Detection of Escherichia coli, rotavirus, and Cryptosporidium spp. from drinking water, kitchenware, and flies in a periurban community of Lusaka, Zambia

Water, sanitation, and hygiene (WASH) are the basic needs for healthy living. However, many people are still living under inadequate WASH conditions, despite the global efforts under the Millennium Development Goals and Sustainable Development Goals. Poor WASH conditions are the major cause of diarrhea. The mortality rate due to diarrhea has been the highest in low- and middle-income countries, particularly in sub-Saharan Africa (UNICEF & WHO 2009). More than 100 million people in sub-Saharan Africa are estimated to be using unimproved sanitation and drinking water contaminated with feces (Bain et al. 2014; UNICEF & WHO 2019). These statistics have largely been contributed by the slow progress in WASH aspects in countries with high poverty and a dense population in urban informal settlements (UN-HABITAT 2016). The reduction of WASH-related health risks should be addressed, particularly in the urban informal settlements in sub-Saharan Africa.

By 2015, 60% of the informal settlements in sub-Saharan Africa only had access to unimproved sanitation, and were subjected to poorly managed environments (UN 2017). Urban informal settlements have been struggling with access to adequate WASH facilities. For example, in urban Uganda (Katukiza et al. 2014), 87.8 and 11.8% of the population at the study site used tap water and unprotected spring water for daily living, respectively. However, Escherichia coli, a major indicator bacterium of fecal contamination, was detected from tap water and unprotected spring water at the point-of-source (mean: 0.5 and 2.5 log₁₀ CFU/100 mL, respectively). In another case study in urban Kenya (Bauza et al. 2019), E. coli was detected in 20% of stored water and in 65% of kitchenware used for dietary purposes.

Enteric pathogens are detected from stool samples owing to poor WASH conditions in sub-Saharan Africa. In sub-Saharan Africa, diarrheal deaths in children aged <5 years were mostly attributable to rotavirus and Cryptosporidium infection (Troeger et al. 2017). Rotavirus is the main cause of diarrhea among young children in sub-Saharan Africa. Investigations of rotavirus diarrhea associated with children aged <5 years are frequently conducted. Forty percent of stool samples from hospitalized patients with acute gastroenteritis among children aged <5 years in eight sub-Saharan countries were positive for rotavirus (Mwenda et al. 2010). Rotavirus infection in sub-Saharan Africa was responsible for a high rate (range: 38.9–45.5%) of diarrheal deaths in 2000–2013 (Tate et al. 2016). Thirteen percent of stool samples from children aged <5 years presenting to outpatient departments with diarrhea or a history of diarrhea were positive for Cryptosporidium species (Krumkamp et al. 2021). Detection rates of Cryptosporidium oocysts in stool samples from 19,033 patients at all ages and 2,762 patients in the 1- to 4-year age group in Zambia were 1.46 and 3.8%, respectively (Mulunda et al. 2020). Cryptosporidiosis is one of the AIDS-defining illnesses and people living with HIV infections in sub-Saharan Africa, responsible for 70% of the global burden of HIV infections (Kharsany & Karim 2016), are especially vulnerable to diarrhea due to cryptosporidiosis. Although the presence of rotavirus and Cryptosporidium spp. in stool samples has been investigated in many studies, little is known about the contamination of important exposure media, such as drinking water and kitchenware, in the sub-Saharan African context, although the quantitative data through transmission-exposure pathways of such enteric pathogens need to be essentially examined.

In addition to the contamination of the exposure media, flies may act as vectors in the transmission of fecal pathogens, as described in the F-diagram of Wagner & Lanoix (1958). After contacting feces, flies may carry pathogens to the surfaces of exposure media, such as food, and then the pathogens are ingested by a person (Wagner & Lanoix 1958). Previous studies on urban informal settlements have shown the effect of flies in transmitting fecal microorganisms.

For example, in urban Nigeria, Oyeyemi et al. (2016) revealed that flies were commonly found in indoor and outdoor environments, and were suggested to be important agents of pathogen transmission to humans at the study site. In urban Bangladesh, Lindeberg et al. (2018) reported that the risk of rice contamination by E. coli was significantly higher when there were flies landing on the rice (odds ratio (OR): 5.4; 95% confidence interval (CI): 2.5–11.7; p < 0.001), and diarrheagenic E. coli and Shigella species were detected on the surface of the contaminated rice. Thus, flies are potential vectors that transmit enteric pathogens to exposure media in the kitchen. Although enteric pathogen transmission from feces to flies has been studied in laboratory-scale experiments (Tan et al. 1997; Graczyk et al. 1999, 2003), little is known about the contamination of wild flies with enteric pathogens in the field.

In the present study, we aimed to investigate enteric pathogen contamination in the living environment, with a focus on exposure media in kitchens, in a periurban informal settlement of sub-Saharan Africa. Together with E. coli, the contamination levels of rotavirus and Cryptosporidium spp. in household stored drinking water and cups in kitchens, as exposure media, were investigated in a periurban community (Chawama and Kanyama) of Lusaka, the capital city of Zambia, with large informal settlements in periurban areas having limited access to WASH. As a potential vector of rotavirus and Cryptosporidium spp., we also investigated the contamination of these enteric pathogens from wild flies caught in the community. Rotavirus was quantified and Cryptosporidium spp., which are assumed to have low contamination in the living environment, were detected by RT-qPCR. To the best of our knowledge, this is the first study to quantify rotavirus in flies and on kitchenware and to detect Cryptosporidium spp. contamination on the kitchenware.

The study was conducted in Lusaka City, Zambia, with a population of approximately 2.5 million people in 2018. The focus of the study was on two densely populated urban slums called the Chawama (440 ha; 188,000 inhabitants; 40,000 households) and Kanyama compounds (3,000 ha; 360,000 inhabitants; 76,000 households) (Central Statistics Office 2012).

According to a study conducted on the same compounds (Nyambe et al. 2020), the community had limited access to improved water sources and sanitation facilities. Among the residents, 84.9% had access to improved drinking water sources, such as public taps and boreholes with hand pumps, whereas 15.1% used an unimproved water source, open dug wells with or without covers, linings and slabs as an alternative. The majority (80.5%) used toilets shared with neighbors where pit latrines with or without solid wall/roofing were commonly found. However, signs of open defecation were still observable, as human feces were scattered on open grounds. Stored water collected from public stand taps shared with several households and located outside was kept in plastic pails in kitchens for drinking and cooking. An open dug well is an alternative water source used for flushing toilets and cleaning. After washing and drying, kitchenware, such as cups and plates ready for use, were kept in kitchens located inside houses. At the study site, stagnant water was commonly found near water sources, such as taps, open dug wells, and outdoor bathing sites, where water could accumulate in potholes on the ground. Flies were also observed moving freely in kitchens, outside houses, near toilets and near the waste bag of each household where they dispose of daily waste outside the house.

This study was approved by the Research Institute for Humanity and Nature in Japan (ref: 2018-8) and the Excellence in Research Ethics and Science (ERES) Converge in Zambia (ref: 2019-Feb-009). All households were notified of the purpose and the survey process, and written consent was obtained prior to conducting the survey in each household.

All the samples were collected during 16 October–14 November 2019. Nine types of environmental media were targeted for the analysis of E. coli as a fecal indicator, rotavirus, and Cryptosporidium spp. as enteric pathogens. Water samples were obtained using sterilized sampling bags. Stored water was sampled from 20 L plastic pails in the kitchens of 10 households; they were originally drawn from taps by local people. Open dug well water was sampled from four wells using buckets that were regularly used by the residents. Some of the open dug well water was sampled repeatedly, and in total, seven samples were collected from four wells. Stagnant water was sampled at 10 locations: near taps (6), open dug wells (3), and a bathing site (1). All water samples were used for the analyses of both E. coli and enteric pathogens.

From 16 kitchens, 33 cups ready for use were collected: 20 for E. coli analysis and 13 for enteric pathogen analysis. The inner surfaces of the cups were carefully swabbed on the site using swab test kits (Pro-media ST25 PBS, ELMEX, Japan); swabs were placed inside a plastic tube containing phosphate buffer solution (PBS) for the following laboratory analysis.

Fly tapes were hung at five locations for each of the seven households to catch flies on the surface of the sticky tape: inside the toilet (7), outside toilet (7), inside kitchen (7), outside house entrance (7), and around waste bag (7). After 1 h of hanging, the tapes were kept in sterilized plastic bags for the following laboratory analysis. After collection, all samples were transported on ice to the laboratory within 4 h. In the laboratory, a maximum of five flies were collected from each tape using sterilized forceps; each fly was transferred into a 50-mL Falcon tube and vortexed to rinse the surface of flies with 40 mL of phosphate-buffered saline. These solutions were used as fly samples for subsequent analyses.

Each of the samples was agitated rigorously. Of stored tap water samples, 100 mL was filtrated to test by the culture method for detection of E. coli following the WHO guideline for drinking-water quality (World Health Organization 2017). Other types of water samples were filtered in the following different volumes: 10 mL of 100 mL open dug well water and 1 mL of 100 mL stagnant water. For cup swab samples, 4 mL of 10 mL PBS solution in the swab was filtered. For fly samples, 10 of 40 mL of PBS solution was filtered. The tested volume of water and PBS solution was decided based on a preliminary survey to have appropriate concentration for the following test. The test for E. coli was conducted via membrane filtration (0.45 μm pore size, EMD Millipore Microfil V Filtration Device, Fisher Scientific, USA) and agar cultivation (XM-G agar, Nissui, Japan) with incubation at 37 °C for 22 h. Colonies of E. coli appeared blue on the filter. As the maximum reliable count of E. coli on the filter was 300, the upper detection limits for water samples were 300 CFU/100 mL for stored tap water, 3,000 CFU/100 mL (equivalent to 300 CFU/10 mL) for open dug well water, and 30,000 CFU/100 mL for stagnant water (300 CFU/mL). For the cup swab samples, the upper detection limit was 750 CFU/cup (300 CFU/4 ml for 10 mL). For the fly samples, the upper detection limit was 1,200 CFU/fly (300 CFU/10 mL for 40 mL).

Pretreatment to concentrate rotavirus was conducted as described by Katayama et al. (2002). For water samples, MgCl₂ was added and mixed with 100 mL of water at a concentration of 25 mM. For fly samples, each individual fly was collected from fly tapes using sterilized forceps, mixed with 40 mL of PBS in a 50-mL Falcon tube, and MgCl₂ was added at a concentration of 25 mM. These solutions were filtered through a mixed cellulose ester membrane (Millipore, HA mixed cellulose, pore size of 0.45 μm and diameter of 47 mm). The membranes were frozen until the analysis. For swab samples, the tubes containing swab and solution were kept frozen and used for the following analysis.

Viral and protozoan RNA were extracted from the membranes and swab samples using the NucliSENS kit (bioMérieux, Lyon, France) according to Miura et al. (2019). A murine norovirus (MNV) was used as a process control virus to analyze rotaviruses. Briefly, MNV was added to the membranes and swab samples in a 50-mL tube, and the tube was incubated for 5 min at −80 °C and for 2 min at 56 °C, four times. After the incubation, 15 mL of lysis buffer was added to the tube. The tube was vortexed for 15 s and incubated at 56 °C for 30 min. The tube was centrifuged at 2,300 × g for 5 min. The supernatant was collected in a 15-mL tube, and 140 μL of magnetic silica particles was added to the supernatant. All washing steps for the supernatant were performed using the NucliSENS miniMAG (bioMérieux, Lyon, France) according to the manufacturer's instructions, and the extracted RNA was collected in 100 μL of elution buffer.

For rotavirus, a LightCycler^® 480 System II (Roche) was used. The viral RNA was amplified using an RNA Ultrasense™ One-Step Quantitative RT-PCR System (Thermo Fisher Scientific, Japan) with previously reported primers, probes, and PCR conditions for MNV and rotavirus A (Miura et al. 2019). For Cryptosporidium spp., RT-PCR was performed on a Veriti Thermal Cycler (Applied Biosystems), and real-time PCR was performed on a LightCycler^® 480 System II. Protozoan RNA was amplified using a Cycleave RT-PCR Cryptosporidium (18S rRNA) (Takara, Japan) according to the manufacturer's instructions. Mulunda et al. (2020) identified four species (C. hominis, C. parvum, C. felis, and C. meleagride) of Cryptosporidium from stool samples of patients in Lusaka, Zambia. This kit can detect 10 species of Cryptosporidium spp., including these four species.

Undiluted and 10-fold diluted RNA extracts were tested to verify inhibition in real-time RT-PCR with TaqMan probe detection in each sample. The number of genome copies in each sample was calculated from the cycle threshold (C_T) values less than 40 and a standard curve generated with serial dilutions of plasmid DNA containing each target region (Miura et al. 2019). Standard curves with amplification efficiencies of 85–110% were used for quantification (Miura et al. 2019). When the MNV recovery rate of the sample was above 1%, the virus concentration in the sample was calculated based on the volume of RNA extracts and samples (Miura et al. 2019). Non-specific amplification was checked by 1.5% agarose gel electrophoresis.

A summary of E. coli concentrations on nine types of samples is presented in Tables 1–3. E. coli was detected in 68 of 102 samples. Among the water samples (Table 1), E. coli was detected in all 10 samples of stagnant water on the ground at high levels (>30,000 CFU/100 mL). Although the samples of open dug well water were directly collected from the wells, E. coli was detected in all seven samples, among which the concentration in five was >3,000 CFU/100 mL. The results for E. coli concentration in stagnant water indicated heavy fecal contamination of the living environment, potentially causing fecal contamination of open dug well water.

For stored tap water samples, which were used for direct drinking, E. coli was detected in six of 10 samples. In addition, the concentration in three samples was >300 CFU/100 mL, indicating fecal contamination of drinking water tested. In addition to stored tap water, E. coli was detected on half of the 20 cups (Table 2); these cups were used for drinking or collecting stored tap water from plastic pails. Out of 20 cups, the concentration on seven cups was >750 CFU/cup.

For fly samples (Table 3), the number of flies caught and the E. coli concentrations in them varied among the five locations. The average number of flies caught per tape for an hour was 0.9 fly inside the toilet space, 3.1 flies outside the toilet space, 4.3 flies in the kitchens, 4.9 flies at the house entrance, 9.7 flies near waste bags. The proportion of E. coli-positive flies inside the kitchen was the lowest (40.0%; 6 of 15 flies), although the average number of flies in the kitchen was higher than that of flies inside and outside the toilet space. Nevertheless, two flies from the kitchen had E. coli concentrations of >1,200 CFU/fly. All the four flies inside the toilet were positive for E. coli, with three having concentrations of >1,200 CFU/fly, although the average number of flies caught on a tape was the lowest (0.9 flies per tape). The average number of flies caught per tape near waste bags located outside was the highest in all five locations, with 11 of 17 flies being positive for E. coli, of which the concentration in seven flies was >1,200 CFU/fly.

Amplification efficiencies of standard curves by real-time RT-PCR with TaqMan probe detection of rotavirus, MNV and Cryptosporidium spp. were 97, 94, and 86%, respectively (Supplementary Figure S1). Rotavirus and Cryptosporidium spp. were detected by real-time RT-PCR in eight and three of the nine sample types, respectively (Table 4). The genotypes of rotavirus and the species of Cryptosporidium spp. were not identified from amplification products by real-time RT-PCR. All detection samples gave C_T values less than 40 and recovery rates above 1% estimated from C_T values of RNA extracts of MNV. But inhibition was detected in three water samples and one cup sample, comparing C_T values of undiluted RNA extracts to those of 10-fold diluted RNA extracts. The concentrations of these samples were calculated from C_T values of 10-fold diluted RNA extracts. Also, specific amplification was detected from all detection samples (Supplementary Figure S2). A summary of the concentrations of rotavirus and Cryptosporidium spp. in water, flies, and cups is presented in Table 4. Of the 27 water samples, 7 and 2 were positive for rotavirus and Cryptosporidium spp., respectively, with a maximum concentration of 2.2 × 10⁵ copies/L (stored water) and 3.6 × 10⁴ copies/L (stored water), respectively. Rotavirus was detected from all of the three types of water: stagnant water near water sources, water from open dug wells, which were unimproved drinking water sources, and stored water from public taps and boreholes, which were improved water sources for drinking and cooking. In contrast, Cryptosporidium spp. was detected only from the stored water. Of the 13 cup samples, 2 and 1 were positive for rotavirus and Cryptosporidium spp., respectively. These exposure media were contaminated with rotavirus and Cryptosporidium spp.

Of the 17 fly samples, 5 and 1 were positive for rotavirus and Cryptosporidium spp., respectively, with the concentrations ranging from 5.0 × 10¹ to 2.0 × 10² copies/fly at four locations (inside toilet, outside toilet, in the kitchen, and on waste bag) and a concentration of 8.5 × 10² copies/fly on the waste bag. Many flies were observed in the living environment of the targeted area. The flies might be contaminated with rotavirus and Cryptosporidium spp. in various parts of the living environment, where people had limited access to improved sanitation facilities.

Transmission routes of enteric pathogens include contaminated hands, drinking water, utensils, and flies (Brown et al. 2013). In this survey, we focused on stored drinking water and cups, as important exposure media in kitchens, and flies, as potential vectors of enteric pathogens.

E. coli was detected in most of the water samples, and the E. coli concentrations in some water samples were remarkably high. The presence of E. coli in various types of water, including stored tap water for drinking, well water and stagnant water, indicated that water might have been contaminated with human and/or animal feces. The contamination of uncovered open well water and stagnant water may be due to their exposure to the surrounding ground surface that was potentially contaminated with feces. As a common practice at the study site, plastic buckets and cups were used to collect open dug well water and stored tap water, respectively. Although we did not sample the surface of the plastic buckets used for collecting open dug well water, the cups for the stored tap water were contaminated (Table 2). This suggests potential cross-contamination between the point-of-source and point-of-use water sources, as revealed in some areas such as Bolivia (Rufener et al. 2010) and Bangladesh (Harada et al. 2018).

The number of flies varied at different locations and in kitchens, and they carried high E. coli concentrations that could potentially contaminate the living environment. Flies may carry feces from the source of feces in toilets and transmit them to the kitchen environment, where food and water are prepared, as observed in the flies in kitchens (Table 3). Similarly, previous studies showed that E. coli concentrations in stored food in the kitchen were positively associated with the presence of flies in urban Bangladesh (Lindeberg et al. 2018) and with E. coli concentrations in flies in urban Bangladesh (Doza et al. 2018). As Cryptosporidium spp. and rotavirus were detected in flies, our results suggest that flies may act as potential vectors of enteric pathogens in the kitchen. Furthermore, a previous study at the same study site found that certain households disposed of feces with solid wastes (Nyambe et al. 2018). This WASH practice may attract or breed more flies with contamination of enteric pathogens. WASH management at dumpsites could be important not only in toilets and kitchens, but also in controlling the transmission of enteric pathogens in the living environment.

Recent studies have investigated rotavirus in drinking water sources in rural areas of sub-Saharan Africa (Verheyen et al. 2009; Kiulia et al. 2010). Although the data are outdated, Kelly et al. (1997) investigated the presence of C. parvum in water sources using 100 L of water samples in Zambia. Oocysts of C. parvum were identified in five (Chipata supply, Chawama borehole, Misisi quarry, George zone (LWSC), and George JICA borehole) of six water sources in Zambia. However, there are no studies on the detection of these pathogens with regard to the use of drinking water. In the present study, rotavirus was detected in all three water types including stored drinking (tap) water in the kitchens (point-of-use); Cryptosporidium spp. was in stored drinking (tap) water in the kitchen (point-of-use). This point-of-use contamination indicates the exposure of rotavirus and Cryptosporidium spp. through contaminated drinking water.

Rotavirus and Cryptosporidium spp. were also detected on the surfaces of cups. These pathogens may be transferred from contaminated sources, such as human hands, water, or flies. For example, hands and flies contaminated with these pathogens frequently contact kitchenware surfaces and kitchenware is washed with contaminated water. Drying of cups with cloth is also one of the factors associated with E. coli contamination (Benwic et al. 2018). However, no information is available on the detection of rotavirus and Cryptosporidium spp. on cups. We showed the contamination of kitchenware with rotavirus and Cryptosporidium spp. The concentrations of rotavirus on the kitchenware are also shown. These findings suggest that people living in the target area are also exposed to enteric pathogens from the kitchenware.

Rotaviruses (Rzezutka & Cook 2004) and Cryptosporidium oocysts (Robertson et al. 1992) can survive for months in some types of water, such as tap water and river water. Rotaviruses can also survive and remain infectious on nonporous materials (glass, stainless steel, a smooth or rough plastic) (Sattar et al. 1986). In a previous study, Cryptosporidium oocysts were found to be viable in dry soils at 32 °C for 10 days (Fayer et al. 1998). Cryptosporidium oocysts have the potential to survive under high temperatures and dry conditions. Therefore, these enteric pathogens may maintain infectivity in stored drinking water and on kitchenware.

The 50% infection doses (ID₅₀s) for rotavirus and Cryptosporidium spp. are 6.17 focus-forming units (FFU) (approximately 9.6 × 10⁴ particles) (Haas et al. 1999) and 12.1; 132; 2,066 oocysts (UCP, IOWA, TAMU strain) (Messner et al. 2001), respectively. Detection of rotavirus and Cryptosporidium spp. by real-time RT-PCR in the present study do not necessarily indicate the existence of infectious viral particles of rotavirus and oocysts of Cryptosporidium spp. Considering the detection of rotavirus and Cryptosporidium spp. in stored drinking water and on kitchenware, we suggest that these exposure media may promote infection with these pathogens. The detection rate of rotavirus in stool samples from children in Zambia was the highest, being 67.6% (Chisenga et al. 2018). In addition, Cryptosporidium spp. was detected in 1.46% of 19,033 stool samples from patients, collected between 2017 and 2019, in Lusaka, Zambia (Mulunda et al. 2020). The findings of this study are supported by the detection rates 67.6% (rotavirus) (Chisenga et al. 2018), 1.46% (Cryptosporidium spp.) (Mulunda et al. 2020) of these enteric pathogens in stool samples from diarrheal patients in the same region.

Enteric pathogens are detected in flies during field and laboratory experiments. In addition, these pathogens can survive in flies for some days (Motarjemi et al. 1993). The role of flies as vectors of infectious diseases in humans has previously been suggested (Tan et al. 1997; Graczyk et al. 1999, 2003). There is no field study on the detection of rotavirus and Cryptosporidium spp. in wild-caught flies at various locations of the living environment, although rotavirus was detected in a field study at households in or close to food preparation areas in India (Collinet-Adler et al. 2015). Many flies were observed in the living environment of the targeted area. Detection of rotavirus and Cryptosporidium spp. on flies caught from various locations of the living environment indicated that flies play a potential role in the contamination of enteric pathogens.

There are some limitations to the present study. Because the RT-qPCR assay was applied to achieve greater sensitivity, no quantitative information on Cryptosporidium spp. was collected. Considering the small sample size, it was not possible to compare sufficient quantitative data among the exposure media and vectors of enteric pathogens. The present study investigated the stored drinking water at the point-of-use since the contamination of this water has direct impact on humans, who use the water; water contamination at the source was out of the scope of this study. It is necessary to track the sources of enteric pathogens to evaluate transmission routes. Nevertheless, our study in a periurban context contributes to society by providing essential information on enteric pathogen contamination in living environments, especially in poor WASH environments. Our future studies will focus on the evaluation of transmission and exposure routes for these pathogens by analysis of more samples determined from the findings of the present study and employing genome sequence analysis.

E. coli, rotavirus, and Cryptosporidium spp. in water and on cups, as exposure media, and on flies, as potential vectors of enteric pathogens, in the living environment were investigated in a periurban community of Lusaka. Widespread contamination of E. coli, rotavirus, and Cryptosporidium spp. has been observed from the detection of these microorganisms in stored tap water for drinking, dug well water for domestic use, stagnant water on the ground, cups, and flies. To our knowledge, the present study provides the first quantitative information on rotavirus in the kitchenware and flies and the first information on Cryptosporidium spp. contamination in the kitchenware. These data indicate that drinking water and kitchenware contributed to enteric pathogen exposure. Detection of flies in a living environment implies the potential role of flies as vectors of rotavirus and Cryptosporidium spp.

This work was supported by JSPS KAKENHI Grant Number JP19H02274 and Research Institute for Humanity and Nature (Project No. 14200107). The authors acknowledge Dr Meki Chirwa for supporting laboratory works in Lusaka.

Y.A. designed the experiment of enteric pathogens, analyzed the data of enteric pathogens. M.L.C. and M.T. conducted the field work, sampling, and E. coli analysis. M.L.C. analyzed the data of E. coli. Y.A. and M.L.C. wrote the first draft of the manuscript. T.Y. and I.N. supported and supervised the field work. H.H. designed the overall study and edited the manuscript. All authors reviewed and approved the final manuscript.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.