Carios Capensis (Acari: Argasidae) in the Nests of the Yellow-Legged Gull (Larus Michahellis) in the Aguéli Island of Réghaia, Algeria

International Journal of Botany and Research (IJBR) ISSN(P): 2277-4815; ISSN(E): 2319-4456 Vol. 4, Issue 3, Jun 2014, 23-30 © TJPRC Pvt. Ltd.

CARIOS CAPENSIS (ACARI: ARGASIDAE) IN THE NESTS OF THE YELLOW-LEGGED GULL (LARUS MICHAHELLIS) IN THE AGUÉLI ISLAND OF RÉGHAIA, ALGERIA

FADHILA BAZIZ–NEFFAH 1,6, TAHAR KERNIF 1,2,3,6, ASSIA BENELJOUZI 2, AMINA BOUTELLIS 3,4,6, AMIROUCHE MORSLI 5, ZOUBIR HARRAT 2, SALAHEDDINE DOUMANDJI 1 & IDIR BITAM 3,4,6 1Department of Zoology, Agronomic graduate school, El Harrach, Algiers, Algeria 2Parasite Ecology and Population Genetics, Pasteur Institute of Algiers, Algeria 3Aix Marseille University, URMITE, UM63, CNRS 7278, IRD 198, Inserm 1095, Marseille, France 4Faculty of Biological Sciences, University of Science and Technology Houari Boumediene, Algiers, Algeria 5Réghaïa Cynegetic centers, Algiers, Algeria 6VALCORE laboratory. University M'hamed Bougara, Boumerdes, Algeria

ABSTRACT

During the two last years 2012 and 2013, we conducted a surveillance to identify the soft ticks species (Acari: Argasidae) found in nests of Yellow-legged Gull (Larus michahellis ), in Wetland from Réghaïa (Algeria) more specifically on the island Agueli, 1 km of the beach Réghaïa. We collected 227 ticks on 31 nests. Carios capensis , soft tick species, was identified by morphological and molecular methods using the polymerase chain reaction (PCR) targeting the mitochondrial 16S rRNA gene. Prevalence, intensity and abundance of this ectoparasite species as well as its potential vector role are discussed to survey the risk factors for human populations.

KEYWORDS: Carios capensis , Larus michahellis , Nests, Reghaïa, Algeria

INTRODUCTION

In the Mediterranean basin, the Laridae species are numerically abundant, the Yellow-legged Gull (Larus michahellis ) experiencing strong population growth over the past forty years as well as these nests along the coasts, particularly in the northwestern Mediterranean (Thibault et al ., 1996; Oro and Martinez-Abrain, 2007). Indeed, although non-breeding birds may travel long distances, breeding adults show more restricted movements and high breeding site fidelity Baaloudj et al. (2012). Yellow-legged Gulls form dense colonies and reuse breeding sites year after year; they may be exposed to a number of pathogens and arthropod vectors, such as ticks (Bosch and Figuerola, 1999; Dietrich et al. 2011). Argasid ticks are vectors of viral and bacterial agents of humans and animals. Carios capensis , a soft tick of seabirds are known as reservoirs of pathogenic bacteria of medical importance that are found around the world Wilkinson et al. (2014). The study by Arnal et al (2014) shows that legged Gulls in the Mediterranean basin are exposed to MEABAN virus transmitted by soft ticks and mosquitoes, which emphasizes the need to study the spatial and temporal distribution of the flavivirus and its potential pathogenicity for animals and humans. The objective of this study was to show the presence of soft ticks Carios capensis in the nests of Larus michahellis in the island Aguéli and evaluate the potential risk of pathogen transmission from birds to humans.

www.tjprc.org [email protected] 24 Fadhila Baziz–Neffah, Tahar Kernif, Assia Beneljouzi, Amina Boutellis, Amirouche Morsli, Zoubir Harrat, Salaheddine Doumandji & Idir Bitam

METHODS Sites study The Yellow-legged Gull frequents the swamps of Lake and Aguéli Island from Réghaïa. The wet zone of Réghaïa (36° 47' N, 3° 20' E) is a complex specific ecosystem. It is limited to the North by the Mediterranean Sea and by the oriental part of Mitidja in the South which the first hills announced of Kabyle massive Mutin (1977). Its climate is sub-humid to winter warm and precipitation fluctuates from year to year between 200 and 800 mm. This study was conducted on the Aguéli Island, which has an area of 29, 705 m² with rare herbaceous plants growing between boulders Ouarab et al. (2014). It is located at 1 km off Réghaïa beach (Figure. 1).

Figure 1: Presentation of the Aguéli Island from Réghaia Region (Algiers)

Nests Collection This work was carried out after the bird nesting period of 2012 and 2013. The nests are recovered when the chicks leave their nests. The Yellow-legged Gull nests are collected between the ratchets in the Aguéli Island. Subsequently, the nests are placed in individual bags to avoid loss of parasites; these bags are identified by bird species.

Conservation of Ectoparasites

The ticks were kept in 70% ethanol and sent to the WHO Collaborative Centre for Rickettsial Diseases and Other Arthropod-borne Bacterial Diseases (Marseille, France)

Morphological Identification of Ticks

Ectoparasites were identified by their morphological characteristics using a stereomicroscope SFC-11 (MOTIC ®) and an optical microscope (OPTECH ®). For ticks, we used the key (Hoogstraal H (1952); Hoogstraal H et (1956)). Pictures were made with a binocular microscope ZEISS AXIO ZOOM. V16 (URMITE, France). Finally, the identification was confirmed by molecular biology (see next section).

Impact Factor (JCC): 1.6913 Index Copernicus Value (ICV): 3.0 Carios capensis (Acari: Argasidae) in the Nests of the Yellow-legged Gull ( Larus michahellis ) in the Aguéli Island of Réghaia, Algeria 25

Molecular Identification of Ticks DNA Extraction Ticks are rinsed with distilled water, dried on sterile filter paper, and then crushed individually in sterile Eppendorf tubes. Thereafter, each tick was incised using a scalpel and then incubated overnight at 56°C in 180 µL of Buffer G2 (30 mM Tris·Cl; 30 mM EDTA; 5% Tween 20; 0.5% Triton X-100; 800 mM GuHCl) containing 20 µL of proteinase K (activity = 600 mAU/mL solution or 40 mAU/mg of protein) until lysis and then homogenized Kernif et al., (2012). After the pre-lysing steps, DNA extraction for 200 µL of homogenization was performed using an automatic EZ1 kit (QIAGEN-BioRobot ® EZ1, Tokyo, Japan), according to the manufacturer’s instructions (EZ1 DNA Tissue Kit, QIAGEN ®, Hilden, Germany).

Amplification by Standard Polymerase Chain Reaction (PCR) and Sequencing

DNAs were subjected to PCR by thermocycler PTC-200 Peltier model (MJ Research Inc., Watertown, Mass.) with amplification of mitochondrial 16S rRNA gene according to Mangold et al. (1998). The PCR product was subsequently verified by the migration of the amplicons on agarose gel 1%. The DNA sequencing reactions were performed for all samples amplified by PCR, as described previously (Kernif et al., 2012). The Data were collected using a capillary sequencer genetic analysis ABI Prism 3130xl (ABI PRISM, PE Applied Biosystems, USA). The sequences were edited and assembled using Chromas Pro 1.34 (Technelysium Pty Ltd, Tewantin, Australia) software. The sequences obtained were compared to existing sequences in the database NCBI-BLAST (basic local tool alignment mark) obtained from the internet server (http://blast.ncbi.nlm.nih.gov/Blast.cgi ).

Quantitative Real Time PCR Assay

All samples were screened by using a quantitative real-time PCR (qPCR) performed on the CFX (Applied biosystem) incorporated primers and probes targeting a portion of the Bartonella 16S–23S intergenic spacer region (ITS), to detect DNA of Borrelia species, a real time PCR with primers and probe targeting the 16S rRNA gene was used as previously described (Socolovschi et al. 2012), For a specific Rickettsia and Anaplasma phagocytophylum gene, we used the previously described primer and probes (Socolovschi et al. 2012).

DATA ANALYSIS Parasitic Indications We calculated prevalence, abundance and the intensity of ticks found at the nests. Parasitic indications proposed by Margolis and al. (1982).

Prevalence (P)

Prevalence is the percentage ratio of the number of infected nests (N) by parasite given by the number of nests examined (H).

P (%) = N/H * 100

Abundance (A)

The abundance is the ratio of the total number of individuals of a species of parasite (n) on the total number of nests examined (H).

www.tjprc.org [email protected] 26 Fadhila Baziz–Neffah, Tahar Kernif, Assia Beneljouzi, Amina Boutellis, Amirouche Morsli, Zoubir Harrat, Salaheddine Doumandji & Idir Bitam A = n / H

Intensity (I)

The intensity is the ratio of the total number of individuals of a species of parasite (n) on the number of infected nests (N) I = n / N

RESULTS

During the visits to Agueli Island in June 2012, about 15 nests were recovered. At last June 2013, 16 nests were recovered. For the both periods, we haven’t recovered the nests with eggs or chick and those with difficult accession. The nests consisted mainly of plant debris recovered by Yellow-legged Gull around the marshes of the lake Réghaia and near habitations from Réghaia beach. The birds added others debris including the feathers, bones and anything that could be found near the nest. Among the 31 nests examined in laboratory, 18 nests were infested by the ticks including 6 of 2012 and 12 of 2013. We collected a total of 227 ticks. The abundance values of these ticks are 10.86 and 4 in 2012 and 2013 respectively. We were noted the intensity value at 27.16 and 5.33 in 2012 and 2013 respectively (table 1). All ticks collected were morphologically identified as soft ticks Carios capensis species. Additionally, these ticks were identified through PCR of the tick mitochondrial 16S rDNA gene. Sequence analysis showed 100% similarity to the corresponding 16S rDNA of Carios capensis (GenBank accession no. JQ824316.1). The quantitative PCR assay showed that all of the samples were negative for all the pathogenic tested bacteria whereas, the positive controls amplify normally.

Table 1: Prevalence, Abundance and Intensity of Carios capensis in the Nests of Larus Michahellis in the Island Aguéli

Number of Nests Infested by Ticks / Year P% Abundance Intensity Carios capensis Collected Total of Nests Recovered 2012 163 6/15 40 10.86 27.16 2013 64 12/16 75 4 5.33 P: prevalence

DISCUSSIONS

This study has reported the first identification of Carios capensis in the nests of Larus michahellis in the island Aguéli (Reghaia, Algiers, ALGERIA). All the molecular experiments were done on the reference center for Rickettsial Diseases and Other Arthropod-borne Bacterial Diseases (Marseille, France). The experiments were done on a place free of soft ticks DNA and all negative controls remain negative.

As confirmed by Reeves et al (2006), our results show that Carios capensis was found in marine bird's nests of the Gull leucophée (Larus michahellis ) and it seems that it is it’s the only ecological nich in the marine environment. As brought back previously , C. capensis is considered as a tick of this type of birds. On the islands of the county of Charleston (U.S.A ), C. capensis can even cause the abandonment of the nest by pelicans (Reeves and al 2002 & Williams and al. 1999).

The role of seabirds in the ecology and epidemiology of pathogens transmitted by ticks is rarely considered, they have a potentially important role in the maintenance and the dissemination worldwide. We know that these ticks of the birds’ sea systems can harbor a variety of pathogens cited previously by Dietrich et al (2011). Such as the study of Reeves et al (2006) where it was reported that C. capensis can convey pathogenic germs as Borrelia, Coxiella and Rickettsia.

Recently Duh et al (2010) have isolated a rickettsia, “Rickettsia hoogstraalii” from C. capensis in Georgia, in the United States. Hutcheson et al (2005) proved experimentally that the West Nile virus can be transmitted by C. capensis. Larvas of C. capensis can prick the man and pets during the visits of colonies (summer camps) of pelicans Estrada-pena & Jongejan (1999). In Japan on the island of Torishima Carios capensis was collected on Albatross Diomedea nigripes black Ushijima et al (2003), in the same country Kishimoto et al (2006) reporeted a Rickettsia detected for the first time in seabirds transmitted by Carios capensis and C. sawaii .

Moreover, Wilkinsson et al (2014) have demonstrated that Cell lines from C. capensis will be useful on investigating interactions between soft ticks and microorganisms Mattila et al. (2007). Then; Based on a combined pyrosequencing approach with a conventional molecular analysis targeting the bacteria of an interest medical potential to describe the bacterial community in two ticks of tropical seabirds, Carios capensis and Amblyomma loculosum Reeves et al (2005) in South Carolina, United States of America have found a new agent “Coxiella” by PCR and sequencing the amplicons of 64 ticks pelican Carios capensis , of Devoux Bank. Sequences of certain metabolic genes and housekeeping genes shared 92-98 % similarity to the C. burnetii. Thus, all the visitors can be exposed to infected ticks by going on similar islands with bird's nests.

CONCLUSIONS

During the two years of our study, we confirmed the presence of Carios capensis in the nests of Larus michahelis with a prevalence that is higher in 2013 than in 2012. As Confirmed by the literature; C. capensis is a potential vector of the West Nile virus and Rickettsia. Thus, further epidemiological studies with soft tiks collected from more nests and from different countries should be performed to determine whether the invasion of pathogenic bacteria is a general phenomenon around the Mediterranean.

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