Dermatomycosis caused by Paranannizziopsis australasiensis in five tuatara (Sphenodon punctatus) and a coastal bearded dragon (Pogona barbata) in a zoological collection in New Zealand.

CASE HISTORY: Health monitoring of tuatara (Sphenodon punctatus) at Auckland Zoo between 2001 and 2009 showed that 58/93 tuatara had been affected by dermatitis of unknown origin. From 2011 onwards, cases of suspected fungal dermatitis underwent extensive diagnostic investigations. CLINCAL FINDINGS: Six cases of dermatomycosis were attributed to Paranannizziopsis australasiensis, five in tuatara and one in a coastal bearded dragon (Pogona barbata). Cases presented typically as raised, yellow to brown encrustations on the skin. Severe cases progressed to necrotising ulcerative dermatitis, and in the bearded dragon to fatal systemic mycosis. Following topical and systemic treatments, lesions resolved in all five tuatara. LABORATORY FINDINGS: Histopathological examination of skin biopsy samples revealed dermatitis with intralesional septate branching hyphae. Fungal culture yielded isolates morphologically resembling Chrysosporium species, and isolates were submitted for molecular confirmation and sequencing of DNA. DIAGNOSIS: All six cases were confirmed as dermatitis due to infection with P. australasiensis, on the basis of fungal culture and DNA sequencing of isolates. CLINICAL RELEVANCE: These are the first reported cases of dermatomycosis associated with P. australasiensis infection in tuatara, and the first cases in which systemic therapeutic agents have been used in the treatment of such disease. Tuatara at the Auckland Zoo are now routinely examined every 3 months and tissue samples from any lesions sent for histopathology and fungal culture. Further work to elucidate the epidemiology and significance of P. australasiensis infections in reptiles in New Zealand is important for both welfare and conservation purposes.

The first reported outbreak of equine herpesvirus myeloencephalopathy in New Zealand.

CASE HISTORY AND CLINICAL FINDINGS: On 9 January 2014 (Day 0) a mare from a stud farm in the Waikato region presented with urinary incontinence without pyrexia. Over the following 33 days 15 mares were clinically affected with neurological signs. All but one mare had a foal at foot. The most commonly observed clinical signs were hind limb paresis and ataxia. In some cases recumbency occurred very early in the course of disease and seven mares were subject to euthanasia for humane reasons. LABORATORY FINDINGS: Equid herpesvirus (EHV) type 1 was detected using PCR in various tissues collected post mortem from two mares with neurological signs. DNA sequencing data from the DNA polymerase gene of the virus showed a nucleotide transition at position 2254, a mutation encoding amino acid D752 that is highly associated with the neuropathogenic genotype of EHV-1. In total 12/15 mares were confirmed positive for EHV-1 on PCR. Results from a virus neutralisation test and ELISA on paired serum samples, and PCR on whole blood and nasal swabs, indicated that of four paddocks in a high-risk area where a cluster of cases had occurred, 20/21 (95%) horses were likely to have been exposed or were confirmed infected with EHV-1. Subsequent to the outbreak two mares aborted, one at 9 months and one at 10 months of gestation. The cause of abortion was confirmed as EHV-1 with the same genotype as that involved in the outbreak. DIAGNOSIS: Equine herpesvirus myeloencephalopathy. CLINICAL RELEVANCE: The outbreak described shows the considerable impact that can occur in outbreaks of equine herpesvirus myeloencephalopathy in New Zealand. Early biosecurity controls not only reduced the effect on the farm but mitigated the potential for the virus to spread to other horse enterprises.

Development of real-time PCR assays for detection of megalocytiviruses in imported ornamental fish.

Megalocytiviruses have been associated globally with severe systemic disease and economic loss in farmed food fish and ornamental fish. The viruses have been spread internationally by translocation of live fish. In New Zealand, megalocytiviruses are regarded as exotic. A potential pathway for introduction has been identified, namely imported ornamental fish. In the present study, real-time PCR assays were developed for detection of megalocytiviruses using a conserved major capsid protein gene. A SYBR green assay was developed to target all known megalocytiviruses. A second real-time PCR assay using a molecular beacon was developed to specifically target gourami, Trichogaster trichopterus, iridovirus, a species of iridovirus previously linked to ornamental fish imports in Australia. The analytical sensitivity for the SYBR green and molecular beacon assays were 10 and 100 fg, respectively. The analytical specificity of the real-time PCR assays determined using genomic DNA templates from three target viruses, 12 non-target viruses and 25 aquatic bacterial species were 100%. The intra-run and inter-run coefficients of variation of both assays were <5%. The real-time PCR assays developed in this study provide rapid, sensitive, and specific detection of megalocytiviruses and gourami iridovirus.

Outbreaks of pleuritis and peritonitis in calves associated with Pasteurella multocida capsular type B strain.

CASE HISTORY: Three dairy calf-rearing properties experienced high mortality in calves during 2008 and 2009. Affected calves were aged 13-18 weeks (Farm I), 6 months (Farm II), and 2-11 weeks (Farm III), and the mortality rate was 22/175 (13%), 5/80 (6%), and 60/900 (7%), respectively. CLINICAL AND LABORATORY FINDINGS: Affected calves rapidly became moribund, were in respiratory distress, and had a fever (40-41°C). Post-mortem examination of nine calves revealed fibrinopurulent pleuritis, pericarditis, and peritonitis. This was confirmed histopathologically on tissues from three calves, one from each farm; aggregates of small Gram-negative coccobacilli were evident on Gram stain. Pasteurella multocida was cultured from tissues from affected calves on the three farms, and PCR of DNA extracted from tissue samples amplified cap-sular type B-specific DNA. Multi-locus sequence typing (MLST) demonstrated that all capsular type B isolates belonged to the same sequence type (ST), ST62, but did not belong to serotype B:2, the only B serotype classified as causing haemorrhagic septicaemia by the Office International des Epizooties (OIE). DIAGNOSIS: Pleuritis and peritonitis due to infection with P. multocida capsular type B strain. CLINICAL RELEVANCE: Haemorrhagic septicaemia was excluded as a cause of disease from the three farms, however P. multocida was the primary agent in the affected calves. It is possible the agent has been present in New Zealand for some time but not reported, as there had been no transfer of animals between affected farms. Emergence of the syndrome could potentially be a result of factors other than just the presence of the organism, such as changing management. The syndrome described may be of increasing importance in the future.

Epitope location of 13 anti-gag HIV-1 monoclonal antibodies using oligopeptides and their cross reactivity with HIV-2.

A series of 15-mer oligopeptides which overlapped by five amino acids (AA) across the p24 of HIV-1SF2 and a similar series across the p18 of HIV-1SF2 were used to identify the locations of 13 anti-gag monoclonal antibodies (MAbs). Three anti-p24 MAbs recognized sequences within the first 50 AA of the amino-terminal. Another anti-p24 recognized a conformational epitope in the centre of the protein and this MAb cross-reacted with two HIV-2 isolates suggesting conservation of this epitope between HIV-1 and HIV-2. One anti-p24 MAb recognized a linear sequence in the carboxy-terminal 100 AA and one p24 antibody was assumed to recognize a truly conformational epitope as it did not react with any of the linear peptides. Four anti-p18 MAbs were located at the carboxy-terminus of p18 with another MAb mapping slightly inwards from the carboxy-terminus and one anti-p18 MAb failed to bind to the p18 peptides. The carboxy-terminal distribution of the p18 MAbs indicated a highly immunogenic nature for this region in mice. None of the anti-p18 MAbs showed cross-reactivity with HIV-2 isolates, confirming the greater sequence variability of p18 over p24.

The cloning and expression in Escherichia coli of sequences coding for p24, the core protein of human immunodeficiency virus, and the use of the recombinant protein in characterizing a panel of monoclonal antibodies against the viral p24 protein.

The sequences encoding the p24 core protein of human immunodeficiency virus type 1 were identified in a cDNA library made from infected CEM cells. The nucleotide sequence of the DNA coding for p24 was shown to be very similar but not identical to the sequences of lymphadenopathy virus and human T-cell leukaemia virus type IIIb. These sequences were expressed in Escherichia coli at the amino terminus of beta-galactosidase and used to screen a panel of monoclonal antibodies raised against virus-expressed p24. Regions containing the epitopes of five of the monoclonal antibodies were located using a series of amino- and carboxy-terminal deletion mutants of the recombinant p24 protein.

The expression in Escherichia coli of sequences coding for the p18 protein of human immunodeficiency virus and the use of the recombinant protein in characterizing a panel of monoclonal antibodies against the viral p18 protein.

The sequences coding for the p18 protein of CBL-1, a British human immunodeficiency virus (HIV) type 1 isolate, were expressed in Escherichia coli as beta-galactosidase fusion proteins. The recombinant proteins were used to screen a panel of five monoclonal antibodies (MAbs) raised against p18 expressed in CBL-1-infected cells. The regions containing the epitopes for four of the MAbs were located using carboxy deletion mutants and synthetic peptides. The epitope of one of the MAbs (1D9) was reconstructed as part of an unfused, E. coli-expressed p18 protein using the polymerase chain reaction technique. Four different HIV strains and one lymphadenopathy virus type 2 strain were analysed by fluorescence-activated cell sorting of live infected cells using the p18-reactive MAbs.

Detection and subtyping of HIV-1 isolates with a panel of characterized monoclonal antibodies to HIV p24gag.

A panel of monoclonal antibodies (Mabs) was used to analyze the number and localization of B-cell epitopes on human immunodeficiency virus (HIV) p24gag and the variability of these epitopes in sequential HIV isolates and in isolates from different geographical origin. The specificity of these Mabs was demonstrated by immunoblotting and radioimmunoprecipitation assays. Cross-inhibition experiments indicated the presence of at least five different epitopes on p24. Analysis with p24 recombinant products revealed that three of the Mabs to p24 were directed to epitopes localized on the C-terminal part. Four other Mabs were directed to epitopes localized on the N-terminal half of the protein. Anti-p24 Mabs were used to develop HIV p24 antigen-capture assays. Application of these assays in HIV isolation resulted in more efficient recovery of HIV. Serotyping of HIV-1 isolates with five anti-p24 Mabs demonstrated that 55/65 isolates recovered from Dutch and Belgian individuals, but only 4/9 HIV-1 African isolates, were recognized by all five Mabs. Five of nine Central African HIV-1 isolates were not reactive with at least one of these Mabs. The variability of p24 appeared to be predominantly localized on the N-terminal part of the protein. Lack of expression of antigenic determinants on p24 was shown to be independent of culture conditions. Moreover, an infectious molecular clone was shown to have the same serotype as the corresponding HIV isolate. The serotype of sequential isolates obtained from 17 individuals over a 1 1/2- to 2 1/2-year period did not change, suggesting a limited in vivo p24 variation over time.

Analysis of human papillomavirus sequences in cell lines recently derived from cervical cancers.

The DNA and RNA from four cell lines recently derived from cervical carcinomas (HX151c, HX155c, HX156c, and HX160c) were analyzed for the presence of human Papillomavirus DNA. Each contained HPV-16 DNA in a multicopy integrated form of varying complexity. Each also expressed RNA transcripts of similar sizes to CaSki cell transcripts. The splice acceptor position within the E6 coding region of the cell line HX156c was identical to CaSki, SiHa, and HeLa cells as well as a cell line derived from mouse fibroblasts transformed with HPV-16 sequences in a retrovirus vector. Immunoprecipitation with an anti-HPV-16 E6 polyclonal antiserum demonstrated that HX160c cells contain the E6 polypeptide derived from unspliced transcripts.

A comparison of the induced polypeptides and RNAs of three orbiviruses isolated from ticks (Ixodes uriae) collected in seabird colonies on the Isle of May, Scotland.

Three orbiviruses of the Kemerovo serogroup, isolated from ticks from two sites on the Isle of May in Scotland, were plaque purified and the proteins and RNA induced in Vero cells compared. Two viruses (Mill Door/79 and Mill Door/81) from the same site differed in the migration of at least 7 segments of dsRNA. The third virus (North Clett/81) was clearly distinguished from Mill Door/79 and Mill Door/81 viruses in the migration of 6 dsRNA segments. Analysis of virus-induced polypeptides demonstrated minor molecular weight differences, but partial proteolysis failed to show significant variations in the proteins. Precipitation of radiolabelled virus-induced proteins by hyperimmune ascitic fluids did not distinguish between the isolates.

Proteins expressed by Mill Door/79 virus, a Kemerovo serogroup orbivirus transmitted by the ticks Ixodes uriae.

Mill Door/79 virus, an orbivirus of the Kemerovo serogroup, Great Island Complex, was shown to induce in infected cells 10 segments of double-stranded RNA with a total molecular weight of 11.46 X 10(6) daltons. Using methyl mercuric hydroxide as a denaturing agent the double-stranded RNA was translated in a cell-free system producing 11 polypeptides, 10 of which co-migrated with those produced in Mill Door/79 virus infected Vero cells. The segments were separated and individually translated in a cell-free system allowing their coding assignments to be made. These assignments were confirmed by partial proteolysis.

The isolation of Kemerovo group orbiviruses and Uukuniemi group viruses of the family bunyaviridae from Ixodes uriae ticks from the Isle of May, Scotland.

Viruses isolated from ticks (Ixodes uriae) from a seabird colony on the Isle of May, Scotland, were shown by complement fixation tests to be related to the Uukuniemi and Kemerovo serogroups. Electron microscopic studies on the Uukuniemi viruses showed them to have a morphology characteristic of the bunyaviridae, and the Kemerovo group to be characteristic of orbiviruses. Separate isolates from the two serogroups were distinguished from each other by neutralization tests.

Replication and polypeptide synthesis of Mill Door/79, an orbivirus isolated from ticks from a seabird colony in Scotland.

The replication and polypeptide synthesis of orbivirus isolate Mill Door/79, a member of the Kemerovo serogroup, were studied. In Vero cells cell-associated virus exceeded cell-free virus by about 2 log10 PFU/ml. Attempts to purify the virus resulted in the demonstration of five polypeptides. Thirteen virus-induced polypeptides and 10 segments of double-stranded RNA were identified in infected cells. Partial proteolysis demonstrated homology between some polypeptides.