In September 2003, three Barbary macaques (Macaca sylvanus) at a sanctuary for exotic animals in Almere, the Netherlands, showed multifocal gingival, buccal, and lingual lesions. Typical intranuclear inclusions were found by histologic analysis, and poxlike particles were found by transmission electron microscopy of 6 biopsy specimens from buccal lesions of the same animals. Because of concerns that these macaques were infected with MPXV, additional biopsy specimens of poxlike lesions were obtained for virus isolation and polymerase chain reaction (PCR) studies.

Vero cells were infected with homogenized biopsy samples from the 3 macaques, and cells were monitored daily for appearance of cytopathic changes. Three days after infection, cells showed cytopathic effects characterized by plaques of rounded cells with prominent cytoplasmic bridging and syncytia formation. To confirm the isolation of a poxvirus, an immunofluorescence test was conducted with human antivaccinia serum (diluted 1:1,000) and goat antihuman immunoglobulin G (IgG) (diluted 1:500, Dako, Roskilde, Denmark). Diffuse cytoplasmic fluorescence confirmed an orthopoxvirus.

Figure

Figure. Sequence alignment of the partial hemagglutinin gene of cowpox viruses (CPXV) isolated from Barbary macaques and brown rats. CPXV-2001 strain was isolated from a rat in 2001 from the Netherlands (<>

Isolates were further characterized by PCR and sequence analysis with primers for the hemagglutinin gene (9). Melting curve and sequence analyses confirmed the presence of an orthopoxvirus, most likely CPXV (Figure). Because this PCR assay was designed to differentiate variola virus from other orthopoxviruses but not among nonvariola orthopoxviruses, we developed a CPXV-specific PCR by using nested primer sets within the A-type inclusion protein (ATI) gene. PCR was conducted by using external primers (ATIF1) 5´-GAACTTAATAAGTGTTTCGATA-3´> (forward primer) and (ATIR1) 5´-CAGTAACGTCGGACGATGGAGG-3´ (reverse primer) with nested forward primer ATIF2 5´-GAGGAAGTTAAGAGATTGCGTC-3´ and reverse primer ATIR1. The nucleotide sequences are available from GenBank. Nucleotide sequencing confirmed that the virus isolated from Barbary macaques was a CPXV.

Since all macaques were in the center before disease manifested and they had not been in contact with other animals, other monkeys were tested for CPXV infection by serologic analysis. Serum samples from 16 Barbary macaques (Macaca sylvanus), 2 pig-tailed macaques (M. nemestrina), 2 squirrel monkeys (Saimiri sciureus), 2 Japanese macaques (M. fuscata), 6 cynomolgus macaques (M. fascicularis), 2 Hamadryas baboons (Papio hamadryas), 4 rhesus macaques (M. mulatta), and 1 vervet (Cercopithecus aethiops) were tested with a virus neutralization test (VNT) using a CPXV strain isolated in this study (CPXVmac). Neutralizing titers were determined after 5 days on the basis of complete reduction of a cytopathic effect. At the end of the outbreak, neutralizing serum antibodies were detected in 9 Barbary macaques, 2 pig-tailed macaques, 1 Japanese macaque, 3 cynomolgus macaques, and 1 rhesus monkey. This finding suggested that that all of these animals had been exposed to CXPV. Retrospective serosurveillance showed that only 1 Barbary macaque was seropositive at the start of the outbreak. No swab samples from the animals were available for culture or PCR analysis.

To identify the possible reservoir of the CPXV infection, animals known to be susceptible to CPXV and housed at the sanctuary at the time of the outbreak were tested. These included 4 domestic cats (Felis catus), 2 red squirrels (Sciurus vulgaris), and 6 prairie dogs (C. ludovicianus). In addition, 32 wood mice (A. sylvaticus) and 34 rats (R. norvegicus) trapped in the area of the sanctuary were tested by serologic analysis and PCR. Only 1 of the tested cats had neutralizing serum antibodies (titer 20) to CPXV. Throat swabs of all 4 cats obtained during the outbreak were negative by both PCRs. These results suggest that cats were not infected by CPXV at the time of the outbreak. Prairie dogs housed at the sanctuary at the time of the outbreak were not infected, as shown by negative PCR results on throat swabs and the absence of orthopoxvirus-specific antibodies.

In contrast, PCR and virus culture showed that 56% of rats tested were infected with CPXV (Table). Sequence comparison of the hemagglutinin and ATI genes of 19 CPXV rat isolates with the Barbary macaque isolates showed identical sequences, which indicated that rats were the most probable source of infection. No significant differences were observed between the genes of CPXVs isolated during this outbreak and previous isolates (Figure).

Since serum samples collected from trapped dead rats could not be tested by VNT, we developed an indirect enzyme-linked immunosorbent assay (ELISA) based on vaccinia virus, which was validated with sera of monkeys infected with vaccinia virus. Briefly, vaccinia virus was treated with 2% Triton X-100, and 100 ng was added to wells of high-binding plates (Costar, Corning Inc., Corning, NY, USA). Two-fold dilutions of sera starting at 1:20 were added to the plates, and antibodies were detected with horseradish peroxidase–labeled protein A (1:1,000 dilution, Zymed Laboratories, South San Francisco, CA, USA). Titers were expressed as the reciprocal of the highest serum dilution for which an optical density at 450 nm (OD₄₅₀) was >3× the OD₄₅₀ of the negative control. Of 19 rats that tested positive by PCR, only 3 were seropositive, which suggested that CPXV was spreading actively in the rat population. Swabs of all mice tested were negative by both PCRs, and only 2 of 32 mice tested were seropositive by ELISA, indicating that mice were not the source of infection.

This is the first report describing CPXV infection in captive monkeys. Wild brown rats captured at the sanctuary were infected with CPXV and assumed to be the most probable source of infection. Whether multiple contacts of infected rats with the monkeys contributed to the outbreak or monkey-to-monkey transmission occurred efficiently is not clear. Furthermore, whether monkeys were infected by rats through direct contact or infected excreta is unclear (10). Longitudinal field studies are required to clarify if rats could be the principal reservoirs for CPXV. Circulation of CPXV in wild and captive animals, together with decreased immunity against orthopoxviruses in the community, may put animal trappers and handlers at risk for CPXV infection. Our findings show the threat of orthopoxviruses that can cross species barriers, which indicates the importance of developing new vaccines and antiviral drugs against orthopoxvirus infection of humans.