Diverse Genotypes of Cryptosporidium in Sheep in California, USA

Cryptosporidium spp. is a parasite that can infect a wide variety of vertebrate species. The parasite has been detected in sheep worldwide with diverse species and genotypes of various levels of zoonotic potential and public health concern. The purpose of this study was to determine the distribution of genotypes of Cryptosporidium in sheep in California, USA. Microscopic positive samples from individual sheep from central and northern California ranches were genotyped by sequencing a fragment of the 18S rRNA gene and BLAST analysis. Eighty-eight (63.8%) of the microscopic positive samples were genotyped, and multiple genotypes of Cryptosporidium were identified from sheep in the enrolled ranches. Approximately 89% of isolates (n = 78) were C. xiaoi or C. bovis, 10% of isolates (n = 9) were C. ubiquitum, and 1% of isolates (n = 1) were C. parvum. The C. parvum and C. ubiquitum isolates were detected only from lambs and limited to four farms. Given that the majority of Cryptosporidium species (i.e., C. xiaoi and C. bovis) were of minor zoonotic concern, the results of this study suggest that sheep are not a reservoir of major zoonotic Cryptosporidium in California ranches.


Introduction
Cryptosporidium spp. parasites virtually infect all vertebrate animals, including humans, livestock species, companion animals, and a wide range of mammalian wildlife [1,2]. Among the nearly forty named species of Cryptosporidium [3], the majority of species are host-specific with an additional subset of zoonotic species and genotypes that are infectious to humans [4,5]. Cryptosporidium spp. that are considered zoonotic in alphabetical order include (major vertebrate host in parenthesis): C. andersoni (cattle), C. bovis (cattle), C. canis (dogs), C. cuniculus (rabbits), C. erinacei (tree squirrels), C. fayeri (kangaroo), C. felis (cats), C. meleagridis (turkeys), C. muris (mice), C. parvum (cattle), C. scrofarum (pigs), C. suis (pigs), C. tyzzeri (mice), C. ubiquitum (cattle), and C. xiaoi (sheep and goats). In addition, Cryptosporidium spp. chipmunk genotype I (chipmunk), horse genotype (horse), mink genotype (mink), and skunk genotype (skunk) have also been associated with human infections [4]. Among these zoonotic species and genotypes, C. hominis and C. parvum are responsible for the majority of human infections [5,6] as well as the majority of waterborne outbreaks in human communities [7]; therefore, these two species are considered major zoonotic species of public health concern. Livestock species infected with zoonotic Cryptosporidium species and genotypes are considered a public health risk due to the possibility of transmitting infective oocysts to humans through direct contact [8] or by contaminating sources of drinking or recreational water leading to human waterborne cryptosporidiosis [9,10].
Cryptosporidium infections in sheep have been reported globally from numerous countries [11]. The most common Cryptosporidium species reported in sheep are C. ubiquitum, C. xiaoi, and C. parvum [12]. However, infections with other species such as C. andersoni,

Genotypes of Cryptosporidium in Sheep in California
Among the 138 microscopic positive samples across all sheep ranches, 88 (63.8%) samples from infected individual animals were successfully genotyped by sequencing a fragment of the 18s rRNA gene. The alignment of the 88 sequences resulted in four genogroups of Cryptosporidium in sheep in California. Except for genogroup 1, which contained only one isolate, sequences in genogroups 2, 3, and 4 were composed of multiple variants (i.e., a, b, c, d, e, and f) due to several nucleotide differences between the sequences. Genogroup 1 contained one isolate; genogroup 2 contained nine isolates; genogroup 3 contained 34 isolates; and genogroup 4 contained 44 isolates (Table 1). To avoid redundancy of submitting identical sequences for each variant, fifteen sequences were selected to represent these four genogroups and within-genogroup variants and were deposited into GenBank with accession numbers ON245368-ON245383.

Distribution of Cryptosporidium by Sheep Age, Breed, Fecal Characteristics, and Ranch Location
Approximately 93% (82/88) of the genotyped Cryptosporidium isolates were from lambs. Among these lamb isolates, only one (2%) was C. parvum and nine (10%) were C. ubiquitum; the remaining 88% (72/82) of Cryptosporidium isolates were C. xiaoi (i.e., genogroup 3) or C. bovis/C. xiaoi (i.e., genogroup 4). Only one Cryptosporidium isolate was from a yearling ewe and was identified as C. xiaoi-c; the remaining five isolates were from ewes and were identified as either C. xiaoi or C. bovis. Because none of the genotyped samples were from diarrheic sheep, no association was found between the Cryptosporidium species and fecal characteristics ( Table 2). Stratified by sheep breed, the only C. parvum isolate was detected from Dorper; the nine isolates of C. ubiquitum were found in Capay Red (n = 3), Suffolk (n = 2), and mixed breeds (n = 4) ( Table 3). C. xiaoi was distributed among Dorset, Rambouillet, Suffolk, Targhee, and mixed breeds, while C. xiaoi/bovis was distributed among Capay Red, Dorper, Hampshire, Rambouillet, Suffolk, and mixed breeds (Table 3). The single C. parvum isolate was detected from ranch No. 1 in Sonoma County in northern California. The nine isolates of C. ubiquitum were distributed across four ranches (No. 5, 6, 7, and 11) located in two counties in northern California. All Cryptosporidium isolates in sheep from other farms were either C. xiaoi or C. bovis (Table 4).       The phylogenetic relationships between C. ubiquitum from California sheep and C. ubiquitum strains from other geographical locations are shown in Figure 1. The Californian C. ubiquitum (genogroup-a) is close to the strain isolated from Iraq; the genogroup-b and c formed a clade with strains from the UK, China, and Ghana; and the genogroup-d formed another clade with strains from Iran, the UK, Maryland, and Spain ( Figure 1). These phylogenetic results indicate that variant strains of C. ubiquitum are widely distributed across diverse geographical locations.

Discussion
Given that the sequencing of the 18S rRNA gene is generally the most common method for the genotyping and speciation of Cryptosporidium spp. [6], the present study focused on the 18S rRNA sequences to compare Cryptosporidium from sheep throughout California with Cryptosporidium sequences in GenBank. Using the nucleotide BLAST's default setting of targeting 100 sequences, genogroup 1 was 100% identical to 100 sequences of C. parvum; variants of genogroup 2 were 99.63-100% identical to 8 to 57 sequences of C. ubiquitum; variants of genogroup 3 were 99.49-100% identical to 3 to 7 sequences of C. xiaoi in GenBank. Because of the high sequence similarity, it is highly likely that the single isolate of genogroup 1 is C. parvum, the 9 isolates of genogroup 2 are C. ubiquitum, and the 34 isolates in genogroup 3 are C. xiaoi. For genogroup 4, given that the isolates with maximum sequence similarity were equivalent for both C. xiaoi and C. bovis from sheep and goats (Table 1), it is difficult to determine the species of Cryptosporidium for these 44 iso-

Discussion
Given that the sequencing of the 18S rRNA gene is generally the most common method for the genotyping and speciation of Cryptosporidium spp. [6], the present study focused on the 18S rRNA sequences to compare Cryptosporidium from sheep throughout California with Cryptosporidium sequences in GenBank. Using the nucleotide BLAST's default setting of targeting 100 sequences, genogroup 1 was 100% identical to 100 sequences of C. parvum; variants of genogroup 2 were 99.63-100% identical to 8 to 57 sequences of C. ubiquitum; variants of genogroup 3 were 99.49-100% identical to 3 to 7 sequences of C. xiaoi in GenBank. Because of the high sequence similarity, it is highly likely that the single isolate of genogroup 1 is C. parvum, the 9 isolates of genogroup 2 are C. ubiquitum, and the 34 isolates in genogroup 3 are C. xiaoi. For genogroup 4, given that the isolates with maximum sequence similarity were equivalent for both C. xiaoi and C. bovis from sheep and goats (Table 1), it is difficult to determine the species of Cryptosporidium for these 44 isolates in genogroup 4; they could be either C. xiaoi or C. bovis.
This confusion over which species of Cryptosporidium is present in a single fecal sample may also be the result of a mixed infection with more than one Cryptosporidium species in sheep; for example, C. bovis and C. ubiquitum mixed infection was observed in sheep in the UK [19], and C. parvum and C. xiaoi mixed infections were observed in sheep in Australia [20]. However, because the sequences were identical to more isolates of C. xiaoi than C. bovis, the genogroup 4 isolates could be more related to C. xioai. This assertion is supported by the phylogenetic analysis because genogroup 4 isolates were in clades closer to C. xiaoi than C. bovis ( Figure 2). In summary, the combination of BLAST and phylogenetic analyses allowed us to identify Cryptosporidium species in sheep in California. Our results agree with previous reports that C. xiaoi, C. ubiquitum, and C. parvum are the most common Cryptosporidium species infecting sheep.
In addition to geographical locations, the distribution of Cryptosporidium species in sheep can also vary by farm, sheep age, and season [11]. In our study, based on genotyping of >60% (88/138) of all the microscopic positive samples, nearly 90% (78/88) of Cryptosporidium from the California sheep were identified as C. xiaoi or C. bovis. C. ubiquitum comprised only 10% (9/88) of these isolates and C. parvum comprised only 1% (1/88). Given that C. xiaoi, C. bovis, and C. ubiquitum are of minor zoonotic concern due to few human cases being attributable to these species, our results indicate that sheep in California ranches are not a major reservoir of major zoonotic Cryptosporidium of public health concern. Our findings are in agreement with the reports of Cryptosporidium in sheep in Western Australia [13], which were also not a major reservoir of major zoonotic Cryptosporidium, based on the observation that the majority of genotyped Cryptosporidium from sheep were C. ubiquitum, which is not commonly found in humans. These findings suggest that sheep-derived Cryptosporidium might have been overestimated in the past as a significant cause of waterborne human cryptosporidiosis.
The single C. parvum isolate and all the isolates of C. ubiquitum were detected in lambs ( Table 2). This could be due to the majority of the microscopic positive samples being from lambs (87.7% or 121/138); subsequently, the majority genotyped isolates were from lambs (93.2% or 82/88), in part due to lambs being more susceptible than yearlings or ewes to zoonotic infections with C. parvum and C. ubiquitum. In our previous work, we found a higher prevalence and higher intensity of oocyst shedding in lambs compared to yearlings and ewes; in addition, contact with cattle increased fecal oocyst shedding significantly [16]. Beneficial management practices, such as avoiding contact between sheep and cattle, and accessing surface water as drinking water, may help reduce the transmission of zoonotic Cryptosporidium species within and between livestock species.
Using existing knowledge of Cryptosporidium species of different zoonotic potential, this study assessed the zoonotic risks of Cryptosporidium from sheep in California. The findings of our studies suggest that diverse Cryptosporidium species are prevalent in different ages and breeds of sheep on California ranches, and that the majority of cryptosporidial species are not of significant public health concern. This work also contributes to the research of species and genotypes of Cryptosporidium infection in sheep worldwide.

Sample Collection
An epidemiological study was conducted to investigate the prevalence of Cryptosporidium and intensity of fecal shedding of oocysts in sheep, and to identify risk factors for sheep infection in California, USA [16]. Through collaborations with livestock and natural resource advisors of the University of California Cooperative Extension, 16 sheep ranches located in Northern and Central California (Figure 3) were enrolled in this study based on voluntarily participation. Four ranches were located in the Mountain North region, four in the Central Valley North region, five in the San Francisco Bay Area, and three in the Central Coast region (Figure 3). A total of 798 fecal samples from 372 adult ewes, 31 yearlings, and 395 lambs were collected and tested for Cryptosporidium spp. We found that the overall prevalence of Cryptosporidium in California sheep was 17.3% (138/798), with access to surface sources of drinking water and contact with cattle being significantly associated with a higher risk of oocyst shedding in sheep of all ages [16]. Using archived DNA samples from this epidemiological study, the objective of the current work was to determine the genotypes of Cryptosporidium in sheep in California, USA.

DNA Extraction, PCR, and Sequencing
All fecal samples that were microscopic positive of Cryptosporidium oocysts were subjected to genotyping of Cryptosporidium. A 0.2 g of fresh feces was exposed to 5 cycles of freeze (−80 • C) and thaw (+70 • C), and then used for DNA extraction by using the DNA Stool Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. All DNA samples were stored at −20 • C until further analysis. A nested PCR was performed on DNA samples using primers and reaction conditions amplifying an~830 bp fragment of the 18S rRNA gene according to methods previously described [49,50]. A DNA template of C. parvum isolated from calves from a local dairy farm was used as a positive control, and a negative control without DNA template was included. PCR products were verified by electrophoresis in 2% agarose gel stained with ethidium bromide. Products of the secondary PCR were purified using Qiaquick spin columns (Qiagen) and sequenced at the UC Davis DNA Sequencing Facility using an ABI 3730 capillary electrophoresis genetic analyzer (Applied Biosystems Inc., Foster City, CA, USA). Primers of the secondary PCR were used for sequencing in both forward and reverse directions. Consensus sequences were generated from the forward and reverse sequences of each isolate using Vector NTI Advanced 11 software (Invitrogen Corporation, Carlsbad, CA, USA).

BLAST Analysis
To compare Cryptosporidium spp. isolates with existing reference species and genotypes of Cryptosporidium in GenBank, selected representative sequences of each genogroup were aligned with other Cryptosporidium sequences in GenBank using the NCBI's online nu-cleotide basic local alignment search tool (BLAST). The BLAST analysis was optimized for highly similar sequences using default algorithm parameters and 100 maximum targeting sequences (6 April 2022, as last day accessed). located in Northern and Central California (Figure 3) were enrolled in this study based on voluntarily participation. Four ranches were located in the Mountain North region, four in the Central Valley North region, five in the San Francisco Bay Area, and three in the Central Coast region (Figure 3). A total of 798 fecal samples from 372 adult ewes, 31 yearlings, and 395 lambs were collected and tested for Cryptosporidium spp. We found that the overall prevalence of Cryptosporidium in California sheep was 17.3% (138/798), with access to surface sources of drinking water and contact with cattle being significantly associated with a higher risk of oocyst shedding in sheep of all ages [16]. Using archived DNA samples from this epidemiological study, the objective of the current work was to determine the genotypes of Cryptosporidium in sheep in California, USA.  The rationale for conducting this BLAST analysis was that comparative genotyping is commonly used to broadly characterize the zoonotic or human-infection risk for a novel isolate of Cryptosporidium. For example, if the DNA sequence for a reasonably long section of the 18S rRNA gene from a Cryptosporidium isolate is either highly related (≥99.5%) or has 100% sequence homogeneity to a known zoonotic species or genotype, the isolate is typically considered to be zoonotic and infectious to humans. In contrast, if the DNA sequence for an isolate is not highly related to any known zoonotic species or genotypes of this parasite, it is generally considered not zoonotic. Although this decision process is not perfect, it is a current convention used by many researchers and regulatory agencies around the world to assign zoonotic disease risk of an isolate of Cryptosporidium found either in water, food, or animals.

Phylogenetic Analysis
Because of the diverse genotypes observed of C. bovis/C. xiaoi and C. xioai in sheep in California, we conducted a phylogenetic analysis to compare C. bovis/C. xiaoi and C. xiaoi from our study to C. bovis and C. xiaoi from sheep worldwide. Similarly, a phylogenetic analysis was conducted to compare C. ubiquitum from our study to C. ubiquitum from sheep worldwide. Sequence alignments were conducted using the online 'Multiple Sequence Alignment' tool at Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/ (accessed on 6 May 2022)). Phylogenetic trees were constructed using the online 'Simple Phylogeny' tool (https://www.ebi.ac.uk/Tools/phylogeny/simple_phylogeny/ (accessed on 10 May 2022)) using the neighbor-joining method. Depending on the availability of sequences of Cryptosporidium from sheep in GenBank, reference sequences for the phylogenetic analyses were selected based on: (1) sequences of the 18s rRNA genes; (2) sequences of C. bovis, C. ubiquitum, and C. xiaoi from sheep/goat; (3) sequences representative of different geographical locations; and (4) sequence length (longer sequences available for each species, i.e.,~500 bp or longer) [51,52]. Information of Cryptosporidium species, locations, and GenBank accession numbers of selected sequences is available in Figures 1 and 2.

Conclusions
The results of our study demonstrate that C. xiaoi was the dominant Cryptosporidium species isolated from sheep in California, which indicates that California sheep do not appear to be a major reservoir of zoonotic Cryptosporidium species of major public health concern in California ranches (i.e., not a major source of C. parvum or C. hominis). The findings of this work and our previous studies suggest that managing lamb health, avoiding contact with cattle, and using secure sources of drinking water for sheep may help to reduce the shedding of zoonotic Cryptosporidium in sheep in California ranches. Future studies are warranted to further investigate the geographical distributions and epidemiology of Cryptosporidium species in small ruminants.