Int.J.Curr.Microbiol.App.Sci (2019) 8(9): 422-430 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 8 Number 09 (2019) Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2019.809.051 Pathology of Avipoxvirus Isolates in Chicken Embryonated Eggs Bhavesh Sharma1, Nawab Nashiruddullah1*, Jafrin Ara Ahmed2, Sankalp Sharma1 and D. Basheer Ahamad3 1Division of Veterinary Pathology, 2Division of Veterinary Physiology & Biochemistry, Faculty of Veterinary Sciences and Animal Husbandry, Sher-e- Kashmir University of Agricultural Science and Technology of Jammu, RS Pura-181102, India 3Department of Veterinary Pathology, Veterinary College and Research Institute, Tamil Nadu University of Veterinary and Animal Sciences, Tirunelveli, India *Corresponding author ABSTRACT K e yw or ds Fowlpox virus (FWPV) and Pigeonpox virus (PGPV) isolates from domesticated fowls and Avipoxvirus , pigeons from Jammu region were inoculated on chorioallantoic membrane (CAM) of Chicken chicken embryonated eggs (CEE) through three passage levels to observe their cytopathic embryonated eggs effects (CPE) and underlying pathology. Development of PGPV induced lesions was (CEE), earlier than FWPV, and more severe. PGPV progressed from small white opaque lesions Chorioallantoic from first passage; to CAM thickening, haemorrhages and very prominent and extensive membrane (CAM), pock lesions at second; and by third passage, focal areas of necrosis was evident. With Cytopathic effect FWPV, opaque thickening with haemorrhages at first passage; to tiny, discrete, white foci (CPE) , Fowlpoxvirus on second; and at third passage, small, round, raised circular white pock were distinctly visible. CAM histology revealed PGPV induced hyperplasia of the chorionic epithelium; (FWPV), Pigeonpoxvirus oedema, thrombosis, massive fibroblastic proliferation, necrosis and islands of vacuolated (PGPV) epithelial cells containing eosinophilic intra-cytoplasmic inclusions in the mesoderm. FWPV induced lesions were similar but necrosis of the epithelial layers was more Article Info widespread; vascular lesions in the mesoderm were more profound, proliferation of fibrous Accepted: tissue was less, and the absence of inclusion bodies. It was observed that pock morphology 15 August 2019 on CAM may not be a reliable basis of Avipoxvirus species differentiation, although such Available Online: variations both grossly and histologically exist. 10 September 2019 Introduction They have been observed in more than 230 of the known 9000 species of birds, spanning 23 Avian pox is a group of DNA viruses of the orders (van Riper and Forrester, 2007), yet genus Avipoxvirus, under family Poxviridae, little is known about the genome diversity, and subfamily Chordopoxvirinae, and has host range and host specificity of the causative long been observed in several avian species. agents. It is characterized by the formation of 422 Int.J.Curr.Microbiol.App.Sci (2019) 8(9): 422-430 proliferative lesions, scabs on the skin, and belong to clade 2. Since differences in CPE on diphtheritic lesions in the upper digestive and CAM by different Avipoxviruses (APV) have respiratory tracts. The genus currently been earlier reported (Manarolla et al., 2010; comprises of ten recognized species (Smith et Abdallah and Hassanin, 2013; Offerman et al., al., 2008), of which Fowlpox virus (FWPV) is 2013) it was proposed to attempt isolation of the type species. Antigenic relationship exists the viruses on CEE. Whereas the TKPV among Avipoxvirus, and some avian samples comprised of formalin preserved and poxviruses are more closely related to some archival specimens, growth of only the FWPV than others. There is evidence of considerable and PGPV isolates were tried in the present heterogeneity among species of avian study to observe their differential pathology, if poxviruses. any. Avipoxvirus replicates in the cytoplasm of Materials and Methods infected cells and depending on the isolate- produces typical cytopathic effect (CPE) 4 to Suspected clinical cases 6 days post infection. The virus can be grown on the chorioallantoic membrane (CAM) of Suspected samples of Avipoxvirus infection embryonated chicken egg and in a variety of were collected from backyard poultry (fowl) cell culture models. Pox virus culture on and pigeons from Rajouri and Jammu districts. chorioallantoic membrane (CAM) of chicken Samples were collected at different times from embryonated eggs (CEE) is one of the April 2016 to May 2017. Fowlpox (FP) identification tools of choice (Woodruff and outbreaks were covered from six select areas Goodpasture, 1931; Cunningham 1973). Since of which five were from Rajouri district and the chicken embryo at 10–15 days old lacks a one from Jammu district. Similarly, samples functional specific immune system (Eerola et of Pigeonpox (PP) were taken from four select al., 1987; Dibner et al., 1998) it provides an places in Ranbir Singh Pura in Jammu which opportunity to analyze virus-induced host was very near to the Indo-Pak International responses in the absence of specific adaptive border. The open-ranged pigeons although immune responses (Fredrickson et al., 1992). domesticated, sometimes crossed over to each Thickening of the infected CAM produces side of the border. typical focal and diffuse lesions. These lesions are fairly diagnostic and can be readily Collection of clinical samples confirmed by histological observations or electron microscopy (OIE, 2008). Clinical materials included scabs, skin lesions, or occasional incisional biopsies, and lesions In our laboratory, domesticated fowls, pigeons from oral membranes from Avipox suspected and turkey birds were screened for live or dead birds. avipoxvirus infection from different areas in Jammu region, India, and on the basis of the Isolation of putative APV (FWPV and phylogenetic analysis of the partial P4b gene PGPV) from clinical samples sequences, three different isolates were identified as Fowlpox virus (FWPV), Clinical scab/skin samples of affected birds Pigeonpox virus (PGPV) and Turkeypox virus were processed for virus isolation. A 10-20% (TKPV) respectively (Sharma et al., 2019); solution of the scab samples in PBS was and it was also found that the FWPV and prepared by mechanical homogenization using TKPV were phylogenetically 99% related a sterilized mortar pestle. Homogenized within clade 1, while PGPV was found to samples were centrifuged at 3,000 × g for 15 423 Int.J.Curr.Microbiol.App.Sci (2019) 8(9): 422-430 min. The supernatant was then passed through each of the three passage levels are depicted in a 0.45 μm syringe filter (Millipore) and Figure 1. incubated with streptomycin, penicillin antibiotic solution (@ 200 IU of penicillin and FWPV 200 µg of streptomycin per ml suspension) for 1 hr at 37°C. Processed samples were used for On the first and second passage, the CAM infecting CAM for virus isolation. appeared opaque, thick oedematous and haemorrhagic (Fig. 1A, B). Tiny, discrete, Chorio-allantoic membrane inoculation of white foci were observed on CAM that was virus in chicken embryonated eggs not always discernible (Fig. 1C, D). During third passage, small, round, raised circular For virus isolation in CAM, the protocol white lesions or ‘pocks’, of varying described in Cunningham (1973) by the morphology were distinctly visible (Fig. 1E, artificial air sac route was employed. F). Size of lesions was approximately 1-3 mm Approximately 10-12 day old chicken in diameter. No CPE was observed in control embryonated eggs from Government Poultry CEE incubated at each passage level. Hatchery Unit, Belicharana, under Animal Husbandry Department, Jammu were procured PGPV for virus inoculation. The eggs were acquired after ensuring that there was no history of On first passage, in some instances the CAM Fowlpox infection in the parent flock. Out of showed small white opaque lesions (Fig. 2A) various field samples, one each of FWPV and or appeared opaque, thick, oedematous and PGPV were adopted (in triplicate) on CAM often with petechial haemorrhagic (Fig. 2B). through three passage levels. The isolates have Subsequent passage revealed very prominent been earlier verified by partial amplification of and extensive pock lesions (Fig. 2C). In one P4b gene and confirmed as FWPV and PGPV instance a substantial thickening and diffuse by alignment with published sequences opaqueness of the CAM was observed, (Sharma et al., 2019). The eggs were candled completely obscuring any details (Fig. 2D). daily and opened on 5 d.p.i. Cutaneous lesions By 3rd passage, they were characterized by were processed and used as inoculum for first focal areas of necrosis and opacity, thickening passage. Blind passages or CAM showing and oedema (Fig. 2E, F). Size of pock lesions confluent growth of pocks was harvested as a was nearly 1-5 mm in diameter. Development source of Avipoxvirus for next passage. The of lesions was earlier and more severe in development of characteristic lesions if any Pigeonpox samples than those of Fowlpox. No was noted for each passage level. For each CPE was observed in control CEE incubated Avipox inoculation and at every passage level, at each passage level. a negative control CEE was incubated alongside, with injection of sterile PBS on the CAM histopathological lesion CAM. PGPV Results and Discussion The Pigeonpox lesion in the chorio-allantoic Chicken embryonated egg inoculation membrane is noticeable with hyperplasia of the epithelial cells of the ectoderm (chorionic