Zoonotic Abbreviata caucasica in Wild Chimpanzees (Pan troglodytes verus) from Senegal

Abbreviata caucasica (syn. Physaloptera mordens) has been reported in human and various non-human primates including great apes. The identification of this nematode is seldom performed and relies on egg characterization at the coproscopy, in the absence of any molecular tool. Following the recovery of two adult females of A. caucasica from the feces of wild Senegalese chimpanzees, morphometric characteristics were reported and new data on the width of the esophagus (0.268–0.287 mm) and on the cuticle structure (0.70–0.122 mm) were provided. The molecular characterization of a set of mitochondrial (cox1, 16S rRNA, 12S rRNA) and nuclear (18S rRNA and ITS2) partial genes was performed. Our phylogenetic analysis indicates for the first time that A. caucasica is monophyletic with Physaloptera species. A novel molecular tool was developed for the routine diagnosis of A. caucasica and the surveillance of Nematoda infestations. An A. caucasica-specific qPCR targeting the 12S gene was assessed. The assay was able to detect up to 1.13 × 10−3 eggs/g of fecal matter irrespective of its consistency, with an efficiency of 101.8% and a perfect adjustment (R2 = 0.99). The infection rate by A. caucasica in the chimpanzee fecal samples was 52.08%. Only 6.19% of the environmental samples were positive for nematode DNA and any for A. caucasica. Our findings indicate the need for further studies to clarify the epidemiology, circulation, life cycle, and possible pathological effects of this infestation using the molecular tool herein developed.


Introduction
Physalopteriasis is caused by parasitic nematodes from the genus Physaloptera (Spirurida, Physalopteridae) [1], which has been distributed in Africa and the Middle East (i.e., Iran) since Pathogens 2020, 9,517 3 of 22

Morphological Characteristics of Adult A. caucasica
Comparative measurements of A. caucasica adult females from our study with A. caucasica (Linstow, 1902) and its synonymous species P. mordens (Lipper, 1908) [8] are detailed in Table 1. Two female complete specimens measuring 54.7 mm and 59.6 mm in length and 2.08 mm and 2.13 mm in width, respectively, were examined. The nematodes were characterized by the anterior end with a short buccal cavity (Figure 1(1a)) and by a cuticle reflecting over the lips to form a cephalic collarette (Figure 1(1b)) with two lateral pseudolabia undivided (Figure 1(1c,1d)). Nerve ring at 0.430 mm from the anterior end.
The esophagus consisting of two parts: muscular esophagus 0.75 mm long (Figure 1(2a)) and 0.287 mm wide, 0.79 mm long, and 0.268 mm wide, respectively. The esophagus total length was 5.52 mm and 4.82 mm, respectively in two samples.

A. caucasica Eggs from Positive Feces
Eggs of A. caucasica were identified morphologically from two fecal samples taken from animals found infested with adult worms. The eggs were apparently identical to the micrograph of A. caucasica eggs reported elsewhere [7]. Eggs were embryonated and had a characteristic thick shell and hyaline coat ( Figure 2).

A. caucasica Eggs from Positive Feces
Eggs of A. caucasica were identified morphologically from two fecal samples taken from animals found infested with adult worms. The eggs were apparently identical to the micrograph of A. caucasica eggs reported elsewhere [7]. Eggs were embryonated and had a characteristic thick shell and hyaline coat ( Figure 2). Pathogens 2020, 9, x FOR PEER REVIEW 6 of 21

A. caucasica Eggs from Positive Feces
Eggs of A. caucasica were identified morphologically from two fecal samples taken from animals found infested with adult worms. The eggs were apparently identical to the micrograph of A. caucasica eggs reported elsewhere [7]. Eggs were embryonated and had a characteristic thick shell and hyaline coat ( Figure 2).
BLASTn analyses of 16S rRNA (416 bp) and 12S rRNA (573 bp) sequences identified the first 60 sequences that corresponded to those of Filarioidea and Thelazidae without any Physaloptera. Nucleotide identity of about 75% with a query coverage of more than 99% were observed among these Spiriruds. Finally, the BLASTn analysis of the partial (675 bp) sequence of the ITS2 showed an identity of 93.37% (67/71) and a coverage of 10% with the unique GenBank sequence of Physaloptera alata (AY702694) isolated from birds.
The interspecific nucleotide pairwise (INP) distance of the 18S rRNA, cox1, 16S rRNA, 12S rRNA, and ITS2 of A. caucasica within Physalopteridae members are shown in Table S1. All sequences were well resolved in the chromatograms. The partial cox1 sequence was correctly aligned against the complete cox1 sequence (MH931178) of P. rara and no stop codon was observed in the translated amino-acid sequences, suggesting the absence of co-amplified numts. Furthermore, sequence alignment of COI with those of Physaloptera species showed nineteen amino-acid changes specific for A. caucasica ( Figure S1). The interspecific nucleotide pairwise (INP) distance among the 645 bp of cox1 corroborated with the IaaP distance, among the corresponding 208 amino acid ( Figure 3) and was substantially higher (ten times) between A. caucasica and Physalopteridae members in comparison with the 18S rRNA sequences. The Bayesian trees inferred from cox1, nucleotide, and protein sequences, and from 18S rRNA genes are shown in Figures 4A,B and 5, respectively. All phylograms provide evidence that A. caucasica is an integral part of the genus Physaloptera. In particular, on the cox1 tree, A. caucasica clustered with Physaloptera sp. (MH752202) and P. retusa (KT894803) isolated respectively from Anolis sagrei in the USA and Tupinambis teguixin in Brazil ( Figure 4A). Similarly, on the COI tree, A. caucasica clustered with P. rara (QDF64304), P. retusa (AMX28288), Physaloptera spp. (QEQ27063, AYA23053), P. turgida (AMX28293), and Turgida sp. (AFZ99495) ( Figure 4B), while on the 18S rRNA tree, A. caucasica clustered together with Physaloptera apivori (EU004817) and Physaloptera alata (AY702703) isolated from birds in Germany ( Figure 5). The Bayesian trees inferred from cox1, nucleotide, and protein sequences, and from 18S rRNA genes are shown in Figure 4A,B and Figure 5, respectively. All phylograms provide evidence that A. caucasica is an integral part of the genus Physaloptera. In particular, on the cox1 tree, A. caucasica clustered with Physaloptera sp. (MH752202) and P. retusa (KT894803) isolated respectively from Anolis sagrei in the USA and Tupinambis teguixin in Brazil ( Figure 4A). Similarly, on the COI tree, A. caucasica clustered with P. rara (QDF64304), P. retusa (AMX28288), Physaloptera spp. (QEQ27063, AYA23053), P. turgida (AMX28293), and Turgida sp. (AFZ99495) ( Figure 4B), while on the 18S rRNA tree, A. caucasica clustered together with Physaloptera apivori (EU004817) and Physaloptera alata (AY702703) isolated from birds in Germany ( Figure 5).
In addition, all Physaloptera and A. caucasica haplotypes shared a Euler circuit in the Templeton-Crandall-Sing (TCS) network tree for cox1 sequences. Abbreviata caucasica was connected by three-step branches to the Euler circuit, while all Physaloptera haplotypes were connected to the circuit by one to three-step branches ( Figure 6). Hence, the TCS network analysis replicates the same results observed in the Bayesian inferences.
caucasica is an integral part of the genus Physaloptera. In particular, on the cox1 tree, A. caucasica clustered with Physaloptera sp. (MH752202) and P. retusa (KT894803) isolated respectively from Anolis sagrei in the USA and Tupinambis teguixin in Brazil ( Figure 4A). Similarly, on the COI tree, A. caucasica clustered with P. rara (QDF64304), P. retusa (AMX28288), Physaloptera spp. (QEQ27063, AYA23053), P. turgida (AMX28293), and Turgida sp. (AFZ99495) ( Figure 4B), while on the 18S rRNA tree, A. caucasica clustered together with Physaloptera apivori (EU004817) and Physaloptera alata (AY702703) isolated from birds in Germany ( Figure 5).   In addition, all Physaloptera and A. caucasica haplotypes shared a Euler circuit in the Templeton-Crandall-Sing (TCS) network tree for cox1 sequences. Abbreviata caucasica was connected by threestep branches to the Euler circuit, while all Physaloptera haplotypes were connected to the circuit by one to three-step branches ( Figure 6). Hence, the TCS network analysis replicates the same results observed in the Bayesian inferences.

Molecular Investigation of A. caucasica and Nematode Infestation from Biological Samples
The molecular tool developed in the present study was specific for the target DNA without any amplification from the negative controls.
Results of the molecular screening for A. caucasica and nematode DNA are detailed in Table 2. Among the 48 fecal samples tested, 52.08% (n = 25) were positive for A. caucasica, while all environmental samples tested negative. In addition to the samples that tested positive for A. caucasica, the pan-Nematoda qPCR assay allowed for the detection of 29.17% (n = 14) of other fecal samples and 6.2% (n = 7, three soil samples from termite mounds and four termite specimens) of positive environmental samples.
Fisher's exact test showed that there were no significant effects of localities and fecal consistency on nematodes and A. caucasica prevalences ( Table 2). Abbreviata caucasica cox1 species-specific primers successfully amplified a partial sequence (504 bp) from 84% (21/25) of samples identified as positive by the qPCR targeting the 12S rRNA gene. There was no significant difference between both assays according to the McNemar test (p = 0.25). All

Molecular Investigation of A. caucasica and Nematode Infestation from Biological Samples
The molecular tool developed in the present study was specific for the target DNA without any amplification from the negative controls.
Results of the molecular screening for A. caucasica and nematode DNA are detailed in Table 2. Among the 48 fecal samples tested, 52.08% (n = 25) were positive for A. caucasica, while all environmental samples tested negative. In addition to the samples that tested positive for A. caucasica, the pan-Nematoda qPCR assay allowed for the detection of 29.17% (n = 14) of other fecal samples and 6.2% (n = 7, three soil samples from termite mounds and four termite specimens) of positive environmental samples. Fisher's exact test showed that there were no significant effects of localities and fecal consistency on nematodes and A. caucasica prevalences ( Table 2).
Abbreviata caucasica cox1 species-specific primers successfully amplified a partial sequence (504 bp) from 84% (21/25) of samples identified as positive by the qPCR targeting the 12S rRNA gene. There was no significant difference between both assays according to the McNemar test (p = 0.25). All sequences were identical to each other and showed 100% similarity to those from adult specimens amplified with pan-Nematoda primers.
All sequences were deposited in the GenBank database under the following accession numbers: MT231296-MT231316.

The Analytical Sensitivity of A. caucasica 12S rRNA qPCR and Egg Counting
The performance characteristics of the 12S rRNA qPCR are shown in Table S2 and Figure S2. The assay was species specific and was able to detect up to 1.13 × 10 −3 eggs/g of positive fecal samples (i.e., corresponding to 1.13 × 10 −5 eggs/5 µL of DNA). The qPCR efficiency was 101.8% with −3.28 and 28.68 as a Slope and Y-intercept values, respectively, allowing a perfect adjustment (R 2 = 0.99). Table 3 compares the A. caucasica eggs quantified by qPCR in terms of fecal consistency (fresh or degraded samples). Egg concentration in degraded feces (n = 4) was low (<1/g), but was higher (mean = 1.4 egg/g) in fresh feces (n = 21), while no effect of fecal consistency on egg concentration was observed (ANOVA, R2 = 0.032, Pr > F = 0.403).

Discussion
In this study, we report on the presence of A. caucasica (adults and eggs) in the feces of western chimpanzees from Senegal. Our data indicate that this population of chimpanzees is exposed to a high nematode infestation (81.3%), particularly A. caucasica (52.1%). This corroborates previous data from chimpanzees in southeastern Senegal, in which the reported nematode species-specific prevalence was between 0.78% to 31% where Physaloptera sp. was often the most prevalent species (13.26 to 31%) [12,14]. However, it was not specified whether these Physaloptera sp. were A. caucasica or author Physaloptera species. Perhaps the use of molecular assays, which were not applied in these studies, could offer a better species resolution.
The adult worms were designated as A. caucasica after careful identification based on the morphological and morphometric features, which was strengthened by previous descriptions by Schulz, (1926), Ortlepp, (1926) and Brede and Burger, (1977) [8,9,21]. In addition to the morphological and morphometric features previously listed, we reported the width of the esophagus (0.268-0.287 mm) and that of the cuticle (0.70-0.122 mm), which may help in the future identification of A. caucasica. Morphologically, Abbreviata species are closely related to each other [22]. In 1945, Morgan described the utility of uterine morphology (number and mode of origin of the uteri in the female worm) in the taxonomic classification. He classified species from the genus Abbreviata (n = 27) into more than three classes with two (didelphys), four (tetra-delphys), or more than four (polydelphys) branches. Of those, three were associated with monkeys: A. caucasica (Linstow, 1902), A. poicilomeira (Sandground, 1936), and A. multipapillata (Kreis, 1940) [4]. Based on the uterine morphology, A. poicilomeira and A. multipapillata are listed in class 5-15 G (5-15 uteri with common trunk), and 9-13 H (9-13 uteri without common trunk), respectively. However, A. caucasica can be easily differentiated by the fact that it is in class 4-D (4 uteri with common trunk).
The morphologic-based classification of Physalopteridae members (e.g., Skrjabinoptera, Abbreviata, and Physaloptera) exclude some morphometric measurements from the taxonomic characters such as the length of the esophagus, vulva position, and egg dimensions. These features seem to variate in the same species and are used only in exceptional cases such as P. squamatae (Harwood 1932), S. chamaeleontis (Gedoelst 1916), and S. simplicidens (Ortlepp 1922) [23]. As expected, our data confirmed the variability of these parameters within the A. caucasica (Table 1). This reduced the utility of some commonly used indexes (a, b, and c) in nematode taxonomy [24].
In addition to the important taxonomic characters highlighted by Fain and Vandepitte (1964) (e.g., morphological features of the anterior end posterior ands, the number of uterine branches), the two adult females measured in the present study exhibited morphometric features of body and egg size close to those of P. mordens (Lipper, 1908), a species synonymous with A. caucasica (Linstow, 1902), where the body size of the female is 41-100 × 1.8-2.8 mm with a small egg of 45-49 × 32-34 µm [7]. Eggs were also similar to those reported by Poinar et al., 1972, where the size is 35-40 × 25-35 µm [5]. However, A. caucasica (Linstow, 1902) has been described as having a small body size of 24.75-23.84 × 1.12-1.18 mm and larger eggs of 57-62 × 42-45 µm. In contrast, the measurements from the study of Fain and Vandepitte (1964) showed that the A. caucasica (syn. P. mordens) adult females had a big body size of 108-117 mm and larger eggs of 60-65 × 45-55 µm (Table 1). Furthermore, the same authors confirmed and described the inconsistency of some measurements within this species [7]. Traditional taxonomic keys are known to be inconclusive for the taxonomic classification of nematodes [25] and should be confirmed by molecular barcoding, which circumvents the limitations of classical morphology-based classification [26]. The question then arises of whether A. caucasica (Linstow, 1902) is the same specie as P. mordens (Lipper, 1908), as indicated by Ortlepp (1926) and Fain and Vandepitte (1964) using morphologic-based taxonomy [7,9]. To address this question, a molecular comparative characterization of the specimens from the studies of Schulz (1926) and Fain and Vandepitte (1964) [7,8] should be performed to confirm or refute the synonymy of these two species.
In our study, we expanded the genetic data available for A. caucasica with sequences of mitochondrial and nuclear DNA (i.e., cox1, 12S rRNA, 16S rRNA, 18S rRNA, and ITS2 genes), though the genetic characterization was based on cox1 and 18S rRNA genes, due to the limited data on other gene sequences of Physalopterida members in the GenBank database.
The molecular analyses carried out in this study such as the phylogenetic comparisons of cox1 and 18S rRNA genes, the TCS network analysis of the cox1 gene, and the Bayesian inference of both cox1 and COI sequences confirmed that A. caucasica is monophyletic with Physaloptera species (Figures 4A,B and 5). cox1 and 18S rRNA genes are widely used as markers for the molecular barcoding of nematodes [27] with cox1 sequences of relevance in resolving taxonomic relationships among nematode species [27,28]. This gene is described by an interspecific nucleotide pairwise distance (INPD) of 16% to 27.8% between nematodes species [29].
The description of new species from the genus Physaloptera as well as the recording of new hosts has quickly evolved over the last decade [30][31][32][33][34][35][36][37]. However, there is a lack of additional data on the epidemiology, life cycle, clinical signs, and description of larval stages in intermediate hosts, which impedes progress in the understanding of these parasites. This is also related to the limited diagnostic and monitoring methods, which has for long time been exclusively based on the identification of eggs in feces [1].
Abbreviata caucasica appears to be capable of living attached to the wall of the digestive tract between the esophagus and the small intestine in human and non-human primates [1,33]. However, clinical features of A. caucasica infestation in chimpanzees remain unknown at this time and further studies are needed to identify such features [6].
We developed a specific 12S qPCR-based assay for the detection of A. caucasica from biological samples and potential intermediate hosts, though the unique Abbreviata species DNA and target sequence from A. caucasica used to confirm the assay specificity may represent a limitation of the assay. In contrast, the newly cox1 A. caucasica specific PCR could be used to assess the identification of A. caucasica from hosts exposed to a wide range of nematode infestations. Since the PCR replicated the same result as the qPCR (p = 0.25), both tools were highly sensitive and specific in detecting A. caucasica, even the presence of coinfestations, avoiding the hard diagnosis based on egg identification. These tools can resolve problems related to the detection of larval stages from the intermediate and paratenic hosts and therefore avoid the sequencing identification by nematode generic primers. A detection limit as low as 1.13 × 10 −3 eggs per gram of positive feces, regardless of consistency, solves the problems associated with conventional protocols requiring fresh equipment [38]. Data generated by qPCR showed a rate ranging from 0.2 to 1.4 eggs/g according to the fecal consistency, the best record being 113 egg/g. Appleton and Henzi (1993), reported the same results from baboons in Natal, South Africa, where egg output of A. caucasica ranged from 0.32 to 1.48 eggs/g with 215 eggs/g as the best record [39]. These observations highlight the usefulness of the qPCR quantification protocol we developed to evaluate the load of A. caucasica eggs. We therefore developed a 5S pan-Nematoda qPCR for the global exploration of nematode infestations from different biological samples.
The absence of A. caucasica DNA from all environmental samples could be explained by the fact that they were not contaminated by the feces of infested hosts. However, despite the absence of A. caucasica DNA in the termite (Isoptera spp.) specimens that we tested, we cannot be sure if they are involved in the life cycle of A. caucasica or not. Termites (Isoptera) are the intermediate host of several nematodes such as A. antarctica, achanthocephalans (Thorny-headed worms), and Heterakis gallinarum [40][41][42]. Poinar and Quentin, (1972) experimentally demonstrated the ability of Blatella germanica and Schistocerca gregaria to develop the infective stage of A. caucasica. However, the life cycle of this nematode remains largely unknown. More than 28 paratenic and second intermediate hosts are also suspected [6]. However, we cannot be sure whether the environmental samples from species included in the diet of the chimpanzee population in our study, screened here, are not implemented in the life cycle of A. caucasica even in the absence of its DNA from all specimens. Termites are known to be the most prevalent arthropod in the chimpanzee diet [43].

Study Site and Study Subjects
Samples were collected at the Dindefelo Community Natural Reserve, located in the Kedougou region, southeastern Senegal, about 35 km from the town of Kedougou. The vegetation of the reserve is a sudano-guinean savanna woodland [44], one of the driest and more open habitats occupied by the species [45]. All chimpanzees live in multi-female/multi-male communities composed of flexible groups that fission and fuse [46]. At the time of data collection, some individuals were semi-habituated to observers, but the rest remained unhabituated and thus the exact community composition and size were unknown. Although the total home range of Dindefelo chimpanzees was not known, conspecifics living in savanna woodland habitats have extremely large home ranges (e.g., >85 km 2 , [47]). Based on size, the fecal samples analyzed in this study were assumed to belong to adults.

Fecal, Worms, and Environmental Samples
Two expeditions to the Dindefelo Community Natural Reserve in Senegal were undertaken in order to collect the samples. During the first trip (August 2016), 49 fecal samples of the western chimpanzee (Pan troglodytes verus) ( Figure 7A) were collected from three localities in the reserve: Locality 1, three decomposing "degraded" fecal samples (12.382539, −12.287977); Locality 2, six degraded fecal samples (12.381437, −12.290776); and Locality 3, thirty-eight fresh fecal samples (12.379919, −12.296830). The fecal samples were collected and stored at −80 • C. Two adult worms were recovered from two fresh feces in the field and stored in 70% ethanol. In shape and general appearance, these worms resembling Ascaris to the naked eye ( Figure 7B). All samples were transported to our laboratory at the Institut Hospitalo-Universitaire (IHU) Méditerranée Infection for further examination, and the adult worms were sent to the parasitology laboratory of the Department of Veterinary Medicine (University of Bari, Italy), where they were subjected to morphological identification. During the second expedition (August 2019), we targeted the potential contamination of these parasitic nematodes for the chimpanzees (e.g., the possible intermediate hosts that chimpanzees could eat or their water sources). A total of 113 environmental samples including the main species from the chimpanzee's diet were collected in the vicinity of chimpanzee sleeping sites and other areas frequented by the apes. These included 47 termites, 42 soil samples from termite mounds, 21 plant species, one sample from a water source used by the chimpanzees, a centipede, and one maggot. All samples were preserved in 70% ethanol and were transported to our laboratory for analysis.
Pathogens 2020, 9, x FOR PEER REVIEW 14 of 21 and other areas frequented by the apes. These included 47 termites, 42 soil samples from termite mounds, 21 plant species, one sample from a water source used by the chimpanzees, a centipede, and one maggot. All samples were preserved in 70% ethanol and were transported to our laboratory for analysis.

Morphological Analysis of A. caucasica Adult Worms
The female worms were processed for morphometric analysis. The body of the nematodes were measured and then cut into three pieces. The central part was subjected to DNA extraction for molecular identification. The cephalic and caudal ends of the worms were fixed and stained in lactophenol solution to observe anatomical structures. Digital images and measurements were made with an optic microscope Leica ® DM LB2 with differential interference contrast. The software Leica ® LASAF 4.1 was used for the image analysis process including the measuring of nematodes, which are provided in micrometers. The identification was carried out following the description made by Schulz, (1926), Ortlepp, (1926) and Brede and Burger (1977) [8,9,21].
The observation of structures in the cephalic region, the stout size of the nematode, a thick cuticle finely striated, a large cephalic collarette, the total length, and the distance from the anterior end to the vulva all confirmed the identification of this helminth as A. caucasica.

Identification of A. caucasica Eggs from Positive Feces
The exploration of A. caucasica eggs was investigated from two fecal samples from which the adult worms were collected. A formol-ether sedimentation method of fecal concentration was used [48]. Egg identification was carried out according to the key of Fain and Vandepitte (1964) [7], while the differential diagnosis was performed as described elsewhere [49].

DNA Extraction
Genomic DNA was extracted from 200 mg of fecal samples, adult worms of A. caucasica, and environmental specimens using the QIAGEN DNA tissue kit (QIAGEN, Hilden, Germany) following the manufacturer's recommendations. The extraction was performed after two lysis steps: (i) mechanical lyses performed on a FastPrep-24™ 5G homogenizer using high speed stirring for 40 s in the presence of the powder glass, and (ii) enzymatic digestion using the proteinase K in the

Morphological Analysis of A. caucasica Adult Worms
The female worms were processed for morphometric analysis. The body of the nematodes were measured and then cut into three pieces. The central part was subjected to DNA extraction for molecular identification. The cephalic and caudal ends of the worms were fixed and stained in lactophenol solution to observe anatomical structures. Digital images and measurements were made with an optic microscope Leica ® DM LB2 with differential interference contrast. The software Leica ® LASAF 4.1 was used for the image analysis process including the measuring of nematodes, which are provided in micrometers. The identification was carried out following the description made by Schulz, (1926), Ortlepp, (1926) and Brede and Burger (1977) [8,9,21].
The observation of structures in the cephalic region, the stout size of the nematode, a thick cuticle finely striated, a large cephalic collarette, the total length, and the distance from the anterior end to the vulva all confirmed the identification of this helminth as A. caucasica.

Identification of A. caucasica Eggs from Positive Feces
The exploration of A. caucasica eggs was investigated from two fecal samples from which the adult worms were collected. A formol-ether sedimentation method of fecal concentration was used [48]. Egg identification was carried out according to the key of Fain and Vandepitte (1964) [7], while the differential diagnosis was performed as described elsewhere [49].

DNA Extraction
Genomic DNA was extracted from 200 mg of fecal samples, adult worms of A. caucasica, and environmental specimens using the QIAGEN DNA tissue kit (QIAGEN, Hilden, Germany) following the manufacturer's recommendations. The extraction was performed after two lysis steps: (i) mechanical lyses performed on a FastPrep-24™ 5G homogenizer using high speed stirring for 40 s in the presence of the powder glass, and (ii) enzymatic digestion using the proteinase K in the appropriate buffer (QIAGEN, Hilden, Germany) for 12 h at 56 • C. The extracted DNA was then eluted in a total volume of 100 µL and stored at −20 • C.

Development of PCR Primer Sets
The primer sets used in this study are listed in Table 4. First, sequences of the cytochrome c oxidase I (cox1), 16S rRNA, 12S rRNA, and the internal transcribed spacer 2 (ITS2) genes were used to design primer sets targeting nematodes. For each PCR system, a fasta file was constructed from nematode sequences retrieved from the GenBank database. Sequences were aligned using BioEdit v7.0.5.3 software [50]. The highly conserved areas were submitted in Primer3 software v. 0.4.0 [51]. PCRs standardization was performed as described elsewhere [52]. Primers designed are reported in Table 4. In addition, primers Fwd.18S.631 and Rwd.18S.1825r, recently designed to amplify a partial fragment of the 18S rRNA gene of nematodes, were also used in this study (Table 4) [53]. These genes were chosen in order to compare the relatedness with Physaloptera and parasitic nematodes available in the GenBank database.

Polymerase Chain Reaction, Sequencing and Phylogenetic Analysis
All PCR reactions were carried out in a total volume of 50 µL, consisting of 25 µL of AmpliTaq Gold master mix (Thermo Fisher Scientific), 18 µL of ultra-purified water DNAse-RNAse free, 1 µL of each primer, and 5 µL of genomic DNA. PCR reactions with all systems were run using the following protocol: incubation step at 95 • C for 15 min, 40 cycles of 1 min at 95 • C, 30 s for the annealing at a different melting temperature for each PCR assay, and elongation for 45 s to 1 min and 30 s (Table 4)  The amplicons obtained from each gene examined were purified using the filter plate Millipore NucleoFast 96 PCR kit following the manufacturer's recommendations (Macherey Nagel, Düren, Germany). Purified DNAs were subjected to the second amplification using the BigDye™ Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). Then, BigDye PCR products were purified on the Sephadex G-50 Superfine gel filtration resin prior to sequencing on the ABI Prism 3130XL.
First, all nucleotide sequences were assembled and edited by ChromasPro 2.0.0. The absence of co-amplification of nuclear mitochondrial genes (numts) was verified for the cox1 DNA sequences, wherein the alignment was performed with the complete sequence of cox1 DNA from the P. rara mitochondrion sequence (MH931178) using the ClustalW application within Bioedit v.7.2.5. [50]. In addition, the visual verification of sequence chromatograms ambiguities, indels and stop codons of the translated sequences was performed using Chromas Pro 2.0.0 software as recommended [54]. Sequences amplified from the cox1, 16S, 12S, 18S rRNA, and ITS2 genes were subjected separately to a preliminary analysis using the Basic Local Alignment Search Tool (BLAST) [55].
The closely related sequences of Physaloptera and nematode species retrieved from the GenBank database were included in the study and a fasta file was constructed for each gene and then subjected to the alignment. In addition, alignment of nematode COI protein sequences was also performed. All alignments were conducted using the ClustalW application within Bioedit v.7.2.5. [50]. The conservation of amino acids between the COI sequences of A. caucasica relative to the sequences of Physaloptera was visualized on CLC Sequence Viewer 7 (CLC Bio Qiagen, Aarhus, Denmark).
From the alignment of each gene examined, the interspecific nucleotide pairwise distance (INPD) was evaluated to estimate the genetic divergence between all species included. Furthermore, the interspecific amino acid pairwise distance (IaaPD) was reproduced for COI-protein sequence alignment. standard errors were obtained by a bootstrap procedure with 1000 replicates using the maximum composite likelihood model [56] and Poisson correction model [57] for nucleotide and protein sequence alignments, respectively. Evolutionary analyses were inferred on MEGA6 software [58].
DNA sequences of Necator americanus (AJ920348) and Ascaris sp. (KC839986) were chosen as out-groups for18S rRNA and cox1, respectively, according to the fast-minimum evolution tree on BLAST [55]. The corresponding COI protein sequence of Ascaris sp. (AGN72537) was maintained as an out-group for the COI-protein alignment. The best model parameters with the lowest score were selected to generate phylogenetic trees of aligned 18S and cox1 sequences as well as COI protein sequences by running the MrBayes algorithm on each model using Topaliv2.5 software [59]. The Bayesian phylogenetic tree [60] was inferred for nucleotide sequence alignments using the K80 (+G) [61] and GTR (+G, +I) [62] models, respectively, while the Bayesian phylogenetic tree was inferred on the COI protein sequence alignment using Mtmam (+G) [63]. All phylograms were generated with five runs for 1,100,000 generations, 25% of burn-in length, and 1000 for sample frequencies.
In order to resolve the haplotype variations of Physaloptera species and A. caucasica, the Fasta file of the cox1 sequences was converted to the Nexus format using an online converter [64]. During the second time, a Templeton-Crandall-Sing (TCS) network phylogram [65] was inferred with a 95% connection limit and drawn with 1000 iterations using the PopArt software [66].
All qPCR reactions included 5 µL of DNA template, 10 µL of Master Mix Roche (Eurogentec), and 3 µL of ultra-purified water DNAse-RNAse free. Concentration of each primer, UDG, and each probe was 0.5 µL. The TaqMan reaction of both systems was run using the same cycling conditions. This included two hold steps at 50 • C and 95 • C for 2 and 15 min, respectively, followed by 40 cycles of two steps each (95 • C for 30 s and 60 • C for 30 s). The qPCR reaction was performed in a CFX96 Real-Time system (Bio-Rad Laboratories, Foster City, CA, USA) after activating the appropriate dye readers for each qPCR system.
A protocol for the quantification of eggs has been established to assess the analytical sensitivity of qPCR in the detection of fecal infestation. A 10-fold serial dilution of DNA extracted from 200 mg of fecal matter containing 113 eggs ( Figure 2) per gram (i.e., 22.6 eggs/100 µL of eluted DNA and 1.13 eggs/5 µL of qPCR reaction). Standard curves and derived parameters (PCR efficiency, Slope, Y-intercept, and correlation coefficient) were generated using CFX Manager Software Version 3 [70].
The molecular approaches described above were used to screen the presence of A. caucasica and other nematodes in chimpanzee fecal and environmental samples collected in a chimpanzee dormitory.

Conventional PCR Specific for A. caucasica
The use of universal pan-Nematoda primers does not allow for the identification of species-specific DNA sequences due to a non-specific amplification in co-infestations. A specific cox1-based PCR was developed in order to complete the identification of A. caucasica from fecal samples. The specific region for A. caucasica was analyzed for the design of the primers COI.51f and COI.601r, targeting 550 bp of the cox1 gene (Table 1). A. caucasica cox1 partial sequences herein amplified by the pan-Nematoda primers from the adult worms were aligned with Heliconema longissimum (AN: GQ332423) and Gongylonema pulchrum (AN: AP017685), representative members of Physalopteroidea and Gongylonematidae, respectively.

Molecular Survey of A. caucasica and Nematode Infestations in a Chimpanzee Population and the Environmental Samples
DNA from fecal samples of chimpanzee (n = 48) and environmental samples (n = 113) were screened for the DNA of A. caucasica and nematode using the 12S rRNA A. caucasica and the 5S rRNA pan-Nematoda qPCR assays, respectively. Positive samples for A. caucasica were also subjected to amplification and sequencing using the cox1 A. caucasica-specific primers.

Statistical Analysis
XLSTAT Addinsoft version 4.1 (XLSTAT 2019: Data Analysis and Statistical Solution for Microsoft Excel, Paris, France) was used for the statistical analysis. Results of qPCRs analysis were used to set a database using the Microsoft Excel ® program (Microsoft Corp., Redmond, Washington, USA). The effect of localities and fecal consistency on the infestation rates were tested using the Khi2 test or exact Fisher test. One-way analysis of variance (ANOVA) was performed to compare the predicted eggs from fresh and degraded feces. Negative samples and those with a studentized residual higher than 2.9 were removed before discarding the ANOVA test. McNemar's test was used to compare the detection accuracy of the qPCR and conventional PCR of A. caucasica from the chimpanzee samples. Significance level was considered at alpha ≤ 0.05 for all analyses.

Conclusions
A. caucasica measurements indicated the inconsistencies of certain indexes such as index a, b, and c ( Table 1) within this nematode, while it remains distinguishable from other Physaloptera species by the morphological features of the anterior and posterior ends as well as the presence of four uteri with a common trunk. However, the phylogenetic analyses showed that A. caucasica are clustered together with other monophyletic species of the Physaloptera genus. In the absence of strong morphological and epidemiological data, the species of Abbreviata may be re-classified as Physaloptera and a revision of the genus is needed. We developed specific and reliable molecular tools for the detection and egg quantification of A. caucasica from fecal samples. The tests can ultimately help to identify possible intermediates as well as paratenic hosts involved in the life cycle of A. caucasica. We therefore investigated its prevalence in a chimpanzee population from Senegal. Further studies are needed to clarify the epidemiology, circulation, life cycle, and possible pathological effects of A. caucasica, and the role of paratenic hosts or arthropods as intermediate hosts.
Supplementary Materials: The following are available online at http://www.mdpi.com/2076-0817/9/7/517/s1. Figure S1. Abbreviata caucasica partial COI-protein sequences alignment against Physaloptera species. The sequences of A. caucasica (selected box) obtained from adult worms were aligned against Physaloptera sequences available in the GenBank database. Residues were matched as dots. Conserved areas are indicated in blue, while the intensity of mutations is indicated by a foreground color (red to black). Figure S2. Quantification protocol of the 12S A. caucasica-specific qPCR. (A) Determination of detection limits and efficiency (eggs/g of fecal matter). (B) Standard curves generated from a serial 10-fold dilution of DNA.