Immunohistochemical demonstration of Ranavirus Antigen in the tissues of infected frogs (Rana temporaria) with systemic haemorrhagic or cutaneous ulcerative disease.

Ranavirus disease emerged as a cause of annually recurring epidemic mortality of common frogs (Rana temporaria) in Britain in the late-1980s. Affected frogs present with a peracute disease characterized by systemic haemorrhage, or with a chronic disease characterized by skin ulceration, but no internal gross lesions. Common toads (Bufo bufo) have also been found with haemorrhagic ranavirus disease. In order to investigate possible differences in the pathogenesis of ranavirus infection for each main disease syndrome, we studied a range of tissues from both naturally and experimentally infected frogs using anti-ranavirus immunohistochemistry. Ranavirus was located in a variety of cells, including fibrocytes, epithelial cells, lymphocytes, hepatocytes and melano-macrophages, but fewer tissues were infected in frogs with the skin ulcerative syndrome than in frogs with systemic haemorrhagic disease. Specifically, and in contrast to frogs with haemorrhagic syndrome, there was no labelling for viral antigen in the splenic lymphocytes, pancreas or gastrointestinal epithelium in frogs with ulcerative syndrome. Intracytoplasmic virus inclusions were seen in the liver, kidney, pancreas and stomach of frogs with systemic haemorrhagic disease, but not in frogs with the ulcerative syndrome. Immunohistochemical labelling of selected tissues from an affected toad demonstrated ranavirus antigen in the skin and viscera. This technique demonstrates that, in comparison to ranavirus ulcerative syndrome, the haemorrhagic form of ranavirus disease is associated with virus infection of a wider range of internal organs and identifies the infection of certain tissues, such as the spleen, which might be important in the pathogenesis of the haemorrhagic disease.

Eye-drop DNA can induce IgA in the tears and bile of chickens.

DNA vaccines protect chickens against lethal virus infections but whether they induce local antibody which is associated with preventing viral entry, is unknown. We were able to show how avian DNA vaccines can induce local IgA. 65 microg plasmid DNA encoding the reporter protein beta-galactosidase induced antigen-specific IgA in the tears of 6/10 birds, IgA in the bile of 4/10 birds and IgG in the serum of 2/10 birds. Giving the DNA by the intramuscular route, as is more usual, induced lacrimal IgA in 2/8 birds, biliary IgA in no birds and serum IgG in 4/8 birds. Eye-drop DNA therefore favoured local IgA whereas intramuscular DNA favoured serum IgG. Further to this preliminary work eye-drop DNA should be improved by adjuvants and cytokines as a way of inducing protective IgA at the mucosal surfaces of the alimentary and respiratory tracts.

Naked DNA when co-administered intranasally with heat-labile enterotoxin of Escherichia coli primes effectively for systemic B- and T-cell responses to the encoded antigen.

In this study a novel prime-boost immunisation strategy was evaluated. Priming of BALB/c mice by the intranasal route with plasmid DNA encoding beta-galactosidase (LacZ) with or without heat-labile enterotoxin (LT) of Escherichia coli as a mucosal adjuvant, resulted in the induction of weak serum antibody and proliferative T-cell responses. However, following an intraperitoneal booster injection with the beta-galactosidase protein (beta-gal), strong antibody and proliferative T-cell responses were induced in all the mice. These responses were highest in mice primed intranasally with a mixture of LacZ+LT as compared to those mice primed with DNA (LacZ) or protein (beta-gal) alone. Moreover, LacZ+LT primed mice produced high avidity antibodies and the subclasses of serum antibodies were IgG1 and IgG2a, suggesting a mixed Th1/Th2-type response. Priming of mice with either protein (beta-gal) or DNA (LacZ) alone, produced predominantly IgG1 antibodies, suggesting a Th2-type response. These findings suggest that the use of a heterologous DNA-prime, protein-boost immunisation scheme combining different routes of administration, might be an advantageous strategy for the induction of accelerated immune responses.

Antibody responses of goldfish (Carassius auratus L.) to DNA-immunisation at different temperatures and feeding levels.

DNA vaccines which work in winter are needed for fish. At 18 weeks after intramuscular injection of a plasmid containing lacZ goldfish at 15 degrees C had five-fold more antibody to betagalactosidase than those at 25 degrees C or those at

The safety and longevity of DNA vaccines for fish.

A plasmid that contained the cytomegalovirus (CMV)-promoter-driven lacZ reporter gene (pCMV-lacZ) remained in the epaxial muscle of five of eight goldfish as covalently closed circles, the most functional form of plasmid, for at least 70 days at 22 degrees. It was not present in the gills or elsewhere by polymerase chain reaction and was not integrated. Its expressed protein, Escherichia coli beta-galactosidase (beta-gal), which was in the injected myofibres, was detected in all the fish at 4-21 days and in about half the fish from 28 days until the end of the experiment at 70 days. The numbers of cells that secreted antibody to beta-gal in the kidney peaked at 14 days. Serum antibody and proliferating kidney cells to beta-gal were in all fish from 14 days with a plateau of the responses from 21 days onwards. The plasmid did not induce autoimmune-like antibodies to itself or to single- or double-stranded salmon testis DNA. Plasmids can therefore induce long-term foreign protein expression whilst inducing humoral and cell-mediated immunity without autoimmunity or integration in goldfish.

Mammalian granulocyte-macrophage colony-stimulating factor and some CpG motifs have an effect on the immunogenicity of DNA and subunit vaccines in fish.

A eukaryotic plasmid DNA carrying the AACGTT CpG motif in its ampR gene is a 'danger' signal for mice and caused an increase in the specific antibody titres of fish and mice after immunization with beta-galactosidase (beta-gal). A second pUC-based plasmid, which is inactive in mice and contains the GACGTC CpG motif in its cytomegalovirus (CMV) promoter, had no effect on antibody responses to beta-gal in either fish or mice. A synthetic oligonucleotide, which contains the GACGTT motif, potentiated antibody responses to co-administered beta-gal protein in mice, but not in fish. This is early evidence that lower and higher vertebrates recognize different unmethylated CpG motifs as 'danger' signals. In addition, plasmid DNA expressing mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) had a marked effect on cytotoxic T-cell-like activity in fish by reducing the average number of myofibres that expressed beta-gal, 28 days after co-injection with plasmid DNA expressing beta-gal. Although the mechanism by which the mouse GM-CSF exerted its biological effects in fish is unknown, this finding might have important implications for fish vaccination, particularly when cytotoxic T cells may play a critical role.

DNA is as effective as protein at inducing antibody in fish.

Antiviral vaccines are needed for fish. 50 microg plasmid DNA in saline by the intramuscular route and 10 microg beta-gal protein in a commercial oil adjuvant by the peritoneal route induced serum antibody of the same titre and avidity in goldfish. The DNA expressed beta-gal under control of the immediate early promoter/enhancer gene of human cytomegalovirus. Commercial bacterin vaccines are administered to fish by the intraperitoneal route with oil and this route for DNA induced only 2-fold less antibody than DNA by the intramuscular route. Bacterin vaccines and antiviral plasmid DNA could therefore be co-injected into the peritoneum of fish in an oil adjuvant as a single dose.

Tropism of subgroup J avian leukosis virus as detected by in situ hybridization.

The HPRS-103 strain of avian retrovirus is the prototype of subgroup J avian leukosis virus (ALV-J) and causes myeloid leukosis in meat-type chickens. Using immunohistochemical detection of the viral groupspecific antigen (Gag) we have previously demonstrated that the induction of myeloid leukosis by ALV-J is associated with viral tropism for myelomonocytic cells. In this paper we describe an in situ hybridization (ISH) technique using digoxigenin (DIG)-labelled probes for detecting RNA transcripts in tissues from chickens infected with avian leukosis viruses (ALV) of subgroups J (HPRS-103 strain) and A (RAV-1 strain). Virus-specific RNA was detected mainly in the heart, kidney, proventriculus and adrenal in locations similar to those of the Gag protein. Viral gene expression could not be detected in the bone marrow or tumour tissues using this test. Higher levels of viral gene expression in the bursa of Fabricius infected with RAV-1, but not with HPRS-103, might help explain the inability of the latter virus to induce lymphoid leukosis.

The potential transmission of infectious agents by semen packaging during storage for artificial insemination.

Plastic straws, of a type widely used for semen cryopreservation, sealed using three different methods, (PVA powder, plastic spheres and plasticine modelling clay) were tested for leakage of low molecular weight dye (methylene blue), bacteria (Escherichia coli) and virus (Newcastle disease virus). Leakage was found to be dependent on the method used to fill the straws. Straws filled using a traditional 'dip and wipe' method and sealed with PVA powder demonstrated a significant degree of methylene blue leakage (0.0269% of the total straw contents) probably associated with contamination of the powder sealing plug. Straws filled using an aseptic filling technique showed no detectable leakage of any agent with any of the sealing methods. This study highlights the need to establish good-practice guidelines for the packaging of semen collected for freezing and future AI from non-domestic livestock where disease-free status cannot be guaranteed and unsophisticated technology is used.

Some chickens which are viraemic with subgroup J avian leukosis virus have antibody-forming cells but no circulating antibody.

New immunoperoxidase-based assays for splenic IgG -antibody-forming cells (AFC) and serum IgG-antibody were used to look for antibody to HPRS-103 in meat-type birds. Meat type birds are known to be less likely to produce neutralising antibody and to be less likely to clear virus from their serum than layer-type birds after infection at hatch. In this work all 12 of the brown leghorn layer-type birds and 5/12 of the line 21 meat-type birds had produced AFC and serum antibody and had cleared serum virus at 63, 82 and 110 days of age. None of the seven viraemic line 21 birds contained serum antibody but three produced AFC. The four viraemic line 21 birds which lacked AFC occurred later in the experiment and had a higher level of virus than the three viraemic line 21 birds which possessed AFC. This suggests that most line 21 birds do not control HPRS-103 and eventually become anergic.

Antigenic diversity and similarities detected in avian paramyxovirus type 1 (Newcastle disease virus) isolates using monoclonal antibodies.

Newcastle disease (ND) virus (APMV-1) isolates submitted to the International Reference Laboratory for ND were characterised antigenically by their ability to cause binding of mouse monoclonal antibodies (mAbs) to cell cultures infected with the isolate. Since the availability of the mAbs 1526 viruses have been examined using a panel of nine mAbs and 818 with an extended panel of 26 mAbs. Using the nine mAb panel a total of 14 different patterns was seen and viruses grouped by the same pattern showed relationships with each other which were either biological, temporal or geographical or more than one of these. There was a marked tendency of viruses placed in the same group to show similar virulence for chickens. Extension of the panel to 26 mAbs produced 39 distinct patterns, although some of these were seen with only a single virus. Again, viruses inducing similar binding patterns shared similar properties and some binding patterns were specific for viruses causing discrete epizootics. Cluster analysis of the mAb binding patterns did not produce concise, discrete groupings, but did emphasise some relationships between virus properties and antigenicity. Examples of the usefulness of this approach were the ability to link two important outbreaks to the contamination of stored food by infected feral pigeons, and the demonstration of two separate viruses responsible for outbreaks in countries of the European Union during 1991 to 1994 thus preventing erroneous epizootiological tracing.

Tissue tropism of the HPRS-103 strain of J subgroup avian leukosis virus and of a derivative acutely transforming virus.

The tissue tropism was studied for the HPRS-103 strain of avian leukosis virus, which belongs to a new envelope subgroup, designated J. Studies were conducted in blood monocyte and bone marrow cell cultures and in chickens from six lines that had been shown previously to differ in susceptibility to induction by this virus of myeloid leukosis and other tumors. Using an immunohistochemical technique to detect expression of viral group-specific antigen (Gag) in various tissues, we detected no major differences among the six lines of chickens at 3 and 7 weeks of age following infection as embryos. Thus, Gag expression did not correlate with differences in tumor susceptibility. Of the tissues examined, greatest Gag expression was observed in cells specific to the adrenal gland, heart, kidney, proventriculus and especially in smooth muscle cells and connective tissue. After infection of 1-day-old chicks, greater tissue expression was observed in line 21 chicks, which mostly developed a tolerant viremic infection, than in Brown Leghorn chicks, which developed virus-neutralizing antibodies. An acutely transforming virus, strain 966, derived from HPRS-103-induced myeloid leukosis, showed a tropism similar to HPRS-103. The HPRS-103 strain showed a lower propensity to replicate in the medullary region of the lymphoid follicles of the bursa of Fabricius than did the RAV-1 strain of subgroup A avian leukosis virus. This low bursal tropism may be a factor in why HPRS-103 does not induce lymphoid leukosis. The HPRS-103 and 966 virus replicated in blood monocyte cultures from chickens from the six lines, indicating a tropism for the myelomonocytic cell lineage. In comparison, as previously reported, RAV-1 did not replicate well in the monocyte cultures, whereas RAV-2, a subgroup B avian leukosis virus, did replicate. The tropism of HPRS-103 for monocytes may relate to its ability to cause myeloid leukosis. Monocyte and bone marrow cell cultures from the six lines ranked similarly in differences in susceptibility to transformation by 966 virus and showed evidence that their relative susceptibilities correlated with susceptibility of chickens from these lines to induction of myeloid leukosis by HPRS-103, suggesting common tissue-specific viral and host factors involved in oncogenesis by these two viruses.

The effects of cyclosporin A and cyclophosphamide on the populations of B and T cells and virus in the Harderian gland of chickens vaccinated with the Hitchner B1 strain of Newcastle disease virus.

The cellular response to conjunctival vaccination with the Hitchner B1 strain of Newcastle disease virus was studied in the Harderian gland (HG) by immunohistochemistry. Bu-1+ cells and all subpopulations of T cells, (CD3+, CD4+, CD8+, TCR gamma delta, TCR alpha beta 1, and TCR alpha beta 2) were in the interstitial tissue between the ducts and the acini. Plasma cells with cytoplasmic IgM were more dispersed than the other cells and outlined the acini. Bu-1+ cells and all subpopulations of T cells increased at least three-fold after vaccination when compared to uninfected birds on the basis of the average cell counts in sections taken at 3, 5, 7, 10, 14, and 20 days after vaccination. The most marked increase was in the CD8+ cells which increased six-fold. Virus replicated for 10 days in cyclophosphamide (Cy) treated birds and for 7 days in cyclosporin A (CsA) treated birds compared with 5 days in untreated birds. Cy treatment prevented an antibody response to NDV and reduced Bu-1+ and IgM cells in the HG by 20-fold. Cy treatment resulted in a doubling of the number of T cells in the HG but these T cells may have been transiently disabled because it also caused a poor response of the lymphocytes in whole blood to the T cell mitogen concanavalin A (ConA). CsA reduced the T cell numbers in the HG and whole-blood responses to ConA by about 4-fold but T cell numbers rebounded to normal resting values after vaccination with NDV. The clearance time was prolonged either by T cells being less numerous than normal after CsA or being disabled after Cy. T cells, but not B cells, may therefore be essential for virus clearance. CD8+ cells expanded more than CD4+ cells after the vaccination of untreated and CsA-treated birds indicating that CD8+ cells may be key players in vaccinal immunity to NDV.

The Hitchner B1 strain of Newcastle disease virus induces high levels of IgA, IgG and IgM in newly hatched chicks.

Reaseheath line C chicks produced IgA, IgG and IgM in their serum, tears, spleen and Harderian gland (HG) as a consequence of oculotopical vaccination with the Hitchner B1 strain of Newcastle disease virus. The IgM response was seen first, at 5 days after vaccination, and antiviral IgM levels in the tears and serum were negatively correlated to the level of virus in HG over days 4 to 10 postinfection. The frequency of virus-specific IgM-antibody forming cells in the HG of 10-day-old birds that had been vaccinated at 1 day old could reach 1/30th of the total lymphoid cells prepared from the HG. Young chicks made an irregular or low response to inactivated virus by the intravenous route whereas 40-day-old birds made a high serum response to both live and inactivated virus. This emphasizes how day-old chicks respond well to live virus vaccination and that their IgM response is likely to have a role in the clearance of virus.

Newcastle disease virus vaccines: differences between Line C and Line 15I chickens with respect to virus replication and IgA responses in the gut and Harderian gland.

The Ulster 2C and Hitchner B1 strains of Newcastle disease virus were inoculated into inbred White Leghorn birds of the Reaseheath-C and 15I lines by the oculonasal route. Both viruses replicated in the Harderian gland (HG) and induced virus-specific IgA in the tears and bile. Ulster 2C but not Hitchner B1 replicated in the small intestine and induced virus-specific antibody forming cells (AFC) in the small intestine. Line 15I birds produced 120-fold more virus and 13-fold more IgA-AFC in the small intestine than Line C birds. Line C birds produced 20-fold more virus in the HG and at least three-fold higher titres of lacrimal IgA than Line 15I birds. The level of local virus replication in the HG or small intestine, which varied according to the line of bird, positively predicted the local antibody response in the same organ. When low doses of Ulster 2C were inoculated into Line C birds virus replication was low and irregular in birds older than 18 days whereas low doses of Hitchner B1 replicated in all ages of bird.

Newcastle disease virus: virus replication in the harderian gland stimulates lacrimal IgA; the yolk sac provides early lacrimal IgG.

An indirect immunoperoxidase monolayer assay was used to measure the location of virus and class-specific antibodies to Newcastle disease virus (NDV) of chickens. The intra-ocular vaccination of chicks with the Ulster and Hitchner B1 strains of NDV resulted in the highest titre of virus being recovered from the Harderian gland (HG) at 10(6) iu/0.1 g tissue without any accompanying virus in the faeces. Maternal IgG antibody which was detected in the lacrimal fluid at 1/50 of serum levels of IgG antibody could prevent this replication. Lacrimal IgA antibody to NDV was shown to be absolutely dependent on local virus replication for its production because it reached titres of 10(3) after local intra-ocular infection compared with < 10(1.5) after two intravenous inoculations of inactivated virus. Biliary IgA, by contrast, reached 10(3.5) after either immunisation schedule. Lacrimal IgG and IgM antibody to NDV occurred at 1-9% of the titres of serum antibody to NDV after immunisation with inactivated virus or the passive transfer of NDV-immune serum between chickens. This percentage increased to 13-33% of serum titres after intra-ocular infection with NDV as if the local replication of NDV in the HG stimulated lacrimal antibody of all classes. This is the first report of NDV replication in the HG and of virus-specific IgM in the lacrimal fluid.

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

The Hitchner B1 and Ulster strains of Newcastle disease virus (NDV) replicated to high titre in the Harderian gland (HG) 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, HG 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 Iscoms of NDV could stimulate the spleen, but not the Harderian gland to the same extent as a live virus.

The 36K polypeptide synthesized in Newcastle disease virus-infected cells possesses properties predicted for the hypothesized 'V' protein.

Newcastle disease virus (NDV) virions possess two proteins which react strongly with monoclonal antibody 688 following separation by high resolution two-dimensional (isoelectric focusing/SDS) PAGE and detection by Western blotting. One is the phosphorylated nucleocapsid-associated 53K [P (NAP)] protein, the other comigrates with the 36K protein detected by radiolabelling NDV-infected chick embryo fibroblasts. [35S]Cysteine/[3H]leucine dual-labelling experiments show that the 36K protein is very rich in cysteine compared to the P (NAP) protein. In the Beaudette C strain it comigrates on one-dimensional SDS-polyacrylamide gels with the matrix protein (M); however, it is resolved from the slower migrating M protein from the Ulster strain of NDV. The size, strain-specific isoelectric point, high cysteine content and antigenic relatedness to the P (NAP) protein suggest that the 36K protein is the 'V' protein of NDV, the counterpart of which is found in other Paramyxoviridae.

Monoclonal antibodies to avian paramyxovirus type 2.

Three mouse monoclonal antibodies, raised against PMV2/chicken/California/ Yucaipa/56, which caused haemagglutination inhibition of the homologous virus were tested against 53 isolates which had been placed in the PMV2 serogroup using polyclonal antisera. The results allowed the viruses to be placed in four distinguishable groups consisting of 34, 4, 2 and 13 isolates.

Antibody-forming cell assays of avian paramyxoviruses: immunization of chickens demonstrates two serotype groups.

Chickens were immunized with eight different serotypes of avian paramyxovirus (PMV). Ninety percent of splenic IgG antibody-forming cells (AFC) were serotype-specific with respect to the glycoprotein and nucleoprotein-polymerase antigens in an indirect immunoperoxidase binding system. The IgG AFCs which cross-reacted between serotypes divided the serotypes into two mutually exclusive super serogroups composed of PMV-1, -3, -4, -7 and -9, and PMV-2, -6 and -8. PMV-5 was not tested.