in much of rural india , 
 local surface water is used for personal 
 and domestic hygiene and local groundwater is used for drinking and 
 cooking , with over half of rural households getting their drinking 
 water from tubewells . when contaminated 
 with fecal pathogens , these water sources can be a transmission route 
 for diarrheal disease . among fecal pathogens , the protozoa cryptosporidium and giardia are responsible 
 for the majority of detected waterborne disease outbreaks worldwide , and cryptosporidium has been 
 identified as a leading cause of moderate - to - severe diarrhea in indian 
 children < 2 years old . to reduce 
 diarrhea and other disease burdens , the government of 
 india has made significant investments to improve rural sanitation 
 through widespread promotion and construction of household pour - flush 
 latrines . under india s total sanitation 
 campaign ( 20002012 ) , an estimated 95 million rural household 
 latrines were installed with government support ; millions more are 
 planned under its total sanitation for all campaign ( 20122022 ) . leachate from pour - flush latrines and other on - site 
 sanitation , however , can contaminate local groundwater . evidence exists 
 for bacterial and viral transport laterally up to 25 and 50 m , respectively , 
 from latrines . public health guidelines 
 for the distance between latrines and water points vary by country , 
 with india recommending 310 m and others a distance equal 
 to 25 days travel time for leachate , but these guidelines can be difficult 
 to enforce in rural areas . to our knowledge , no published 
 studies have examined the impact 
 of pit latrines on groundwater contamination for cryptosporidium and giardia as indicated by a recent systematic 
 review . one explanation for the gap is 
 an assumption that their larger size ( 418 m ) , compared 
 to bacteria ( typically < 5 m ) and viruses ( typically < 1 
 m ) , precludes cryptosporidium and giardia from being transported in groundwater over distances 
 required to reach wells . although experimental 
 column studies of groundwater transport indicate a high rate of short - term 
 removal of cryptosporidium in the first few meters 
 of saturated sandy soils , they also show long - term , 
 low - level transport of cryptosporidium at longer 
 distances , attributed to remobilization mechanisms that reverse initial 
 filtration / straining . because both cryptosporidium and giardia can persist 
 for extended periods of time in soil and water ( > 3 months ) and both pathogens can cause infection at very 
 low dose , the public health threat from 
 long - term , low - level subsurface transport of cryptosporidium and giardia from latrines to wells requires further 
 examination in general , and particularly in the rural indian context 
 of rapid latrine expansion . identifying the causes and implications 
 of environmental contamination 
 for cryptosporidium and giardia requires 
 consideration of additional contamination sources and processes beyond 
 groundwater contamination from latrines . cryptosporidium and giardia can be shed by livestock and domestic 
 animals species , making it essential 
 to account for animal host sources and their proximity to water sources . human open defecation which remains widespread in rural india , and postdefecation anal cleansing in surface 
 water bodies , present other nonpoint human sources and mechanisms 
 of surface and groundwater contamination . additionally , environmental 
 processes , involving climate , hydrogeology , vegetation and soil for 
 example , mediate cryptosporidium and giardia transport and distribution in the environment , while the type , quality , and condition of the water point 
 ( e.g. , private and public , damaged vs intact ) can have important impacts 
 on water source quality . prior studies 
 have examined some of these processes , most often in isolation and 
 using fecal indicators or tracers , for either surface or groundwater 
 contamination , but rarely for both at the same time within the same 
 setting . joint evaluation of local sources and processes of fecal protozoa 
 pathogen contamination for surface and groundwater together , accounting 
 for both animal and human fecal pollution , multiple transport pathways , 
 and environmental and other mediators , has not been undertaken . such 
 integrated study designs are needed to fully assess the public health 
 implications of protozoal contamination and contributions of latrines 
 as both a source and sink . in this study , we investigate potential 
 causes of previously reported cryptosporidium and giardia contamination 
 observed concurrently in community surface and groundwater sources 
 during two monsoon seasons in 60 villages in puri district , odisha , 
 india . these villages were part of a 
 large - scale cluster randomized controlled trial ( the odisha sanitation 
 trial ) of health impacts of improved household sanitation under a 
 total sanitation campaign intervention conducted in the district during 
 2011 . we develop a conceptual hierarchical 
 model of local factors and processes causing protozoal contamination 
 for cryptosporidium and giardia in 
 surface and groundwater sources , and apply it to guide multivariable 
 modeling to test relationships between protozoal contamination in 
 a tested water source and water source characteristics , meteorological 
 conditions prior to sampling , the density of nonpoint sources of human 
 and animal fecal loading around each water source , including potential 
 subsurface leaching from household pour - flush latrine pits , and village - level 
 socio - economic ( ses ) characteristics as proxies for other mediating 
 factors . much of the groundwater is held in shallow 
 unconfined and deeper semiconfined and confined aquifers , both primarily 
 composed of unconsolidated gravel and sands . unconfined aquifer depth is variable , but can 
 reach 135 m below ground ( blg ) , whereas confined aquifers can reach 
 602 m blg . other significant formations 
 include porous laterites and jointed / faulted formations susceptible 
 to weathering at depths of 20 m. the 
 climate is tropical and characterized by wet summers ( june to september ) 
 and dry winters ( october to may ) , with much of the region experiencing 
 annual flooding during the southwest monsoon from june to september . data and water samples for the 60 villages analyzed in this study 
 were collected as part of the odisha sanitation trial . details of 
 the trial design , village selection , population characteristics , sanitation 
 intervention and pour - flush pit latrine design and construction can 
 be found elsewhere . briefly , trial villages had similar 
 size , infrastructure , geography , and ses characteristics . most households 
 ( 62% ) lived below the indian poverty line and owned livestock ( 59% ) , 
 with cattle an important host of zoonotic protozoa and the predominant 
 species owned ( 56% ) . access to improved drinking water sources in 
 the form of government - installed ( deep ) or private ( shallow ) tubewells 
 was high ( 82% ) . deep tubewells ( dtws ) were fitted with india mark 
 ii style handpumps ( maximum lift : 50 m ) , shallow tubewells ( stws ) 
 had no . 6 style handpumps ( maximum lift : 7 m ) and both were expected 
 to be fully cased . public ponds were 
 used for daily hygiene activities by more than 50% of households . government subsidized pour - flush latrines had a single 
 circular leach pit of 1 m diameter , installed at ground level , 
 extending 1 m deep . self - financed 
 pour - flush latrines were similar , except with deeper pits ( 12 
 m ) , or two in series . during the monsoon season in some areas of puri 
 district see supporting information ( si ) , figure s1 , map of villages . we previously reported results 
 of testing six water sources in each 
 village on a single day for cryptosporidium and giardia , comprising two public ponds , and two each of dtws 
 and stws ( when present ) , and protozoal shedding rates among humans , 
 livestock and domestic animals in the region . each source was sampled once in either 2012 or 2013 between june 
 and august . samples were collected during the morning ( 8 am to 11 
 am ) and gps location and site observations recorded . cryptosporidium was detected in 37% , 14% , and 5% , respectively , of ponds ( n = 94 ) , dtws ( n = 110 ) , and stws ( n = 96 ) and giardia was detected in 74% , 
 12% , and 17% of sources , respectively . see si , section s1 , for water processing 
 details and si , table s1 for pathogen concentrations . we developed a conceptual model of multilevel 
 factors involved in cryptosporidium and giardia contamination of community ponds and tubewells ( figure 1 ) to aid investigation . factors are categorized 
 as meteorological , loading from local human and animal sources of 
 fecal protozoa , water source - specific characteristics , or village - level 
 ses characteristics . the top - level outlines factors that remain essentially 
 constant within each sampling season ( i.e. , 2012 or 2013 ) , such as 
 village ses characteristics , annual climatic pattern , and village - level 
 population of host animals , open defecators , latrines and latrine 
 users . factors in the second level are those that vary during the 
 sampling season and affect day - to - day pollution levels , such as seasonal 
 cumulative rainfall , recent rain , or an extreme event . the lowest 
 level represents factors that can vary hour - to - hour and affect pollution 
 levels at the time of sample collection , such as number of people 
 using the site . using the conceptual model we identified predictor 
 variables from data sets collected as part of the odisha sanitation 
 trial and available meteorological data . conceptual model of multilevel 
 factors involved in cryptosporidium and giardia contamination of community water sources . the three large boxes group factors hierarchically based of their 
 time scale or temporal stability . arrows indicate direct and indirect associations / causality 
 between factors at different time scales and the outcome ( e.g. , level 
 of open defecators in a village has an effect on the local uses of 
 ponds , which in turn has an effect on the direct observations of human 
 uses at a pond ) . cryptosporidium and giardia from 
 humans and from host species of domestic livestock in each village 
 were considered , accounting for the most common livestock host species 
 in puri district ( cattle , buffalo , goat , and sheep ) . two local processes by which protozoal pathogens shed in 
 human feces can contaminate a pond are overland flow from nearby open 
 defecation fields and directly by people anal cleansing and bathing 
 in ponds after defecation . people using the pond at the time of sampling 
 could also stir up settled microorganisms . processes by which local 
 human feces can contaminate tubewell groundwater are through leaching 
 and groundwater transport from nearby latrine pits and from direct 
 infiltration of above - ground contaminated water at the tubewell head 
 via a damaged well pad ( platform ) or inadequate seal . similar to human 
 fecal inputs , unmanaged livestock feces can reach ponds via overland 
 flow and directly if animals enter them and can reach tubewell groundwater 
 via direct surface infiltration at the well head if improperly sealed 
 or damaged . weather conditions and events are presumed to mediate 
 the impacts of human and livestock fecal pollution sources on pond 
 and tubewell contamination , through mechanisms such as flushing or 
 dilution from rainfall or removal by ultraviolet decay . ses factors 
 can also influence and mediate impacts of human and livestock sources 
 as ses groups may differ in their health status , affecting pathogen 
 shedding rates , or levels of access to improved sanitation and private 
 water sources , affecting practices at ponds and public tubewells , 
 for example . we obtained 
 rainfall data ( mm 
 day ) from four weather stations in puri district 
 beginning june 1 ( historical start of monsoon in odisha is june 10th ) 
 for 2012 and 2013 . to estimate rainfall in each village , we used the 
 thiessen polygon method as implemented in arcgis , version 10.2 ( esri ) . the following rainfall variables were compiled and considered in analyses : 
 cumulative seasonal rainfall from june 1 until the day prior to sampling ; 
 occurrence of any rainfall and of an extreme daily precipitation event 
 during the 1 , 2 , and 3 days prior to sampling . an extreme daily event 
 was defined as rainfall exceeding the 90th percentile of daily totals 
 between june and august in 2012 and 2013 ( i.e. , > 4 cm day ) . variables were also developed for other meteorological 
 factors 
 from daily data from a regional station ( see si , tables s24 ) . a village - wide 
 census and mapping of the location of each household and the sludge 
 pit of each pour - flush latrine was conducted between december 2012 
 and february 2013 as previously described . during the census the following were collected : number of members 
 of each household , whether the household had a cattleshed at / near 
 the house , whether they owned a functional latrine , the household 
 members using the latrine , and the age of the latrine . because latrine 
 use by those with household access in the region is suboptimal , to account for potentially large differences 
 in loading rates of each census latrine in a village , we used reported 
 number of users and age of each household latrine at the time of the 
 census relative to the village s water sampling date to calculate 
 the number of person - years of latrine use as an estimate for fecal 
 loading at the time of water source sampling for each latrine pit 
 ( details in si , section s3 ) . using 
 arcgis , version 10.2 ( esri ) , the numbers of households , cattlesheds , 
 and latrines pits around each tested water source , within a series 
 of increasing buffer distances from the source , were counted ( si , figure s4 ) . calculations were made at 5 m 
 intervals from 050 and 50 m intervals from 50200 m , 
 followed by a single buffer at 500 m. using extracted data at each 
 buffer distance combined with village census data , the number of open 
 defecators ( associated with each household location ) , and person - years 
 of latrine use ( associated with each latrine pit location ) were calculated 
 ( see si , sections s3 and 4 ) . using 
 baseline data on animal ownership from a representative sample 
 of households in each trial village , we 
 estimated village livestock populations from the average number of 
 animals owned by species per baseline household and the total number 
 of census households . trial baseline 
 data was also 
 used to estimate the following village ses characteristics : ( 1 ) illiteracy 
 rate among household heads , ( 2 ) fraction of households belonging to 
 a scheduled caste , and ( 3 ) fraction of households self - reporting ownership 
 of a poverty ration card ( i.e. , living below the indian poverty line ) 
 as indicators of health status and behavioral factors . characteristics and observations 
 recorded when sampling each water source and considered in the analysis 
 were number of people ( by age category and gender ) and of livestock 
 ( by species ) at the site , types of uses of the water source , condition 
 of the tubewell pad ( intact , cracked , or missing ) , and color of the 
 water ( clear and not clear ) . we 
 developed separate 
 multivariable models of protozoa contamination for cryptosporidium and giardia in each water source type to account 
 for different transport characteristics , sources , shedding rates , 
 and survival of each microorganism , and different pathways involved 
 in contamination of each water source type . to build our statistical 
 models , we started with univariable analysis ( i.e. , one predictor 
 variable ) ( see lists of predictors , si , tables s216 ) . initial multivariable models were constructed 
 by including predictor variables with a p - value 0.2 
 from univariable results . when greater than one predictor variable 
 representing the same factor qualified for inclusion , the one with 
 the smallest p - value was chosen , except for fecal 
 loading spatial variables , where both a near ( < 100 m ) and far ( 100 
 m ) distance were included . we then used a backward selection approach and sequentially dropped each predictor variable 
 with a p - value > 0.2 , starting with the variable 
 with the largest p - value , to produce a final multivariable 
 model with all variable p - values  0.2 . we 
 accounted for village clustering in final models using general estimating 
 equations ( gee ) . statistical analyses 
 were performed using sas software , version 9.4 ( sas institute inc . ) . prevalence of protozoa 
 contamination of ponds was high and oocyst / cyst concentrations varied 
 by up to 5 orders of magnitude , thus pond contamination was modeled 
 as concentration level in univariable and multivariable modeling using 
 gee for proportional odds cumulative logit regression ( also known 
 as ordered logistic regression).cryptosporidium and giardia concentrations 
 ( oocysts / cysts 20 l ) were converted to log10 levels 
 ( i.e. , < 1 parasite = 0 ; 110 = 1 ; 11100 = 2 ; so 
 on to 4 ) . adjusted 
 odds ratios ( or ) were calculated to estimate the marginal likelihood 
 of one log - level higher concentration from a defined change in each 
 predictor variable . cryptosporidium and giardia in 
 dtws and stws were modeled as binary 
 ( presence / absence ) outcomes due to their much lower prevalence and 
 low concentrations and because tubewells are used for drinking . indicate the marginal likelihood of tubewell protozoa contamination 
 for a defined change in each predictor variable . we projected 
 future densities of person - years of latrine loading ( for the 2013 
 population density ) at critical distances around each sampled surface 
 and groundwater source in study communities and used these with final 
 multivariable models to estimate the impacts on water source protozoa 
 contamination of 100% pour - flush latrine usage in each study community 
 by 2017 ( the government of india s target ) and at the end of 
 the total sanitation for all campaign ( 2022 ) . all other variables 
 were held constant ( see details , si , section s6 ) . predictors of cryptosporidium and giardia contamination and their adjusted or 
 and p - values 
 in the final gee regression models for dtws and stws are shown in table 1 and for public ponds 
 in table 2 . mean value of predictor variable 
 from subset of records with a value greater than zero . mean value of predictor variable 
 from subset of records with a value greater than zero . increased risk of protozoa detection in 
 stws was significantly correlated with human loading into nearby latrine 
 pits : 10 more person - years of latrine loading within 15 m of a stw 
 increased the odds of detecting cryptosporidium by 
 21% ( 95% or : 1.061.38 ) and , when occurring within 10 m , increased 
 the odds of detecting giardia by 44% ( 95% or : 1.121.85 ) . 
 weaker evidence for latrine leaching effects on dtws was also found . each 10 person - years more of latrine loading within 500 m increased 
 the odds of cryptosporidium detection in a dtw by 
 1% ( p = 0.139 ) , while the same increase within 150 
 m increased the odds of giardia detection by 1% ( p = 0.050 ) . only giardia detection in stws 
 was associated with any village - level ses characteristics . a 5-percentage 
 point increase in the proportion of illiterate household heads decreased 
 the odds of detecting giardia by 8% ( 95% or : 0.890.97 ) . specifically , cryptosporidium in stws was positively associated with the goat population ( or = 
 1.09 , 95% or : 1.031.14 , per three additional goats ) . no other 
 associations were found between protozoa in shallow or deep tubewells 
 and livestock animal loading variables . both protozoa were significantly 
 more likely to be detected in 
 groundwater from a dtw with a damaged well pad , at 7.10 times ( 95% 
 or : 1.9220.57 ) for cryptosporidium and 5.91 
 times ( 95% or : 1.1829.60 ) for giardia , than 
 from one with an intact pad . we did not find statistically greater 
 odds of detecting protozoal pathogens in a damaged stw , however , stws 
 with a damaged pad had higher detection frequencies of both protozoa 
 ( si , table s19 ) . stws which were being 
 used immediately prior to sampling had significantly lower odds of 
 detecting giardia ( 0.26 , 95%or : 0.071.00 ) . in stws cryptosporidium was significantly more 
 likely to be detected within 2 days after an extreme rain event ( or 
 = 12.91 , 95% or : 3.2651.10 ) , while giardia was significantly less likely to be detected as seasonal rainfall 
 increased ( or = 0.20 , 95% or : 0.110.39 , each 25 cm of seasonal 
 rainfall prior to sampling ) . no rainfall variables were associated 
 with dtw contamination , however , both protozoa were detected in dtws 
 significantly more often in 2012 than in 2013 ( cryptosporidium or = 5.24 , 95% or : 1.3420.57 ; giardia or 
 = 36.14 , 95% or : 3.52371 ) . protozoa 
 concentration levels in ponds were positively associated 
 with the number of people practicing open defecation living within 
 50 m of the pond but not at distances beyond that , and with village 
 illiteracy rates , recent use by cattle , populations of specific species 
 of livestock , sampling year , and antecedent rainfall patterns . each 
 six additional people ( average household size ) practicing open defecation 
 and living within 50 m significantly increased the odds of a pond 
 having one log ( 10 times ) more cryptosporidium oocysts 
 ( 20 l ) by 1.13 ( 95% or : 1.021.26 ) while 
 each additional 5% of households with an illiterate head significantly 
 increased the odds of one log more giardia concentration 
 by 1.22 ( 95% or : 1.061.42 ) . neither signs of regular or recent 
 human use ( e.g. , paths ) nor human use observed at a pond prior to 
 sampling were associated with increased protozoa levels . when 
 cattle were observed at a pond prior to sampling , we found some evidence 
 that the cryptosporidium concentration was likely 
 to be 10 times higher than when cattle were absent ( or = 5.33 , 95% 
 or : 0.6543.50 ) and when there were more cattlesheds within 
 200 m of a pond , the giardia concentration was likely 
 to be higher ( or = 1.06 , 95% or : 0.991.13 , each five additional 
 cattlesheds ) . we also observed associations between a village s 
 sheep and buffalo populations and pond parasite levels . three additional 
 sheep significantly increased the likelihood of a higher cryptosporidium level ( or = 1.06 , 95% or : 1.011.12 ) , while three additional 
 buffalo ( mean number per buffalo - owning household ) had a nearly significant 
 protective effect on giardia concentrations ( or = 
 0.84 , 95% or : 0.701.01 ) . conceptually , rainfall could 
 either flush oocysts / cysts into ponds 
 through overland flow or dilute concentrations by flooding or filling 
 ponds ; our analyses showed evidence of the effects of overland flow / flushing 
 on cryptosporidium concentrations and of dilution 
 on giardia concentrations in ponds , consistent with 
 the results for stws . when rainfall occurred anytime during the 2 
 days prior to sampling , a pond s cryptosporidium concentration was 5.38 times ( 95% or : 1.2523.21 ) more likely 
 to be an order of magnitude higher than when it had been dry , while 
 each additional 25 cm of seasonal rainfall increased the probability 
 of a pond s giardia concentration being a 
 log10 level lower by 3% ( 95% or : 0.921.01 ) . lastly , as found 
 for cryptosporidium in dtws , ponds in 2012 were significantly 
 more likely to have higher cryptosporidium concentration 
 levels than those sampled in 2013 ( or = 2.91 , 95% or : 1.207.05 ) 
 all other effects constant . model - derived predictions of local surface 
 and groundwater source 
 contamination rates for each protozoa during the monsoon season are 
 shown in table 3 for 
 projected latrine loading densities under 100% latrine coverage by 
 2017 and through 2022 . under projected full coverage , small predicted 
 reductions occur in the pond contamination rate for cryptosporidium ( 16% ) concurrently with large predicted increases in shallow and 
 deep tubewell groundwater contamination for each protozoa ( 1.94.1 
 times by 2022 ) over baseline 201213 rates , with the greatest 
 increases expected for cryptosporidium contamination 
 in stws . calculated using predicted probabilities 
 from multivariable models in tables 1 and 2 for projected full latrine 
 use ( for tubewells ) and elimination of open defecation rates ( for 
 ponds ) at critical distances around each sampled water source in study 
 communities , by 2017 , and at end of total sanitation for all campaign 
 ( 2022 ) . predicted probability 
 of either cryptosporidium or giardia contamination 
 estimated by summing the predicted detection probability of each protozoa 
 at a given water source we investigated multiple sources and mechanisms for cryptosporidium and giardia contamination of community tubewells 
 and ponds across 60 villages in a coastal district in rural india 
 including pour - flush household latrines as a potential protozoa source 
 ( or sink , via reduced open defecation ) . multivariable modeling showed 
 that protozoa contamination of local groundwater used for drinking 
 was positively correlated with the density of human fecal loading 
 into latrine pits , literacy rates , livestock populations , damaged 
 tubewells , antecedent rainfall patterns and annual variability . levels 
 of contamination of local ponds used daily for bathing and hygiene 
 were positively correlated with the number of residents practicing 
 open defecation nearby , illiteracy rates , livestock populations , antecedent 
 rainfall patterns and annual variability . previous observational and experimental 
 studies have identified bacteria and virus groundwater contamination 
 from latrines up to 25 and 50 m away , respectively . this is the first study to examine latrines simultaneously 
 as a source and sink of environmental protozoa contamination . we found 
 strong evidence of protozoa contamination of shallow groundwater from 
 pour - flush latrines within 15 m and some limited evidence for deeper 
 groundwater contamination from pour - flush latrines up to 500 m away . shallow tubewells in the study area draw water from < 7 m bgl and 
 during the monsoon period the water table can rise as high as 02 
 m bgl , making a hydraulic connection 
 between latrine pits ( 1 m bgl ) and shallow groundwater likely . 
 intermittent hydraulic connection between latrines pits and groundwater , 
 persistence of protozoa in water and soil , and potential for long - term 
 protozoal transport support our finding of nearby latrine loading 
 as an important risk for shallow groundwater protozoal contamination 
 in this setting . pathogen transport in deeper groundwater at 
 distances > 100 m from a pollution source , as detected in our study , 
 is less often examined . waterborne outbreaks of cryptosporidiosis 
 and giardiasis in north america linked to use of groundwater for drinking , 
 however , provide evidence of deep groundwater protozoal contamination . further evidence for the plausibility of deep 
 groundwater contamination at distance > 100 m comes from a study 
 of 
 artificial groundwater recharge using treated wastewater in which 
 both cryptosporidium and giardia were detected in groundwater at 320 and 500 m , respectively , from 
 recharge zones in fractured limestone . the mostly porous gravels and sand aquifers in our study area may provide sufficient pore space for protozoal 
 transport , supporting the limited evidence ( 0.05 < p < 0.14 ) we also found for local latrines as a source of deeper 
 groundwater protozoal contamination . research on overland transport 
 of protozoa shows a clear connection 
 between animal fecal loading on land and contamination of nearby surface 
 water , with factors such as slope , soil type , and vegetation density 
 affecting transport . while human fecal loading on 
 land also poses risks for protozoa contamination of nearby surface 
 water , the circumstances and magnitude of benefits on surface water 
 protozoa contamination from ending open defecation in countries where 
 the practice remains prevalent have not been examined . we observed 
 a significant relationship between the number of people living nearby 
 a pond who practiced open defecation ( within 50 m , not beyond ) and 
 the pond cryptosporidium concentration level . genotyping 
 of contaminated pond samples in this and another similar rural setting 
 in bangladesh identified cryptosporidium hominis ( human - specific 
 genotype ) but not other genotypes . together , 
 these findings suggest that promoting latrine use by people living 
 nearby , and protective buffers around local water bodies , could reduce 
 human host - specific cryptosporidium pond contamination 
 in this setting . reduced cryptosporidium contamination 
 of ponds from increased latrine uptake predicted in this setting is , 
 however , overshadowed by the large expected negative effects of increased 
 uptake on protozoal contamination of local groundwater drinking sources 
 ( table 3 ) . village sheep , goats , and cattle appear to 
 be important local animal host contributors of cryptosporidium contamination detected in community water sources in this setting , 
 while cattle in particular appear to contribute to giardia contamination . unexpectedly , we observed a trend where villages 
 with more buffalo had lower levels of pond giardia contamination . we know of no biological explanation for the association , 
 which may be confounded . among livestock and domestic animal populations 
 in india and globally , however , evidence from a systematic review and other studies of 
 zoonotic disease transmission from livestock and cattle to humans 
 in this and similar settings is mixed . overall , the public 
 health significance of exposure to protozoa shed by livestock and 
 domestic animals is case - specific since these animals can shed zoonotic 
 protozoa , infectious to humans , as well as host - specific genotypes . rainfall 
 effects on protozoal surface water contamination have been studied , but few have considered human and nonhuman protozoa sources and 
 examined impacts on surface and groundwater contamination . over short 
 time scales ( i.e. , within the monsoon season ) our results indicate 
 that rainfall is an important mediator of local environmental sources 
 of human and animal fecal protozoal loading on contamination of ponds 
 and shallow groundwater . positive associations between rainfall events 
 ( i.e. , > 90th and > 80th percentile of monthly rainfall ) and 
 waterborne 
 disease outbreaks and fecal contamination , have been previously reported . in our modeling of stw and pond 
 contamination , rainfall variables associated with cryptosporidium contamination were different from those associated with giardia contamination , implying different physical processes 
 of importance . cryptosporidium in stws and higher 
 concentrations in ponds were more likely after a recent event ( within 
 2 days ) suggesting short - term cyclical processes of environmental 
 loading and accumulation followed by contaminant flushing and transport 
 pulses . on the other hand , giardia detection in stws 
 and concentration levels in ponds were negatively associated with 
 increasing cumulative rainfall ( monsoon season ) , suggesting dilution 
 of widespread background contamination . these hypothesized contrasting 
 environmental processes are consistent with finding giardia to be much more ubiquitous ( higher endemic infection rates and orders 
 of magnitude more parasites shed per host ) than cryptosporidium in this setting and a similar environment . after controlling for within - season shorter time - scale meteorological 
 effects , sampling year ( 2012 vs. 2013 ) , a potential proxy for longer 
 time - scale variability , remained associated with significant differences 
 in cryptosporidium and giardia contamination 
 in dtws , but not stws , and cryptosporidium in ponds . characteristics and population - level patterns of infection for each 
 protozoa , as well as larger scale climatic and hydrogeological 
 conditions for which we were unable to account , may have played a 
 role . village illiteracy rates 
 were the strongest predictor of increased giardia contamination levels in ponds , while at the same time were associated 
 with lower giardia risk in stws . livestock ownership 
 in our study population at baseline was nearly equal among illiterate 
 and literate households ( 57% vs 60% ) , however , latrine ownership was 
 greater among literate households ( 13% ) compared to illiterate 
 households ( 3% ) and more illiterate households bathed at ponds 
 ( 72% ) compared to literate households ( 55% ) . given similar animal 
 ownership but lower latrine use ( i.e. , higher open defecation ) and 
 higher pond bathing rates among illiterate vs. literate households , 
 the link between illiteracy and increased pond giardia contamination would seem to point to a human source behind elevated giardia levels in ponds , that is , from higher overall rates 
 of open defecation and of anal cleansing and bathing in ponds in villages 
 with higher illiteracy rates . the protective effect of illiteracy 
 on stw contamination may arise similarly from the lower overall village - wide 
 latrine uptake rate and its associated loading into shallow groundwater , 
 or may be confounded . wellhead 
 protection is 
 critical for preserving groundwater quality , and poor well condition has previously been linked to well water 
 fecal bacterial contamination . our finding that dtws with damaged pads ( cracked or missing ) were 
 significantly associated with detectable levels of cryptosporidium and giardia , extends this link to larger protozoal 
 organisms in low - income settings . interestingly , we did not find a 
 significant increased risk of contamination in stws with well pad 
 damage , possibly because private household stws are used less intensely 
 than public dtws . stws used immediately prior to sampling were 
 significantly less likely to have detectable levels of giardia than those that had been idle . the sample collected from such stws 
 may have included a larger volume of more distant and deeper cleaner 
 groundwater , which may have diluted the impact from nearby latrine 
 leaching . we did not observe an effect on cryptosporidium , possibly because the subsample of cryptosporidium - positive stws was too small to detect an effect . future studies examining water body contamination 
 and fecal pollution sources in similar settings should consider the 
 potential effects of fecal sources located outside defined study boundaries . mapping in this analysis was limited to the point locations of local 
 pollution sources in odisha sanitation trial villages . we were thus 
 unable to account for the possibility of unmapped villages having 
 open defecator households , latrine pits or cattlesheds within the 
 largest buffer distance ( 500 m ) considered around community water 
 sources . this limitation is likely to mainly affect our analyses of 
 ponds , which were often located on the outskirts , rather than of tubewells , 
 located within the village . we also relied on self - report , which is 
 subject to recall bias , for estimating 
 usage rates and ages of latrines . repeated sampling of each water 
 source would have provided greater accuracy on protozoal prevalence 
 and concentration levels in each source and allowed for more fully 
 characterizing spatial and temporal variations in water source contamination 
 than was possible in our staggered cross - sectional study design . however , 
 because site visits for this study were randomized spatially across 
 a relatively homogeneous geographic study area and temporally within 
 the first 23 months of each monsoon season , and because study 
 villages were similar in terms of size , types of water and sanitation 
 infrastructure , ses characteristics , and economic activity , and all 
 samples were collected in the morning hours , we do not foresee significant 
 bias stemming from our single point sample design when used to estimate 
 area - level associations within the context of monsoon season contamination 
 patterns , as we have done here . better information on soils , 
 weather , and hydrogeology , such as groundwater recharge zones and 
 detailed lithology , would have allowed for better characterization 
 of protozoal transport into each tubewell or pond , but was not available 
 and beyond the scope of data collection for the odisha sanitation 
 trial . however , all study villages were within 50 km of each 
 other , and the geology of the study region is defined primarily as 
 a delta plane with predominant alluvium geological formation , reducing potential bias from these limitations . we can not preclude 
 the possibility that cryptosporidium and giardia detected in a tubewell sample could have come from 
 a contaminated spout / mouth , however , we flushed each tubewell prior 
 to sample collection for 30 s and then rinsed each 2 l sample 
 bottle with tubewell water 3 times prior to filling to minimize this 
 possibility . our village - level ses variables are imprecise and subject 
 to residual confounding from measurement error or miss - specification 
 of a category . for example , beyond literacy status of the household 
 head , educational attainment level may have improved the association 
 between education status and water contamination . finally , although 
 our conceptual model represents causal relationships grounded in scientific 
 literature , cross - sectional data and empirical modeling can only identify 
 correlations and our sample size may be under - powered to detect some 
 postulated associations . these limitations must be kept in mind when 
 interpreting the associations we found between outcomes and predictor 
 variables . further research 
 and better guidelines are needed to ensure the protection of critical 
 water sources used for drinking and to ensure household access to 
 microbiologically safe protozoa - free drinking water in rural india . based on our findings , guidelines would include repairing damaged 
 deep tubewells to protect public groundwater drinking sources and 
 placing pour - flush latrine pits in india at distances > 15 m from 
 shallow 
 tubewells used for drinking . however , these recommendations may be 
 ineffective in areas like this where water tables can rise within 
 2 m of ground - level and given the marginal evidence for and plausibility 
 of deeper groundwater contamination from latrine leaching densities 
 at greater distances . alternatively , changing the below - ground design 
 of wet latrines may be needed to reduce or prevent leaching rates 
 and associated pathogen loads , but these increase sanitation costs . 
 in view of already widespread contamination of local water sources , 
 anticipated rapid growth in latrine coverage , and difficulties achieving 
 high compliance and effective use of household water treatment , centrally treated and reliable piped water supplies with house connections