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Mechanical Transmission of Vesicular Stomatitis New Jersey Virus by Simulium vittatum (Diptera: Simuliidae) to Domestic Swine (Sus scrofa)

Paul F. Smith, Elizabeth W. Howerth, Deborah Carter, Elmer W. Gray, Raymond Noblet, Daniel G. Mead
DOI: http://dx.doi.org/10.1603/033.046.0643 1537-1540 First published online: 1 November 2009


Biting flies have been suggested as mechanical vectors of vesicular stomatitis New Jersey Virus (family Rhabdoviridae, genus Vesiculovirus, VSNJV) in livestock populations during epidemic outbreaks in the western United States. We conducted a proof-of-concept study to determine whether biting flies could mechanically transmit VSNJV to livestock by using a black fly, Simulium vittatum Zetterstedt (Diptera: Simuliidae), domestic swine, Sus scrofa L., model. Black flies mechanically transmitted VSNJV to a naïve host after interrupted feeding on a vesicular lesion on a previously infected host. Transmission resulted in clinical disease in the naïve host. This is the first demonstration of mechanical transmission of VSNJV to livestock by insects.

  • black fly
  • mechanical transmission
  • Simulium
  • vesicular stomatitis virus

The role of insects in the transmission of vesicular stomatitis New Jersey virus (family Rhabdoviridae, genus Vesiculovirus, VSNJV) to livestock during epidemics has not been fully investigated. Vesicular stomatitis New Jersey virus is a causative agent of vesicular stomatitis, an economically important livestock disease in the United States that affects primarily cattle, horses, and swine (Wunner et al. 1995). The virus has been isolated from biting (Theiler and Downs 1973, Calisher et al. 1983, Walton et al. 1987, Francy et al. 1988, Kramer et al. 1990, Schmidtmann et al. 1999) and nonbiting (Jenney 1967, Francy et al. 1988) Diptera during outbreaks, which suggests that insects serve as mechanical and biological vectors of VSNJV.

Biological transmission of VSNJV by black flies to mice (Mead et al. 1999, 2000), domestic swine (Mead et al. 2004a), and domestic cattle (Mead et al. 2009) has been demonstrated. In addition, colonized biting midges, Culicoides sonorensis Wirth & Jones, are susceptible to VSNJV infection (Nunamaker et al. 2000, Drolet et al. 2005), and experimentally infected midges have been shown to transmit the virus to guinea pigs (Perez de Leon et al. 2006) and cattle (Perez de Leon and Tabachnick 2006).

Although biological transmission of VSNJV by insects has been clearly demonstrated, little has been done to investigate mechanical transmission of VSNJV by insects. Ferris et al. (1955) evaluated mechanical transmission of VSVNJ by biting Diptera using an embryonated chicken egg model and found that members of the genera Aedes, Chrysops, Culex, and Tabanus could mechanically transmit the virus. These results suggest that this form of transmission may occur during epidemics, or even in endemic regions, but such transmission has not been validated in livestock. We conducted a proof-of-concept study to evaluate mechanical transmission of VSNJV by biting Diptera to livestock.

Materials and Methods

Three juvenile domestic swine, Sus scrofa L., weighing 15.9–22.7 kg (35–50 lbs) were obtained from a purpose bred supplier (Valley Brook Farm, Madison, GA) and acclimated for 1 wk in the Animal Health Research Center, a BSL-3Ag large animal containment facility at the College of Veterinary Medicine, University of Georgia, before initiation of the study. Animals were held individually in 1.524- by 1.524-m (5- by 5-ft) dog runs (Britz and Co., Wheatland, WY). For the purposes of this study, the animals were designated as pig 40, pig 46, and pig 51. Standard BSL-3Ag biosecurity and biosafety protocols were observed throughout the study. The use of animals was approved by the University of Georgia's Institutional Animal Care and Use Committee (approval no. 2008-08-22).

Pig 51 was tranquilized (2 mg/kg Telazol and 2 mg/kg xylazine intramuscularly) and infected with a 1982 Arizona bovine VSNJV isolate as described previously, by allowing VSNJV-infected black flies to feed on the planum rostrale (Mead et al. 2004a). Ten VSNJV-infected black flies fed on this individual as determined by visual observation of blood in the crop. On postinfection day (PID) 1, this animal was tranquilized, and clean feeding cages containing 25–30 uninfected black flies (Fig. 1) were allowed to initiate feeding or probing on developing lesions on the planum rostrale. After ≈5 min, these cages were transferred to two naïve, separately housed pigs (pigs 40 and 46), which had been tranquilized as described above. The clean sides of the feeding cages (those not in contact with the developing lesion) were placed against the planum rostrale of the naïve pigs. Black flies were allowed to feed to repletion on these animals (≈15 min).

Fig. 1

Feeding cages were constructed with 5-cm-diameter polyvinyl chloride or polycarbonate tubing cut into 1.3-cm sections and enclosed on the two sides with polyester mesh (12 squares per cm) or nylon organdy.

All animals were observed daily for lesion development, and temperatures were recorded. Blood was collected on PID 1–6 through vena cava puncture for virus isolation on African green monkey kidney (Vero) cell monolayers. Additional samples taken for virus isolation included swabs of the planum rostrale, nasal cavity, and tonsil of the soft palate. Swabs were placed in individual cryovials containing 1 ml of virus transport medium (minimal essential medium supplemented with 3% fetal bovine serum, 1000 U of penicillin G, 1 mg of streptomycin, 0.25 mg of gentamicin sulfate, 0.5 mg of kanamycin monosulfate, and 2.5 μg/ml amphotericin B) and processed as described previously (Mead et al. 2004a). All animals were euthanized by administration of barbiturates on PID 6, and tissue was collected from lesion sites and tonsil of the soft palate for virus isolation. Titration of virus isolation-positive samples was performed via end-point titration, and all isolates were confirmed as VSNJV using a previously described reverse transcription-polymerase chain reaction (rt-PCR) with specific primers (Rodriguez et al. 1993). Planum nasale, tonsil, and lateral retropharyngeal lymph node were collected in 10% buffered formalin, processed routinely for histology, and sections stained with hematoxylin and eosin for histopathology and for VSNJV by immunohistochemistry (IHC) as described previously (Howerth et al. 2006).


Pig 51, which was inoculated by infected black flies developed a large vesicular lesion on the planum rostrale on PID 1, and spiked a fever of 105.3° F. By PID 2, the lesion had covered the entire surface of the planum rostrale and started to crust over. Swab samples from surface of the planum rostrale, nasal cavity, and tonsil of the soft palate were positive for VSNJV by virus isolation and confirmed by rt-PCR on PID 1–3. The highest virus titer for this animal, 104.45 50% tissue culture infective dose (TCID50) per 25 μl, was from the planum rostrale surface swab on PID 1. By PID 6, the lesion on the planum was starting to heal and characterized by extensive epidermal erosion and ulceration that was beginning to re-epithelialize and a dermal infiltration of lymphocytes, plasma cells, histiocytes, and a few eosinophils histopathologically. Lymphoid hyperplasia was present in lateral retropharyngeal lymph node and tonsil. A few epithelial cells lining tonsillar crypts were positive for VSNJV by IHC, but no virus was detected in planum lesion or lymph node. Viremia was not detected during the course of the study.

Pig 46 developed lesions on the nasal planum starting 1 d after exposure to flies that had first been allowed to feed on the lesion of pig 51. Initially, there was a pinpoint raised reddened planum lesion on PID 1 (Fig. 2A), which grew to an ≈1-cm-diameter vesicle by PID two (Fig. 2B). A second planum lesion was observed on PID 3, which reached a similar size as the initial lesion. Unlike pig 51, these lesions did not coalesce to cover the entire planum rostrale surface and fever was not detected in this animal. Virus was isolated from the planum rostrale surface and nasal cavity swabs on PID 1–4 and from the tonsil of the soft palate swabs on PID 1–5. The highest swab titer for this animal was 103.66 TCID50 per 25 μl from a planum rostrale surface swab on PID 4. Tonsil of the soft palate collected at necropsy was positive for VSNJV by virus isolation and a small number of epithelial cells lining tonsillar crypts were positive for VSNJV by IHC. Microscopically, the planum had small erosions filled with serocellular crust and acantholytic cells with underlying epidermal hyperplasia and a dermal infiltration of lymphocytes and histiocytes, consistent with healing vesicles, and tonsil and lymph node had lymphoid hyperplasia; planum lesions and lymph node were negative for VSNJV by IHC. Pig 40 did not have signs of VSNJV infection, virus was not recovered from any swabs of this animal at any time, and tissues collected at necropsy were negative for virus by virus isolation and IHC and did not have microscopic changes consistent with VSNJV infection or lymphoid hyperplasia in tonsil or lymph node as seen in pigs 51 and 46.

Fig. 2

(A) Pinpoint lesion of pig 46 on PID 1 (indicated by arrow). (B) Lesion of pig 46 on PID 2.


For a virus to be mechanically transmitted by insects at least two factors have to be considered: There must be sufficient virus available in blood, vesicles, or skin; and the virus should be somewhat resistant to varying environmental conditions. In VSNJV, viremia is absent in infected livestock; however, virus titers ranging from <102.3 to 104.6 TCID50 per swab have been recovered from the surface of vesicular lesions (Stallknecht et al. 2001, 2004) and 109.15 plaque-forming particles per ml in fluid aspirated from vesicular lesions (Marcus et al. 1998). The importance of lesions for insect infection or contamination with VSNJV was confirmed by Mead et al. (2004a,b), who demonstrated under experimental conditions that female black flies ingest infectious VSNJV when they feed on virus rich-lesions and that VSNJV can routinely be isolated from male black flies (which do not blood feed) that had surface contact with vesicular lesions of infected livestock. In regard to VSNJV stability in the environment, the virus is inactivated when exposed to 56°C for 30 min (Watson 1981) or intense irradiation with UV light (Weck et al. 1979). Under more natural conditions, VSNJV can remain viable in infected saliva on pails or food buckets for 3–4 d (Hanson 1952). Drolet et al. (2009) demonstrated that viable virus can be recovered from plant surfaces up to 24 h after surface inoculation and maintenance at room temperature. Stability of VSNJV on the mouthparts or exterior surfaces of insects has not been fully investigated. In the chicken embryo model investigated by Ferris et al. (1955), transmission of virus by horse flies and Aedes mosquitoes was detected up to 72 h after exposure to infected embryos; however, Mead et al. (2004b) were unable to recover viable VSNJV from contaminated male black flies 5 d after exposure. This level of stability and virus survival would be sufficient to allow mechanical transmission by biting flies, particularly in feedlot or dairy facilities, where a high density of susceptible hosts could be found, and interrupted feeding by biting flies would occur frequently.

Mechanical transmission of VSNJV by insects has long been suspected but has never been demonstrated using an insect-animal model. The hypothesis that VSNJV can be transmitted mechanically by insects is supported by entomological data collected during epidemics (Jenney 1967, Francy et al. 1988), by experimental studies in livestock using transmission routes consistent with mechanical insect transmission (Stallknecht et al. 1999, 2001), and by an experimental VSNJV transmission study using biting flies and an embryonated egg model (Ferris et al. 1955). In the current study, we demonstrated that VSNJV can be mechanically transmitted from one livestock host to another by biting insects.

In the current study, one test animal developed clinical disease, whereas the other animal did not. Previous studies of livestock infection with VSNJV have demonstrated animal-to-animal variation in susceptibility to infection as well as clinical outcome after infection. Variation in lesion size as well as the extent and duration of virus shedding in experimentally infected animals has been reported previously (Stallknecht et al. 2001, Mead et al. 2004a, Howerth et al. 2006). It is also possible that VSNJV was never inoculated into pig 40. Fly-bite marks were visualized on the planum rostrale of pig 40 after exposure to flies that were allowed to feed on the virus-rich lesion of the primary infected animal. However, it is impossible to determine whether any of these flies had fed on the primary animal before feeding on the test animal. Either of these scenarios could explain the differences observed in this study.

The goal of this research was to demonstrate proof-of-concept and not to determine the relative importance of this transmission route compared with the other routes that have been previously investigated (e.g., animal-to-animal contact or biological insect transmission). Therefore, further research into the efficiency of mechanical VSNJV transmission is needed to elucidate the importance of insects as mechanical vectors.


We thank Gary Doster for critically reviewing this manuscript. Funding for this research was provided by the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service, grant 2005-35204-16102. Additional support was provided by the Georgia Research Alliance and USDA, APHIS, Veterinary Services Cooperative Agreement 08-9613-0032-CA.

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