Phylogenetic analysis of nasal avian schistosomes (Trichobilharzia) from aquatic birds in Mazandaran Province, northern Iran

    Phylogenetic analysis of nasal avian schistosomes (Trichobilharzia) from aquatic birds in Mazandaran province, northern Iran Mahdi Fakhar, Maryam Ghobaditara, Sara V. Brant, Mehdi Karamian, Shaban Gohardehi, Reza Bastani PII: DOI: Reference:

S1383-5769(15)00193-2 doi: 10.1016/j.parint.2015.11.009 PARINT 1433

To appear in:

Parasitology International

Received date: Revised date: Accepted date:

24 July 2015 10 November 2015 24 November 2015

Please cite this article as: Fakhar Mahdi, Ghobaditara Maryam, Brant Sara V., Karamian Mehdi, Gohardehi Shaban, Bastani Reza, Phylogenetic analysis of nasal avian schistosomes (Trichobilharzia) from aquatic birds in Mazandaran province, northern Iran, Parasitology International (2015), doi: 10.1016/j.parint.2015.11.009

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Phylogenetic analysis of nasal avian schistosomes (Trichobilharzia)

RI

PT

from aquatic birds in Mazandaran Province, Northern Iran

a

NU

MA

Shaban Gohardehia, Reza Bastania

SC

Mahdi Fakhara, Maryam Ghobaditaraa, Sara V. Brantb,c, Mehdi Karamiand,*,

Molecular and Cell Biology Research Center, Department of Parasitology and Mycology, School of

Medicine, Mazandaran University of Medical Sciences, Sari, Iran

D

Museum of Southwestern Biology Division of Parasites, Department of Biology, University of New

Mexico, Albuquerque, USA d

TE

b,c

AC CE P

Cellular and Molecular Research Center, Department of Microbiology, Birjand University of Medical

Sciences, Birjand, Iran

*Corresponding author: Mehdi Karamian

Tel: 00985632433004 Fax: 00985632433004 E-mail: [email protected]

1

ACCEPTED MANUSCRIPT ABSTRACT Nasal schistosomes are trematodes in the family Schistosomatidae, many members of which are

PT

causative agents of human cercarial dermatitis (HCD). Little is known about the species diversity

RI

and distribution of nasal dwelling schistosomes of water birds, particularly in countries outside

SC

of Europe; even less is known in countries like Iran. Nasal schistosomes are of particular interest since these species migrate via the central nervous system to the nasal cavity once they penetrate

NU

their host. Thus, there must be efforts to determine the incidence of HCD due to nasal schistosomes. HCD outbreaks are reported seasonally in Mazandaran Province, northern Iran, an

MA

area well known for rice cultivation leading to increased person contact with water and infected snails. Such places include favorable habitat for both domestic ducks year round, and wild

TE

D

migratory ducks in the winter through spring. Recent reports have detected the presence of both nasal and visceral schistosomes in ducks in this area but with little species characterization. In

AC CE P

this study, we examine a diversity of aquatic birds to determine the distribution, prevalence and bird host use of nasal schistosomes. We apply for the first time a molecular identification and phylogenetic analysis of these schistosomes. From 2012 to 2014, the nasal cavity of 508 aquatic birds from Mazandaran Province were examined that included species in Anseriformes, Gruiformes, Charadriiformes and Phoenicopteriformes. Nasal schistosomes were found in 45 (8.9 %) birds belonging to Anseriformes (Anas platyrhynchos and A. clypeata). Phylogenetic analysis of the nuclear internal transcribed spacer 1 rDNA and the mitochondrial cytochrome oxidase1 gene of isolated eggs revealed that all samples grouped in a sister clade to the European Trichobilharzia regenti. However, Trichobilharzia from this study were more similar to a unique haplotype of Trichobilharzia, isolated from the nasals of an A. clypeata in France. The genetic

2

ACCEPTED MANUSCRIPT and phenotypic differences between the species found herein and T. regenti from Europe, may

PT

prove with additional data to be a distinct species of Trichobilharzia.

RI

1. Introduction

SC

Schistosomes (Schistosomatidae) are blood trematodes that infect aquatic snails as intermediate hosts and birds and mammals as definitive hosts. Mammalian species are well

NU

known for causing the most devastating helminth disease in people, schistosomiasis (WHO

MA

2013). Most of the species diversity within Schistosomatidae is found in avian definitive hosts and much of that diversity is found the venous system (liver, mesenteric veins), and less

D

commonly in the nasal tissue and arterial system habitat of their hosts [1]. Though avian

AC CE P

in the Eastern Hemisphere.

TE

schistosomes are distributed globally, species inhabiting in the nasal cavity have been found only

Within the avian schistosomes, there are 8 described species known to occur in the nasal tissue, 5 from Africa, 2 from Australia, and one from Eurasia. Trichobilharzia regenti [2,3] is the only nasal species for which there are well described morphological, genetic, pathological, and life cycle features. The life cycle of the nasal schistosomes in Australia have been detailed [4,5], and the species in Africa have been reported once to a few times [6,7]. However, in the absence of substantiating genetic data, it is difficult to know the species and phylogenetic relationships among them. This question of relationships is of interest since these regions are connected by the migratory flyways of the avian hosts. Other than Western Europe, limited investigations have included avian schistosomes in Eurasia, most of it conducted in China and Russia [8,9,10,11,12].

3

ACCEPTED MANUSCRIPT Both mammalian and avian schistosomes are the causative agents of human cercarial dermatitis (HCD), but avian schistosomes species are most often implicated [13]. HCD is an

PT

allergic skin reaction at the penetration site of the larvae or schistosome cercariae [13] in both

RI

freshwater and marine environments. These cercariae do not mature and usually die in the skin. Studies on the pathology of an avian nasal schistosome, Trichobilharzia regenti, have shown that

SC

the larvae migrate via the central nervous system to reach the nasal tissue [14]. In a mammalian

NU

host, mice infected experimentally with T. regenti have shown that the worms attack the central nervous system and create neuromotor disorders such as leg paralysis [14,15]. The potential for neurological

symptoms

occurring

in

MA

similar

humans,

particularly

the

young

and

immunocompromised, cannot be ignored. Thus, there is a need to know if there are nasal

TE

D

schistosomes responsible for outbreaks of HCD, particularly in areas where people spend a majority of their time in water in contact with these parasites. Thus our broader goal is to

AC CE P

describe the species and life cycles of schistosomes causing HCD in northern Iran, and this paper will focus on defining a species found in nasal cavities of birds [16]. Until recently, almost nothing was known about avian schistosomes and HCD in the Middle East, much of which includes areas at high risk of HCD. Only recent investigations in Iran have contributed much of what we know today [17,18,19,20]. In a study in northern Iran, Mazandaran Province, where HCD has been well established [21], infections with a nasal species of Trichobilharzia in the duck A. clypeata was confirmed by molecular methods [19]. That work motivated this study to include a more rigorous survey of nasal schistosome occurrence. Northern Iran remains a place of interest for HCD, as it is a major stopover for migratory birds and has a high incidence of HCD, particularly in rice fields [22]. Investigations continue to establish the prevalence and species of mammalian (domestic animals used for aquiculture) 4

ACCEPTED MANUSCRIPT versus avian (large populations of migratory birds) schistosomes, since both hosts are present and cause HCD [22]. In the present study, efforts were made to determine the prevalence and

PT

distribution of nasal avian schistosomiasis in Mazandaran Province in species of wild migratory

RI

aquatic birds.

SC

2. Materials and methods

NU

2.1. Study area and sampling

The study was conducted in two main wintering areas for migratory birds in Mazandaran

MA

province (Sari and Fereydun kenar; 36.6544 N; 52.4928 E) in northern Iran to determine the distribution of nasal schistosomes in bird hosts. From December 2012 to February 2014, a total

TE

D

of 508 heads of aquatic birds were collected. The birds belonged to 10 species from four orders (see Table 1): Anseriformes—Anas platyrhynchos (mallard), A. clypeata (northern shoveler), A.

duck),

Anser

AC CE P

crecca (common teal), A. acuta (northern pintail), A. strepera (gadwall), Aythya fuligula (tufted anser

(graylag

goose);

Gruiformes

—Fulica

atra

(common

coot);

Charadriiformes— Tringa ochropus (green sandpiper); Phoenicopteriformes — Phoenicopterus roseus (greater flamingo). Most of the birds were bought legally from the local hunters during the hunting season or otherwise found dead by the local authorities. The samples were transported to the helminthology research lab at Mazandaran University of Medical Sciences for parasitological and molecular evaluation.

2.2 Parasitological examination Birds for dissection were usually fresh from the field, though some heads were kept frozen until examination. Their heads were examined for nasal schistosomes by dissection of the 5

ACCEPTED MANUSCRIPT nasal tissue according to Skírnisson and Kolářová [23]. When eggs, miracidia or adults of the schistosomes were found, they were preserved in 96% ethanol and stored at -20°C. The

PT

measurements of the schistosome eggs were made with fresh material, prior to preservation in

RI

ethanol.

SC

2.3. Molecular and Phylogenetic analysis

NU

DNA was extracted from fresh or 96% ethanol preserved samples using the modified salting-out procedure [24]. When genetically characterizing parasites, it is ideal to use a

MA

minimum of two genes of different origin [25], particularly in the absence of independent data, such as adult worm morphology, to identify specimens. Thus, sequences of two gene regions, the

TE

D

nuclear internal transcribed spacer region 1(ITS1) and the mitochondrial cytochrome oxidase 1 (cox1) were used for the identification and molecular phylogenetic analysis of avian

AC CE P

schistosomes in naturally infected birds. These regions are also the most often reported for other species of avian schistosomes in GenBank, particularly species of Trichobilharzia. Primers BD1 (5′-GTCGTAACAAGGTTTCCGT-3′) and 4S (5′-TCTAGATGCGTTCGAARTGTCGATG-3′) [26] were used to amplify partial ITS1 nuclear rDNA (several bases of the 5’ and 3’were not sequenced

with

those

primers)

and

the

primer

pairs

CO1F6(5′-

TTTGTYTCTTTRGATCATAAGCG-3′) and Cox1_schist_3′ (5′-TAATGCATM GGA AAA AAACA-3′) were used for amplification of partial mitochondrial cox1 gene that includes the 824 bp of the 5’ end of the gene [27,28]. Polymerase chain reactions (PCR) were performed in a Mastercycler gradient thermal cycler (Eppendorf, Germany). The PCR fragments were submitted to Bioneer Company (South Korea) for sequencing by the Applied Biosystem automated sequencer (3730 XL). Sequencing was performed in both 6

ACCEPTED MANUSCRIPT directions using the same PCR primers using BigDye Terminator v3.1 cycle sequencing kit. The sequences of ITS1 and cox1 genes were deposited in GenBank database under the accession

PT

numbers KR108323 to KR108326.

RI

To reveal the putative species identification of these egg samples and their close relatives, a molecular phylogenetic analysis of species of Trichobilharzia was reconstructed using the ITS1

SC

and the mitochondrial cox1 region identified in this study and compared to those of T. regenti

NU

and other species of Trichobilharzia available in the GenBank database (Table 1,2). Phylogenetic analyses were reconstructed with Bayesian inferences (BI) using MrBayes v3.1 [29]. The BI

MA

analyses were as follows: For ITS1 dataset we used Nst = 6; rates = invgamma;ngammacat = 4; and for cox1 dataset (all parameters were unlinked) we used for codons one and two, Nst = 2,

D

and for codon three Nst = 6; rates = gamma;,ngammacat = 4. Four chains were run

TE

simultaneously for 5 × 105 generations, with 4 incrementally heated chains sampled at intervals

AC CE P

of 100 generations. The first 5000 trees with pre asymptotic likelihood scores were discarded as burnin, and the retained trees were used to generate 50% majority-rule consensus trees and posterior probabilities.

For the cox1 dataset we used Trichobilharzia stagnicolae, T. szidati and T. mergi as outgroups [30,31]. For the ITS1 dataset, species other than those related to T. regenti could not be aligned (to many and/or presence of large indels), therefore the analysis is ingroup only. Because the cox1 dataset confirmed that our samples were related to T. regenti, we wanted to use the most variable regions of ITS1 to more accurately determine how the clade of T. regenti samples grouped, which include the regions with many indels, thus excluding other closely related species of Trichobilharzia.

7

ACCEPTED MANUSCRIPT 2.4. Statistical analysis To summarize the observed infection patterns among the two species of hosts as well as between

RI

PT

males and females, a Fisher's exact test with the significance limit of 0.05 was used.

SC

3. Results

The number of birds examined and the prevalence of infection by nasal schistosomes for

NU

each species are presented in Table 1. Nasal schistosomes (eggs and/or adult fragments) were found in 45 out of 508 (8.9 %) heads of aquatic birds, which represented only two anseriform

MA

species (A. clypeata, A. platyrhynchos) out of the 10 aquatic birds examined. Worms were found in 29/89 (32.6%) A. clypeata and 16/186 (8.6%) A. platyrhynchos (Table 1). Interestingly,

TE

D

among these two bird species, the infection rate of male birds was significantly higher than females (P < 0.05) and the prevalence of nasal schistosomes in A. clypeata was significantly

AC CE P

higher than in A. platyrhynchos (P<0.0001). Microscopic examinations revealed a morphological polymorphism among the eggs isolated from A. clypeata and A. platyrhynchos. In A. platyrhynchos, the eggs measured 300 ± 15 × 68 ± 7 μm (n=30) that were larger than the eggs from A. clypeata (270 ± 10 × 63 ±4 μm; n=30) (based on t-test) and had a different shape (Figs 1,2; Table 4). For our phylogenetic reconstruction, we obtained partial ITS1 (937bp) and partial mitochondrial cox1 (824 bp) sequences and included Trichobilharzia species with ITS1 and cox1 sequence data available in GenBank. The phylogenetic analysis confirmed that the nasal schistosomes in our study from A. clypeata and A. platyrhynchos were included in the genus Trichobilharzia and grouped as close relatives to Trichobilharzia regenti. Interestingly, based on our phylogenetic results our samples were more closely related to Trichobilharzia JIT11 France 8

ACCEPTED MANUSCRIPT (cox1; HM439505) and Trichobilharzia JIT10 France (ITS1; HM439493) also both from A. clypeata, and this clade formed a sister group to T. regenti (Figs. 3,4). This clade also included

PT

ITS1 samples from Poland and Czech Republic that are likely not T. regenti. However, there is

RI

not as much of a marked genetic difference between the sample from A. clypeata versus A. platyrhynchos in our samples as was seen in the egg measurements (uncorrected p-distance 0.9%

SC

and 0.6%, cox1 and ITS1, respectively) and seems to be in the ranges of within species variation

NU

of other species of Trichobilharzia (Table5). Thus our samples are referred to as Trichobilharzia

MA

cf. regenti.

4. Discussion

TE

D

The data presented herein are part of the larger goal to understand the epidemiology of HCD as an endemic parasitic disease in northern Iran [19]. The initial steps to realizing this goal

AC CE P

are to systematically survey and identify the hosts and their schistosomes to make the critical life cycle connections. In addition to very little survey data, few molecular studies had been undertaken to identify the species of schistosomes and hosts, particularly in the Middle East regions. The northern provinces of Iran include part of a major migratory route for birds (about one million birds annually migrate to and from the Mazandaran Province), thus it is expected that the bird schistosomes, rather than mammalian schistosomes, are the major agents of HCD in this area. Most of the migratory birds spend the summer in Europe. The results of this paper illustrate for the first time a detailed molecular phylogenetic position of a species of Trichobilharzia that occurs in the nasal passages of two species of wild ducks in northern Iran, Trichobilharzia cf. regenti. Based on our survey of several orders of aquatic birds, only A. platyrhynchos and A. clypeata had nasal schistosomes, duck species that 9

ACCEPTED MANUSCRIPT are also hosts to T. regenti in Europe. In general, these two species of ducks are locally common and are frequently found as hosts for species of Trichobilharzia. These two species of ducks,

PT

among a few others in Europe, are often the most common species encountered in areas where

RI

there are outbreaks of HCD [13]. Along those lines, we also examined many (112) A. fuligula, also a common host for T. regenti in Europe (50% in France) [32] as well as a common shared

SC

migrant, yet none of these birds were infected by nasal schistosomes. Some non-mutually

NU

exclusive explanations might be that: a) in Iran, A. fuligula does not often reside in areas where the preferred snail host is common, like A. clypeata and A. platyrhynchos, b) the snail host in

MA

Iran has a different ecology from Radix balthica (the most common transmitting host), and/or c) prevalence in these birds is very low in this area and infected birds were not detected. Bird

TE

D

surveys in Europe have shown that T. regenti is commonly found in A. clypeata, swans (Cygnus olor) and Mergus merganser, and the highest prevalence has been observed in A. platyrhynchos

AC CE P

[32,33]. It may not be a surprise that nasal Trichobilharzia were not found in birds other than anseriforms (ducks, geese, swans) since this order of birds appears to be their preferred host [2,4,5,7,32,34]. But a few nasal schistosomes species other those in Trichobilharzia have been reported from non-anseriform birds [6] thus there remains a critical need to continue to survey all aquatic birds.

In our study the prevalence of nasal Trichobilharzia was 32.6% for A. clypeata and 8.6% for A. platyrhynchos. In two previous studies conducted in the Mazandaran Province area, Gohardehi et al. [22] reported a higher (4 times) prevalence of infection by nasal Trichobilharzia in A. clypeata than in A. platyrhynchos and Maleki et al. [19] reported the prevalence of nasal Trichobilharzia in A. clypeata as 33.3%, in concordance with our results. These studies, like ours, were conducted during the hunting season (winter). The prevalence values could indicate 10

ACCEPTED MANUSCRIPT that A. clypeata is the main host, or at least the host that spends the most time in contact with the snail host. If the prevalence values in Iran are compared to European surveys, the results are

PT

reversed. Rudolfova et al. [33] reported infection prevalence of T. regenti in A. clypeata as 4.5%

RI

and in A. platyrhynchos as 73.3% from Czech Republic and Poland. In a study conducted in Iceland [23], the prevalence of the T. regenti in A. platyrhynchos was 73.3%. In France, the

SC

prevalence of T. regenti in A. platyrhynchos and A. clypeata had been reported 55% and 40%

NU

respectively [32].

It appears that these two species of ducks are common hosts for T. regenti and T. cf.

MA

regenti. In Europe, the studies suggest that A. platyrhynchos maintains a higher prevalence than A. clypeata, yet in northern Iran, our results and others suggest that A. clypeata might be the

TE

D

main host [19,21]. However, some non-mutually exclusive explanations might be that: a) in Europe, the specific overlapping ecologies of both A. platyrhynchos and Radix balthica exist

AC CE P

more often thus favoring A. platyrhynchos as the principal definitive host relative to A. clypeata. The same would be in Iran, where perhaps A. clypeata is more often in the same ecological conditions as the preferred snail host species thus the probability that this species is more in contact with infected snail increases over other species of ducks, b) that the host with lower prevalence is an accidental host and/or an artifact of sampling and season , and/or c) in general, these two species of birds are in high densities, particularly in areas where humans are using the same water, thus maybe explaining why they are often infected: in Europe, most people are in contact with A. platyrhynchos in recreation areas where they are very common. But in Iran, most people are in contact with A. clypeata in rice fields for work where this bird is common, a habitat not as favored by A. platyrhynchos. As no samples of T. regenti from Europe were recovered thus far from ducks in Iran, it can be suggested that the nasal species common in Europe is not 11

ACCEPTED MANUSCRIPT common in northern Iran, again likely because of the absence of Radix balthica. While this snail is not the only permissive host, it is usually the species with higher prevalence of infection and

PT

likely more often shares the habitat of the particular duck hosts. However, surveys and molecular

RI

identities of the snails and the cercariae from snails needs to be implemented in northern Iran. Jouet et al. [34] proposed a possible origin of their T. regenti-like haplotype (JIT) in A.

SC

clypeata from migratory areas outside Europe, since their haplotype had not been isolated

NU

previous from any intermediate host snails in Europe, at least since molecular identification of avian schistosomes from European snail hosts [32]. Thus it is a reasonable hypothesis to suggest

MA

that for the ITS1 data, the clade containing the T. cf. regenti from France, Poland and Iran, may be a lineage specific to the region that includes northern Iran, possibly cycling through a

TE

D

common lymnaeid snail, R. gedrosiana [21,35]. But with the lack of molecular studies on cercariae from the local snails and snail systematic studies, the genetic association between them

AC CE P

and what we found in this study remains unclear. The infected duck hosts in this survey are common migrants between Europe and Iran, and northern Africa, and thus it was expected that the nasal schistosome species would be T. regenti, since it is the most common and only nasal schistosome reported from Europe. The schistosome species recovered in this survey is interesting because there is variation in egg morphology between this species and the T. regenti descriptions, as well as variation even between the eggs from the two species of duck in Iran. Furthermore, genetically, it is similar to T. regenti, yet different enough to question its status as a conspecific until more data are available. Egg morphology as a diagnostic feature of schistosomes is a somewhat reliable feature, though egg morphology does change with time, host and season and must be interpreted with caution [34,36]. 12

ACCEPTED MANUSCRIPT The eggs isolated from A. clypeata are most similar in size and shape to those eggs from A. clypeata described in Jouet et al. [34, Figure 1D], which were predicted not to belong to the T.

PT

regenti clade. Our samples were also similar in size and shape to T. spinulata and T. duboisi

RI

described by Fain [6,7] and T. arcuata [5]. They are similar in size to T. regenti but not in shape [2] and they are similar in shape to T. nasicola (also from an anseriform bird), but those eggs are

SC

larger than the ones found here [6,7]. Egg comparisons can be found in Table 4. In our samples,

NU

the crescent and bi-spindled shape of the eggs from A. clypeata distinguish them from the elongated oval shaped eggs in A. platyrhynchos (Figs. 1 and 2). Jouet et al. [34] also found that

MA

eggs from A. platyrhynchos were polymorphous and suggested that perhaps host and season may have contributed to egg polymorphism as was shown by Bayssade-Dufour et al. [36] in other

TE

D

genera of avian schistosomes. Because of the extreme fragmentation of adult worms, their morphological characteristics were not distinguishable and could not be compared to T. regenti

AC CE P

or other nasal species in the genus.

In addition to the egg polymorphism, there were distinct genetic difference between the European samples of T. regenti and those from Iran. Our cox1 phylogenetic results show two monophyletic clades. One clade includes T. regenti from Europe from both bird and snail hosts [e.g. Jouet et al., 2010]. The second sister clade includes the samples from this study, in addition to an unnamed species of Trichobilharzia, from France [34], 2 haplotypes of putative T. regenti from Poland [33] and other samples of nasal Trichobilharzia from different regions of northern Iran [19] (Figs. 3,4). All samples in this second clade, Trichobilharzia cf. regenti, came from either A. clypeata or A. platyrhynchos. While these two species of hosts are not exclusive to the clade that includes the Iranian samples, and are common host for several species of Trichobilharzia, it is interesting to note that in the three geographic areas where they have been 13

ACCEPTED MANUSCRIPT reported (Iran, France, Poland), they were the only species found thus far to host this Trichobilharzia cf. regenti clade. But clearly more collecting is necessary, particularly from the

PT

wintering grounds in northern Iran. Since we did not have adult morphology and do not yet know

RI

the snail host, we used genetic distances and phylogenetics as a yardstick to estimate species identities.

SC

There is no exact margin for the upper bounds of what distinguishes genetically closely

NU

related species in the absence of independent data or even comparative data, but there have been found reasonable ranges of differences in some trematode families [25,37]. We compared not

MA

only our samples to other samples of T. regenti, but also to the other well delineated species, as well as those species to each other (Table 5). Relative consistency of values may suggest

TE

D

reasonable hypotheses for species boundaries. Both the ITS1 and cox1 data sets revealed distinct clades of the Iranian samples with three of the European samples for T. cf. regenti (Figs. 3,4).

AC CE P

The genetic diversity among the samples in the ITS1 dataset is quite high (Table 5), but should be interpreted with caution [25]. The genetic distance values for cox1 were lower than other between-species comparison values, yet higher than any of the within species comparisons (Table 5). More sampling and a life cycle description is necessary to determine the significance of the diversity values, as it appears the ITS1 genetic distance suggest there may be multiple species of nasal schistosomes [30,37] and the cox1 data suggest they are very closely related species, but clearly there is some isolation of species (e.g. host use, geography and/or reproductive). Low mitochondrial genetic differences relative to nDNA genetic differences might suggest a much more complex evolutionary history. The genetic distances (relative to comparisons within and among other species in Trichobilharzia) alone argue in favor of congeners, separated by geography or more likely, snail 14

ACCEPTED MANUSCRIPT host species use. In Europe, T. regenti is most often transmitted by Radix balthica (=Radix peregra) and in Iran, it is speculated to use Radix gedrosiana (Lymnaea gedrosiana) [35,38] but

PT

this has not been confirmed. In this instance, it could be proposed that in the switch to a new host

RI

species of snail, there was a subsequent divergence in the lineages of T. regenti [39]. Other than Europe, Africa and Australia [3,4,5,40], there have been no other continents that report nasal

SC

schistosomes. There are no molecular data to accompany the morphological descriptions of the

NU

species of nasal schistosomes from Africa or Australia, and thus, there remains the possibility that the clade of nasal schistosomes delineated here might match one of those named species,

MA

particularly those from Africa with shared migratory flyways. While there has been a diversity of visceral species of Trichobilharzia described from the Americas (that also include Anas clypeata

TE

D

and Anas platyrhynchosas hosts), thus far no nasal species have been found [30]. One explanation for this might again be the absence of appropriate snail host, since the duck host is

AC CE P

present. There is one species of Radix in North America, R. auricularia, which may not be a permissive host for T. regenti, unlike in the eastern hemisphere, which maintains the species diversity of Radix, some of which can serve as hosts for T. regenti [41]. These questions will be resolved in future studies with genetic analyses of the schistosome cercariae and snail host to verify species and compare with other studies.

5. Conclusions The results herein delineated and confirmed a clade of samples closely related to the nasal schistosome Trichobilharzia regenti that suggest they are a distinct species. Because the samples found in our study form a sister clade to T. regenti, it is hypothesized that Trichobilharzia cf. regenti has a focal point in this geographic area, including northern Iran. 15

ACCEPTED MANUSCRIPT Until this study, there were no sequences of Trichobilharzia cf. regenti except those from France and Poland, even after a decade or more of molecular surveys of snails and ducks in Europe. Our

PT

work in northern Iran is contributing to our understanding of the evolution and diversification of

RI

avian schistosomes, parasites characterized by highly vagile definitive hosts. Future work will need to include the details of the life cycle, adult/egg/cercarial morphology, snail host species

SC

and use and as well as including additional genes for molecular comparison. The implications of

NU

a nasal schistosome in Iran are concerning in regards to HCD, as this may be the main etiological

MA

agent of dermatitis.

Acknowledgements

TE

D

The authors would like to thank Mr. AbdolSattar Pagheh from the Mazandaran University of Medical Sciences, Sari, Iran, for his kind help; as well as the Deputy of Research and

AC CE P

Technology of Mazandaran University of Medical Sciences for generous support of this study. This study has been supported by the Mazandaran University of Medical Sciences (project No. 143-92).

References [1] Horák P, Kolárová L, Adema CM. Biology of the schistosome genus Trichobilharzia. Adv Parasitol 2002;52:155-233. [2] Horák P, Kolárová L, Dvorák J. Trichobilharzia regenti n. sp. (Schistosomatidae, Bilharziellinae), a new nasal schistosome from Europe. Parasite 1998;5:349-357.

16

ACCEPTED MANUSCRIPT [3] Horák P, Dvorák J, Kolárová L, Trefil L.Trichobilharzia regenti, a pathogen of the avian and mammalian central nervous systems.Parasitology 1999;119:577-581.

PT

[4] Blair D, Islam KS. The life-cycle and morphology of Trichobilharzia australis n. sp. (Digenea: Schistosomatidae) from the nasal blood vessels of the black duck (Anas superciliosa) in

RI

Australia, with a review of the genus Trichobilharzia. Syst Parasitol 1983;5:89–117.

SC

[5] Islam KS. The morphology and life-cycle of Trichobilharzia arcuata n. sp. (Schistosomatidae: Bilharziellinae) a nasal schistosome of water whistle ducks (Dendrocygna arcuata) in

NU

Australia. Syst Parasitol 1986;8:117–128.

[6]Fain A. Une nouvelle bilharziose des oiseaux: la trichobilharziose nasale. Remarque sur

MA

l’importance des schistosomesd’oiseaux en pathologiehumaine. Note préliminaire. Ann Soc Belge Med Trop 1955;35:323–327.

TE

D

[7] Fain A. Les schistosomes d'Oiseaux du genre Trichobilharzia Skrjabin et Zakharow, 1920 au Ruanda-Urundi. Revue de Zoologie et de Botanique Africaines 1956;54:147-178.

AC CE P

[8] Berezantsev A, Kurochkin YV. Investigation of schistosomatic cercarioses in the USSR. I. Distribution of cercarioses and their causative agents. 1966 Meditsinskaia Parazitologiia i Parazitarnye Bolezni;35:332-337.

[9] Guo J, Li CP, Wang KX. Survey on natural nidus of Trichobilharzia in Huainan area. Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi 2007;25:60-61. [Chinese] [10] Korsunenko AV, Chrisanfova GG, Ryskov AP, Movsessian SO, Vasilyev VA, Semyenova SK. Detection of European Trichobilharzia schistosomes (T. franki , T. szidati , and T. regenti ) based on novel genome sequences. J Parasitol 2010;96:802-806. [11] Korsunenko A, Chrisanfova G, Lopatkin A, Beer SA, Voronin M, Ryskov AP ET AL. Genetic differentiation of cercariae infrapopulations of the avian schistosome Trichobilharziaszidati based on RAPD markers and mitochondrial cox1 gene. Parasitol Res 2012;110:833-841. [12] Lopatkin AA, Chrisanfova GG, Voronin MV, Zazornova OP, Beer, SA, Semyenova SK. 17

ACCEPTED MANUSCRIPT Polymorphism

of

the

cox1

gene

in

cercariae

isolates

of

bird

schistosomes

(Trematoda:Schistosomatidae) from ponds of Moscow and Moscow region. 2010 Russian Journal

PT

of Genetics;46:873-880. [13] Horák P, Mikeš L, Lichtenbergová L, Skála V, Soldánová M, Brant SV. Avian schistosomes

RI

and outbreaks of cercarial dermatitis. Clin Microbiol Rev 2015;28:165-190.

SC

[14] Horák P, Dvorák J, Kolárová L, Trefil L. Trichobilharzia regenti, a pathogen of the avian and mammalian central nervous systems. Parasitology 1999;119:577-581.

NU

[15] Hrádková K, Horák P. Neurotropic behaviour of Trichobilharzia regenti in ducks and

MA

mice. J Helminthol2002;76:137-141.

[16] Horák P, Mikes L, Rudolfová J, Kolárová L. Penetration of Trichobilharzia cercariae into mammals: dangerous or negligible event? Parasite 2008;15:299-303.

TE

D

[17] Farahnak A, Essalat M. A study on cercarial dermatitis in Khuzestan province, south western Iran. 2003 BMC Public Health;3:35-38.

AC CE P

[18] Karamian M, Aldhoun JA, Maraghi S, Hatam G, Farhangmehr B, Sadjjadi SM. Parasitological and molecular study of the furcocercariae from Melanoides tuberculata as a probable agent of cercarial dermatitis. Parasitol Res 2011;108:955-962. [19] Maleki SH, Athari A, Haghighi A, Taghipour N, Gohardehi SH, Tabaei SS. Species identification of birds nasal Trichobilharzia in sari, north of Iran. Iran J Parasitol 2012;7:82-85. [20] Mahdavi S, Farahnak A, Mobedi I, Rad MM, Azadeh H. Survey of Migratory Birds (Anatidae: Anas platyrhynchos) for Schistosome Parasites from Mazandaran Province, Northern Iran. Iran J Parasitol 2013;8:333-336. [21] Sahba GH, Malek EA. Dermatitis caused by cercariae of Orientobilharzia turkestanicum in the Caspian Sea area of Iran. Am J Trop Med Hyg 1979;28:912-913. [22] Gohardehi S, Fakhar M, Madjidaei M. Avian schistosomes and human cercarial Dermatitis in a wildlife refuge in Mazandaran Province, northern Iran. Zoonoses Public Health 18

ACCEPTED MANUSCRIPT 2013;60:442-447. [23] Skírnisson K, Kolárová L. Diversity of bird schistosomes in anseriform birds in Iceland

PT

based on egg measurements and egg morphology. Parasitol Res 2008;103:43-50. [24] Poljak M, Barlic J, Seme K, Avsic-Zupanc T, Zore A. Isolation of DNA from archival

RI

Papanicolaou stained cytological smears using a simple salting-out procedure. Clin Mol Pathol

SC

1995;48:M55-56.

[25] Locke SA, Al-Nasiri FS, Caffara M, Drago F, Kalbe M, Lapierre AR. Et al. Diversity,

NU

specificity and speciation in larval Diplostomidae (Platyhelminthes: Digenea) in the eyes of

MA

freshwater fish, as revealed by DNA barcodes. Int J Parasitol 2015;pii:S0020-7519(15)00189-7. [26] Bowles J, McManus DP. Rapid discrimination of Echinococcus species and strains using a polymerase chain reaction-based RFLPmethod. Mol Biochem Parasitol 1993;57:231-239.

TE

D

[27] Lockyer AE, Olson PD, Østergaard P, Rollinson D, Johnston DA, Attwood SW, et al. The phylogeny of the Schistosomatidae based on three genes with emphasis on the interrelationships

AC CE P

of Schistosoma Weinland, 1858. Parasitology 2003;126:203–224. [28] Morgan JA, DeJong RJ, Kazibwe F, Mkoji GM, Loker ES. A newly-identified lineage of Schistosoma. Int J Parasitol 2003;33:977–985. [29]

Huelsenbeck

JP, Ronquist

F.

MRBAYES: Bayesian inference of phylogenetic trees.

Bioinformatics 2001;17:754-755.

[30] Brant SV, Loker ES. Molecular systematics of the avian schistosome genus Trichobilharzia (Trematoda: Schistosomatidae) in North America. J Parasitol 2009;95:941-963. [31] Kolářová L, Skírnisson K, Ferté H, Jouet D.Trichobilharzia mergi sp. nov. (Trematoda: Digenea: Schistosomatidae), a visceral schistosome of Mergus serrator (L.) (Aves: Anatidae). Parasitol Int 2013;62:300-308. [32] Jouet D, Ferté H, Hologne C, Kaltenbach ML,.Depaquit J. Avian schistosomes in French aquatic birds: a molecular approach. J Helminthol 2009;83:181-189. 19

ACCEPTED MANUSCRIPT [33] Bayssade-Dufour C, Jouet D, Rudolfova J, Horák P, Ferté H. Seasonal morphological variations in bird schistosomes. Parasite 2006;13:205-214.

regenti under natural conditions. Parasitol Res 2010;107:923-930.

PT

[34] Jouet D, Skírnisson K, Kolárová L,Ferté H. Final hosts and variability of Trichobilharzia

RI

[35] Athari A, Gohar-Dehi S, Rostami-Jalilian M. Determination of definitive and

SC

intermediate hosts of cercarial dermatitis-producing agents in northern Iran. Arch Iran Med 2006;9:11-15.

NU

[36] Rudolfová J, Littlewood DT, Sitko J, Horák P. Bird schistosomes of wildfowl in the

MA

Czech Republic and Poland. Folia Parasitol (Praha) 2007;54:88-93. [37] Vilas R, Criscione CD, Blouin MS. A comparison between mitochondrial DNA and the

TE

Parasitology 2005;131:839-846.

D

ribosomal internal transcribed regions in prospecting for cryptic species of platyhelminth parasites.

[38] Imani-Baran A, Yakhchali M, Malekzadeh Viayeh R, Paktarmani R. Molecular study for

AC CE P

detecting the prevalence of Fasciola gigantica in field-collected snails of Radix gedrosiana (Pulmonata: Lymnaeidae) in northwestern Iran. Vet Parasitol 2012;189:374-377. [39] Brant SV, Loker ES. Discovery-based studies of schistosome diversity stimulate new hypotheses about parasite biology. Trends in Parasitology 2013;29:449-459. [40] Fain A. Nasal trichobilharziasis: a new avian schistosomiasis. Nature 1956;177:389. [41] Huňová K1, Kašný M, Hampl V, Leontovyč R, Kuběna A, Mikeš L et al. Radix spp.: Identification of trematode intermediate hosts in the Czech Republic.Acta Parasitol 2012;57:273284.

20

ACCEPTED MANUSCRIPT Fig 1. The eggs of T. regenti isolated from nasal tissues of A. clypeata. Scale bar 100 µm.

RI

PT

Fig 2. The eggs of T. regenti in nasal mucosa of A. platyrhynchos. Scale bar 100 µm.

Fig. 3. Phylogenetic tree based on a Bayesian analysis of cox1. The shaded grey box

NU

SC

highlights the species of Trichobilharzia found in nasal tissue of their duck hosts. Taxa collected from this study are in bold. All taxa in the tree indicate their GenBank Accession number. Nodal support is indicated above the branches as Bayesian posterior probabilities.

Fig. 4. Phylogenetic tree based on a Bayesian analysis of ITS1. The shaded grey box

AC CE P

TE

D

MA

highlights the relationship the samples of Trichobilharzia found in this study with that from France. Taxa collected from this study are in bold. All taxa in the tree indicate their GenBank Accession number. Nodal support is indicated above the branches as Bayesian posterior probabilities.

21

AC CE P

Figure 1

TE

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

22

TE AC CE P

Figure 2

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

23

AC CE P

TE

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

Figure 3

24

AC CE P

TE

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

Figure 4 25

ACCEPTED MANUSCRIPT Table 1

16

Anas clypeata

89

29

Anas acuta

25

0

Anas crecca

36 7

Aythya fuligula

141

Fulica atra

12 2

Phoenicopterus roseus

6

8.6

32.6 0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

AC CE P

Tringa ochropus

TE

4

D

Anas strepera

Anser anser

Prevalence (%)

NU

186

MA

Anas platyrhynchos

No.infected

RI

No. examined

SC

Host

PT

Prevalence of nasal Trichobilharziain aquatic birds from this study.

26

ACCEPTED MANUSCRIPT Table 2

Host

Locality

Trichobilharzia physellae T. physellae T. physellae Trichobilharzia sp. T. franki T. franki T. franki T. franki Trichobilharzia sp. Trichobilharzia sp. Trichobilharzia sp. T. querquedulae T. querquedulae T. querquedulae Trichobilharzia sp. Trichobilharzia sp. Trichobilharzia sp. T. regenti T. regenti T. regenti T. regenti T. regenti T. regenti Trichobilharzia sp. JIT11 Trichobilharzia sp. Trichobilharzia sp. Trichobilharzia sp. T. mergi T. mergi Trichobilharzia sp. Trichobilharzia sp. T. szidati T. szidati T. szidati Trichobilharzia sp. Trichobilharzia sp. Trichobilharzia sp. Trichobilharzia sp. T. stagnicolae T. stagnicolae T. stagnicolae

Physa gyrina Aythya affinis Bucephala albeola Physa marmorata Radix auricularia Radix auricularia Radix auricularia Radix auricularia Anas americana Anas americana Anas americana Anas clypeata Anas cyanoptera Anas discors Lophodytes cucullatus Aix sponsa Radix luteola Anas platyrhynchos Anas platyrhynchos Radix peregra Mergus merganser Cygnus olor Radix peregra Anas clypeata Radix peregra Radix peregra Radix peregra Mergus serrator Mergus serrator Radix ampla Radix ampla Lymnaea stagnalis Stagnicola elrodi Lymnaea stagnalis Stagnicola sp. Anas acuta Stagnicola sp. Stagnicola sp. Stagnicola emarginata Mergus merganser Stagnicola emarginata

USA USA USA Brazil France France France France USA USA USA USA USA USA USA USA Nepal France Iceland Czech Republic France France France France France France France Iceland Iceland Belarus Belarus USA USA Czech Republic Canada Canada Canada Canada USA USA USA

SC

NU

MA

D

TE

AC CE P

Reference

FJ174523 FJ174518 FJ174514 KJ855995 HM131202 HM131200 HM131201 HM131197 FJ174471 FJ174525 FJ174528 FJ174497 FJ174499 FJ174511 FJ174529 KJ855996 KF672863 HM439502 HM439504 AY157190 HM439501 HM439500 HM439499 HM439505 HM131205 HM131204 HM131203 JX456172 JX456171 JQ681540 JQ681535 FJ174496 FJ174495 AY157191 FJ174483 FJ174487 FJ174485 FJ174485 FJ174494 FJ174490 FJ174491

Brant and Loker 2009 Brant and Loker 2009 Brant and Loker 2009 Pinto et al. 2014 Jouet et al. 2010 Jouet et al. 2010 Jouet et al. 2010 Jouet et al. 2010 Brant and Loker 2009 Brant and Loker 2009 Brant and Loker 2009 Brant and Loker 2009 Brant and Loker 2009 Brant and Loker 2009 Brant and Loker 2009 Pinto et al. 2014 Devkota et al. 2014 Jouet et al. 2010 Jouet et al. 2010 Lockyer et al. 2003 Jouet et al. 2010 Jouet et al. 2010 Jouet et al. 2010 Jouet et al. 2010 Jouet et al. 2010 Jouet et al. 2010 Jouet et al. 2010 Kolarova et al. 2013 Kolarova et al. 2013 Chrisanfova et al. 2003 Chrisanfova et al. 2003 Brant and Loker 2009 Brant and Loker 2009 Lockyer et al. 2003 Brant and Loker 2009 Brant and Loker 2009 Brant and Loker 2009 Brant and Loker 2009 Brant and Loker 2009 Brant and Loker 2009 Brant and Loker 2009

GenBank Accession Number

RI

Species

PT

Samples of Trichobilharzia available in GenBank used for the mitochondrial cox1 phylogenetic analysis and the reference.

27

ACCEPTED MANUSCRIPT Table 3 Samples of Trichobilharzia regenti available in GenBank and used for phylogenetic analysis by ITS1 rDNA region Locality

Accession Number

Anas platyrhynchos

Czech Republic

GU233740

Anas platyrhynchos

Czech Republic

EF094538

Anas clypeata

Czech Republic

EF094540

Anas platyrhynchos

Poland

EF094535

Aythya fuligula

Poland

EF094534

Rudolfova 2006

Anas platyrhynchos

France

HM439494

Jouet et al. 2010

Anas platyrhynchos

Switzerland

AJ312049

Jousson 2001

Lymnaea ovate

Switzerland

AJ312047

Jousson 2001

Lymnaea ovate

Switzerland

Mergus merganser

France

Radix peregra

France

Radix peregra

France

Cygnus olor Anas clypeata

Aldhoun and Horak 2009

RI

Rudolfova 2006

SC

NU

MA

reference

PT

Host

Rudolfova 2006 Rudolfova 2006

Jousson 2001

HM439498

Jouet et al. 2010

HM439495

Jouet et al. 2010

HM439496

Jouet et al. 2010

France

HM439497

Jouet et al. 2010

France

HM439493

Jouet et al. 2010

AC CE P

TE

D

AJ312048

Anas platyrhynchos

Poland

EF094537

Rudolfova 2006

Anas clypeata

Poland

EF094533

Rudolfova 2006

Iran

AB594831

Maleki et al. 2010

Iran

AB594834

Maleki et al. 2010

Iran

AB594830

Maleki et al. 2010

Iran

AB594832

Maleki et al. 2010

Iran

AB594833

Maleki et al. 2010

Anas clypeata Anas clypeata Anas clypeata Anas clypeata Anas clypeata

28

ACCEPTED MANUSCRIPT

Table 4

PT

Comparison of egg measurements of described species of nasal Trichobilharzia. Species are grouped by bird hosts Anseriformes (ducks, geese, swans)

Egg width (um)

Avian Host

NU

Egg length (um)

SC

RI

and then other birds.

Anseriform avian hosts

Snail Host

General region

Reference

270 ±10

63 ±4

Anas clypeata

unknown

Iran

This study

Trichobilharzia cf. regenti

300 ±15

68 ±7

Anas platyrhynchos

unknown

Iran

This study

Trichobilharzia cf. regenti

259 ±17

62 ±6

Anas clypeata

unknown

France

Trichobilharzia regenti

274 ±94

MA

Trichobilharzia cf. regenti

Radix peregra

Europe

45

Dendrocygna viduata

unknown

Rwanda

Fain, 1956

300 - 325

70

Sarkidiornis melanotos

unknown

Rwanda

Fain, 1956

280 - 330

50 - 70

Anas undulata

unknown

Rwanda

Fain, 1955, 1956

250 - 300

50 - 70

Alopochen aegyptiaca

215

Trichobilharzia sp. Trichobilharzia nasicola Trichobilharzia spinulata

225 - 400

40 - 70

Trichobilharzia australis

230

48

Trichobilharzia arcuata

260

57

175 - 220

32 - 40

AC

Trichobilharzia duboisi

unknown

Rwanda

Fain, 1955, 1956

Plectropterus gambensis

unknown

Rwanda

Fain, 1956

Nettapus auritus

unknown

Rwanda

Fain, 1959

Anas platyrhynchos domesticus

Austropeplea lessoni

Australia

Blair and Islam, 1983

Dendrocygna arcuata

Austropeplea lessoni

Australia

Islam, 1986

Podiceps cristatus infuscatus

unknown

Rwanda

Fain, 1956

unknown

Rwanda

Fain, 1955, 1956

CE

Trichobilharzia sp.

PT ED

ducks, geese, swans

Jouet et al. 2010 Horak et al., 2002; Jouet et al., 2010

Other avian hosts Trichobilharzia aureliani

Poliocephalus ruficollis capensis Trichobilharzia rodhaini

280 - 325

55 - 70

Bostrychia hagedash

29

ACCEPTED MANUSCRIPT Table 5 Average pairwise uncorrected p-distance between and within species of Trichobilharzia. Bolded

RI

PT

samples are those collected for this study.

ITS1

cox1

0.18%*

1.0%

0.7%*

1.1%

-

1.0%

0.97% (0.2%-1.5%)

0.3%

0.6%

0.9%

0.6%

0.7%

0%

0.9%

2.1% (0.7%-3.2%)

-

T. regenti - Iran samples this study

4.1% (3.6%-4.3%)

3.1%

T. regenti - Iran Trichobilharzia previous

3.2% (2.5%-3.8%)

-

4.3%

3.2%

-

5.9%

3.0%*

9.2%

T. querquedulae - T. franki

3.1% - 3.4%*

8.3%

T. physellae - T. franki

0.82% - 1.3%*

9.4%

SC

Taxon Trichobilharzia querquedulae (within)

Trichobilharzia mergi (within) Trichobilharzia regenti (within)

MA

T39 A. clypeata - T44 A. platyrhynchos

NU

Trichobilharzia franki (within)

T39 A. clypeata - Trichobilharzia JIT11

D

T44 A. platyrhynchos - Trichobilharzia JIT11

AC CE P

TE

Iran samples this study- IranTrichobilharzia previous

T. regenti - Trichobilharzia JIT11 T. physellae - Trichobilharzia Brazil T. querquedulae - T. physellae

* Brant and Loker, 2009

30

ACCEPTED MANUSCRIPT

AC CE P

TE

D

MA

NU

SC

RI

PT

Graphical abstract

31

ACCEPTED MANUSCRIPT

PT

Highlights

Report of the first molecular phylogenetic data for a nasal Trichobilharzia in Iran



Our isolates were homologous with unique haplotypes of putative T. regenti in France, Poland and Czech Republic.



Anas clypeata seems to be the main final host of this nasal schistosome in Northern Iran.



This may be one of the major species causing cercarial dermatitis, particularly in rice fields.

AC CE P

TE

D

MA

NU

SC

RI



32