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RESEARCH ARTICLE
Common occurrence of Cryptosporidium
hominis in asymptomatic and symptomatic
calves in France
Romy Razakandrainibe, El Hadji Ibrahima Diawara, Damien Costa, Laetitia Le Goff,
Denis Lemeteil, Jean Jacques Ballet, Gilles Gargala, Loïc Favennec*
Normandie Universite
´, UNIROUEN, EA3800, CNR laboratoire expert Cryptosporidiose, Rouen, France
*loic.favennec@chu-rouen.fr
Abstract
Background
Cryptosporidium spp. are infections the most frequent parasitic cause of diarrhea in humans
and cattle. However, asymptomatic cases are less often documented than symptomatic
cases or cases with experimentally infected animals. Cryptosporidium (C.) hominis infection
accounts for the majority of pediatric cases in several countries, while C.parvum is a major
cause of diarrhea in neonatal calves. In cattle Cryptosporidium spp. infection can be caused
by C.parvum,C.bovis,C.andersoni and C.ryanae, and recently, reports of cattle cases of
C.hominis cryptosporidiosis cases suggest that the presence of C.hominis in calves was
previously underestimated.
Methodology/Principal findings
From February to November 2015, Cryptosporidium spp. infected calves were detected in
29/44 randomly included farms from 5 geographic regions of France. C.hominis and C.par-
vum were found in 12/44 and 26/44 farms, respectively with higher C.hominis prevalence in
the western region. In 9 farms, both C.parvum and C.hominis were detected. Eighty-six of
412 (73/342 asymptomatic and 13/70 symptomatic) one to nine-week-old calves shed C.
hominis or C.parvum oocysts (15 and 71 calves, respectively), with no mixed infection
detected. The predominant C.hominis IbA9G3 genotype was present in all regions, and
more frequent in the western region. An incompletely characterized Ib, and the IbA13G3,
IbA9G2 and IbA14G2 genotypes were present only in the western region. For C.parvum,
the most frequent genotype was IIaA16G3R1 with no geographic clustering. Most C.homi-
nis infected calves were asymptomatic, with some exceptions of IbA9G2 and IbA9G3 iso-
lates, while C.parvum IIaA16G3R1 was associated with symptoms.
Conclusions/Significance
Present results indicate for the first time that in several geographic regions of France, C.
hominis was present in about one fifth of both asymptomatic and symptomatic infected
calves, with isolated genotypes likely associated with human infection. Further
PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0006355 March 29, 2018 1 / 12
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OPEN ACCESS
Citation: Razakandrainibe R, Diawara EHI, Costa D,
Le Goff L, Lemeteil D, Ballet JJ, et al. (2018)
Common occurrence of Cryptosporidium hominis
in asymptomatic and symptomatic calves in
France. PLoS Negl Trop Dis 12(3): e0006355.
https://doi.org/10.1371/journal.pntd.0006355
Editor: Christine A. Petersen, University of Iowa,
UNITED STATES
Received: September 14, 2017
Accepted: February 28, 2018
Published: March 29, 2018
Copyright: ©2018 Razakandrainibe et al. This is an
open access article distributed under the terms of
the Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: Sequences derived
from this study have been deposited in the
GenBank database with accession numbers
MG676495 - MG676505 and MG676506 -
MG676576.
Funding: This work was partly supported by a
grant from Re
´gion Normandie (GRR Chimie
Biologie Sante
´, re
´seau Se
´curite
´sanitaire, grant
#16b06801GR). The funders had no role in study
design, data collection and analysis, decision to
publish, or preparation of the manuscript.
investigations are aimed at documenting direct or indirect transmissions between livestock
and humans.
Author summary
Symptomatic infection by the Apicomplexan Cryptosporidium spp. is presently considered
the most frequent parasitic cause of acute diarrhea in both humans (especially severe in
immunocompromised individuals and infants in both developed and developing coun-
tries) and cattle (calves), while asymptomatic infections are less often documented. Cryp-
tosporidium (C.) hominis once considered to be restricted to humans accounts for the
majority of pediatric cases in several countries. C.parvum can also infect cattle as well as
C.bovis,C.andersoni, and C.ryanae. Recently, cattle C.hominis cryptosporidiosis has
been reported, suggesting that the presence of C.hominis in calves was previously under-
estimated. The aim of this work was to characterize Cryptosporidium spp. infection in
both asymptomatic and symptomatic dairy and beef calves from Metropolitan France.
From February to November 2015, C.parvum or C.hominis infected calves were detected
in farms from 5 geographic regions of France. Surprisingly, C. hominis was present in
about one fifth of Cryptosporidium spp. infected calves, and exhibited genotypes which
were previously reported in human and nonhuman primate. Further investigations are
aimed at documenting direct or indirect C.hominis transmissions between and among
livestock and humans.
Introduction
Cryptosporidium spp. are Apicomplexa which include parasite species causing asymptomatic
to severe gastrointestinal infections in a wide range of vertebrate hosts, and exhibiting varying
degrees of host adaptation [1]. Previously, information on cryptosporidial host restriction of
natural cryptosporidial infection was usually obtained from animal and human cases for
which clinical symptoms, ages and immune statuses were recorded. However, evidence of
asymptomatic sustained or transitory infection, and the role of additional parameters such as
parasite detection methods, host’s genetic background, co-infection and environmental factors
such as climate, seasons and socioeconomic status were less documented [2]. For some cryp-
tosporidial host specialisations, information is presently limited to experimentally infected
immunocompetent or immunosuppressed laboratory animals [3].
In humans, cryptosporidiosis is presently identified as the most frequent zoonotic cause of
parasitic diarrhea, especially severe in immunocompromised individuals and infants in both
developed and developing countries [4–7]. In addition to 8 others sporadically observed spe-
cies, C.hominis, once considered to be restricted to humans, and C.parvum, of which some
isolate genotypes also infect ruminants, account for more than 90% of reported human cases
worldwide [8–10]. There is equal or higher prevalence of C.hominis than C.parvum in
humans in many parts of the world except in Europe where C.parvum largely prevails, likely
reflecting the ratios of human to animal sources of anthroponotic C.hominis and anthropo-
zoonotic C.parvum contamination, respectively [11–15].
In cattle, the main symptom of cryptosporidiosis is watery and profuse acute diarrhea
which can be associated with dehydration, anorexia, and impaired growth [16]. It was previ-
ously established that cattle can be infected by at least 4 Cryptosporidium species, i.e.C.
Cryptosporidium hominis in calves
PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0006355 March 29, 2018 2 / 12
Competing interests: The authors have declared
that no competing interests exist.
parvum,C.bovis,C.andersoni, and C ryanae [17,18]. In France, C.parvum,C.bovis and C.
andersoni predominate in newborn and older calves, respectively, C.parvum infection is iden-
tified as a major cause of diarrhea in newborn calves of less than one month old, with econom-
ically significant morbidity and mortality. However, detailed epidemiology on the occurrence
of viable oocysts from normal feces of asymptomatic calves is unknown [19,20]. Recently, a
limited number of observations reported of cattle cryptosporidiosis due to C.hominis have
been reported in Australasia, Asia, Africa and Europe, suggesting that the presence of C.homi-
nis in calves was previously underestimated in studies on diarrheic and adult animals [21–27].
The aim of this work was to document the prevalence of Cryptosporidium spp oocysts in
calves from five different geographic regions of Metropolitan France. Farms were randomly
included in the study, the clinical status of each animal was recorded, and the presence of
calves with Cryptosporidium spp. oocysts in feces was investigated. Isolates were genetically
characterized for their synzootic and zoonotic potentials.
Methods
Calves and fecal samples
From February to November 2015, 412 calves aged from 1 to 9 weeks were selected in 44 farms
from a national list of farms under regular veterinarian survey (16 veterinary offices, from 1 to
4 farm(s) per office). Farms were randomly selected, and within farms, calves aged from 1 to 9
weeks were randomly selected. Selected farms were situated in 14 “de
´partements” (an adminis-
trative sub-region) distributed in 5 geographic regions of Metropolitan France: western (Co
ˆtes
d’Armor, Ille-et-Vilaine, Morbihan), central western (Vende
´e, Deux Sèvres, Mayenne), north-
eastern (Pas-de- Calais, Moselle), southwestern (Landes, Pyre
´ne
´es atlantiques, Tarn, Hautes-
Pyre
´ne
´es), and central (Puy-de-Do
ˆme, Allier). For each calf, the clinical status was evaluated
and recorded by all veterinarians at the time of sampling as follows: presence or absence of
digestive symptoms such as diarrhea and abdominal bloating and/or respiratory symptoms,
and evaluation of the general condition as follows: "Normal general condition, shiny hair coat,
regular appetite Impaired general condition, delayed growth, dull hair coat, capricious appetite
Poor general condition, dull hair coat, capricious appetite, marked growth retardation"
From each farm (housing from 11 to 50 calves), feces samples were obtained by veterinari-
ans from 5–10 calves by rectal stimulation. Most farms were dairy farms and breeds consisted
of Salers, Holstein, Charolais, Montbeliard, Blonde d’Aquitaine, Parthenaise, Limousine and
the Belgian Blue Breed (BBB). In all farms, calves were kept in semi-intensive farming systems
and separated from their dams.
Microscopic detection of Cryptosporidiumspp. oocysts in calf feces
The presence of Cryptosporidium spp. oocysts was microscopically determined by the same
experienced clinical parasitologists using Bailenger type feces concentration method [28] and
Heine staining [29]. The presence of other intestinal parasites (Giardia,Strongyloides, and coc-
cidia) detected in some of the calves using various methodologies was not considered in the
present study.
Cryptosporidiumspeciation and genotyping
All samples were subjected to molecular analysis for speciation and genotyping of speciation
positive samples. Before DNA isolation, feces were subjected to a pre-treatment with a
mechanical lysis in Lysing Matrix A Tubes (garnet matrix and ¼ceramic spheres) (Qiagen,
CA, USA) with the Fastprep-24 device and transferred into 2 ml Eppendorf tube prior to
Cryptosporidium hominis in calves
PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0006355 March 29, 2018 3 / 12
thermal shock lysis (6 freeze-thaw cycles). Samples were placed in an ultrasonic bath for soni-
cation (3x20 sec bursts). In accordance with the manufacturer’s instructions, a modified
QIAamp Stool Mini Kit (Qiagen, CA, USA) was used to isolate DNA from the pre-treated
samples. All centrifugation steps were performed at RT (20–25˚C), at 14.000 rpm. Eight
hundred μL/tube of ASL buffer was added, and tubes were heated at 99˚C for 15 min.
For speciation, a 18S rRNA gene sequence was amplified using a nested PCR and restriction
digestion of the secondary product with SspI (NEB, MA, USA) and VspI (NEB, MA, USA) was
performed [30]. Briefly, for the primary PCR step, a PCR product (about 1,325 bp long) was
amplified by using primers 5-TTCTAGAGCTAATACATGCG-3 and 5-CCCTAATCCTTC
GAAACAGGA-3. For the secondary PCR step, by using 5 μl of the primary PCR product and
primers 5-GGAAGGGTTGTATTTATTAGATAAAG-3’ and 5-AAGGAGTAAGGAACAAC
CTCCA-3 a PCR product (819 to 825 bp long, depending on the species) was amplified.
Each PCR mixture (total volume, 50 μl) contained 5 μl of 10X DreamTaq Buffer, each deox-
ynucleoside triphosphate at a concentration of 0.2mM, each primer at a concentration of 100
nM, 2.5 U of DreamTaq polymerase, and 5μL of DNA template. Then, 1.25μL of DMSO
(100%) was added to the mixture.
A total of 40 cycles, each consisting of 94˚C for 45 s, 55˚C for 45 s, and 72˚C for 1 min,
were performed. An initial hot start at 94˚C for 3 min and a final extension step at 72˚C for
7 min were also included. Each amplification run included a negative control (PCR water)
and two positive controls (genomic DNA from C.parvum oocysts purchased from Water-
borne Inc., and C.hominis genomic DNA from fecal specimen collected at Rouen University
Hospital). Products were visualized in 2% agarose gels using ethidium bromide staining and
identification was confirmed by sequencing. Positive samples were further genotyped by
DNA sequencing of the gp60 gene amplified by a nested PCR following the protocol
described by Sulaiman et al. [31] (2005). All Amplification experiments were repeated at
least thrice to check reproducibility.
Purified PCR products were sequenced in both directions on an ABI 3500 sequencer ana-
lyzer (Applied Biosystems, CA, USA) by using the secondary PCR primers and the BigDye
Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, CA, USA). The obtained
sequences were inspected using the 4 peaks software (https://nucleobytes.com/4peaks/index.
html), edited with the BioEdit sequence alignment editor (version 7.2.5), and analyzed for
DNA database search and comparisons using the BLAST server (www.ncbi.nlm.nih.gov/
BLAST). Genotypes were named using the established gp60 genotype nomenclature [31].
Statistical analysis
Statistical analyses were performed using the chi-square (χ2) test or Fisher’s exact test as
appropriate using the Number Cruncher Statistical System (NCSS), version 2000 to determine
the association between the prevalence of Cryptosporidium infection vs regions, and sampling
periods. A p value <0.05 was considered statistically significant.
Ethics statement
Before carrying out this work, informed written authorization to perform and anonymously
publish the present epidemiological study was obtained from all cattle owners and veterinari-
ans. Clinical examination of calves and stool harvest were part of routine breeding and veteri-
nary procedures, without any invasive, traumatic or specific containment method. Such
procedures are not qualified as animal experimentation involving vertebrate according to
French laws, and no specific ethical clearing was required.
Cryptosporidium hominis in calves
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Results
As shown in Table 1, infected calves were detected in 29/44 farms from all geographic regions
and “de
´partements” except Puy-de-Do
ˆme and Mayenne, with no inter-regional difference in
the ratios of the number of infected farms to the number of included farms. C.hominis and C.
parvum were found in calves from 12/44 and 26/44 farms, respectively. In 9/44 farms, both C.
parvum and C.hominis infected calves were found with no mixed infection in any animal. No
mixed infection of C.hominis and C.parvum was noted in any animal.
Eighty-six of 412 included calves exhibited Cryptosporidium spp. oocysts in feces, of which
15 and 71 had C.hominis and C.parvum infection respectively. There were no inter-regional
differences in the ratio of the number of infected calves to the number of included calves
(p = 0.839). In the western region, the ratio of C.hominis infected farms (8/13) was higher
than in all other geographic regions (p p = 0.018), while no interregional difference was found
for C.parvum (p = 0.122). Infections were found in calves from 2 weeks to 7 weeks of age (3 to
7 weeks and 2 to 7 weeks for C.hominis and C.parvum cases, respectively).
As shown in Table 2,C.hominis IbA9G3 genotype isolates were predominant and present
in all geographic regions. The incompletely characterized Ib and the IbA13G3, Ib A9G2 and
IbA14G2 genotypes were only represented in the western region.
For C.parvum, IIaA16G3R1 genotype was the most frequent with no geographic clustering
(p = 0.574), and the limited number of isolates exhibiting other genotypes precluded further
investigation on their geographic representation (Table 3).
Table 1. Geographic distribution of 15 Cryptosporidium hominis and 71 Cryptosporidium parvum infected calves from 44 farms in 5 regions.
Region De
´partement/
Region subtotal
Included
farms
Included
calves
C.hominis
infected
farms
C.parvum
infected
farms
Cryptospordium
infected farms
C.hominis
infected
calves
C.parvum
infected
calves
Cryptosporidium
infected calves
Central Allier 3 31 1 3 3 1 12 13
Puy-de-Do
ˆme 4 40 0 0 0 0 0 0
region subtotal 7 41 1 3 3 1 12 13
Western Co
ˆtes d’Armor 1 10 0 1 1 0 2 2
Ile-et-Vilaine 6 51 4 2 4 5 4 9
Morbihan 6 58 4 4 4 6 7 13
Region subtotal 13 119 8 7 9 11 11 22
Southwestern Pyre
´ne
´es
Atlantiques
2 17 0 1 1 0 2 2
Hautes-
Pyre
´ne
´es
1 10 0 1 1 0 2 2
Landes 2 19 0 1 1 0 2 2
Tarn 4 39 1 3 3 1 4 5
Region subtotal 9 85 1 6 6 1 10 11
Central
western
Mayenne 1 5 0 0 0 0 0 0
Deux-Sèvres 4 35 0 3 3 0 9 9
Vende
´e 3 30 1 2 2 1 5 6
Region subtotal 8 70 1 5 5 1 14 15
Northwestern Moselle 4 37 0 3 3 0 13 13
Pas-de Calais 3 30 1 2 3 1 11 12
Region subtotal 7 67 1 5 6 1 24 25
Total 44 412 12 26 29 15 71 86
Results given in numbers of farms or calves.
https://doi.org/10.1371/journal.pntd.0006355.t001
Cryptosporidium hominis in calves
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No C.hominis infected calves exhibited diarrhea during the week before or at the time of
stool sampling (Table 4). Two infected calves, one with genotype IbA9G2 and one with geno-
type IbA9G3 presented with general state alteration and non-diarrheal digestive symptoms.
Table 2. Geographic distribution of 15 Cryptosporidium hominis genotypes from infected calves in 5 regions.
Geographic region De
´partement Calves with C.hominis genotype
Region IbA9G3 Ib IbA13G3 IbA9G2 IbA14G2
Central Allier 1
Puy-de-Do
ˆme
Western Co
ˆtes d’Armor
Ille-et-Vilaine 2 1 1 1
Morbihan 2 3 1
Southwestern Pyrenees-Atlantiques
Landes
Tarn 1
Central western Mayenne
Deux-Sèvres
Vendee 1
Northwestern Moselle
Pas-de-Calais 1
Total 8 4 1 1 1
Results given in numbers of calves
https://doi.org/10.1371/journal.pntd.0006355.t002
Table 3. Geographic distribution of 71 Cryptosporidium parvumgenotypes from infected calves in 5 regions.
Region De
´partement IIaA15G3R1 IIaA16G1R1 IIaA16G3R1 IIaA17G2R1 IIaA18G3 IIaA17G1 IIaA17G2 IIaA17G3 IIaA14GR1 IIaA19G2R1 IIaA15G2R1
Central Allier 1 3 5 2 1
Puy-de-Do
ˆme
Region subtotal 1 3 5 2 1
Western Co
ˆtes d’Armor 1 1
Ille-et-Vilaine 1 1
Morbihan 1 3 1 1 1
Region subtotal 2 4 2 1 2
South-
western
Pyrenees-
Atlantiques
2
Hautes-Pyrenees 2
Tarn 4
Landes 1 1
Region subtotal 1 8 1
Central
western
Mayenne
Deux-Sèvres 6 1 2
Vendee 1 1 1 1 1
Region subtotal 1 6 1 1 2 1 1 1
North
western
Moselle 9 1 1 2
Pas-de-Calais 10 1
Region subtotal 19 1 1 1 2
Total 2 6 42 5 3 1 3 4 1 1 3
Results given in numbers of calves.
https://doi.org/10.1371/journal.pntd.0006355.t003
Cryptosporidium hominis in calves
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Ten calves infected with the most frequent C.parvum IIaA16G3R1 genotype presented with
digestive symptoms, respiratory symptoms, or both, and 12 exhibited an impaired or poor
general state. The limited number of observations, precluded from investigating further associ-
ations between symptoms and genotypes.
The ratios of the number of infected calves to the number of sampled calves observed dur-
ing the August-September (50/216) and October-November (28/106) periods were higher
than those during February to March (3/45) and April to June (5/42) periods (p = 0.010) with
no difference in the respective C.hominis and C.parvum representations (the corresponding
values for C.hominis were 9/50, 4/28, 2/3, and 0/5, respectively, p>0.05). No seasonal effect on
Cryptosporidium infection prevalence, however, could be unambiguously established, taking
into account that due to the yearly calving cycle, most calves were sampled during summer
and autumn, and that no feces sample was obtained in January, July and December. The age
distribution of C.parvum infected calves is similar to that of C.hominis infected calves (Fig 1)
Discussion
In this work, the presence of C.hominis in calves was addressed for the first time in France by
investigating both asymptomatic and symptomatic calves from randomly chosen farms in sev-
eral geographic regions. Surprisingly, C.hominis was identified in about one fifth of Cryptospo-
ridium spp. infected animals, a figure which to our knowledge has not been reported
previously in Europe.
The random selection of farms resulted in a variety of geographic regions corresponding to
different geologic and climatic characteristics with oceanic, or humid continental influences.
Due to breeding procedures, most calves from all regions were sampled during summer and
Table 4. Symptoms and general conditions of Cr yptosporidium hominis and Cryptosporidium parvum infected calves.
Isolate genotype Infected calves Symptoms General condition
Asymptomatic Digestive Respiratory Digestive and respiratory Normal Impaired Poor
C.hominis Ib 4 4 4
IbA13G3 1 1 1
IbA14G2 1 1 1
IbA9G2 1 1 1
IbA9G3 8 7 1 7 1
Total 15 13 2 13 2
C.parvum IIaA15G3R1 2 2 2
IIaA16G1R1 7 6 1 6 1
IIaA16G3R1 42 34 3 4 1 38 2 2
IIaA17G2R1 5 4 1 4 1
IIaA18G3 3 3 2 1
IIaA15G2R1 3 3 1 2
IIaA17G1 1 1 1
IIaA17G2 2 2 2
IIaA17G3 4 3 1 2 2
IIaA14G1R1 1 1 1
IIaA19G2R1 1 1 1
Total 71 60 6 4 1 59 10 2
Results given in number(s) of calves. Each number corresponds to one calf except for calves with genotype IIaA16G3R1 of which 2/3 presented with both symptoms and
impaired general state or poor conditions.
https://doi.org/10.1371/journal.pntd.0006355.t004
Cryptosporidium hominis in calves
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autumn. For each calf, age, clinical condition, farm and sampling date were recorded by veteri-
narians. The study was focused on calves, and unbiased sampling was confirmed by similar C.
hominis infection rates of calves aged between 2 and 7 weeks. Most animals were clinically
asymptomatic, none of them presenting diarrhea in the week before, or at the time of sam-
pling, and a exhibiting digestive or respiratory symptoms and/or impaired or poor general
condition consistent with clinical cryptosporidiosis. Sampling and preservation of samples
were adapted to oocyst detection in calf feces: Heine-stained oocysts were detected microscop-
ically by experienced parasitologists, and oocyst species and genotypes were determined using
previously validated DNA amplification methods. C.hominis was unexpectedly detected in 15
calves. Four out of 15 C.hominis positive samples could not be identified at the genotype level,
with unreadable superimposition of electrophoregrams which might result from amplification
and sequencing of different genetic fragments from several genotypes present in the same sam-
ple. For both C.hominis and C.parvum, several subtypes were not seen in the same sample
(32).
Farms from all “de
´partements” except 2 presented both C.hominis and C. parvum-infected
animals. C.hominis was present in about half of the “de
´partements” with Cryptosporidium spp.
infected calves, and C.parvum was present in all of them. Both the ratios of C.hominis infected
farms and animals were higher in the western geographic region compared with other regions.
In 9/44 farms, both C.parvum and C.hominis were detected in different calves. Although no
data concerning the origin of water given to calves was available, a waterborne transmission of
C.hominis is possible as illustrated by the recent occurrence of a C.hominis waterborne out-
break in France. All samples were obtained during the calving period; thus, no seasonal varia-
tion of Cryptosporidium species nor genotypes could be ascertained. The present unexpected
high proportion of C.hominis infected calves compares with previously reported figures in
New-Zealand Australia, Africa, China and Europe [21,24,25,32,33]. Several studies have
established that cattle can be infected with at least 4 species (C.parvum,C.bovis,C.andersoni,
and C.ryanae). C.hominis and C.parvum were the only species detected in the present study,
likely due to the age of the calves, and possibly to the masking of concurrent infections by C.
bovis,C.ryanae or C.andersoni by the predominant shedding of oocysts of other species, or to
preferential PCR amplification of predominant species [17,18,35].
It was once generally accepted that C hominis primarily infects humans while C.parvum
infects both human and non-human hosts, and reports of cattle C.hominis infections are few.
Early observations based on 18S rDNA gene analysis revealed mixed cases with C.hominis in
Fig 1. Age distribution of uninfected, C.parvum and C.hominis infected calves.
https://doi.org/10.1371/journal.pntd.0006355.g001
Cryptosporidium hominis in calves
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heifers and cows from 2001 to 2003, and C.hominis cases in domestic cattle and goats [22,26].
More recently, limited numbers of C.hominis isolates from cattle have been genotyped in
Africa, China, New Zealand, Australia, the UK and Italy [21,23–25,34,36,37]. Besides cattle,
C.hominis has been reported in sheep and goats in the UK and in foals in Brazil [38,39].
In addition to species identification, gp60 genotyping was performed to provide clues to
symptomatic, epidemiologic, and zoonotic characteristics of isolates. Genotypes included
IbA13G3, IbA9G2, and IbA9G3 and the newly described IbA14G2, and for 4 isolates, only
attribution to the Ib genotype family was obtained, due to unreadable superimposition of elec-
trophoregrams. No association between genotypes and symptoms could be determined. While
1bA9G3 was only detected in one human patient from the western region, no evidence for C.
hominis case clustering and no seasonal variation could be established using genotypes. To our
knowledge, none of the above gp60 genotype sequences (IbA13G3, IbA9G2, and IbA9G3) has
been previously reported in cattle. C.hominis gp60 IbA10G2 genotype has been detected in
Australia and New Zealand [21,34]. The present genotyping data suggest the existence of
potentially zoonotic C.hominis isolates in calves, since the 3 genotypes mentioned above have
been previously described in human and non-human primates. Genotype IbA13G3 was
reported in wastewaters from a densely populated urban region (Shanghai, China) [40], geno-
type IbA9G2 was reported in humans and genotype IbA9G3 was found in non-human pri-
mates in Kenya and China [41–43]. Such data aim at investigating associations between
genotypes found in cattle and humans from the same geographic region, as reported in India
for C.hominis genotype IdA15G1 found in one calf, and children from the same geographic
region [44].
Conclusions
Present results indicate for the first time that in several geographic regions of France, C.homi-
nis was present in about one fifth of both asymptomatic and symptomatic calves, and exhibited
genotypes likely linked to human infection. Cattle have been considered to be a primary reser-
voir for Cryptosporidium spp. and to play a role in transmitting zoonotic C.parvum organisms
to humans [44,45]. Results of the present study suggest that calves in France also frequently
harbor C.hominis isolates which might be cause of human infections. Further investigations
are aimed at determining whether the source of cattle infections was other livestock or
humans, and whether the transmission was direct or indirect.
Acknowledgments
The authors are grateful to Nikki Sabourin-Gibbs for her help in editing the manuscript.
Author Contributions
Conceptualization: Loïc Favennec.
Data curation: Romy Razakandrainibe, Laetitia Le Goff.
Formal analysis: Romy Razakandrainibe, Loïc Favennec.
Funding acquisition: Loïc Favennec.
Investigation: Romy Razakandrainibe, El Hadji Ibrahima Diawara, Damien Costa, Laetitia Le
Goff, Denis Lemeteil, Gilles Gargala.
Methodology: Romy Razakandrainibe, El Hadji Ibrahima Diawara, Damien Costa, Laetitia Le
Goff, Denis Lemeteil.
Cryptosporidium hominis in calves
PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0006355 March 29, 2018 9 / 12
Project administration: Damien Costa, Gilles Gargala, Loïc Favennec.
Resources: Loïc Favennec.
Supervision: Romy Razakandrainibe, Loïc Favennec.
Validation: Romy Razakandrainibe, Jean Jacques Ballet, Gilles Gargala.
Visualization: Jean Jacques Ballet.
Writing – original draft: Romy Razakandrainibe, Jean Jacques Ballet, Loïc Favennec.
Writing – review & editing: Jean Jacques Ballet, Loïc Favennec.
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