Orthopoxvirus Antibodies in Grey Squirrels (Sciurus aureogaster) in Mexico City, Mexico

Chordopoxviruses (Family Poxviridae) infect a wide range of vertebrates (Damon 2006) and have been important in terms of global economy, wildlife conservation, and public health. These pathogens are currently divided into 10 accepted genera, distinguished by serologic cross-reaction (Fenner 2000). The Orthopoxvirus (OPXV) genus is widely distributed among Europe, Asia, and Africa. Since the 1960s, three OPXV (raccoonpox, skunkpox, and volepox [VPXV] viruses) have been isolated from North American wild mammals (Alexander et al. 1972; Regnery 1987; Knight et al. 1992). This group of viruses is endemic to North America, monophyletic, and the most genetically divergent within the OPXV genus (Emerson et al. 2009). Information regarding OPXV ecology, distribution, and host competence and their role as zoonotic pathogens is lacking. No OPXV has been demonstrated in Mexico.

Several mammalian taxa are susceptible to OPXV infection, but rodents are the most frequently associated taxon and are considered the primary reservoir for some OPXV (Emerson et al. 2009). Some species of rodents, such as mice, rats, and squirrels, which live in close proximity with humans, constitute a potential risk for pathogen transmission (Salzer et al. 2013). In Mexico City, one of the largest metropolises in the world, the Mexican grey squirrel (Sciurus aureogaster) is the most common nonvolant native mammal. Although their presence enriches the aesthetic and ecologic value of the urban landscape, the interest generated in humans increases the risk of direct contact and interspecific pathogen transmission. We evaluated grey squirrel serum for the presence of OPXV antibodies in Mexico City.

From February to June 2011, we captured 366 grey squirrels in 18 urban parks in southwestern Mexico City. We captured squirrels during 3 days per park using Tomahawk live-traps (number 204, Tomahawk Live Trap, Tomahawk, Wisconsin, USA) placed at the base of mature trees and baited with peanut butter, oats, and vanilla. Traps were opened 1 hr after sunrise and remained open for 8 hr. The number of traps per park was established depending on the size of the area occupied by grey squirrels (nine to 54 traps per park). Captured squirrels were immobilized with physical restraint using a canvas handling bag (Koprowski 2002) and ear-tagged with metal rings for identification. Individuals were weighed, and sex and age were determined. Oral swabs, 1 mL of blood, and skin lesions or scabs (if present) were collected from all individuals; swabs and scabs were preserved in sterile microtubes on dry ice. Blood samples were obtained from the retro-orbital sinus following the Guidelines of the American Society of Mammalogists for the use of wild mammals in research (Sikes et al. 2011). Sampled individuals were released safely at capture site. Blood was centrifuged at 1,500 × G for 10 min to obtain serum. All samples were preserved at −20 C until transfer to the Centers for Disease Control and Prevention (CDC, Atlanta, Georgia, USA) for laboratory testing. Samples were collected under the Mexican environmental ministry license (FAUT- 0250) after approval by the animal-care committee of Universidad Nacional Autónoma de México (UNAM).

A modified enzyme-linked immunosorbent assay (ELISA) was used for detection of anti- orthopoxvirus immunoglobulins (Karem et al. 2005). Microtiter plates were coated with crude VPXV (from the CDC Poxvirus Library) at 1.36×10⁷ plaque-forming units/mL (1.1 µg/well) on one half of the plate and an equal volume of BSC-40 cell lysate (African green monkey kidney cell line used to grow stock of VPXV) on the other half. Squirrel sera were tested at a 1∶50 dilution, and 100 µL/well of a 1∶30,000 dilution of ImmunoPure A/G conjugate (Pierce ImmunoPure Protein A/G Peroxidase conjugated; Pierce, Rockford, Illinois, USA) was used. We added each serum dilution for duplicate wells on the VPXV half and on the half of BSC-40 cell lysate. Positive and negative rabbit anti-vaccinia sera were used as assay controls on every plate. Optical densities (OD) were read at 450 nm. The average of the BSC-40 cell lysate half of each plate plus three standard deviations was used to generate a cutoff value (COV). Samples were considered positive if the average of the duplicate wells was above the COV (0.001 to 2.047; median = 0.280); thereby we discarded false positives due to lysate background OD.

To determine whether the serologic reaction in ELISA was due a previously described North American OPXV, DNA was extracted from oral swabs (n = 366) and from skin lesions or scabs (n = 65), using the DNA EZ1 tissue kit (Qiagen, Valencia, California, USA). DNA samples were screened with the North American Orthopoxvirus real time-PCR assay, which targets the myristoylated protein gene ortholog of the Vaccinia virus Copenhagen G9R (Gallardo-Romero et al. 2011). DNA samples were all negative.

We captured 366 Mexican grey squirrels. General antibody prevalence and prevalence by sex and age are presented in Table 1. Our serologic evidence (prevalence = 30%) provides the first evidence of OPXV antibodies in Mexican wildlife. In particular, their presence in grey squirrel populations from Mexico City suggests squirrels have been in contact with a circulating OPXV and developed an immune response. We could not identify which OPXV grey squirrels were exposed to because all members of OPXV genus cause cross-reaction in serologic assays (Karem et al. 2005). Previous molecular evidence of OPXV infection was from ill animals during the acute phase of infectious (Knight et al. 1992; Thomas et al. 2003). Our lack of molecular evidence may result from sampling individuals that were clinically healthy with no signs of a recent poxlike infection. Additionally, some OPXVs can cause subtle immunologic or even asymptomatic processes in rodents, presumably due to a long history of coevolution (Saluzzo et al. 1986).

Other species of Orthopoxvirus were involved in the near extinction of endemic wildlife species in other world regions (Sainsbury et al. 2008) or represent substantial zoonotic risks in other countries (Parker et al. 2007), hence future long-term studies are needed to expand our knowledge of North American OPXV geographic distribution, host range, pathogenesis, phylogenetics, and ecology.

We acknowledge the support from urban parks authorities from Benito Juárez, Coyoacán, Alvaro Obregón, Miguel Hidalgo, and Tlalpan delegations in Mexico City (Distrito Federal), for allowing us to conduct this research. We are grateful to the students of the veterinary and biology schools from UNAM for their valuable assistance in the field.