Morphological, molecular, and histopathological studies on Hadjelia truncata from Columba livia domestica and its role as an environmental biological indicator
Article Category: Research Paper
Data publikacji: 16 lis 2023
Zakres stron: -
Otrzymano: 13 lip 2023
DOI: https://doi.org/10.2478/jofnem-2023-0050
Słowa kluczowe
© 2023 Mohammed Albeshr et al., published by Sciendo
This work is licensed under the Creative Commons Attribution 4.0 International License.
Domestic pigeons (
Diagnosis is currently determined by the presence of nematodes under the koilin layer of the gizzard during necropsy.
The basic discriminating tool for the
This study was designed to shed more light on habronematid parasites, especially
A total of 48 domestic pigeons,
Pigeons were autopsied after being euthanized with lethal chloroform, and then alimentary tracts were removed to be examined under a stereomicroscope (Nikon SMZ18, NIS ELEMENTS software). The esophagus, crop, proventriculus, gizzard, duodenum, jejunum, ileum, ceca, and rectum were separated and placed into Petri dishes. Longitudinal incisions were made in all parts to expose their contents, and then nematode parasites were gently collected using fine-tip forceps in separate Petri dishes with normal saline (0.9%) and then fixed in either 70% ethanol (for light microscopic study) or 95% ethanol (for molecular study), or stored in −20°C (for heavy metal detection). Parasitological terms of the prevalence and mean intensity were calculated using equations per Bush
Parasites were cleared in lactophenol and then different body parts were photographed with the aid of a Leica DM 2500 microscope (NIS ELEMENTS software, ver. 3.8). Identification of the parasites was done according to Yamaguti (1961). Measurements were taken using ImageJ 1.53e software (Wayne Rasband and contributors, National Institute of Health, USA). Dimensions were given in micrometers (µm) and expressed as a range followed by mean in parentheses.
Genomic DNA (gDNA) was extracted using QIAamp® DNA Mini Kit (Qiagen, Germany) from ethanol-preserved samples with consideration of protocol steps. A partial fragment of the 18S rDNA region was amplified by PCR using the primer pair Nem 18SF, 5′-CGC GAA TRG CTC ATT ACA ACA GC-3′, and Nem 18SR, 5′-GGG CGG TAT CTG ATC GCC-3′, following the conditions described by Floyd et al. (2005). A partial fragment of the COX I gene was amplified using LCO1490, 5′-GGT CAA CAA ATC ATA AAG ATA TTG G-3′, and HC02198, 5′-TAA ACT TCA GGG TGA CCA AAA AAT CA-3′, with the recommended conditions of Folmer et al. (1994).
The PCR products were verified on a 1.5% agarose gel (Sigma-Aldrich, Missouri, USA) in 1 × Tris-acetate–EDTA (TAE) and post-stained with SYBR Safe DNA gel dye (Thermo Fischer Scientific, Ottawa, Canada) against the GeneRuler 100 bp Plus ready-to-use DNA ladder as a molecular weight marker (Fermentas, Lithuania) (Catalano et al., 2010). They were then visualized using a gel documentation system (Image Analyzer, Malvern, UK). PCR products were sent to Macrogen (Seoul, South Korea) to sequence the DNA. To identify related sequences, a BLAST search was conducted on the NCBI database. The DNA sequences were aligned using multiple alignments of CLUSTAL-X software (Thompson et al. 1997). Phylogenetic trees were constructed with maximum parsimony using MEGA ver. 7.0 (Kumar et al., 2018). Bootstrap analysis was performed based on 1,000 replicates to assess the robustness of the tree topologies.
Tissue specimens from the gizzards of pigeon-containing parasites were processed by paraffin embedding technique after fixation in 10% neutral buffered formalin for 24 hr according to the method of Banchroft and Stevens (1982). Specimens were washed in tap water and dehydrated in serial dilutions of ethyl alcohol. They were then cleared in xylene and embedded in paraffin. A cross-section of the gizzard was made 5 µm thick using a microtome, followed by deparaffinization, staining with hematoxylin, and eosin (H&E) stain examination. Sections were examined and photographed using a Leica DM 2500 microscope (NIS ELEMENTS software, version 3.8).
Pigeon tissues (liver, muscle, and gizzard) and parasites were analyzed to detect heavy metals according to the procedure described by UNEP/FAO/IOC/IAEA (1984). The samples were dried at 105°C for 1 hr in an oven. Samples were digested with 2 ml of concentrated hydrochloric acid overnight. After complete digestion, the samples were diluted with distilled water and then analyzed for trace elements using inductivity-coupled plasma iCAP-6500 Duo (Thermo Scientific, UK). Values of all monitored heavy metals are presented in mg/g wet weight. According to Sures et al. (1999), the bioaccumulation factor (BF) was determined as a ratio of the metal concentration in the parasite to that in the host tissue. Values from chemical analyses were presented as mean ± SE. Data obtained were analyzed using the SPSS v.18 software package (SPSS, Chicago, Illinois, USA).
Fourteen specimens of the examined domestic pigeons out of 48 (29.16%) were found to be naturally infected with
Adult worms are elongated with slender bodies, with the cuticle striated transversally and lateral alae absent. The mouth is surrounded by two well-developed trilobed lips. Interlabia are present. Cephalic papillae and amphidial pores are present on the outer surface of the lips. The pharynx is tubular. The oesophagus is divided into an anterior muscular portion and a glandular portion.
(Figure 1, Table 1): The body is 6.201–8.023 (7.270) mm long and 0.191–0.219 (0.204) mm wide. The pharynx is 0.036–0.041 (0.038) mm long and 0.074–0.093 (0.085) mm wide. The muscular oesophagus is 0.153–0.176 (0.167) mm long and the glandular portion ranges from 1.599–1.758 (1.623) mm long. The nerve ring is located at 0.184–0.201 (0.197) mm from the anterior end of the body. Testes are coiled and reflexed, extending through four-fifths of the body. The posterior extremity is provided with six pedunculated pairs of caudal papillae (4 preanal and 2 postanal). Two unequal spicules are present, the left one measuring 1.332–1.511 (1.416) mm long, and the right one 0.270–0.320 (0.284) mm long. Gubernaculum is absent. The tail is slightly twisted and bent in the ventral direction, carrying a well-developed caudal ala on each side.
Figure 1:
Photomicrographs for the
Morphological characteristics of male worms for
| Cram, 1927 | 5–7 | – | – | – | – | 0.220 | 1.6 | Six pairs (4 pre- & 2 post-) of caudal papillae | – |
| Baer, 1954 | 8 | 0.285 | 0.033 | – | – | 0.225–0.300 | 1.320–1.600 | – | |
| Ibrahim et al., 1995 | 4–8 (6.4) | – | 1.8–2.7 (2.2) | – | – | – | – | ||
| Junker & Boomker, 2007 | 7–8 | 0.145–0.160 | 0.042–0.044 | 1.750–1.927 | 0.208–0.212 | 0.254–0.271 | 1.346–1.434 | – | |
| Razmi et al., 2007 | 7–9 | – | – | – | – | 0.34 | 1.26 | – | |
| Sentíes-Cué et al., 2011 | 6.5–9 | – | – | – | – | 0.35 | 1.27 | – | |
| Naem et al., 2013 | 6.5–9 | 0.151 | – | – | – | 0.35 | 1.27 | – | |
| Nabavi et al., 2013 | 1–2 | – | – | – | – | 0.320–0.350 | 1.410–1.470 | – | |
| Al-Moussawi, 2015 | 7–8 (7.5) | 0.204–0.258 (0.231) | 0.374–0.386 (0.380) | 1.620–1.680 (1.650) | 0.198–0.211 (0.204) | 0.240–0.270 (0.255) | 1.512–1.620 (1.566) | 0.127–0.138 (0.132) | |
| Oryan et al., 2016 | 7.5 | – | – | – | – | – | – | – | |
| Jameel et al., 2016 | 6.5–9 | – | – | – | – | – | – | – | |
| Khordadmehr et al., 2018 | 7–11 | – | – | – | – | – | – | – | |
| Present study, 2023 | 6.201–8.023 (7.270) | 0.191–0.219 (0.204) | 0.353–0.376 (0.367) | 1.599–1.758 (1.623) | 0.184–0.201 (0.197) | 0.270–0.320 (0.284) | 1.332–1.511 (1.416) | 0.118–0.127 (0.122 | |
(Figure 1, Table 2): The body is 12.270–18.583 (17.792) mm long and 0.256–0.314 (0.287) mm wide. The pharynx is 0.041–0.044 (0.042) mm long and 0.099–0.012 (0.11) mm wide. The muscular oesophagus is 3.515–3.759 (3.601) mm long and its glandular portion is 2.160–2.211 (2.181) mm long. The nerve ring is located at 0.253–0.295 (0.282) mm from the anterior end of the body. The vulva is located in front of the posterior end of the oesophagus at a distance of 3.011–3.054 (3.027) mm from the anterior extremity of the body, and appears as a rounded aperture with a thickened cuticular edge. The vagina is elongated and divided into two divergent branches of the uteri that are filled with numerous oval and thick-shelled eggs, which become embryonated
Morphological characteristics of female worms for
| Cram, 1927 | 10–16 | 0.300 | – | – | – | 2.6–16 | 0.027 | – | Numerous embryonated eggs covered with thick-shelled | – |
| Baer, 1954 | 8–16 | 0.143–0.285 | – | – | – | 1.640–3.000 | – | – | – | |
| Ibrahim et al., 1995 | 17–22 (20) | – | 2.0–2.4 (2.4) | – | 1.5–2.5 (2) | 0.050 | 0.030 | – | ||
| Junker & Boomker, 2007 | 10–11 | 0.140–0.217 | 0.005–0.007 | 1.948–2.076 | 0.159–0.185 | 1.691–2.238 | 0.050–0.053 | 0.032–0.035 | 0.121–0.138 | |
| Razmi et al., 2007 | 13–17 | – | – | – | – | – | – | – | – | |
| Al-Moussawi, 2008 | 14–15.22 (14.62) | 0.168–0.199 (0.183) | 0.512–0.57 (0.54) | 0.589–0.622 (0.605) | 0.003–0.024 (0.015) | – | – | – | 0.113–0.150 (0.132) | |
| Mohammad & Al-Moussawi, 2011 | 11.10–14.88 (13.15) | 0.18–0.28 (0.23) | 0.450–0.566 (0.461) | 1.47–3.32 (2.435) | – | 1.575–2.730 (2.281) | 0.033–0.057 (0.044) | 0.015–0.041 (0.034) | – | |
| Sentíes-Cué et al., 2011 | 12–16.5 | – | – | – | – | – | – | – | – | |
| Naem et al., 2013 | 12 | 16.5 | 0.229 | – | – | 2.155 | – | – | – | |
| Nabavi et al., 2013 | 3–5 | – | – | – | – | 0.054–0.059 | 0.030–0.032 | |||
| Al-Moussawi & Jassim, 2015 | 23.226–26.156 (24.594) | 0.231–0.312 (0.2782) | 3.510–3.666 (3.588) | – | 0.260–0.312 (0.291) | 3.276–3.413 (3.364) | 0.052–0.104 (0.078) | 0.021–0.312 (0.026) | 0.104–0.234 (0.148) | |
| Al-Moussawi, 2015 | 8–17 (12) | 0.147–0.270 (0.224) | 2.750–3.648 (3.379) | 2.002–2.835 (2.215) | 0.178–0.206 (0.192) | 1.501–3.022 (2.108) | 0.046–0.048 (0.047) | 0.024–0.0408 (0.047) | 0.145–0.230 (0.196) | |
| Oryan et al., 2016 | 19.8 | – | – | – | – | – | 0.045 | 0.031 | – | |
| Jameel et al., 2016 | 12–16.5 | – | – | – | – | – | – | – | – | |
| Khordadmehr et al., 2018 | 15–20 | – | – | – | – | – | 0.043–0.045 | 0.020–0.030 | – | |
| Present study, 2023 | 12.270–18.583 (17.792) | 0.256–0.314 (0.287) | 3.515–3.759 (3.601) | 2.160–2.211 (2.181) | 0.198–0.225 (0.211) | 3.011–3.054 (3.027) | 0.046–0.051 (0.048) | 0.029–0.033 (0.031) | 0.118–0.132 (0.124) | |
(Figures 2, 3) Amplification of both partial 18S rDNA and cytochrome oxidase C subunit 1 (COX I), of the nematode
Figure 2:
A consensus phylogenetic tree constructed with neighbor joining (NJ) and maximum likelihood (ML) methods, showing phylogenetic relationships between
Figure 3:
A consensus phylogenetic tree constructed with neighbor joining (NJ) and maximum likelihood (ML) methods, showing phylogenetic relationships between
Only three sequences of the 18S rDNA related to
Three sequences were also obtained from the COX I region and were given the accession numbers OR122277 to OR122297 in GenBank. There are no COX I sequences related to
Macroscopic examination showed enlargement of the infected gizzard in the domestic pigeon (Figure 4). Moreover, there is noticeable damage to the koilin layer of the gizzard, with necrosis of the mucosal cells and interstitial infiltration of inflammatory cells in the lamina propria and muscular layer, and the presence of numerous cross-sections of nematode parasites between the koilin layer and glandular layers (Figure 5).
Figure 4:
Photographs of the gizzards of the domestic pigeons. (A) Non-infected gizzard. (B) Enlargement of the infected gizzard. (C) Normal koilin layer. (D and E) Nematodes infection under the koilin layer of the gizzard.
Figure 5:
(A–C) Histopathological sections from the gizzard’s wall infected with
Significant differences in concentrations of analyzed metals were observed among the different pigeon tissues (Tables 3–5). These metals were classified as essential metals of iron (Fe), copper (Cu), and zinc (Zn) and non-essential metals of cadmium (Cd), chromium (Cr), and cobalt (Co). There was a significant decrease in the concentration of metals in the pigeon tissues infected by nematode parasites compared to non-infected ones. The liver was considered as the main site of heavy metal storage, especially for Fe, while the gizzard had the lowest levels of analyzed metals.
Heavy metals in the liver of the domestic pigeons and its parasite
| Fe | 5.907 ± 0.02 | 1.022 ± 0.02 a | 1.463 ± 0.02 ab |
| Cu | 0.608 ± 0.02 | 0.132 ± 0.01 a | 0.313 ± 0.00 ab |
| Zn | 0.935 ± 0.02 | 0.253 ± 0.02 a | 0.522 ± 0.02 ab |
| Cd | 0.046 ± 0.001 | 0.011 ± 0.001 a | 0.036 ± 0.001 ab |
| Cr | 0.617 ± 0.02 | 0.341 ± 0.01 a | 0.518 ± 0.02 ab |
| Co | 0.212 ± 0.01 | 0.104 ± 0.01 a | 0.114 ± 0.01 ab |
Values are means ±SD.
significance at p ≤ 0.05 against a control group,
significant at p ≤ 0.05 against the infected group.
Heavy metals in the muscle of the domestic pigeons and its parasite
| Fe | 2.222 ± 0.02 | 0.873 ± 0.02 a | 1.463 ± 0.02 ab |
| Cu | 0.582 ± 0.02 | 0.128 ± 0.01 a | 0.313 ± 0.01 ab |
| Zn | 0.974 ± 0.02 | 0.324 ± 0.02 a | 0.522 ± 0.02 ab |
| Cd | 0.050 ± 0.001 | 0.016 ± 0.001 a | 0.036 ± 0.001 ab |
| Cr | 0.592 ± 0.02 | 0.092 ± 0.001 a | 0.518 ± 0.02 ab |
| Co | 0.473 ± 0.01 | 0.085 ± 0.01 a | 0.114 ± 0.01 ab |
Values are means ±SD.
significance at p ≤ 0.05 against a control group,
significant at p ≤ 0.05 against the infected group.
Heavy metals in the gizzard of the domestic pigeons and its parasite
| Fe | 2.176 ± 0.02 | 0.741 ± 0.02 a | 1.463 ± 0.02 ab |
| Cu | 0.690 ± 0.01 | 0.120 ± 0.01 a | 0.313 ± 0.01 ab |
| Zn | 0.911 ± 0.02 | 0.409 ± 0.01 a | 0.522 ± 0.02 ab |
| Cd | 0.034 ± 0.001 | 0.010 ± 0.001 a | 0.036 ± 0.001 ab |
| Cr | 0.559 ± 0.01 | 0.267 ± 0.01 a | 0.518 ± 0.02 ab |
| Co | 0.166 ± 0.01 | 0.086 ± 0.001 a | 0.114 ± 0.01 ab |
Values are means ±SD.
significance at p ≤ 0.05 against a control group,
significant at p ≤ 0.05 against the infected group.
Parasites were also capable of accumulating higher levels of metals than those in the tissues of infected pigeons. The bioaccumulation factor was more significant in muscles than in other tissues of infected pigeons with
Bioaccumulation of heavy metals concerning nematode parasite/pigeon model
| Fe | 1.431 | 1.675 | 1.974 |
| Cu | 2.371 | 2.445 | 2.608 |
| Zn | 2.063 | 1.611 | 1.276 |
| Cd | 3.272 | 2.250 | 3.600 |
| Cr | 1.519 | 5.630 | 1.940 |
| Co | 1.096 | 1.341 | 1.325 |
Domestic pigeons play an essential role in the social economy throughout the world (Aldamigh et al., 2022). Endoparasites, especially gastrointestinal nematodes, are responsible for severe health problems in domestic pigeons resulting in severe economic losses (Adang et al., 2008). This study explored the
In this study, 29.16% (14/48) of
This study represents the first report determining the prevalence of this parasite in Riyadh, Saudi Arabia. Previously, few studies have reported pigeon infection with this parasite in Egypt (Tadros and Iskander, 1975; Ibrahim et al., 1995; Abd-Alhadi and Al-Awady, 2020), Iraq (Al-Attar and Abdul-Aziz, 1985; Shubber, 2010; Mohammad and Al-Moussawi, 2011; Al-Moussawi, 2015; Al-Moussawi and Jassim, 2015; Jameel et al., 2016), Cyprus (Appleby et al., 1995), Iran (Razmi et al., 2007; Radfar et al., 2012; Nabavi et al., 2013; Khordadmehr et al., 2018), and California (Sentíes-Cué et al., 2011; Naem et al., 2013).
The morphological characteristics of the habronematid species recovered from the gizzard of pigeons were consistent with the key descriptions previously presented for the
Analysis of the 18S rDNA sequences obtained in the present study showed two haplotypes with only one transition at position 116 of the alignment. Sequences obtained from the present study grouped with Habronematidae confirming the morphological identity of the worm detected in the present study. Kelly et al. (2013) obtained partial 18S rDNA sequences from
One of the partial 18S rDNA sequences of
The current study confirms the harmful effects on the host pigeon from nematode parasites that have been reported previously (Appleby et al., 1995). The most prominent pathological findings in the gizzards of the infected
One of the biggest issues facing the earth today is environmental pollution. To identify pollutants in specific contaminants, biological indicators such as microorganisms are exploited (Zaghloul et al., 2020; Baghdadi et al., 2023). Our study displays the extent of metal accumulation in domestic pigeons and their parasites. Our findings supported the previous study of Al Quraishy et al. (2019), which found that accumulation of heavy metals in various organs was higher in non-infected pigeons than in infected ones. Our results regarding heavy metal accumulation in pigeon tissue also corroborate the findings of Ghita et al. (2009), who reported high concentrations of trace elements in avian feed, which not only accumulated in various body tissues but also polluted the soil and water on the avian litter.
In addition, Boncompagni et al. (2003) reported that higher concentrations of these metals may affect metabolic processes through the replacement of essential elements at the active sites of biologically important molecules, thus indirectly inducing nutritional deficiencies. Our study found significantly higher concentrations of six metals (Fe, Cu, Zn, Cd, Cr, and Co) in parasitic species
The present study provides valuable information about the occurrence of a habronematid species identified as