The first study of molecular prevalence and species characterization of Cryptosporidium in free-range chicken ( Gallus gallus domesticus ) from Brazil Primeiro estudo de prevalência molecular e caracterização de espécies de Cryptosporidium em galinhas ( Gallus gallus domesticus ) colonial / caipiras

Rearing free-range chicken is based on grazing feeding patterns, and these animals could be potential environmental contaminants of Cryptosporidium oocysts for humans and other animals. Therefore, the present study aimed to evaluate the molecular prevalence of Cryptosporidium spp. in free-range chickens from Brazil. A total of 351 fecal samples from chickens were examined from 20 farms. For detection of Cryptosporidium spp., 18S rRNA gene fragments were amplified using a nested PCR reaction. Positive samples were sent for sequencing. The overall prevalence of Cryptosporidium was 25.6% (95% CI = 21.2% 30.6%). Sequencing of the amplified fragments allowed for the identification of three species: C. meleagridis in 57 (62.6%), C. baileyi in 15 (16.4%), C. parvum in 3 (3.2%) samples, and a new Cryptosporidium genotype (C. genotype BrPR1) in 3 (3.2%) samples. Cryptosporidium genotype BrPR1 has not yet been classified as a species, and its host spectrum is not known. Cryptosporidium, including zoonotic species, exists at a high prevalence in free-range chickens within the region studied.

Cryptosporidiosis can be acquired through several routes, including person-to-person contact, ingestion of contaminated food, drinking, and recreational water, as well as contact with companion and farm animals (GRIFFITHS, 1998;MAcKENZIE et al., 1994;SHIELDS et al., 2008;SMITH et al., 2007;TANGERMANN et al., 1991).Therefore, because the production of free-range chickens is based on grazing feeding patterns, these animals are potential biological vectors for the environmental contamination of oocysts (THAMSBORG et al., 1999).
In considering the demands of consumers in industrialized nations who desire meat raised without chemical agents in welfare-friendly and organic systems, there is an association with the popular concept that animals raised under free-range conditions are sanitary and safer than those reared indoors (WANG et al., 2009).However, considering host-pathogen interactions, outdoor animals may have inherently more risk to be infected with zoonotic parasites or act as infection sources for several animals and humans (THAMSBORG et al., 1999).Despite this perception, there is no study focusing on characterizing Cryptosporidium spp.from free-range chicken production systems.
Therefore, the present study is aimed at evaluating the molecular prevalence of Cryptosporidium spp. in chickens raised under free-range systems in southern Brazil.

Study population and sampling
Fecal samples from 351 free-range chickens, aged between 90 and 190 days, were collected between August 2011 to February 2014.Samples were acquired from 20 farms located in 10 different cities from the northern Parana state, southern Brazil.All animals were raised in free-range system and slaughtered at farms for human consumption.Conglomerate sampling was carried out proportional to the number of animals slaughtered in the period.The sample size was calculated assuming a prevalence of 35% (BAROUDI et al., 2013) with a confidence level of 95% and efficacy of 5% by Epi Info 3.5.4.A questionnaire including several factors, such as, presence of other animals, water source, presence of fence, and population density, was applied to all farmers to determine possible risk association.
Fecal samples were collected from the distal portion of the rectum and stored at -20 °C until DNA extraction.This study was approved by Ethics Committee of the Universidade Estadual de Londrina (Protocol n° 206/12).

DNA extraction and nested-PCR
DNA extraction was performed using the NucleoSpin  Tissue Kit (Macherey-Nagel, Düren, Germany) according to the manufacturer's instructions.For detection of Cryptosporidium spp., 18S rRNA gene fragments were amplified using a nested PCR (n-PCR) reaction using primers previously described by (XIAO et al., 1999).The first PCR reaction consisted of 9.25 μL of autoclaved ultrapure water, 2.5 μL of buffer 10X concentrate, 0.5 μL dNTP (10 mM), 1.25 μL MgCl 2 (50 mM), 0.5 μL of each primer 20 pmol/uL (forward and reverse), Platinum™ Taq DNA Polymerase (Invitrogen™, Carlsbad, CA, USA) 0.25 μL (5 U/μL), and 2.0 μL of genomic DNA, reaching a final volume of 25 μL.To reduce unused primers from the primary PCR, the first round of PCR products was diluted 1:5 with dH 2 O.The conditions for each second-round reaction were identical to the first.Initial heating was carried out at 95 °C for 5 minutes followed by 35 cycles, each cycle consisting of 94 °C for 45 seconds (denaturation), 55 °C for 45 seconds (annealing) and 72 °C for 60 seconds (extension).One final extension step at 72 °C for 5 minutes was included.The PCR products were subjected to electrophoresis on 1.5% agarose gel (Ultrapure™ Agarose, Invitrogen™, Carlsbad, CA, USA) stained with SYBR  Safe (Invitrogen, Carlsbad, CA, USA), visualized by ultraviolet light and photodocumented using program LPix Image ST (Loccus Biotecnologia, Cotia, SP, Brazil).Negative and positive controls consisting of ultrapure water and C. muris DNA respectively, were included in each batch.

Sequence analysis of SSU rRNA gene
The positive PCR products were purified from agarose gel using a PureLink™ Quick Gel Extraction Kit (Invitrogen™, Carlsbad, CA, USA) and quantified using a Picodrop™ Spectrophotometer (Invitrogen™, Carlsbad, CA, USA).The sequencing was performed using the BigDye Terminator™ v.3.1 Cycle Sequencing kit (Applied Biosystems, Carlsbad, USA) with second reaction forward and reverse primers using a 3500 Genetic Analyzer (Applied Biosystems™, Carlsbad, USA) according to the manufacturer's instructions.The obtained sequences were analyzed in Chromas Lite v.2.6.1, and the contigs were generated in BioEdit (Biological Sequence Alignment Editor) v.7.2.5 (HALL, 1999).dentity was verified with sequences deposited in the GenBank © using BLAST algorithm (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and by manual alignment using the program BioEdit (Biological Sequence Alignment Editor).Phylogenetics analysis were carried out using neighbor-joining method with bootstrap values derived from 1000 replicates in MEGA v7.0.14, evolutionary distances were calculated by Kimura 2-parameter method.When uncommon Cryptosporidium species were identified on 18S n-PCR, we performed n-PCR for actin and hsp70 genes and posterior sequencing at the same conditions as described above for 18S rRNA n-PCR (SULAIMAN et al., 2000(SULAIMAN et al., , 2002)).

Statistical analysis
Association of the risks between variables and positivity to Cryptosporidium spp. was performed using the chi-square test or Fisher's exact test, and a 5% significance level was adopted.The measure of association Odds Ratio was calculated with a 95% confidence interval.The statistical package Epi 3.5.4.(DEAN et al., 2011) was used for analysis.
The association of risk between the positivity from the 18S rRNA n-PCR, and characteristics of the raising system, such as water source, population density, and presence of livestock, showed no significant differences in all variables analyzed (p>0.06).

Discussion
To the best of the author's knowledge, this study shows, for the first time, the molecular prevalence and characterization of Cryptosporidium spp. in fecal samples from free-range chickens.Here,   the 18S rRNA n-PCR reactions were positive for Cryptosporidium spp. in 91 (25.9%) of 351 fecal samples There are few reports of Cryptosporidium molecular characterization from chickens in Brazil (HUBER et al., 2007;NAKAMURA et al., 2009) and worldwide (HSU et al., 2008;NG et al., 2006;SILVERLÅS et al., 2012;WANG et al., 2014), however, none of the previously studies reported studies worked with free-range chickens.In Brazil, Huber et al. (2007) and Nakamura et al. (2009) worked with chickens from indoor public markets and chickens under captivity, respectively.The authors from other regions of world isolated Cryptosporidium spp.from commercial poultry farms, layer chickens, and pet shops.
In the present study, all positive samples tested at 18S rRNA n-PCR were submitted to sequencing, and 62.6%, 16.4%, 3.2%, and 3.2% were detected as C. meleagridis, C. baileyi, C. parvum, and a new genotype named C. genotype BrPR1, respectively.Similar results were observed by Baroudi et al. (2013), who studied 90 broiler chickens from 23 farms in Algeria; which found by 18S rRNA gene 28.9% of C. meleagridis, and 5.5% of C. baileyi.Conversely, C. baileyi had been previously reported as the most common Cryptosporidium species in chickens (FAYER, 2010).There are few reports in Brazil on the occurrence of C. baileyi infecting domestic chickens (CARDOZO et al., 2005;MEIRELES & FIGUEIREDO, 1992).Herein, we observed two clusters in 18S rRNA gene phylogenetic tree from C. baileyi, this can be explained by genetic diversity observed in sequences of GenBank.
In a previous study by Nakamura et al. (2009), C. meleagridis was found in one sample of a domestic chicken in Brazil.Previous studies performed outside Brazil affirm that infections by C. baileyi, C. galli and other parasite genotypes are present in several avian cases.While the number of reports of infection by C. meleagridis was low (SRÉTER & VARGA, 2000;WANG et al., 2014), Ng et al. (2006) did not find this species in 430 avian fecal samples, and described that C. meleagridis infection has low prevalence in avian hosts.This finding is the opposite what we observed in our results.C. meleagridis has a wide host range, and it is the third most prevalent species infecting humans (CAMA et al., 2003;ELWIN et al., 2012;GATEI et al., 2003;GUYOT et al., 2001;LEONI et al., 2006;PEDRAZA-DÍAZ et al., 2000;XIAO, 2010).This species is considered an important human pathogen, accounting for several reports of human infection (DARABUS, 1997;DARABUS & OLARIU, 2003;SRÉTER & VARGA, 2000;XIAO & FENG, 2008).A study in England found C. meleagridis in 0.9% of 2,414 human cases (LEONI et al., 2006).Sequence analysis of the 18S rRNA gene and hsp70 loci has been used to provide evidence of zoonotic transmission of C. meleagridis from chickens to humans on a Swedish farm (SILVERLÅS et al., 2012).
C. parvum was detected in three animals from two farms; therefore, animals could be acting as an environment spread host, considering that viable oocysts of C. parvum can pass undamaged through the digestive system of several avian hosts (GRACZYK et al., 1996(GRACZYK et al., , 1997)).Oocysts of C. parvum were detected in feces of wild Canada geese (Branta canadensis) (GRACZYK et al., 1998), and it was demonstrated experimentally to cause a weak tracheal infection in 1-and 7-day-old chickens (LINDSAY et al., 1987).However, molecular detection of C. parvum in feces of chickens is uncommon (BAROUDI et al., 2013;HUBER et al., 2007;NAKAMURA et al., 2009;NG et al., 2006) In the present work, sequencing of 18S rRNA amplicons allowed the identification of a new Cryptosporidium genotype in three samples, and consequently we performed actin and hsp70 n-PCR and sequencing of the identified isolates.The results showed identities of these genes with C. bovis between 95% to 98%.Thus, because of such genetic distance with C. bovis, C. ryanae, and C. xiaoi we named this new genotype as Cryptosporidium genotype PR1.
There were no significant differences in all variables studied here that could be associated to cryptosporidiosis.It is known that water can act as a transmission route of oocysts to birds and acts as a major source of oocysts and human outbreaks in developed countries, including North America, the United Kingdom, and Japan (FAYER, 2004).In the present work, although no significance was observed, the farms that were provided with deep water showed a reduction in positive animals (20%) when compared with surface water (30%).

Conclusion
Cryptosporidium, including zoonotic species, exists at a high prevalence in free-range chickens.A high prevalence of farms positive for Cryptosporidium, mainly C. meleagridis, shows us the importance of the chicken as an environmental indicator for this zoonotic protozoa.Other studies must be performed to better elucidate the role of chickens in the dissemination of Cryptosporidium species that infect cattle.We described a new genotype of Cryptosporidum which was named C. genotype BrPR1.

Figure 1 .
Figure 1.Phylogenetic relationships between Cryptosporidium spp.found in this study by 18S rRNA and selected Cryptosporidium spp.from GenBank inferred by Neighbor-joining (ML).The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches.Cryptosporidium galli and C. proliferans were used as outgroup.Isolates obtained in the present study were marked as CryBrFRCh# (Cryptosporidium Brazil Free Range Chickens and the number of respective sample).*New genotype (Cryptosporidium genotype BrPR1).

Figure 2 .
Figure 2. Phylogenetic relationships between Cryptosporidium spp.found in this study by partial DNA sequences of actin (A), and HSP70 (B) loci and selected Cryptosporidium spp.from GenBank inferred by model Kimura 2-parameter.The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches.Cryptosporidium galli was used as outgroup.Isolates obtained in the present study were marked as CryBrFRCh# (Cryptosporidium Brazil Free Range Chickens and the number of respective sample).*New genotype (Cryptosporidium genotype BrPR1).

Table 1 .
Results of positivity in 18S rRNA nested-PCR for Cryptosporidium from fecal samples in free-range chickens from the northern Paraná state, Brazil.
*NAQ: Not sequenced due to low quality of DNA.