Introduction

Orf virus belongs to the genus Parapoxvirus, family Poxviridae, and subfamily Chordopoxvirinae. It is the cause of contagious ecthyma infection in sheep and goats (Smith and Sherman 2011). The infection results in the formation of erythematous spots at the beginning and subsequently results in papules, vesicles, pustules, and scabs, which finally become dry and shaded (Spyrou and Valiakos 2015). Because of its contagious nature, zoonotic potential, and economic importance, it is considered a very important infection in ruminants. The economic importance of ORFV infection is seen as reduced food intake, impaired growth, and a drop in the market value of the animals (Abdullah et al. 2015; Sadiq et al. 2017). It is transmitted via direct contact with damaged skin and replicates in epidermal cells. Transmission usually occurs during grazing and through abrasions developed on the lips, nostrils, and mouth (Spyrou and Valiakos 2015). The disease affects primarily sheep and goat, but camels, steenboks, deer, reindeer, bighorn sheep, Dall sheep, musk oxen, mountain goats, dogs, cats, and squirrels have been reported to be affected as well (Spyrou and Valiakos 2015).

The incubation period is 3–8 days; the proliferative lesions are typically formed on the lips, face, ears, coronary band, scrotum, teats, or vulva as seen in Fig. 1 (Smith and Sherman 2011). The lesions are more severe in animals less than 2 months old (Spyrou and Valiakos 2015; Sadiq et al. 2017). Affected lambs and kids have difficulty in suckling and grazing, whereas most adults with lesions on the lips continue to eat and drink. In addition, lesions can also be seen in the esophagus, abomasum, intestines, or respiratory tract of lambs and kids (Chan et al. 2007). Its diagnosis is based on the clinical signs and history; however, confirmation can be made through viral isolation and molecular detection (Spyrou and Valiakos 2015). IgM and IgG antibodies are good indicators of infection status in farm animals (Jesse et al. 2018). In vitro cell culture lines used in the isolation of ORFV include primary chicken fibroblast, lamb testis, Madin-Darby canine kidney cell line (Abdullah et al. 2015), and Madin-Darby bovine kidney (Klein and Tryland 2005). Polymerase chain reaction (PCR), loop mediated isothermal amplification assay, and phylogenetic analysis are important molecular detection methods that are used in the confirmation of ORFV infection from skin biopsy samples (Abdullah et al. 2015; Klein and Tryland 2005).

Fig. 1
figure 1

Skin lesions at the dorsum of the rats in the inoculation groups. a Moderate hyperemia of the dorsum of a rat in the dexamethasone group; b scabs and hyperemia of the dorsum of a rat inoculated with ORFV UPM 1/14 isolate; c moderate hyperemia (black arrow) of the ear pinna of a rat inoculated with ORFV UPM 1/14 isolate; d mild hyperemia (black arrow) of the ear pinna of a rat inoculated with ORFV UPM 2/14 isolate; e skin lesion at the labial commissure of a rat in the treatment group; f ruffled hair coat at the cranial and middle parts of the body

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Haig and McInnes (2002) reported that the immune reaction to Orf virus infection involves neutrophils, dermal dendritic cells, T cells, B cells, and antibody response. CD4+ T cells, IFN-γ, and CD8+ T cells are also involved in partial protection against ORFV infection (Haig and McInnes 2002); it also involves local inflammatory response in the skin and cytokine response in the lymph. Repeated infection with ORFV may result in a reduced lesion size and time of resolution. This is due to the interference of immune response caused by immune-modulatory protein encoding (Haig and McInnes 2002). Neutralizing antibodies have been detected with enzyme-linked immunosorbent assay (ELISA), immunofluorescent antibody test (IFAT), and viral neutralization test (VNT) in several Orf virus studies, confirming their involvement in ORFV infection (Gameel and Homaida 2008; Zeedan et al. 2015). Detailed studies on the biology of Orf virus are imperative in order to elucidate its ability to re-infect the same host; therefore, an experimental animal model is important in order to explore this. However, there is a paucity of pathogenicity studies using ORFV in the rat model. Besides, dexamethasone administration in experimental animal studies has been shown to mimic field stress conditions which make the animals more susceptible to infection. Thus, this study was carried out in order to investigate the pathogenicity of Malaysian isolates of Orf virus in a rat model with specific emphasis on the effects of different routes of inoculation and immunosuppression using parenteral dexamethasone administration.

Materials and methods

Animal management

Male Sprague Dawley rats weighing 200–250 g were used in this study. The rats were housed at the Faculty of Veterinary Medicine animal house facility, Universiti Putra Malaysia. Acclimatization was done for 2 weeks prior to commencement of the experiments. Standard chow and water were used to feed the rats ad libitum.

Preparation of virus suspension

Two Malaysian strains of Orf virus, UPM1/14 and UPM2/14, were obtained from the Virology Laboratory, Faculty of Veterinary Medicine, Universiti Putra Malaysia, and used as the viral inocula; the strains were previously isolated and characterized (Abdullah et al. 2015) following the standards for the virus isolation protocol from skin tissue (scab) of the infected animal prepared as 10% virus suspension. PCR using the B2L gene and F1L gene as target templates was carried for the detection of positive samples prior to virus isolation in cell culture (Abdullah et al. 2015). A 10% virus suspension was prepared by homogenizing 1 g of scabs with 1 g of sterile sand and 1 mL of sterile phosphate-buffered saline (PBS). The mixture was centrifuged at 1000 rpm for 10 min and the supernatant collected and filtered using a 0.45-μm syringe filter. Penicillin, streptomycin, and nystatin were added following filtration, and the suspension was stored at − 80 °C until use.

The virus suspension was subjected to titration by tissue culture infective dose 50% (TCID50) in order to determine the TCID50 where ten dilutions of the suspension (10−1 to 10−10) were made and 100 μL of each dilution was added onto a confluent lamb testis (LT) monolayer in a 24-well culture plate. The plate was incubated with the virus suspension at 37 °C for 1 h and washed with sterile PBS before addition of fresh DMEM media containing 1% FBS. The plate was incubated at 37 °C and observed daily for evidence of cytopathic effect (CPE) in the cells. After 1 week of incubation, the wells showing visual evidence of CPE in all dilutions were recorded and the 50% tissue culture infective dose (TCID50) was estimated using a method previously described by Reed and Muench (1938).

$$ \mathrm{proportionate}\ \mathrm{distance}=\frac{\left(\%\mathrm{CPE}\ \mathrm{at}\ \mathrm{dilution}\ \mathrm{next}\ \mathrm{above}\ 50\%\right)-50\%}{\left(\%\kern0.75em \mathrm{CPE}\ \mathrm{at}\ \mathrm{next}\ \mathrm{dilution}\ \mathrm{above}\ 50\%\right)-\left(\%\mathrm{CPE}\ \mathrm{at}\ \mathrm{next}\ \mathrm{dilution}\ \mathrm{below}\ 50\%\right)} $$

Experimental design and viral inoculation

Two experiments were conducted in this study. In the first experiment, the pathogenicity of two Malaysian Orf virus isolates was determined through the inoculation of mice at different sites (dorsal skin, ear pinna, and labial commissure). Here, thirty-five 7- to 8-week-old Sprague Dawley rats were grouped into 3; the control group had 5 rats, while inoculation groups 1 (ORFV UPM 1/14 isolate) and 2 (ORFV UPM 2/14 isolate) had 15 rats each. Inoculation group 1 (ORFV UPM 1/14 isolate) was subdivided into 1A, 1B, and 1C with 5 rats each and inoculated with 0.5 mL of ORFV UPM 1/14 isolate at the dorsal skin, ear pinna, and labial commissure, respectively. Inoculation group 2 (ORFV UPM 2/14 isolate) was subdivided into 2A, 2B, and 2C with five rats each and inoculated with 0.5 mL of ORFV UPM 2/14 isolate at the dorsal skin, ear pinna, and labial commissure, respectively.

The second experiment investigated the effect of dexamethasone administration on the pathogenicity of ORFV UPM 1/14. Dexamethasone has been used to induce immunosuppression in laboratory animals which mimics field stress conditions in farm animals. Here, 15 rats were divided into 3 groups of 5 rats each: the dexamethasone group (n = 5), which was administered dexamethasone at a concentration of 5 mg/kg, intramuscularly, two times daily for two consecutive days prior to virus inoculation and for 5 days post virus inoculation; the non-dexamethasone group (n = 5); and the control group (n = 5). Both the dexamethasone and non-dexamethasone groups were inoculated with ORFV UPM 1/14 isolate at the dorsal skin, ear pinna, and labial commissure, while the control was injected with distilled water at all the sites above. All the animals were monitored daily for clinical signs and scoring was performed.

Clinical monitoring and histological examination

General clinical and lesion examinations were performed daily. Four clinical signs were evaluated and scored and recorded accordingly. These clinical indicators/parameters were level of alertness, ruffled hair coat, ocular discharge, and skin lesions (Table 1). The rats were euthanized, and skin specimens were fixed in 10% neutral buffered formalin, processed, stained with Harris hematoxylin and eosin, and examined microscopically.

Table 1 Scoring for clinical signs
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Molecular detection

DNA extraction was carried out from infected rat skin tissues using a viral nucleic acid extraction kit (Vivantis GF-1 tissue DNA) as instructed by the manufacturer. Detection of ORFV by PCR was carried out using B2L and F1L viral genes. The primer sequences used were B2L forward primer (5-ATG TGG CCG TTC TCC TCT ATC-3), B2L reverse primer (5-TTA ATT TAT TGG CTT GCA G-3), F1L forward primer (5-ATG GAT CCA CCC GAA ATC CAG-3), and F1L reverse primer (5-TCA CAC GAT GGC CGT GAC CAG-3). The PCR amplification and gel electrophoresis protocols used were as previously described (Abdullah et al. 2015).

Statistical analysis

Data obtained from clinical scores and histopathological lesion scores were summarized as mean ± SEM and comparisons done using one-way analysis of variance (ANOVA) and Tukey’s post hoc test. Data that were not normally distributed were analyzed using the Mann-Whitney U test and Kruskal-Wallis H test. All the statistical analyses were conducted using IBM SPSS software version 20.

Results

Clinical signs

Ruffled hair coat, reduced alertness, and skin lesions were observed in the inoculation groups. Ocular discharge was absent in all the inoculation groups. Mild hyperemia was observed at the dorsum, ear pinna, and labial commissure of the rats in both groups 1 (UPM 1/14) and 2 (UPM 2/14) (Fig. 1). The dexamethasone group showed mild to moderate hyperemia at the dorsum, ear pinna, and labial commissure, while the non-dexamethasone group showed only mild hyperemia at these inoculation sites. Macules, raised rash on the skin resembling papules, and crusts were also observed in both the dexamethasone- and non-dexamethasone-treated groups. The control group showed transient skin redness after inoculation, which disappeared after 1 day.

The percentage of hyperemia on the skin of the rats in groups 1 and 2 was 80 and 40%, respectively, on the dorsum, while scab formation was 20% in both groups. Hyperemia on the ear pinna was 60 and 80%, respectively, in groups 1 and 2, and 60 and 40% on the labial commissure. The dexamethasone- and non-dexamethasone-treated groups had 60 and 40% hyperemia on the dorsum, 40 and 80% hyperemia on the ear pinna, and 80 and 40% hyperemia on the labial commissure with 20% macule on the ear pinna.

At 14 days post inoculation, there was a significant difference in the mean score of ruffled hair coat between groups 1A, 1B, 1C, and control (0.007, 0.000, 0.000, and 0.000). However, Mann-Whitney U tests showed no significant differences in the mean ruffled hair coat scores between groups 1A and 1B (U = 77, P = 0.072), between groups 1A and 1C (U = 77, P = 0.072), and between groups 1B and 1C (U = 98, P = 1.000). There was no ruffled hair observed in all the rats in group 2A, 2B, 2C, and control.

The mean scores of clinical signs between the two Malaysian ORFV inoculation groups showed no difference (P > 0.05) in ruffled hair coat and ocular discharge. However, the level of alertness and skin lesion scores were higher (P < 0.05) in rats in the ORFV UPM 1/14 inoculation group (Table 2).

Table 2 Mean total scores of clinical signs observed at 14 days post virus inoculation of group 1 (ORFV UPM 1/14), group 2 (ORFV UPM 2/14), and the control
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The mean scores of clinical signs in the dexamethasone-treated and non-treated groups showed a higher (P < 0.05) level of alertness and skin lesions in the dexamethasone-treated group (Table 3).

Table 3 Mean total scores of clinical signs observed at 7 days post inoculation in the dexamethasone-treated and non-dexamethasone-treated groups
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Ocular discharge

No ocular discharges were observed in all treatment groups; therefore, there were no comparisons between groups.

Level of alertness

The mean scores for level of alertness in rats inoculated with two different isolates of ORFV showed a higher (P < 0.05) level of alertness in rats inoculated with UPM 1/14 at all sites as compared to those inoculated with UPM 2/14 (Table 4).

Table 4 Mean scores of alertness observed at 14 days post inoculation of group 1 (ORFV UPM 1/14), group 2 (ORFV UPM 2/14), and the control
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Skin lesions

The mean skin lesion scores in rats inoculated with two different isolates of ORFV were higher (P < 0.05) in the ORFV UPM 1/14 inoculation group as compared to the ORFV UPM 2/14 group following dorsum and labial commissure injection and lower following ear pinna inoculation (Table 5).

Table 5 Mean scores of skin lesions observed at 14 days post inoculation of group 1 (ORFV UPM 1/14), group 2 (ORFV UPM 2/14), and the control
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Histopathological changes

Keratosis, acanthosis, and ballooning degeneration were observed in all treatment groups, and occasionally, intracytoplasmic eosinophilic inclusion bodies were seen. Other findings were vasculitis and congestion, which were observed in some sections. Infiltration of inflammatory cells was observed in the dexamethasone-treated group.

There was evidence of keratosis, acanthosis, and ballooning degeneration of cells at the stratum spinosum and stratum basale in groups 1A, 1B, and 1C (Fig. 2). Group 2A also showed evidence of keratosis, acanthosis, and ballooning degeneration of cells of the stratum spinosum and stratum basale, group 2B had acanthosis and keratosis, while group 2C had keratosis, acanthosis, and ballooning degeneration of cells of the stratum spinosum and stratum basale (Fig. 3).

Fig. 2
figure 2

Histopathological changes observed in rats inoculated with ORFV UPM 1/14 through different routes. a Group 1A showing acanthosis (black arrow) and keratosis (red arrow). b Group 1B showing acanthosis (black arrow) and keratosis (red arrow). c Group 1C showing acanthosis of the stratum spinosum (black arrow). d Group 1C showing ballooning degeneration of cells in the stratum spinosum (black arrow). H&E, × 200 and × 400

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Fig. 3
figure 3

Histopathological changes observed in rats inoculated with ORFV UPM 2/14 through different routes. a Group 2A showing keratosis (black arrow) and ballooning degeneration (red arrow) of cells at the stratum spinosum. b Group 2B showing acanthosis (black arrow) at the stratum spinosum of the upper ear pinna. c Group 2C showing ballooning degeneration (black arrow). d Group 2C ballooning degeneration (black arrow) and acanthosis (red arrow). H&E, × 400

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The dexamethasone-treated group showed evidence of keratosis, acanthosis, and ballooning degeneration of cells at the stratum spinosum and stratum basale with the presence of a circumscribed area of suppurative dermatitis containing leucocytes and dead tissue. There was also vascular congestion in the dermis (Fig. 4). The non-dexamethasone-treated group also had evidence of keratosis, acanthosis, and ballooning degeneration of cells at the stratum spinosum and stratum basale. A few inflammatory cells were also seen in the dermis of the upper ear pinna (Fig. 5).

Fig. 4
figure 4

Histopathological changes observed in rats in the dexamethasone-treated group. a Bulging of epidermis due to suppurative dermatitis with infiltration of leucocytes in the labial commissure (black arrow) and congestion at dermis (red arrow); H&E, × 100. b Presence of acanthosis and ballooning of epidermal cells (black arrow) at the dorsum and keratosis (red arrow); H&E, × 200

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Fig. 5
figure 5

Histopathological changes observed in rats in the non-dexamethasone-treated group. a Acanthosis (black arrow) at the stratum spinosum of the upper ear pinna, with presence of few inflammatory cells (red arrow); H&E, × 200. b Keratosis (red arrow) and acanthosis (black arrow) at the dorsum; H&E, × 200

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Mean thickness of the stratum spinosum and basale

The mean thickness of the stratum spinosum and stratum basale was measured and compared between the different inoculation groups. There was no difference (P > 0.05) in the mean thickness of the stratum spinosum and stratum basale between ORFV UPM 1/14 and ORFV UPM 2/14 groups inoculated through all routes (dorsum, ear pinna, and labial commissures) (Table 6).

Table 6 Mean thickness of the stratum spinosum and stratum basale in the dorsum, ear pinna, and labial commissure of the rats inoculated with two different Malaysian isolates of ORFV
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The mean thickness of the stratum spinosum in the dorsum, ear pinna, and labial commissure was higher (P < 0.05) in the dexamethasone-treated group. However, the mean thickness of the stratum spinosum is higher (P < 0.05) in the dorsum of the dexamethasone-inoculated group and not different (P > 0.05) in the ear pinna and labial commissure between the two groups (Table 7).

Table 7 Mean thickness of the stratum spinosum and stratum basale in the dorsum, ear pinna, and labial commissure of dexamethasone- and non-dexamethasone-treated groups
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Molecular detection of Orf virus by PCR

PCR-positive amplification of ORFV was detected using F1L and B2L genes from the skin tissues of rats from all inoculation groups with and without dexamethasone administration (Fig. 6).

Fig. 6
figure 6

Gel electrophoresis of positive amplifications from rats experimentally inoculated with ORFV UPM isolate. Positive detection of two ORFV genes, B2L and F1L, was confirmed. M, 100-bp marker; 1, +ve control (F1L); 2, +ve control (B2L); 3, group 1A (F1L); 4, group 1A (B2L); 5, group 1B (F1L); 6, group 1B (B2L); 7, group 2A (F1L); 8, group 2A (B2L); 9, group 2B (F1L); 10, group 2B (B2L)

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Discussion

This study evaluated the effect of Malaysian ORFV isolates in rats. The clinical signs observed on the different inoculation sites were generally mild with presence of hyperemia and macules. These findings concur with a previous report in rabbits and mice inoculated with Orf virus in an experimental study in Brazil (Cargnelutti et al. 2011). Thickening of the skin and redness at the sites of injections observed in this study were consistent with findings of erythema, macules, papules, and vesicles or pustules in dried sheep and goat scabs in India (Hosamani et al. 2006). There is, however, species variation with respect to the clinical course of Orf virus infection. Cargnelutti et al. (2011) indicated that maculopapular, vesicular, and pustular lesions were only observed in rabbits, while other lesions are typical in goat and lambs (Gallina et al. 2008).

Dexamethasone has the ability to suppress both cell-mediated and humoral immunity (Coutinho and Chapman 2011). It had been used in some researches and results in an increase in disease severity. Studies have shown that in immunocompromised animals, an ORFV lesion will be more extensive. The dexamethasone group had mild to moderate hyperemia at all inoculation sites, while the non-dexamethasone group had only mild hyperemia. These together with other changes observed in the mean level of alertness, mean skin lesion score, and mean thickness of the stratum spinosum and basale indicate that the dexamethasone induced immune suppression which resulted in more lesion severity. A similar finding was reported by Haig and McInnes (2002), who described the relationship between Orf virus infection and immune-modulators in sheep. The authors reported that this process involved neutrophils, dermal dendritic cells, T cells, B cells, and antibody production during the infection phase. Similarly, immunosuppression was also reported to potentiate ORFV infection (Haig and McInnes 2002).

The histopathological changes observed in this study such as acanthosis, ballooning degeneration, and keratosis are similar to the pathological changes observed in ORFV infection in sheep and goats (Gallina et al. 2008; Pompei 2010). Similar histopathological changes were observed in rabbits and mice intradermally inoculated with ORFV (Cargnelutti et al. 2011). However, the mild pathogenicity recorded in this study might be due to a lower viral load in the suspension used or due to slow ORFV growth resultant from a short experimental period as natural incubation of the ORFV is usually 3 weeks and above (Zamri-Saad et al. 1992) in sheep and goats and about 1–4 weeks in humans (Kumar et al. 2015). Other studies reported failures in inducing ORFV infection through inoculation with scab suspension in guinea pigs, mice (Asakawa et al. 1952; Greig 1956), and rabbits (Wheeler et al. 1956).

In this study, rats inoculated with ORFV UPM 1/14 showed more pathogenicity than ORFV 2/14. However, there was no significant difference in the clinical signs and mean thickness of the stratum spinosum and stratum basale between these two groups. Therefore, this study suggests that ORFV UPM 1/14 is more pathogenic than ORFV UPM 2/14 in rats. Based on the results from this study, the dorsum, ear pinna, and labial commissure showed varying lesions which also suggests that inoculation sites play a critical role in the development of ORFV infection (Asakawa et al. 1952; Cargnelutti et al. 2011; Gallina et al. 2008; Wheeler et al. 1956). Intradermal inoculation of rats with ORFV UPM 1/14 did not show significant difference among the dorsum, ear pinna, and labial commissure, thus suggesting that any of the three inoculation sites can be used for inoculation in the rat model.

This study detected ORFV through PCR amplification from the skin tissues of all infected rats, confirming that the lesions observed were induced by the virus. PCR is a powerful diagnostic method for the detection of Orf virus from the infected host (Abdullah et al. 2015). The virus had been isolated and detected from the lesions in many experimental studies (Cargnelutti et al. 2011); (Arya et al. 2014).

Conclusion

This study suggests that Malaysian strains ORFV UPM 1/14 and ORFV UPM 2/14 are pathogenic in rats, as shown by the presence of clinical lesions similar to what was observed in the natural host. Thus, the rat model is an acceptable model to study the pathogenesis of ORFV. This study also showed that the dorsum, ear pinna, and labial commissure can be used as inoculation sites in the study ORFV in rats. Dexamethasone treatment showed increased pathogenicity in the inoculated groups as evidenced by higher mean lesion scores in thickness of the stratum spinosum and stratum basale in the dorsum.