Grasshoppers (Orthoptera: Acrididae) Could Serve as ... - PubAg - USDA

VECTOR/PATHOGEN/HOST INTERACTION

Grasshoppers (Orthoptera: Acrididae) Could Serve as Reservoirs and Vectors of Vesicular Stomatitis Virus RICHARD A. NUNAMAKER,1 JEFFREY A. LOCKWOOD,2 CHARLES E. STITH, COREY L. CAMPBELL, SCOTT P. SCHELL,2 BARBARA S. DROLET, WILLIAM C. WILSON, DAVID M. WHITE, AND GEOFFREY J. LETCHWORTH3 USDAÐARS, Arthropod-borne Animal Diseases Research Laboratory, P.O. Box 3965, Laramie, WY 82071

J. Med. Entomol. 40(6): 957Ð963 (2003)

ABSTRACT Vesicular stomatitis (VS) is an economically devastating disease of livestock in the Americas. Despite strong circumstantial evidence for the role of arthropods in epizootics, no hematophagous vector explains the Þeld evidence. Based on the spatiotemporal association of grasshopper outbreaks and VS epizootics, we investigated the potential role of these insects as vectors and reservoirs of the disease. The critical steps in the grasshopperÐ bovine transmission cycle were demonstrated, including 1) 62% of grasshoppers [Melanoplus sanguinipes (F.)] fed vesicular stomatitis virus (VSV) from cell culture became infected, with titers reaching 40,000 times the inoculative dose; 2) 40% of grasshoppers that cannibalized VSV-infected grasshopper cadavers became infected, amplifying virus up to 1,000-fold; 3) one of three cattle consuming VSV-infected grasshopper cadavers contracted typical VS and shed virus in saliva; and 4) 15% of grasshoppers became infected when fed saliva from this infected cow. The ecological conditions and biological processes necessary for these transmissions to occur are present throughout much of the Americas. Field studies will be required to show these Þndings are relevant to the natural epidemiology of VSV. KEY WORDS vesicular stomatitis virus, VSV, grasshopper, bovine, Melanoplus sanguinipes

VESICULAR STOMATITIS (VS) IS an economically devastating disease of horses, cows, and pigs, but it also affects many wildlife species, as well as humans. In the United States, two vesicular stomatitis virus (VSV) serotypes, NJ and Indiana, cause disease. The disease is enzootic from Mexico to Colombia and periodically spreads into temperate regions. Northern movements originating in southern Mexico (Rodriguez et al. 2000) may develop into epizootics in the western United States, occasionally spreading as far north as Canada. Each outbreak of VS in the United States provokes quarantines designed to slow the spread of the disease and allow time to distinguish it from foot-and-mouth disease (FMD), which causes similar vesicular lesions of the mouth, teats, and coronary bands of cattle, sheep, and pigs. Unlike FMD, VSV can spread to livestock owners and veterinarians, causing a short-lived but debilitating disease (Hanson and Karstad 1958, Fields and Hawkins 1967, Johnson et al. 1969, Tesh et al. 1969, Reif et al. 1987). Infection of people in rural Central America is relatively common (Tesh et al. 1969).

Current address: 640 Leon St., Delta, CO 81416. Entomology Section, Department of Renewable Resources, University of Wyoming, Laramie, WY 82071. 3 E-mail: [email protected]. 1 2

VS spreads quickly within a herd of animals by direct contact and fomites contaminated with virus shed in saliva and vesicular exudates (Fig. 1, step 1). However, this does not explain the onset of widespread epizootics or the rapid long-distance spread during VS outbreaks. Field observations and circumstantial evidence strongly suggest VSV is spread by one or more arthropod species. The pathogen has even been proposed as a well-adapted insect virus that inadvertently spreads to vertebrates (Tesh and Chaniotis 1975). In tropical and subtropical zones, VS is most common at the end of the rainy season or early in the dry season when many insect species are most abundant (Mason et al. 1976, Orrego et al. 1987, Rodriguez et al. 1990). Similarly, in temperate regions VS is associated with periods of insect activity, occurring in the summer, usually disappearing after the Þrst hard frost, and often sparing horses that are protected from insects (Hanson 1981, Carver 1984, Webb et al. 1987). Outbreaks seem to follow terrestrial features such as rivers or distinct ecological features such as open woodlands or savannas (Acree et al. 1964, Hanson et al. 1968, Hanson 1981). The 1982 VS outbreak in the western Unites States seemed to spread long distances in a windward direction (Sellers and Maarouf 1990). The transmission of VSV only by hematophagous vectors and fomites is not consistent with the ecological evidence. Experimentally infected hematopha-

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Vol. 40, no. 6

Fig. 1. Hypothesized VSV transmission cycle between cows and grasshoppers.

gous insects can transmit virus to mammals during blood feeding (Muggsay and Suarez 1962; Rao et al. 1967; Bergold et al. 1968; Donaldson 1970; Tesh et al. 1971, 1972). However, VSV does not usually circulate in blood at levels required to infect blood-feeding insects (Johnson et al. 1969; Yuill 1981; Carbrey 1982; Arbelaez 1983; Arbelaez and Valbuena 1987; Orrego et al. 1987; Thurmond et al. 1987; Comer et al. 1995a, b), so it is not evident how hematophagous insects could be infected under natural conditions, except by probing skin lesions. Oral lesions shed copious amounts of virus that could also serve as a source of infection for detritivorous and herbivorous insects (Patterson et al. 1955, Thurmond et al. 1987). Reviewing all data available in the mid-1960s, Jonkers (1967) proposed the general outline of an epizootiological model that was entirely consistent with grasshoppers being a source of VSV. Although he never hypothesized the role of herbivorous insects, Jonkers posited that 1) the virus source is already present in pastures before the occurrence of the Þrst cases and is brought into contact with livestock only if proper conditions prevail; 2) the virus is infrequently spread from premise to premise during an epizootic; 3) the virus source is able to cause infection only during a limited period; 4) the virus source is passive, and the prospective host contacts virus-infected material; 5) the virus source is close to the ground, in or on the soil, or vegetation; and 6) livestock are indicator hosts only, the proportion infected giving some indication of the availability of virus in the pasture. Indeed, passive invertebrate reservoirs have also been shown to transmit another livestock disease, Potomac horse fever (Madigan et al. 2000). JonkersÕ astute observations led Vernon to hypothesize that grasshoppers play a role in the VSV enzootic cycle, based on her observations of a temporal and spatial correlation between grasshoppers and VS outbreaks (Vernon 1989). Grasshoppers graze the same pastures as livestock, often reach extremely high population densities, are occasionally consumed by livestock, presumably consume vegetation contaminated by livestock saliva, and migrate long distances (Chapman and Joern 1990, Gangwere et al. 1997). Although other herbivorous insects, such as leafhoppers, have been shown to replicate VSV in the laboratory (Lastra

and Esparza 1976), no plausible means exists for horizontal transmission to other leafhoppers, as might occur with a cannibalistic insect, such as grasshoppers (Lockwood 1988). Horizontal transmission would provide a means of virus ampliÞcation in the absence of a susceptible mammalian host. Although ample evidence supports the existence of several species of hematophagous vectors (Tesh et al. 1972, Tesh and Chaniotis 1975, Travassos da Rosa et al. 1984, Mead et al. 2000, Nunamaker et al. 2000), the intent of this study was to propose and test the novel hypothesis that VSV would cycle within a passive reservoir, in this case grasshoppers (Fig. 1, step 2) and between grasshoppers and cattle (Fig. 1, steps 3Ð 4). Because grasshoppers are cannibalistic (Lockwood 1988), they would provide a unique mechanism for maintenance of viral ampliÞcation in the absence of a susceptible host, unlike other herbivores. Materials and Methods Infection of Grasshoppers with Cultured Virus. Grasshopper [Melanoplus sanguinipes (F.)] eggs were acquired from the USDAÐARS laboratory in Sidney, MT, and reared according to standard protocol. Adult grasshoppers were provided with a 30-␮l meal of 105 median tissue culture infectious doses (TCID50) of VSV-New Jersey, Hazelhurst strain on a 0.5-cm2 piece of Whatman Þlter paper that had been treated with a solution of molasses:water (1:1). A maintenance diet of bran, dried dandelions, and fresh barley or lettuce was offered to the insects after they had consumed the Þlter paper, or after 48 h, whichever came Þrst. They were maintained at 22⬚C with 40 Ð 60% RH and a photoperiod of 13:11 (L:D) h for a period of 2 or 4 wk and then assayed for virus. Grasshoppers were homogenized in 1 ml of antibiotic cell culture medium (199E cell culture medium, 200 U/ml penicillin, 200 ␮g/ml streptomycin, 100 ␮g/ml gentamycin, 100 ␮g/ml neomycin, and 5 ␮g/ml amphotericin B), spun to pellet debris, and used to inoculate Vero cell monolayers in 96-well plates. Cytopathic effects (CPEs) were used as an indicator of infection. A sample was scored as positive if more than two wells exhibited CPE. Virus titers were determined by endpoint dilution (Ka¨rber 1931). Virus was con-

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NUNAMAKER ET AL.: GRASSHOPPERS AS RESERVOIRS AND VECTORS OF VSV

Þrmed as VSV by a direct ßuorescent antibody staining using a rabbit polyclonal anti-VSV antibody conjugated to ßuorescein isothiocyanate and Evans blue counterstain. Grasshopper-to-Grasshopper Transmission. Individual grasshoppers were allowed to feed on cadavers of grasshoppers that had been fed VSV-laden Þlter paper, held for 2 wk, and then killed by freezing. The cannibals were held for an additional 2 wk. Infections were quantiÞed as described above. Grasshopper-to-Cattle Transmission. The inner cheeks of three yearling Hereford cows were scariÞed with a curette to simulate epithelial tissue damage commonly associated with feeding on arid rangeland vegetation. During a 15-min period, each animal was given 33 infected grasshoppers (see above) that had been killed by freezing. Each aliquot contained 11 grasshoppers and 150 g of Roll Mix (ⱖ9% protein, ⱖ2% crude fat, and ⱖ5% crude Þber). The cattle were held in a BL-3 biocontainment facility during the experiment, during which time they were maintained on alfalfa hay and water, ad libitum, Roll Mix, and Range Cubes (ⱖ20% crude protein, ⱖ2% crude fat, and ⱖ6% crude Þber). Swabs of the oral cavity and serum samples were collected daily; the animalsÕ body temperatures were recorded twice daily, and biopsies were taken of oral lesions. Maintenance and care of animals conformed to the National Institutes of Health guidelines for the humane use of laboratory animals. To detect VSV in the saliva and sloughed epithelial cells of VSV-exposed cattle, cotton-tipped swabs were saturated with saliva and submersed in 0.5 ml of Vero maintenance media and freeze-thawed (⫺80 to 4⬚C). The presence of virus in oral swab media was determined by standard plaque assay on Vero cells. To detect virus in free-ßowing saliva from the cow (no. 602) showing clinical signs, saliva was collected with a disposable pipette and used in plaque assay on Vero cells. Plaques from cotton swabs, free-ßowing saliva and tongue biopsies were conÞrmed as VSV-speciÞc by immunocytochemistry (Drolet et al. 1993) using a rabbit anti-VSV-New Jersey IgG polyclonal antibody (Lee Biomolecular Research, San Diego, CA). To detect virus in tongue tissue samples (one from a sloughing area, one from a previously sloughed area) from cow 602, the tissue samples were diluted 1:5 (wt:vol) with Vero maintenance media and homogenized. The presence of virus in tongue sample supernatants was determined by standard plaque assay on Vero cells. Total RNA was extracted from samples using the Purescript RNA extraction kit as described previously (Gentra Systems, Inc., Minneapolis, MN). Reverse transcriptase-polymerase chain reaction (PCR) was performed using primers speciÞc for the 22Ð 661-base pair region of VSV-New Jersey N gene (forward primer, GTCAAGAGAATCATT; reverse primer, GTTCCGTATCTGA) using standard conditions. Cattle-to-Grasshopper Transmission. Naõ¨ve grasshoppers were fed saliva from an uninfected cow, spiked with 0.00, 0.05, 0.75, 1.40, or 3.50 log10 TCID50 of VSV. The saliva was delivered by placing 20 ␮l of liquid directly onto the grasshopperÕs mouthparts with

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a micropipette. Naõ¨ve grasshoppers were allowed to consume 0, 5, 10, 15, or 20 ␮l of saliva from the VSVinfected cow (see above) provided on a Þlter paper, as described above. The grasshoppers were maintained for 14 d and assayed as described previously. VSV was identiÞed directly on the cell culture plates by immunocytochemistry (Drolet et al. 1993). Virus Isolation from Field-Collected Samples. Grasshoppers were collected from farms located in Can˜ as and the folds of the Poas volcano in Costa Rica during the VSV outbreak season, DecemberÐMay 2001. These collections were composed of ⬇60% Acrididae (shorthorned grasshoppers) and 40% Tettigoniidae (longhorned grasshoppers). Collected samples were ßash frozen in liquid nitrogen and shipped on dry ice. Two hundred Þfty six grasshoppers were processed individually or in pools. The abdomen was ground in 1 ml of grinding medium (199E medium base, 400 U/ml penicillin, 400 ␮g/ml streptomycin, 200 ␮g/ml gentamycin, 400 ␮g/ml neomycin, and 5 ␮g/ml amphotericin B); the remainder of the insect was reserved for taxonomic identiÞcation. Virus isolation was performed by three blind passages of duplicate ground insect suspensions on Vero cells. Culture ßasks showing overt signs of bacterial or fungal contamination were ßash frozen at ⫺80⬚C, passed through a sterile 0.2-␮m syringe Þlter, and the ßow-through was added back to a culture ßask with maintenance medium.

Results Infection of Grasshoppers with Cultured Virus. The initial step in our proposed transmission cycle of VSV was investigated by feeding grasshoppers (M. sanguinipes) substrate treated with virus. Of the 29 grasshoppers provided a virus meal, 14 (48%) became infected with VSV, as conÞrmed by ßuorescent antibody assay (Table 1). Of the 14 infected grasshoppers, eight (57%) yielded titers greater than the meal provided. The amount of virus in the grasshoppers increased with time, demonstrating the presence of a productive infection. Those that consumed ⱖ50% of the treated substrate in the Þrst 24 h and were examined after 28 d had mean titers nearly 16,000 times greater than those that consumed the same amount of substrate and were assayed after 14 d. Virus titers increased as a function of the total amount and rate of virus-laden substrate consumed. Of the grasshoppers held for 14 d after inoculation, those that ate ⱖ50% of the substrate at 48 h had mean titers 630 times greater than those that consumed less, and those that consumed this amount within the Þrst 24 h had mean titers 25 times greater than those that did not consume this dose until 48 h. These differences were most likely due to the rapid deterioration of the virus on the substrate. Assays of the treated substrate revealed that the original dose of 105 TCID50 declined to 102.3 TCID50 by 24 h and was undetectable by 48 h. However, even with this rate of deterioration, one grasshopper that consumed no substrate for the Þrst

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Table 1.

VSV titers in grasshoppers (M. sanguinipes) fed filter paper treated with 105 TCID50

Days, postfeeding 14 28

Vol. 40, no. 6

% of substrate consumed dose 105 TCID50

Titer of VSV (log10 virus/individual)

24 h

48 h

Range

Mean

n

⬍50 ⬍50 ⱖ50 ⬍50 ⬍50 ⱖ50

⬍50 ⱖ50

0.0Ð3.1 0.0Ð5.8 4.8Ð6.8 0.0 0.0Ð8.8 8.6Ð9.6

2.3 5.1 6.5

1/8 6/10 2/2 0/2 1/3 4/4

⬍50 ⱖ50

8.3 9.2

Positives/Total

Mean-fold titer increase

1.3⫻ 32⫻ 2,000⫻ 16,000⫻

Values reported as % substrate consumed refer to the amount of the virus-laden Þlter paper that the grasshoppers ingested in the stated time. VSV titers are expressed in terms of log10.

24 h and ate only 50% of the Þlter paper by 48 h produced 108.8 TCID50 after 28 d of incubation. Grasshopper-to-Grasshopper Transmission. Given that grasshoppers became infected by VSV, the next question was whether it is biologically possible for the virus to be maintained in a grasshopper population (Fig. 1, step 2). The two most obvious mechanisms for grasshopper-to-grasshopper transmission that would allow these insects to function as a reservoir are vertical transmission via transovarial transfer (TOT) and horizontal transmission via necrophagy or cannibalism. TOT would only allow transmission once per year in most of North America, where most grasshoppers are univoltine. It has been shown that TOT is inadequate for the maintenance of related viruses in nature without concomitant horizontal transfer within the population (Tesh and Modi 1987, Ciufolini et al. 1989, Tesh et al. 1992). Cannibalism within the passive reservoir population would allow continuous transmission and ampliÞcation of the virus over the course of a single generation. Grasshoppers were fed virus-laden Þlter paper, as described above, and held for 14 d; these served as cadavers for cannibalism by a second group of grasshoppers. All four of the grasshoppers fed cadavers that had consumed ⬎50% of their inoculum, and thus estimated from Table 1 to contain an average of ⬇105.6 TCID50, became infected. Two of these cannibalistic grasshoppers developed VSV titers 63 or 200 times greater than their estimated dose, one reaching 108.8 TCID50. It should be noted that the cannibalistic grasshoppers consumed from a trace to 25% of the cadavers, with a mean of ⬇10%. As such, the effective inoculative dose was ⬇104.5 TCID50, suggesting ampliÞcation in one of cannibals was ⬎1,000-fold. None of the six cannibalistic grasshoppers that ate cadavers who had consumed ⬍50% of their virus inoculum, and thus estimated to contain an average of 102.3 TCID50, became infected. Grasshopper-to-Cattle Transmission. Given that grasshoppers can become infected with VSV, the next question was whether it is biologically possible for these insects to transmit virus to cattle (Fig. 1, step 3). One of three Hereford yearlings contracted the disease when fed VSV-infected grasshoppers. The infection was evidenced by increased body temperature, virus isolation from oral swabs, and development of serum antibody titers after a typical disease course.

The clinical signs over a 2-wk period were consistent with a classical VSV infection. Excessive salivation began 3 d after exposure (DAE) and persisted for 6 d; the body temperature peaked at 40.7⬚C (normal ⬇38.6⬚C) at 5 DAE; the epidermis sloughed from the tongue at 7 DAE, with vesicular lesions persisting in the oral cavity until 12 DAE. Reduced consumption of feed was observed from 4 to 10 DAE. The animal recovered fully by 13 DAE, typical of VSV infections. Virus was detected by standard plaque assay in two of the three oral swabs taken at 4 DAE. The highest titers detected in saliva were from samples taken 5 h apart on four DAE, with 4.92 ⫻ 104 and 4.85 ⫻ 104 plaqueforming units (pfu)/ml. All plaques were conÞrmed as VSV by immunocytochemistry. A 4-mg tissue biopsy taken from a lesion on the tongue at 7 DAE contained 3.2 ⫻ 105 pfu. The presence of VSV nucleic acid in this animal was conÞrmed by reverse transcriptase PCR analysis of RNA extracted from saliva and tissue (lesion) biopsies. By 2 wk after infection, the serum neutralization titer had increased from undetectable to 1:1,280. Cattle-to-Grasshopper Transmission. To complete the hypothesized cycle of VSV transmission, we investigated whether grasshoppers could be infected by ingesting infected bovine saliva (Fig. 1, step 4). Susceptibility of the grasshoppers to VSV infection was variable (Table 2). One of the grasshoppers fed 5 ␮l of saliva (101.35 TCID50) from the infected cow (no. 602) yielded a titer of 104.5 TCID50, a 1,400-fold increase. Although none of the grasshoppers fed 10 ␮l (102.7) or 15 ␮l (103.0) of saliva from the infected cow became infected, 15% of those fed 20 ␮l (103.2) became infected and yielded titers greater than the virus meal provided. Immunocytochemistry of infected cell culture conÞrmed VSV as the cytopathic agent. In addition, feedings of normal saliva spiked with known concentrations of VSV were performed to ascertain the minimum infectious dose. Doses of 101.4 and 103.5 TCID50 led to infections of 30 and 46% of grasshoppers, respectively (Table 2). Titers conÞrmed ampliÞcation of the virus up to 1,250 times (106.6) the inoculative dose, with the greater initial dose (103.5) yielding higher Þnal titers of virus. As described above, immunocytochemistry conÞrmed that the grasshoppers were infected with VSV. Virus Isolation from Field Specimens. As a Þrst effort to isolate VSV from Þeld-collected grasshop-

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Table 2. VSV titers in grasshoppers (M. sanguinipes) 14 d after being fed saliva from an uninfected cow spiked with virus grown in cell culture or saliva from the cow infected by feeding on VSV-infected grasshoppers Saliva

Carrier

Doses

Spiked

Liquid

1

Solid

1

Infected

Inoculation titer (log10 virus/10 ␮l)

n

Infected (%)

0.00 0.05 0.75 1.40 3.50 0.00 1.35 2.70 3.00 3.18

10 23 21 23 13 5 10 10 10 20

0 0 0 30 46 0 1 0 0 15

Grasshopper virus Titer (log10/individual) Range

Mean

NA NA NA 2.3Ð4.3 2.1Ð6.6 NA NA NA NA 3.5Ð3.8

NA NA NA 3.6 4.5 NA 4.5 NA NA 3.6

NA, not applicable. Infected percentage refers to the grasshoppers containing a cytopathogenic agent. Titers of VSV are expressed in terms of TCID50.

pers, virus isolation assays were performed on 212 pools representing 256 Þeld-collected grasshoppers and katydids from VSV endemic regions of Costa Rica. No live virus was isolated from these samples. Discussion Our results demonstrated the biological potential of grasshoppers to serve as a source of VSV. Three critical questions remain. 1) Are all of the essential transmissions ecologically plausible? 2) Are VSV-infected grasshoppers found in pastures alongside infected livestock? and 3) Can VSV spread be controlled by reducing grasshopper populations? The transmission of virus among grasshoppers within a population via cannibalism is reasonable, given the frequency of necrophagy in rangeland species (Lockwood 1988, 1989). Typically 5Ð15% of a grasshopper population engages in necrophagy in a 24-h period (Lockwood 1988). This behavior begins in the third instar and increases with development, reaching the highest levels in adult females, which apparently use the nutrients for egg production. Lockwood (1988) found that rangeland grasshoppers begin to arrive at a cadaver within 5 min of its being placed in the Þeld, and several individuals are commonly seen feeding upon a single cadaver (Lockwood 1989). Many species of grasshoppers use the volatile fatty acids emitted from cadavers to locate these valuable food sources (Bomar and Lockwood 1994). Hence, sustaining or amplifying VSV within a grasshopper community via necrophagy seems to be an ecologically plausible process. Oral transmission of VSV from grasshoppers to cattle under natural conditions would initially seem to be a rare, even implausible, event. For this to happen, two conditions must coincide. First, cattle must have a viral route of entry, presumably in the form of a wound to the oral cavity, because infection cannot be established by virus contact with intact epithelium of the gum, tongue, coronary band, or teats (Webb et al. 1987) or by instilling virus in the eye (Stallknecht et al. 1999). Rather, infection seems to require skin scariÞcation (Patterson et al. 1955, Jonkers 1967). Coarse roughage, hard feed pellets, and poor hygiene were

associated with VS on California dairies (Hansen et al. 1985). Oral injuries commonly occur in cattle that consume dry hay, rough vegetation (e.g., thistles), and other abrasive substances (Smith 1996), and may be ubiquitous among cattle feeding on dry vegetation, with 75Ð90% of rangeland cattle having cactus spines in their tongues (Migaki et al. 1969, Smith 1996). Although such wounding is common in dry, rangeland vegetation (e.g., southwestern United States), this phenomenon is relatively unusual in lush pastures (e.g., southeastern United States), a pattern that reßects the geographic distribution of VS in the United States. Second, given a wound in the oral cavity, the cattle would have to consume infected grasshoppers. In pasture and rangeland habitats, grasshopper populations often reach densities of 40 individuals per square meter, and it is not unusual to Þnd 60 Ð 80 individuals per square meter during high density years (Chapman and Joern 1990, Gangwere et al. 1997). Extremely high population densities, exceeding 100 individuals per square meter, can develop in mesic, low-lying habitats when the upland vegetation dries out. These moist low-lying areas are also heavily used by cattle. Thus, high densities of grasshoppers may be concentrated in areas of cattle feeding. There are three modes by which grasshoppers may be consumed by cattle. When considered together, these natural conditions provide a scenario in which oral transmission of VSV from grasshoppers to cattle may be not only plausible but also a cogent means of maintaining the transmission cycle. First, grasshoppers typically climb the vegetation before ecdysis and become immobile for a period of 1.5Ð2.5 h during the molting process, which normally occurs in the midmorning or late afternoon (Uvarov 1977, Tsyplenkov 1978). Under these conditions, the grasshopper would be prone to consumption by livestock. Given that a molt normally occurs every 5 d during nymphal development (Uvarov 1977, Tsyplenkov 1978), this means that a grasshopper would be highly susceptible to consumption for 1.2Ð2.1% of the time. If we assume a moderate population density of 30 grasshoppers per square meter on a grassland habitat producing 450 kg of dry forage per hectare, there would be one vulner-

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able (molting) grasshopper for every 150 g of dry forage. A cow consuming 7 kg of dry forage per day would therefore encounter, and likely consume, 47 molting grasshoppers per day. In a second pathway, healthy, nonmolting grasshoppers may also be consumed by cattle. A very low level of incidental feeding can be expected as cattle graze in grasslands heavily infested by grasshoppers. Upon disturbance, grasshoppers commonly jump or ßy in an undirected manner, sometimes bringing them into contact with the source of disturbance. Hence, it is almost certain that some grasshoppers will enter the mouth or nose of feeding cattle. A third mode of transmission is via processed forage or feed for cattle. In this context, grasshoppers commonly reach high population densities (⬎60 individuals per square meter) in irrigated alfalfa and grass Þelds. After these Þelds are cut for hay, the forage is either fed directly to livestock or is left in windrows to dry. These windrows functionally concentrate grasshoppers, which are subsequently incorporated into the bales. Based on our observations, many of the grasshoppers will be crushed during cutting or baling, but some individuals survive in the bales for several days and ranchers have reported Þnding live grasshoppers in bales 3 wk after harvesting. Cattle are also fed blocks of feed mixed with molasses. Molasses is both a potent attractant and feeding stimulant for grasshoppers, and these insects are often seen nearby and adhered to these blocks in the Þeld. Also, we have recovered grasshopper fragments from Roll Mix, a livestock feed supplement comprised of rolled grain and cane molasses. This study documents a herbivorous insect functioning as a possible source of a viral disease for domestic animals. Two additional critical observations must be made to show that grasshoppers are relevant to VSV epizootiology. First, infected grasshoppers must be identiÞed in pastures alongside infected livestock. A preliminary survey of a small number of grasshoppers from a VSV enzootic area failed to reveal naturally infected insects but was too small to give a deÞnitive answer. A larger study is in progress. Second, speciÞc control of grasshopper populations must repress vesicular stomatitis in pastured livestock. Testing the effect of removing grasshoppers without altering other insect populations is made possible by bacterial and fugal pathogens speciÞc for grasshoppers. Only Þeld evidence can conÞrm the transmission cycle proposed here. Acknowledgments We thank S. Lonning for design of PCR primers, M. Muth and L. Santistevan for assistance during the biocontainment phase of this research, B. Taro and S. Kontour for technical assistance, and C. Viets for providing grasshoppers. We appreciate the help provided by the research team (M. Herrero, A. Jimenez, R. Pereira, and J. Rodriguez) in the Laboratory of Entomology at the School of Veterinary Medicine, UNA, Costa Rica, for providing grasshoppers from a VSV-endemic area.

Vol. 40, no. 6 References Cited

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