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PLOS One logoLink to PLOS One
. 2022 Apr 14;17(4):e0267103. doi: 10.1371/journal.pone.0267103

Genetic diversity of Cryptosporidium spp. in non-human primates in rural and urban areas of Ethiopia

Ambachew W Hailu 1,*, Abraham Degarege 2, Beyene Petros 1, Damien Costa 3,4, Yonas Yimam Ayene 5, Ven-ceslas Villier 3, Abdelmounaim Mouhajir 3, Loic Favennec 3,4, Romy Razakandrainibe 3,4,#, Haileeysus Adamu 6,#
Editor: Benjamin M Rosenthal7
PMCID: PMC9009656  PMID: 35421188

Abstract

Non-Human Primates (NHPs) harbor Cryptosporidium genotypes that can infect humans and vice versa. NHPs Chlorocebus aethiops and Colobus guereza and humans have overlapping territories in some regions of Ethiopia, which may increase the risk of zoonotic transmission of Cryptosporidium. This cross-sectional study examined the molecular prevalence and subtypes of Cryptosporidium spp. from 185 fecal samples of Chlorocebus aethiops and Colobus guereza in rural and urban areas in Ethiopia. Samples were tested for Cryptosporidium infection using nested polymerase chain reaction (PCR), and subtypes were determined by sequencing a fragment of the 60-kDa glycoprotein gene (gp60). Of the 185 samples, fifty-one (27.56%) tested positive for Cryptosporidium infection. The species detected were C. parvum (n = 34), C. hominis (n = 12), and C. cuniculus (n = 3). Mixed infection with C. parvum and C. hominis were detected in 2 samples. Four C. hominis family subtypes (Ia, Ib, Id, and Ie) and one C. parvum family subtype (IIa) were identified. C. hominis IaA20 (n = 7) and C. parvum IIaA17G1R1 (n = 6) were the most prevalent subtypes detected. These results confirm that Chlorocebus aethiops and Colobus guereza can be infected with diverse C. parvum and C. hominis subtypes that can also potentially infect humans. Additional studies could help to understand the role of NHPs in the zoonotic transmission of Cryptosporidium in Ethiopia.

Introduction

Cryptosporidiosis is a zoonotic parasitic disease that cause diarrhea in humans, farm animals and nonhuman primates (NHPs); water and foodborne outbreaks [1,2]; and malnutrition and cognitive deficits in children [3]. Cryptosporidiosis causes considerable morbidity and mortality in untreated acquired immunodeficiency syndrome (AIDS) patients and production loss in developing countries [4,5]. Globally, over 550 water-borne and foodborne outbreaks have been linked to cryptosporidiosis [6,7].

NHPs coexist with humans and livestock in an anthropogenically altered habitat [8,9]. This spatial proximity and close interaction between humans and NHPs may increase the risk of potential cross-species Cryptosporidium transmission [1012]. Indeed, several species of Cryptosporidium that infects humans have also been identified from NHPs in Asia, America, and Africa [1315]. In western Uganda, potential cross-species Cryptosporidium transmission between humans and NHPs was documented [16]. Similarly, a study conducted in Tanzania also highlighted the potential of Cryptosporidium for cross-species transmission between humans and NHPs [17]. Studies conducted in Rwanda, China, and Thailand also reported eight Cryptosporidium species including, C. hominins, C. parvum, C. felis, C. muris, C. ubiquitum, C. meleagridis, C. bovis, and C. andersoni in NHPS which can also infect human [14,18,19].

Chlorocebus aethiops (vervet monkey) and Colobus guereza (mantled guereza) are the most common NHPs in Ethiopia and they coexist in shared habitats with humans and other animals [20,21]. While vervet monkeys are regarded as a nuisance in some Ethiopian regions, mainly due to crop-raiding activities, mantled guereza is known for their cohabitation behavior with human settlement adjoining the forest areas in the southern part of Ethiopia [22,23]. Hence, identifying genotype and subtype of Cryptosporidium in NHPs is of paramount importance to understand their role as a reservoir of infections to humans and other animals. However, studies on the prevalence and distribution of Cryptosporidium species/genotype/subtypes in NHPs and their zoonotic potential are scanty. As there are no vaccines to prevent infection with Cryptosporidium and there are limited drug to treat infection with this parasite [1], accurate identification of species and subtypes of Cryptosporidium in vast arrays of host species, including NHPs is crucial for the characterization of transmission patterns and potential control options. We, therefore, carried out this study to shed new light on the genetic diversity of Cryptosporidium infection in NHPs in Ethiopia.

Methods

Study area description

The study was conducted in urban (i.e., a recreational site on the eastern shore of Lake Hawassa city) and rural areas (small village called Wurgissa) from June through September 2018. Subjects were free-ranging wild vervet and colobus monkeys found in two localities. Hawassa urban area offer colobus and vervet monkeys easy access for interacting with visitors, and people in the open recreation area often feed the monkeys with leftover food. However, only vervet monkeys were found in the rural area and participated in this study from the vervet monkeys in the rural site move throughout the village, spending substantial amounts of time feeding, defecating, and sleeping near the human villagers, and picking food off the ground contaminated with cattle faeces.

Stool sample collection and identification of NHPs

Verbal approval was obtained from the residents of Wurgissa and Hawassa, who live near the sampling site, and from local authorities (Hawassa city agriculture office and Wurgissa Kebele administration) before collecting samples from the NHPs. A single fresh fecal sample was collected from each monkey’s feces dripping off the floor following standard noninvasive sampling procedure [24]. Briefly, when a NHPs troop were encountered, first the total number of monkeys in a troop, the species and their geospatial location, age (adult, infant) and sex (male, female) information were recorded and followed until they defecate. Then the monkeys in the troop were followed until they defecate or lost from the connection. When monkeys defecated, the appearance of the faeces identified (diarrheic, formed), and an approximately 1 gram placed into 100ml plastic containers and mixed thoroughly with a 2.5% potassium dichromate solution. Samples within the plastic containers were stored at +4°C and transported to the biomedical laboratory of the Addis Ababa University. At the end, samples transferred to the laboratories of expert Cryptosporidiosis at the Charles Nicole University Hospital in France.

DNA extraction, molecular detection, and subtyping

Before the DNA isolation was performed, the stool samples had their preservative (2.5% potassium dichromate solution) removed in centrifugation process at 3000 X g for 10min at 4°C with PBS (pH = 7.2) repeated thrice. Total genomic DNA from each fecal sample was extracted using QIAamp Power Fecal DNA Kit (Qiagen). DNA extraction was performed with 250 μL of the fecal sample in accordance with the manufacturer’s instructions. Eluted DNA was stored at −20°C prior to PCR analysis [25]. Sequence-based characterization of Cryptosporidium has been performed to determine species and genotype. Cryptosporidium species were detected by nested PCR amplification of the SSU rRNA gene as described by Koehler et al., 2017 [26], PCR amplifications were performed with positive (C. parvum and C. hominis) and negative controls (no DNA water). PCR products were visualized on a UV transilluminator following electrophoresis on 2% agarose gels stained with ethidium bromide. All samples positive for Cryptosporidium species at the 18S locus were further subtyped at the 60 kDa glycoprotein (gp60) locus using a nested-PCR producing a ~ 364 bp secondary product, as previously described elsewhere [26]. The amplified DNA from secondary PCR products was separated by gel electrophoresis and sequenced using an ABI3500 sequencer analyzer (Applied Biosystems, Foster City, CA). Sanger sequencing chromatogram files were imported into Bioedit (http://www.mbio.ncsu.edu/BioEdit/bioedit.html), edited, analyzed, and aligned with reference sequences from GenBank.

Statistical analysis

Statistical analysis was performed using IBM SPSS Statistics software (version 26). Chi-square test was used to test the association of Cryptosporidium infection with the age (adult vs infant), sex (male vs female), species (vervet vs colobus), stool appearance (non-diarrheic vs diarrheic), and location (rural vs urban) of the NHPs. Because the number of Cryptosporidium infected cases in colobus monkey, and non-Diarrheic category were less than 5 infections, fisher exact test was used to test the association of Cryptosporidium infection with species and stool appearance used for cell values less than 5. P values < 0.05 were considered statistically significant.

Ethic statement

This study was part of another study which was approved by the ethical review board of Addis Ababa university. permission to collect fecal samples was obtained from the residents of Wurgissa and Hawassa, who live near the sampling site, and from local authorities (Hawassa city agriculture office and Wurgissa Kebele administration) before collecting samples from the NHPs.

Results

Prevalence of Cryptosporidium in NHPs

Faeces from 185 NHPs (177 Chlorocebus aethiops and 8 Colobus guereza) located in rural Wurgissa (n = 145) and urban Hawassa (n = 40) were examined. Out of the 185 NHPs, fifty-one (51) were infected with Cryptosporidium species based on a nested PCR test (Table 1). The prevalence of Cryptosporidium infection was significantly higher in female (35.4%) than in male (14.70%) monkeys(p = 0.002). A significantly greater proportion of samples collected from monkeys in urban (i.e., Hawassa, 42.5%) area were positive for Cryptosporidium compared to samples obtained from monkeys in the rural area (i.e., Wurgissa, 23.44%) (P = 0.017). In terms of NHPs species, Cryptosporidium infection was detected in 47 out of 177 Chlorocebus aethiops and 4 out of 8 Colobus guereza. The monkeys were asymptomatic carriers of Cryptosporidium (p = 0.001). However, the prevalence of Cryptosporidium infection did not vary with the age of the monkey (Table 2).

Table 1. Distribution of Cryptosporidium species infection in NHPs from Wurgissa and Hawassa areas in Ethiopia and their geospatial localization.

Areas Troop s ID Geospatial location C.cunicul us C.hominis C.parvum C.hominis
/
C.parvum
Negative Total
Vervet


Colobus


Vervet


Colobus


Vervet


Colobus


Vervet


Colobus


Vervet


Colobus


Urba
n area
HA Amora Gedel park
(7°02’37.4"N
38°27’24.1"E)

1

10

3

8

4

26
HB Wabishebel Hotel
(7°02’55.6"N
38°27’34.7"E)
1 2 11 14
Rural area WA Gollo
(11°32’22.1"N
39°36’28.7"E)
2 2 15 19
WB Gorarba
(11°32’10.1"N
39°40’45.4"E)
1 3 2 11 17
WC Worekalu
(11°34’15.1"N
39°40’03.2"E)
4 1 15 20
WD Gatira Georgis church
(11°33’10.2"N
39°36’51.6"E)
2 2 2 1 26 33
WE Goda
(11°31’55.4"N
39°37’18.9"E)
3 2 14 19
WF Burka
(11°33’21.6"N
39°39’39.6"E)
6 15 21
WG Gebriel
(11°32’43.8"N
39°37’13.9"E)
1 15 16
Total 3 11 1 31 3 2 134 185

Table 2. Prevalence of Cryptosporidium infection by age, sex, and stool character among NHPs.

Attributes Categorie
s
Cryptospridiu m infection rate
SSU-rRNA
PCR % (n/N)
Cryptospridium spp (n) Subtype (n)
P-value* C.parvum


C. hominis


C.cuniculus


Mixed


Prevalence (n/N) 27.56 (51/185)
Location Wurgissa
(rural)
23.44 (34/145) 0.017 19 10 3 2 IIaA17G1R1(6), IIaA19G2R1(3),
IIaA15G2R1(2), IIaA16G2R1(1)
IIaA16G1R1(1) IIaA17G2R1(1),
IIaA20G1R1(1), and IaA20 (5),
IdA21(1) lbA10G2(1), IeA11G3T3(1), IaA26(1)
Hawassa (urban) 42.50 (17/40) 15 2 IaA20(2), IIaA15G2R1(1),
Sex Male 14.70 (10/68) 0.002 6 2 1 1 IIaA19G2R1(1), IIaA16G2R1(1), lbA10G2(1)
Female 35.04 (41/117) 28 10 2 1 IIaA17G1R1(6), IIaA19G2R1(2),
IIaA15G2R1(3), IIaA16G1R1(1),
IIaA17G2R1(1), IIaA20G1R1(1),
IaA20(7), IdA21(1), IeA11G3T3(1) IaA26(1)
Age group Infants 23.52 (20/85) 0.26 12 4 2 2 IIaA17G1R1(3), IIaA16G1R1(1)
IIaA15G2R1(1), IIaA20G1R1(1),
IaA20(3), lbA10G2(1), IaA26 (1),
Adult 31 (31/100) 22 8 1 IIaA17G1R1 (3), IIaA19G2R1(3),
IIaA17G2R1(1), IIaA15G2R1(2),
IaA20 (4), IdA21(1), IeA11G3T3(1)
Species colobus 50(4/8) 0.218 3 1 IaA20(1)
vervet 26.6(47/177) 31 11 3 2 IIaA17G1R1 (6), IIaA19G2R1 (3),
IIaA15G2R1(3) IIaA16G1R1 (1),
IIaA17G2R1(1), IIaA20G1R1(1)
(IaA20 (6), IaA26 (1), lbA10G2 (1), IdA21 (1) IeA11G3T3 (1)
Appearance of the stool Diarrheic 0 (0/32) 0.001
Non-
Diarrheic
33.33 (51/153) 34 12 3 2 IIaA17G1R1 (6), IIaA19G2R1 (3),
IIaA15G2R1(3) IIaA16G1R1 (1),
IIaA17G2R1(1), IIaA20G1R1(1)
IaA20 (7), IaA26 (1), lbA10G2 (1), IdA21 (1) IeA11G3T3 (1)

Cryptosporidium species and genotypes distribution

Sequence analysis of 51 positive samples from Gp60 and 18s RNA gene revealed three species: C. parvum (n = 34), C. hominis (n = 12) and C. cuniculus (n = 3). Two samples with mixed infections of C. hominis and C. parvum were detected. Out of the 51 positive samples, 16 of the 34 C. parvum and 11 of 12 C. hominis positive isolates were successfully subtyped. Diverse subtype families, including IIa, Iba, IId and Ie, were seen in the rural study area, but only the IIa subtype family was identified in the urban area.

Subtyping analysis of the C. parvum and C. hominis isolates from Chlorocebus aethiops identified five family subtypes for C. hominis {IaA20 (n = 6), IaA26 (n = 1); lbA10G2 (n = 1), IdA21 (n = 1) IeA11G3T3 (n = 1)}, and six family subtypes for C. parvum {IIaA17G1R1 (n = 6), IIaA19G2R1 (n = 3), IIaA15G2R1(n = 3) IIaA16G1R1 (n = 1), IIaA17G2R1(n = 1), IIaA20G1R1(n = 1)}. Subtyping analysis of the four Cryptosporidium positive samples obtained from Colobus guereza revealed the subtype ‘IaA20’. A total of 12 subtypes {IIaA17G1R1(6), IIaA19G2R1(3), IIaA15G2R1(2), IIaA16G2R1(1) IIaA16G1R1(1) IIaA17G2R1(1), IIaA20G1R1(1), IaA20 (5), IdA21(1) lbA10G2(1), IeA11G3T3(1), IaA26(1))} were seen from NHPs located in rural Wurgissa but only two subtypes {IaA20 (2), IIaA15G2R1 (1)}were identified from monkeys in the Hawassa town (Table 2).

Discussion

There is limited information about the molecular prevalence and diversity of Cryptosporidium spp. in NHPs in Ethiopia. This study examined the molecular prevalence of Cryptosporidium spp. subtypes in Chlorocebus aethiops (vervet monkey) and Colobus guereza (mantled guereza) in Ethiopia. Out of 185 NHPs samples examined, C parvum, C. hominis and C. cuniculus were found in 34, 12 and 3 samples, respectively. Amongst multiple subtype families identified, C. hominis IaA20 was the most frequently seen.

The prevalence of Cryptosporidium infection among vervet monkeys (47/177, 26.6%) detected in the current study area was higher than the prevalence reported among vervet monkeys in other regions of Ethiopia (3.5% to 9.5%) and Tanzania (16%) [17,20]. A study in Indonesia (2.7%) and Central African Republic (0.5%) also reported lower prevalence of Cryptosporidium infection in Gorilla beringei (Mountain gorillas) [11,27]. However, a study in Uganda showed a higher prevalence of Cryptosporidium spp. among free-ranging mountain gorillas (73%, 8/13) [28]. The variation in the prevalence of infection in the different regions could be due to the difference in the NHPs hosts examined; encroachment habits of NHPs to humans and domestic animals; presence of habitats for feeding and ingestion of water. The sensitivity and specificity of the diagnostic test used could also contribute to the differences in the magnitude of the prevalence of infection reported by the studies. Some of the studies used microscope to check infection [19,25] which is less sensitive compared to the molecular methods applied in the current study. The size of the sample examined could also be a source of variation in the prevalence of infection reported in the studies. Most of these studies involved a sample size of lower than 100 [15,19,25,29] and even some lower than 20 samples [20] compared to the 185 samples examined in the current study.

In agreement with the previous studies, C. hominis and C. parvum have been detected in NHPs [13,15,3032]. These reports suggest that C. hominis, which was initially described as infectious to humans, may potentially expand its hosts from humans to NHPs. Indeed, C. hominis has been reported in several domestic livestock, wildlife hosts, rabbits, other mammals (marsupials) [3337]. Among the NHPs examined, C. cuniculus was also detected in three samples. To the best of our knowledge, C. cuniculus has not been previously reported in NHPs. However, the zoonotic potential of C. cuniculus was apparent when it was responsible for a drinking-water-associated outbreak of cryptosporidiosis in the United Kingdom [38]. The likely hood of an increase in close contact between humans and NHPs due to the incursion of NHPS into agricultural fields, homes, and recreational areas may contribute to the increased prevalence of C. hominis infections in NHPs in Ethiopia.

This study documented multiple subtype families of C. hominis including Ia, Ib, Id, and Ie. Studies conducted in China have also shown diverse C. hominis subtype families including Ia, Id, Ie and If in NHPs [13,30]. Also, a survey in Kenya showed subtype families of C. hominis including Ib, If and Ii [39]. In Ethiopia, C. hominis subtype families Id, Ie, and Ib have been identified in humans [40]. Due to some level of similarity in their genetics make up, humans and NHPs could probably be susceptible to the same pathogens, including C. hominis [41]. Moreover, the incursion behaviors of non-human primates to the human habitat may enhance the probabilities of spread through faeces contaminated food and water.

C. parvum IIaA17G1R1 (n = 6) was the most common subtype found in this study. This subtype has been reported in human samples in Wales and England [42,43], Romania [44], Iranian [45], The Netherlands [46] suggesting that the subtype has a potential to expand its hosts from humans to NHPs. This could be linked to the subtype’s infectivity, pathogenesis, transmissibility, and host adaption potential, which are shaped by genetic re-combination and selective pressure of the C. parvum population structure [1]. The predominant isolate of C. parvum (IIaA17G1R1) was frequently detected in the rural area. Domestication of livestock, animal manure, or excreta as a fertilizer, poor latrine uses, and conducive temperature and rainfall patterns infection may increase the likelihood of fecal-oral transmission C. parvum in rural areas. The transmission dynamic remains obscure, reinforcing the need for multi-locus genotype analysis of the parasite (isolated from human, livestock, and water samples) to properly elucidate the host population structure and the dynamic of Cryptosporidium transmission.

A higher prevalence of Cryptosporidium infection was seen among NHPs living in the urban area compared to the NHPs living in the rural village. This difference might have resulted from ecologic factors (e.g., fecal contaminated water and close habitations), which may facilitate transmission of the parasite between NHPs and humans. Similarly, the higher prevalence Cryptosporidium infection in adult monkeys might have been related to greater longevity, increasing the risk of acquiring Cryptosporidium infection from contaminated drinking water and contaminated food.

A significantly higher prevalence of Cryptosporidium infection was seen in females than males NHPs. Studies in Sri-Lanka [47] and China [30] also reported an increased prevalence of Cryptosporidium spp. in female NHPs than males. This observed difference in the prevalence could be due to a difference in the risk of infections between males and females. Male and female NHPs may not contact with humans and other domestic animals and ingest fecal contaminated water equally. Male and female NHPs may also have different preferences in their dwelling area, affecting susceptibility to Cryptosporidium infection.

Cryptosporidium species can infect a broad range of hosts, including humans, domestic and wild animals worldwide, causing asymptomatic or mild to severe gastrointestinal disease in their host species. In this study, all the Cryptosporidium infected NHPs were asymptomatic. Although Cryptosporidium infections can lead to watery diarrhea in infected hosts, no Cryptosporidium parasite was detected among 32 diarrheic stool samples. The absence of Cryptosporidium infections in diarrheic stool samples could be explained by the varying degree of pathogenicity and virulence isolates of the same species of Cryptosporidium, replication of the parasite, and the resulting immune response [48].

This study is not without limitations. Although the nested PCR method has been described as very sensitive and specific [49], out of the 51 Cryptosporidium-positive samples identified at SSUrRNA level, Gp60 PCR amplification and sequencing failed during this study for 25 samples. This lower efficiency of PCR for Gp60 (as compared to 18S rRNA gene) is due to the lower gene copy number and longer length of the amplified fragment [25,50]. In addition, sequence analysis was unsuccessful for C. cuniculus due to lower gene copy number and longer length of the amplified fragment.

In conclusion, findings from this study suggest that Chlorocebus aethiops and Colobus guereza can be infected with diverse C. parvum and C. hominis subtypes, which are likely associated with human infection. Additional studies involving the characterization of parasites from livestock, drinking, and recreational water sources could help understand the direct or indirect transmissions between NHPs, livestock, and humans and elucidate the role of NHPs in the zoonotic transmission of Cryptosporidium.

Acknowledgments

The authors are grateful, Rouen University Hospital (France), and Addis Ababa University (Ethiopia) support.

Data Availability

All relevant data are within the manuscript.

Funding Statement

The authors received no specific funding for this work.

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Decision Letter 0

Benjamin M Rosenthal

26 Jan 2022

PONE-D-21-37489Genetic diversity of Cryptosporidium spp. in non-human primates in rural and urban areas of EthiopiaPLOS ONE

Dear Dr. Hailu,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Reviewer #1: Partly

********** 

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

********** 

3. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #1: Yes

********** 

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Reviewer #1: No

********** 

5. Review Comments to the Author

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Reviewer #1: The manuscript “Genetic diversity of Cryptosporidium spp. in non-human primates in rural and urban areas of Ethiopia” describes the results of a molecular survey of Cryptosporidium from two species of NHPs. The study is straightforward, methods seems appropriate, and the data from this study are of interest and value. However, the manuscript needs improvement in how the results have been presented and described before it is suitable for publication. My specific comments are as follows:

1. Data are from 2 species of NHP, Chlorocebus aethiops (vervet monkey) and Colobus guereza (mantled guereza). Methods seems to only describe collection of samples for vervet monkey. Also there are far more samples from vervet monkeys (177) than mantled guerezas (8). But description of results combines data from both species. Overall the manuscript would be improved by more clearly describing the data from each species included in the study.

2. Like for comment 1, descriptions of results and discussion of the data do not delineate between rural and urban populations in a clear manner. The manuscript would be improved by more clearly describing the data from the two sampling locations.

3. Results: when describing prevalence results, you describe a significant difference between males and females but don’t give the prevalence in each of these groups. Update text to include these values.

4. The tree does not seem to be necessary? It is not contributing to the results or discussion of the manuscript as data from the tree are not described or used to explain any aspects of this study.

5. What is the purpose of the last sentence of the results section? Those subtypes are described above. Why are they listed as additional here? I think this could be resolved by better describing data from each species and study site as suggested in comments 1 and 2.

6. In the discussion the language that parvum and hominis have broadened their hosts from humans to NHPs should be revised. As you point out the presence of these organisms in NHPs is not well surveyed at the molecular level, and you can not distinguish between shifts in host range versus observations in new hosts based on this study alone.

********** 

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Reviewer #1: No

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PLoS One. 2022 Apr 14;17(4):e0267103. doi: 10.1371/journal.pone.0267103.r002

Author response to Decision Letter 0


10 Mar 2022

PONE-D-21-37489

Genetic diversity of Cryptosporidium spp. in non-human primates in rural and urban areas of Ethiopia

PLOS ONE

Dear Dr. Benjamin M. Rosenthal

Thank you for your comments and sending us the reviewer's comments. We thank also the reviewer for the constructive comments. We have revised the manuscript following the reviewer's suggestions. We described these changes in the below paragraphs. We hope that you will find our responses are acceptable, and we are looking forward to hearing your decision.

Editors

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Response: We have checked the formatting of the manuscript. The revised manuscript meets the Plos one’s style requirements.

2. In your Methods section, please provide additional information regarding the permits you obtained for the work. Please ensure you have included the full name of the authority that approved the field site access and, if no permits were required, a brief statement explaining why.

Response: We acknowledge the editor’s comment and included the text below in the methods section.

“We performed non-invasive sampling [24] and collected only a fresh sample from the monkey's feces dripping off the floor. Verbal approval was obtained from the residents of Wurgissa and Hawassa, who live near the sampling site, and from local authorities (i.e., Hawassa city agriculture office and Wurgissa Kebele administration).” (See line 64-66 pages 5, 6)

3. Please include your tables as part of your main manuscript and remove the individual files. Please note that supplementary tables (should remain/ be uploaded) as separate "supporting information" files.

Response: We have included the table as part of the main manuscript in the revised submission.

4. Thank you for stating the following financial disclosure:

“NO”

At this time, please address the following queries:

a) Please clarify the sources of funding (financial or material support) for your study. List the grants or organizations that supported your study, including funding received from your institution.

b) State what role the funders took in the study. If the funders had no role in your study, please state: “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

c) If any authors received a salary from any of your funders, please state which authors and which funders.

d) If you did not receive any funding for this study, please state: “The authors received no specific funding for this work.”

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

Response: This study didn’t receive funding support. So, we have included the text “The authors received no specific funding for this work” in the cover letter.

5. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

You may need to state your data availability statement here and mention that you will specify this in the additional information section when you submit the revised manuscript.

Response: we have included data availability statement which reads ‘All relevant data are within the manuscript.” in the cover letter. We have also included the same statement as additional information during the online submission

6. We note that Figure 1 in your submission contain map images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software (Google Maps, Street View, and Earth). For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:

a. You may seek permission from the original copyright holder of Figure 1 to publish the content specifically under the CC BY 4.0 license.

We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:

“I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.”

Please upload the completed Content Permission Form or other proof of granted permissions as an "Other" file with your submission.

In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].”

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The following resources for replacing copyrighted map figures may be helpful:

USGS National Map Viewer (public domain): http://viewer.nationalmap.gov/viewer/

The Gateway to Astronaut Photography of Earth (public domain): http://eol.jsc.nasa.gov/sseop/clickmap/

Maps at the CIA (public domain): https://www.cia.gov/library/publications/the-world-factbook/index.html and https://www.cia.gov/library/publications/cia-maps-publications/index.html

NASA Earth Observatory (public domain): http://earthobservatory.nasa.gov/

Landsat: http://landsat.visibleearth.nasa.gov/

USGS EROS (Earth Resources Observatory and Science (EROS) Center) (public domain): http://eros.usgs.gov/#

Natural Earth (public domain): http://www.naturalearthdata.com/

Response: We acknowledge the editor’s suggestion and removed Fig 1 from the revised submission.

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Response: We acknowledge the lack of clarity of the details in Fig 2, but we have removed the figure from the revised submission at the reviewer’s recommendation.

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Response: We have checked the references to meet to Plos one’s style requirement. There are no retracted references but have included one new reference to the list (reference # 24).

Reviewers' comments:

Comments to the Author

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Reviewer #1: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

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Reviewer #1: Yes

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Reviewer #1: No

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5. Review Comments to the Author

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Reviewer #1

The manuscript “Genetic diversity of Cryptosporidium spp. in non-human primates in rural and urban areas of Ethiopia” describes the results of a molecular survey of Cryptosporidium from two species of NHPs. The study is straightforward, methods seems appropriate, and the data from this study are of interest and value. However, the manuscript needs improvement in how the results have been presented and described before it is suitable for publication. My specific comments are as follows:

1. Data are from 2 species of NHP, Chlorocebus aethiops (vervet monkey) and Colobus guereza (mantled guereza). Methods seems to only describe collection of samples for vervet monkey. Also there are far more samples from vervet monkeys (177) than mantled guerezas (8). But description of results combines data from both species. Overall, the manuscript would be improved by more clearly describing the data from each species included in the study.

Response: We have made clear that samples were collected from vervet monkey and mantled guereza in the method section and presented the data separately for the two species. We have copied below the added text in the methods and results section of the revised submission

Method:

“The study subjects were free-ranging wild vervet and colobus monkeys found in Hawassa town and the Vervet monkeys living in a rural village ‘Wurgissa’ when collecting the sample. Visitors, and people in the open recreation area of Hawassa town often feed the monkeys with leftover food. (see line 75-77; page 5 )

Results

“Out of 51 fecal samples tested positive for Cryptosporidium infection, 47 were obtained from Chlorocebus aethiops and 4 from Colobus guereza. ( see line 144-145 page 8)

“Subtyping analysis of the C. parvum and C. hominis isolates from Chlorocebus aethiops identified five family subtypes for C. hominis {IaA20 (n=6), IaA26 (n=1); lbA10G2 (n=1), IdA21 (n=1) IeA11G3T3 (n=1)}, and six family subtypes for C. parvum {IIaA17G1R1 (n=6), IIaA19G2R1 (n=3), IIaA15G2R1(n=3) IIaA16G1R1 (n=1), IIaA17G2R1(n=1), IIaA20G1R1(n=1)}. Subtyping analysis of the four Cryptosporidium positive samples obtained from Colobus guereza revealed the subtype ‘IaA20’. ( see line 148-153 page 8 )

2. Like for comment 1, descriptions of results and discussion of the data do not delineate between rural and urban populations in a clear manner. The manuscript would be improved by more clearly describing the data from the two sampling locations.

Response: - We have presented and discussed the data stratifying it by the urban/rural regions where the sample was collected. “

Added text in the results and discussion

Result

A significantly greater proportion of samples collected from monkey in urban (i.e. Hawassa, 42.5% ) area was positive for Cryptosporidium compared to samples obtained from monkey in the rural area (i.e., Wurgissa , 23.44%) (P= 0.017) (see page 8-9 line 127-130)

A total of 12 subtypes { IIaA17G1R1(6), IIaA19G2R1(3), IIaA15G2R1(2), IIaA16G2R1(1) IIaA16G1R1(1) IIaA17G2R1(1), IIaA20G1R1(1), IaA20 (5), IdA21(1) lbA10G2(1), IeA11G3T3(1), IaA26(1) )} were seen from NHPs located in Rural Wurgissa but only two subtypes { IaA20(2), IIaA15G2R1 (1)}were identified from monkeys in the Hawassa town ( Table 2 ). (” See page 9 line 145 -149)

Discussion

“A higher prevalence of Cryptosporidium infection was seen among NHPs living in the urban area compared to the NHPs living in the rural village. This difference might have resulted from ecologic factors (e.g., fecal contaminated water and close habitations), which may facilitate transmission of the parasite between NHPs and humans.” (See line 206-209, page 21).

3. Results: when describing prevalence results, you describe a significant difference between males and females but don’t give the prevalence in each of these groups. Update text to include these values.

Response: We have provided prevalence data for males and females in the revised submission. It reads “The prevalence of Cryptosporidium infection was higher in female (35.4%) than male (14.70%) monkeys and the difference was statistically significant (p=0.002) (see line 124-126, page 11.”

4. The tree does not seem to be necessary? It is not contributing to the results or discussion of the manuscript as data from the tree are not described or used to explain any aspects of this study.

Response: - We acknowledge the reviewer’s comment and removed the phylogenetic tree from the revised manuscript.

5. What is the purpose of the last sentence of the results section? Those subtypes are described above. Why are they listed as additional here? I think this could be resolved by better describing data from each species and study site as suggested in comments 1 and 2.

Response : - We have revised the text in the last paragraph of the result section. We have presented data stratifying by monkey spp. and region as described above.

The revised text in the last paragraph reads “ “ Subtyping analysis of the C. parvum and C. hominis isolates from Chlorocebus aethiops identified five family subtypes for C. hominis {IaA20 (n=6), IaA26 (n=1); lbA10G2 (n=1), IdA21 (n=1) IeA11G3T3 (n=1)}, and six family subtypes for C. parvum {IIaA17G1R1 (n=6), IIaA19G2R1 (n=3), IIaA15G2R1(n=3) IIaA16G1R1 (n=1), IIaA17G2R1(n=1), IIaA20G1R1(n=1)}. Only one family subtype (IaA20) was identified from Colobus guereza. A total of 12 subtypes {IIaA17G1R1(6), IIaA19G2R1(3), IIaA15G2R1(2), IIaA16G2R1(1) IIaA16G1R1(1) IIaA17G2R1(1), IIaA20G1R1(1), IaA20 (5), IdA21(1) lbA10G2(1), IeA11G3T3(1), IaA26(1) )} were seen from NHPs located in Rural Wurgissa but only two subtypes { IaA20(2), IIaA15G2R1 (1)}were identified from monkeys in the Hawassa town ( Table 2 ) (line 148-157, page8 ).

6. In the discussion the language that parvum and hominis have broadened their hosts from humans to NHPs should be revised. As you point out the presence of these organisms in NHPs is not well surveyed at the molecular level, and you cannot distinguish between shifts in host range versus observations in new hosts based on this study alone.

Response: - We thank the reviewer for the suggestion. We have revised the text. It reads as “These reports suggest that C. hominis, which was initially described as infectious to humans, may potentially expand its hosts from humans to NHPs. (lines 181, 182 ; page 10)

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Attachment

Submitted filename: Response to Reviewers.docx

Decision Letter 1

Benjamin M Rosenthal

4 Apr 2022

Genetic diversity of Cryptosporidium spp. in non-human primates in rural and urban areas of Ethiopia

PONE-D-21-37489R1

Dear Dr. Hailu,

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Reviewers' comments:

Acceptance letter

Benjamin M Rosenthal

7 Apr 2022

PONE-D-21-37489R1

Genetic diversity of Cryptosporidium spp. in non-human primates in rural and urban areas of Ethiopia

Dear Dr. Hailu:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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