Detection of Chlamydia infection in Peromyscus species rodents from sylvatic and laboratory sources.

To determine if Chlamydia muridarum, or other chlamydiae, are enzootic in rodents, we probed a serum bank of wild Peromyscus spp. mice for immunoglobulin G-antibody reactivity to ultraviolet light-inactivated C. muridarum elementary bodies (EBs) using an enzyme-linked immunoassay. Applying a cut-off for a positive reaction of OD(405) nm = 0.1 at a 1:20 dilution, we found titratable antibody reactivity in 190 of 247 specimens surveyed (77%, mean OD(405) = 0.33 ± 0.26, range = 0.11-1.81, median = 0.25). In addition, serum samples were obtained from a colony of specific pathogen-free Peromyscus spp. maintained at the University of South Carolina and six of 12 samples were reactive (50%, mean OD(405) = 0.19 +/- 0.08, range = 0.1-0.32, median = 0.18). Lastly, 40 additional wild Peromyscus spp. were captured in a disparate region of Midwestern USA and 22 serum specimens were reactive (55%, mean OD(405) = 0.22 +/- 0.11, range = 0.1-0.48, median = 0.2). Specificity of selected reactive sera for chlamydial antigen was confirmed on Western blot using resolved purified EBs as the detecting antigen. From tissues removed from several mice at necropsy, the gene for chlamydial 16S ribosomal ribonucleic acid (rRNA) was amplified by polymerase chain reaction (PCR). Positive samples of 16S rRNA were subjected to additional PCR for the major outer membrane protein gene (ompA). The amplicons of three select ompA positive samples were sequenced with ≥99% homology with C. muridarum. Our findings indicate that chlamydial infection is enzootic for Peromyscus spp., and that C. muridarum, or a closely related species or strain, is likely the agent in the tested rodent species.

Susceptibility of fox squirrels (Sciurus niger) to West Nile virus by oral exposure.

Fox squirrels (Sciurus niger) (five of eight) were infected with West Nile virus (WNV) when challenged by the oral route with 10(2.3) or 10(3.4) plaque forming units (PFU). The mean maximum serum WNV titer of infected fox squirrels was 10(5.1) PFU/mL and ranged from 10(4.6) to 10(5.6) PFU/mL. These levels of viremia are infectious for several mosquito vectors of WNV. This virus was also isolated from swabs of the oral and rectal cavities, and urine swabs between day 5 and 9 postexposure (p.e.) in amounts as high as 10(2.0), 10(2.8), and 10(2) PFU, respectively. WNV RNA was detected in salivary gland and/or kidney tissue of three squirrels between day 65 and 72 p.e. in the presence of WNV neutralizing antibody, suggesting that long-term persistent infection occurs in fox squirrels. These observations justify further studies to determine if nonarthropod transmission and long-term persistent infection occur naturally in fox squirrels and contribute to trans-seasonal maintenance of WNV.

Antibodies to West Nile virus in raccoons and other wild peridomestic mammals in Iowa.

Surveillance for evidence of West Nile virus (WNV) infection in small- and medium-sized wild mammals was conducted in Iowa, USA, from May 2005 to June 2007. Sera were collected from 325 mammals belonging to nine species and tested for antibodies to WNV and other flaviviruses by epitope-blocking enzyme-linked immunosorbent assay (ELISA). All sera that had antibodies to flaviviruses by blocking ELISA were further examined by plaque reduction neutralization test (PRNT). Thirteen mammals were seropositive for WNV by PRNT, including 10 raccoons (Procyon lotor). The seroprevalence for WNV in raccoons was 34%. Although a moderately high seroprevalence for WNV has been detected in raccoons in other surveillance studies in the United States, this has not been reported previously in Iowa or most bordering states. Together, these data indicate that raccoons are exposed to WNV at high rates throughout the United States. Two Virginia opossums (Didelphis virginiana) and one fox squirrel (Sciurus niger) were also seropositive for WNV. Nineteen mammals had antibodies to an undetermined flavivirus(es). In summary, we provide serologic evidence that raccoons in Iowa are commonly exposed to WNV.

Genomic sequence and phylogenetic analysis of Culex flavivirus, an insect-specific flavivirus, isolated from Culex pipiens (Diptera: Culicidae) in Iowa.

Adult mosquitoes (Diptera: Culicidae) were collected in 2007 and tested for specific viruses, including West Nile virus, as part of the ongoing arbovirus surveillance efforts in the state of Iowa. A subset of these mosquitoes (6,061 individuals in 340 pools) was further tested by reverse transcription-polymerase chain reaction (RT-PCR) using flavivirus universal primers. Of the 211 pools of Culex pipiens (L.) tested, 50 were positive. One of 51 pools of Culex tarsalis Coquillet was also positive. The flavivirus minimum infection rates (expressed as the number of positive mosquito pools per 1,000 mosquitoes tested) for Cx. pipiens and Cx. tarsalis were 10.3 and 1.2, respectively. Flavivirus RNA was not detected in Aedes triseriatus (Say) (52 pools), Culex erraticus (Dyar & Knab) (25 pools), or Culex territans Walker (one pool). Sequence analysis of all RT-PCR products revealed that the mosquitoes had been infected with Culex flavivirus (CxFV), an insect-specific virus previously isolated in Japan, Indonesia, Texas, Mexico, Guatemala and Trinidad. The complete genome of one isolate was sequenced, as were the envelope protein genes of eight other isolates. Phylogenetic analysis revealed that CxFV isolates from the United States (Iowa and Texas) are more closely related to CxFV isolates from Asia than those from Mexico, Guatemala, and Trinidad.

Detection of RNA from a novel West Nile-like virus and high prevalence of an insect-specific flavivirus in mosquitoes in the Yucatan Peninsula of Mexico.

As part of our ongoing surveillance efforts for West Nile virus (WNV) in the Yucatan Peninsula of Mexico, 96,687 mosquitoes collected from January through December 2007 were assayed by virus isolation in mammalian cells. Three mosquito pools caused cytopathic effect. Two isolates were orthobunyaviruses (Cache Valley virus and Kairi virus) and the identity of the third infectious agent was not determined. A subset of mosquitoes was also tested by reverse transcription-polymerase chain reaction (RT-PCR) using WNV-, flavivirus-, alphavirus-, and orthobunyavirus-specific primers. A total of 7,009 Culex quinquefasciatus in 210 pools were analyzed. Flavivirus RNA was detected in 146 (70%) pools, and all PCR products were sequenced. The nucleotide sequence of one PCR product was most closely related (71-73% identity) with homologous regions of several other flaviviruses, including WNV, St. Louis encephalitis virus, and Ilheus virus. These data suggest that a novel flavivirus (tentatively named T'Ho virus) is present in Mexico. The other 145 PCR products correspond to Culex flavivirus, an insect-specific flavivirus first isolated in Japan in 2003. Culex flavivirus was isolated in mosquito cells from approximately one in four homogenates tested. The genomic sequence of one isolate was determined. Surprisingly, heterogeneous sequences were identified at the distal end of the 5' untranslated region.

Fox squirrels (Sciurus niger) develop West Nile virus viremias sufficient for infecting select mosquito species.

The West Nile virus (WNV) viremia and shedding profiles of 11 adult fox squirrels (Sciurus niger) infected by needle inoculation or mosquito bite were characterized. Daily mean titers (95% confidence intervals) for all squirrels on days 1 through 6 postexposure (p.e.) were: 10(1.7 (1.32.1)), 10(4.4 (4.04.8)), 10(5.3 (5.05.6)), 10(4.4 (3.94.9)), 10(2.7 (2.03.4)), and 10(1.1 (0.52.1)) plaque-forming units (PFU)/mL. The highest WNV serum titers of individual squirrels infected by needle inoculation or mosquito bite ranged from 10(4.5) to 10(6.1) and from 10(5.1) to 10(5.3) PFU/mL, respectively. Nine (82%) squirrels, including all 4 squirrels infected by mosquito bite, had WNV serum titers > or =10(5.1) PFU/mL that persisted on average for 1.6 +/- 0.3 days. Infection and dissemination rates of Culex pipiens (L.) that fed on squirrels with serum titers of 10(4.4 +/- 0.1) PFU/mL were 56% and 13%, respectively. Both of these rates increased to over 80% when fed on squirrels with a mean WNV titer of 10(5.5 +/- 0.1) PFU/mL. Infection and dissemination also occurred in Aedes triseriatus (Say) but at a much lower rate. WNV was isolated from the oral and rectal cavities of all squirrels and from urine that was opportunistically collected from 5 squirrels. The largest quantity of WNV recovered from swabs of the oral cavity and urine was 10(3.1) PFU. The longest periods after exposure that WNV was isolated from the oral cavity and urine from a squirrel were 22 and 17 days p.e., respectively. WNV RNA was also detected in kidney tissue in 1 squirrel 29 days p.e., suggesting that fox squirrels can be persistently infected. Collectively these observations provide further evidence that squirrels can contribute to the natural history and epidemiology of WNV, especially in peridomestic environments.

West Nile virus viremia in eastern chipmunks (Tamias striatus) sufficient for infecting different mosquitoes.

In eastern chipmunks (Tamias striatus) inoculated intramuscularly with 101.5 to 105.7 PFU of West Nile virus (WNV), serum titers developed sufficient to infect Aedes triseriatus (Say), Ae. vexans (Meigen), and Culex pipiens (L.). Mean titers (95% confidence interval) of 8 chipmunks were 103.9(3.3-4.5), 106.7(6.4-7.0), and 105.8(4.1-7.5) PFU/mL on days 1-3 postinoculation (p.i.) and 105.8 PFU/mL in 1 chipmunk on day 4 p.i. Mean estimated days that WNV titers were >104.8 and >105.6 were 1.7 (1.1-2.3) and 1.4 (1.0-1.6). The longest period of viremia >104.8 PFU/mL was 3-4 days. WNV antigen was detected in the small intestine of 2 chipmunks and the kidneys of 4 chipmunks by immunohistochemistry. WNV also was detected in urine, saliva, and feces of some chipmunks. These data suggest chipmunks might play a role in enzootic WNV cycles and be an amplifying host for mosquitoes that could infect humans.

A comparision of West Nile Virus transmission by Ochlerotatus trivittatus (COQ.), Culex pipiens (L.), and Aedes albopictus (Skuse).

Transmission of West Nile virus (WNV) by Ochlerotatus trivittatus, Culex pipiens, and Aedes albopictus were compared 14 days after taking blood meals from viremic chickens with titers ranging from 10(2.5) to 10(9.5) cell infective dose (50)s (CID50s)/mL serum. Transmission occurred in one of four (25%) Oc. trivittatus and one of 25 (4%) Cx. pipiens that fed on chickens with titers of 10(5.5) CID50s/mL. No transmission occurred among two of 16 (13%) Oc. trivittatus or one of 25 (4%) Cx. pipiens that became infected after blood meals with titers of 10(5.0) and 10(4.5) CID50s/mL, the next lowest blood meal titers evaluated. Seventeen of 28 (61%) Ae. albopictus transmitted WNV after blood meals with titers of 10(7.0) CID50s/mL, but no infection or transmission was observed among 21 Ae. albopictus that fed on chickens with titers of 10(5.0) CID50s/mL, the next lowest titer evaluated. Transmission by all three species increased dramatically after blood meals with WNV titers of > or = 10(5.5) CID50s/mL. No significant differences occurred in dissemination and transmission rates of the three species after taking blood meals with titers of > 10(7.0) CID50s/mL. The cumulative mean +/- SE transmission rates of Oc. trivittatus, Cx. pipiens, and Ae. albopictus after blood meals with titers of > or = 10(7.0) CID50s/mL were 45.5 +/- 4.1%, 46.8 +/- 4.5%, and 72.4 +/- 5.5%. The cumulative mean dissemination rates of the three species were 78.3 +/- 6.7%, 74.8 +/- 2.6%, and 88.6 +/- 2.1%. The rates of transmission by the three species that developed disseminated infections after blood meals with titers of > or = 10(7.0) CID50s/mL were 58.8 +/- 4.4%, 62.6 +/- 5.8%, and 81.6 +/- 5.4%, respectively. In a previous study, we found that susceptibility of the three species to WNV was essentially the same when fed on chickens with WNV titers of > 10(7.0) CID50s/mL, but Oc. trivittatus and Cx. pipiens were more susceptible than Ae. albopictus to WNV at lower virus titers. The current study strongly suggests that Ae. albopictus is a more efficient vector than Oc. trivittatus and Cx. pipiens when fed blood meals with titers of > 10(7.0) CID50s/mL. However, Oc. trivittatus and Cx. pipiens might be more efficient as vectors when infected by blood meals with titers of < 10(7.0) CID50s/mL.

Eastern cottontail rabbits (Sylvilagus floridanus) develop West Nile virus viremias sufficient for infecting select mosquito species.

The potential of the eastern cottontail rabbit (CTR; Sylvilagus floridanus) to contribute to an enzootic West Nile virus (WNV) cycle was demonstrated by characterizing the WNV viremia profile of 15 CTRs and demonstrating that mosquitoes could become infected by feeding on these CTRs. Eight CTRs were infected with a titer of 10(5.0) cell-infectious dose 50% endpoints (CID50s) of WNV (NY99-Crow) by needle and seven CTRs by bite of one or more WNV-infected mosquitoes. There were no marked differences between the WNV viremia profiles of CTRs infected by either method. West Nile virus was detected in serums of all CTRs by 24 h p.i. The daily mean titers of all 15 CTRs on days 1-4 p.i. were 10(4.1+/-0.4), 10(4.7+/-0.3), 10(4.1+/-0.6), and 10(3.7+/-0.6) respectively, declining to 10(1.2+/-0.1) CID50s/ml of serum by day 6 p.i. No virus was detected in the blood of any CTR on day 7 p.i. The average duration of WNV titers of >or=10(4.3) and <10(5.0) CID50s/mL for all CTRs was 2.2 +/- 0.6 and 1.0 +/- 0.1 days, respectively. The minimum estimated infection rates (MEIRs) of Culex pipiens (L.) and Culex salinarius (Coq.) that fed on CTRs with titers of >or=10(4.3) and >10(5.0) were 11.5 +/- 5.5 and 21 +/- 6.0%, respectively. These rates increased to 20.5 +/- 6.4% and 25.0 +/- 3.0% when CTR serum titers were >10(5.0) CID50s/mL. Neither Aedes aegypti (L.) nor Aedes albopictus (Skuse) were infected by feeding on CTRs with titers of <10(5.0) CID50s/mL. The MEIRs of these two species were 11.5 +/- 3.5% and 1.5 +/- 0.5% after feeding on CTRs with titers of >10(5.0) CID50s/ml. None of the CTRs infected by mosquito bite or by needle showed any symptoms of WNV disease.

Susceptibility of Ochlerotatus trivittatus (Coq.), Aedes albopictus (Skuse), and Culex pipiens (L.) to West Nile virus infection.

The susceptibility of Ochlerotatus trivittatus (Coq.) to West Nile virus (WNV) was assessed by comparing it to the susceptibility of Aedes albopictus (Skuse), a likely bridge vector, and Culex pipiens (L.), a primary WNV amplifying species. The three species were infected with WNV (NY crow-1999) by feeding on 2-3-day-old chickens with serum virus titers ranging from 10(2.5) to 10(9.5) cell culture infective dose (CID) 50s/mL. The lowest infective titer for Oc. trivittatus and Cx. pipiens was 10(4.5) CID50s/mL. Thirteen percent (4/32) and 2% (1/45) of each species became infected postprandially. Infection rates of the two species increased to 43% (6/14) and 15% (6/40) after blood meals with a titer of 10(5.5) CID50s/mL. In contrast no infection was observed in nine Ae. albopictus that fed among three chickens with titers of 10(4.5) CID50s/mL nor in 41 Ae. albopictus that fed among three chickens with titers of 10(5.0) CID50s/mL. The infective dose 50s for Oc. trivittatus, Cx. pipiens and Ae. albopictus were 10(6.0), 10(6.2), and 10(6.6) CID50s/mL, respectively. Collectively these observations suggest that Oc. trivittatus and Cx. pipiens are more susceptible than Ae. albopictus to WNV when they feed on hosts with WNV titers of <10(7.5) CID50s/mL, but nearly as susceptible with blood meal titers of > or =10(7.5) CID50s/mL. Unpublished studies in our laboratory showed that cottontail rabbits fed on by WNV-infected Oc. trivittatus developed viremias as high as 10(5.5) CID50s/mL serum which exceeds 10 (4.2 (3.4-4.6)) CID50s/mL, the predicted ID10+/-95% CI of Oc. trivittatus. Consequently this mosquito, which also feeds on humans and birds has the potential to serve as a bridge vector and as a maintenance vector among mammals.

Identification and characterization of auto-anti-idiotypic antibodies specific for antibodies against porcine reproductive and respiratory syndrome virus envelope glycoprotein (GP5).

Auto-anti-idiotypic antibodies (Aab-2s) were detected from pigs experimentally infected with porcine reproductive and respiratory syndrome virus (PRRSV). The Aab-2s were specific against the idiotypic antibodies (Ab-1s) to the envelope glycoprotein GP5 of PRRSV and were detected from serum samples collected between 21 and 98 days post-infection (DPI). Serological characterization indicated that the Aab-2s recognized the idiotype located within or near the antigen-combining sites of the anti-GP5 antibodies, which was shared by both mouse MAb anti-GP5 and swine polyclonal antibodies. The fact that the Aab-2 inhibited the anti-GP5 antibodies from binding to PRRSV and that they were detected at different time periods in pigs that cleared the infection prior to 98 DPI versus pigs in which virus was detected at 98 DPI suggests that Aab-2 antibodies may play a role in immunity to PRRSV infection.