Molecular characterization and phylogenetic analysis of orf virus isolated from goats in Sokoto metropolis, Nigeria

Aim: The aim of this study was to molecularly characterize orf virus isolated from clinical infections in goats in Sokoto metropolis. Materials & methods: Embryonated chicken eggs were used to isolate orf virus according to the established protocol. Viral DNA was extracted and full coding region of B2L gene was amplified by polymerase chain reaction, sequenced and blasted for identification and phylogenetically analyzed. Results and discussion: The B2L gene sequences of the isolate showed slight variability (96–98.7%) with the reference sequences as it clustered within the same clade with Korean, Zambian and Ethiopian strains, signifying a close genetic relationship. Unique amino acid substitutions were noted. This is the first genetic characterization of B2L gene of orf virus circulating in Nigeria. Conclusion: This study has provided in sight into the genetic diversity of orf virus in the study area.

Natural Infection of Goats with Orf (Contagious ecthyma) and Its Diagnosis

Background: The Study was intended to evaluate some common diagnostics that could supplement the clinical and histological identification of orf in goats.Methods: Samples from suspected clinical cases of orf (contagious ecthyma) were collected from various organized and unorganized goat herds around Guwahati, Assam. Presumptive diagnosis was based on the typical signs and lesions. For confirmatory diagnosis, various molecular and immunoassays were employed for the detection of orf virus or circulating antibodies.Result: Cutaneous lesions observed included solitary to multifocal erythema, papules, vesicles, pustules and scab stages on the lips, ears, gums, tongue, udder and perineal region. No mortality was observed and the morbidity rate varied between 35-60%. Microscopic lesions in skin biopsies were typically marked by epidermal hyperplasia and ballooning degeneration of the keratinocytes along with other changes. Eosinophilic intracytoplasmic inclusion bodies were demonstrable within keratinocytes only in early papulo-vesicular stages. Dermis was infiltrated with polymorphonuclear and mononuclear cells in varying proportions. Initial screening of samples was done with PCR systems to specifically detect parapoxvirus DNA employing a semi-nested PCR targeted against the partial B2L gene (with reported primers yielding 594 bp and 235 bp amplified products) and an uniplex PCR targeting the whole B2L gene (with reported primer set to yield an amplified product of 1206 bp). Agar gel immuno-diffusion (AGID) failed to develop positive immunoprecipitation with hyperimmune serum against scab lysates; however, counter-immuno-electrophoresis (CIE) showed positive immunoprecipitation in the same samples. For rapid and in situ detection of ORFV antigen in tissues, immunofluorescent and immunoperoxidase techniques were successfully employed both on cryosections and formalin fixed skin biopsies. Immunofluorescent technique on cryosections was found to be easy, rapid and more specific. A dot-ELISA was also developed for successful confirmation of orf virus from clinical samples. For antibody detection in convalescing goats, an indirect-ELISA and a dot-ELISA was also successfully tested to demonstrate for antibody detection in convalescing goats, but protective titres later after infection was not addressed. Monoclonal antibody employed in the assays was found to be specific, sensitive and versatile for virus detection from direct clinical samples. It is contemplated that assays employing hyperimmune sera or detection of circulating antibody against orf virus may have limited diagnostic applications owing to its partial and transient humoral immunity and the inherent property of the virus to modulate and interfere with the host response and evade immune mechanisms.

First detection and molecular characterisation of pseudocowpox virus in a cattle herd in Zambia

Background Pseudocowpox virus (PCPV) of the genus Parapoxvirus in the family Poxviridae causes pseudocowpox in cattle worldwide and presents a zoonotic concern. Most poxviruses produce diseases of similar clinical signs in affected animals, which are impossible to differentiate clinically or by serology. It is, therefore, vital to use molecular assays to rapidly identify the causative agents of poxvirus infections. This study aimed to detect, diagnose, and characterize the causative agent of pox-like skin lesions in a cattle herd in Zambia, initially suspected to be infected with Lumpy Skin Disease virus. Methods We used a High-Resolution Melting (HRM) analysis assay to detect the PCPV genome and sequenced the major envelope protein (B2L gene) for comparative sequence and phylogenetic analysis. Results Our field investigations showed cattle presenting atypical skin lesions and high morbidity within the herd. The laboratory diagnosis, based on the HRM assay revealed PCPV DNA in the samples. Phylogenetic and comparative sequence analyses confirmed PCPV in the samples and revealed genomic differences between samples collected in 2017 and 2018 from the same farm. Conclusion Our work is the first documented report of PCPV in Zambia. It shows the strength of molecular methods to diagnose pox-like infections in cattle and discriminate between diseases causing similar clinical signs. This rapid and accurate diagnosis improves the response time for more accurate veterinary interventions.

Molecular Characterization and Phylogenetic Analysis of Orf Virus Isolated From Goats in Sokoto Metropolis, Nigeria

Aim: Despite the endemic nature of contagious ecthyma in Nigeria, there is limited report on the molecular characterization of the isolates responsible for disease outbreaks. The aim of this study was to molecularly characterize ORFV isolated from clinical infections in goats in Sokoto metropolis. Materials and Methods: Seronegative embryonated chicken eggs were used to isolate ORFV via the chorio allantoic membrane (CAM) route according to the established protocol. Viral DNA was extracted from infected CAM and the full coding region of B2L gene was amplified by PCR and subsequently sequenced by Sanger’s method. The nucleotide sequence results were blasted for identification and phylogenetically analyzed using MEGA and Bioedit softwares. Results and Discussion: The results showed that B2L gene sequences of the ORFV UDUS/01/19/More strain showed slight variability (96- 98.7%) with the reference sequences. Our isolate clustered within the same clade with Korean strain signifying a close genetic relationship. Unique amino acid substitutions were noted in our isolate when compared with other references. This is arguably the first genetic characterisation of B2L gene of ORFV circulating in Nigeria. Conclusion: Our study has provided in sight into the genetic diversity of ORFV in the study area. This is crucial for the design of effective vaccines against the disease which are currently lacking in the country.

First Detection and Molecular Characterisation of Pseudocowpox Virus in a Cattle Herd in Zambia

Background: Pseudocowpox virus (PCPV) of the genus Parapoxvirus in the family Poxviridae causes pseudocowpox in cattle worldwide and presents a zoonotic concern. Most poxviruses produce diseases of similar clinical signs in affected animals, which are impossible to differentiate clinically or by serology. It is, therefore, vital to use molecular assays to rapidly identify the causative agents of poxvirus infections. This study aimed to detect, diagnose, and characterize the causative agent of pox-like skin lesions in a cattle herd in Zambia, initially suspected to be infected with Lumpy Skin Disease virus.Methods: We used a High-Resolution Melting (HRM) analysis assay to detect the PCPV genome and sequenced the major envelope protein (B2L gene) for comparative sequence and phylogenetic analysis. Results: Our field investigations showed cattle presenting atypical skin lesions and high morbidity within the herd. The laboratory diagnosis, based on the HRM assay revealed PCPV DNA in the samples. Phylogenetic and comparative sequence analyses confirmed PCPV in the samples and revealed genomic differences between samples collected in 2017 and 2018 from the same farm.Conclusion: Our work is the first documented report of PCPV in Zambia. It shows the strength of molecular methods to diagnose pox-like infections in cattle and discriminate between diseases causing similar clinical signs. This rapid and accurate diagnosis improves the response time for more accurate veterinary interventions.

Epidemiological Survey, Molecular Detection and Risk Factors of Camel ORF Virus in Arero District, Ethiopia

Background: While camels (dromedaries) were traditionally believed to be resistant to mostlivestock diseases, research has demonstrated that they are susceptible to a large number ofinfectious agents. Based on the clinical appearance of typical lesions, Camel contagiousecthyma (CCE), caused by a Orf virus, is thought to be one of the most common viraldiseases of camelids in Ethiopia. However, the epidemiology of the disease has not beenformally described and the causative agent has never been molecularly confirmed.Methods: a cross-sectional study was conducted from November 2013 to April 2014 inArero district of Borena Zone, to assess morbidity and mortality rates of the of diseaseconsistent with CCE, isolate and molecularly identify the causative agents and to find out thepotential risk factors. Molecular technique, namely, PCR based on B2L gene-specific primers ofORFV was used for the confirmatory diagnosis of CCE virus from the skin lesions.Results: Majority (86.8%) of the respondents indicated occurrence of CCE outbreaks in theirherds in the past one year (a year preceding the start of the study). The overall morbidity andmortality rates attributed to CCE was 43.6% (95 % CI: 41.2%–46%) and 6.3% (95 % CI: 5.2–7.6%) respectively. Confirmatory diagnosis of the suspected Orf virus isolates usingconventional PCR techniques generated the expected amplification product of 1200bp for oneof the samples. No product was amplified from the DNA samples of the negative control.This study showed that young camels (calves) had higher odds of becoming affected by CCEthan adults [OR=3.44 (95 % CI: 2.29 –4.09); (p<0.05)]. The disease had marked seasonalitywith most of the cases occurring during rainy season. Acacia trees significantly contribute tovirus dissemination through damaging the lips of browsing camels.Conclusions: This study confirms the presence and importance of CCE in Ethiopia andestablishes the basis for further research.

First Detection and Molecular Characterisation of Pseudocowpox Virus in a Cattle Herd in Zambia

Background: Pseudocowpox virus (PCPV) of the genus Parapoxvirus in the family Poxviridae causes pseudocowpox in cattle worldwide and presents a zoonotic concern. Most poxviruses produce diseases of similar clinical signs in affected animals, which are impossible to differentiate clinically or by serology. It is, therefore, vital to use molecular assays to identify the causative agents of poxvirus infections rapidly. This study aimed to detect, diagnose, and characterize the causative agent of pox-like skin lesions in a cattle herd in Zambia, initially suspected to be infected with the Lumpy Skin Disease virus.Methods: We used a high resolution melting (HRM) analysis assay to detect the PCPV genome and sequenced the major envelope protein (B2L) gene for comparative sequence and phylogenetic analysis. Results: Our field investigations showed cattle presenting atypical skin lesions and high morbidity within the herd. The laboratory diagnosis based on an HRM assay revealed the PCPV genome in the samples. Phylogenetic and comparative sequence analyses confirmed PCPV in the samples. They revealed genomic differences between samples collected in 2017 and 2018 from the same farm.Conclusion: Our work is the first documented report of PCPV in Zambia. It shows the strength of molecular methods to diagnose pox-like infections in cattle and discriminate between diseases causing similar clinical signs for better veterinary interventions.

Molecular characterization of contagious ecthyma virus (CEV) isolated from goat and cross-protection studies

Contagious ecthyma (CE) is a zoonotic viral infection caused by the Parapoxvirus in sheep and goats which is classified in the family of Poxviridae. In this study, the Penorf CE vaccine strain that originated from lambs (PKCE1 strain) and three CE strains (O-CEV1, O-CEV2, O-CEV3) originated from kids were used for molecular characterization and cross-protection studies. A phylogenetic similarity has been investigated by comparing the B2L gene of CE viruses originated from kids and lambs. It was observed that the isolates O-CEV1 and O-CEV2 had a similar DNA sequence (100%) whereas the other isolate, O-CEV3 had a different DNA sequence from the others, and the proportion of the difference between them was 2.6% as stated in the similarity index. The phylogenic evaluation revealed that CE viruses were not species specific and have different genotypes in lambs and kids in Turkey. Penorf vaccine strain which is still known as lamb origin was found to be also kid origin. In the pathogenity studies in kids and lambs, there was no rise in the body temperatures of lambs and kids and hyperemia, vesicles and pustules occurred in the scarified skin regions from the second day of the epruvation. In addition to these findings, it was determined that the healing in lesions occurred after the scabs fell off beginning from the 38th to 55th days of the study. At the end of the this study, the presence of CE strains with different pathogenicity properties was revealed. In goats vaccinated with Penorf vaccine, protection to O-CEV3 field isolate has been observed, but not obtained protection to PK-CK1 strain. As a result, the phylogenic evaluation revealed that CE viruses were not species specific and have different genotypes in lambs and kids in Turkey. The Penorf vaccine strain, which is still known to be of lamb origin, was found to be of kid origin and it was not seen to protect lambs against CE disease due to the genomic differences. Therefore, it was concluded from this data that at least a bivalent CE vaccine containing lamb and kid isolates should be prepared and used for effective immunity against CE infection, especially in lambs and kids.

Molecular characterization of Orf virus isolates from Kodai hills, Tamil Nadu, India

Aim: The present study was carried out to find out the causative agent of exanthematous skin lesions in sheep maintained by Southern Regional Research Centre, Mannavanur, Kodai hills, Tamil Nadu. Materials and Methods: Polymerase chain reaction (PCR) with Orf virus (ORFV) B2L gene-specific primers was carried out by employing the total genomic DNA isolated from the scabs as the template. The ORFV isolates from Kodai hills were characterized by the use of bioinformatics tools. Results: The amino acid identity of ORFV isolate 1 from Kodai hills is having 98.14%, 96.29%, and 83.59% identity with reference strains of ORFV, Pseudocowpox virus, and bovine papular stomatitis virus, respectively. Phylogenetic analysis revealed that ORFV isolates from Kodai hills clustered with the other ORFV isolates from different geographical areas of India. Conclusion: The etiological agent of exanthematous skin lesion among sheep of Kodai hills is ORFV.