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Am. J. Trop. Med. Hyg., 77(4), 2007, pp. 753-756
Copyright © 2007 by The American Society of Tropical Medicine and Hygiene

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Detection of Four Adenovirus Serotypes within Water-Isolated Strains of Acanthamoeba in the Canary Islands, Spain

Jacob Lorenzo-Morales, Nieves Coronado-Álvarez, Enrique Martínez-Carretero, Sutherland K. Maciver, AND Basilio Valladares*
Instituto Universitario de Enfermedades Tropicales y Salud Pública de Canarias, University of La Laguna, La Laguna, Canary Islands, Spain; Centre for Integrative Physiology, School of Biomedical Sciences, University of Edinburgh, Edinburgh, Scotland


ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We surveyed 236 potentially pathogenic Acanthamoeba strains, isolated from water sources in the Canary Islands, for the presence of human adenoviruses (HAdV) using a polymerase chain reaction (PCR)-based typing assay. A total of 34 of these strains were found to be positive for adenovirus belonging to four different HAdV serotypes (HAdV-1, 2, 8, and 37). We found that HAdV-2 was the most frequently encountered serotype amongst the Acanthamoeba strains, and their identification was confirmed by a nested PCR specific for this serotype. We showed that Acanthamoeba genotype T4 was highly associated with serotype HAdV-2, whereas Acanthamoeba genotype T3 was most often associated with adenovirus serotypes related to ocular diseases. Based on these data, we suggest that Acanthamoeba should be considered as a potential reservoir and perhaps even a transmitter of adenoviruses to human and other secondary hosts.


INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human adenoviruses (Ads) are responsible for a wide spectrum of clinical disease, including respiratory illness, pharyn-goconjunctival fever, conjunctivitis, cystitis, gastroenteritis, and neurologic and sexually transmitted diseases.1 Most infections are clinically unapparent or associated with mild, self-limited disease; however, severe and sometimes fatal infections can occur, particularly among the very young, elderly, and the immunocompromised. Ads are classified within the family Adenoviridae, genus Mastadenovirus, and are further divided into six species (A–F) and 51 currently recognized serotypes, of which about one third are responsible for Ad-associated human disease.2 Among the pathogenic adenoviruses, particular species and serotypes are more commonly associated with disease syndromes, epidemiologic settings, and demographic risk groups.3

Acanthamoeba spp. are free-living protozoans that pervade the entire biosphere and can be isolated from tap, fresh, coastal, and bottled mineral water; contact lens solutions and eyewash stations; soil, dust, and air; sewage; heating, ventilation, or air-conditioning units; and gastrointestinal washings.4 Genetic studies have led to the identification of 15 different genotypes (T1–T15) of Acanthamoeba based on rRNA gene sequencing.5–8 To date, ~90% of Acanthamoeba isolates that produce infections belong to the T4 genotype, suggesting that the abundance of T4 isolates in infections is most likely caused by their great virulence and/or properties that enhance their transmissibility, as well as their decreased susceptibility to chemotherapeutic agents.9,10

Together with their ability to harbor bacteria, the ubiquity of Acanthamoeba in the environment makes it a potential incubator for infectious agents.11–13 This association remained something of an interesting curiosity until a connection was proposed between amoebae and pathogenic bacteria, in particular, Legionella pneumophila.14

The relationship between Acanthamoeba and viruses is less well characterized. Previous studies have indicated that Acanthamoeba could not transmit poliovirus, vesicular stomatitis virus,15 or echoviruses,16,17 but recently, a large DNA virus (mimivirus) was identified from A. polyphaga,18 and it is known that coxsackie virus can infect Acanthamoeba.17 This study investigated the possibility that Acanthamoeba could play a role in the survival and transmission of adenovirus because some studies have reported patients with coinfections with Acanthamoeba and human pathogenic viruses,19 including adenovirus.20 We found that some of our Acanthamoeba isolates harbor adenoviruses in support of the suggestion that these amoeba may act as reservoirs for adenoviruses.


MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Acanthamoeba strains were isolated from samples collected from tap water in locations throughout the Canary Islands (Table 1Go) and grown without shaking in 712 PYG medium (ATCC; 0.75% proteose peptone [wt/vol], 0.75% yeast extract [wt/vol], and 1.5% glucose [wt/vol]) at 25°C as previously described.21 Briefly, the amoebae were grown axenically in PYG medium containing 50 µg/mL gentamicin (Sigma, Madrid, Spain). Acanthamoeba trophozoites were harvested at a density of 2 x 107 cells/mL. The cells were pelleted (500g) for 10 minutes at room temperature and washed three times with phosphate-buffered saline (PBS), pH 7.2. Cell pellets were resuspended in lysis buffer (50 mmol/L NaCl, 10 mmol/L EDTA, and 50 mmol/L Tris-HCl, pH 8.0) and incubated at 55°C for 1 hour with 0.25 mg/mL Proteinase K. The DNA samples were purified from amoebae isolates by the phenol-chloroform method.22 The DNA amplification reactions were performed, using the specific primer pair for adenovirus typing as previously described,23 ~15 days after the amoeba strains were isolated from the tap water samples.


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TABLE 1
Adenovirus serotypes isolated from Acanthamoeba strains in this study
 
Ad2 nested polymerase chain reaction (PCR) was performed as previously described to corroborate the Ad2 serotype identification24 with minimal modifications in a 50 µL volume containing 0.04 U Taq DNA polymerase (Applied Biosystems, Madrid, Spain), 0.1–1.0 ng DNA, 1.5 mmol/L MgCl2, 250 µmol/L dNTP, and 0.08 µmol/L each primer. The cycling conditions were an initial denaturing phase of 95°C for 2 minutes and 30 repetitions at 95°C for 15 seconds, 55°C for 15 seconds, and 72°C for 10 seconds. The primer extension phase was prolonged for 5 minutes at 72°C in the last cycle, using a MyCycler Thermal Cycler (Biorad, Hercules, CA). Amplification products were fractionated by 3% agarose electrophoresis, and gels were stained with a solution of 0.5 µg/mL ethidium bromide and visualized under UV light.

Amplified products were purified using a QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions and sequenced by Sistemas Genómicos (Valencia, Spain). Obtained sequences were aligned and compared with the adenovirus sequences previously deposited in the GenBank database.


RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
A total of 236 previously isolated and genotyped Acanthamoeba strains21,25 representing 5 of the presently known 15 groups (T1–T15) were screened for the presence of adenovirus. This collection was made up of 32 strains from T2, 43 from T3, 83 from T4, 21 from T6, and 57 from T7. A total of 34 of the collection proved positive for the adenovirus typing-specific primer pair (Table 1Go). Four different Ad serotypes were identified from the PCR products: Ad1, Ad2, Ad8, and Ad37. Nested PCR specific for the Ad2 serotype confirmed Ad2 identification (Table 1Go). Ad2 serotype was identified in 24 of 25 (96%) within the T4 Acanthamoeba genotype. These results indicated that 24 of 83 (29%) of the previously isolated T4 Acanthamoeba genotypes were carrying adenovirus. Moreover, ocular adenovirus serotypes (six of seven; 96%) seemed to be highly associated with genotype T3. Regarding the prevalence of adenovirus within the T3 Acanthamoeba genotypes cluster, 7 of 43 (16%) harbored adenovirus. Adenovirus DNA was amplified ~15 days after the initial isolation of the Acanthamoeba strains, after the population had undergone many doublings. This suggests that the adenoviral DNA originated from the amoebal genomic DNA and did not originate from cell surface attached virions.

Finally, Acanthamoeba genotype T7, a non-pathogenic genotype, was found to harbor Ad2 serotype (2 of 57, a 4% of the previously genotyped strains; Table 1Go), whereas no adenovirus was detected in T2 and T6 genotypes. New adenovirus sequences were deposited in the GenBank database under accession numbers EF205209–EF205323.


DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Free-living amoebae of the genus Acanthamoeba have been isolated from many habitats worldwide and have been reported to feed mainly on bacteria, fungi, and algae by phagocytosis, with digestion occurring within phagolysosomes.10 Some bacteria have evolved to become resistant to protists by either failing to become internalized or if they are internalized escape digestion. They are thus able to survive, grow, and exit free-living amoebae after internalization.26,27 Among the amoeba-resistant bacteria, some are obligate intracellular bacteria,12 and others are considered endosymbionts,13 because a stable host–parasite ratio is maintained,28 whereas others such as the Legionella-like amoebal pathogens actively prey on Acanthamoeba.14

In light of the fact that bacteria can become internalized without digestion, it is possible that viruses are also capable of entering the cell to cause infection and propagation. Indeed, a large DNA virus, a mimivirus, measuring 400 nm in diameter, was identified from A. polyphaga,18,29 and it has also been reported that coxsackie virus can also infect Acanthamoeba.17 Patients whose corneas were simultaneously infected with Acanthamoeba and pathogenic viruses such adenovirus and herpesvirus have been reported,20 and this situation would be expected to increase the probability of viral infection of the amoebae. Coinfections are likely to be underestimated because of the similarities between the kerato-conjunctivitis and the Acanthamoeba keratitis clinical symptoms.20

Our study indicates that pathogenic genotypes of Acanthamoeba isolated from water sources in the Canary Islands harbor adenovirus serotypes that may be able to cause human infections such as ocular and gastrointestinal diseases. It is important to mention the observed relationships between Acanthamoeba genotype T4 and Ad2 and between genotype T3 and ocular adenovirus serotypes. The fact that we detected HAdV-1 and 2 most frequently in Acanthamoeba from the Canaries, is consistent with a human involvement because adenovirus C (that include HAdV-1 and 2) is the most commonly encountered group in human infection. Also, HAdV-8 and 37 (the real ocular types) belong to another very frequent species: human adenovirus D. It therefore seems that human activity may be responsible for the frequency of association of these adenoviruses and Acanthamoeba. The possibility of ocular coinfection with adenovirus and Acanthamoeba should be considered in non-bacterial keratitis cases.

Our study indicates that pathogenic strains of Acanthamoeba could be acting as potential promoters for the transmission of gastrointestinal and ocular adenovirus through water sources in the Canary Islands, Spain. Further studies are needed to establish if virus is actually released from infected amoebae.

Received April 4, 2007. Accepted for publication May 8, 2007.

Financial support: This work was funded by the projects RICET (Project C03/04 of the Programme of Redes Temáticas de Investigación Cooperativa), Spanish Ministry of Health, Madrid, Spain, and the Dirección General de Universidades e Investigación del Gobierno de Canarias Project PI042005/049. J. Lorenzo-Morales was supported by a postdoctoral contract from the Dirección General de Fomento e Innovación Tecnológica, Consejería de Industria, Comercio y Nuevas Tecnologías of the Canary Islands Government IDT-TF-06/055. Biopolis-Interreg IIIB (Canarias, Acores, Madeira).

* Address correspondence to Basilio Valladares, Instituto Universitario de Enfermedades Tropicales y Salud Pública de Canarias University of La Laguna, Av. Astrofísico Fco. Sánchez s/n 38203 La Laguna, Canary Islands, Spain. E-mail: bvallada{at}ull.es Back

Authors’ addresses: Jacob Lorenzo-Morales, Instituto Universitario de Enfermedades Tropicales Y Salud Püblica de Canarias, Av. Astrofísico Fco. Sánchez, S/N, 38203 La Laguna, Tenerife, Canary Islands, Spain, Phone: 34-922318490, Fax: 34-922318514, E-mail: jmlorenz{at}ull.es. Nieves Coronado-Álvarez, Instituto Universitario de Enfermedades Tropicales Y Salud Püblica de Canarias, Av. Astrofísico Fco. Sánchez, S/N, 38203 La Laguna, Tenerife, Canary Islands, Spain, Phone: 34-922318490, Fax: 34-922318514, E-mail: nicoal{at}ull.es. Enrique Martínez-Carretero, Instituto Universitario de Enfermedades Tropicales Y Salud Pública de Canarias, Av. Astrofísico Fco. Sánchez, S/N, 38203 La Laguna, Tenerife, Canary Islands, Spain, Phone: 34-922318490, Fax: 34-922318514, E-mail: emartine{at}ull.es. Sutherland K. Maciver, Centre for Integrative Physiology, School of Biomedical Sciences, Hugh Robson Building, University of Edinburgh, George Square, Edinburgh EH8 9XD, Scotland, Phone/ Fax: 44-131-650-3714, E-mail: smaciver{at}staffmail.ed.ac.uk. Basilio Valladares, Instituto Universitario de Enfermedades Tropicales y Salud Pública de Canarias University of La Laguna, Av. Astrofísico Fco. Sánchez s/n 38203 La Laguna, Canary Islands, Spain, Telephone: 34-922-318484, Fax: 34-922-318514, E-mail: bvallada{at}ull.es.


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 DISCUSSION
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