Local antibody forming cell responses to the Hitchner B1 and Ulster strains of Newcastle disease virus

Veterinary Immunology and Immunopathology, 37 ( 1993 ) 165-180

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Elsevier Science Publishers B.V., A m s t e r d a m

Local antibody forming cell responses to the Hitchner B 1 and Ulster strains of Newcastle disease virus P.H.

R u s s e l l a a n d G. K o c h b

aDepartment of Veterinary Pathology, The Royal Veterinary College, Royal CollegeStreet, London, NWI OTU, UK bCentral VeterinaryInstitute-DLO, Department of Virology, PO Box 365, 8200 AJ, Lelystad, Netherlands (Accepted 24 August 1992 )

ABSTRACT Russell, P.H. and Koch, G., 1993. Local antibody forming cell responses to the Hitchner BI and Ulster strains of Newcastle disease virus. Vet. Immunol. lmmunopathol., 37:165-180. The Hitchner B1 and Ulster strains of Newcastle disease virus ( N D V ) replicated to high titre in the Harderian gland ( H G ) after eye-drop infection. The Harderian gland then became the major site of antiviral IgA-antibody-forming cells (AFC) in the body and their number correlated to the level of antiviral IgA antibody in the tears. The spleen, H G and femoral bone marrow all contained comparable levels of antiviral IgG-AFC and IgM-AFC after two intra-ocular inoculations of virus, whereas the caecal tonsil and bursa contained few AFC despite the local replication of the Ulster strain of NDV leading to high titres of virus in the faeces. Vaccines of the Hitchner B1 strain of NDV were much less effective at inducing antibody by the intranasal compared with intra-ocular route and no virus was re-isolated after intranasal vaccination. The intravenous inoculation of inactivated lscoms of NDV could stimulate the spleen, but not the Harderian gland to the same extent as a live virus. ABBREVIATIONS AFC, antibody forming cells; gmt, geometric mean titre; HG, Harderian gland; IPMA, immunoperoxidase monolayer assay; iu, infectious unit; NDV-Newcastle disease virus; flPL, fl-propiolactase.

INTRODUCTION

The Harderian gland (HG) has long been known to contain high numbers of plasma cells (Bang and Bang, 1968; Wight et al., 1971; Bienenstock, 1973; Koch and J.ongenelen, 1988; Baba et al., 1988). Antigen-specific antibody Correspondence to. D r P.H. Russell, The Royal Veterinary College, Royal College Street, London, N W I 0TU, UK.

© 1993 Elsevier Science Publishers B.V. All rights reserved 0 1 6 5 - 2 4 2 7 / 9 3 / $ 0 6 . 0 0

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production by lymphoid cells of the HG was detected after local immunisation with sheep red blood cells using the haemolytic plaque assay (Mueller et al., 1971 ) and antibodies directed against bovine serum albumin in tears and plasma cells in the HG were detected after both local and intravenous immunisation with bovine serum albumin (Bums, 1976 ). Davelaar et al. ( 1982 ) suggested that HG plasma cells produced specific IgA antibodies and the HG facilitated the transport of circulating IgG into the serum after eye-drop vaccination with infectious bronchitis virus. In non-stimulated, young specific-pathogen-free chickens, HGs contain mainly IgM-secreting cells. In contrast, HGs contain more IgG and IgA than IgM immunoglobulin-secreting cells at older ages (Koch and Jongenelen, 1988; Mansikka et al., 1989 ). Ocular stimulation with tetanus toxoid induces lacrimal IgG and IgA but no IgM antibody responses (Mansikka et al., 1989). These results indicate that antibody-secreting cells quickly switch from IgM to IgG and IgA production or that high numbers of IgG- and IgA-secreting cells immigrate from elsewhere. Contradictory data exists on the contribution of avian bone marrow to antibody formation. Using a reverse haemolytic plaque assay, Lawrence et al. ( 1981 ) detected large numbers of Ig-secreting cells in the bone marrow and spleen whereas Jeurissen et al. ( 1988 ) detected low numbers of bone marrow cells with surface IgM and none with surface IgA or IgG when using a panel of class-specific monoclonal antibodies for immunohistochemistry (Jeurissen et al., 1988 ). In other species, bone marrow has been found to be a major site of antibody formation (reviewed by Benner, 1981 ). The Hitchner B 1 strain of N D V replicates in the HG (Russell, 1993 ) and elicits neutralising antibody in this organ and the lacrimal fluid (Powell et al., 1979 ). Most of the antibody is likely to result from local synthesis and some from transudation as discussed by Aitken and Parry (1976) and Parry and Aitken ( 1977 ). The transfer of maternal IgG into the tears has recently been demonstrated (Russell, 1993). N D V vaccines are usually applied by the intra-ocular or aerosol route, although manufacturers often recommend the intranasal route. Yoshida et al. ( 1971 ) and Zakay-Rones et al. ( 1971 ) indeed elicited an antibody response in the trachea and lungs after intranasal immunisation with inactivated virus. In contrast, Parry and Aitken ( 1977 ) could not detect a local antibody response after intratracheal administration of heatinactivated NDV. We compared quantitatively the AFC-response to different strains of NDV in spleen, HG, bone marrow, caecal tonsils, and bursa. In addition, the effect of route of immunisation and of adjuvant formulation on the i m m u n e response and Ig-class distribution in the different organs was tested.

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MATERIALS AND METHODS

Birds Reaseheath C birds which were negative for antibodies to NDV and other infectious agents have been detailed in the accompanying paper (Russell, 1993 ). In some experiments Birmingham B2 birds, which were also bred from Houghton stock and negative for virus exposure, were used according to availability of stock at the time of experiment. Changing flocks did not alter results in preliminary experiments. Birds received virus grown in eggs of the same flock. Virus infected birds were kept in cages inside negative-pressure plastic isolators. Control birds remained in cages in a conventional animal house. Birds were weighed before they were killed by cervical dislocation. The lacrimal fluid and HG were collected as described in the accompanying paper (Russell, 1993 ).

Viruses Allantoic-grown Ulster and Hitchner B 1 strains of N D V and the vaccinal stock of Hitchner B1 were those used in the accompanying paper and had titres of 108-108.6 infectious units (iu) 0.1 ml-1 (Russell, 1993 ). To establish the antibody response to a commercial vaccine, the Hitchner B 1 vaccine from Salsbury Laboratories was used. It contained 100 to 400-fold less iu than the above allantoic fluid stocks.

Immune-stimulatory complexes (Iscoms) Iscoms were prepared after consultation with Dr N. Mackenzie (Coopers Pitman-Moore, Crewe, U K ) . Virus glycoproteins in Tris-EGTA saline, pH 7.2, were further purified on a lentil-lectin column using 0.1% mega-9 (Cantock, Bristol, U K ) as detergent and then made into Iscoms by adding a final concentration of 0.05% Quil-A (Superfos, Vedbaek, Denmark) and 0.05% phosphotidyl-choline and 0.01% cholesterol. The Iscoms were then washed three times in Tris-saline EGTA pH 7.2 by ultra filtration (M.50 Amicon) and then centrifuged through 10% sucrose, filtered through a 0.22/zm sterilising filter and stored at + 4 ° C . 10 mg virus resulted in 1.6 mg Iscoms. The resultant Iscoms had a diameter of 30 nM and contained both virus envelope proteins of NDV. During preliminary experiments the immunogenicity of 5 #g of Iscoms compared closely with that of 5/tg purified virus providing two intravenous injections were given.

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The detection of antibody directed against ND V by immunoperoxidase monolayer assay (IPMA) The method described by Russell in the accompanying paper was used to detect antiviral IgA, IgG and IgM in the serum, tears and bile (Russell, 1993 ). The geometric mean titre (gmt) of the group was calculated and expressed as loglo of IgA, IgG or IgM to NDV.

The detection of AFC to ND V by IPMA The method described by Russell (1989) was further modified: lymphocytes were always kept in basic salts solution containing 5% horse serum; 0.75% carboxymethyl cellulose was added before incubation for the 3 h at 39°C; Hela or Hep-2 cells instead of MDBK cells were used as antigen monolayer in most experiments since preliminary experiments showed that the same number of AFC were detected when these cells were used. Cell suspensions were prepared from organs to give approximately 107, 106 and 105 lymphocytes in triplicate wells of NDV-infected and uninfected cells for detecting IgA-AFC, IgG-AFC and IgM-AFC. Foci of secreted IgA, lgG and IgM were visualised by immunoperoxidase as described for serum antibody in the accompanying paper (Russell, 1993). The same NDV strain was used to immunise and to detect AFCs. Whenever two strains of NDV were used to immunise birds in the same experiment the Ulster strain was used to prepare the antigen-containing monolayers because splenic IgG-AFC responses did not distinguish between strains (Russell, 1989 ). The majority of AFC secreted antibody which bound to the plasma membrane and cytoplasm. These AFC were counted. By comparison to mouse hybridomas they bind to the envelope glycoproteins of NDV (Russell et al., 1987). The number of AFC in each organ of each bird was adjusted for body weight of that bird and expressed as logw AFC/organ/kg body weight. The geometric mean and standard errors of each group of 3-4 birds was calculated from 1Oglo values. Significance tests (Student's) were performed using the body weight-adjusted loglo values and used to compare AFC responses between immune and control birds, between different organs and between different routes of inoculation. In this work the relative contribution which each organ made to the AFC response was expressed as logm AFC/organ/kg body weight because 1 kg was near to the average weight of the older birds sampled here. NDV-immune and control birds sometimes contain IgA-AFC and IgM-AFC which bound to mock-infected monolayers (see Russell, 1989). AFC assays were therefore done always in parallel on NDV-infected monolayers and mockinfected monolayers which had been prepared at the same time. The loglo AFC on mock-infected cells was subtracted from loglo AFC on infected cells

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for each bird and a mean value then calculated for the group of birds. When this mean value exceeded 10°5, AFC were termed specific. In other words during a specific AFC response the difference between the number of AFC on infected and uninfected cells was more than three-fold and during a non-specific AFC response the difference was less than three-fold. IgG-AFC were always specific because they did not secrete antibody which bound to uninfected monolayers.

The sites of replication of Ulster and Hitchner B1 strains of ND V Three or six 7-week-old Reaseheath C chickens were infected either by intra-oculonasal or by the intravenous administration of 0.4 ml undiluted allantoic fluid containing NDV Ulster or NDV (Hitchner B1 ) (Table 1 ). Three uninfected control birds were used. Chickens were killed and organs were collected 3 days after infection. In a second experiment, 25/tl ofHitchner B 1 vaccine was inoculated either intra-ocularly or intranasally, as recommended by the manufacturers, into four 4-week old Reaseheath C birds and organs were collected 3 days later. Three hatch-matched controls were used.

The primary and secondary AFC responses after immunisation with the Ulster and Hitchner B1 strains of ND V by the intravenous or oculonasal route Forty Reaseheath C birds 42 days of age were used. Eight were controls and 32 received 0.4 ml of undiluted allantoic-fluid virus. Of the 32, eight received Ulster by the intravenous route, eight received Ulster by the intra-oculonasal route, eight received Hitchner B 1 by the intravenous route and eight received Hitchner B 1 by the intra-oculonasal route. Half the birds in each group were killed at 52 days of age and sampled for AFC in their spleen, HG and bone marrow (see Table 2). The other half were reinfected at 72 days of age and killed at 76 days of age (see Table 3).

The secondary AFC response in the spleen, bone marrow, HG caecal tonsil and bursa after N D V immunisation by the oculonasal route Three separate experiments were performed in which four birds received two intra-oculonasal inoculations of 1 ml undiluted allantoic-fluid virus and were killed 4 days later. At the same time, four untreated control birds were killed (see Table 4 ). In the first two experiments, Birmingham B2 birds received the Ulster strain of NDV. In the first experiment, the birds were 120 days of age at inoculation, 144 days at the second inoculation and 148 days when killed. In the second

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P.H. RUSSELL AND G. KOCH

experiment the birds were 90 days of age at inoculation, 111 days at the second inoculation and I 15 days when killed. In the third experiment, half the birds were Reaseheath C because insufficient numbers of Birmingham B2 were available. The birds received the Hitchner Bl strain of NDV and were 30 days of age at inoculation, 90 days of age at the second inoculation and 94 days when killed.

Vaccination with the Hitchner B1 strain o f ND V by the intranasal compared with intra-ocular route Reaseheath C birds were used in each experiment when small groups of two to four birds received 30/tl of intra-ocular vaccine or 30/zl of intra-nasal vaccine, or remained as controls (see Table 5 ). Each group was housed in a separate isolator according to route of inoculation and killed 10 days after inoculation. In Experiments 1, 2 and 3 birds were aged 12, 10 and 7 weeks respectively at inoculation. All birds were sampled for lacrimal and serum antibody in Experiments 1, 2 and 3 and for splenic and HG AFC in Experiments 1 and 2.

The A F C response to inactivated virus or Iscoms Three different methods of immunising birds with inactivated virus were tested in three separate experiments. Birds were tested for AFC in the spleen and HG (see Table 7). In the first experiment, eight Reaseheath C birds 140 days of age received 0.4 ml allantoic-fluid containing fl-propiolactone (flPL)-inactivated NDV (Ulster) by the intravenous route. Four birds were killed at 150 days of age. Four received a second inoculation at 170 days of age and were killed at 174 days of age. Four controls were killed at 174 days of age. The second experiment was the same as the first experiment except that 5 #g Iscoms was used instead offlPL-inactivated virus. In the third experiment, eight Reaseheath C birds were immunised at 50 days of age and killed at 60 days of age. Four birds received 0.4 ml allantoicfluid containing flPL-NDV (Ulster) by the intravenous route, four birds received the same inoculum by the intra-ocular route, and four untreated birds remained as controls. RESULTS

The sites o f replication o f Ulster and Hitchner B1 strains o f ND V Both strains replicated in the conjunctiva and HG after local instillation into the eye and nose. However, the Hitchner B 1 strain reached ten-fold higher

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titres than the Ulster strain in conjunctiva, HG and trachea but did not replicate in the lungs (Table 1 ). The Ulster strain was regularly recovered from the small intestine, caecal tonsil and faeces. After intra-oculonasal immunisation with the Hitchner B 1 strain, no virus was isolated from the faeces. Both strains were recovered from the kidney and bursa at low titre. The Hitchner B 1 strain therefore replicated preferentially in the upper respiratory tract and the Ulster strain in the gut (Table 1 ). After intravenous inoculation, both strains replicated in a wider range of tissues including irregularly the bone marrow, spleen and brain. Both strains were also recovered from the faeces. The Hitchner B1 strain was still recovered in the highest titre in the upper respiratory tract (Table 1 ). A commercial Hitchner B 1 vaccine, which contained 4000-fold less virus than the allantoic fluid inoculum was used next. Virus was regularly recovered to high titre from the Harderian gland and conjunctiva 3 days after intra-ocular administration but no virus was recovered after intranasal administration (Table 1 ).

The primary A F C response after immunisation with the Ulster and Hitchner B1 strains o f ND V by the intravenous or oculonasal route Lacrimal IgA titres and Harderian gland IgA-AFC responses to N D V Ulster correlated very well to each other after either route o f inoculation (Fig. TABLEI

The sites of replication of the Ulster and Hitchner B 1 strains of NDV 3 days after infection Tissue

Log~o virus in 0.1 g tissue ~ (proportion of positives) Experiment 1

Inoculum2: Route3: Conjunctiva Harderian gland Trachea Lung Faeces Small intestine Caecal tonsil Bursa Kidney Spleen Femoral bone marrow Cerebrum Cerebellum

Experiment 2

109.2 B1

109.2 Uls

109.2 B1

109.2 Uls

10 5.6 BI

10 s-6 B1

i/o-n

i/o-n

i/v

i/v

vaccinei/o

vaccine i/n

4.5(3/3) 4.4(3/3) 3.7(3/3) <1 <1 <1 1.0(1/3) 2.5(2/3) 2.1(3/3) <1 <1 <1 < 1

3.3(6/6) 3.5(4/6) 2.0(3/6) 2.1(1/6) 4.0(6/6) 3.8(6/6) 3.0(6/6) 2.2(3/6) 2.1(4/6) <1 <1 <1 < 1

5.4(3/3) 4.8(3/3) 5.5(3/3) 2.5(3/3) 4.2(3/3) 2.5(1/3) 3.0(3/3) <1.0 3.2(2/3) 2.0(1/3) 3.0(1/3) 2.1(1/3) 2.0(1/3)

3.8(4/6) 4.9(4/6) 3.0(4/6) 1.3(1/6) 3.0(6/6) 3.4(5/6) 2.1(2/3) 1.0(1/3) 1.5(3/3) 1.8(1/3) 1.0(1/3) <1 < 1

4.7(3/4) 4.8(4/4) 2.2(1/4) <1 <1 <1

<1(0/4) <1.3 < 1 <1 <1 1

<1 <1

<1 <1

tGeometric mean infectivity titre where SEM < 10°7. 2B1, the Hitchner Bl strain o f N D V , Uls, the Ulster strain of NDV. 3Route: i/o-n, intra-oculonasal; i/v, intravenous; i/o, intra-ocular; i/n, intranasal.

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P.H. RUSSELL A N D G. KOCH

1 ). Both IgA responses were four-fold higher after local compared to intravenous administration of virus (Table 2 ). Lacrimal IgA titres did not correlate to splenic IgA-AFC (Fig. 1 ). Of the organs tested, most of the IgG-AFC/kg were always localised in the spleen, and most of the IgA-AFC were localised in the HG after local but not intravenous immunisation. The bone marrow did contain virus-specific IgGAFC after intravenous immunisation, but not after local immunisation (Table 2). Virus-specific IgM-AFC were above background in the spleen and bone marrow after intravenous, but not local administration of virus and in the HG after local, but not intravenous administration of the Ulster strain. This emphasised the importance of the HG as a source of each class of AFC after local immunisation 10 days earlier and of the spleen as a source of antibody after intravenous virus. The secondary A F C responses after immunisation with the Ulster and Hitchner B1 strains o f ND V by the intravenous or oculonasal route Local immunisation increased the numbers of IgA-AFC in the spleen and HG by five to 20-fold compared with intravenous immunisation and this was accompanied by the AFC in the HG switching from producing non-specific IgA after intravenous virus to producing specific IgA after local stimulation (Table 3 ). The AFC which secreted IgG were virus specific. The spleen contained sigSpleen

Harderian Gland 5.0

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Lacrimal IgA (Log 10) Fig. 1. Three or four 6-week old C4 birds were infected by intra-oculonasal or intravenous administration of 0.4 ml of undiluted allantoic fluid containing 108.6 NDV (Ulster) 0.1 ml '. Spleens, HG and sera were collected 10 days later. LOglo AFC/organs k g - ' body weight of each bird was compared with its lacrimal IgA titre by indirect immunoperoxidase assay on virusinfected cells. The lacrimal IgA titre correlated to the IgA-AFC in Harderian gland ( r = 0.93), but not to the IgA AFC in the spleen (r=0.63).

CELL RESPONSESTO NEWCASTLEDISEASEVIRUS

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TABLE2 The primary AFC and lacrimal antibody responses to infection by the Ulster and Hitchner B1 strains of NDV at 10 days after inoculation Organ/secreta

Spleen HG Bone marrow Tears Spleen HG Bone marrow Tears Spleen HG Bone marrow Tears

Class of antibody

A A A A G G G G M M M M

Logto AFC + SEM kg- ~body weight or log~o titre in tears after inoculation with none

i/v Uls t

i/o-n Uls I

i/v B11

i/o-n B 11

<2.4 2.5_+0.13 <1.1 < 1.5 <2.1 <1.0 < 1.1 <1.5 3.8+0.13 3.0+-0.13 1.9+-0.23 < 1.5

4.0+_0.12 3.3+-0.22 3.5+-0.2 2.4_+0.2 l 5.1_+0.2 1.9+-0.22.4 4.7+-0.12 3.5+-0 5.3+-0.2 3.3+-0.14'5 4.4+-0.12 2.9+-0.3

3.1+-0.1 4.3+-0.24 <2.04 3.0+-0.2 4.2+-0.2 2.9_+0.34 <2.04 3.7+-0.1 3.9+-0.25 4.6+-0.2 2.5+-0.24.5 3.7+-0.3

2.9+-0.2 3.5+-0.12 2.2+-0.1 1.5(2/4) 3.6+-0.1 2.2+-0.24 3.0+-0.42 4.0+-0 4.3+-0.1 3.2+-0.14,5 3.5+-0.24,5 4.0+-0

3.4+-0.1 4.8+-0.24 2.3+-0.14 2.0+-0.2 <3.3 2.6-+0.3 < 1.85 2.8(3/4) 4.0+_0.2 s 3.1+-0.35 <2.5 s 2.8+0.2

~See Table 1 for abbreviations. ZSignificant difference in AFC or antibody after intravenous compared to intra-ocular inoculation of the same virus, P < 0.05. 3Non-specific AFC: the mean value oflogto AFC on infected cells minus loglo AFC on uninfected cells was less than 0.5; i.e. AFC on infected and uninfected monolayers were within three-fold of each other. 4Significant difference in number of AFC in this organ as compared with spleen, P < 0.05 by paired t test. 5No significant increase in number of AFC compared with control birds, P > 0.05.

nificantly more IgG-AFC than the HG after intravenous immunisation, but not after local immunisation (Table 3 ). The femoral bone marrow became a major site of AFC and contained significantly more IgA-AFC than the spleen or HG after intravenous immunisation (Table 3 ). Local immunisation produced responses in all three organs and this was re-examined later using a wider range of organs.

The secondary AFC response of the spleen, bone marrow, HG, caecal tonsil and bursa after immunisation with the Ulster or Hitchner B1 strain of N D V by the oculonasal route The HG was the only organ to give IgA-AFC and IgM-AFC responses significantly above background in all three experiments. Its content of IgA-, IgGand IgM-AFC was always within three-fold of each other. The HG contained four to 11-fold more IgA-AFC than the spleen ( P < 0 . 0 2 5 ) (Table 4). It also contained marginally more IgM AFC than either spleen or bone marrow in all experiments and marginally more IgG-AFC in two out of the three exper-

174

P.H. RUSSELLAND G. KOCH

TABLE 3 The secondary AFC responses to infection by the Hitchner B1 and Ulster strains of NDV at 4 days after boosting Organ

Spleen HG Bone marrow Spleen HG Bonemarrow Spleen HG Bonemarrow

Class of antibody

A A A G G G M M M

Log~o AFC_+ SEM kg- ' body weight after inoculation with: none

i/v Uls ~

i/o-n Uls I

i/v BI 1

<2.2 2.1 _+0.14 <1.2 <2.0 <1.2 < 1.2 3.2_+0.14 2.5_+0.24 2.5_+0.14

3.7+0.12 3.0_+0.12,4`5 4.7_+0.25 6.1_+0.22 3.9_+0.25 6.2+0.12 4.5_+0.14 3-3_+0.13,4,5 4.7_+0.1

4.5+0.2 <2.53 4.3+0.2 2.4+0.33'4 5.1_+0.2 3.2+0.15 5.5_+0.1 5.7_+0.5 4.8_+0.2 3.2_+0.25 5.0_+0.2 5.7_+0.3 4.3_+0.2 4 4.0_+0.1 2.8_+0.13,4.5 2.8_+0.12,3,4`5 4.1_+0.2 3.3_+0.22,3

i/o-n ~ B1 3.3+0.1 3.3_+0.1 3.0_+0.2 4.3+0.2 3.7+0.1 4.4+0.4 3.9_+0.2 4 3.7_+0.1 3.3b0.1

~See Table 1 for abbreviations. 2Significant difference in AFC after intravenous compared to intra-ocular inoculation of the same virus, P < 0.05. 3No significant increase in AFC compared with control birds, P > 0.05. 4Non-specific AFC: the mean value oflog~o AFC on infected cells minus log~oAFC on uninfected cells was less than 0.5; i.e. AFC on infected and uninfected monolayers were within three-fold of each other. 5Significant difference in number of AFC in this organ compared with spleen, P < 0.05 by paired t test.

iments (Table 4). This dominance of the HG was further emphasised because it contained 200-fold fewer lymphocytes than the spleen and 25-fold fewer lymphocytes than the femoral bone marrow (Table 4). The concentration of IgA-AFC per 107 HG lymphocytes therefore exceeded that of the spleen by 800 to 2200-fold. The bursa yielded the same average number of lymphocytes as the bone marrow but produced a weak response in the third experiment only (Table 4). Although the number of lymphocytes in caecal tonsil and HG were about the same, the number of AFC in the former were 0.01% or less of the number in the latter organ (Table 4).

Vaccination with the Hitchner B1 strain of ND V by the intranasal compared with intra-ocular route. No virus could be isolated after vaccination via the intranasal route (Table 1 ). More birds produced detectable levels of IgA, IgG or IgM in serum and tears after intra-ocular than after intranasal vaccination P < 0.04 (Table 5 ). The serum and lacrimal IgA, IgG and IgM titres of antibody-positive birds

CELL RESPONSESTO NEWCASTLEDISEASEVIRUS

175

TABLE 4 The organ distribution of AFC during the secondary response to the Ulster and Hitchner B l strains of NDV administered by the oro-nasal route at four days after boosting Expt.

Virus inoculum

Class of antibody

Loglo AFC kg- i body weight Spleen ~

HG ~

Femoral bone marrow

1 2 3 1 2 3 1 2 3

Uls Uls B1 Uls Uls B1 Uls UIs B1

A A A G G G M M M

2.6_+0.22 3.2+0.03 3.4_+0.1 3.9_+0.2 4.1+0.3 3.6_+0.2 3.3-+0.12,4 3.9+0.1 3.8+0.2

3.7_+0.13 3.8_+0.13 4.3_+0.13 4.1_+0.1 3.9_+0.2 4.7_+0.23 4.0_+0.13 4.1 _+0.1 4.3_+0.3

Bursa I

Caecal tonsil ~

1.5_+0.14 1.6_+0.3 2.9_+0.4 <1.52 <1.92 2.6_+0.8 <1.93,4 2.5_+0.24 3.2_+0.5

< 1.22.4 1.9_+0.2 NC 5 <1.52 <1.42 NC 5 1.5_+0. 24 < 1.12 NC 5

~

2.6_+0.2 2.9_+0.2 3.5_+0.1 3.8+0.2 3.9-+0.4 3.8-+0.2 3.5+0.13,4 3.8_+0.1 3.7_+0.1

tMean lymphocyte count in these organs (logm) spleen 9.2, HG 6.9, bone marrow 8.3, bursa 8.4, caecal tonsil 6.9. 2No significant increase in AFC compared with control birds, P > 0.05. 3Significant difference between AFC in HG compared with AFC in spleen and bone marrow, P < 0.025 by paired t test. Other differences insignificant P > 0.05. aNon-specific AFC: the mean value oflog~o AFC on infected cells minus logm AFC on uninfected cells was less than 0.5; i.e. AFC on infected and uninfected monolayers were within three-fold of each other. NC, not collected.

were three to 30-fold higher after intra-ocular than after intranasal vaccination (Table 5 ). This was also reflected by two to ten-fold higher IgA, IgG and IgM-AFC responses in both spleen and HG after intra-ocular compared to intranasal virus (Table 6).

The AFC response to the Ulster strain o f ND V after flPL-inactivation or to Iscoms o f the same virus

The intravenous inoculation of inactivated virus or Iscoms stimulated splenic IgA-AFC and IgG-AFC responses (Table 7) which compared with those levels obtained earlier with intravenous infectious virus (Tables 2 and 3 ). HG responses to inactivated preparations were, however, either non-specific or within background levels except a low IgG AFC response after intravenous immunisation with inactivated virus. No specific AFC responses were induced in spleen and HG after intra-ocular immunisation with inactivated virus (Table 7).

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P.H. RUSSELLAND G. KOCH

TABLE 5 The primary antibody response to intra-ocular vaccination compared with intranasal vaccination with the NDV Hitchner BI strain of NDV at 10 days after vaccination Fluid

Serum 2 Tears 2 Bile Serum 2'5 Tears 2"s Bile Serum 2'5 Tears 2'5 Bile

Class Log~o mean titre of antibody Expt. 1J

Expt. 2~

Route:

Ocular

Nasal

Ocular

Nasal

Ocular

Nasal

A A A G G G M M M

1.7(3/4) 3 <1.5(0/4) 3.0(4/4) 3.8(4/4) 3.0(4/4) 2.0(1/4) 3.9(4/4) 2.9(4/4) 1.5(1/4)

<1.5(0/3) <1.5(0/3) <1.5(0/3) 2.8(2/3) 1.5(1/3) <1.5(0/3) 3.0(2/3) <1.5(1/3) <1.5(0/3)

1.5(1/4) 2.5(4/4) 2.5(4/4) 3.3(4/4) 3.0(4/4) <1.5(0/4) 3.3(4/4) 2.8(4/4) 2.5(1/4)

1.5(1/4) 2.0(1/4) 2.5(3/4) 2.8(2/4) 2.0(2/4) <1.5(0/4) 2.7(3/4) 2.0(2/4) 1.8(2/4)

1.5(2/2) 2.1(2/2) ND 4 3.5(2/2) 2.0(2/2) ND 3.0(2/2) 2.7(2/2) ND

<1.5(0/2) <1.5(0/2) ND 1.5(0/2) <1.5(0/2) ND <1.5(0/2) <1.5(0/2) ND

Expt. 3

Expts. 1 and 2 as in Table 6. 2More birds responded after intra-ocular compared with intranasal virus by Fisher's test, P < 0.04. 3Between brackets, proportion of responder birds. 4ND, not done. 5Difference in titre between responders in intra-ocular groups compared with intranasat groups, P<0.025.

TABLE 6 The primary AFC response to intra-ocular vaccination and intranasal vaccination with the Hitchner B1 strain of NDV Organ

Class of antibody

Loglo AFC kg ~body weight Experiment 1

Spleen HG Spleen HG Spleen HG

A A G G M M

Experiment 2

Control

Intra-ocular

Intranasal

Control

<2.1 2.9_+0.12 <2.1 <0.9 3.3+_0.12 2.8+_0.2 2

4.0+0.1 4.0+0.1 4.5+0.2 3.7_+0.1 4.7-+0.1 4.5-+0.1

3.5+_0.23 2.0+0.2 3.6_+0.2 <2.0 3 . 5 _ + 0 . 3 3 <2.4 2.2+0.23 <0.9 3.9-+0.13 4.7_+0.2 2 3.7-+0.2 3 2.6_+0.3 2

Intra-ocular

lntranasal

4.1+0.2 4.2_+0.1 5.0_+0.1 3.7+0.1 4.6+_0.3 4 4.3-+0.1

4.3_+0.1 ~ 3.7+0.13 4.4+_0.3 ~ 2.5_+0.13 4.4_+0.51.4 4.1 +0.1

~No response in one bird (Experiment 2 intranasal group ), AFC of the three responders shown. 2Non-specific AFC: the mean value oflog~o AFC on infected cells minus logj0 AFC on uninfected cells was less than 0.5; i.e. AFC on infected and uninfected monolayers were within three-fold of each other. 3AFC significantly different between intra-ocular and intranasal groups, P < 0.05. 4No significant increase in AFC compared with control birds, P> 0.05.

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TABLE 7 The primary and secondary AFC responses to inactivated Ulster strain o f N D V (Uls) or to Iscoms of the same virus Organ

Class of antibody

Log~oAFC kg ~ BW after this inoculum and route ~ Experiment 1

Spleen HG Spleen HG Spleen HG

A A G G M M

Experiment 2

Experiment 3

None

Uls ivxl

Uls ivx2

None

Iscoms ivx2

None

Uls ivxl

Uls ioxl

< 1.9 < 1.6 < 1.9 <0.8 3.92 2.32

3.5 1.12'3 5.0 2.1 4.6 1.93

4.5 1.73 6.2 2.8 5.9 3.1

< 1.8 2.22 <2.2 0.82 2.92'4 3.92

4.1 2.03'4 5.6 1.23 5.54 3.83

<2.1 2.72 <2.1 < 1.1 4.62 3.52

3.6 2. 72,3 5.1 1.9 5.22`3 3.92

<2.62`3 < 2.72"3 2.82 2.12 4.72'3 3.62'3

~Routes: iv, intravenous; io, intra-ocular, × 2, two inoculations separated by 30 days. 2Non-specific AFC: the mean value ofloglo AFC on infected cells minus logto AFC on uninfected cells was less than 0.5 i.e. AFC on infected and uninfected monolayers were within three-fold of each other. 3No significant increase in AFC compared with control birds, P > 0.05. 4SEM between 0.2 and 0.5, otherwise less than 0.2

DISCUSSION

In this paper we show that, in addition to the spleen, the Harderian gland and bone marrow contribute to both the local and systemic antibody response to infectious NDV depending on the route of immunisation a n d / o r the replicative capability of the antigen. Thus, after virus-inactivation, the response in both spleen and HG did depend on the route o f i m m u n i s a t i o n (Table 7 compared with Tables 2 and 3 ). The splenic AFC response is greatly reduced after intra-ocular immunisation with inactivated NDV only, whereas HG AFCs are greatly reduced during intra-ocular and intravenous routes of immunisation with inactivated NDV. These results agree with those of other groups using live and heat-inactivated NDV (Parry and Aitken, 1977), or non-infectious antigens like sheep erythrocytes (Mueller et al., 1971 ) and bovine serum albumin (Burns, 1976) but are in contrast to those of Powell et al. (1979), who could not detect NDV antibody in the HG after intravenous immunisation with live virus. The latter group used however 104 fold lower doses of NDV Hitchner Bl vaccine than we used in this study. The difference of route of immunisation may be explained by different antigen handling. Replicating antigens which are applied locally still reach the circulation and thus will stimulate lymphoid cells in systemic lymphoid organs (Table 1 ), whereas intra-ocular inactivated antigens would drain into the saliva and fail to reach the circulation. An important finding of this study was that the IgA antibody titres of indi-

178

P.H. RUSSELL A N D G. K O C H

vidual birds both after intravenous and local immunisation correlated with the IgA-AFC in HGs, but not to IgA-AFC in the spleen. This indicates that the lacrimal IgA is produced locally in the HG and not in systemic lymphoid organs like the spleen. Similar conclusions were drawn from the surgical removal of the HG leading to absence of IgA (Baba et al., 1988 ) or neutralising antibodies directed against NDV (Survashe and Aitken, 1978) and infectious bronchitis virus (Davelaar et al., 1980). This result also makes it unlikely that the response in the HG is dependent on immigration of AFC precursors which are triggered in the spleen. Such a mechanism could have explained the rapid Ig-isotype switch of AFCs in the HG which occurred after three intra-ocular instillations of tetanus toxoid (Mansikka et al., 1989 ). Since the AFC response to NDV was not skewed to IgA and IgG to the same extent (Tables 2, 3 and 4) as the anti-tetanus toxoid response, this phenomenon may depend on the antigen used. The high frequency of AFCs among mononucleated cells, the low content of T-cells (Sundick et al., 1973), and the scantiness of lymphoid follicles with separated T and B cell regions in the glands proper, indicate that AFC precursors probably are triggered at a remote site. One such site may be the walls of the draining ducts which contain abundant lymphoid infiltrates and IgA-producing cells (Albini et al., 1974; Survashe and Aitken, 1978 ). The avian bone marrow is a major site of antibody formation (Lawrence et al., 1981, Table 4). Whether bone marrow is the major site of antibody formation as in mice (Benner et al., 1981 ) is uncertain, since the contribution of lymphoid cell content in the femoral to the content in the total bone marrow is not known in chickens. When it is assumed that the femoral bone marrow forms 10% of the total marrow (Lawrence et al., 1981 ), then the femoral marrow usually is the major site of AFC (Tables 2 and 4). The response in murine bone marrow is dependent on memory B cells that are triggered in peripheral lymphoid organs and then emigrate to the bone marrow (Benner et al., 1981 ). We obtained no evidence whether the antibody response in chicken bone marrow is also dependent on memory B cells. The inability of Jeurissen et al. ( 1988 ) to detect bone marrow IgA cells by immunochemistry may be related to their use of normal specific pathogen free birds with low antigen exposure as compared with conventional chickens (Lawrence et al., 1981 ) or to the present NDV-immune chickens. No virus could be recovered from any organ after intranasal instillation of Hitchner BI vaccine virus (Table 1 ), which probably led to the significant decrease or, in one out of ten chickens, no antibody responses of any Ig-class in serum, tears and HG (Table 5 ). The use of the intranasal route may therefore be less appropriate than the intra-ocular route for the delivery of Hitchnet B 1 vaccines because it stimulated fewer AFC in the HG (Table 6). This study, therefore, emphasises how the chicken AFC responses are relatively less spleen-centred than in the mouse because the HG and femoral bone

CELLRESPONSESTO NEWCASTLEDISEASEVIRUS

179

marrow can make comparable responses with the spleen. This is consonant with chickens containing about ten-fold fewer splenocytes on bodyweight basis than mice. The avian HG is of prime importance for producing lacrimal IgA to N D V and represents a very concentrated source of specific antibodyforming cells when stimulated by a replicating virus. ACKNOWLEDGEMENTS

This work was funded by grants from The Wellcome Trust and The Agricultural & Food Research Council to P.H. Russell. I thank J. Collier, E. Miles and B. McRandle for technical assistance and Dr G. Ezefeika for help with the last experiment, and Anna John for typing the manuscript.

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Mansikka, A., Sandberg, M., Veromaa, T., Vainio, O., Granfors, K. and Toivanen, P., 1989. B cell maturation in the chicken Harderian gland. J. Immunol., 142: 1826-1833. Mueller, A.P., Sato, K. and Glick, B., 1971. The chicken lacrimal gland, gland of Harder, caecal tonsil, and accessory spleens as sources of antibody-producing cells. Cell. Immunol., 2: 140152. Parry, S.H. and Aitken, I.D., 1977. Local immunity in the respiratory tract of the chicken. II. The secretory immune response to Newcastle disease virus and the role of IgA. Vet. Biol., 2: 143-165. Powell, J.R., Aitken, I.D. and Survashe, B.D., 1979. The response of the Harderian gland of the fowl to antigen given by the ocular route. II. Antibody production. Avian Path., 8: 363-373. Russell, P.H., 1989. Antibody forming cell assays of avian paramyxoviruses: immunization of chickens demonstrates two serotype groups. J. Gen. Virol., 70:2645-2651. Russell, P.H., 1993. Newcastle disease virus: virus replication in the Harderian gland stimulates lacrimal lgA; the yolk sac provides early lacrimal IgG. Vet. Immunol. Immunopathol., 37: 151-163. Russell, P.H., Griffiths, P.C.R. and Cannon, M.J., 1983. A microwell immunoperoxidase test for screening hybridomas and for diagnosing Newcastle disease virus and Sendai virus. J. Immunol. Methods, 61: 105-170. Russell, P.H., Mackay, D.J., Ozdemir, I. and Mackenzie, N.M., 1987, A rapid enzyme-semimicro assay for the enumeration of antibody-forming cells to viral and bacterial antigens in domestic animals. J. Immunol. Methods, 101: 229-233. Sundick, R.S., Albini, B. and Wick, G., 1973. Chicken Harder's gland: evidence for a relatively pure bursa-dependent lymphoid cell population. Cell Immunol., 7: 332-335. Survashe, B.D. and Aitken, I.D., 1978. Further observations on functional deletion of paraocular glands in the fowl (Gal/us domesticus). Res. Vet. Sci., 23:217-223. Wight, P.A.L., Burns, R,B., Rothwell, B. and Mackenzie, G.M., 1971. The Harderian glands of domestic fowl. I. Histology, with reference to genesis of plasma cells and Russell bodies. J. Anat. Lond., 110:307-315. Yoshida, 1., Oka, M., Shimisu, F., Yuasa, N. and Tsubahara, M., 1971. Neutralising antibody in the respiratory tract of chickens inoculated with Newcastle disease vaccines. Nat. Inst. Anim. Health Q., 11: 75-82. Zakay-Rones, Z., Levy, R. and Spira, G., 1971. Local immunologic response to immunization with inactivated Newcastle disease virus. J. lmmunol., 4:1180-1183.