Genetic Diversity of Cryptosporidium Spp. in Njoro Sub County, Nakuru, Kenya

Cryptosporidium spp. cause Cryptosporidiosis in humans through zoonotic and anthroponotic transmission. Previous studies have illustrated the signicance of domestic animals as reservoirs of this parasite. However, there is no information on the Cryptosporidium spp. and genotypes circulating in Njoro Sub County. A total of 2174 samples from humans, cattle, chicken, sheep and goats were assessed for presence of Cryptosporidium spp. Thirty-three positive samples were successfully sequenced. The sequences obtained were compared to Cryptosporidium sequences in the GenBank using NCBI’s (National Center for Biotechnology Information) online BLAST (Basic Local Alignment Search Tool) algorithmic program. Sequence alignment was done using the Clustal W program and phylogenetic analysis was executed in MEGA 6 (Molecular Evolutionary Genetics Analysis version 6.0). The Cryptosporidium spp. present in the watershed showed great genetic diversity with nine (9) Cryptosporidium spp. namely: C. parvum, C. hominis, C. ubiquitum, C. meleagridis, C. andersoni, C. baileyi, C. muris, C. xiaoi and C. viatorum. Cattle were the biggest reservoirs of zoonotic Cryptosporidium spp. hence a potential source of zoonosis in humans while goats had the least species. This is the rst study that reported presence of C. viatorum in Kenya.


Introduction
Cryptosporidium is a common zoonotic parasite responsible for global outbreaks of waterborne gastrointestinal diarrhea (Zahedi et al. 2016). Cryptosporidiosis contributes to a high number of unreported child morbidity and mortality, especially in Africa (Shirley et al. 2012). However, the accurate global load of this disease remains a mystery not only due to lack of a simple and affordable diagnostic tool but also due to unacknowledged frequency and severity of the disease in immunocompetent patients (Shirley et al. 2012). Recent studies have demonstrated that anthroponotic transmission of Cryptosporidium predominates over zoonotic transmission in regions with poor hygiene (King et al. 2019). Domestic animals such as cattle, goats and sheep are vital sources of zoonotic Cryptosporidium spp. (Walter et al. 2021). However, there are other animals, such as poultry and rabbits, which happen to be important sources of emerging zoonotic species, for instance C. meleagridis and C. cuniculus respectively (Robertson et al. 2020). This highlights the signi cance of evaluating livestock production systems in Africa for their potential in zoonotic transmission of cryptosporidiosis.
Although Putignani and Menichella (2010) documented Cryptosporidium parvum as the species that causes most zoonotic infections in developed countries and slums in developing countries, other species circulate in farm animals, rodents and humans in rural areas (Robertson et al. 2020). Therefore, immense genetic diversity occurs among Cryptosporidium spp. in various hosts and geographical areas (Feng et al. 2018). This survey focused on the identi cation of the genetic characteristics of the Cryptosporidium spp. circulating in a rural setting, Njoro Sub County, in Kenya.
River Njoro is a source of drinking water for both humans and domestic animals within the Njoro river watershed. The river could be polluted by waterborne pathogens shed by domestic animals, which can then infect humans (Jenkins and Maina-Gichaba 2009). The prevalence and distribution of zoonotic species of Cryptosporidium in Njoro Sub County is a public health concern. Therefore, the aim of this study was to determine the prevalence and genetic diversity of Cryptosporidium isolates in humans and domestic animals in Njoro Sub County.

Study area
The study was conducted in River Njoro watershed (Fig.1), in Njoro Sub County in Nakuru County, Kenya, which lies between longitudes 35°05′E and 36°05′E, and latitudes 0°15′S and 0°25′S (Mainuri and Owino 2013). River Njoro has its origin in the Eastern Mau, covers about 50km in length and an estimated surface area of about 270km 2 . River Njoro watershed comprises of forested, agricultural lands and urban settlements and terminates in Lake Nakuru (Mainuri and Owino 2013)

Study design
The study employed a Complete Randomized Design to ensure each region sampled within the Njoro Sub County had an equal chance of being included. Purposive sampling design was utilized when sampling humans, where only patients who showed signs of diarrhea were recruited as study participants due to their higher probability of cryptosporidiosis infection. Hospitals and homesteads were randomly selected.
Another inclusion criterion was based on consideration of individuals who resided within Njoro Sub County during the entire study period.

Samples and sample collection
The units of sampling were humans and domestic animals such as cattle, sheep, goats and chicken while the samples used in the study were human and animal fecal matter. A total of 2174 fecal samples were randomly collected, each weighing 10g, and distributed as follows: 378 from human, 1000 cows, 388 sheep, 88 from goats and 320 from chicken. The animal owners were interviewed on the source of water given to the animals. The samples collected were transported to Egerton University, Biological Science laboratory and preserved at 4°C.

Identi cation of Cryptosporidium spp. in stool samples
All the fecal samples were analyzed for the presence of Cryptosporidium spp. oocysts using the Ziehl -Neelsen staining technique as described by Henriksen and Pohlenz (1981). Smear slides were air dried and then examined under the microscope at 40 magni cation. Cryptosporidium spp. oocysts appear as pink to red, spherical to ovoid bodies against a green to purple background. Samples were considered positive if at least one morphologically distinct Cryptosporidium spp. oocyst was observed.
DNA extraction, Quantitation and PCR DNA extraction was done using the Zymo gDNA extraction kit following the manufacturer's protocol with modi cations. Modi cations included; increasing the lysis temperature to 80 o C to enhance denaturation of nucleases, deactivation of PCR (Polymerase Chain Reaction) inhibitors and lysis of cryptosporidium cysts. Initial centrifugation time was increased from 5 minutes to 10 minutes to increase e ciency of separation. Additionally, the incubation time was increased to 6 hours to increase DNA precipitation.
DNA recovery was measured using Nano drop spectrophotometer and agarose gel electrophoresis. Gel electrophoresis involved comparison of the intensity of ethidium bromide-stained DNA bands on agarose gels and Lambda DNA of molecular weight 50ng.
DNA sequencing DNA amplicons were sequenced on contract by Inqaba Biotech Limited (Pretoria, South Africa), using Sanger sequencing platform. The sequencing raw data (chromatographs) were subjected to format conversion, quality control, trimming and ltering processes, all executed in Bioedit. The edited sequences were compared to Cryptosporidium sequences in the GenBank using NCBI's online BLAST tool with the default algorithm parameters to target 100 sequences. Sequence identity was characterized in terms of percentage identity, query coverage, Maximum Score, Total Score and E value.

Phylogenetic analysis
The sequences were aligned using the Clustal W program (Xiao et al. 2001) with manual adjustment. Phylogenetic analysis involved construction of a neighbor-joining (NJ) tree (Spano et al. 1997). The ). The relative distances among different Cryptosporidiumspp. were calculated using the Kimura twoparameter method. In order to assess the relatedness of the genus Cryptosporidium with other members of the phylum Apicomplexa, Babesia microti (GenBank accession no. U53448) sequence was used as the outgroup. B. microti was used as the outgroup because it has been reported to be the most divergent member of this group.

DNA extraction and PCR
Gel electrophoresis comparing the intensity of ethidium bromide-stained DNA bands on agarose gels and Lambda DNA of molecular weight 50ng appeared as shown in Fig 2. Genotyping of Cryptosporidium isolates in Njoro Sub County A total of 33 samples were successfully sequenced; 11 from chicken, 8 from cattle, 6 from humans, 5 from sheep and 3 from goats. In the study, nine Cryptosporidium species were identi ed from all the sources evaluated; human, cattle, sheep, goats and chickens. C. parvum was the predominant species having been detected in 27.27% (9/33) of the samples evaluated and from four sources; cattle, humans, chickens and sheep. C. hominis was detected in 15.15% (5/30) and humans were the only reservoir for this species. C. ubiquitum was identi ed in 18.18% (6/33) of the samples and it was detected in four sources; humans, sheep, goats and chicken. C. meleagridis was detected in 12.12% (4/33) samples exclusively from chicken. C.andersoni was identi ed in 12.12% (4/33) of the samples evaluated and in chicken and humans. C. baileyi, C. muris, C. Xiaoi and C. viatorum were each identi ed in only one sample and from a single source. The results of the 18 rRNA gene sequences of Cryptosporidium spp. in humans and animals in Njoro Sub County was summarized in table 2.

Proportion of Cryptosporidium spp. in domestic livestock
Evaluation of animal sources revealed that cattle were the highest reservoirs of Cryptosporidium spp. having four different species (C. andersoni, C. bovis, C. muris and C. parvum), two of which were identi ed as being zoonotic (C. andersoni, and C. parvum). Three species were detected in chicken (C. andersoni, C. maleagridis and C. parvum) all of which documented as capable of zoonotic transmission. Goats harbored three species of Cryptosporidium (C. baileyi, C. ubiquitum, and C. xiaoi) only one of which was identi ed as being capable of zoonotic transmission (C. ubiquitum). Sheep were identi ed to be the source of two species; C. ubiquitum and C. parvum, both of which are capable of being transmitted from animals to humans (Table 3). Three species were detected to be circulating in humans in Njoro Sub County (C. hominis, C. andersoni and C. parvum).

Zoonotic Cryptosporidium spp. circulating in Njoro Sub County
Maximum likelihood analysis of the sequences grouped the 33 isolates into two evolutionary clusters and three sub clusters (Fig. 2). Clustering was random and no signi cant host clustering among the 33 isolates was observed (Fig. 2).

Discussion
The prevalence of Cryptosporidium spp. reported in this study (6.99%) is consistent with results from other studies, which demonstrated its prevalence in Africa to occur in the range of 3-20% (Current and Garcia 1991). Our study show that Cryptosporidium is one of the most common gastrointestinal pathogen in humans, with a prevalence of 9.8%, almost similar to the 9% infection rates reported among children in Tanzania (Cegielski et al. 1999 This study identi ed nine species of Cryptosporidium through sequencing. These species include; C. parvum, C. hominis, C. andersoni, C. ubiquitum, C. meleagridis, C. bovis, C. muris, C. viatorum and C. xiaoi. In our study, majority of human infections were caused by C. hominis and the cattle genotype, C. parvum. These results are in agreement with those of a documented genotypic survey on the prevalence of Cryptosporidiosis among children with persistent diarrhea at Mulago Hospital in Uganda (Tumwine et al. 2003). The afore-mentioned Ugandan study demonstrated that 73.7% of the infections were caused by C. hominis, 18.4% were due to C. parvum while 3.9% were mixed infections with both species. Elsewhere, an epidemiologic study of cryptosporidiosis among children in Malawi illustrated that out of 43 cases, C. hominis was responsible for two with C. parvum ( According to Pumipuntu and Piratae (2018), transmission of cryptosporidiosis occurs through direct or indirect contact with stools of animals. Outbreaks occur through various routes of transmission: personto person contact in institutions, animal contact during farm visits, and contact with recreational waters, swimming pools, municipal drinking water and food (Chalmers 2012). Previous studies identi ed human-tohuman contact as the most common means of transmission (Cordell and Addiss 1994). This is illustrated by the increased risk of outbreaks in areas where there is routine crowding, such as day-care centers and schools, or patient-patient and patient-staff transmission in hospitals and the ultimate spread to the family members of the attending children or staff (Casemore 1990; Cordell and Addiss 1994). C. parvum is the most documented Cryptosporidium spp. involved in zoonotic transmission (Zahedi et al. 2016). Most of the reported cases of outbreaks of cryptosporidiosis in schoolchildren after exposure to calves or lambs are because of C. parvum (Casemore 1990;Casemore et al. 1997). C. parvum cryptosporidiosis has also been implicated in infection resulting from occupational exposure to infected animals (Current, 1994;Casemore et al. 1997). Furthermore, evidence from genetic analysis has proven that only the cattle genotype of C. parvum is capable of zoonotic transmission (Sulaiman et al. 1999). However, this genotype has also been found in many other host species such as humans, cattle, pigs and sheep (Putignani and Menichella 2010).
The high prevalence of the C. parvum in cattle and sheep coupled with the high numbers of oocysts shed by infected animals, especially newborns, make cattle and sheep important sources of environmental pollution with Cryptosporidium oocysts, which are capable of infecting humans (Uga et al. 2000).
The present study also detected C. parvum in sheep and chickens. This is in contrast with many past studies which had demonstrated a rare occurrence of C. parvum in small ruminants and birds in Africa Several studies in Africa have reported the presence of C. meleagridis infections in both immunocompromised and non-immunocompromised individuals, especially children (Hunter and Nichols 2002;Robertson et al. 2020). In this study, chicken were the sources of C. meleagridis and because this species has been identi ed as a zoonotic Cryptosporidium sp, (Zahedi et al. 2016 This study identi ed C. baileyi, C. muris, and C. xiaoi, each from a single source. Species such as C. muris and C. xiaoi have been previously detected and identi ed in immunocompromised individuals (Chappell et al. 2015); while C. bovis, and C. muris have been detected in immunocompetent humans, especially children (Azami et al. 2007).

Conclusions And Recommendations
This study con rmed that cryptosporidiosis is prevalent in Njoro Sub County and domestic animals are important reservoirs and a potential source of zoonosis in humans. The Cryptosporidium spp. present in the Njoro river watershed show great genetic diversity with C. viatorum having been detected for the rst time in Kenya. Extensive epidemiological and genomic studies of animal reservoirs of C. viatorum in Kenya are therefore required in order to clarify whether the transmission of this species is zoonotic or anthroponotic.

Declarations Funding
The authors did not receive nancial support from any organization for the submitted work.

Con ict of Interest/Competing interest
The authors declare that there is no con ict of interest and have no competing interest in this study.

Availability of data and material
The datasets that support the ndings of the current study have been deposited in a repository, GenBank, awaiting publication and accession numbers.  Geographical location of River Njoro water shed in Njoro Sub County. Njoro Sub County is in Nakuru County, Kenya (Map of Kenya shown on the left). River Njoro watershed is situated along River Njoro, which originates from the Eastern part of Mau forest (around Logomon and Nessuit areas) and terminates into Lake Nakuru (Mainuri and Owino 2013) Note: The designations employed and the presentation of the material on this map do not imply the expression of any opinion whatsoever on the part of Research Square concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. This map has been provided by the authors.