The llama (Lama glama) is a South American camelid used as a pack and meat animal by Andean cultures since pre-Hispanic times. Today, llama breeding is spreading in North America where the animals are used for wool production and as livestock guards. In Italy, llamas are raised in the northern and central regions to produce meat and wool, but they are more commonly considered companion animals or used as pack animals for trekking tours in the mountains.

Viral diseases of llamas are becoming better known as a result of extensive research in North America (1) prompted by the recent growth in commercial breeding of New World camelids. Many of the viral diseases that affect camelids are related to bovine, equine, ovine, and swine virus infections. When examining skin lesions on llamas, viral diseases to consider as differential diagnoses include vesicular stomatitis, rabies, poxvirus (contagious ecthyma and cowpox virus [CPXV]) (2), foot-and-mouth disease, bluetongue, and mucocutaneous fibropapillomas (3).

Figure 1

Figure 1. A) Skin lesions showing a crater morphologic appearance typical of poxvirus around the anus of a llama from a farm near Calcata (Viterbo) in Northern Latium, Italy. B) Electron micrography image...

In July 2009, five of 7 llamas at a farm near Calcata (Viterbo) in Northern Latium, Italy, had skin lesions at different sites (palpebral conjunctiva, auricles, teats, mouth, and anus) that evolved from nodules to crusts; some had a crater morphologic appearance typical of poxvirus lesions (Figure 1, panel A). Within 10 days, 2 males showed depression and lethargy, anorexia, and recumbency until death. A short time later, another llama, a 7-year-old female, became ill and was euthanized. Necropsy was conducted on the euthanized animal, and samples were collected for laboratory investigation. No other animals belonging to numerous species of birds (local and exotic) and mammals (goats, cattle, swine, donkeys, and horses) living at the farm showed any of the above-mentioned symptoms.

Because mice and rats are considered carriers of CPXV (4) and birds of prey at the farm were fed frozen mice imported from a large farm in Germany, 2 lots of these mice were sampled (31 animals in total) along with 44 wild gray rats (Rattus rattus) captured on the farm for molecular analysis and virus isolation. Blood samples were collected for serologic analysis from 4 cattle, 25 goats, 18 donkeys, 3 horses, the 4 living llamas, and 4 humans (the farmer and 3 workers).

At necropsy, mandibular lymphoadenopathy; hemorrhagic gastroenteritis; and congestion of stomach, spleen, and kidney were observed in the euthanized female llama, signs consistent with a bacterial infection. In fact, a strain of Salmonella enterica serovar Typhimurium was isolated from lung, liver, spleen, kidney, and lymph node samples from the dead female llama and from chicken feces.

Samples of skin lesions were fixed in 10% buffered formalin, embedded in paraffin, cut 4 µm thick, and stained with hematoxylin and eosin. Histologically, the skin lesions showed eosinophilic granular intracytoplasmic inclusion bodies in basal and spinous layers of epidermis, compatible with poxvirus infection; superficial ulceration and extensive intraepidermal abscesses were also found.

Because we suspected a poxvirus, the skin lesions were processed (5) for electron microscopy techniques. Brick particles of ≈160–220 nm were observed, consistent with orthopoxvirus (Figure 1, panel B).

Two mammal cell lines (Vero and BHK₂₁) were used for virus isolation from skin lesions of the euthanized animal. During incubation of the cell cultures at 37°C, focal areas of cytopathogenic effect appeared within 3 days from injection, with complete lysis of the monostrate within 5 days. All the samples from mice and rats (pool of liver, spleen, and kidney) had negative results after 3 passages on both cell lines.

Figure 2

Figure 2. Phylogenetic tree based on nucleotide sequences of the complete hemagglutinin open reading frame (921 bp) from the llama orthopoxvirus isolate (arrow) and additional orthopoxvirus sequences available in GenBank. The tree has...

Total viral DNA was extracted from homogenized crusts and Vero cells supernatant by using conventional chemistry (QIAamp DNA Mini Kit; QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. Five μL of DNA was used as template for a 20 μL total volume real-time PCR (6) targeting a region of the cytokine response–modifying protein B (crmB) gene, identifying an orthopoxvirus. A wider region of crmB (7) was amplified by conventional PCR and sequenced to confirm orthopoxvirus identity (data not shown). Phylogenetic analysis was performed by using a complete gene of the hemagglutinin sequence. The phylogenetic tree shows that the llama hemagglutinin sequence is identical to clusters with the cowpox virus strains isolated in Germany in 2008 (CPXV-MonKre08/1–2-3) (Figure 2).

A stock virus suspension of the viral strain isolated from the llama was produced and titrated on Vero cells to use as viral antigen in an in-house seroneutralization (SN) test. The blood of the 4 surviving llamas collected 19 days after the female llama’s euthanasia was assayed by CPXV SN test, as well as for antibodies against bluetongue virus (by ELISA), infectious rhinotracheitis virus (ELISA), Brucella (Rose Bengal plate test), and neosporidia (ELISA), all with negative findings. The SN test for detection of CPXV antibodies was also conducted in llama serum collected ≈3.5 months later (day 105), in parallel with samples collected from various animal species (bovine, equine, and caprine). Only the 4 llama samples collected at day 105 showed CPXV antibodies (Table). The antibody titers (immunoglobulin [Ig] G and IgM) in the farm personnel serum samples were determined by indirect immunofluorescence assay by using in-house slides with Vero-E6 cells infected with a Lancy-Vaxina smallpox vaccine virus. Serum from a person vaccinated 4 years previously was used as a positive control. All serum samples had negative results for IgM (<20), while 3 persons had different IgG titers (Table). Only 1 of the 3 persons (titer 640) had been previously vaccinated against smallpox.

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