Alterations of hemogram, serum biochemistry, oxidative/nitrosative balance, and copper/zinc homeostasis in dromedary camels naturally infected with poxvirus.

Camel pox (CMLP), a contagious viral disease of camels, causes considerable economic loss in terms of milk, meat, wool, and leather production besides reduction of draught power. The effect of spontaneous CMLP infection on hemogram, oxidative/nitrosative imbalance, and trace mineral homeostasis has not been studied earlier in dromedary camels. In the current study, hemogram, serum biochemistry, oxidant/antioxidant imbalance, and zinc (Zn)-copper (Cu) homeostasis were evaluated in healthy and pox-infected camels. The CMLP was confirmed from pooled samples of vesicular fluid, oral mucosa, and skin samples by polymerase chain reaction (PCR) targeting the C18L gene of CMLP virus. Hemogram was performed manually in whole blood. The serum was analyzed for biochemistry. The oxidative/nitrosative imbalance was measured by determining the concentrations of malondialdehyde (MDA), nitrite and nitrate (NOx), and glutathione S-transferase (GST) activity in serum. Simultaneously, copper (Cu) and zinc (Zn) concentrations were measured in serum. A pronounced leucopenia (p = 0.019), lymphopenia (p = 0.005), and hypoproteinemia (p = 0.014) were noted in CMLP-infected camels compared to healthy animals. The significant elevation of the MDA (p = 0.005) and NOx (p = 0.044) concentrations in serum of CMLP-infected indicated marked oxidative stress during the disease. The zinc concentration (p = 0.014) in CMLP-infected camels was significantly lower than healthy camels. The study supports that oxidative/nitrosative imbalance and Cu-Zn homeostasis are compromised and related to the pathophysiology of CMLP infection. The finding will be helpful to veterinary clinicians to adopt effective therapeutic strategies using antioxidants and trace minerals during CMLP outbreak. The timely vaccination and bio-security will be the mainstay for prevention of the diseases.

Putative parapoxvirus-associated foot disease in the endangered huemul deer (Hippocamelus bisulcus) in Bernardo O'Higgins National Park, Chile.

The huemul (Hippocamelus bisulcus) is an endangered cervid endemic to southern Argentina and Chile. Here we report foot lesions in 24 huemul from Bernardo O'Higgins National Park, Chile, between 2005 and 2010. Affected deer displayed variably severe clinical signs, including lameness and soft tissue swelling of the limbs proximal to the hoof or in the interdigital space, ulceration of the swollen tissues, and some developed severe proliferative tissue changes that caused various types of abnormal wear, entrapment, and/or displacement of the hooves and/or dewclaws. Animals showed signs of intense pain and reduced mobility followed by loss of body condition and recumbency, which often preceded death. The disease affected both genders and all age categories. Morbidity and mortality reached 80% and 40%, respectively. Diagnostics were restricted to a limited number of cases from which samples were available. Histology revealed severe papillomatous epidermal hyperplasia and superficial dermatitis. Electron microscopy identified viral particles consistent with viruses in the Chordopoxvirinae subfamily. The presence of parapoxvirus DNA was confirmed by a pan-poxvirus PCR assay, showing high identity (98%) with bovine papular stomatitis virus and pseudocowpoxvirus. This is the first report of foot disease in huemul deer in Chile, putatively attributed to poxvirus. Given the high morbidity and mortality observed, this virus might pose a considerable conservation threat to huemul deer in Chilean Patagonia. Moreover, this report highlights a need for improved monitoring of huemul populations and synergistic, rapid response efforts to adequately address disease events that threaten the species.

[Rapid immunochemical method for the detection of orthopoxviruses (Orthopoxvirus, Chordopoxvirinae, Poxviridae).]

INTRODUCTION: The abolition of smallpox vaccination has led to the disappearance of population immunity to pox viruses. However, the threat of infection by pathogenic orthopoxviruses persists and determines the need to develop sensitive and operational methods for indicating pathogens. OBJECTIVES: Development of a sensitive, fast and easy-to-use immunochemical test for the detection of orthopoxviruses in the «point of care¼ format. MATERIAL AND METHODS: We used preparations of cultural vaccinia virus (VV) with varying degrees of purification, polyclonal antibodies from hyperimmune rabbit serum, and equipment from a previously developed autonomous kit for dot-immunoassay on flat protein arrays. RESULTS AND DISCUSSION: It has been established that rabbit polyclonal antibodies can be used in a single-stage dotanalysis, both as a capture agent immobilized on a substrate and as a detection reagent bound with colloidal gold particles. It is shown that the effectiveness of the detection of VV is inversely related to the degree of purification of viruses from sub-viral structures. The sensitivity of the rapid detection of viruses in a crude preparation was about 30 times higher than in pure viral material. The increase in sensitivity, presumably, occurs due to binding to the capture antibodies of subviral structures, which form large aggregates of sensitized gold particles. The test does not detect cross-reactions with heterogeneous viruses (measles, rubella and chickenpox) that cause exantematous diseases. CONCLUSION: The one-stage variant of the dot-immunoassay reduces the analysis time to 40 minutes and improves the detection sensitivity of orthopoxviruses in crude viral preparations to the range of 105-104 PFU / ml. Full makeup, ease of analysis and the ability to visually accounting for results allow the test to be used outside of laboratories.

Seroprevalence and participatory epidemiology of camelpox in Afar region of Ethiopia.

Camelpox is endemic in most camel rearing regions of the world, causing significant economic losses. However, its epidemiology is not extensively investigated. We conducted a cross sectional seroprevalence study of camelpox in Amibara and Awash Fentale districts in Afar region of Ethiopia from November 2014 to May 2015. In addition, participatory epidemiology (PE) was conducted to identify seasonal occurrence of the disease in the study districts. Blood samples were collected from 384 dromedary camels from 31 herds distributed in five pastoral associations (PAs) in the two districts. Serum samples were separated from the blood samples and tested for the presence of viral antibodies using virus neutralization test. Seroprevalence data were analyzed using multilevel mixed effects logistic regression models accounting for the 4-level hierarchical data structure (camels nested in herds-herds in PA, and PA in district). For the participatory data, Kendall's coefficient of concordance was used to assess agreements between the informants in identifying seasonal occurrences of the top five camel diseases. Camelpox antibodies were detected in 19.3% of camels (n = 384), 81% of herds (n = 31), and in all five PAs from the two districts in the Gabi Rasu zone of Afar region, Ethiopia. The seroprevalence did not significantly vary between herds, PAs or districts suggesting the widespread occurrence of the disease. Estimated age stratified basic reproduction number (R0) was 1.25 (95% CI: 0.62-2.19). Camelpox was identified as one of the top five common camel diseases in the area. The widespread occurrence of the disease can be attributed mainly to the commingling of camels from many herds during seasonal migration in search of feed and water, a practice very common under pastoral production systems. Although the PE informants indicated the clinical disease to be more common in young animals, seropositivity was higher in older animals. Camelpox commonly occurs during the minor and major rainy seasons. In conclusion, camelpox is found to be endemic in Afar pastoral region with sporadic outbreaks occurring during rainy seasons. Vaccination and improved camel management practices particularly during the high-risk period can be viable strategies to reduce the burden of the disease.

Expression and evaluation of recombinant P32 protein based ELISA for sero-diagnostic potential of capripox in sheep and goats.

The study is aimed to develop and evaluate a recombinant P32 protein based ELISA for sero-monitoring and sero-surveillance using known and random/suspected serum samples for capripox infections from sheep and goats. Truncated P32 gene of goatpox virus (with an ORF of 750 bp) was expressed in E. coli BL-21 CodonPlus (DE3)-RIPL cells using pET32a vector and characterized by SDS-PAGE analysis and confirmed by western blotting as 48 kDa polyhistidine-tagged fusion protein. The protein was purified under denaturing conditions using 8M urea and characterized by SDS-PAGE and immunoblotting. The purified protein was used for optimizing ELISA in a chequerboard titration method using anti-GTPV serum as known positive. The optimized conditions were found to be 300 ng of protein/well, 1:10 dilution of antibody, 1:10000 dilution of rabbit anti-goat/sheep conjugate with 3% skim milk powder and 2% gelatin in phosphate buffer saline containing tween-20 as blocking buffer. The expressed protein was specific only for goatpox virus and sheeppox virus but did not react with related viruses of sheep and goats namely orf virus, peste de petits ruminants virus, bluetongue virus and foot and mouth disease virus. The optimized ELISA was evaluated using pre-vaccinated, post-vaccinated and also post-challenge sera. The assay was found to have a diagnostic specificity of 100/98.7% and sensitivity of 97.1/98.1% when compared to whole virus antigen based ELISA/SNT by receiver operating characteristic (ROC) analysis. The optimized ELISA is able to determine the progression of antibody response against GTPV and SPPV following vaccination and challenge in sheep and goats. The rP32 protein based ELISA was evaluated using random field serum samples (n = 1008) suspected for sheeppox and goatpox and it has shown positivity rate as 24.4%. The rP32 protein based ELISA was found to be specific and sensitive for sero-evaluation of sheeppox virus and goatpox virus following vaccination and infection in sheep and goats.

Seroprevalence of Orthopoxvirus in rural Brazil: insights into anti-OPV immunity status and its implications for emergent zoonotic OPV.

BACKGROUND: Bovine vaccinia (BV) is a zoonosis caused by Vaccinia virus, a virus from Orthopoxvirus genus (OPV) that affects mainly cattle herds and humans in rural areas in Brazil. Because most studies have focused on outbreaks situations, data on BV epidemiology is limited. A cross sectional study in Brazilian rural areas during 2012-2013 was conducted to determine the neutralizing antibodies seroprevalence and risk factors for BV. METHODS: A structured questionnaire was applied to elicit demographics data and farming practices considered risk factors for BV exposure. Neutralizing anti-OPV antibodies were investigated using plaque reduction neutralization test. The neutralizing antibodies prevalence rates were calculated and the risk factor analysis was performed using multivariate logistic regression. RESULTS: Two hundred and forty participants were enrolled in this study with a prevalence of neutralizing antibodies of 30.8 % (95 % confidence interval [CI], 25.3-36.9). In multivariate analysis, age > 35 years (Odds Ratio [OR] = 18.2; CI 95 % = 7.7 - 43.2) and previous outbreak in property (OR = 3.9; C I95 % = 1.2 - 12.6) were independently associated with anti-OPV neutralizing antibodies. CONCLUSIONS: In this study, anti-OPV protective immunity (neutralizing antibody titers) was assessed in an endemic BV Brazilian rural area. Our findings indicate that epidemiological surveillance is required and should be applied by public health authorities to create interventions and/or prevention strategies to avoid viral spread causing future outbreaks among individuals who are under risk of infection.

Effects of inactive parapoxvirus ovis on cytokine levels in rats.

The aim of this study is to determine the effects of iPPOV on pro-inflammatory and anti-inflammatory cytokine levels in rats. iPPOV (1 ml/rat) was administered intraperitoneal route to 49 rats, except for 7 rats (Control, 0 group). Serum samples were collected from 7 rats at 1st, 2nd, 4th, 8th, 12th, 16th and 24th hr after treatments. Levels of TNF-α, IL-6, IL-12 and IL-10 were determined using ELISA. Administration of iPPOV stimulated TNF-α (16th and 24th hr) and IL-6 (12th, 16th and 24th hr) synthesis and caused fluctuations in IL-10 and IL-12 concentrations. In conclusion, increased cytokine levels could be attributed to immunomodulatory activity of iPPOV, however, detailed studies are required to fully understand effects of iPPOV on immune system.

Squirrelpox virus: assessing prevalence, transmission and environmental degradation.

Red squirrels (Sciurus vulgaris) declined in Great Britain and Ireland during the last century, due to habitat loss and the introduction of grey squirrels (Sciurus carolinensis), which competitively exclude the red squirrel and act as a reservoir for squirrelpox virus (SQPV). The disease is generally fatal to red squirrels and their ecological replacement by grey squirrels is up to 25 times faster where the virus is present. We aimed to determine: (1) the seropositivity and prevalence of SQPV DNA in the invasive and native species at a regional scale; (2) possible SQPV transmission routes; and, (3) virus degradation rates under differing environmental conditions. Grey (n = 208) and red (n = 40) squirrel blood and tissues were sampled. Enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qPCR) techniques established seropositivity and viral DNA presence, respectively. Overall 8% of squirrels sampled (both species combined) had evidence of SQPV DNA in their tissues and 22% were in possession of antibodies. SQPV prevalence in sampled red squirrels was 2.5%. Viral loads were typically low in grey squirrels by comparison to red squirrels. There was a trend for a greater number of positive samples in spring and summer than in winter. Possible transmission routes were identified through the presence of viral DNA in faeces (red squirrels only), urine and ectoparasites (both species). Virus degradation analyses suggested that, after 30 days of exposure to six combinations of environments, there were more intact virus particles in scabs kept in warm (25 °C) and dry conditions than in cooler (5 and 15 °C) or wet conditions. We conclude that SQPV is present at low prevalence in invasive grey squirrel populations with a lower prevalence in native red squirrels. Virus transmission could occur through urine especially during warm dry summer conditions but, more notably, via ectoparasites, which are shared by both species.

Efficacy of tecovirimat (ST-246) in nonhuman primates infected with variola virus (Smallpox).

Naturally occurring smallpox has been eradicated but remains a considerable threat as a biowarfare/bioterrorist weapon (F. Fleck, Bull. World Health Organ. 81:917-918, 2003). While effective, the smallpox vaccine is currently not recommended for routine use in the general public due to safety concerns (http://www.bt.cdc.gov/agent/smallpox/vaccination). Safe and effective countermeasures, particularly those effective after exposure to smallpox, are needed. Currently, SIGA Technologies is developing the small-molecule oral drug, tecovirimat (previously known as ST-246), as a postexposure therapeutic treatment of orthopoxvirus disease, including smallpox. Tecovirimat has been shown to be efficacious in preventing lethal orthopoxviral disease in numerous animal models (G. Yang, D. C. Pevear, M. H. Davies, M. S. Collett, T. Bailey, et al., J. Virol. 79:13139-13149, 2005; D. C. Quenelle, R. M. Buller, S. Parker, K. A. Keith, D. E. Hruby, et al., Antimicrob. Agents Chemother., 51:689-695, 2007; E. Sbrana, R. Jordan, D. E. Hruby, R. I. Mateo, S. Y. Xiao, et al., Am. J. Trop. Med. Hyg. 76:768-773, 2007). Furthermore, in clinical trials thus far, the drug appears to be safe, with a good pharmacokinetic profile. In this study, the efficacy of tecovirimat was evaluated in both a prelesional and postlesional setting in nonhuman primates challenged intravenously with 1 × 10(8) PFU of Variola virus (VARV; the causative agent of smallpox), a model for smallpox disease in humans. Following challenge, 50% of placebo-treated controls succumbed to infection, while all tecovirimat-treated animals survived regardless of whether treatment was started at 2 or 4 days postinfection. In addition, tecovirimat treatment resulted in dramatic reductions in dermal lesion counts, oropharyngeal virus shedding, and viral DNA circulating in the blood. Although clinical disease was evident in tecovirimat-treated animals, it was generally very mild and appeared to resolve earlier than in placebo-treated controls that survived infection. Tecovirimat appears to be an effective smallpox therapeutic in nonhuman primates, suggesting that it is reasonably likely to provide therapeutic benefit in smallpox-infected humans.

The seroprevalence of avipoxvirus and its association with avian malaria (Plasmodium spp.) infection in introduced passerine birds in the southern regions of the North Island of New Zealand.

Blood samples were collected from 65 free-ranging birds from six species in the southern North Island of New Zealand. Sera from the birds were tested for the presence of avipoxvirus (APV) antibodies by enzyme-linked immunosorbent assay (ELISA), and blood cells from 55 birds were also tested for Plasmodium spp. by PCR. Forty-five birds (69.2%) tested seropositive to APV. Song thrushes (Turdus philomelos) presented the highest seroprevalence at 100% (4/4), followed by Eurasian blackbirds (Turdus merula) (96.86%, 31/32), chaffinches (Fringilla coelebs) (54.55%, 6/11), starlings (Sturnus vulgaris) (25%, 3/12), greenfinches (Carduelis chloris) (25%, 1/4), and European goldfinches (Carduelis carduelis) (0%, 0/2). Plasmodium spp. DNA was detected in 15/55 birds (27.3%), including 11 Eurasian blackbirds, one song thrush, and three starlings. Eight Eurasian blackbird isolates (73%) grouped within the subgenus Novyella. Two Eurasian blackbird isolates and the song thrush isolate clustered within a different group with previously reported lineages LINN1 and AFTRU5. In addition, all three starling isolates clustered within the well-characterized lineage Plasmodium (Huffia) elongatum GRW06. All Plasmodium-positive Eurasian blackbirds and the song thrush were seropositive to APV, whereas only 67% of Plasmodium-positive starlings showed evidence of previous exposure to APV. A significant relationship between birds seropositive to APV and birds infected by Plasmodium spp. was observed (chi2 = 5.69, df = 1, P = 0.0086). To the authors' knowledge this is the first report describing the seroprevalence of APV and its association with Plasmodium spp. infection in introduced bird species in New Zealand.

The pox in the North American backyard: Volepox virus pathogenesis in California mice (Peromyscus californicus).

Volepox virus (VPXV) was first isolated in 1985 from a hind foot scab of an otherwise healthy California vole (Microtus californicus). Subsequent surveys in San Mateo County, CA, revealed serological evidence suggesting that VPXV is endemic to this area, and a second viral isolate from a Pinyon mouse (Peromyscus truei) was collected in 1988. Since then, few studies have been conducted regarding the ecology, pathology, and pathogenicity of VPXV, and its prevalence and role as a potential zoonotic agent remain unknown. To increase our understanding of VPXV disease progression, we challenged 24 California mice (Peromyscus californicus) intranasally with 1.6 × 10(3) PFU of purified VPXV. By day five post infection (pi) we observed decreased activity level, conjunctivitis, ruffled hair, skin lesions, facial edema, and crusty noses. A mortality rate of 54% was noted by day eight pi. In addition, internal organ necrosis and hemorrhages were observed during necropsy of deceased or euthanized animals. Viral loads in tissues (brain, gonad, kidney, liver, lung, spleen, submandibular lymph node, and adrenal gland), bodily secretions (saliva, and tears), and excretions (urine, and/or feces) were evaluated and compared using real time-PCR and tissue culture. Viral loads measured as high as 2 × 10(9) PFU/mL in some organs. Our results suggest that VPXV can cause extreme morbidity and mortality within rodent populations sympatric with the known VPXV reservoirs.

Orthopox virus infections in Eurasian wild rodents.

The genus Orthopoxvirus includes variola (smallpox) virus and zoonotic cowpox virus (CPXV). All orthopoxviruses (OPV) are serologically cross-reactive and cross-protective, and after the cessation of smallpox vaccination, CPXV and other OPV infections represent an emerging threat to human health. In this respect CPXV, with its reservoir in asymptomatically infected wild rodents, is of special importance. In Europe, clinical cowpox has been diagnosed in both humans and animals. The main objective of this study was to elucidate the prevalence of OPV infections in wild rodents in different parts of Eurasia and to compare the performance of three real-time polymerase chain reaction (PCR) methods in detecting OPV DNA in wildlife samples. We investigated 962 wild rodents from Northern Europe (Finland), Central Europe (Germany), and Northern Asia (Siberia, Russia) for the presence of OPV antibodies. According to a CPXV antigen-based immunofluorescence assay, animals from 13 of the 17 locations (76%) showed antibodies. Mean seroprevalence was 33% in Finland (variation between locations 0%-69%), 32% in Germany (0%-43%), and 3.2% (0%-15%) in Siberia. We further screened tissue samples from 513 of the rodents for OPV DNA using up to three real-time PCRs. Three rodents from two German and one Finnish location were OPV DNA positive. The amplicons were 96% to 100% identical to available CPXV sequences. Further, we demonstrated OPV infections as far east as the Baikal region and occurring in hamster and two other rodent species, ones previously unnoticed as possible reservoir hosts. Based on serological and PCR findings, Eurasian wild rodents are frequently but nonpersistently infected with OPVs. Results from three real-time PCR methods were highly concordant. This study extends the geographic range and wildlife species diversity in which OPV (or CPXV) viruses are naturally circulating.

Occurrence of dual infection of peste-des-petits-ruminants and goatpox in indigenous goats of central India.

Peste-des-petits-ruminants (PPR), bluetongue (BT) and goatpox (GP) have been well recognized as causes of significant economic losses in the small ruminant population of Asia and Africa. We describe here the occurrence of these three in an outbreak noticed in non-descript goats from a subtropical region of central India. An investigation was carried out to confirm the aetiology of the heavy mortality in goats (74.6%, 112/150), with testing of samples from 12 surviving animals exhibiting mixed clinical signs indicative of PPR, BT and GP. Sandwich ELISA was used to detect PPR virus antigen and competition ELISA to detect PPR virus and BT virus antibodies. GP was confirmed on the basis of nodular lesions and an immunodiffusion assay. Eight of the 12 affected animals (66.7%) were positive for PPR virus and BT virus antibodies, and two goats (16.7%, 2/12) exhibiting clinical lesions of pox were also found positive for PPR virus/antibodies and BT virus antibodies, respectively. Although BT virus could not be identified in any sample, detection of BT virus antibodies indicated previous or possibly concurrent infection with BT virus in these goats. The N-gene-based RT-PCR was used to confirm the PPR infection in these goats, and one of the amplicons was sequenced. The sequence and phylogenetic analysis revealed close proximity to PPR virus isolates from Tibet and China, with sequence homology of up to 96.9%. The sequence homology was relatively low with the majority of other Indian isolates (72.7-93.5%). The detection of this new PPR virus sequence indicates the circulation of cross-border strains in this region of India. It is presumed that the heavy mortality observed in goats is possibly attributable to the occurrence of mixed infection of PPR and GP, or PPR, BT and GP.

Emerging Capripoxvirus disease outbreaks in Himachal Pradesh, a northern state of India.

Both sheep and goat pox are contagious viral diseases and affect small ruminants and are caused by sheep pox virus and goat pox virus respectively that belong to genus Capripoxvirus of Poxviridae family. Huge economic losses emanating from the disease outbreaks are the results of the wool and hide damage, subsequent production losses and also the morbidities and mortalities associated with the disease. This communication highlights clinico-epidemiological observations from the two sheep pox and one goat pox outbreaks. Grossly, multisystemic nodular lesions, mucopurulent nasal discharges and respiratory symptoms were observed in the affected animals. The morbidity, mortality and case fatality rates were 5.18%, 2.45% and 32.37%, respectively. Histopathological, haematological, molecular and serological techniques and also isolation of virus in embryonated chicken eggs were used for the diagnosis of the diseases. The spatial distribution of the disease signifies the role of common pasturelands used for grazing the animals while temporally all three outbreaks occurred in winters and were probably associated with cold stress and fodder scarcity. This is the first recorded report of Capripoxvirus infection in recent times and it highlights the disease as one of the emerging diseases in the northern state of Himachal Pradesh in India.

A silent enzootic of an orthopoxvirus in Ghana, West Africa: evidence for multi-species involvement in the absence of widespread human disease.

Human monkeypox has never been reported in Ghana, but rodents captured in forested areas of southern Ghana were the source of the monkeypox virus introduced into the United States in 2003. Subsequent to the outbreak in the United States, 204 animals were collected from two commercial trapping sites in Ghana. Animal tissues were examined for the presence of orthopoxvirus (OPXV) DNA using a real-time polymerase chain reaction, and sera were assayed for antibodies against OPXV. Animals from five genera (Cricetomys, Graphiurus, Funiscirus, and Heliosciurus) had antibodies against OPXV, and three genera (Cricetomys, Graphiurus, and Xerus) had evidence of OPXV DNA in tissues. Additionally, 172 persons living near the trapping sites were interviewed regarding risk factors for OPXV exposure, and their sera were analyzed. Fifty-three percent had IgG against OPXV; none had IgM. Our findings suggest that several species of forest-dwelling rodents from Ghana are susceptible to naturally occurring OPXV infection, and that persons living near forests may have low-level or indirect exposure to OPXV-infected animals, possibly resulting in sub-clinical infections.

Patterns of orthopox virus wild rodent hosts in South Germany.

Although cowpox virus (CPXV) infections in a variety of dead-end hosts have been investigated in Germany for more than 50 years, data on species and geographical distribution of CPXV in reservoir hosts are sparse. Here we present the first comprehensive study of 825 rodents that have been collected in Bavaria, Southern Germany. In summary, six different rodent species (Apodemus flavicollis, Myodes glareolus, Microtus arvalis, Apodemus sylvaticus, Microtus agrestis, and Arvicola amphibius) were trapped at three main trapping sites and investigated using a serum neutralization test (SNT). Prevalence of orthopox virus (OPV)-neutralizing antibodies was (with exception of one trapping site) highest in bank voles, ranging from 24.5% to 42.4%; often with SNT titers > or =96. Two up to 25% of yellow-necked mice were OPV sero-positive, but wood mice only at one site with 5.5%. Up to 7.7% of common voles were found to be OPV seroreactive, while M. agrestis and A. amphibius only sporadically showed seroreactivity. Further analyses of a subset of 450 bank voles and yellow-necked mice trapped at one site over a 18-month period revealed that male yellow-necked mice and female gravid yellow-necked mice had significantly more OPV-neutralizing antibodies. Mean body weight and OPV-seroreactivity were significantly negatively associated in male A. flavicollis. This was not due to shorter body length or smaller body mass index, but previously OPV-infected male A. flavicollis had dramatically reduced mean kidney weights. Seroreactivity in female bank voles was positively related to lung weights. We also found that both male yellow-necked mice and male bank voles with positive SNT titers had higher infestation rates with ectoparasites. We here show for the first time that A. flavicollis beside M. glareolus is a hypothetic host for CPXV, and that there are big geographical and spatial variations concerning the seroprevalence in rodent populations in South Germany.

First cases of squirrelpox in red squirrels (Sciurus vulgaris) in Scotland.

Squirrelpox, caused by a poxvirus, is a major threat to the remaining UK red squirrel population. The spread of antibody-positive grey squirrels has been monitored in the UK for the past decade. In 2005 grey squirrels that had been exposed to the virus appeared in the south of Scotland for the first time, followed approximately two years later by the appearance of squirrelpox disease in the local red squirrels. Four squirrels were examined. They all had gross external lesions and histological lesions typical of squirrelpox disease, but no significant internal lesions. The diagnosis was confirmed by PCR, electron microscopy and serology.

Comparative efficacy of conventional and taqman polymerase chain reaction assays in the detection of capripoxviruses from clinical samples.

Sheeppox and goatpox are economically important viral diseases of sheep and goats, respectively. Both diseases are reportable to the World Organization for Animal Health. To implement a control and eradication program for these diseases, a rapid and user-friendly diagnostic tool is imperative for screening. Therefore, in the present study, TaqMan quantitative polymerase chain reaction (qPCR) and conventional PCR assays targeting the DNA polymerase (DNA pol) gene were developed for the detection of Capripoxvirus DNA from clinical specimens of sheep and goats. The 2 assays used different primer sets. Conventional PCR yielded a specific product of 134 bp, whereas qPCR yielded a 180-bp product. The specificity of amplified DNA pol gene products was confirmed by their size and by sequence analysis. The 2 assays were specific for Sheeppox virus and Goatpox virus. However, in comparison to conventional PCR, the qPCR was more rapid, specific, and 100 times more sensitive, with a detection limit as low as 0.042 pg of purified DNA. The qPCR assay was more sensitive (84.05%) than conventional PCR (76.06%) when used on clinical samples (n = 71) from sheep and goats.

A polymerase chain reaction strategy for the diagnosis of camelpox.

Camelpox is a contagious viral skin disease that is mostly seen in young camels. The disease is caused by the Camelpox virus (CMLV). In the present study, a polymerase chain reaction (PCR) assay based on the C18L gene (encoding ankyrin repeat protein) and a duplex PCR based on the C18L and DNA polymerase (DNA pol) genes were developed. The former assay yields a specific amplicon of 243 bp of the C18L gene, whereas the duplex PCR yields 243- and 96-bp products of the C18L and DNA pol genes, respectively, in CMLV, and only a 96-bp product of the DNA pol gene in other orthopoxviruses. The limit of detection was as low as 0.4 ng of viral DNA. Both PCR assays were employed successfully for the direct detection and differentiation of CMLV from other orthopoxviruses, capripoxviruses, and parapoxviruses in both cell culture samples and clinical material. Furthermore, a highly sensitive SYBR Green dye-based, real-time PCR was optimized for quantitation of CMLV DNA. In the standard curve of the quantitative assay, the melting temperature of the specific amplicon at 77.6 degrees C with peak measured fluorescence in dissociation plot was observed with an efficiency of 102%. To the authors' knowledge, this is the first report to describe a C18L gene-based PCR for specific diagnosis of camelpox infection.

Pathological, serological, and virological findings in sheep infected simultaneously with Bluetongue, Peste-des-petits-ruminants, and Sheeppox viruses.

In this study, pathological, serological and virological examinations were performed on 15 sheep from a flock of 250 sheep and lambs that suffer from simultaneous naturally occurring BTV, PPRV and SPV outbreaks. SPV was diagnosed macroscopically and histopathologically, BTV was diagnosed by ELISA, and PPRV was diagnosed pathologically and by ELISA. Clinically fever, diarrhea, depression, polypnea, conjunctivitis, lacrimation, rhinitis, erosive stomatitis, edema of eyelids, photophobia, cutaneous eruption with erythematous areas especially noticeable in wool-free parts of the body and axilla lesions evolving into papules were observed. At necropsy, the most effected organs were lungs and gut. Subepicardial hemorrhages were also commonly seen. While typical pox lesions were observed in some lambs, usually fibrinous pleuropneumonia was more prominent lung lesion. SPV and PPRV lesions were seen at the histopathological examination of the lesioned tissues, BT lesions were mild than SPV and PPRV microscopically. Serum and leukocyte samples of 15 animals were examined for PPRV and BTV by ELISA; 5 samples were positive for PPRV and 6 BTV, 4 were positive for both PPRV and BTV simultaneously. One hundred animals died, most were lambs. Mortality rates were 100% in lambs and 80% in the herd.