MALDI mass spectrometry-based identification of antifungal molecules from endophytic Bacillus strains with biocontrol potential of Lasiodiplodia theobromae, a grapevine trunk pathogen in Peru.

Lasiodiplodia theobromae, a grapevine trunk pathogen, is becoming a significant threat to vineyards worldwide. In Peru, it is responsible for Botryosphaeria dieback in many grapevine-growing areas and it has spread rapidly due to its high transmissibility; hence, control measures are urgent. It is known that some endophytic bacteria are strong inhibitors of phytopathogens because they produce a wide range of antimicrobial molecules. However, studies of antimicrobial features from endophytic bacteria are limited to traditional confrontation methods. In this study, a MALDI mass spectrometry-based approach was performed to identify and characterize the antifungal molecules from Bacillus velezensis M1 and Bacillus amyloliquefaciens M2 grapevine endophytic strains. Solid medium antagonism assays were performed confronting B. velezensis M1 - L. theobromae and B. amyloliquefaciens M2 - L. theobromae for antifungal lipopeptides identification. By a MALDI TOF MS it was possible identify mass spectra for fengycin, iturin and surfactin protoned isoforms. Masses spectrums for mycobacillin and mycosubtilin were also identified. Using MALDI Imaging MS we were able to visualize and relate lipopeptides mass spectra of fengycin (1463.9 m/z) and mycobacillin (1529.6 m/z) in the interaction zone during confrontations. The presence of lipopeptides-synthesis genes was confirmed by PCR. Liquid medium antagonism assays were performed for a proteomic analysis during the confrontation of B. velezensis M1 - L. theobromae. Different peptide sequences corresponding to many antifungal proteins and enzymes were identified by MALDI TOF MS/MS. Oxalate decarboxylase bacisubin and flagellin, reported as antifungal proteins, were identified at 99 % identity through peptide mapping. MALDI mass spectrometry-based identification of antifungal molecules would allow the early selection of endophytic bacteria with antifungal features. This omics tool could lead to measures for prevention of grapevine diseases and other economically important crops in Peru.

Genetic structure, phylogeography, and demography of Anadara tuberculosa (Bivalvia) from East Pacific as revealed by mtDNA: Implications to conservation.

Wild populations of the pustulose ark, Anadara tuberculosa (Bivalvia), an emblematic species of the East Pacific mangrove ecosystem declined in South American countries (Colombia, Ecuador, and Peru) mainly due to overharvesting and habitat loss or degradation. Understanding the genetic aspects of geographic variations and population structure of A. tuberculosa, currently unknown, appears as a priority to fishery authorities in order to elaborate integrated and collaborative conservation policies for fishery management, aquaculture, and stock enhancement programs. We used mtDNA sequence data to investigate haplotype diversity, genetic structure, and demography of A. tuberculosa. Results indicate genetic homogeneity of populations distributed north and south of the equator, respectively. However, statistically significant differentiation emerged between northern and southern populations with pairwise ф ST values ranging between 0.036 and 0.092. The oceanic current system acting in the area (Panama Current and Humboldt Current) might play a role in limiting the larval dispersal of the species, still poorly understood. Demography reconstruction supported recent population expansion, possibly started after last glacial maximum. Our results would suggest separate and independent management of populations north and south of the equator.

[Molecular characterization of pathogenic bacteria of the respiratory tract in peruvian patients with cystic fibrosis]. / Caracterización molecular de bacterias patógenas de las vías respiratorias de pacientes peruanos con fibrosis quística.

OBJECTIVES.: To molecularly characterize the pathogenic bacteria of the respiratory tract isolated from patients with cystic fibrosis (CF) in Peru. MATERIALS AND METHODS.: Bacterial communities cultured from sputum samples of pediatric and adult patients with CF admitted to the Edgardo Rebagliati Martins National Hospital and the National Institute of Child Health were characterized. Standard microbiological techniques were used for bacterial culture, and gene sequencing of 16S rRNA and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry and tandem MALDI-TOF mass spectrometry (MALDI TOF/TOF) were used for molecular characterization. RESULTS.: Seventeen bacterial strains were characterized by 16S rRNA sequencing, and the identified pathogenic bacteria were Pseudomonas aeruginosa (31.5%), Staphylococcus aureus (12.6%), Pseudomonas spp. (11.8%), and Klebsiella oxytoca (3.1%). MALDI-TOF analysis generated a series of spectra representative of each isolated bacterial species, whereas MALDI TOF/TOF analysis identified the peptides and proteins of the most common strains and provided data on pathogenicity and sensitivity to antibiotics. CONCLUSIONS.: The primary pathogenic microorganisms found in the respiratory tract of patients with CF in Peru were the same as those found in other countries. This study is the first to perform 16S rRNA sequencing as well as MALDI-TOF and MALDI-TOF/TOF analysis of the bacterial pathogens circulating in Peru. The inclusion of proteomic analysis further allowed for the identification of native microorganisms involved in CF.

OBJETIVOS.: Caracterizar a nivel molecular las bacterias patógenas de las vías respiratorias de pacientes peruanos con fibrosis quística (FQ). MATERIALES Y MÉTODOS.: Se caracterizaron las comunidades bacterianas cultivables a partir de muestras de esputo de pacientes pediátricos y adultos con FQ registrados en el Hospital Nacional Edgardo Rebagliati Martins y el Instituto Nacional de Salud del Niño (INSN). Para el cultivo bacteriano se utilizaron técnicas microbiológicas estándares, y para la caracterización molecular la secuenciación del gen ARNr 16S y espectrometría de masas de tipo desorción/ionización con láser asistido por matriz con tiempo de vuelo (MALDI TOF) y MALDI TOF/TOF. RESULTADOS.: Por secuenciación del ARNr 16S se identificaron 127 cepas, encontrando las bacterias patógenas Pseudomonas aeruginosa (31,5%), Staphylococcus aureus (12,6%), Pseudomonas spp. (11,8%), Klebsiella oxytoca (3,1%), otras especies mostraron baja prevalencia. El análisis por MALDI TOF permitió obtener una serie de espectros representativos de cada especie aislada, mientras que el análisis por MALDI TOF/TOF reveló péptidos y proteínas de las especies más comunes con informaciones complementarias que revelarían datos sobre su patogenicidad o sensibilidad a antibióticos. CONCLUSIONES.: Los principales microorganismos patógenos encontrados en las vías respiratorias son similares a los reportados en otros países. Estos son los primeros hallazgos en Perú que muestran la caracterización bacteriana por secuenciación del ARNr 16S, por MALDI TOF y MALDI TOF TOF. Los hallazgos permiten la identificación bacteriana de microorganismos nativos relacionados con la FQ basada en el análisis de su proteoma.

Caracterización molecular de bacterias patógenas de las vías respiratorias de pacientes peruanos con fibrosis quística / Molecular characterization of pathogenic bacteria of the respiratory tract in peruvian patients with cystic fibrosis

RESUMEN Objetivos. Caracterizar a nivel molecular las bacterias patógenas de las vías respiratorias de pacientes peruanos con fibrosis quística (FQ). Materiales y métodos. Se caracterizaron las comunidades bacterianas cultivables a partir de muestras de esputo de pacientes pediátricos y adultos con FQ registrados en el Hospital Nacional Edgardo Rebagliati Martins y el Instituto Nacional de Salud del Niño (INSN). Para el cultivo bacteriano se utilizaron técnicas microbiológicas estándares, y para la caracterización molecular la secuenciación del gen ARNr 16S y espectrometría de masas de tipo desorción/ionización con láser asistido por matriz con tiempo de vuelo (MALDI TOF) y MALDI TOF/TOF. Resultados. Por secuenciación del ARNr 16S se identificaron 127 cepas, encontrando las bacterias patógenas Pseudomonas aeruginosa (31,5%), Staphylococcus aureus (12,6%), Pseudomonas spp. (11,8%), Klebsiella oxytoca (3,1%), otras especies mostraron baja prevalencia. El análisis por MALDI TOF permitió obtener una serie de espectros representativos de cada especie aislada, mientras que el análisis por MALDI TOF/TOF reveló péptidos y proteínas de las especies más comunes con informaciones complementarias que revelarían datos sobre su patogenicidad o sensibilidad a antibióticos. Conclusiones. Los principales microorganismos patógenos encontrados en las vías respiratorias son similares a los reportados en otros países. Estos son los primeros hallazgos en Perú que muestran la caracterización bacteriana por secuenciación del ARNr 16S, por MALDI TOF y MALDI TOF TOF. Los hallazgos permiten la identificación bacteriana de microorganismos nativos relacionados con la FQ basada en el análisis de su proteoma.

ABSTRACT Objectives. To molecularly characterize the pathogenic bacteria of the respiratory tract isolated from patients with cystic fibrosis (CF) in Peru. Materials and methods. Bacterial communities cultured from sputum samples of pediatric and adult patients with CF admitted to the Edgardo Rebagliati Martins National Hospital and the National Institute of Child Health were characterized. Standard microbiological techniques were used for bacterial culture, and gene sequencing of 16S rRNA and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry and tandem MALDI-TOF mass spectrometry (MALDI TOF/TOF) were used for molecular characterization. Results. Seventeen bacterial strains were characterized by 16S rRNA sequencing, and the identified pathogenic bacteria were Pseudomonas aeruginosa (31.5%), Staphylococcus aureus (12.6%), Pseudomonas spp. (11.8%), and Klebsiella oxytoca (3.1%). MALDI-TOF analysis generated a series of spectra representative of each isolated bacterial species, whereas MALDI TOF/TOF analysis identified the peptides and proteins of the most common strains and provided data on pathogenicity and sensitivity to antibiotics. Conclusions. The primary pathogenic microorganisms found in the respiratory tract of patients with CF in Peru were the same as those found in other countries. This study is the first to perform 16S rRNA sequencing as well as MALDI-TOF and MALDI-TOF/TOF analysis of the bacterial pathogens circulating in Peru. The inclusion of proteomic analysis further allowed for the identification of native microorganisms involved in CF.

Respuesta fisiológica y molecular de Anadara tuberculosa (Arcoida: Arcidae) al estrés de salinidad / Physiological and molecular response of Anadara tuberculosa (Arcoida: Arcidae) to salinity stress

Resumen: La concha negra Anadara tuberculosa es una especie emblemática del ecosistema manglar que está actualmente en condición vulnerable. El desarrollo de su acuicultura requiere identificar biomarcadores moleculares, en particular asociados al estrés por salinidad en mira al inicio de programas de mejoramiento genético. Se recolectaron ejemplares de Anadara tuberculosa del manglar colindante a la Bahía de Puerto Pizarro (Tumbes, Perú) entre enero 2015 y febrero 2016. Estos individuos fueron sometidos a condiciones de estrés hipo-osmótico (extremo: 5 y 10 ppt); (moderado: 15 y 25 ppt) y sin estrés (grupo control: 33 ppt) por 16 días después de haber sido separados en grupos de diez animales y por triplicado. La presencia de biomarcadores del estrés por salinidad fue evaluada a nivel genético con la detección por PCR de 19 genes reportados como actores claves de la osmorregulación en bivalvos como ostras y mejillones y a nivel proteomico con la secuenciación de péptidos expresados en tejidos de animales expuestos a diferentes salinidades por espectrometría de doble masa. Ninguno de los marcadores genéticos probados pudo ser amplificado por PCR lo que sugiere que A. tuberculosa presente diferencias genéticas significativas en comparación con otros moluscos. El análisis proteómico realizado por MALDI TOF/TOF a nivel de tejido branquial de A. tuberculosa permitió identificar 26 péptidos expresados de formas presenciales y diferenciales a las diferentes salinidades evaluadas, resaltando posibles marcadores como la HSP70 y una proteína transmembrana de transporte de cloruro que están relacionadas con la adaptación a la salinidad. Estas secuencias aminoacídicas permitirán diseñar iniciadores nucleotidicos específicos a A. tuberculosa para la puesta en marcha de futuras investigaciones en ecofisiología de este importante recurso.

Abstract: The pustulose ark A. tuberculosa is an emblematic species of mangrove ecosystem that is currently in a vulnerable condition. The development of its aquaculture, to begin with genetic breeding programs, requires the identification of molecular biomarkers, particularly those associated with salinity stress. With this purpose, specimens of A. tuberculosa were collected from the adjacent mangroves of Puerto Pizarro bay (Tumbes, Perú), from January 2015 to February 2016. Different assays (groups of ten animals in triplicate) were undertaken in separated periods of 16 days: hypo-osmotic stress (extreme: 5, 10 ppt); (Moderate: 15, 25 ppt) and no stress (control group: 33 ppt). The presence of salinity stress biomarkers was assessed at the genetic level throughout PCR detection of 19 genes reported to be key actors in osmoregulation, and at the proteomic level with the sequencing of peptides (tandem mass spectrometry MALDI TOF/TOF), expressed in ark tissues exposed to different salinities. None of the tested genetic markers could be amplified by PCR, suggesting that A. tuberculosa has significant genetic differences compared to other mollusks. Proteomic analysis by mass spectrometry on A. tuberculosa gill tissue, allowed to identify 26 peptides expressed in presential and differential forms at different salinities, highlighting possible markers such as HSP70 and trans-membrane chloride channel transportation protein, to be related with salinity adaptation. These amino acid sequences will allow the design of target specific primers for A. tuberculosa, to implement future research in ecophysiology of this important fishery resource. Rev. Biol. Trop. 65 (3): 1142-1151. Epub 2017 September 01.

Biogeography of Parasitic Nematode Communities in the Galápagos Giant Tortoise: Implications for Conservation Management.

The Galápagos giant tortoise is an icon of the unique, endemic biodiversity of Galápagos, but little is known of its parasitic fauna. We assessed the diversity of parasitic nematode communities and their spatial distributions within four wild tortoise populations comprising three species across three Galápagos islands, and consider their implication for Galápagos tortoise conservation programmes. Coprological examinations revealed nematode eggs to be common, with more than 80% of tortoises infected within each wild population. Faecal samples from tortoises within captive breeding centres on Santa Cruz, Isabela and San Cristobal islands also were examined. Five different nematode egg types were identified: oxyuroid, ascarid, trichurid and two types of strongyle. Sequencing of the 18S small-subunit ribosomal RNA gene from adult nematodes passed with faeces identified novel sequences indicative of rhabditid and ascaridid species. In the wild, the composition of nematode communities varied according to tortoise species, which co-varied with island, but nematode diversity and abundance were reduced or altered in captive-reared animals. Evolutionary and ecological factors are likely responsible for the variation in nematode distributions in the wild. This possible species/island-parasite co-evolution has not been considered previously for Galápagos tortoises. We recommend that conservation efforts, such as the current Galápagos tortoise captive breeding/rearing and release programme, be managed with respect to parasite biogeography and host-parasite co-evolutionary processes in addition to the biogeography of the host.

Host selection and parasite infection in Aedes taeniorhynchus, endemic disease vector in the Galápagos Islands.

Host selection in blood-sucking arthropods has important evolutionary and ecological implications for the transmission dynamics, distribution and host-specificity of the parasites they transmit. The black salt-marsh mosquito (Aedes taeniorhynchus Wiedemann) is distributed throughout tropical to temperate coastal zones in the Americas, and continental populations are primarily mammalphilic. It is the only indigenous mosquito in the Galápagos Islands, having colonised the archipelago around 200,000 years ago, potentially adapting its host selection, and in the process, altering the dynamics of vector mediated pathogen interactions in the archipelago. Here, we use blood-meal analysis and PCR-based parasite screening approach to determine the blood-feeding patterns of A. taeniorhynchus in the Galápagos Islands and identify potential parasite transmission with which this mosquito could be involved. Our results show that A. taeniorhynchus feeds equally on mammals and reptiles, and only one avian sample was observed in 190 successful PCR amplifications from blood meals. However, we detected endemic filarial worms and Haemoproteus parasites known to infect various Galápagos bird species in mosquito thoraces, suggesting that feeding on birds must occur at low frequency, and that A. taeniorhynchus may play a role in maintaining some avian vector-borne pathogens, although more work is needed to explore this possibility. We also isolated three different DNA sequences corresponding to hemogregarine parasites of the genus Hepatozoon from mosquito and iguana blood samples, suggesting that more than one species of Hepatozoon parasites are present in Galápagos. Phylogenetic analysis of Hepatozoon 18sRNA sequences indicates that A. taeniorhynchus may have facilitated a recent breakdown in host-species association of formerly isolated Hepatozoon spp. infecting the reptile populations in the Galápagos Islands.

Adaptation, isolation by distance and human-mediated transport determine patterns of gene flow among populations of the disease vector Aedes taeniorhynchus in the Galapagos Islands.

The black salt-marsh mosquito (Aedes taeniorhynchus) is the only native mosquito in the Galapagos Islands and potentially a major disease vector for Galapagos wildlife. Little is known about its population structure, or how its dynamics may be influenced by human presence in the archipelago. We used microsatellite data to assess the structure and patterns of A. taeniorhynchus gene flow among and within islands, to identify potential barriers to mosquito dispersal, and to investigate human-aided transport of mosquitoes across the archipelago. Our results show that inter-island migration of A. taeniorhynchus occurs frequently on an isolation by distance basis. High levels of inter-island migration were detected amongst the major ports of the archipelago, strongly suggesting the occurrence of human-aided transport of mosquitoes among islands, underlining the need for strict control measures to avoid the transport of disease vectors between islands. The prevalence of filarial nematode infection in Galapagos flightless cormorants is correlated with the population structure and migration patterns of A. taeniorhynchus, suggesting that A. taeniorhynchus is an important vector of this arthropod-borne parasite in the Galapagos Islands. Therefore mosquito population structure in Galapagos may have the potential to influence mosquito-borne parasite population dynamics, and the subsequent impacts of such pathogens on their host species in the islands.

Seasonal effects and fine-scale population dynamics of Aedes taeniorhynchus, a major disease vector in the Galapagos Islands.

Characterization of the fine-scale population dynamics of the mosquito Aedes taeniorhynchus is needed to improve our understanding of its role as a disease vector in the Galapagos Islands. We used microsatellite data to assess the genetic structure of coastal and highland mosquito populations and patterns of gene flow between the two habitats through time on Santa Cruz Island. In addition, we assessed possible associations of mosquito abundance and genetic diversity with environmental variables. The coastal and highland mosquito populations were highly differentiated from each other all year round, with some gene flow detected only during periods of increased precipitation. The results support the hypothesis that selection arising from ecological differences between habitats is driving adaptation and divergence in A. taeniorhynchus, and maintaining long-term genetic differentiation of the populations against gene flow. The highland and lowland populations may constitute an example of incipient speciation in progress. Highland populations were characterized by lower observed heterozygosity and allelic richness, suggesting a founder effect and/or lower breeding site availability in the highlands. A lack of reduction in genetic diversity over time in highland populations suggests that they survive dry periods as dormant eggs. Association between mosquito abundance and precipitation was strong in the highlands, whereas tide height was the main factor affecting mosquito abundance on the coast. Our findings suggests differences in the infection dynamics of mosquito-borne parasites in the highlands compared to the coast, and a higher risk of mosquito-driven disease spread across these habitats during periods of increased precipitation.

Tracking acquired antibiotic resistance in commensal bacteria of Galápagos land iguanas: no man, no resistance.

BACKGROUND: Antibiotic resistance, evolving and spreading among bacterial pathogens, poses a serious threat to human health. Antibiotic use for clinical, veterinary and agricultural practices provides the major selective pressure for emergence and persistence of acquired resistance determinants. However, resistance has also been found in the absence of antibiotic exposure, such as in bacteria from wildlife, raising a question about the mechanisms of emergence and persistence of resistant strains under similar conditions, and the implications for resistance control strategies. Since previous studies yielded some contrasting results, possibly due to differences in the ecological landscapes of the studied wildlife, we further investigated this issue in wildlife from a remote setting of the Galapagos archipelago. METHODOLOGY/PRINCIPAL FINDINGS: Screening for acquired antibiotic resistance was carried out in commensal enterobacteria from Conolophus pallidus, the terrestrial iguana of Isla Santa Fe, where: i) the abiotic conditions ensure to microbes good survival possibilities in the environment; ii) the animal density and their habits favour microbial circulation between individuals; and iii) there is no history of antibiotic exposure and the impact of humans and introduced animal species is minimal except for restricted areas. Results revealed that acquired antibiotic resistance traits were exceedingly rare among bacteria, occurring only as non-dominant strains from an area of minor human impact. CONCLUSIONS/SIGNIFICANCE: Where both the exposure to antibiotics and the anthropic pressure are minimal, acquired antibiotic resistance traits are not normally found in bacteria from wildlife, even if the ecological landscape is highly favourable to bacterial circulation among animals. Monitoring antibiotic resistance in wildlife from remote areas could also be a useful tool to evaluate the impact of anthropic pressure.

Uneven frequency of Vibrio alginolyticus-group isolates among different populations of Galápagos marine iguana (Amblyrhynchus cristatus).

The presence of Vibrio isolates was investigated in cloacal swabs from the Galápagos marine iguana (Amblyrhyncus cristatus). Such unique iguana is endemic to the Galápagos Archipelago, it is listed as vulnerable in the IUCN Red List (2009), and is strictly protected by CITES and Ecuador laws. Our results revealed an uneven isolation frequency of vibrios from animals living in different settings: maximal among the Santa Fe population, scarce at Bahía Tortuga but practically absent in the samples from Puerto Ayora and Plaza Sur. A 16S sequencing confirmed that the isolates belonged to the genus Vibrio, placing them within the V. alginolyticus group; the biochemical identification was, indeed, consistent with V. alginolyticus features. The reason of the observed discrepancy is not clear, but could be either linked to a higher pollution in the inhabited or more touristic places or to differential influence of chemical and physical parameters at a local scale. As V. alginolyticus is an opportunistic pathogen for man and it is known to cause disease in sea-living animals, the ability of these vibrios to enter and persist to a certain extent in the marine iguana gut should be regarded as a risk for health of both the animals and the human personnel involved in monitoring activities.

Evidence for regular ongoing introductions of mosquito disease vectors into the Galapagos Islands.

Wildlife on isolated oceanic islands is highly susceptible to the introduction of pathogens. The recent establishment in the Galápagos Islands of the mosquito Culex quinquefasciatus, a vector for diseases such as avian malaria and West Nile fever, is considered a serious risk factor for the archipelago's endemic fauna. Here we present evidence from the monitoring of aeroplanes and genetic analysis that C. quinquefasciatus is regularly introduced via aircraft into the Galápagos Archipelago. Genetic population structure and admixture analysis demonstrates that these mosquitoes breed with, and integrate successfully into, already-established populations of C. quinquefasciatus in the Galápagos, and that there is ongoing movement of mosquitoes between islands. Tourist cruise boats and inter-island boat services are the most likely mechanism for transporting Culex mosquitoes between islands. Such anthropogenic mosquito movements increase the risk of the introduction of mosquito-borne diseases novel to Galápagos and their subsequent widespread dissemination across the archipelago. Failure to implement and maintain measures to prevent the human-assisted transport of mosquitoes to and among the islands could have catastrophic consequences for the endemic wildlife of Galápagos.

Natural colonization and adaptation of a mosquito species in Galapagos and its implications for disease threats to endemic wildlife.

Emerging infectious diseases of wildlife have been recognized as a major threat to global biodiversity. Endemic species on isolated oceanic islands, such as the Galápagos, are particularly at risk in the face of introduced pathogens and disease vectors. The black salt-marsh mosquito (Aedes taeniorhynchus) is the only mosquito widely distributed across the Galápagos Archipelago. Here we show that this mosquito naturally colonized the Galápagos before the arrival of man, and since then it has evolved to represent a distinct evolutionary unit and has adapted to habitats unusual for its coastal progenitor. We also present evidence that A. taeniorhynchus feeds on reptiles in Galápagos in addition to previously reported mammal and bird hosts, highlighting the important role this mosquito might play as a bridge-vector in the transmission and spread of extant and newly introduced diseases in the Galápagos Islands. These findings are particularly pertinent for West Nile virus, which can cause significant morbidity and mortality in mammals (including humans), birds, and reptiles, and which recently has spread from an introductory focus in New York to much of the North and South American mainland and could soon reach the Galápagos Islands. Unlike Hawaii, there are likely to be no highland refugia free from invading mosquito-borne diseases in Galápagos, suggesting bleak outcomes to possible future pathogen introduction events.

Predicting pathogen introduction: West Nile virus spread to Galáipagos.

Emerging infectious diseases are a key threat to conservation and public health, yet predicting and preventing their emergence is notoriously difficult. We devised a predictive model for the introduction of a zoonotic vector-borne pathogen by considering each of the pathways by which it may be introduced to a new area and comparing the relative risk of each pathway. This framework is an adaptation of pest introduction models and estimates the number of infectious individuals arriving in a location and the duration of their infectivity. We used it to determine the most likely route for the introduction of West Nile virus to Galápagos and measures that can be taken to reduce the risk of introduction. The introduction of this highly pathogenic virus to this unique World Heritage Site could have devastating consequences, similar to those seen following introductions of pathogens into other endemic island faunas. Our model identified the transport of mosquitoes on airplanes as the highest risk for West Nile virus introduction. Pathogen dissemination through avian migration and the transportation of day-old chickens appeared to be less important pathways. Infected humans and mosquitoes transported in sea containers, in tires, or by wind all represented much lower risk. Our risk-assessment framework has broad applicability to other pathogens and other regions and depends only on the availability of data on the transport of goods and animals and the epidemiology of the pathogen.

Characterization of canarypox-like viruses infecting endemic birds in the Galápagos Islands.

The presence of avian pox in endemic birds in the Galápagos Islands has led to concern that the health of these birds may be threatened by avipoxvirus introduction by domestic birds. We describe here a simple polymerase chain reaction-based method for identification and discrimination of avipoxvirus strains similar to the fowlpox or canarypox viruses. This method, in conjunction with DNA sequencing of two polymerase chain reaction-amplified loci totaling about 800 bp, was used to identify two avipoxvirus strains, Gal1 and Gal2, in pox lesions from yellow warblers (Dendroica petechia), finches (Geospiza spp.), and Galápagos mockingbirds (Nesomimus parvulus) from the inhabited islands of Santa Cruz and Isabela. Both strains were found in all three passerine taxa, and sequences from both strains were less than 5% different from each other and from canarypox virus. In contrast, chickens in Galápagos were infected with a virus that appears to be identical in sequence to the characterized fowlpox virus and about 30% different from the canarypox/Galápagos group viruses in the regions sequenced. These results indicate the presence of canarypox-like viruses in endemic passerine birds that are distinct from the fowlpox virus infecting chickens on Galápagos. Alignment of the sequence of a 5.9-kb region of the genome revealed that sequence identities among Gal1, Gal2, and canarypox viruses were clustered in discrete regions. This indicates that recombination between poxvirus strains in combination with mutation led to the canarypox-like viruses that are now prevalent in the Galápagos.