Detection of Parapoxvirus in goats during contagious ecthyma outbreak in Ceara State, Brazil by transmission electron microscopy techniques
Abstract
Contagious ecthyma or contagious pustular dermatitis, is a viral skin disease that occurs in sheep, goats and wild ruminants and is characterized by the formation of papules, nodules or vesicles that progress into thick crusts or heavy scabs on the lips, gingiva and tongue, caused by a member of the Parapoxvirus genus. Human s are occasionally affected constituting important zoonosis. The disease not only has an economic impact on farmers worldwide but also has a considerable negative effect on animal welfa re. In this study, a contagious ecthyma outbreak which occurred in one flock with 90 goats located in the Ceará State , Brazil, was described. Twenty -two goats older than 6 months were affected. The animals presented crusted lesions on the buccal region, to ngue, udder and teat s, which began with swelling in the mouth area. Dri ed crusts and serum collected were processed for transmission electron microscopy utilizing, negative staini ng (rapid preparation), Immunocytochemistry (immunol abelling with colloidal gold particles) and resin embedding techniques.
At the Philips EM 208 tr ansmission electron microscopy all the samples were analyzed by negative staining technique and a great number of parapoxvirus particles ovoid or cylindrical, showing two morphological forms, a mulberry (M ) form with a distinctive criss cross filament patte rn derived from the superimposition of upper and lower virion surfaces and a capsular (C) form caused by stain penetration and distention of the virion core, measuring 300 x 180 nm was observed. Antigen antibody reaction was increased by the colloidal gold particles. In the ultrathin sections of crusts, we verified the presence of three types of intracytoplasmic inclusion bodies, type A or Bollinger inclusion bodies, outlined by membrane, presented in it is interior, oval, mature or complete viral particles , measuring on the average, 225nm x 130 nm, showing an inner dumbell - shaped core, two lateral bodies and an external envelope, or cigar shaped core. In the type B electron dense inclusions bodies, were visualized parapoxvirus particles budding of dense and amorphous material. Fibrillar intracytoplasmic inclusions were also found located between the virions, consisting of groups of fibrils, arranged in groups or concentrically in the middle of the granular material. Intracytoplasmic vesicles outlined by memb ranes, measuring 560 x 420 nm, containing granular material in its interior were also observed. The nuclei showed an aspect deformed.
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Introduction
Genus Parapoxvirus has four members, Orf virus (ORFV), Bovine papular stomatitis virus (BPSV) , Pseudocowpoxvirus (PCPV) and Parapoxvirus of red deer in New Zealand (PVNZ). The ORFV is the prototype member.
Enveloped virions present with an ovoid shape and the spiral tubule surrounding the virion su rface can be clearly distinguished by electron microscopy from orthopoxviruses (OPV) because of their regular surface structure. The particles of ORF virus are 260 nm length by 160 nm wide (Damon, 2007; Moss, 2007) .
The virus genome includes linear double -stranded DNA about 138 kb length with 64% G+C content, which contains 132 putative genes that included 89 highly conserved genes and some variable genes. The envelope gene (P2L) of the virus encodes a highly immunogenic major envelope protein widely used f or molecular characterization and phylogenetic analysis of strains of the virus (Inoshima et al., 2000; Delhon et al., 2004; Mercer et al., 2006; Zhang et al., 2010).
ORF virus is the etiological agent of the contagious ecthyma or contagious pustular derm atitis, a viral skin disease that occurs in sheep, goats and wild ruminants, characterized by the formation of papules, nodules or vesicles that progress into thick crusts or heavy scabs on the lips, gingiva, tongue, eyelids and feet and occasionally on th e teats (Vikoren et al., 2008).
Susceptible animals usually develop the first signs of the disease 4 to 7 days after exposure that persists for 1 to 2 weeks or Page | 2 for large periods (Haig & Mercer, 1998).
The mortality occurs, especially in young sucking lam bs, due to an incidence of dehydration and starvation, as the pain and distortion of the lips and mouth preclude the lamb from sucking (Mombeni et al., 2012).
The mortality rate is usually low but it can reach 93% in lambs with secondary bacterial or funga l infections (Haig & Mercer, 1998; Zhao et al., 2010).
The infection is spread by direct and indirect contact from infected animals or by contact with infected tissue or saliva containing the virus (Lojkic et al., 2007).
Outbreaks occur more frequently du ring periods of extreme temperatures such as late summer and winter (Robinson & Balassu, 1981; Gokce et al., 2005).
Some infected animals become carriers and shed the virus for a long period (Mombeni et al., 2012).
Humans are occasionally affected constitu ting important zoonosis ( Al Salam et al., 2008; Kitchen et al., 2013; Turan et al., 2013).
The disease not only has an economic impact on farmers worldwide but also has a considerable negative effect on animal welfare. Infected animals are sickly, fail to thrive, and are more susceptible to bacterial infections (Gallina et al., 2008).
Outbreaks of the disease have been reported in several countries, such as China (Zhang et al., 2010; Li et al., 2012), Croatia (Lojkic et al., 2010), Italy (Gallina et al., 2 008), Japan (Inoshima et al, 2001), Taiwan (Chen et al., 2007) and Greece (Billinis et al., 2012).
In Brazil, a few reports were described in the states of Mato Grosso, MT (Abrahão et al., 2009); Pará, PA (Oliveira et al., 2012); Paraíba, PB (Nóbrega et al ., 2008); Minas Gerais, MG, Rio Grande do Sul, RS e Pernambuco, PE (Mazur et al., 2000).
Considering the efficiency and speed of transmission electron microscopy techniques, this study aimed to detect the presence of parapoxvirus in skin lesions of goats d uring outbreak of contagious ecthyma first occurred in Ceará state, Brazil.
Conclusion
Considering that contagious ecthyma is a highly contagious, zoonotic, viral skin disease that affects sheep and goats , causing fail to thrive and decreases in production, the application of transmission electron microscopy techniques in routine or during outbreaks of the disease may help to develop measures for prevention and control of contagious ecthyma , collaborating with the National Goat and Sheep Health Program.
