Gill-associated virus of Penaeus monodon prawns: an invertebrate virus with ORF1a and ORF1b genes related to arteri- and coronaviruses.

A 20089 nucleotide (nt) sequence was determined for the 5' end of the (+)-ssRNA genome of gill-associated virus (GAV), a yellow head-like virus infecting Penaeus monodon prawns. Clones were generated from a approximately 22 kb dsRNA purified from lymphoid organ total RNA of GAV-infected prawns. The region contains a single gene comprising two long overlapping open reading frames, ORF1a and ORF1b, of 4060 and 2646 amino acids, respectively. The ORFs are structurally related to the ORF1a and ORF1ab polyproteins of coronaviruses and arteriviruses. The 99 nt overlap between ORF1a and ORF1b contains a putative AAAUUUU 'slippery' sequence associated with -1 ribosomal frameshifting. A 131 nt stem-loop with the potential to form a complex pseudoknot resides 3 nt downstream of this sequence. Although different to the G/UUUAAAC frameshift sites and 'H-type' pseudoknots of nidoviruses, in vitro transcription/translation analysis demonstrated that the GAV element also facilitates read-through of the ORF1a/1b junction. As in coronaviruses, GAV ORF1a encodes a 3C-like cysteine protease domain located between two hydrophobic regions. However, its sequence suggests some structural relationship to the chymotrypsin-like serine proteases of arteriviruses. ORF1b encodes homologues of the 'SDD' polymerase, which among (+)-RNA viruses is unique to nidoviruses, as well as metal-ion-binding and helicase domains. The presence of a dsRNA replicative intermediate and ORF1a and ORF1ab polyproteins translated by a-1 frameshift suggests that GAV represents the first invertebrate member of the Order NIDOVIRALES:

Detection of Australian gill-associated virus (GAV) and lymphoid organ virus (LOV) of Penaeus monodon by RT-nested PCR.

A highly sensitive test based on reverse transcription followed by nested polymerase chain reaction (RT-nPCR) was developed to detect the Australian yellow-head-like viruses, gill-associated virus (GAV) and lymphoid organ virus (LOV) of Penaeus monodon. The RT-nPCR detected viral RNA in as little as 10 fg lymphoid organ total RNA isolated from GAV-infected P. monodon. Amplification of serial dilutions of a GAV cDNA clone showed that the nested PCR was sufficiently sensitive to detect a single genome equivalent using a DNA template. The specificity and sensitivity of the RT-nPCR was also demonstrated using experimentally infected P. (Marsupenaeus) japonicus, where GAV sequences could be amplified from lymphoid organ and haemocyte RNA as early as 6 h post infection (p.i.), and from gills by 24 h p.i. In contrast, transmission electron microscopy (TEM) identified nucleocapsids and virions in lymphoid organ cells and haemocytes from Days 3 and 6 p.i., respectively, while there was no evidence of infection in gill cells at any time. The practical application of the RT-nPCR was demonstrated by screening healthy wild-caught P. monodon broodstock. The high prevalence (>98%) of broodstock that were positive by RT-nPCR suggests that LOV is endemic in northern Queensland. In addition, results with lymphoid organ, gill and haemocyte RNA suggest that small gill biopsies may be best suited to the non-sacrificial testing of valuable broodstock. The speed and sensitivity of the RT-nPCR make it a useful adjunct to TEM for diagnosing LOV/GAV infection of P. monodon, with the additional benefit that screening of gill biopsies may facilitate selection of LOV-free broodstock.

Yellow head virus from Thailand and gill-associated virus from Australia are closely related but distinct prawn viruses.

Corresponding genomic regions of isolates of yellow head virus (YHV) from Thailand and gill-associated virus (GAV) from Australia were compared by RT-PCR and sequence analysis. PCR primers designed from sequences in the GAV ORF1b polyprotein gene amplified the corresponding 577 nucleotide region of the YHV genome. Comparison of the amplified region indicated 85.1% nucleotide and 95.8% amino acid sequence identity. YHV PCR primers designed to amplify a 135 nucleotide product previously described as a YHV diagnostic probe failed to amplify the corresponding product from GAV RNA. However, the cognate GAV sequence for this and another recently reported YHV sequence were located in an upstream region of the ORF1b gene. A comparison of these sequences indicated identities of 83.0 and 80.9% at the nucleotide level and 86.7 and 86.5% at the amino acid level, respectively. The data indicate that GAV and YHV are closely related but distinct viruses for which differential diagnostic probes can be applied.

Anaplasma marginale: effect of the treatment of cattle with an interferon gamma-neutralizing monoclonal antibody or the nitric oxide synthetase inhibitor aminoguanidine on the course of infection.

Cattle undergoing initial infection with the rickettsia Anaplasma marginale were treated with either a monoclonal antibody (MoAb) with neutralizing activity for bovine interferon gamma (IFN-gamma) or aminoguanidine (AG), a specific inhibitor of the inducible form of nitric oxide synthetase (iNOS). Plasma levels of MoAb and AG were measured over the time of administration. The course of A. marginale infection was not altered in the MoAb-treated cattle relative to untreated controls. In cattle treated with AG however, A. marginale infection was significantly ameliorated, as judged by lower parasite levels and decreased anaemia in these cattle relative to the controls. The implications of these findings in relation to the basis for immunity against this economically important haemoparasite are discussed.

Increased resistance to Anaplasma marginale infection in cattle chronically infected with Theileria buffeli (syn. T. orientalis).

Calves chronically infected with the benign haemoprotozoan parasite Theileria buffeli (syn. T. orientalis) and T. buffeli-free calves were experimentally infected with virulent Anaplasma marginale. The daily mean maximum parasitaemia in the T. buffeli-carrier calves was lower and delayed relative to that of the Theileria-free calves. Anaemia was less marked in the Theileria infected calves, although this difference was not statistically significant. The susceptibility of Theileria-carrier and Theileria-free older cattle to virulent A. marginale infection was also investigated. The mean maximum parasitaemia observed in the Theileria-infected cattle was significantly lower than that of the Theileria-free cattle and the time to maximum parasitaemia was increased significantly in the Theileria-infected relative to the Theileria-free cattle. Of the Theileria-carrier cattle, 33% exhibited maximum parasitaemias of less than 0.1% infected erythrocytes and no clinical anaemia as a result of A. marginale infection. In contrast, the lowest maximum parasitaemia observed in the Theileria-free cattle was 7%. The percentage of cattle requiring treatment to prevent mortality due to anaemia was 50% and 91% in the Theileria-infected and Theileria-free cattle respectively. For the duration of increasing A. marginale parasitaemia, the level of Theileria in carrier cattle was significantly depressed or undetectable. Following the resolution of peak A. marginale parasitaemia, the level of Theileria parasites increased rapidly to become significantly higher than that prior to infection and then decreased gradually to a level similar to that prior to infection. The mechanism of the increased resistance to A. marginale infection conferred by T. buffeli-carrier state is unknown, but is likely to involve non-specific cell-mediated immunity, as no serological cross-reactivity exists between these two highly divergent parasite species. The susceptibility of relatively mature cattle to clinical anaplasmosis under field conditions is likely to be significantly affected by the widespread distribution and common occurrence of T. buffeli throughout the range of A. marginale in Australia, Africa and southeast Asia.

Vaccination against Babesia bovis: T cells from protected and unprotected animals show different cytokine profiles.

Vaccination of cattle against the haemoprotozoan parasite, Babesia bovis, with the recombinant antigen 11C5 resulted in 9 of 15 cattle being protected against challenge infection. The cellular immune responses of protected and unprotected cattle were compared in order to identify differences in response. No differences were observed in the pattern of change in various blood leukocyte populations throughout challenge infection. FACScan analysis revealed an increase in the proportion of cells bearing the CD2 marker in both protected and unprotected cattle over the course of infection. There were no observable differences in the frequency of various cell-surface markers between the unprotected and protected cattle. During the period of patent parasitaemia, in vitro cultures of peripheral blood mononuclear cells (PBMC) from protected cattle produced significantly more TNF-alpha (P < 0.05) than cultures from unprotected cattle. TNF-alpha concentrations remained at pre-challenge levels until day 10, when levels in the unvaccinated control and vaccinated/unprotected animals dropped. By peak parasitaemia, TNF-alpha production in vitro was significantly greater (P < 0.05) in cultures of PBMCs from protected cattle. Interferon production showed an initial peak at day 5 in all cattle, followed by a decrease and a second peak at days 10-13 in protected cattle only, which coincided with resolution of the infection.

Anaplasma marginale: detection of carrier cattle by PCR-ELISA.

A highly sensitive and specific polymerase chain reaction (PCR) based assay for the detection of the minute levels of Anaplasma marginale present in the blood of long-term carrier cattle was developed. A simple lysis method was used to remove most of the haemoglobin from the blood to facilitate direct input of samples into the PCR reactions without prior purification of the DNA. PCR product was detected by enzyme-linked immunosorbent assay (ELISA) to simplify the processing of large numbers of samples. The sensitivity limit of the PCR-ELISA was 0.00015% parasitaemia (24 infected erythrocytes per microlitre of blood). No cross-reactivity of the assay was observed when A. marginale-negative blood infected with Babesia bovis or Theileria orientalis was tested. The PCR-ELISA was shown to be 92% efficient in the detection of long-term A. marginale carrier cattle. No false-positive results were obtained. These results compared favourably with 2 serological assays for detection of A. marginale carrier cattle (card agglutination test and ELISA) which were applied to the same experimental animals.

Peripheral blood lymphocyte proliferative responses in cattle infected with or vaccinated against Anaplasma marginale.

An assay was developed for measurement of the peripheral blood lymphocyte proliferative response (PBLPR) in cattle infected with or immunised against Anaplasma marginale. PBLPR was not evident in all cattle that had recovered from A. marginale infection. However, A. marginale-sensitised lymphocytes were detected in the spleens of all immune cattle tested in the absence of detectable PBLPR. During the course of initial infection, cattle exhibited detectable PBLPR for a period corresponding with and up to 2 weeks after patent parasitaemia, followed by a second, usually larger peak in PBLPR corresponding to the time of sub-clinical relapse of cattle. Analysis of the PBLPR of A. marginale chronically infected cattle demonstrated highly variable PBLPR between individuals and over time. A positive PBLPR was induced in cattle by vaccination using a crude A. marginale antigen preparation. The PBLPR of vaccinated cattle subsequently infected with A. marginale was markedly different from that of naive cattle, with reduced PBLPR being associated with the onset of parasitaemia. The antigen used in the PBLPR assay was inactivated by proteolysis. Proteolysis also abolished immunity that had been induced in cattle vaccinated using the antigen preparation. A marginale-sensitised PBL did not proliferate in response to antigen from the heterologous species A. centrale. A. centrale-sensitised PBL, however, responded to A. marginale antigen. Interferon-gamma (IFN-gamma) was detected in PBLPR-assay supernatants and was associated with a strong PBLPR.

Characterisation of a family of multi-copy genes encoding rhoptry protein homologues in Babesia bovis, Babesia ovis and Babesia canis.

A monoclonal antibody that had been raised against a protease-containing fraction of Babesia bovis, and shown to bind to a protein located in the rhoptries, was used to screen a B. bovis cDNA expression library. The sequence of the protein encoded by a positive clone was almost identical to the equivalent region of a previously described B. bovis 60-kDa rhoptry protein (Bv60). A tandem repeat of the gene encoding Bv60 was identified in all Australian isolates of B. bovis examined. Genes encoding homologous of Bv60 were cloned from Babesia ovis and Babesia canis. In B. ovis, 5 closely linked genes were identified. Four of these genes appeared to encode very similar proteins (Bo60.1-4). The protein (Bo60.5) encoded by the fifth B. ovis gene had 72% amino acid identity to Bo60.1-4 in the amino-terminal 306 amino acids, but no significant similarities in the carboxy-terminal region. In B. canis one gene (Bc60.2) was sequenced and a second closely linked gene was identified. A further member of the family, p58, has also been described previously from Babesia bigemina. Tandemly repeated genes subject to extensive gene conversion appear to be a feature of this family of babesial rhoptry protein homologous. No proteins significantly related to any members of the gene family were identified in a search of translated DNA and protein sequence databases. Thus the function of this family of proteins remains a matter for speculation.

Babesia bovis, Babesia bigemina, Babesia canis, Babesia microti and Babesia rodhaini: comparison of ribosomal RNA gene organization.

The three ribosomal DNA (rDNA) units have been cloned from an Australian isolate of Babesia bigemina. The organization of the units is very similar to that reported for a Mexican isolate of B. bigemina. In Babesia canis four rDNA units have been identified. Both Babesia rodhaini and Babesia microti contain two different rDNA units. A small number of different rDNA units appears to be a common feature of this group of Protozoa. Restriction enzyme analysis of the rDNA units form these species and B. bovis suggests that the genus Babesia as currently defined does indeed include two distinct groups of organisms namely, B. bovis, B. bigemina and B. canis and B. rodhaini and B. microti.

Anaplasma marginale: failure of sera from immune cattle to confer protection in passive-transfer experiments.

High levels of immunity to Anaplasma marginale were induced in cattle either by vaccination using sonically disrupted A. marginale-infected erythrocytes or by repeated infection with different strains of the rickettsia. In both instances, high levels of anti-A. marginale antibody were detected in the sera of the immune cattle by immunoblotting. Serum from one animal that had been made immune by repeated infection was transferred intravenously to A. marginale-susceptible calves (three non-splenectomised and two splenectomised) undergoing initial A. marginale infection at serum doses of 2-10 ml/kg. Neither the course nor the outcome of infection as indicated by the parasite levels attained or the level of anaemia induced was altered in the calves that received the immune serum relative to the course or outcome of infection in control calves (two non-splenectomised and two splenectomised) that received serum from an two splenectomised) that received serum from an A. marginale-naive donor animal. In a similar experiment, a pool of sera from four steers that had been vaccinated with sonically disrupted A. marginale initial bodies was transfused into two intact A. marginale-susceptible calves during the early stage of A. marginale infection at a dose of 10 ml/kg. No difference was observed in the course or outcome of infection in these calves relative to the course or outcome of infection in the two non-splenectomised calves that were transfused with non-immune serum.(ABSTRACT TRUNCATED AT 250 WORDS)