INTRODUCTION
Giardia spp. and Cryptosporidium spp. are protozoan parasites that infect a wide range of vertebrate hosts, including humans, domestic and wild animals (Xiao, Reference Xiao2010; Feng and Xiao, Reference Feng and Xiao2011). Currently, 2 of the 6 species of Giardia are recognized in avian hosts on the basis of the morphology of trophozoites and/or cysts: Giardia ardeae and Giardia psittaci (Ryan and Cacciò, Reference Ryan and Cacciò2013). Likewise, 3 species of Cryptosporidium have been reported in birds on the basis of biological and genetic differences: Cryptosporidium galli, Cryptosporidium baileyi and Cryptosporidium meleagridis. Furthermore, some genetically distinct Cryptosporidium genotypes have recently been described in avian hosts (Ryan, Reference Ryan2010; Ryan et al. Reference Ryan, Fayer and Xiao2014). Among these species/genotypes, only C. meleagridis is of moderate public health significance due to its zoonotic and anthroponotic spread as well as its documented high infectivity rate (Wang et al. Reference Wang, Yang, Cama, Wang, Cabrera, Ortega, Bern, Feng, Gilman and Xiao2014).
Cryptosporidium is considered as an emerging pathogen in the field of avian medicine and is currently one of the most prevalent parasites affecting domestic, caged and wild birds worldwide. Infection of avian flocks with this parasite may lead to important economic losses (Pagès-Manté et al. Reference Pagès-Manté, Pagès-Bosch, Majó-Masferrer, Gómez-Couso and Ares-Mazás2007; Ryan and Xiao, Reference Ryan, Xiao, Fayer and Xiao2008). Most studies usually focus on captive species of commercial or economic interest (Lim et al. Reference Lim, Rohela and Shukri2007; Nakamura et al. Reference Nakamura, Simões, Antunes, da Silva and Meireles2009; Qi et al. Reference Qi, Wang, Ning, Li, Zhang, Jian, Sun and Xiao2011; Quah et al. Reference Quah, Ambu, Lim, Mahdy and Mak2011; Gomes et al. Reference Gomes, Huber, da Silva and do Bomfim2012; Papini et al. Reference Papini, Girivetto, Marangi, Mancianti and Giangaspero2012; Wang et al. Reference Wang, Wang, Zhao, Qi, Ning, Zhang and Xiao2012; Baroudi et al. Reference Baroudi, Khelef, Goucem, Adjou, Adamu, Zhang and Xiao2013; Nguyen et al. Reference Nguyen, Fukuda, Tada, Huynh, Nguyen and Nakai2013), and fewer studies have involved wild birds (Kuhn et al. Reference Kuhn, Rock and Oshima2002; Papazahariadou et al. Reference Papazahariadou, Diakou, Papadopoulos, Georgopoulou, Komnenou and Antoniadou-Sotiriadou2008; Yong et al. Reference Yong, Ambu, Devi and Maung2008; Abreu-Acosta et al. Reference Abreu-Acosta, Foronda-Rodríguez, López and Valladares2009; Plutzer and Tomor, Reference Plutzer and Tomor2009; Sevá et al. Reference Sevá, Funada, Richtzenhain, Guimarães, Souza, Allegretti, Sinhorini, Duarte and Soares2011). Various studies have demonstrated the presence of the zoonotic species Cryptosporidium parvum in avian wildlife, suggesting that birds may play a role in disseminating this parasite (Graczyk et al. Reference Graczyk, Majewska and Schwab2008). On the other hand, studies concerning the presence and genetic identity of Giardia in avian hosts are scarce. Thus far, zoonotic assemblages A and B of Giardia duodenalis (syn. Giardia intestinalis, Giardia lamblia) have been found, particularly in aquatic birds (Kuhn et al. Reference Kuhn, Rock and Oshima2002; Majewska et al. Reference Majewska, Graczyk, Slodkowicz-Kowalsk, Tamang, Jȩdrzejewski, Zduniak, Solarczyk, Nowosad and Nowosad2009).
The Iberian Peninsula is particularly rich in bird life. However, no studies of the presence of Giardia and Cryptosporidium in wild birds have been carried out in this region, with the exception of a clinical case of cryptosporidiosis in Eurasian Scops-Owl (Otus scops) (Molina-López et al. Reference Molina-López, Ramis, Martín-Vázquez, Gómez-Couso, Ares-Mazás, Cacciò, Leiva and Darwich2010). Cryptosporidium spp. have only been described in a few studies of farm birds, such as chickens (Fernández et al. Reference Fernández, Quezada, Gómez, Navarro, Rodríguez and Sierra1990), rheas (Ponce Gordo et al. Reference Ponce Gordo, Herrera, Castro, García Durán and Martínez Díaz2002) and red-legged partridges (Pagès-Manté et al. Reference Pagès-Manté, Pagès-Bosch, Majó-Masferrer, Gómez-Couso and Ares-Mazás2007), or in ornithological gardens (Pérez Cordón et al. Reference Pérez Cordón, Hitos Prados, Romero, Sánchez Moreno, Pontes, Osuna and Rosales2008). The aim of the present study was to detect and molecular characterize Giardia and Cryptosporidium in faecal samples from wild birds in Galicia (Northwest Spain).
MATERIALS AND METHODS
Sample collection and processing
Between February 2007 and September 2009, 433 faecal samples from wild birds were provided by 4 wildlife recovery centres (WRC) located in Galicia (NW Spain). The birds belonged to 64 species in 17 different orders, as follows: 138 were Accipitriformes (31·9%), 125 were Strigiformes (28·9%), 31 were Falconiformes (7·2%), 25 were Passeriformes (5·8%), 23 were Charadriiformes (5·3%), 18 were Columbiformes (4·2%), 13 were Ciconiiformes (3·0%), 13 were Pelecaniformes (3·0%), 13 were Suliformes (3·0%), 7 were Apodiformes (1·6%), 6 were Caprimulgiformes (1·4%), 5 were Procellariiformes (1·2%), 4 were Anseriformes (0·9%), 4 were Galliformes (0·9%), 4 were Gruiformes (0·9%), 3 were Piciformes (0·7%) and 1 was a Coraciiforme (0·2%) (Table 1). At the time of the admission to the WRC, the birds were isolated in individual cages. Faecal droppings were collected from the floor of the cages, stored at 4 °C and sent to the Laboratory of Parasitology, Faculty of Pharmacy, University of Santiago de Compostela for analysis. Data regarding the age, sex or health status of the animals were not provided by the WRC.
Table 1. Prevalence and genotyping results for Giardia and Cryptosporidium detected in free-ranging birds in Galicia (Northwest Spain).
a Raptors.
b Aquatic species.
n.d., not determined.
The samples (0·52 ± 0·29 g) were diluted in 10–20 ml of 0·04-M phosphate buffered saline (PBS) pH 7·2, filtered through 2 sieves (mesh size 150 and 45 μm), shaken with diethyl ether (2:1, v/v) and centrifuged at 1250 g for 15 min at 4 °C. The resulting uppermost 2 layers were carefully removed and discarded, the sediment was washed in PBS by centrifugation at 1250 g for 15 min at 4 °C, and the pellet was resuspended in 500 μl of 0·04-M PBS, pH 7·2.
Detection of Giardia cysts and Cryptosporidium oocysts by epifluorescence microscopy
A direct immunofluorescence antibody test (IFAT) was performed on 50 μl aliquots of the sediments by using the AquaGlo™ G/C Direct test (Waterborne, Inc., New Orleans, LA, USA), according to the manufacturer's instructions. The cysts/oocysts were identified by epifluorescence microscopy (400 × magnification) on the basis of their shape, size and the pattern and intensity of immunofluorescence staining. The intensity of infection by Giardia and/or Cryptosporidium was determined by counting the number of cysts/oocysts in 50 μl of concentrated sample.
Molecular characterization of Giardia spp. and Cryptosporidium spp
Nucleic acids were extracted from the remaining 450 μl of sediment by using the QIAamp® DNA Stool Mini Kit (QIAGEN®, Hilden, Germany). The extraction was carried out according to the manufacturer's instructions and the DNA was stored at −20 °C until use.
A two-step nested-PCR technique was used to amplify a ~175-bp fragment of the small subunit ribosomal gene (SSU-rDNA) and of a ~315-bp fragment encompassing the ITS1-5·8S-ITS2 region in the ribosomal unit of Giardia (Read et al. Reference Read, Walters, Robertson and Thompson2002; Cacciò et al. Reference Cacciò, Beck, Almeida, Bajer and Pozio2010). For Cryptosporidium, a two-step nested-PCR technique was used to amplify a ~587-bp fragment of the SSU-rDNA gene (Ryan et al. Reference Ryan, Xiao, Read, Zhou, Lal and Pavlasek2003); moreover, a protocol for amplifying a ~325-bp fragment of the HSP-70 gene (Morgan et al. Reference Morgan, Monis, Xiao, Limor, Sulaiman, Raidal, O'Donoghue, Gasser, Murray, Fayer, Blagburn, Lal and Thompson2001) was tested on 8 samples. Positive and negative controls were included in all PCR experiments. The PCR products were subjected to electrophoresis on 2% agarose/ethidium bromide gels.
Positive PCR products were purified using the QIAquick® PCR Purification Kit (QIAGEN®, Hilden, Germany) and sequenced in both directions using the ABI PRISM® BigDye™ Terminator Cycle Sequencing Kit (Applied Biosystems®, Life Technologies™, Carlsbad, CA, USA) according to the manufacturer's instructions. The sequencing reactions were analysed using the ABI PRISM® 3100 automatic sequencer (Applied Biosystems®) and sequences were assembled using SeqMan™ 7·0 (DNASTAR®, Madison, WI, USA) and BioEdit 7·2·3 (©1997–2013 Tom Hall, Ibis Therapeutics, Carlsbad, CA, USA) software. The resulting sequences were compared with those deposited in GenBank® (National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD, USA) using the public web interface of the BLAST® 2·2·29 program (http://blast.ncbi.nlm.nih.gov/Blast.cgi, National Centre for Biotechnology Information).
Nucleotide sequence accession numbers
Representative nucleotide sequences of the isolates analysed in this study have been deposited in the GenBank® database under accession numbers KJ939300–KJ939308.
RESULTS
Prevalence
By IFAT and PCR methods, Giardia spp. cysts and Cryptosporidium spp. oocysts were detected in 9 (2·1%) and 36 (8·3%) of the 433 faecal samples, respectively, being observed both pathogens in 6 samples. All positive samples contained small number of parasitic forms, i.e. 1–5 cysts/oocysts per 50 μl of concentrated faecal sample (Table 1).
Among 433 faecal samples analysed, 294 samples belonged to 20 raptor species (see Table 1). Cryptosporidium spp. oocysts were detected in 21 of these faecal samples (7·1%) from 9 species (45·0%): Buteo buteo (7/84, 8·3%), Strix aluco (1/55, 1·8%), Tyto alba (6/49, 12·2%), Accipiter nisus (1/19, 5·3%), Falco tinnunculus (1/13, 7·7%), Athene noctua (2/8, 25·0%), Pernis apivorus (1/7, 14·3%), Milvus migrans (1/4, 25·0%) and the sample from Parabuteo unicinctus (100%). Giardia spp. cysts were observed only in 1 sample from B. buteo (1·2%) and in other from T. alba (2·0%) (Table 1).
On the other hand, 70 (16·1%) of the faecal samples analysed in the present study belonged to aquatic species and of those, 2·8 and 5·7% were positive for Giardia and Cryptosporidium, respectively. Cryptosporidium spp. oocysts were detected in faecal samples from Ciconia ciconia (1/13, 7·7%) and Morus bassanus (1/13, 7·7%), and both Cryptosporidium and Giardia were detected in samples from Anas platyrhynchos (2/4, 50·0%) (Table 1).
In the order Passeriformes, only some species of the family Corvidae were positive (Corvus corone, Corvus monedula, Garrulus glandarius and Pica pica), showing an overall prevalence of 12·5% for Giardia and 37·5% for Cryptosporidium (Table 1).
Moreover, Giardia and/or Cryptosporidium were also detected in Caprimulgus europaeus, Coturnix coturnix, Streptopelia turtur, Apus apus and Columba palumbus (Table 1).
PCR and sequencing analyses
For Giardia spp., 8 samples were PCR-positive. Partial sequences of the SSU-rDNA locus revealed the presence of G. duodenalis assemblage A (KJ027407) in 3 samples (A. platyrhynchos, B. buteo and C. europaeus), assemblage B (JX972180) in 1 sample (C. coturnix), assemblage D (KJ027400) in 1 sample (G. glandarius) and assemblage F (AB569366) in another sample (A. platyrhynchos). Moreover, the results obtained for the ITS1-5·8S-ITS2 region revealed the presence of assemblage B (GU126436) in 2 more samples (P. pica and S. turtur) and confirmed those obtained for the SSU-rDNA locus, except in 1 sample (A. platyrhynchos) in which the sequencing results obtained for both loci were inconsistent (Table 1).
With respect to Cryptosporidium spp., partial sequences of the SSU-rDNA and HSP70 genes were obtained in 4 and 3 isolates, respectively. Sequencing analyses revealed that 6 sequences obtained from A. nisus, B. buteo, C. corone, M. migrans and P. apivorus, were identical to sequences of the SSU-rDNA and HSP70 genes of C. parvum deposited in GenBank® (KJ569798), and another one (S. turtur) was identical to sequence KC734572 corresponding to C. meleagridis (Table 1).
DISCUSSION
This is one of the largest parasitological studies involving detection of Giardia and Cryptosporidium carried out on wild birds worldwide and the first performed in the Iberian Peninsula, covering 42% of the bird species described in the region of Galicia (approximately 152 bird species are known to inhabit this area) (Anonymous, 2014). Little is known about the presence of Giardia and/or Cryptosporidium in wild birds. Most previous studies have focused on species that play a potential role in the mechanical transmission of these enteropathogens, such as aquatic and migratory birds (Kuhn et al. Reference Kuhn, Rock and Oshima2002; Graczyk et al. Reference Graczyk, Majewska and Schwab2008; Majewska et al. Reference Majewska, Graczyk, Slodkowicz-Kowalsk, Tamang, Jȩdrzejewski, Zduniak, Solarczyk, Nowosad and Nowosad2009; Plutzer and Tomor, Reference Plutzer and Tomor2009).
The overall prevalence of Giardia in the samples was 2·1%, as determined by IFAT and PCR analysis. This value is lower than in previous studies carried out in several species of wild birds, in which the prevalence ranged from 5·0 to 28·0% (Kuhn et al. Reference Kuhn, Rock and Oshima2002; Papazahariadou et al. Reference Papazahariadou, Diakou, Papadopoulos, Georgopoulou, Komnenou and Antoniadou-Sotiriadou2008; Majewska et al. Reference Majewska, Graczyk, Slodkowicz-Kowalsk, Tamang, Jȩdrzejewski, Zduniak, Solarczyk, Nowosad and Nowosad2009; Plutzer and Tomor, Reference Plutzer and Tomor2009). The prevalence of Cryptosporidium was 8·3%, which is consistent with the data reported in the international literature: 5·8% in Hungary (Plutzer and Tomor, Reference Plutzer and Tomor2009), 6·3% in Australia (Ng et al. Reference Ng, Pavlasek and Ryan2006), 7·2% in USA (Ziegler et al. Reference Ziegler, Wade, Schaaf, Stern, Nadareski and Mohammed2007) and 13·0% in Greece (Papazahariadou et al. Reference Papazahariadou, Diakou, Papadopoulos, Georgopoulou, Komnenou and Antoniadou-Sotiriadou2008).
Most of the faecal samples examined in the present study corresponded to raptor species (68·0%). The prevalence of Giardia in these species was 0·7% and that of Cryptosporidium, 7·1%. To our knowledge, Giardia spp. is described for the first time in the Barn Owl (T. alba) and G. duodenalis assemblage B in the Common Buzzard (B. buteo). Likewise, Cryptosporidium spp. is described for the first time in the Tawny Owl (S. aluco), Little Owl (A. noctua), Common Kestrel (F. tinnunculus) and Harris Hawk (P. unicinctus). In addition, C. parvum was found for the first time in the Common Buzzard (B. buteo), Sparrowhawk (A. nisus), Honey Buzzard (P. apivorus) and Black Kite (M. migrans).
Raptors are located at the top of the food chain and detection of both parasite species, especially Cryptosporidium, in this group indicates that the parasites are present in the environment. Food sources play a major role in the transmission of cryptosporidiosis in birds of prey, because most raptor species feed on small mammals, other birds or fish and reptiles, which may be infected (Ryan, Reference Ryan2010).
With respect to aquatic species, Cryptosporidium spp. oocysts were observed in one faecal sample from a White Stork (C. ciconia). Both C. parvum and G. duodenalis have been found in this host species in Poland (Majewska et al. Reference Majewska, Graczyk, Slodkowicz-Kowalsk, Tamang, Jȩdrzejewski, Zduniak, Solarczyk, Nowosad and Nowosad2009). Cryptosporidium spp. oocysts were also detected in a sample of a Northern Gannet (Morus bassanus) in the present study, which as far as we know constitutes the first description of Cryptosporidium in this species and in the order Suliformes.
Moreover, G. duodenalis assemblages A and B cysts and Cryptosporidium sp. oocysts were detected in 2 of the 4 samples of Mallard Duck (A. platyrhynchos), suggesting that there is a high chance that this species will act as a carrier of these pathogens (Majewska et al. Reference Majewska, Graczyk, Slodkowicz-Kowalsk, Tamang, Jȩdrzejewski, Zduniak, Solarczyk, Nowosad and Nowosad2009; Plutzer and Tomor, Reference Plutzer and Tomor2009). These cases of co-detection are according to the findings made by other authors (Kuhn et al. Reference Kuhn, Rock and Oshima2002; Plutzer and Tomor, Reference Plutzer and Tomor2009). Ducks are more likely than other birds to become infected from water contaminated with faecal material of human or domestic animal origin, because of their strict aquatic habit (Cacciò et al. Reference Cacciò, Thompson, McLauchlin and Smith2005).
Environmental contamination with human and domestic-animal faecal material is recognized as a potential pathogen pathway for wildlife infections with zooanthroponotic parasites such as Giardia and Cryptosporidium (Appelbee et al. Reference Appelbee, Thompson and Olson2005; Kutz et al. Reference Kutz, Thompson, Polley, Ortega-Pierres, Cacciò, Fayer, Mank, Smith and Thompson2009; Thompson, Reference Thompson2013). Indeed, previous studies of environmental samples, wild otters, bivalve molluscs and freshwater macroinvertebrates in Galicia provided evidence that streams in this region are widely contaminated with Giardia and Cryptosporidium (Gómez-Couso et al. Reference Gómez-Couso, Méndez-Hermida, Castro-Hermida and Ares-Mazás2005, Reference Gómez-Couso, Méndez-Hermida, Castro-Hermida and Ares-Mazás2006; Méndez-Hermida et al. Reference Méndez-Hermida, Gómez-Couso, Romero-Suances and Ares-Mazás2007; Reboredo-Fernández et al. Reference Reboredo-Fernández, Prado-Merini, García-Bernadal, Gómez-Couso and Ares-Mazás2014). Moreover, the rate of contamination with these enteropathogens in Galician water bodies has been positively correlated with levels of livestock activity (Castro-Hermida et al. Reference Castro-Hermida, García-Presedo, González-Warleta and Mezo2010). As livestock constitute a potential source of environmental contamination with these pathogens, other animals that live on or close to farms are at risk of being infected (Ziegler et al. Reference Ziegler, Wade, Schaaf, Stern, Nadareski and Mohammed2007; Castro-Hermida et al. Reference Castro-Hermida, García-Presedo, Almeida, González-Warleta, Correia Da Costa and Mezo2009; Gracenea et al. Reference Gracenea, Gómez and Ramírez2011). Moreover, different studies have shown that invertebrates such as snails can act as bioindicators of soil contamination (Neira et al. Reference Neira, Muñoz, Stanley, Gosh and Rosales2010) and that non-biting synanthropic flies can transport infectious oocysts of C. parvum in their digestive tracts and/or on external surfaces in a transmission route that may occur in rural areas (Clavel et al. Reference Clavel, Doiz, Morales, Varea, Seral, Castillo, Fleta, Rubio and Gómez-Lus2002; Conn et al. Reference Conn, Weaver, Tamang and Graczyk2007).
As it has mentioned previously, in birds belonging to the order Passeriformes, all the positive samples were included in the family Corvidae. The prevalence of Cryptosporidium was 37·5% in Corvidae, which is very similar to the prevalence of 33·9% reported for these birds in Malaysia (Yong et al. Reference Yong, Ambu, Devi and Maung2008). In the same way, the prevalence of Giardia (13·0%) is also relatively high for this family of birds. Moreover, zoonotic genotypes/species of both parasites were identified in these hosts. Thus, C. parvum was identified in a Carrion Crow (C. corone) and in a Eurasian Magpie (P. pica). C. parvum has been reported in C. corone in Poland (Majewska et al. Reference Majewska, Graczyk, Slodkowicz-Kowalsk, Tamang, Jȩdrzejewski, Zduniak, Solarczyk, Nowosad and Nowosad2009); however, to our knowledge, this is the first time that C. parvum has been detected in P. pica, although C. baileyi has been described in this species in China (Qi et al. Reference Qi, Wang, Ning, Li, Zhang, Jian, Sun and Xiao2011). G. duodenalis assemblage B was found in the Eurasian Magpie (P. pica) constituting the first description of this species/assemblage of Giardia in this avian host. Also, G. duodenalis assemblage D (canine genotype) was identified in the faecal sample from a Eurasian Jay (G. glandarius). This is the first time that this assemblage of Giardia has been detected in birds. Corvids are widely distributed and are very common in urban landscape, and they may therefore constitute an important source of transmission of the parasite forms (Yong et al. Reference Yong, Ambu, Devi and Maung2008).
Interestingly, some other species were positive for Giardia and/or Cryptosporidium. G. duodenalis assemblage B and Cryptosporidium sp. oocysts were detected in a faecal sample from Common Quail (C. coturnix). This is the first time that G. duodenalis assemblage B has been found in this host. However, Cryptosporidium was previously reported in a study carried out in farm quails in China, in which C. baileyi was more prevalent than C. meleagridis (Wang et al. Reference Wang, Wang, Zhao, Qi, Ning, Zhang and Xiao2012). A faecal sample from the European Nightjar (C. europaeus) contained cysts of the zoonotic G. duodenalis assemblage A as well Cryptosporidium sp. oocysts. Moreover, Cryptosporidium sp. oocysts were also found in Common Swift (A. apus). To our knowledge, this the first report of Giardia in the order Caprimulgiformes and of Cryptosporidium in the orders Apodiformes and Caprimulgiformes.
The presence of C. meleagridis in the Turtle Dove (S. turtur) extends the range of this enteropathogen in avian hosts and raises questions about the potential zoonotic transmission of cryptosporidiosis from doves to humans, as C. meleagridis is an emerging human pathogen and constitutes the third most common Cryptosporidium parasite in humans in some geographical locations (McLauchlin et al. Reference McLauchlin, Amar, Pedraza-Díaz and Nichols2000; Matos et al. Reference Matos, Alves, Xiao, Cama and Antunes2004; Cama et al. Reference Cama, Bern, Roberts, Cabrera, Sterling, Ortega, Gilman and Xiao2008).
Birds are important components of the ecosystem because they act as pollinators, agents of seed dispersal and natural pest controllers, as well as scavengers and environmental cleaners. They are therefore often used as environmental health indicators (Carignan and Villard, Reference Carignan and Villard2002; Gregory and Strien, Reference Gregory and Strien2010). Moreover, anthropogenic alterations to the environment caused by modern agricultural practices and urban sprawl may impact wildlife health through stress and exposure to introduced pathogens (Daszak et al. Reference Daszak, Cunningham and Hyatt2001; Kutz et al. Reference Kutz, Thompson, Polley, Ortega-Pierres, Cacciò, Fayer, Mank, Smith and Thompson2009). A better understanding of how bird populations may be affected by infections and the potential impact that pathogens have on wild populations is therefore required. Finally, the results obtained in this study demonstrate the wide dissemination of Giardia and/or Cryptosporidium in wild birds. Further studies are needed to confirm whether the presence of the cysts/oocysts in the wild bird species is due to mechanical transport or to a true infection.
ACKNOWLEDGEMENTS
The authors thank the personnel and volunteers at the four Wildlife Recovery Centres of Galicia (Xunta de Galicia) for collecting the samples used in this study.
FINANCIAL SUPPORT
This study was funded by the Spanish Ministry of Education and Science (grant no. CGL-2007-60656) and by the Department of Culture, Education and University of the Autonomous Government of Galicia through the Program of Consolidation and Structuring of Competitive Research Units in Consolidated Technological Centres from Galicia (grant no. GPC2014/069).
CONFLICT OF INTEREST
None of the authors have any commitments, consultancies or contracts that could be considered as conflicts of interest with respect to this study.
