Document Type : Infectious agents- Diseases- Surgery

Authors

1 Department of Parasitology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran

2 Department of Pathobiology, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran

3 Department of Parasitology, Faculty of Veterinary Medicine, University of Tehran, Tehran- Iran

4 Department of Internal Medicine, Faculty of Veterinary Medicine, University of Tehran, Tehran- Ira

Abstract

 
BACKGROUND: Anaplasmosis is caused by an obligate intracellular, gram-negative microorganism, which be-longs to the family Anaplasmatacea and can be transmitted by ticks and other arthropods.

OBJECTIVES: The present study aimed to investigate the status of Anaplasma spp. infection by microscopy and molecular methods in dromedary camels in Bushehr province, Iran.

METHODS: A total of 139 blood samples were collected from dromedary camels in Bushehr province. Giemsa staining and nested-polymerase chain reaction (PCR) were conducted to detect Anaplasma infection in the drome-dary camels.

RESULTS: We found that 27 (19.4%) out of the total 139 blood samples were suspected for the presence of Ana-plasma spp. by morphological study. The PCR and nested-PCR sequencing results showed 111 (80%) and 134 (96%) samples positive for Anaplasma spp. and BLAST search in NCBI GenBank presented 100% identity with Candidatus Anaplasma camelii.

CONCLUSIONS: The molecular results presented the high frequency of Candidatus Anaplasma camelii in camels, in Bushehr city.

Keywords

Article Title [فارسی]

بررسی مورفولوژیک و مولکولی عفونت آناپلاسما در شتر یک کوهانه در استان بوشهر، ایران

Authors [فارسی]

  • زهرا مرادی 1
  • الهه ابراهیم زاده 2
  • پرویز شایان 3
  • فیصل ضرغامی 4

1 گروه انگل شناسی، دانشکده دامپزشکی دانشگاه تهران، تهران-ایران

2 گروه پاتوبیولوژی، دانشکده دامپزشکی دانشگاه فردوسی مشهد، مشهد، ایران.

3 گروه انگل شناسی، دانشکده دامپزشکی دانشگاه تهران، تهران-ایران

4 گروه بیماری های داخلی، دانشکده دامپزشکی دانشگاه تهران، تهران-ایران.

Abstract [فارسی]

 
زمینه مطالعه: آناپلاسموزیس توسط یک جرم داخل سلولی اجباری گرم منفی متعلق به خانواده آناپلاسماتاسهآ ایجاد میشود که میتواند توسط کنه ها و
سایر بندپایان منتقل شود.
هدف: هدف مطالعه حاضرتعیین وضعیت آلودگی آناپلاسما در شترهای یک کوهانه استان بوشهر با روش مولکولی و میکرسکوپی است.
روش کار: در تحقیق حاضر 139 نمونه خون از شترهای استان بوشهر جمع آوری شد. رنگ آمیزی گیمسا و PCR-nested به منظور بررسی حضور عفونت
آناپلاسما در شترهای یک کوهانه استفاده شد. همچنین نسبت به شناسایی ملکولی گونه اناپلاسما اقدام گردید.
نتایج:در این بررسی در27) 4/19 (%نمونه خون از مجموع 139 گسترش خون با روش میکروسکوپی اجرام آناپلاسمایی مشاهده گردید. نتایج PCR وجود
آلودگی آناپلاسما را در 111)80 (%نمونه و نتایج PCR-nested وجود آلودگی را در 134) 96 (%نمونه تایید کرد. نتایج تعیین توالی و آنالیز آن نشان دهنده
شباهت 100 %با نمونه های تعیین توالی شده کاندیداتوس آناپلاسما کاملی بود.
نتیجه گیری نهایی: نتایج مولکولی نشان داد فراوانی کاندیداتوس آناپلاسما کاملی در شترهای یک کوهانه در شهرستان بوشهر بالا بود. 

Keywords [فارسی]

  • آناپلاسما
  • آناپلاسموزیس
  • کاندیداتوس آناپلاسما کملی
  • شتر یک‌کوهانه
  • مطالعه مولکولی

Introduction

 

Anaplasma spp. are obligate intracellular organisms that cause anaplasmosis in animals and humans. The members of the Anaplasmatacea family can be transmitted by ticks and other arthropods. The genus Anaplasma entails diverse species, such as A. marginale, A. centrale, A. phagocytophilum, A. bovis, A. ovis, and A. platys (Rymaszewska et al., 2008). Up to now, A. phagocytophilum, A. ovis, and newly diagnosed Candidatus Anaplasma camelii (genetically close to A. platys) have been detected in camels by molecular methods (Bahrami et al., 2018; Bastos et al., 2015; Li et al., 2015; Noaman, 2018).

  1. phagocytophilum propagates in polymorphonuclear leucocytes causing granulocytic anaplasmosis in humans, tick-borne fever in ruminants, in addition to canine and equine granulocytic anaplasmosis (Rymaszewska et al., 2008). A. ovis is an intraerythrocytic pathogen of small ruminants (de la Fuente et al., 2004). A. platys have been reported as an intraerythrocytic rickettsia that causes canine cyclic thrombocytopenia with different clinical symptoms, including fever, anorexia, lethargy, respiratory distress, mucous hypersecretion, purulent ocular discharge, splenomegaly, and muzzle hyperkeratosis (Sainz et al., 1999).

Bastos et al. (2015) reported a new species close to A. platys in Saudi Arabia and named it Candidatus Anaplasma cameli. Limited information is available concerning the presence of Anaplasma species in Iranian dromedaries. A microscopic study demonstrated Anaplasma spp. infection in camels in Iran (Ghazvinian et al., 2017). Some molecular evaluations have revealed A. phagocytophilum, A. ovis, and Candidatus A. cameli in Iranian camels (Bahrami et al., 2018; Noaman, 2018; Sharifiyazdi et al., 2017).

Bushehr province is one of the main camel breeding areas in Iran. Therefore, we investigated the presence of Anaplasma spp. in the dromedarian camels in Bushehr province, Iran.

Materials and Methods

Sampling and Morphological Investigation

Blood samples were collected from 139 healthy or anemic dromedaries throughout Bushehr province with a mean annual rainfall of 237-350 mm in the South of Iran between 28° 58' 59.99" N latitude and 50° 48' 59.99" E longitude. A thin layer of blood was spread on a clean dry microscopic glass slide, allowed to dry, and stained by Giemsa staining. In summary, the smears were immersed in pure methanol for fixation and allowed to air dry for 30 sec. The slide was flooded with 10% Giemsa stain solution for 45 min.

DNA Extraction and PCR

Total DNA was extracted from the blood samples employing a DNA extraction kit (MBST, Tehran, Iran) according to the manufacturer’s instructions. The DNA extraction was evaluated using common primer pairs (camel-act F: 5′-ttacaatgagctgcgtgtgg-3′ and camel-act R: 5′-gagtccatcacgatgccagt-3′ derived from the β actin gene of camels. Nested polymerase chain reaction (PCR) was performed according to the method of Noman (Noaman et al., 2009). Briefly, the samples infected with Anaplasma were assessed for the presence of the 16S rRNA gene of this rickettsia by PCR.

Two sets of primers were designed based on the 16S rRNA gene of Anaplasma (M60313). The first DNA amplification was carried out using primers F1 (5′-agagtttgatcctggctcag-3′) and R1 (5′-agcactcatcgtttacagcg-3′) of 16S rRNA sequences. To control the specificity of the PCR products for the 16S rRNA gene of Anaplasma spp., nested PCR was applied in which the additional primers F2 (5′-gcaagcttaacacatgcaagtc-3′) and R2 (5′-gttaagccctggtatttcac-3′) belonged to the same gene.

In PCR and nested PCR, about 20 ng of DNA solution was utilized in a total volume of 100 μL, including 10x PCR buffer, 2.5 U Taq DNA polymerase (Sinaclon, Iran), 20 μM of each primer (Sinaclon, Iran), 100 μM dNTPs (Fermentas), and 1.5 mM MgCl2 (Sinaclon, Iran). The reaction was completed in a thermal cycler (Bio-Rad) with the following program: 5 min incubation at 95°C followed by 35 cycles of 45 sec at 95°C (denaturation), 45 sec at 59°C or 55°C (annealing), 45 sec at 72°C (extension), and a final extension at 72°C for 5 min. Positive (available from previous work) and negative controls were used in each PCR. The annealing temperature for PCR using primers derived from the β-actin gene of camelids was 50°C. Next, the PCR products were electrophoresed on 1.5% agarose gel, stained with Cybersafe, and visualized under UV light. Purified nested PCR products were sequenced by Kowsar Company (Tehran, Iran). The resultant nucleotide sequences were analyzed by the basic local alignment search tool (BLAST) on the National Center for Biotechnology Information (NCBI) database website (http://www.ncbi.nlm.nih.gov/blast). A phylogenetic tree was created with MEGA 6 software (USA) applying the maximum likelihood method with bootstrap analysis (1,000 replicates).

Results

Anaplasma spp. was detected in 27 (19.4%) of 139 blood smears by microscopy. For molecular characterization, first, the efficient isolation of DNA was ensured from each sample using the specific primers designed from the β-actin gene of camelids. Each sample was analyzed by PCR and nested PCR by two pairs of primers designed based on 16S rRNA and sequencing in the presence of Anaplasma spp. The PCR product was observed at 781 bp in PCR and 543 bp in nested PCR (Figure1). In the present study, Anaplasma spp. infection was detected in 111 (80%) specimens by PCR and in 134 (96%) cases by nested PCR. The similarity between all the sequenced Anaplasma spp. was identified to be 100% with Candidatus A.camelii from Iran (KX765882) and Saudi Arabia (KF843825.1-KF843823). The phylogenetic tree represented two major branches for Anaplasma spp., one of which contained Candidatus A. camelii and A. platys and the other included other Anaplasma spp. Wolbachia was in a separate branch (Figure 2).

 

Figure 1. PCR products and nested-PCR products were used to detect Anaplasma. Lane 1= Neated-PCR product; Lane 2= positive control; Lane 3= DNA marker; Lane 4, 5= PCR product; Lane 6= positive control; Lane 7= negative control

Figure 1. PCR products and nested-PCR products were used to detect Anaplasma. Lane 1= Neated-PCR product; Lane 2= positive control; Lane 3= DNA marker; Lane 4, 5= PCR product; Lane 6= positive control; Lane 7= negative control

 

Figure 2. Molecular Phylogenetic analysis based on the 16S rDNA gene of Anaplasma obtained from dromedary camel in Bushehr province

Figure 2. Molecular Phylogenetic analysis based on the 16S rDNA gene of Anaplasma obtained from dromedary camel in Bushehr province (showed with ●) by using Maximum Likelihood method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. Evolutionary analyses were conducted in MEGA6.

 

 

Discussion

 

Anaplasmosis in camels is more commonly referred to as a subclinical disease and sometimes has symptoms, such as weakness, anorexia, and anemia. In the current investigation, microscopy and molecular techniques were used to detect Anaplasma spp. in dromedary camels in Bushehr province, Iran.

We determined the frequency of Anaplasma spp. microscopically in 19.4% of blood smears. The frequency of Anaplasma spp. infection using microscopy method has been reported as, 40.5% in Saudi Arabia (Ismael et al., 2016), 17% in Tunisia (Ismael et al., 2016; Selmi et al., 2019), and 17.4% in Iran (Ghazvinian et al., 2017). Some other studies did not detect Anaplasma in blood smears in Iran (Sazmand et al., 2019; Sharifiyazdi et al., 2017) and China (Li et al., 2015). The discrepancy in the results may be attributed to climatic conditions or misdiagnosis with Howell-Jolly's body or other pathogens, such as Mycoplasma. Considering the similarity of Anaplasma spp. to Howell-Jolly's body or other pathogens that lead to misdiagnosis, microscopic examination is not an appropriate method especially for the detection of reservoir animals.

Due to the disadvantages of microscopic techniques, the best method of diagnosis is molecular methods. The 16S rRNA gene with a high copy number was found to be convenient and appropriate for the detection of Anaplasma spp. even in small amounts. The molecular examinations (PCR and nested PCR) showed that 80% and 96% of camels were infected with Anaplasma species. Comparison between the results of microscopy and molecular methods revealed that the microscopic method was not enough sensitive (19.4% vs. 96% infection rate).

Similar molecular studies indicated the infection rate of Anaplasma spp. in camels as 15% and 6% in Iran (Sazmand et al., 2019; Sharifiyazdi et al., 2017), 30% in Saudi Arabia (Bastos et al., 2015), 7.2% in China (Li et al., 2015), 17.7% in Tunisia (Belkahia et al., 2015), 39.62% in Morocco (Lbacha et al., 2017), 13.3% in Pakistan (Azmat et al., 2018), and 68.67% in Kenya (Kidambasi et al., 2019). The infection rate of Anaplasma in the present study was higher than previous reports from Iran (Sazmand et al., 2019; Sharifiyazdi et al., 2017) and other parts of the world (Azmat et al., 2018; Bastos et al., 2015; Belkahia et al., 2015; Lbacha et al., 2017; Li et al., 2015). The reason may be that we sampled the camels with the symptoms of anemia.

In this study, sequence analysis showed the highest identity (100%) between our sequences and the Candidatus A. camelii sequence from Saudi Arabia. There is a notable nucleotide difference in the hypervariable region of the 16S rDNA gene between the obtained sequence in the present study and A. platys. In line with other researchers, we believe that Candidatus A. camelii is an independent species. However, more extensive research is required on other Anaplasma genes. Genetic analysis of the 16S rDNA gene of dromedary camels in the hypervariable region revealed 100% identity with Candidatus A. camelii that was previously reported from Fars province, Iran (Sharifiyazdi et al., 2017) and Saudi Arabia (Bastos et al., 2015).

  1. phagocytophilum and A. ovis were also reported in camels in Iran (Bahrami et al., 2018; Noaman, 2018). A phylogenetic tree of the achieved sequences in the current study with sequences submitted in GenBank revealed two major branches for Anaplasma spp. One branch included Candidatus A. camelii and A. platys and the other entailed other Anaplasma spp. Wolbachia was in a separate branch. Li et al. (2015) also generated a separate cluster from A. platys and Candidatus A. camelii in phylogenetic trees based on 16S rDNA.

Conclusion

The molecular results showed the high frequency of Candidatus A. camelii in camels in Bushehr province. Furthermore, molecular examination (96%) demonstrated a higher and more accurate frequency than microscopic examination (19.4%). We believe that Candidatus A. camelii is a different species from A. platys in dromedarian camels.

Acknowledgments

This research was supported by grants from the University of Tehran. We are grateful to the Research Council of the University of Tehran and the Research Council of the Faculty of Veterinary Medicine of the University of Tehran.

Conflict of Interest

There is no conflict of interest.

Azmat, M., Ijaz, M., Farooqi, S. H., Ghaffar, A., Ali, A., Masud, A., ... & Zhang, H. (2018). Molecular epidemiology, associated risk factors, and phylogenetic analysis of anaplasmosis in camel. Microbial Pathogenesis123, 377-384. [DOI:10.1016/j.micpath.2018.07.034] [PMID]
Bahrami, S., Hamidinejat, H., & Tafreshi, A. R. G. (2018). First molecular detection of Anaplasma phagocytophilum in dromedaries (Camelus dromedarius). Journal of Zoo and Wildlife Medicine49(4), 844-848. [DOI:10.1638/2017-0165.1] [PMID]
Bastos, A. D., Mohammed, O. B., Bennett, N. C., Petevinos, C., & Alagaili, A. N. (2015). Molecular detection of novel Anaplasmataceae closely related to Anaplasma platys and Ehrlichia canis in the dromedary camel (Camelus dromedarius). Veterinary Microbiology179(3-4), 310-314. [DOI:10.1016/j.vetmic.2015.06.001] [PMID]
Belkahia, H., Said, M. B., Sayahi, L., Alberti, A., & Messadi, L. (2015). Detection of novel strains genetically related to Anaplasma platys in Tunisian one-humped camels (Camelus dromedarius). The Journal of Infection in Developing Countries9(10), 1117-1125. [DOI:10.3855/jidc.6950] [PMID]
De La Fuente, J., Vicente, J., Höfle, U., Ruiz-Fons, F., De Mera, I. G. F., Van Den Bussche, R. A., ... & Gortazar, C. (2004). Anaplasma infection in free-ranging Iberian red deer in the region of Castilla-La Mancha, Spain. Veterinary Microbiology100(3-4), 163-173. [DOI:10.1016/j.vetmic.2004.02.007] [PMID]
Ghazvinian, K., & Khodaiean, T. (2017). Anaplasmosis among camels in Iran and observation of abnormalities in infected blood films. International Journal of Animal and Veterinary Sciences10(7), 475-478.
Ismael, A. B., Swelum, A. A., Khalaf, A. F., & Alowaimer, A. N. (2016). First evidence of natural anaplasmosis in Camelus dromedarius in Saudi Arabia. Journal of Camel Practice and Research23(1), 95-100. [DOI:10.5958/2277-8934.2016.00014.X]
Kidambasi, K. O., Masiga, D. K., Villinger, J., Carrington, M., & Bargul, J. L. (2019). Detection of blood pathogens in camels and their associated ectoparasitic camel biting keds, Hippobosca camelina: the potential application of keds in xenodiagnosis of camel haemopathogens. AAS Open Research2, 164. [DOI:10.12688/aasopenres.13021.2] [PMID] [PMCID]
Lbacha, H. A., Zouagui, Z., Alali, S., Rhalem, A., Petit, E., Ducrotoy, M. J., ... & Maillard, R. (2017). “Candidatus anaplasma camelii” in one-humped camels (Camelus dromedarius) in Morocco: a novel and emerging anaplasma species?. Infectious Diseases of Poverty6(1), 1-8. [DOI:10.1186/s40249-016-0216-8] [PMID] [PMCID]
Li, Y., Yang, J., Chen, Z., Qin, G., Li, Y., Li, Q., ... & Zhang, L. (2015). Anaplasma infection of Bactrian camels (Camelus bactrianus) and ticks in Xinjiang, China. Parasites & Vectors8(1), 1-6. [DOI:10.1186/s13071-015-0931-1] [PMID] [PMCID]
Noaman, V. (2018). Molecular detection of novel genetic variants associated to Anaplasma ovis among dromedary camels in Iran. Archives of Razi Institute73(1), 11-18.
Noaman, V., Shayan, P., & Amininia, N. (2009). Molecular diagnostic of Anaplasma marginale in carrier cattle. Iranian Journal of Parasitology4(1), 26-33.
Rymaszewska, A., & Grenda, S. (2008). Bacteria of the genus Anaplasma–characteristics of Anaplasma and their vectors: a review. Veterinární Medicína53(11), 573-584. [DOI:10.17221/1861-VETMED]
Sainz, A., Amusategui, I., & Tesouro, M. A. (1999). Ehrlichia platys infection and disease in dogs in Spain. Journal of Veterinary Diagnostic Investigation11(4), 382-384. [DOI:10.1177/104063879901100419] [PMID]
Sazmand, A., Harl, J., Eigner, B., Hodžić, A., Beck, R., Hekmatimoghaddam, S., ... & Joachim, A. (2019). Vector-borne bacteria in blood of camels in Iran: new data and literature review. Comparative Immunology, Microbiology and Infectious Diseases65, 48-53. [DOI:10.1016/j.cimid.2019.04.004] [PMID] [PMCID]
Selmi, R., Dhibi, M., Ben Said, M., Ben Yahia, H., Abdelaali, H., Ameur, H., ... & Mhadhbi, M. (2019). Evidence of natural infections with Trypanosoma, Anaplasma and Babesia spp. in military livestock from Tunisia. Tropical Biomedicine36(3), 742-757.
Sharifiyazdi, H., Jafari, S., Ghane, M., Nazifi, S., & Sanati, A. (2017). Molecular investigation of Anaplasma and Ehrlichia natural infections in the dromedary camel (Camelus dromedarius) in Iran. Comparative Clinical Pathology26(1), 99-103. [DOI:10.1007/s00580-016-2350-x]