Molecular detection of parapoxvirus in Ixodidae ticks collected from cattle in Corsica, France

Abstract Background Several viruses belonging to the family Poxviridae can cause infections in humans and animals. In Corsica, livestock farming (sheep, goats, pigs, and cattle) is mainly mixed, leading to important interactions between livestock, wildlife, and human populations. This could facilitate the circulation of zoonotic diseases, and makes Corsica a good example for studies of tick‐borne diseases. Objectives To gain understanding on the circulation of poxviruses in Corsica, we investigated their presence in tick species collected from cattle, sheep, horses, and wild boar, and characterized them through molecular techniques. Methods Ticks were tested using specific primers targeting conserved regions of sequences corresponding to two genera: parapoxvirus and orthopoxvirus. Results A total of 3555 ticks were collected from 1549 different animals (687 cattle, 538 horses, 106 sheep, and 218 wild boars). They were tested for the presence of parapoxvirus DNA on one hand and orthopoxvirus DNA on the other hand using Pangeneric real‐time TaqMan assays. Orthopoxvirus DNA was detected in none of the 3555 ticks. Parapoxvirus DNA was detected in 6.6% (36/544) of ticks collected from 23 cows from 20 farms. The remaining 3011 ticks collected from horses, wild boars, and sheep were negative. The infection rate in cow ticks was 8.0% (12/148) in 2018 and 6.0% (24/396) in 2019 (p = 0.57). Parapoxvirus DNA was detected in 8.5% (5/59) of Hyalomma scupense pools, 8.2% (15/183) of Hyalomma marginatum pools, and 6.7% (16/240) of Rhipicephalus bursa pools (p = 0.73). We successfully amplified and sequenced 19.4% (7/36) of the positive samples which all corresponded to pseudocowpox virus. Conclusions Obviously, further studies are needed to investigate the zoonotic potential of pseudocowpox virus and its importance for animals and public health.

There are three known zoonotic orthopoxvirus species: monkeypox virus, cowpox virus, and vaccinia virus which are associated with outbreaks in Africa, Europe, South America, and Asia (Singh et al., 2007).
Humans are susceptible to monkeypox virus, cowpox virus, vaccinia virus, bovine popular stomatitis virus, orf virus, and pseudocowpox virus. Although the complete host range of these viruses is unclear, domestic animals such as sheep, goats, cats, dogs, and dairy cows can be infected with orthopoxvirus and/or parapoxvirus .
Infected humans play an important role in the spread of orthopoxvirus and parapoxvirus among domestic animals, especially during milking and other livestock-related occupational activities McFadden, 2005). Clinically, the exanthematous lesions caused by zoonotic orthopoxvirus and parapoxvirus species are very similar, especially in humans and cows, and can be diagnosed in areas of orthopoxvirus/parapoxvirus cocirculation (Inoshima et al., 2000).
Recently, the presence of two parapoxvirus (pseudocowpox virus and bovine popular stomatitis virus) was reported in ticks collected from zebu cattle in Eastern Burkina Faso (Ouedraogo et al., 2020).
Although the natural interaction between ticks and the detected parapoxvirus in that study is unknown, this finding shows that ticks may be a good indicator of the spread of these pathogens.
In Corsica, a French Mediterranean island, ticks of the genus Ixodes, Hyalomma, Dermacentor, Haemaphysalis, and Rhipicephalus have been identified and can act as vectors for a variety of emerging diseases Cicculli et al., 2020;Cicculli, Oscar, et al., 2019;Grech-Angelini et al., 2020

DNA extraction and polymerase chain reaction detection
Ticks were washed once in 70% ethanol for 5 min and then twice in distilled water for 5 min. Ticks were analyzed as pools consisting of 1-6 ticks of the same species, same stage, and collected from the same animal (Table 2). Individual ticks or pools of ticks were crushed in minimal essential medium containing antibiotics and fungicide, using the TissueLaser II (Qiagen, Hilden, Germany) at 30 cycles/s of 3 min. DNA extraction was performed on a QIAcube HT (Qiagen) using a QIAamp Cador Pathogen Minikit according to the manufacturer's instructions.
DNA was eluted in 100 µl of buffer and stored at -80 • C. Extraction was monitored by systematic spiking of each sample with MS2 bacteriophage and subsequent quantitative polymerase chain reaction (qPCR) to assess PCR-inhibitory factors. Individual ticks or tick pools were tested using a set of qPCR assays for the detection of parapoxvirus (Kulesh et al., 2004;Nitsche et al., 2006) (Table 1).

Reactions were performed on a 96-well Applied Biosystems
QuantStudio 3 Real-Time PCR System using QuantiFast Pathogen.
Internal and negative controls were included in each run. Samples with Ct ≥32 were considered as negative. Positive samples detected using qPCR were then analyzed by two different PCR protocols to obtain DNA fragments for sequencing (Table 1)

Sequence alignment and phylogenetic analysis
For comparative analysis, additional partial B2L gene and ORF 032 sequences of other parapoxvirus were retrieved from GenBank and F I G U R E 1 (a) Map of Corsica, France, indicating the tick collection sites and the animal species and farm and (b) tick species and positive pools of ticks collected from cattle in the study area, Corsica. R. sanguineus (n = 6) and H. punctata (n = 4) were not included

Genus or species
Primer and probe 5′ → 3′ Sequence Gene Reference

Statistical analysis
The pathogens detected in pools were expressed as the percentage and minimum infection rate (maximum likelihood estimation (MLE)) method with 95% confidence intervals (CIs) based on the assumption that each PCR-positive pool contained at least one positive tick (Sosa-Gutierrez et al., 2016). Infection rate of DNA viruses was compared by using Fisher exact test (p < 0.05). The analysis was conducted using the R statistical platform (version 3.1.2) (Team, 2015).
Overall, 1566 ticks were collected from 687 cattle from 83 different cattle-breeding farms ( Table 2). The most abundant species was Rhipicephalus bursa (n = 820; 52% of ticks collected in cattle), followed by A total of 1285 ticks were collected from 538 horses from 21 farms.

Detection of pathogens
Overall parapoxvirus DNA was detected in 6.6% (36/544) of tick pools collected from 23 cows from 20 farms (
The seven sequences were obtained from ticks collected from five cows belonging to seven farms (  (Cargnelutti et al., 2012;Ohtani et al., 2017;Ziba et al., 2020), there is no published record of the disease at the human or animal health level in Corsica. Therefore, this report marks the first identification of parapoxvirus and pseudocowpox virus in the island. The detection rate of parapoxvirus DNA in 6.6% of tick pools collected in this study was lower than the detection rate (14% parapoxvirus DNA) reported in ticks collected from cattle in Burkina Faso (Ouedraogo et al., 2020),  The analysis was performed using a maximum-likelihood method with JTT matrix-based model with 1000 replicates (only values higher than 70% are shown). This analysis involved 24 amino acid sequences F I G U R E 3 Phylogenic radiation tree of parapoxvirus-group based deduced of 297 amino acid sequences of B2L gene of parapoxvirus. The analysis was performed using a maximum-likelihood method with JTT matrix-based model with 1000 replicates (only values higher than 70% are shown). This analysis involved 15 amino acid sequences These two viruses have also been detected in milk from affected dairy cows (de Oliveira et al., 2018). In conclusion, this study showed that parapoxvirus circulates in cattle in Corsica. Therefore, a broad surveillance is crucial to provide data that elucidate the origin and dissemination dynamics of parapoxvirus to investigate the prevalence of parapoxvirus infections in the cattle population and identify infection risks for other animals and humans.