POTENTIALLY PATHOGENIC FREE-LIVING AMOEBAE IN SOME FLOOD-AFFECTED AREAS DURING 2011 CHIANG MAI FLOOD

SUMMARY The survey was carried out to investigate the presence of potentially pathogenic free-living amoebae (FLA) during flood in Chiang Mai, Thailand in 2011. From different crisis flood areas, seven water samples were collected and tested for the presence of amoebae using culture and molecular methods. By monoxenic culture, FLA were detected from all samples at 37 °C incubation. The FLA growing at 37 °C were morphologically identified as Acanthamoeba spp., Naegleria spp. and some unidentified amoebae. Only three samples (42.8%), defined as thermotolerant FLA, continued to grow at 42 °C. By molecular methods, two non-thermotolerant FlA were shown to have 99% identity to Acanthamoeba sp. and 98% identity to Hartmannella vermiformis while the two thermotolerant FLA were identified as Echinamoeba exundans (100% identity) and Hartmannella sp. (99% identity). This first report of the occurrence of FLA in water during the flood disaster will provide information to the public to be aware of potentially pathogenic FLA.RESUMO A pesquisa foi feita para investigar a presença de amebas de vida livre (FLA) durante a inundação em Chiang Mai, Tailândia, ano de 2011. A partir de diferentes áreas de inundação sete amostras de água foram coletadas e testadas para a presença de amebas usando métodos moleculares e de cultura. Através da cultura monoxênica, FLA foi detectada em todas as amostras após incubação a 37 °C. As FLA crescendo a 37 °C foram identificadas morfologicamente como Acanthamoeba spp, Naegleria spp e algumas amebas não determinadas. Somente três amostras (42,8%) definidas como FLA termotolerantes continuaram a crescer a 42 °C. Por métodos moleculares duas FLA termotolerantes tiveram 99% de identidade com a Acanthamoeba sp e 98% de identidade com Hartmannella vermiformis enquanto as duas FLA termotolerantes foram identificadas como Echinamoeba exundans (100% de identidade) e Hartmannella sp (99% de identidade). Este primeiro relato da ocorrência de FLA em águas durante inundações informa ao público que ele deve estar atento de FLA potencialmente patogênica

First isolation of Leishmania from Northern Thailand:case report, identification as Leishmania martiniquensis and phylogenetic position within the Leishmania enriettii complex

Since 1996, there have been several case reports of autochthonous visceral leishmaniasis in Thailand. Here we report a case in a 52-year-old Thai male from northern Thailand, who presented with subacute fever, huge splenomegaly and pancytopenia. Bone marrow aspiration revealed numerous amastigotes within macrophages. Isolation of Leishmania LSCM1 into culture and DNA sequence analysis (ribosomal RNA ITS-1 and large subunit of RNA polymerase II) revealed the parasites to be members of the Leishmania enriettii complex, and apparently identical to L. martiniquensis previously reported from the Caribbean island of Martinique. This is the first report of visceral leishmaniasis caused by L. martiniquensis from the region. Moreover, the majority of parasites previously identified as "L. siamensis" also appear to be L. martiniquensis

Genetic and morphological evidence for a new species of the Maculatus Group of Anopheles subgenus Cellia (Diptera: Culicidae) in Java, Indonesia

Abstract Background Anopheles maculatus, a species of the Maculatus Group of subgenus Cellia (Diptera: Culicidae), is an important vector of human malarial protozoa in Java, Indonesia. However, the identity of this species in Indonesia has been questionable because published reports and records are based mainly on morphological identification, which is unreliable for distinguishing members of the Maculatus Group due to overlapping characters. Methods We performed morphological assessments, metaphase karyotype preparations, phylogenetic analyses of ITS2 and cox2 sequence data and cross-mating experiments to determine whether the Javanese form and An. maculatus (s.s.) from Thailand were conspecific. Results The adults of the Java strain are similar to those of An. maculatus (s.s.), but the larvae and pupae exhibit significant differences. The metaphase karyotype of Javanese specimens includes a long acrocentric X chromosome and a small telocentric Y chromosome, which are distinct from other members of the Maculatus Group. Cross-mating of the Java strain with An. maculatus (s.s.) revealed genetic incompatibility. Phylogenetic analysis of ITS2 and cox2 sequences revealed that the Java strain forms a single clade that is distinct from clades of other members of the group (Kimura 2-parameter, K2P, genetic distances 3.1–19.2% and 1.6–9.6%, respectively). Conclusions This study provides evidence that the Javanese form of An. maculatus is not conspecific with An. maculatus (s.s.) and constitutes a previously unrecognized species of the Maculatus Group

Chrysomya chani Kurahashi (Diptera: Calliphoridae), a blow fly species of forensic importance: morphological characters of the third larval instar and a case report from Thailand

Blow flies are worldwide the most important insects from a forensic point of view. In Thailand, aside from the two most common species, Chrysomya megacephala (F.) and Chrysomya rufifacies (Macquart), Chrysomya chani Kurahashi was also found to be of forensic importance. We present a case of a human female cadaver in its bloated stage of decomposition, discovered at Pachangnoi Subdistrict, northern Thailand. Entomological sampling during the autopsy displayed an assemblage of numerous dipteran larvae. Macroscopic observations showed the coexistence of third instar larvae of the three blow flies C. megacephala, Chrysomya villeneuvi Patton, an unknown blow fly species and one muscid, Hydrotaea sp. The minimum post-mortem interval was estimated to be six days, based on the developmental rate of C. megacephala. The ID of the unknown larva, which is the focus of this report, was revealed later as C. chani by DNA sequencing, using a 1205 bp of cytochrome c oxidase subunit I (COI). The occurrence of C. chani on a human body revealed the need to analyse and describe the morphology of its immature stage, to enable forensic entomologists to identify this fly species in future cases. The morphological examination of the third instar was performed, revealing peculiar characteristics: protuberant tubercles encircling abdominal segments; 9–11 lobes on the anterior spiracle; six prominent pairs of tubercles along the peripheral rim of the eighth abdominal segment; a heavily sclerotized complete peritreme of the posterior spiracles. A key to differentiate the third instar of blow flies of forensic importance in Thailand is provided

Wing morphometrics as a tool in species identification of forensically important blow flies of Thailand

Background: Correct species identification of blow flies is a crucial step for understanding their biology, which can be used not only for designing fly control programs, but also to determine the minimum time since death. Identification techniques are usually based on morphological and molecular characters. However, the use of classical morphology requires experienced entomologists for correct identification; while molecular techniques rely on a sound laboratory expertise and remain ambiguous for certain taxa. Landmark-based geometric morphometric analysis of insect wings has been extensively applied in species identification. However, few wing morphometric analyses of blow fly species have been published. Methods: We applied a landmark-based geometric morphometric analysis of wings for species identification of 12 medically and forensically important blow fly species of Thailand. Nineteen landmarks of each right wing of 372 specimens were digitised. Variation in wing size and wing shape was analysed and evaluated for allometric effects. The latter confirmed the influence of size on the shape differences between species and sexes. Wing shape variation among genera and species were analysed using canonical variates analysis followed by a cross-validation test. Results: Wing size was not suitable for species discrimination, whereas wing shape can be a useful tool to separate taxa on both, genus and species level depending on the analysed taxa. It appeared to be highly reliable, especially for classifying Chrysomya species, but less robust for a species discrimination in the genera Lucilia and Hemipyrellia. Allometry did not affect species separation but had an impact on sexual shape dimorphism. Conclusions: A landmark-based geometric morphometric analysis of wings is a useful additional method for species discrimination. It is a simple, reliable and inexpensive method, but it can be time-consuming locating the landmarks for a large scale study and requires non-damaged wings for analysis

Chrysomya chani Kurahashi (Diptera: Calliphoridae), a blow fly species of forensic importance : morphological characters of the third larval instar and a case report from Thailand

Blow flies are worldwide the most important insects from a forensic point of view. In Thailand, aside from the two most common species, Chrysomya megacephala (F.) and Chrysomya rufifacies (Macquart), Chrysomya chani Kurahashi was also found to be of forensic importance. We present a case of a human female cadaver in its bloated stage of decomposition, discovered at Pachangnoi Subdistrict, northern Thailand. Entomological sampling during the autopsy displayed an assemblage of numerous dipteran larvae. Macroscopic observations showed the coexistence of third instar larvae of the three blow flies C. megacephala, Chrysomya villeneuvi Patton, an unknown blow fly species and one muscid, Hydrotaea sp. The minimum post-mortem interval was estimated to be six days, based on the developmental rate of C. megacephala. The ID of the unknown larva, which is the focus of this report, was revealed later as C. chani by DNA sequencing, using a 1205 bp of cytochrome c oxidase subunit I (COI). The occurrence of C. chani on a human body revealed the need to analyse and describe the morphology of its immature stage, to enable forensic entomologists to identify this fly species in future cases. The morphological examination of the third instar was performed, revealing peculiar characteristics: protuberant tubercles encircling abdominal segments; 9–11 lobes on the anterior spiracle; six prominent pairs of tubercles along the peripheral rim of the eighth abdominal segment; a heavily sclerotized complete peritreme of the posterior spiracles. A key to differentiate the third instar of blow flies of forensic importance in Thailand is provided