Diagnóstico de ectima contagioso em pequenos ruminantes através da Reação em Cadeia da Polimerase em Tempo Real

The virus of contagious ecthyma (CEV), also known as orf virus (ORFV) is the etiological agent of contagious ecthyma (CE) in sheep and goat and belongs to the Parapoxvirus genus, family Poxviridae. In some cases, CE can be confused with vesicular diseases so there is need for differentiation especially because, according to the standards of the National Program for the Eradication of FMD (PNEFA), goats and sheep are not vaccinated against Foot and Mouth Disease (FMD), acting as sentinel animals. Although initial studies have demonstrated the usefulness of the polymerase chain reaction (PCR) as a diagnostic test, there are no studies involving its use on Brazilian field samples, which may be genetically distinct from previously studied samples, as described in a study of restriction sites analysis of Brazilian CE samples. This work was conducted with the goal of standardizing a PCR (qPCR) test using SYBR Green I dye for molecular diagnosis of EC in DNA extracted from lesions of affected animal or cell culture inoculated in field samples. The products were detected with qPCR dissociation curve analysis which showed a peak at 88 ºC indicating that positive samples have only one specific amplification product. All DNA samples tested (29 animals crusts and their cell cultures) were positive in the qPCR. The qPCR was able to detect the DNA of at least 10,000 times dilution corresponding to 0.056 ng of DNA. It is believed that with the additional qPCR validations reported in this study, it can be used for differential diagnosis in the health surveillance of PNEFA.

Contagious ecthyma: how serious is the disease worldwide?

Contagious ecthyma (CE) is an infectious disease of small ruminants caused by a parapoxvirus of family Poxviridae subfamily Chordopoxvirinae. The disease is obviously distinguished by an establishment of scabby lesions and ulcerative formation on less hairy areas including muzzle, ears, nostril, and sometimes on genitalia. The disease is endemic in sheep and goats. The virus is transmissible to other ruminants and is a public health concern in humans. Although the disease is known as self-limiting, it may cause a significant economic threat and financial losses due to lower productivity in livestock production. Information with regard to the risk of the disease and epidemiology in most parts of the world is underreported. This paper aims to provide relevant information about the epidemiology of CE in selected regions of Europe, South America, North America, Asia, Africa, and Australia. An in-depth comprehension of virus infection, diagnoses, and management of the disease will enable farmers, researchers, veterinarians, abattoir workers, health personnel, and border controllers to improve their measures, skills, and effectiveness toward disease prevention and control, toward reducing unnecessary economic loss among farmers. A herd health program for significant improvement in management and productivity of livestock demands a well planned extension program that ought to encourage farmers to equip themselves with adequate skills for animal healthcare.

Molecular Insights into the Genetic Variability of ORF Virus in a Mediterranean Region (Sardinia, Italy)

Orf virus (ORFV) represents the causative agent of contagious ecthyma, clinically characterized by mild papular and pustular to severe proliferative lesions, mainly occurring in sheep and goats. In order to provide hints on the evolutionary history of this virus, we carried out a study aimed to assess the genetic variation of ORFV in Sardinia that hosts a large affected small ruminant population. We also found a high worldwide mutational viral evolutionary rate, which resulted, in turn, higher than the rate we detected for the strains isolated in Sardinia. In addition, a well-supported genetic divergence was found between the viral strains isolated from sheep and those from goats, but no relevant connection was evidenced between the severity of lesions produced by ORFV and specific polymorphic patterns in the two species of hosts. Such a finding suggests that ORFV infection-related lesions are not necessarily linked to the expression of one of the three genes here analyzed and could rather be the effect of the expression of other genes or rather represents a multifactorial character.

Evolution of humoral immunity effectors in four flocks of sheep with symptoms of contagious ecthyma, from Santău village, Romania

Contagious ecthyma or contagious pustular dermatitis is a debilitating condition located on the skin of sheep and goats, with a zoonotic character. Contagious ecthyma is a disease with economic and animal welfare implications, rarely being fatal if the host organism suffers from immunosuppression or if secondary infections occur. Similar to other members of the Poxviridae family, immunity to contagious ecthyma is mediated by both innate and adaptive immune responses. Following the invasion of the contagious ecthyma virus, immunoglobulins are mobilized that specifically bind to the contagious ecthyma virus to form immune complexes, which are then eliminated by defense system to protect tissues from damage. The level of circulating immune complexes and immunoglobulins can be quantified and can be an indicator of the stage of infection. In the present study we aimed to investigate whether there is a link between the individual characteristics (sex, age, severity of clinical signs) of sheep with orf symptoms and the evolution of circulating immune complexes and total immunoglobulins. In addition to these investigations, we aimed to follow the evolution of these immunological parameters compared to the evolution in clinically healthy animals. Investigations of the serum revealed that levels of these two parameters (total Ig and CIC), can be influenced by the individual characteristics of sheep affected by the contagious ectima and shows differences compared to clinically healthy animals.

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.