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

Orf is an acute and contagious exanthematous disease of sheep and goats. The disease is caused by orf virus (ORFV) belonging to the genus Parapoxvirus of Poxviridae family and the virus according to the International Committee on Taxonomy of Viruses (ICTV, 2013; http://ictvonline.org/virusTaxonomy.asp) shares many features with three other Parapoxvirus species- bovine papular stomatitis virus (BPSV), pseudocowpox virus (PCPV) and parapoxvirus of red deer in New Zealand (PVNZ).
       
The disease is not particularly devastating (Hosamani et al., 2009), yet is underlined by a high morbidity. Characteristic lesions on predilection sites are conspicuous and therefore usually diagnosed symptomatically, but often sometimes require differentiating from other cutaneous affections of ruminants leading to misdiagnosis. This is particularly true in severe and dramatic outbreaks with atypical lesions, or in the case of aggravation by secondary infection. Lesions may overlap with those in ulcerative dermatosis, mycotic dermatitis, facial eczema, foot and mouth disease, cutaneous papillomatosis (warts) especially in proliferative manifestations, sheeppox and very often with bluetongue (Radostits et al., 2007).
 
Antigenically, parapoxviruses are closely related to each other and exhibit serological cross-reactivity. According to Wittek et al., (1980) and Lard et al., (1991), there is no established serological classification either. For the detection of parapoxvirus DNA in clinical samples, PCR methods detecting a number of target genes have been developed (Karki et al., 2019) and most have been validated that can be used to identify and differentiate parapoxviruses with other pathogens causing similar clinical lesions. However, PCR primers specific to each virus species of the parapoxvirus genus is lacking (Inoshima et al., 2000). For the diagnosis of orf infection, therefore, the epidemiological criterion like mortality and morbidity rates, lesion predilection and distribution, systemic involvement and laboratory diagnostics are best employed together for differential diagnosis and confirmation of the disease.
       
In this context, it was pertinent to evaluate some common diagnostics that could supplement the clinical and histological identification of the disease and those that could be performed with ease and rapidity to provide a reliable diagnosis. The present study was aimed to assess some immunological methods for antigen or antibody detection in spontaneous infections, together with nucleic acid detection methods for the specific diagnosis of orf in goats. 

Animals, presumptive diagnosis and clinical samples
 
Goats from organized and unorganized farms around Guwahati (Kamrup district) during the period 2012-2014 were screened for orf (Contagious ecthyma). A presumptive diagnosis of orf was based on the typical signs and lesions of affected animals. Collection of clinical materials included serum samples, scabs, or occasional incision biopsies. The disease was suspected during multiple outbreaks in two organized farms and various unorganized herds from where a total of 53 clinical samples were collected (52 affected goats and 1 pooled scab sample). All laboratory work was carried out in the Department of Veterinary Pathology, College of Veterinary Science, Assam Agricultural University, Assam, India.
       
All affected clinical cases were thoroughly recorded for history, age, signs, distribution and severity of lesions. Wherever possible the natural course of disease was observed and followed on a regular basis. To study the prevalence of the lesions in affected animals, three age categories of affected goats were considered (0-6 months, 6-24 months and ≥ 24 months).
       
Tissues obtained from shed scabs (n=15), incision skin biopsies (n=15) and the occasional necropsy examination (n=2) were also preserved in 10% buffered formalin and processed for histopathological examination. Serum was collected from affected/convalescent animals (n=8) and stored in -20°C for further use.
       
The study was approved by the Institutional Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) to raise hyperimmune serum (HIS) in a goat with purified vaccine virus for immuno-precipitation application and incision biopsies of skin.
 
Diagnosis based on nucleic acid amplification
 
The highly immunogenic envelope protein p42K of orf virus (homologue of vaccinia virus envelope protein antigen-p37K) encoded by the B2L gene was targeted for virus detection. Preliminary screening of all the samples was carried out using semi-nested PCR (snPCR) with primers PPP1 (GTC GTC CAC GAT GAG CAG CT), PPP3 (GCG AGT CCG AGA AGA ATA CG) and PPP4 (TAC GTG GGA AGC GCC TCG CT) in the primer sequence set PPP1-PPP3 and PPP1-PPP4 which targets putative virion envelope B2L gene (Inoshima et al., 2000).
       
Full-length B2L gene was amplified by PCR on the basis of published sequences of primers OVB2LF1 (TCC CTG AAG CCC TAT TAT TTT TGT G) and OVB2LR1 (GCT TGC GGG CGT TCG GAC CTT C) that bind the flanking region of the open reading frame (ORF) (Hosamani et al., 2006). For extraction of viral DNA, commercially available DNeasy Blood and Tissue Kit (Qiagen USA) was used according to manufacturer’s instructions.
 
Orfvirus (ORFV) antigen detection
 
A list of antigens and antibodies used in various immunochemical assays is summarized in Table 1.
 

       
Live attenuated vaccine virus (OrfMuk 59/05, batch 06/09) isolated from goat and previously propagated in primary lamb testis cells (PLT) produced at poxvirus disease laboratory, Division of Virology, Mukteshwar were used as a reference ORFV strain. Tissue lysates from scab samples were prepared according to Nashiruddullah et al., (2016).
 
a) Agar gel immunodiffusion (AGID) test
 
This test was employed for detecting orf virus precipitating antigen using lysates prepared from clinical scab samples against hyperimmune serum on 1% agarose prepared in borate buffer, pH 8.6 (Kitching et al., 1986).
 
b) Counter immunoelectrophoresis (CIE) test
 
The test was devised with antigen in electrophoresis loading dye (Thermo,). A microslide was placed in an electrophoresis apparatus (Hoefer,) with 1x electrophoresis buffer (HiMedia,) to run the samples at a constant 30V potential for 30-60 minutes with a power supply (Hoefer, PS300B 300V). Since the antiserum migrated relatively slowly, a pre-run with antiserum alone was done to allow the migration of the antibody front in the gel, so that an approximate equidistant precipitation line between antigen and antibody wells could be visible. Gels were stained with Coomassie brilliant blue for visual enhancement of precipitin lines.
 
c) Indirect immuno-peroxidase test (IPT) for detecting antigen in tissues
 
For detection of ORFV antigen in tissues, both cryosections and formalin-fixed, paraffin-embedded (FFPE) sections were prepared and immunohistochemically stained with peroxidase enzyme (HRP).
       
Tissue cryosections were obtained from fresh skin biopsies that were trimmed and embedded in embedding matrix (Shandon™ M-1, Thermo Scientific) and snap frozen in liquid nitrogen, in plastic moulds. The frozen blocks were stored at -80°C till further use. About 4-6 µm thick sections were cut in a cryostat (Thermo Scientific^™ Cryotome^™ E) and mount on poly-L lysine pre-coated glass slides. The slides were stored at -70°C until required. Before staining, the slides were brought to room temperature for 30 minutes and fixed in chilled acetone (-20°C) for 10 minutes. They were then air dried for 30 minutes.
       
FFPE tissue sections were mounted on poly-L lysine coated slides, deparaffinized and rehydrated through descending grades of alcohol. Antigenic determinants masked by formalin-fixation and paraffin-embedding were exposed by epitope unmasking using heat induced epitope retrieval (HIER) in Tris-EDTA antigen retrieval buffer in an autoclave (Pileri et al., 1997).
       
For immunohistochemical staining (http://www.ihcworld.com/general_IHC.htm), cryosections were overlaid with a 1:100 working dilution of the ORFV MAb in blocking buffer and a 1:500 dilution goat anti-mouse peroxidase conjugate in PBS-Tween was used. FFPE sections after antigen retrieval were similarly processed in TBS added with 0.025% Triton X-100.
 
d) Indirect immuno-fluorescent test (FAT) for detecting antigen in tissues
 
Fluorescent dye (FITC) was employed forimmunohistochemical detection of ORFV antigen in tissues in both cryosections and FFPE sections. Cryosectionswere rinsed thrice with washing buffer (PBS, pH 7.4) and overlaid with a 1:500 working dilution of ORFVMAb in washing buffer and incubated for 1 hour at 37°C in a moist chamber. A pre-filtered (0.2 µm syringe filter) 1:500 working dilution of secondary antibody-FITC conjugate in washing buffer was overlaid on the tissue sections and incubated for 60 min at 37°C in a moist chamber, rinsed thrice for 5 min each in washing buffer and once in distilled water. A drop of 50% glycerol in PBS is placed onto the sections and mounted for observation under a fluorescent microscope under UV illumination. Similar protocol was followed for FFPE section after antigen retrieval.
 
e) Indirect dot-ELISA for the detection of viral antigen
 
The test was optimised for detection of ORFV antigen with Orf Virus (2E5) MAb following the procedure described by Yamaura et al., (2003) with modifications. A nitrocellulose membrane (Whatman®Protran® nitrocellulose membranes) was marked with a soft lead pencil into roughly 1 cm² squares. In each test square about 1 µl of antigen (tissue lysate) was dotted and allowed to adsorb and dry. Positive antigen control was dotted with vaccine antigen. Unbound sites were blocked with 1% BSA in washing buffer for 1 hour at 37°C. After blocking, 1:100 working dilution of the monoclonal antibody in blocking buffer and incubated for 1 hour at 37°C. The membrane was then washed three times for 5 min each with washing buffer.  After draining excess buffer, 1 µl of 1:500 dilution of secondary antibody conjugate in blocking buffer was added and incubated for 1 hour at 37°C. The membrane was washed three times as before and bound conjugated antibody was detected by adding freshly prepared chromogen-substrate and the membrane was incubated for about 5 min.
 
Orfvirus (ORFV) antibody detection
 
a) Indirect ELISA
 
The test was carried out as per method of Bhanuprakash et al., (2006) and Balamurugan et al., (2007) with modifications. Plates were coated with 50 µl working vaccine antigen (1:50dilution) in 10 mM PBS incubated overnight at 4°C and washed with 0.001M PBS-T wash buffer. About 50 µl test serum diluted 1:10 in blocking buffer (3 g LAH, 5% skim milk powder, 3% rabbit serum, 0.1% Tween 20 in 0.001 M PBS, pH 7.4) was added in tenfold serial dilution and incubated at 37°C for 1 hour. Antibody conjugate (50 µl) in 1:5000 dilution with blocking buffer was added and incubated at 37°C for 1 hour. After substrate addition, the ELISA plate was observed for colour development and the reaction was read optically using an ELISA reader at 492 nm wavelength. Cut-off value was based on negative serum reactivity at 0.1 OD.
 
b) Indirect dot-ELISA
 
Similar to the dot-ELISA for antigen detection, an indirect dot-ELISA for detection of antibodies was devised with ten-fold serial dilution of convalescent sera against vaccine virus. Orf virus MAb was also evaluated alongside as positive antibody control.

Clinical observations and lesions
 
Generally, the young animals were found to be severely affected. Oral lesions around the lips and muzzle were consistent in all 52 goats, while other lesions were associated only in a limited distribution (Table 2). Solitary to multifocal erythema, papules, vesicles, pustules and scab stages in the lips, ears, gums, tongue, udder and perineal region were observed (Fig 1). Scabs were usually hard, dry, crusty and fissured that often were loosely attached and tended to shed off, exposing the underlying granulation tissue or healed skin. Raw and friable scab lesions tend to become traumatized leaving raw bleeding surfaces. Teat and udder lesions were associated with lactating does. The presence of solitary scabs in four does in the perinea was also observed. The lesions on the gums and tongue were associated with nodular swellings that often formed ulcerations. No mortality attributable to orf was observed during the study. Close to 190 out of 600 animals were found to be affected during numerous outbreaks in the study period and the morbidity rate varied between 35-60% in different herds. Animals as young as 1-2 months of age were also affected.
       

 

 
Scab or incision biopsies collected from clinically affected animals and skin tissues collected at necropsy were routinely processed for histopathological examination. Microscopic changes of old scabs comprised of degenerate and hyalinized tissue (Fig 2A). Relatively recent scabs appear to undergo a uniform coagulative necrosis with very little histological details, a dense accumulation of inflammatory cells that were predominantly polymorphonuclear (Fig 2B) and abundant secondary bacterial infection discernible as cocco-bacillary clumps.
 

       
Microscopic lesions in intact skin biopsies were characterized by marked epidermal hyperplasia and ballooning degeneration of the keratinocytes, often revealing eosinophilic intracytoplasmic inclusion bodies within them(Fig 2C). The inclusions were not consistent in all tissues examined, while relatively abundant in early lesions. Degenerative changes were also evident in stratum spinosum cells showing vacuolation and pyknotic changes. Vesicles were also evident in early stage. Hyperkeratosis and parakeratosis in varying degrees were found in all affected tissues. Acanthotic changes in the epidermis led to deep down growths of rete ridges between the dermal papillae (Fig 2D). The dermis was found to be infiltrated with polymorphonuclear and mononuclear cells in varying proportions. In most cases scabs were present attached superficially to an underlying reactive regenerating epithelium.
 
Confirmatory diagnosis of ORFV
 
A summary of the various assays and test employed for confirmation of ORFV infection and their results are presented in Table 3.
 

 
Amplification of ORFV nucleic acid
 
Semi-nested PCR targeting partial B2L gene
 
Initial detection and screening of samples was done with semi-nested PCR targeted against the Parapoxvirus partial B2L gene. Clinical samples screened along with positive control vaccine virus were found positive by the semi-nested PCR. Amplification with the first set PPP1-PPP4 could detect a specific DNA product size of 594 bp and the second set of primers PPP3-PPP4 successfully amplified the appropriate sized product of 235 bp (Fig 3A).
 

 
Uniplex PCR targeting whole B2L gene
 
The uniplex PCR targeting the whole pan-ParapoxvirusB2L gene with the primer set OVB2L F1-OVB2L R1, was also used to detect parapoxvirus specific DNA to yield a specific product size of 1206 bp (Fig 3B) from the same clinical samples and positive control.
 
Detection of ORFV antigen
 
Agar gel immuno-diffusion (AGID) and counter immuno-electrophoresis (CIE)
 
Scab suspension from eight clinically affected goats used as antigen failed to develop a positive precipitation with hyperimmune sera, while CIE showed positive precipitin line (Fig 4) in two of the same samples tested.
 

 
Immnofluorescence and immunoperoxidase tests
 
Tissue cryosections and FFPE tissue sections were used in select samples for successful and rapid detection of ORFV antigen in tissues by immunofluorescence and also byimmunoperoxidase techniques (Fig 5A-D). Viral antigen was detected in the perinuclear region of affected granulosa and the spinous cells in the epidermis and along the external hair sheaths.
 

 
Dot-ELISA for antigen detection
 
Dot-ELISA showed positive detection of antigen prepared from all eight clinical samples tested (Fig 6A). Brownish spots indicated a positive reaction in test squares and no colouration in negative control squares.


 
Detection of ORFV antibody
 
Indirect ELISA
 
Serum from eight clinically affected animals was screened by indirect ELISA for detection of ORFV antibodies. A cut-off titer of 1:10 was considered as positive. All eight samples tested were found positive by indirect ELISA (Table 3).
 
Dot-ELISA for antibody detection
 
Serum antibody from two convalescent animals (Fig 6B) was faint and not conspicuous  at higher dilutions and negative for 1:1000 dilutions.
       
Apart from typical proliferative and scabby lesions on the lips, labial commissure and nostrils, contagious ecthyma lesions in other parts of the body such as the udder and teats of nursing animals have been reported. They often lead to mastitis (Nandi et al., 2011), or become a source of mouth infection for suckling kids (Said et al., 2013). Generalized lesions have been occasionally reported (Kumar et al., 2015) and those in internal organs such as the tongue and gums are said to be rare (Hosamani et al., 2006; 2009). It is speculated that oral lesions occur commonly because the muzzle comes in direct contact with contaminated sources when the animals eat, scrape, browse, nudge, or suckle, particularly when the skin is injured or abraded. Because of the erythematous lesions localizing around the mouth and nares, affected animals may find it difficult to eat or suckle and become prone to starvation and dehydration. This is particularly true for the affected young. Archetypal lesions reportedly progress through stages of multifocal erythema, papules, vesicles, pustules and scabs (Haig and Mercer, 1998).
       
Microscopic lesions observed in skin tissues were akin to those described in the literature (Haig and Mercer, 1998; McElroy and Bassett, 2007; Abu Elzein and Housawi, 2009; Nandi et al., 2011). Animals may be infected repeatedly, but with limited proliferative lesions (McKeever et al., 1988; Haig and McInnes, 2002). It was observed that characteristic eosinophilic cytoplasmic inclusion bodies were not a consistent feature and is rather associated with the initial stages of infection. Inclusions were relatively abundant particularly during the papulo-vesicular stage with active viral proliferation rather than in older scab lesions. This could have important diagnostic implications for the clinician, futilely seeking to demonstrate inclusions in older lesions. 
       
The pan-parapoxvirus primer set PPP-1 and PPP-4 and the nested inner primer set PPP-3 and PPP-4 are highly specific and have been validated across many laboratories (Abrahao et al., 2009; Oem et al., 2009). The complete or partial B2L sequences have often been used in phylogenetic analysis and therefore this highly specific target gene was chosen. However, the amplified products were not sequenced to draw phylogenetical studies.
       
In the present study, many diagnostics based on ORFV antigen detection have been successfully demonstrated except for a rapid precipitation test (AGID). It is contemplated that assays employing hyperimmune sera or detection of circulating antibody may have limitations, largely owing to the partial and transient humoral immunity elucidated by orf virus. Besides, the virus is also known to modulate and interfere with the host response and evade immune mechanism. Many earlier workers have validated AGPT as a rapid method for detection of ORFV antigen (Papadopoulos et al., 1968; Inoshima et al., 2001), including differentiation with VACV as control (Kuroda et al., 1999). However, Sawhney et al., (1973) opined that although scab suspensions could very well be detected with known positive serum, however he cautioned that sensitivity of antibody detection with convalescent sera was poor and not recommended. Our experiments with CIE yielded better results, perhaps with the enhance diffusion of antigen and antibody. Besides, the type of buffer and pH may also possibly lead to differences in the results. Staining with protein stains like Coomassie blue may also enhance the visualization of the precipitation lines. On the other hand, immunofluorescence and immunoperoxidase detection of antigen in cryosections and FFPE tissue sections were very successful and aided in the rapid detection of ORFV antigen by use of specific monoclonal antibody. The immunofluorescent method was found to be rapid, easier to perform and more sensitive, whereas the immunoperoxidase method gave a better histological relation. Though there is paucity of literature on use of cryosections, it proved simpler, rapid and need not require elaborate epitope unmasking. The dot-ELISA for antigen detection is a fairly rapid and easy methodfor diagnosis from direct clinical samples and if standardized could find useful applicability. Besides the current applications, other confirmatory applications were also employed. In our earlier studies, ORFV has also been successfully cultured in continuous lamb testis cells (OA3.Ts) (Nashiruddullah et al., 2016) and the virus has been demonstrated by immunofluorescence and visualized by Transmission Electron Microscopy (TEM) (Nashiruddullah et al., 2018). ELISA applications for antibody detection showed positive results and were consistent. dot-ELISA was rapid but not quantitative. Indirect ELISA has been employed for serological surveys of orf infection in goats (Begum, 2011). In all cases sera was collected from affected, recovering animals. There is no data on circulating antibody titres of past infections.
       
Orf virus infection elucidates a very weak and transient humoral immune response in the host, either in natural infection, or even after vaccination (Musser et al., 2008). This is one reason perhaps, that animals are prone to repeated infection or require continued vaccinations with a live vaccine for protection against the disease in endemic areas. Besides, the virus also elucidates many immuno-modulator proteins that interfere with host immune and inflammatory responses (Haig and McInnes, 2002). Therefore application of diagnostics using hyperimmune serum would presumably be very limited. On similar lines, a waning humoral immunity may also find limited applicability of serological diagnostics used for antibody detection.
               
In conclusion, monoclonal antibodies are versatile using direct clinical samples and useful for many confirmatory diagnostic applications. The present study explores the applicability of many immunological diagnostics that can be employed for a confirmatory diagnosis of orf infection; the adoption of any method would depend on many factors such as ease of use, rapidity, economics, feasibility and sophistication of the laboratory, etc. Owing to the limited humoral response, serum antibody detection may find limited applicability.