaire de rplication de l'agent BLS ainsi que sa nature.

ZUSAMMENFASSUNG
Dicke Leber und Milzkrankheit bei Broilerzuchtherden
Fnf erwachsene Broilerzuchttiere wurden mit Faeces und Gewebeextrakten von Tieren
mit Dicker Leber und Milzkrankheit (BLS) inokuliert. Nach 8 Wochen wurde an Hand von
Immunreaktion und/oder durch Demonstration des BLS Antigens in Milz und Gewebe-
austrichen, durch Immunofluoreszenztest und durch Agargelimmunodiffusion das
Haften der Infektion bei diesen Tieren und bei vier von fnf Kontakttieren nachgewiesen.
Mit Hilfe von geeigneten Antisera wurde BLS Antigen besonders in phagozytierenden
Leberzellen und in Makrophagen und in Dendritiden der Milz gefunden. Weniger
antigenhaltige Zellen wurden in der Niere gesehen. Die Doppelfrbung von Leber und
Milzgewebe auf BLS Antigen und Hhnderimmunoglobulin ergab, da viele Zellen auf
beide positive reagierten. Mglicherweise handelt es sich bei den BLS Antigen in den
meisten Zeller eher um phagozytiertes Material als um ein sich vermehrendes Agens. Dies
knnte erklren, da virushnliche Partikel nicht beobachtet werden konnten, was
andererseits besagt, da der primre Replikationsort des BLS Agens und seine Natur erst
noch festzustellen sind.

RESUMEN
Enfermedad del hgado y bazo grandes de reproduetores broiler
Se inocol a cinco reproduetores broiler adultos con heces y extractos tisulares procedentes
de aves con la enfermedad del hgado y bazo grandes (BLS). Se situaron a cinco aves en
contacta con las inoculadas. Despus de 8 semanas todas las aves inoculadas y cuatro de
las que estaban en contacta mostraron signos de haber sido infectadas evidenciado por la
deteccin de una respuesta inmune y/o la demonstracin de antgeno BLS en el bazo y en
improntas de tejido mediante inmunodifusin en gel de agar e immunofluorescenica,
respectivamente.
Usando antisueros apropiados se encontr antgeno de BLS principalmente en las clulas
fagocticas del hgado y en los macrfagos y clulas dendrticas esplnicas. Se encontr un
menor nmro de clulas conteniendo antgeno en el rin.
Un doble inmunomarcaje para el antgeno BLS e inmunoglobulinas de polio en el hgado
y el bazo indic que muchas clulas mostraban una doble reactin. Probablemente el
antgeno BLS en la mayora de las clulas pueda representar material fagocitado mas que
una replicacin del agente. Si fuera as este hecho podra explicar el fallo para detectar
partculas virales e implicara que el lugar primario de replicacin del agente del BLS y su
naturaleza se encuentra todava por establecer.