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A qualitative and quantitative assessment of the humoral antibody response of the sheep to orf virus infection.

The serological response of naturally and experimentally infected lambs to orf virus infection was analysed using an enzyme-linked immunosorbent assay (ELISA) together with the Western blotting technique. The combination of these two methods permitted a qualitative and quantitative assessment of the response, which revealed considerable variation between animals. Despite this, all post-exposure sera reacted with a polypeptide (molecular weight 40 kilodaltons) which appears to be a component of the surface tubules that are characteristic of the virus.

Animals↗

Comparison of native and subunit antigens as ELISA reagents for the detection of antibodies against scabby mouth virus.

A simple ELISA test to detect antibodies against scabby mouth virus (SMV) has been developed. Native whole virions and subunits of SMV generated by boiling the virus in the presence of sodium dodecyl sulphate (SDS) detergent and beta-mercaptoethanol were compared as ELISA assay reagents using naive and hyperimmune sera from sheep and rabbits. Approximately 2 x 10(4) intact virus particles per microtiter well were required to generate a positive to negative signal of 0.8:0.3 ELISA O.D. units when the serum was used at a dilution of 1/100. In contrast, total subunit antigen generated by disrupting and coupling of 250-500 virions per well provided a signal ratio of 1.58:0.3 ELISA O.D. units at a serum dilution of 1/250. Total subunit antigens were therefore 400 times more economical to use than intact virions. In addition, subunit antigens could be readily bound to microtiter plates without the need for removal of the SDS. Secondly, it was not necessary to block non-specific binding sites on the plate with blockers such as gelatin and skim-milk, thereby shortening the time needed to complete the ELISA assay. The total subunit antigen ELISA test was used to detect seroconversion in new born lambs where there was an occurrence of SMV infection in housed sheep. Three bleeds were taken at fortnightly intervals and the ELISA results showed that 9 out of 15 lambs were seropositive for all bleed points. Four of the lambs showed a sequential rise in titer while only one lamb failed to seroconvert.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Antiviral activity of HPMPC (cidofovir) against orf virus infected lambs.

(S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine [corrected] (HPMPC, cidofovir, CDV, Vistide) is an acyclic nucleoside analogue with a potent and selective activity against a broad spectrum of DNA viruses including the poxviruses. In this study we present the results of different treatment regimens in lambs experimentally infected with orf virus with different cidofovir formulations prepared in Beeler basis and Unguentum M. Our results show that choice of excipient, concentration of codofovir [corrected] and treatment regimen were all important to the clinical outcome of the therapy. Whilst one particular regimen appeared to exacerbate the lesion, treatment with 1% (w/v) cidofovir cream, prepared in Beeler basis, for 4 consecutive days did result in milder lesions that resolved in milder lesions that resolved [corrected] more quickly than untreated lesions. Furthermore the scabs of the treated animals contained significantly lower amounts of viable virus meaning there should be less contamination of the environment with virus than would normally occur.

Administration, Topical↗

Bacterial infections: uncommon presentations.

The essence of dermatology is morphology. The most important instrument in the practice of dermatology has always been, and still is, the naked eye; however, "We see only what we are ready to see, what we have been taught to see" (Jean Martin Charcot). Although most practitioners will easily correctly diagnose common bacterial skin diseases (such as cellulitis, erysipelas, impetigo, etc), only a trained and updated dermatologist will recognize the unusual forms and rare variants of these diseases. Bacterial skin diseases are sometimes acute and life-threatening. The mortality rates from necrotizing fasciitis range from 20% to 40%, to name just one example. It is not unreasonable to expect that dermatologists, whether in clinical practice or in referral centers, will be the first physicians to be confronted with unusual variants of bacterial skin diseases that have been unrecognized by non-dermatologists. Some of these cases might even be life-threatening, and only prompt and early recognition, diagnosis, and treatment can make the difference between losing and saving a patient's life. In short, we dermatologists should hone our clinical diagnostic skills and expand our knowledge of the rare forms and unusual and atypical variants of skin diseases: the textbook variants will probably be recognized and treated by general practitioners.

Cellulitis↗

Cloning and expression of Lipomyces starkeyi alpha-amylase in Escherichia coli and determination of some of its properties.

The Lipomyces starkeyi alpha-amylase (LSA) gene encoding soluble starch-degrading alpha-amylase was cloned and characterized from a derepressed and partially constitutive mutant for both dextranase and amylase activities. The nucleotide (nt) sequence of the cDNA fragment reveals an open reading frame of 1944 bp encoding a 619 amino acid (aa) mature protein (LSA) with a calculated molecular weight of 68.709 kDa that was estimated to be about 73 kDa, including His tag (4 kDa) based on SDS-PAGE (10% acrylamide gel), activity staining, and the Western blotting, using anti-amylase-Ab. LSA had a sequence similar to other alpha-amylases in four conserved regions of the alpha-amylase family: (I) (287)DIVVNH(292), (II) (372)GLRIDTVKH(380), (III) (399)GEVFD(403), (IV) (462)FLENQD(467). Polymerase chain reaction and sequence analysis showed one intron of 60 nucleotides in the genomic lsa at positions between 966 and 967 of cDNA. The cloned LSA amylase showed a maximum activity at pH 6 and optimum temperature of 40 (o)C, with greater than 90% stability between pH 5 and pH 8 for 16 h. It was inhibited by Cu(2+) and stimulated by Ca(2+) and Mg(2+). Enzyme activity was not affected by 1 mM EGTA but was inhibited by 1 mM EDTA. LSA did not hydrolyze maltodextrins of G2 to G4, yet formed G2+G3 from G5, G2+G4 or G3+G3 from G6, and G3+G4 from G7. LSA did not hydrolyze soluble starch in the present of 2% (w/v) of acarbose. Kinetics of LSA was carried out by using starch as a substrate and the inhibition type of acarbose was the mixed non-competitive type (ki = 3.4 microM).

Amino Acid Sequence↗

A real time PCR assay for the detection and quantification of orf virus.

A real time quantitative PCR assay based on TaqMan technology was developed for orf virus (ORFV) DNA quantification in clinical samples, infected cells and organotypic cultures. This method was based on the amplification of a 70 bp fragment from the ORFV B2L gene (orthologue of the Vaccinia virus Copenhagen F13L gene) that encodes the major envelope protein. Both intra- and inter-assay variability were well within +/-0.25 log(10) S.D. showing the high efficiency and reproducibility of the assay. The TaqMan PCR was subsequently used to determine the titre of several batches of the ORFV strain NZ-2, with it being possible to quantify virus solutions in the range of 1 x 10(1) to 1 x 10(6) TCID(50)/ml. A good correlation between the titre determined by the TaqMan PCR and by conventional endpoint dilution was found. The PCR assay is reproducible and can be used for a rapid quantification of ORFV in vitro and ex vivo, being readily achievable within 1h.

Animals↗

The development of oral lesions in lambs naturally infected with orf virus.

Stomatitis in sheep caused by orf virus can be confused with lesions of more economically significant diseases, including foot-and-mouth disease, but there is no published account of the sequential development of oral orf lesions in the sheep. This report describes the clinical appearance of such lesions during a natural outbreak of the disease in young lambs. Lesions were seen on the gingiva, the tongue and the dental pad/hard palate, and progressed from small erythematous papules to larger, often coalescing papules that in some cases were ulcerated. Resolution started within seven days and was complete within 22 days. The lambs continued to suck and thrive throughout the infection. Lesions at all stages were proliferative, providing a major differentiating factor between orf and other causes of stomatitis in sheep.

Animals↗

Genetic characterization of orf viruses isolated from various ruminant species of a zoo.

In the present study, an outbreak of proliferative dermatitis in musk ox (Ovibos moschatus), Sichuan takin (Budorcas taxicolor tibetana) and domestic Shetland sheep (Ovis aries) in a zoo is described. Skin lesions consisted of severe, persistent, multifocal, proliferative dermatitis in musk ox, and mild, transient, focal, dermatitis in the Sichuan takin and Shetland sheep. Parapoxviruses were isolated from skin lesions, and characterized by restriction enzyme analysis and partial gene sequencing. The results of this investigation indicate that the outbreak of proliferative dermatitis was due to infection by a single parapoxvirus, which is genetically closely related to other orf virus (ORFV) strains but distant to bovine papular stomatitis virus (BPSV) and pseudocowpox virus (PCPV).

Amino Acid Sequence↗

The activation status of ovine CD45R+ and CD45R- efferent lymph T cells after orf virus reinfection.

The dynamics and activation status of CD4+ and CD8+ T-cells differentially expressing the CD45R (220 kDa) antigen were studied in prefemoral efferent lymph draining the site of cutaneous reinfection with orf virus. CD4+, CD45R+ lymphoblasts preceded CD4+, CD45R- lymphoblasts during the first 48 h after reinfection. Thereafter, the output of both total and blast-transformed CD4+, CD45R- T-cells increased in proportion to the CD4+, CD45R+ cells for the duration of the virus reinfection. Output of CD8+, CD45R+ T-cells exceeded that of the CD8+, CD45R+ cells both before and after reinfection. However, within the lymphoblast population, CD8+, CD45R+ and CD8+, CD45R- T-cells increased and decreased in parallel. CD4+, CD45R- and CD8+, CD45R- T-cells produced interleukin-2, interferon-gamma and granulocyte-macrophage colony-stimulating factor after culture for 24 h without exogenous restimulation, whereas CD4+, CD45R+ T-cells produced only interleukin-2. The results show that although both CD45R+ and CD45R- alpha beta receptor+ T-cell subsets are activated as a consequence of virus reinfection in vivo, it is the CD45R- subset that predominates in the later stages of reinfection and is the principal cellular source of lymphokines in the efferent lymph.

Animals↗

Pseudomonas septicemia; necrotizing bowel lesions (NEC) and skin lesions in a 5-mo-old child.

Necrotizing enterocolitis (NEC) that occurs in the high risk neonate is not usually associated with pathogenic organism. In older children the presence of NEC is often due to infection with a specific pathogen. A case of a five month old child with pseudomonas aeruginosa septicemia presented with typical NEC. in the course of two laparotomies, most of the small bowel was resected. Necrotizing skin lesions were also present, and the likely source of both of these lesions were septic embolic.

Ecthyma↗

The immune and inflammatory response to orf virus.

Orf virus is a zoonotic, epitheliotropic DNA parapox virus that principally infects sheep and goats. The fact that the virus can repeatedly reinfect sheep has provoked an interest in the underlying cellular, virological and molecular mechanisms for its apparent escape from the host protective immune response. The local immune and inflammatory response in skin and the cell phenotype and cytokine response in lymph analysed around a single lymph node are characteristic of an anti-viral response. An unusual feature is the dense accumulation of MHC Class II+ dendritic cells in the skin lesion. The function of these cells is not known. Orf virus virulence genes and activities have been identified that may interfere with the development of the host protective immune and inflammatory response.

Animals↗

Erysipeloid infection in a sheep farmer with coexisting orf.

Erysipeloid is a rarely reported zoonotic infection caused by Erysipelothrix rhusiopathiae. We report a case of coexisting erysipeloid and orf infections in a sheep farmer, an association not previously recorded to our knowledge.

Administration, Oral↗