CHAPTER 2
Longitudinal Serosurvey of Wild Rodents for Venezuelan Equine
Encephalitis Virus Antibody in Coastal Chiapas, Mexico
36
ABSTRACT
Venezuelan equine encephalitis virus (VEEV) subtype IE is endemic in southern
Mexico. Until an outbreak of severe and fatal Venezuelan equine encephalitis (VEE)
occurred in 1993, it was thought that only enzootic, or equine avirulent, VEEV strains
circulated there. Since the outbreak, numerous studies have been conducted to
characterize the newly recognized equine-virulent subtype IE VEEV strains. The results
revealed that specific mutations had occurred in the viral genome that led to enhanced
infection of epizootic mosquito vectors. The primary aim of this study was to determine
the ground-dwelling mammal species most likely to participate in the natural
transmission cycle of subtype IE VEEV in coastal Chiapas. Over the course of one year
four habitat types representing the most common forms of land use in the area were
sampled for rodent species composition, abundance and subtype IE VEEV-specific
antibodies. In contrast to previous studies, antibody was rarely detected in a total of 92
mammals captured, representing 5 genera and 5 species. Several factors, including a
major meteorological disturbance, may have contributed to these findings.
INTRODUCTION
The 1993 outbreak of VEE in coastal Chiapas, Mexico was caused by a VEEV
subtype that had never before been associated with equine disease (Oberste, Fraire et al.
1998). Subsequently, studies have been conducted to determine the factors, both
37
ecological and molecular, that accompanied this apparent change. Equine-virulent strains
of subtype IE VEEV phylogenetically cluster more closely together than they do with
strains of subtype IE VEEV isolated prior to the 1993 outbreak and have been shown to
have mutations in the envelope glycoprotein gene that contribute to improved mosquito
infection (Oberste, Fraire et al. 1998; Brault, Powers et al.). Experimental vector studies
have shown that the new, equine-virulent strains infect the epizootic vector Aedes
taeniorhynchus better than the old strains (Brault, Powers et al. 2004). Aedes
taeniorhynchus is a voracious biter of horses and cows and would be favored by changing
patterns of land use as pristine lowland tropical forest is consumed for agricultural
purposes (Cupp, Scherer et al. 1986). If subtype IE VEEV was able to switch vector
species, it is possible that the vertebrate amplifying host range might have changed
accordingly. This study sought to identify the primary mammalian amplifying hosts of
recently emerged, equine-virulent subtype IE VEEV in coastal Chiapas.
Epizootic strains of VEEV typically cause severe disease in horses, which serve
the as primary amplifying host of the viruses. (Wang, Bowen et al. 2001; Weaver, Ferro
et al. 2004). Strains that are not associated with equine disease are referred to as enzootic
strains. These strains are thought to utilize ground dwelling mammals, particularly
rodents as amplifying hosts (Scherer, Dickerman et al. 1985; Salas, Garcia et al. 2001;
Barrera, Ferro et al. 2002; Weaver, Ferro et al. 2004). Laboratory studies have shown that
a variety of wild rodents from several different genera survive experimental infection,
develop high viremia and strong antibody responses after infection with various VEEV
strains (Young, Johnson et al. 1969; Bowen 1976; Coffey, Carrara et al. 2004; Carrara,
38
Coffey et al. 2007). Understanding which species serves as the primary amplifying host
in a particular ecosystem will help to elucidate the natural transmission cycle and how
epizootic arboviruses emerge from enzootic precursors.
A previous VEEV seroprevalence study was performed from 1998 to 2003
(Estrada-Franco, Navarro-Lopez et al. 2004) in and around Mapastepec municipality in
coastal Chiapas. Small serosurveys were conducted on wild and domestic animals as well
as on humans and cattle. Among the wild animals, cotton rats, opossums and rice rats had
the highest VEEV-specific antibody rates with values of 67% (6/9), 25% (2/8) and 17%
(1/6) respectively (table 2.1). Antibody rates among domestic animals dogs, chickens,
turkeys and a goose were 33% (1/3), 20% (1/5), 33% (1/3) and 100% (1/1) respectively
(Estrada-Franco, Navarro-Lopez et al. 2004). These small serosurveys were conducted
sporadically in the areas where antibody rates were highest among humans and therefore
may not be representative of the region as a whole.
Table 2.1: VEEV seroprevalence in animals from coastal Chiapas State, Mexico*
1
Species (common name)
Month (2000)
% pos (n)
Titer (test)
Philander opossum (grey four-eyed opossum)
Apr.
0% (0/2)
na
Nov.
20% (1/5)
640 (HI)
Didelphis marsupialis (common opossum)
Jun.
100% (1/1)
160 (HI)
Oryzomys alfaroi (Alfaro's rice rat)
Apr.
50% (1/2)
20 (PRNT)
Aug.
0% (0/4)
na
Oryzomys couesi (Coues' rice rat)
Apr.
0% (0/2)
na
Aug.
0% (0/1)
na
Nov.
0% (0/6)
na
Sigmodon hispidus (hispid cotton rat)
Apr.
0% (0/1)
na
Aug.
100% (6/6)
20—160 (HI)
Jun.
0% (0/2)
na
Rattus rattus (roof rat)
Aug.
0% (0/2)
Cows
Nov.
70% (14/20) 20—640 (PRNT)
Dogs
Jun.
33% (1/3)
20 (PRNT)
Chickens
Jun.
20% (1/5)
20 (PRNT)
Turkeys
Jun.
33% (1/3)
20 (PRNT)
Goose
Jun.
100% (1/1)
320 (PRNT)
1
titer reported as reciprocal of highest serum dilution to yeild positive result
VEEV, Venezuelan equine encephalitis virus
HI, Hemagglutination inhibition
PRNT, Plaque reduction neutralization test
*adapted w ith permission from Estrada-Franco et al, 2004
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Human serosurveys showed an overall VEEV-specific antibody rate of 42% for
the entire region with higher rates (up to 75%) in coastal villages and lower rates (as low
as 4%) in inland villages (Estrada-Franco, Navarro-Lopez et al. 2004). Additionally, a
serosurvey was performed on 767 cattle. Cattle are thought to make good sentinel
animals because they are never vaccinated, they seroconvert with little or no apparent
illness, they are usually slaughtered at two years of age in this part of Mexico and they
are exposed to a high number of mosquito bites. Seropositivity rates in cattle were higher
(up to 74%) near the coast and lower (as low as 5%) closer to the mountains (EstradaFranco, Navarro-Lopez et al. 2004). These data are consistent with the human serosurvey
results and support continual circulation of VEEV in the region of the 1993 outbreak.
The previously reported rodent serosurvey data were acquired from a small
number of locations and months. These locations were within villages, thus the sampling
included mostly peridomestic rodents. However, it is possible that the primary amplifying
hosts are not peridomestic in nature and are only found away from human settlements. In
order to more fully understand which rodents are most likely to serve as amplifying hosts,
it was important to sample the broadest variety of habitats and seasons as possible.
METHODS
Location
During the 1993 VEE outbreak, equine cases occurred throughout the entire area
flanked by the Pacific Ocean to the west and the Sierra Madre Mountains 25 kilometers
to the east (Figure 2.1). For this reason, field sites were chosen throughout this area based
40
on the four most common types of land use: cattle pasture, mango orchard, palm orchard
and mangrove swamp. Cattle pastures occupy the majority of the land in the region where
this study was performed. The permanent pastures are lined with rows or stands of trees
that divide the properties. Large stands of natural forest are non-existent in these
lowlands. The closest equivalent to forest stands would be the orchards and plantations of
mangos, bananas, and oil palms. These are relatively small in scale (approximately 2—10
hectares) and are not nearly as abundant or extensive in area as cattle pastures.
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Figure 2.1:
Arial image of coastal Chiapas, Mexico. Google Earth image of the area
where the 1993 outbreak of VEEV IE occurred. White stars indicate
approximate locations where confirmed cases of equine infection
occurred. Black stars indicate sites used in this study for rodent trapping.
41
A system of mangroves and lagoons that are fed and maintained by 17 rivers and
numerous streams lies immediately behind the Pacific coast. At the interface between
mangroves and the orchards/pastures, is a mangrove swamp wetland habitat. While these
areas are not continually submerged, they are too wet for agricultural purposes or human
habitation and are thus comparatively unchanged by human activity. We chose trapping
sites in cattle pastures, mango orchards, palm orchards and mangrove swamps in an
attempt to cover the broadest range of habitats in order to correlate land use with VEEV
exposure risk.
Trapping
Two representative sites within each habitat type were chosen for a total of eight
sites within the study area. All trapping was done on a grid system: square grids
measuring one hundred meters on a side (one hectare) were established at each field site.
Trapping was conducted during four five-week field trips over the course of one calendar
year: July/August 2006, October/November 2006, February/March 2007, May/June 2007.
The rainy season lasts from May through November is bimodal: June and September are
the wettest months while July and August receive slightly less rain. Due to limited
resources and personnel trapping was conducted at one site at a time for 2 days in a row
at each site.
At each site a trapping grid was arranged such that 10 lines were spaced 10 meters
apart with 10 trap sites placed 10 meters apart along each line for a total of 100 trap sites
per field site and 800 trap sites total. One hundred Sherman traps (H.B. Sherman Traps,
Tallahassee, FL) for small rodents and nine tomahawk traps (Tomahawk Live Trap Co.,
42
Tomahawk, WI) for larger animals such as opossums were set at each site for two nights
in a row. The tomahawk traps were placed at each of the four corners, along the midlines
of the perimeter and in the very center of the grid (Figure 2.2).
Figure 2.2:
Trapping grid layout used at all eight sites during all four trapping trips.
One hundred Sherman traps were set 10 meters apart for capturing small
animals such as rats and mice. Nine larger Tomahawk traps were set 50
meters apart for capturing larger animals such as opossums, raccoons and
skunks.
Traps were set in the early evening before dark and baited with oats (Sherman traps) or
carrots (tomahawk traps). All traps were checked for captured animals early the
following morning, within two hours of sunrise. Demographic data and capture site were
recorded for each animal caught and reference photographs were taken. Animals were
anesthetized briefly by being placed in a plastic bag with a small amount of ether until
sedated. Once sedated, animals were earmarked for re-capture identification and blood
43
samples were obtained from the retro-orbital sinus using heparinzed glass capillary tubes.
Blood samples were either absorbed into Nobuto blood filter strips (Advantec,
Pleasanton, CA) for later elution and serological testing or were diluted at a ratio of 1:10
in phosphate buffered saline (PBS), centrifuged at 1000 x g for 3 min. to separate blood
cells from diluted plasma and stored frozen for later serological testing. Animals were
returned to the site of capture and observed until fully recovered and were not bled a
second time in the event of immediate re-capture the following day. This study was
conducted under permit number SGPA/DGVS/03858/07 Julio 2 de 2007, issued to Dr.
J.G. Estrada-Franco, by the Secretaria de Salud de Mexico.
Antibody Assays
Diluted rodent plasma were tested for VEEV-specific antibodies using a
hemagglutination inhibition (HI) test. This test was chosen because it allows for
screening for antibodies against a broad array of pathogens with a relatively small
amount of serum or plasma (Clarke and Casals 1958; Calisher, Shope et al. 1980). The
test sera were mixed with added goose erythrocytes and hemagglutinin antigen from a
reference strain of virus. If antibodies were present in the test serum, they bound the
hemagglutinin antigens and prevented them from hemagglutinating, or forming
aggregates of the added erythrocytes. After screening sera by HI, remaining samples were
tested by plaque reduction neutralization test (PRNT) for subtype IE VEEV-specific
antibodies. Briefly, test sera were serially diluted in PBS and heat inactivated at 56°C for
one hour, then mixed with approximately 100 plaque forming units (pfu )of virus and
incubated at 37°C for one hour. The mixture was inoculated onto Vero cells and dilutions
44
resulting in a >80% reduction in virus titer were considered positive, with titers reported
as the reciprocal of the endpoint dilution.
RESULTS
Census
I sampled blood from a total of 92 wild mammals including 4 species of rodent
and 2 species of opossum (Table 2.2). Animal identification was initially based on
morphology (Reid 1997), though Oryzomys couesi, O. alfaroi and O. rostratus are
difficult to distinguish by physical appearance. Cytochrome-b gene PCR amplification
and sequencing was performed on all available blood samples to confirm and clarify
species identity (Boakye 1999). Seven animals that had been identified as either O.
alfaroi or O. rostratus were re-assigned to O. couesi or Oligoryzomys fulvescens. All O.
couesi results matched previously published O. couesi sequences with 97% sequence
identity and were 90% identical to O. palustris. The Oligoryzomys fulvescens animals did
not resolve as well with 91% identity to previously published O. fulvescens sequence and
93% identity to O. vegetus which is thought to be confined to Costa Rica and Panama
(Reid 1997). Voucher specimens were sent to Dr. Robert Bradley at Texas Tech
University and they were confirmed as O. fulvescens (personal communication), though
this species designation is under taxonomical revision and may comprise several distinct
species (Musser and Carlton 1993; Dickerman and Yates 1995). Unfortunately, cellular
blood fractions were not available for 4 samples (3 O. alfaroi and 1 O. rostratus),
rendering PCR testing impossible. However, because all other animals morphologically
45
identified as O. alfaroi and O. rostratus were shown genetically to be O. couesi, these
four have been listed as O. couesi in table 2.2. The highest number of captured mammals
occurred during May/June (n = 35) and the second highest number occurred during
February/March (n = 29). The lowest capture of mammals was encountered in
July/August (n = 11) and the second lowest in October/November (n = 17). For each
trapping trip there was a total of 1,744 trap nights (109 traps x 2 nights x 8 sites) making
the trapping efficiency 0.6—2.0%.
46
47
1
16
3
6
Didelphis virginiana
(Virginia opos sum )
Liom ys salvini
(Salvin's s piny pocket m ous e)
Oligoryz om ys fulvescens
(fulvus pygm y rice rat)
Oryz om ys cousei
(Coues ' rice rat)
2
30
1
Didelphis m arsupialis
(com m on opos sum )
Sigm odon hispidus
(his pid cotton rat)
Total
1
no b lood sam ple collected
1
Feb/Mar
1
Conepatus m esoleucus
(com m on hog-nos ed s kunk)
Mam m al Species
1
35
5
7
18
2
2
May/Jun
12
1
8
2
1
July/Aug
5
17
1
8
2
1
Oct/Nov
8
94
13
10
50
7
4
2
TOTAL
Abundance by s pecies and tim e of year
31
9
8
10
2
1
1
2
13
4
1
1
1
3
1
1
41
1
36
3
5
9
3
1
8
94
13
10
50
7
4
2
Abundance by s pecies and habitat
Mangrove Mango
Palm
Cattle
Swam p Orchard Orchard Pas ture TOTAL
Table 2.2: Distribution and Collections of Wild M ammals Captured in coastal Chiapas over the course of one ye ar.
Distribution
The habitat that yielded the highest number of animals was the palm orchard. Out
of the 42 animals captured in this habitat, 36 were L. salvini captured in just one of the
two palm orchard sites (PALM1), which seems to have been a hotspot for these rodents.
The habitat with the next highest mammal abundance was the mangrove swamp in which
30 animals were captured. The mango orchard habitat yielded 11 animal captures, and the
pasture habitat only yielded 9. The highest number of mammal species was encountered
in the mango orchard habitat and the lowest in the cattle pasture.
Serology
Blood samples from the first trip were stored on paper bloodstrips. Whole blood
was absorbed into the strips, allowed to air dry, then stored in the dark away from
moisture for up to 35 days. Unfortunately, the blood from these strips did not appear to
elute efficiently in the lab for serological testing– based on solution color change.
Bloodstrips are often used in field studies when refrigeration is unavailable or unreliable.
They have been shown to be very efficient for collecting blood/serum samples for virus
isolation and antibody assays (Fortes, Menitove et al. 1989; Guzman, Ding et al. 2005).
In order to elucidate the cause of poor elution, antibody positive mouse blood from
experimental animals was absorbed into blood strips that were then subjected
experimentally to heat, sun or heat plus high humidity for 5 days – these conditions were
chosen to simulate conditions in the field. After elution into PBS all samples eluted
efficiently and were found to be positive by HI test. Liquid plasma that had been diluted
1:10 and stored frozen yielded 2—4 times higher antibody titers than 1:10 rehydrated
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blood sample eluted from dried bloodstrips. In light of this field blood samples were
subsequently stored as frozen liquid rather than dried bloodstrips.
Sera were tested by HI assay against a variety of viral pathogens known to
circulate in Central America and Mexico: VEEV (vaccine strain TC-83), St. Louis
encephalitis virus (strain TBH28, isolated in 1962 in the USA), West Nile Virus (strain
385-99 isolated in 1999 in the USA), Maguari virus (BeAr 7272, isolated in 1957 in
Brazil) and Rio Grande Virus (TBM3-24, isolated in 1973 in the USA). Additionally, 38
sera were tested for antibodies against Eastern equine encephalitis virus (TenBroeck
strain, isolated in 1933 in the USA). The only positive HI test result was a female O.
couesi that tested positive for antibodies against Rio Grande virus with a titer of 40. This
animal was sampled in March from one of the mangrove swamp sites (MNGRV2).
To maximize safety, the HI test used antigen from the VEEV IAB strain that is
used for vaccination (strain TC-83), which was believed to be cross reactive enough to
detect subtype IE VEEV-specific antibodies. However in light of the high
seroprevalences found in previous studies, my lack of VEEV seropositive samples
prompted testing by a second means (Scherer, Dickerman et al. 1971; Scherer,
Dickerman et al. 1985; Estrada-Franco, Navarro-Lopez et al. 2004). Neutralization tests
with subtype IE VEEV (strain 68U201, isolated in 1968 in Guatemala) were performed
on all serum samples for which adequate volume remained after HI testing. By this
method, the only positive result was a female O. couesi that had a titer of 20 and was
captured in June from one of the mangrove swamp sites (MNGRV2). This animal had
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tested negative by HI, raising concern that the subtype IAB VEEV antigen was not
completely cross reactive with subtype IE VEEV-specific antibodies.
DISCUSSION
Seasonality
The variation in trap efficiency between the 4 trapping trips is likely due to
differences in rodent food availability. Most of the animals encountered (Liomys salvini,
Oryzomys couesi and Oligoryzomys fulvescens) are known to feed primarily on seeds
with an occasional beetle larva or green plant while Sigmodon hispidus favors more green
plants and fungi with occasional seeds and insects (Reid 1997). The highest trap
efficiencies were observed during the February/March and May/June trapping trips.
February is the peak of the dry season and there is very little rain so rodents would
perhaps be more adventurous in their search for food. May marks the beginning of the
rainy season and is accompanied by new plant growth but little seed production. The
lower trap efficiencies were observed later in the rainy season when seeds, plants and
insects are most abundant thus rodents are less inclined to enter traps in search of food.
Though overall rodent capture rates fluctuated from site to site, there was no strong
fluctuation in species composition throughout the year.
Antibody prevalence
The only positive test results from screening all 92 animals against 5 classes of
virus were one O. couesi with neutralizing antibodies against subtype IE VEEV and one
O. couesi with HI antibodies against Rio Grande virus. Rio Grande virus is a phlebovirus
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(family Bunyaviridae) that was originally isolated from a wood rat (Neotoma micropus)
in south Texas in 1973 (Calisher, McLean et al. 1977). This area is semi-arid mesquiteacacia savannah with low shrubs and salt flats. This habitat contrasts sharply with the
tropical mangrove swamp where my seropositive O. couesi animal was captured. This
positive result was not likely to be the result of antibody cross reactivity because Rio
Grande virus has been shown to be largely non-cross reactive with other known members
of the Phlebovirus genus (Travassos da Rosa, Tesh et al. 1983). It is possible that Rio
Grande virus is endemic to coastal Chiapas, though it has not previously been reported
from this area and the ecology of the area is starkly different and geographically distant
from the area of original isolation. Alternatively, it could be that there is an as-yet
unidentified closely related phlebovirus circulating in the mangrove swamp that has
cross-reactive properties to Rio Grande virus.
The low number of VEEV-specific antibody positive rodents (<1%) contrasts
previous findings. For example, between April and November of 2000, thirty-four wild
rodents were captured in four different villages in coastal Chiapas (Estrada-Franco,
Navarro-Lopez et al. 2004). Of these, nine were found to have detectable antibodies
specific to VEEV, for a total seroprevalence of 26%. Another study in Veracruz, Mexico,
found 28 individuals from 9 different species of wild mammal with antibodies specific to
VEEV during a three year time period from 1963 to 1966 (Scherer, Dickerman et al.
1971). Aguirre et al. found serological evidence of subtype IE VEEV infection in 9 out of
75 wild rodents captured in Coahuila state in the north of Mexico (Aguirre, McLean et al.
1992). In five years, during the course of a long-term study in coastal Guatemala, an
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average of 8.6 isolations of VEEV were made each year from sentinel hamsters (total of
43 strains) (Scherer, Dickerman et al. 1985). Additionally, the same study found 15
animals in a single year at a single location with subtype IE VEEV-specific antibodies.
These included: S. hispidus (n = 5), D. marsupialis (n = 8), L. salvini (n = 1) and 1 O.
couesi (n = 1).
Ecological Considerations
In light of the historical evidence indicating constant circulation throughout the
entire region and current human and bovine serological evidence supporting continued
VEEV circulation, it seems unlikely that the negative results reported here are an accurate
reflection of true overall rates of infection among wild rodent populations in Chiapas
state (Estrada-Franco, Navarro-Lopez et al. 2004). However, if the rates we found are
considered a true and accurate report on antibody seroprevalence, some explanation is
needed. The effects of hurricane Stan, which hit southern Mexico in October of 2005,
could possibly provide this explanation. According to local landowners, the entire region
where this study occurred was flooded during and after the storm (Figure 2.3). Roads,
houses, fields and bridges were destroyed. The deluge from the storm occurred at the
peak of the rainy season when rivers were already running high and land was already
inundated. Many resident rodents would likely have been either drowned or displaced.
The abundance of available reservoir hosts has been shown to be correlated to the level of
virus circulation in lowland, tropical forests (Shope 1972; Woodall 1972).
Host populations thus may have experienced a reduction and the rebounding populations
of susceptible VEEV hosts had not yet re-established pre-flood seroprevalence by the
52
time my study was conducted. Hurricanes are ecological disturbances that result in finegrained mosaic habitat heterogeneity (Turner, Dale et al. 1997). It is possible that my
trapping sites were coincidentally situated in areas adversely affected by this
heterogeneity. It has been shown that flooding can reduce both rodent abundance and
species diversity for up to two years after a farmland-flooding event (Zhang, Wang et al.
2006). With a large reduction in the number of available amplifying hosts various
lineages of virus can die off resulting in focus fragmentation or elimination. My
collections, which were begun nine months after the storm, could lack the seroprevalence
found in pre-storm collections because of interruption of the natural transmission cycle.
Figure 2.3:
Hurricane Stan making landfall in Southern Mexico, in October of 2005.
The state of Chiapas is shown in black and La Encrucijada biosphere
reserve is highlighted in red along the coast of Chiapas.
53
Other Considerations
Although unlikely, it is possible that frozen serum storage conditions were
inadequate to preserve HI antibodies. Blood samples were placed in a food cooler with
ice packs immediately upon collection. After the samples were diluted with PBS and
blood cells were removed, they were transferred to a conventional kitchen freezer within
two hours after collection and kept there for up to 35 days. For shipment to the University
of Texas Medical Branch, frozen samples were placed, along with ice packs, inside
insulated thermos bottles which were then packed with more ice packs into heavily
insulated shipping boxes and transported by airplane. Upon unpacking they were
confirmed still frozen and immediately placed at -80 until testing could be performed.
We feel that an inadequate cold chain is not likely, as the sera were invariably kept frozen
solid from the day of collection until the day of testing. No evidence of a break in the
cold chain, such as microbial growth, was observed.
Another possibility is that the subtype IAB VEEV antigen used in the HI tests is
not as cross-reactive as we had expected and did not detect all subtype IE VEEV-specific
antibodies. The HI test is known to be relatively cross-reactive and was thus considered a
useful first method of screening (Clarke and Casals 1958). However, the single animal
that tested positive during secondary PRNT was initially found to be negative by HI. This
prompted us to derive new antigen from a subtype IE VEEV strain isolated from the
study area in 2001 and compare it to the conventional IAB antigen in a control HI test.
With plasma from experimentally infected wild rodents, the use of antigen derived from
subtype IE VEEV revealed antibody titers 2—4 times higher than those revealed by the
54
use of antigen derived from the subtype IAB VEEV vaccine strain TC83. Testing by
PRNT revealed a 2—4 fold additional increase in antibody titer over those determined by
HI. Thus, a titer of 80 by PRNT may not be detected by HI, even with the antigen
derived from the subtype IE VEEV strain. It is likely that by screening test sera with the
subtype IAB VEEV antigen, animals that were circulating low levels of antibody (less
than 80) may have been falsely shown to be seronegative.
Subsequent to this study (four months later) 72 wild rodents were collected from
the same area and were imported to the laboratory for experimental infection studies (see
Chapter 3). Two of these animals, were found to have pre-existing antibodies against
subtype IE VEEV. One S. hispidus animal had a titer of 160 by HI with the subtype IE
VEEV antigen and 640 when measured by the more specific and more sensitive PRNT.
The second animal, an O. couesi had a titer of 320 by PRNT and was not tested by HI.
This finding of 2.7% seropositivity (2/72) is more consistent with my finding of ~1%
seropositivity (1/92) than to previously published findings of 26% (Estrada-Franco,
Navarro-Lopez et al. 2004) which suggests that sensitivity issues related to cross-reactive
antigens, although a possible confounding factor, probably had little effect on my
seroprevalence estimates.
Conclusions
The previously published result of 26% VEEV seropositivity resulted from animal
collections in southern Mexican towns known to have high human VEEV seroprevalence.
If these towns were selected because they were near VEEV transmission foci, then the
seroprevalence reported is from a potentially biased sampling of the larger rodent
55
populations in the region. The effects of Hurricane Stan, though impossible to measure
retrospectively, were certainly responsible for altering the ecology of coastal Chiapas, at
least temporarily. It is plausible that a reduction of rodent populations caused an abrupt
interruption to VEEV circulation. By the time my collections were performed, though
rodent populations had rebounded, virus transmission cycles may not yet have fully
recovered. Comparison to an artificially high previous finding and the possible temporary
reduction in transmission caused by the hurricane are the most plausible explanations for
the low seroprevalence reported here.
56