Abstract
A wide range of hosts, especially birds, can be infested with Dermanyssus gallinae (D. gallinae), as an obligate hematophagous mite. In this study, cytochrome oxidase 1 (CO1) gene sequences were employed to perform molecular and phylogenetic analyses of D. gallinae collected from different bird species in Iran. Adult mites were collected from the body surface and cage material of ornamental and wild birds in industrial farms located in the Western and Northwestern regions of Iran. The infestation was identified in layer poultry farming by inspecting the eggs and the whole surfaces of the birds' bodies. The holding area and body surface of the ornamental and wild birds were also thoroughly examined. The D. gallinae samples were assigned to two subgroups of haplogroup A (i.e., A1 and A2). The phylogenetic tree suggested that the D. gallinae samples collected from wild birds in the A1 sub-haplogroup should be placed beside Japanese, Norwegian, Italian, and French samples isolated from wild birds in the A2 sub-haplogroup. Additionally, the highest phylogenetic similarity in the A2 sub-group was observed between mites isolated from ornamental and industrial birds in Australia. The findings of the present study suggest that crows and sparrows may play an important role in the transmission of D. gallinae infestation to other species of wild birds due to their high population, as well as their presence in most areas.
Keywords: CO1, Mite, Phylogenetic relationship, Reservoir host, Wild bird
1. Introduction
As an obligate hematophagous ectoparasite with a wide range of hosts, especially birds and rodents ( 1 ), the poultry red mite, Dermanyssus gallinae (Acari: Dermanyssidae), is globally considered the most damaging parasite of laying hens ( 2 ). Female mites have been known to feed more on blood, compared to male mites ( 3 ). The infestation of farmed birds and poultry with D. gallinae results in their dermatitis, weight loss, irritation, decreased egg production, anemia, and even death in severe cases, which in turn leads to huge economic losses ( 4 ). In the infested layer farms, viral and bacterial poultry diseases, including Colibacillosis, Salmonellosis, Pasteurellosis, and Newcastle ( 2 , 5 ), spread by D. gallinae as their vector ( 6 ). These mites have also caused a growing concern in human medicine since the infestation can cause skin lesions, such as gamasoidosis in humans, especially those in close contact with poultry ( 7 , 8 ).
The determination of the phylogenetic diversity of mites is critical for their tracing (i.e., specifying whether they are transmitted by the infected hosts or wild birds, which transfer between layer farms and breeding facilities ( 9 )). D. gallinae and D. hirundinis have been observed in eight and nine different phylogenetically-distant types of birds, respectively. The phylogenetic reconstruction of birds by Livezey and Zusi ( 10 ) showed the development of D. gallinae in Galliformes and Passeriformes, which are basally and distally situated in a large clade of Neognathae.
In this study, Partial cytochrome c oxidase subunit 1 (CO1) gene sequences were utilized to perform molecular analyses of D. gallinae isolated from different bird species in the West and Northwest of Iran.
2. Materials and Methods
2.1. Sampling
The adult mites were collected from the body surface and cage materials of turkeys, egg-laying poultry, and ornamental birds. In the case of wild birds, the samples were collected either from body surfaces or nests of birds, as shown in table 1 ( 11 - 14 ). The collected samples were placed in 70% ethanol and transferred to the parasitology laboratory of the Faculty of Veterinary Medicine in Urmia University, Urmia, Iran. Mites were identified using diagnostic entomological keys ( 15 ).
Table 1.
Birds and infestation rates of Dermanyssus gallinae regarding to sampling regions in Iran
| Birds: Types and Species | No. of Samples (Positive %) | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| Type | Common name | Scientific name | Ku | Hm | Zn | Wa | Ea | Ar | Total |
| Ornamental birds | Canary | Serinus canaria domestica | 10 (0) | 22 (4.5) | 8 (0) | 7 (14.3) | 36 (13.9)a | 5 (0) | 88 (7.9) |
| Pigeon | Columba livia domestica | 30 (10) | 32 (0) | 19 (5.3) | 23 (4.3) | 45 (6.7) | 10 (0)b | 159 (5) | |
| Budgerigar | Melopsittacus undulatus | 10 (10) | 22 (0) | 8 (0)c | 7 (14.3) | 36 (8.3) | 5 (0) | 88 (5.7) | |
| Finch | Taeniopygia guttata | 10 (0) | 22 (0) | 8 (25) | 7 (14.3)d | 36 (22.2) | 5 (20) | 88 (13.6) | |
| Parrots | Psittacula eupatrica | 10 (10) | 22 (4.5)e | 8 (0) | 7 (0) | 36 (2.8) | 5 (20) | 88 (4.5) | |
| Cockatiel | Nymphicus hollandicus | 10 (0)f | 22 (0) | 8 (0) | 7 (0) | 36 (5.5) | 5 (20) | 88 (3.4) | |
| Wild birds | Sparrow | Passer domesticus | 56 (1.8) | 110 (2.7) | 33 (0) | 193 (4.1) | 340 (4.7)h | 86 (2.3) | 818 (3.7) |
| Owl | Strix nebulosa | 0 | 2 (0) | 0 | 10 (10)i | 18 (11.1) | 0 | 30 (10) | |
| Crow | Corvus corone | 33 (6.1)j | 55 (1.8) | 21 (0) | 110 (3.6) | 189 (6.9) | 56 (1.8) | 464 (4.5) | |
| Partridge | Alectoris chukar | 0 | 0 | 9 (0) | 38 (7.9) | 138 (5.8) | 49 (2)k | 234 (5.1) | |
| Peacock | Pava muticus | 0 | 0 | 0 | 5 (0) | 4 (25)l | 0 | 9 (11.1) | |
| Industrial farms | Turkey | Meleagris gallopavo | 20 (5) | 15 (0) | 88 (6.8)g | 9 (11.1) | 92 (3.7) | 7 (0) | 231 (4.8) |
| Laying hens farm | 6 (16.7)m | 10 (10)n | 13(23.1)o | 19 (10.5)p | 22 (22.7)q | 8 (12.5)r | 78 (16.7) | ||
Sampling sites/regions: West (Ku=Kurdistan, Hm=Hamadan), and Northwest (Zn=Zanjan, Wa=West Azerbaijan, Ea=East Azerbaijan, Ar=Ardabil) in Iran.
Accession numbers: a=MT416629, b=MT416627, c=MT416624, d=MT416625, e=MT416628, f=MT416626, g=MT416608, h=MT416607, i=MT416605, j=MT416606, k=MT416609, l=MT416604, m=MT791527, n=MT791526, o=MT79130, p=MT791529, q=MT791528, r=MT791525.
2.2. DNA Extraction and Molecular Diagnosis
The mites were first crushed in 20-µl phosphate-buffered saline. Genomic DNA was extracted using MBST tissue kits (Molecular Biological System Transfer Company, Iran), according to the instructions given in the manufacturer's manual.
Molecular detection and proliferation of 400 bp fragments were performed using a pair of primers for mitochondrial CO1 gene (i.e., CO1F (5'-TGATTTTTTGGTCACCCAGAAG-3’) and CO1R (5'-TACAGCTCCTATAGATAAAAC-3’) ( 16 ). The reactions were regulated in a 25-µL volume of the polymerase chain reaction (PCR) reaction mixture. The PCR program was as follows: 10 min at 94°C, followed by 44 cycles at 94°C, 53°C, and 72°C for 1 min, and the final extension at 72°C for 4 min. Amplified products were confirmed using 1.2% agarose gel electrophoresis after staining with ethidium bromide (0.3 μg/ml) and visualization under UV light ( 16 ). Finally, the purified PCR products were sent to Takapozist Company (Tehran, Iran) for sequencing.
2.3. Sequencing and Genomic Analysis
All the obtained nucleotide sequences of each host and various mite collection regions were deposited in the GenBank with the assigned accession numbers (Table 1). A bioinformatics analysis was ultimately performed using the Basic Local Alignment Search Tool on the National Center for Biotechnology Information site, and the phylogenetic tree was examined in DNASIS Max 3.0, BioEdit 7.2, and MEGA X. The results were interpreted as follows:
Sequence trimming and editing construction were conducted using DNASIS MAX 3.0 (MiraiBio, Hitachi Ltd.) software. Sequence alignments were performed using BLOSUM matrices and CLUSTAL W tool in BioEdit 7.2 (http://www.mbio.ncsu.edu/BioEdit/bioedit.html) software. The sequences were compared in a table in BioEdit 7.2 based on the percentage of similarity and closeness of the sequence identity matrix and the analysis of maximum composite similarity. Phylogenetic relationship was examined and constructed by Maximum-likelihood method using the MEGA, version 6.0. The reliability of an inferred tree was tested by 1000 bootstrap.
The analysis was performed on the phylogenetic tree based on haplotypes and the classification proposed by Chu, Murano ( 7 ).
3. Results
Table 1 presents the rate of infestation with D. gallinae categorized by bird types and sampling locations. The highest infestation rate with the mites was observed at 16.7% in laying hen farms, followed by 13.6% in finch (i.e., ornamental birds group), and 11.1% in peacock (i.e., wild birds group). Figure 1 show the sequence alignment based on the D. gallinae samples isolated from ornamental, industrial, and wild birds in Urmia, Iran. A transition occurred between nucleotides T and C. This mutation was visible in all D. gallinae specimens isolated in this study, compared to the reference specimens. In nucleotides 617 and 731, transitions were observed both between A and G, as well as between C and T. These two Single Nucleotide Polymorphisms (SNP) were also observed in the D. gallinae isolated from wild birds. The alignment of amino acid sequences was performed in order to investigate the effects of SNP (Figure 2). The sequences, in this study, which were isolated from industrial, ornamental, and wild birds, showed a similarity of 100%. Compared to the isolates of other countries, the isolates in this study showed that SNPs had no effect on gene expression.
Figure 1.
Nucleotide sequence alignments based on D. gallinae samples isolated from ornamental, industrial, and wild birds in Urmia, Iran
Figure 2.
Effects of point mutations and the alignment of the amino acid sequences of the D. gallinae samples isolated from ornamental, industrial, and wild birds
Mites in Iran and other countries were clustered into haplogroups A, B, C, and D, according to the phylogenetic tree constructed based on the 400-bp CO1 nucleotide sequence (Figure 3) ( 7 ). Isolates of D. gallinae lay in A1 and A2 sub-haplogroups of haplogroup A. According to the phylogenetic tree, the D. gallinae samples isolated from wild birds in A1 sub-haplogroup were placed beside the isolates from Japan (LC029559, LC029476, LC029466, LC029463 and LC029553), Norway (MK599418), Italy (KC774792 and KC774766), and France (HQ842161 and FM208732), all of which were isolated from wild birds and assigned to a separate sub-haplogroup. The samples of D. gallinae in the A2 sub-haplogroup, which were isolated from ornamental and industrial birds, were placed beside those in a separate sub-haplogroup, collected from industrial birds in Turkey (KU866541), France (AM921858), and Australia (HQ842356). Based on phylogenetic analysis, the highest similarity (>98%) was observed in A1 subgroup among the isolates in this study (MT416604-09) and wild bird isolates from Italy (KC774766 and KC774792) and France (HQ842161 and FM208732). In addition, in the A2 subgroup, the highest similarity (>99%) was noted between the isolates from this study (MK791525-30 and MT424624-29) and those derived from ornamental and industrial birds in Australia (HQ842356). In the present study, the isolates derived from haplogroup A were classified into two sub-haplogroups (i.e., A1 and A2).
Figure 3.
Phylogenetic tree constructed using neighbor-joining and CO1 gene sequences of D. gallinae in Iran
4. Discussion
The phylogenic diversity of D. gallinae populations was investigated through sequencing mitochondrial CO1 gene amplicons, collected from six different regions in Iran. COI diversity in D. gallinae has demonstrated multiple lineages with comparative analysis, concluding that the presence of cryptic species is essential ( 17 - 19 ). The isolates from different regions in the world were used as reference isolates for comparing with the isolates used in the present study and drawing the phylogenetic tree. The ineffectiveness of these SNPs in the gene expression and the characteristics of functional proteins can be explained by the functional stability of this protein in evolution. There are a limited number of wild birds in Iran due to its climatic condition; therefore, most of the birds for the present study were selected from a zoo. The sequence analysis showed significant correlations between A1 and A2 sub-haplogroups in the CO1 tree and the sequences of haplotypes in France (AM921858), Australia (HQ842356), and Turkey (KU866541). The A2 sub-haplogroup, which is originally from Turkey, was also found in West Azerbaijan, Iran, because of a common geographical border between the two countries. The sequences of the mitochondrial CO1 gene can be used to reveal the history of hybridization between different lineages. In line with the results of a study by Brannstrom, Morrison ( 12 ), the differences in the CO1 sequences showed that two genotypes in the mites were associated with wild birds and domesticated poultry (both laying and ornamental). Limitations of the present study include sampling the mites from the nests of the wild birds, and the fact that although the experimental boxes used as nests were convenient in terms of mating location and ethological research, they were sometimes far from ornamental birds and poultry facilities. Regarding the identical genetic sequences of isolated mites, irrespective of geographical location and bird species, a similar sequence was detected among farm birds and different species of wild birds that nested near the farms.
The findings of the present study suggest the insignificant role of wild birds as a D. gallinae reservoir for commercial poultry production systems at least in Iran. Wild birds appeared to be infested with the D. gallinae acquired from crows and sparrows nesting in poultry farms. Since the population of crows and sparrows is high in all regions, the poultry production management system would be responsible for transmission routes by transporting live birds and eggs in rural regions and farms, as well as wildlife.
The isolates of D. gallinae were generally categorized as at least as two genotypes, one infesting wild birds, and the other egg-producing poultry. Moreover, wild birds were found not to play a major role as a reservoir for D. gallinae. Further studies are recommended to be conducted on CO1 diversity in other countries to gain a more comprehensive understanding of the present subject.
In conclusion, this study pioneered the molecular characterization of D. gallinae in Iran for the first time. These findings could be useful as the basis of future studies on D. gallinae infestation.
Authors' Contribution
Study concept and design: H. R.
Acquisition of data: H. R.
Analysis and interpretation of data: H. R.
Drafting of the manuscript: H. R.
Critical revision of the manuscript for important intellectual content: M. T. and B. E.
Statistical analysis: H. R.
Administrative, technical, and material support: M. T. and B. E.
Ethics
Ethical approval for this study was obtained from the Ethical Review Board in Urmia University, Urmia, Iran.
Conflict of Interest
The authors declare that they have no conflict of interest.
References
- 1.Bhowmick B, Tang Y, Lin F, Oines O, Zhao J, Liao C, et al. Comparative morphological and transcriptomic analyses reveal chemosensory genes in the poultry red mite, Dermanyssus gallinae. Sci Rep. 2020;10(1):17923. doi: 10.1038/s41598-020-74998-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Sigognault Flochlay A, Thomas E, Sparagano O. Poultry red mite (Dermanyssus gallinae) infestation: a broad impact parasitological disease that still remains a significant challenge for the egg-laying industry in Europe. Parasit Vectors. 2017;10(1):357. doi: 10.1186/s13071-017-2292-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Pritchard J, Kuster T, Sparagano O, Tomley F. Understanding the biology and control of the poultry red mite Dermanyssus gallinae: a review. Avian Pathol. 2015;44(3):143–53. doi: 10.1080/03079457.2015.1030589. [DOI] [PubMed] [Google Scholar]
- 4.Sparagano O, Pavlicevic A, Murano T, Camarda A, Sahibi H, Kilpinen O, et al. Prevalence and key figures for the poultry red mite Dermanyssus gallinae infections in poultry farm systems. Exp Appl Acarol. 2009;48(1-2):3–10. doi: 10.1007/s10493-008-9233-z. [DOI] [PubMed] [Google Scholar]
- 5.Sang-lk o, Noh G, Yi S, Do W, Kim E, You J. Molecular epidemiological characterization of poultry red mite (Dermanyssus gallinae) collected from Korea. Korean J Vet Serv. 2019;42(3):161–7. [Google Scholar]
- 6.Valiente Moro C, De Luna CJ, Tod A, Guy JH, Sparagano OA, Zenner L. The poultry red mite (Dermanyssus gallinae): a potential vector of pathogenic agents. Exp Appl Acarol. 2009;48(1-2):93–104. doi: 10.1007/s10493-009-9248-0. [DOI] [PubMed] [Google Scholar]
- 7.Chu TT, Murano T, Uno Y, Usui T, Yamaguchi T. Molecular epidemiological characterization of poultry red mite, Dermanyssus gallinae, in Japan. J Vet Med Sci. 2015;77(11):1397–403. doi: 10.1292/jvms.15-0203. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.George DR, Finn RD, Graham KM, Mul MF, Maurer V, Moro CV, et al. Should the poultry red mite Dermanyssus gallinae be of wider concern for veterinary and medical science? . Parasit Vectors. 2015;8:178. doi: 10.1186/s13071-015-0768-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Marangi M, de Luna CJ, Cafiero MA, Camarda A, le Bouquin S, Huonnic D, et al. Phylogenetic relationship between Dermanyssus gallinae populations in European countries based on mitochondrial COI gene sequences. Exp Appl Acarol. 2009;48(1-2):143–55. doi: 10.1007/s10493-009-9237-3. [DOI] [PubMed] [Google Scholar]
- 10.Livezey BC, Zusi RL. Higher-order phylogeny of modern birds (Theropoda, Aves: Neornithes) based on comparative anatomy. II. Analysis and discussion. Zool J Linn Soc. 2007;149(1):1–95. doi: 10.1111/j.1096-3642.2006.00293.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Alimehr M, Tavassoli M, Yousefian E. Evaluation of poultry red mite (Dermanyssus gallinae) susceptibity isolated from the layer farms to some acaricides. Iran J Vet Clin Sci. 2017;11(2):121–42. [Google Scholar]
- 12.Brannstrom S, Morrison DA, Mattsson JG, Chirico J. Genetic differences in internal transcribed spacer 1 between Dermanyssus gallinae from wild birds and domestic chickens. Med Vet Entomol. 2008;22(2):152–5. doi: 10.1111/j.1365-2915.2008.00722.x. [DOI] [PubMed] [Google Scholar]
- 13.Hobbenaghi R, Tavassoli M, Alimehr M, Shokrpoor S, Ghorbanzadeghan M. Histopathological study of the mite biting (Dermanyssus gallinae) in poultry skin. Vet Res Forum. 2012;3(3):205–8. [PMC free article] [PubMed] [Google Scholar]
- 14.Mohammadi Ghalehjoughi E, Tavassoli M, Naem S. Dermanyssus gallinae (Acari, Mesostigmata) in the Barn Swallow (Hirundo rustica) nests in Urmia suburb, North West of Iran. Persian J Acarol. 2017;6(2) [Google Scholar]
- 15.Wall R, Shearer D. Veterinary entomology: Arthropod ectoparasites of veterinary importance. Netherlands: Springer; 2012. [Google Scholar]
- 16.Marangi M, Cantacessi C, Sparagano OA, Camarda A, Giangaspero A. Molecular characterization and phylogenetic inferences of Dermanyssus gallinae isolates in Italy within an European framework. Med Vet Entomol. 2014;28(4):447–52. doi: 10.1111/mve.12050. [DOI] [PubMed] [Google Scholar]
- 17.Karp-Tatham E, Kuster T, Angelou A, Papadopoulos E, Nisbet AJ, Xia D, et al. Phylogenetic Inference Using Cytochrome C Oxidase Subunit I (COI) in the Poultry Red Mite, Dermanyssus gallinae in the United Kingdom Relative to a European Framework. Front Vet Sci. 2020;7:553. doi: 10.3389/fvets.2020.00553. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Roy L, Buronfosse T. Using mitochondrial and nuclear sequence data for disentangling population structure in complex pest species: a case study with Dermanyssus gallinae. PLoS One. 2011;6(7):22305. doi: 10.1371/journal.pone.0022305. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Rozas J, Ferrer-Mata A, Sanchez-DelBarrio JC, Guirao-Rico S, Librado P, Ramos-Onsins SE, et al. DnaSP 6: DNA Sequence Polymorphism Analysis of Large Data Sets. Mol Biol Evol. 2017;34(12):3299–302. doi: 10.1093/molbev/msx248. [DOI] [PubMed] [Google Scholar]