Validation of molecular diagnostics for the detection of pseudocowpox virus.

The pseudocowpox virus (PCPV) is recognized for causing exanthematic lesions in cattle and humans. The diagnosis is important because it is a zoonosis and its clinical signs can be confused with foot-and-mouth disease, a high-impact bovine disease in livestock. The objective of this work is to validate a SYBR Green qPCR and a conventional PCR for virus detection in bovine samples. Detection limit tests, repeatability, reproducibility, sensitivity, and specificity were compared. When two analysts were compared, results demonstrated that training and pipetting influence the repeatability. The qPCR was more sensitive than conventional PCR but showed nonspecific reactions distinguishable by the melting curve. Both showed high repeatability and reproducibility.

qPCR assay for the detection of pseudocowpox virus.

Pseudocowpox is a zoonosis caused by pseudocowpox virus (PCPV), which mainly affects cows but can be an occupational disease of humans. The aim of the study was to validate a quantitative polymerase chain reaction (qPCR) assay for the detection of PCPV. The assay was able to detect up to 1000 copies of PCPV per µL in field samples, with a sensitivity of 80% and a specificity of 100%. We did not observe any cross-reactivity between PCPV-positive samples and samples that were positive for other genetically similar viruses. The repeatability and reproducibility were adequate according to parameters preestablished in official test validation manuals.

A multiplex PCR for viruses associated with exanthematic and vesicular disease in cattle.

Exanthematic and papulo-vesicular lesions in the udder and teats of milking cows are fairly common in some Brazilian dairies, especially those with poor sanitary conditions and hand milking. The orthopoxvirus Vaccinia virus (VACV) and the parapoxviruses Pseudocowpox virus (PCPV) and Bovine popular stomatitis virus (BPSV) have been frequently associated with such conditions. Elsewhere, Bovine herpesvirus 2 (BoHV-2) has also been associated with similar clinical signs. Thus, we herein describe a conventional multiplex PCR designed to detect the genome of these viruses in clinical samples while differentiating among them by amplicon size. For this, primer sets targeting the orthopoxvirus vascular growth factor (amplicon size 292bp), PCPV (374bp) and BSPV (607bp) B2L genes, and the BoHV-2 DNA polymerase gene (138bp) were selected. The chosen primers anneal within the same temperature range and do not interfere with each other during the PCR amplification. PCR conditions were initially standardized for each agent in individual PCR reactions firstly using the target virus as positive control followed by using a mixture of all four virues. Lastly, a multiplex PCR containing the four sets of primers was set up to amplify all four targeted viruses in one reaction. The multiplex PCR was able to detect DNA extracted from cell culture supernatants containing 20 TCID50 of BoHV-2 and 50 TCID50 of VACV. Further, the test could detect the viral genomes in 1:10, 1:50 and 1:1000 dilutions of total DNA extracted from clinical specimens (e.g. scabs, crusts) of natural cases (PCPV, VACV and BPSV) and 1:10 dilutions of DNA extracted from scabs collected from BoHV-2 experimentally infected cattle. A possible amplification of other orthopoxviruses, predicted by in silico analysis, was considered to not represent an important pitfall since these are exotic in Brazil, very rare, or viruses not associated with cattle. For definitive agent identification amplicon sequencing needs to be conducted. Thus, this multiplex PCR seems suitable for initial detection and identification of the agents involved in exanthematic and vesicular disease, providing a sensitive and specific diagnosis for such conditions in dairy cows.

Occurrence of Pseudocowpox virus associated to Bovine viral diarrhea virus-1, Brazilian Amazon.

In 2011, an outbreak of severe vesicular disease occurred in the state of Pará, Amazon region. Besides proliferative or verrucous lesions, cattle showed atypical clinical signs such as diarrhea and leading to death. The animals were submitted to clinical, pathological and molecular diagnosis, and laboratory tests have confirmed the presence of Pseudocowpox virus (PCPV), a Parapoxvirus genus member, and have also found Bovine viral diarrhea virus-1 (BVDV-1), probably causing persistent infection. The results of molecular diagnostics, followed by sequencing data demonstrated the circulation of both viruses (PCPV and BVDV-1) in an area previously affected by another poxvirus, as Vaccinia virus.The cocirculation between PCPV and BVDV-1 indicates a major concern for animal health because the clinical presentation can be a severe disease. This is the first detection of PCPV in the Brazilian Amazon.

An outbreak of pseudocowpox in fattening calves in southern Brazil.

Pseudocowpox virus is a parapoxvirus frequently associated with papulovesicular and scabby lesions on the teats and udders of milking cows and is often transmitted to human beings. An unusual outbreak of skin disease in fattening calves in southern Brazil is described. Fourteen of 17 male cattle (82%), aged 6-48 months, feeding on grass pastures were affected. Animals developed papules, vesicles, and scabby proliferative lesions on the muzzle in a clinical course of approximately 10-15 days. The scabby lesions often presented with exudation and bleeding. Histological examination of mucocutaneous tissue in detached scabs revealed acanthosis with thickening of the corneal layer and premature keratinization (parakeratotic hyperkeratosis). The dermis had multifocal lymphoplasmacytic infiltrates. Electron microscopic examination of scab specimens revealed typical parapoxvirus particles: oval shaped (260 nm × 160 nm), enveloped, and covered with a helical layer. Polymerase chain reaction using a set of pan-parapoxvirus primers for the B2L gene amplified a 590-bp product out of DNA extracted from scabs. Nucleotide sequencing of the amplicons revealed a nucleotide homology of 97% with Pseudocowpox virus and lower homology with other parapoxviruses: Bovine papular stomatitis virus (84%) and Orf virus (94%). A phylogenetic tree based on the B2L sequence was constructed, showing that the virus clustered with Pseudocowpox virus isolates.

Human Vaccinia virus and Pseudocowpox virus co-infection: clinical description and phylogenetic characterization.

BACKGROUND: Occupational exanthematic diseases represent an important cause of public health impact and economical losses. Among the viral exanthematic diseases, two caused by poxviruses are noteworthy: the bovine vaccinia (BV), caused by the Vaccinia virus (VACV); and the milker's nodule, in which the agent is the Pseudocowpox virus (PCPV). Both agents are zoonotic and have been associated with several cases of bovine infection. In Brazilian rural areas BV has been highly prevalent, particularly in milk herds. Farmers, milkers and their close contacts developed lesions on the hands, forearms, legs and face accompanied by several systemic symptoms. Although VACV and PCPV present with similar epidemiological and transmission patterns, no VACV and PCPV co-infection cases have to date been described. OBJECTIVES: To describe the first case of zoonotic VACV and PCVP co-infection, based on serological and molecular methods. STUDY DESIGN AND RESULTS: In this work we report a case of a Brazilian rural worker who presented with a large severely ulcerated-pustule skin lesion, associated with fever, headache, malaise, myalgia and axillary, inguinal and cervical limphadenopathy. The worker declared occupational contact with cattle that had notable injuries on their teats. Human and bovine clinical samples were collected and submitted to serological and molecular tests. PCR and phylogenetic analysis revealed the presence of VACV DNA and PCPV DNA in the patient's lesion. Serological tests indicated anti-VACV neutralizing antibodies and molecular assays showed the presence of VACV and PCPV DNA in the patient sera. VACV and PCPV also were detected in dairy cattle. CONCLUSION: Together, these results indicate a case of zoonotic VACV/PCPV co-infection. Epidemiological surveillance and appropriate medical treatment are essential for the control of both diseases, especially in the most severe cases, as described in the present study.