Human WNV disease incidence first occurred in significant numbers in Iowa in 2002 and peaked in 2003 with 147 cases. This is consistent with the second year of significant intrusion having the highest number of human cases. Illinois (884), Michigan (614) and Ohio (441) had the highest number of human cases in 2002, while in 2003 Colorado (2947), Nebraska (1036), and South Dakota (1039) had the highest number of human cases in the country. In each of these states, the virus was first detected in a significant amount in the previous year . There are many possible reasons for these reductions after the peak year. These reasons could include public awareness, increased mosquito abatement, and a build up of immunity in bird and mammal populations . Immune birds are considered dead-end hosts like humans . Although none of these states has matched the peak number of cases, there have been a large number of cases since 2004 .
Human cases of West Nile virus have occurred throughout Iowa, with a greater concentration in the western part of the state (Figures 1 and 5). The analysis of spatial autocorrelation for human WNV disease rates showed significant hot-spots in Western Iowa over the course of the study period and significant cool spots in some urban areas. States directly to the west of Iowa, including Nebraska (10.9% of cases, 0.6% of population in continental USA) and South Dakota (6.2% of cases, 0.26% of population), have had some of the highest WNV disease rates in the country [1, 34]. In Iowa, using the east-west split as described earlier, Eastern Iowa has 0.54% of the continental USA population with 0.36% of the WNV disease cases from 2002–2006, while Western Iowa has 0.45% of the continental USA population with 0.9% of the WNV human disease cases . This higher incidence rate and the increasing proportion of Cx. tarsalis trapped in the western half of the state suggest that the western half of Iowa exhibits vector-host ecology more similar to that in the states to the west [10, 35]. Culex tarsalis mosquitoes feed on both mammals and birds and thus are more likely to serve as a bridge vector to humans than mosquitoes of the Cx. pipiens group .
In a novel finding, it was shown that human cases of WNV disease occurred in more rural settings in Iowa. This is opposed to eastern studies which showed more urban/suburban occurrence [7, 19, 20]. Figure 2 demonstrates that census block groups with an increasing number of human WNV disease cases (zero to three cases) had progressively lower population densities, while Figure 3 also demonstrates the rural nature of cases. There have been a limited number of studies that specifically investigated population density [7, 11, 19, 20] in relation to WNV disease incidence. In Chicago and Detroit, areas classified as 'Inner Suburbs' with moderate population density had the highest percentage of WNV disease cases [7, 19]. Similarly in Georgia, moderate housing density was associated with WNV disease incidence . In Nebraska, the highest seroprevalence of WNV, estimated by blood donor data, was in the most sparsely populated part (western) of the state . In Iowa, the western half of the state has a lower population density (0.17 persons/ha) than the eastern half (0.23 persons/ha). However, the census block groups with WNV disease incidence had a lower population density (although not a statistically significant difference) in the eastern part of the state. There were 213 (71.5%) cases in the western part of the state. The nine most densely populated census block groups with WNV disease incidence are in the western part of the state. It is possible this was caused by a higher overall WNV infection rate in mosquitoes in the Western part of the state. This is supported by the fact more positive WNV mosquito pools were found in the western half (64.5%) of the state. Figure 1 demonstrates that several of the cities in Eastern Iowa show up as being cold spots for human WNV disease rates, while none of the western cities do. In Sioux City, which is along the Missouri River on the western border, there were 13 cases in densely populated census block groups. Rural census block groups around this city also had WNV disease incidence. It is possible the movement of vectors or hosts between the rural-urban interface might have led to the urban WNV disease cases. Also, the fact that in the western part of the state both Cx. pipiens group and Cx. tarsalis positive mosquito pools were found extensively, could help explain both the urban and rural human disease occurrence. It must be noted that due to logistical constraints of running a continuous collection regime, the NJLT locations are often located in or near towns or cities and thus sampling of rural areas is likely a bit under-represented. This could lead to an under representation of Cx. tarsalis mosquito populations and positive pools, as they are a very rural mosquito.
The results of the land cover and land use analysis supported the rural nature of WNV disease incidence in Iowa. The most significant difference in land cover mean proportions between census block groups with and without WNV disease was for agricultural/row crop areas (Figure 4). Figure 5 demonstrates the large number of agriculturally dominated census block groups with WNV disease occurrence. In addition, the land use indicators of permitted agricultural irrigation and animal feeding operations were associated with the census block groups with WNV disease incidence. In California, Reisen et al.  suggested that farmhouse environs provide 'islands' of elevated vegetation used by birds for nesting and by Cx. tarsalis for host-seeking and resting. This could help explain the agricultural association in Iowa also. Total crop sales were shown to be a significant independent predictor of WNV disease incidence by county in Colorado, Nebraska, Louisiana, and Pennsylvania in 2002–2003 . However, the number of irrigated acres was not a significant predictor in that study. In the Great Plains region , and in California , large populations of Cx. tarsalis were associated with irrigated areas. In 2003, the year of highest WNV disease incidence in Iowa, census block groups with WNV disease incidence had significantly less precipitation than those without. In the drier years of 2003 and 2005, a higher proportion of census block groups with WNV disease incidence were ones which contained permitted irrigation points compared to the other four years. The interaction of agricultural activity, such as irrigation and intensive animal operations in relation to mosquito abundance, and WNV disease incidence needs to be studied more thoroughly in Iowa. Census block groups having WNV disease incidence encompassed less forested areas than those without. Many heavily forested areas have low population densities. Culex tarsalis larval habitats include newly-created sunlit surface pools surrounded by grasses  and thus forested habitat likely does not support Cx. tarsalis larvae. Also, Cx. pipiens is considered an urban mosquito and thus also might not be prevalent in or near forested areas. If Cx. tarsalis is the primary vector in Iowa, then we might expect there to be higher proportion of grasslands associated with WNV disease, but there was no significant difference in grassland proportions in census block groups with and without WNV disease. Higher proportions of the built environment land cover classes (roads, commercial, residential) were associated with those census block groups without WNV disease. This implies that the urban-centric Cx. pipiens species might play a more minor role in Iowa as compared to the rural Cx. tarsalis species. Urban and suburban land use was an important predictive variable for WNV disease incidence in Georgia  and Illinois  where the more urban Cx. quinquefasciatus and Cx. pipiens are considered the primary vectors respectively. There is not a large amount of wetland area in Iowa (~0.5% of state), and it is often in quite rural areas, but it was still somewhat surprising that wetlands were shown to be less common in census block groups with WNV disease. This could be due to host-vector interactions that are not well understood. In Louisiana, Ezenwa et al.  found that WNV infection rates in Culex mosquitoes declined with increasing wetland cover. They found that the area of wetlands was significantly and negatively correlated with the passerine to non-passerine bird ratio and they theorize that the non-passerine bird species act as dilution hosts. At a state level there was a negative relationship between NDVI score and WNV disease cases as indicated in Figure 6. This is interesting, in that warmer and drier years (2003 and 2002) had more WNV disease than a cooler and wetter year (2004). Given that this was a March-September NDVI average, and for only a four year period, limited conclusions can be made.
Some interesting year-by-year associations were made between climatic data and WNV disease incidence. However, analysis of climatic data from the year of WNV disease observations did not reveal any consistent trends. In 2003, census block groups with WNV disease had significantly less precipitation, lower dewpoint and average annual minimum temperatures. These relationships were very strong in the western half of the state but reversed in the eastern part of the state indicating varying disease dynamics. In 2003, there were relatively wet conditions in May, June, and July, but then a very dry August. The mean and median week of WNV disease occurrence was at the end of August. This is opposite to the pattern described by Shaman et al. , where early season drought followed by later season rainfall led to large outbreaks. The mean annual minimum temperature was always lower in census block groups with WNV disease incidence and significantly so in 2003, 2004, and 2005. This relationship was true for all years in the western part of the state with 2003, 2004, 2005, and 2006 having significantly lower average minimum temperatures in census block groups with WNV disease incidence. This is a bit contradictory to other studies that conclude higher temperatures could potentially increase probability of WNV transmission [23, 40]. The magnitude in differences on an annual basis (e.g. approximately 0.5°C) are not huge and it is unclear in what way this would have an effect on WNV disease incidence. However, given that we considered only annual average climatic variables, it is possible that more detailed temporal data might reveal different associations. Temperatures are generally lower in the northern part of the state, which also has significant agricultural areas and animal feeding operations. The influence of temperature needs to be examined more thoroughly to help understand its effect on human WNV disease incidence in Iowa. The differences between eastern and western halves of the state suggest that different vector-host-disease dynamics have been occurring in different parts of the state. A possible difference is that drier conditions in western Iowa lead to higher levels of irrigation which in turn provides good habitat for Cx. tarsalis mosquitoes. The majority of irrigation points (~71%) were in the western part of the state. Landesman et al.  demonstrated regional difference in precipitation associations with WNV disease incidence for the United States as a whole when split into east and west using the Mississippi River as a dividing line. They point out that this is likely due to the transition to Cx. tarsalis as the primary vector in the western United States. This is supported by the fact that the proportion of Cx. tarsalis positive mosquito pools increased as WNV moved westward in 2001–2004 . Figure 7 demonstrates that in Iowa there is a transition as you move west to a greater proportion of Cx. tarsalis out of total Culex mosquitoes. Numerous studies have used degree days to explain or estimate the timing of mosquito populations and WNV disease occurrence [23, 40, 41], and this is an approach that might be used in Iowa in the future. The timing of vector abundances and WNV disease incidence needs to be studied in greater depth.
Other studies have demonstrated that climatic phenomena from the preceding year can be important for mosquito activity  and WNV disease incidence . In Iowa, in 2003, 2004, and 2005 there were significantly lower mean precipitation values, annual average dewpoint temperatures, and annual average minimum temperatures in the preceding year for census block groups with WNV disease incidence. Similar to this study, Landesman et al.  found that 2003 and 2004 WNV disease incidence was associated with lower amounts of precipitation in the preceding year. They cite the theory put forth by Chase and Knight  that disruption of food-webs in preceding years by drought can lead to large outbreaks in wetland mosquitoes the next year. In Iowa, Cx. tarsalis populations were highest in 2004 , following drought in late summer 2003, but the number of human WNV disease cases and sentinel chicken seroconversions were lowest. In addition, no WNV positive mosquito pools were found in 2004 in Iowa. Similar to this study, Landesman et al.  also found that WNV disease incidence in 2002 was associated with greater precipitation in 2001. In Iowa, those census block groups with WNV disease incidence had greater precipitation totals in 2001 but not significantly so. These findings clearly indicate a need for more detailed temporal study of climatic effects.