Cercarial trematodes in freshwater snails from Bangkok, Thailand: prevalence, morphological and molecular studies and human parasite perspective

We investigated the prevalence, morphological characters and molecular classifications of trematode cercariae in freshwater snails randomly collected from 59 sampling localities in Bangkok from May 2018 to March 2019. We used a crushing technique to observe the cercarial stage inside each snail body and amplified the internal transcribed spacer 2 regions of cercarial DNA using polymerase chain reaction methodology. The associated phylogenetic tree was reconstructed using Bayesian inference analyses. A total of 517 of 15 621 examined snails were infected with trematode cercariae, and the infected snails were classified into 11 species of seven families with a 3.31% overall prevalence of the infection. The Bithynia siamensis siamensis snail displayed the highest prevalence of infection (16.16%), whereas the Physella acuta snail exhibited the lowest prevalence (0.08%) of infection. Eight morphological types of cercariae were observed. The highest prevalence of infection was observed in mutabile cercaria (1.86%). Based on molecular investigations, the phylogram revealed eight cercaria types assigned to at least nine digenean trematode families, of which five belong to groups of human intestinal flukes. Although, with the exception of schistosome cercaria, trematode cercariae are not known to directly damage humans, understanding the general biology of trematode cercariae (including diversity, distribution, infection rates and host range) is important and necessary for the prevention and control of parasitic transmission that impacts aquatic cultivations, livestock farming and human health.

Detection of ‘Candidatus Liberibacter asiaticus’ in Diaphorina citri and Its Importance in the Management of Citrus Huanglongbing in Florida

Citrus huanglongbing (HLB or citrus greening), is a highly destructive disease that has been spreading in both Florida and Brazil. Its psyllid vector, Diaphorina citri Kuwayama, has spread to Texas and Mexico, thus threatening the future of citrus production elsewhere in mainland North America. Even though sensitive diagnostic methods have been developed for detection of the causal organisms, Candidatus Liberibacter spp., the pathogen cannot be detected consistently in plants until symptoms develop, presumably because of low titer and uneven distribution of the causal bacteria in nonsymptomatic tissues. In the present study, TaqMan based real-time quantitative polymerase chain reaction methodology was developed for detection of ‘Ca. L. asiaticus’ in D. citri. Over 1,200 samples of psyllid adults and nymphs, collected from various locations in Florida, from visually healthy and HLB symptomatic trees at different times of the year were analyzed to monitor the incidence and spread of HLB. The results showed that spread of ‘Ca. L. asiaticus’ in an area may be detected one to several years before the development of HLB symptoms in plants. The study suggests that discount garden centers and retail nurseries may have played a significant role in the widespread distribution of psyllids and plants carrying HLB pathogens in Florida.

ICC/PCR detection of enteroviruses and hepatitis A virus in environmental samples

This study applied the integrated cell culture/polymerase chain reaction methodology (ICC/PCR) for rapid and specific detection of both cytopathogenic and noncytopathogenic viruses. Results of this study showed that the use of direct RT-PCR or conventional cell culture alone may yield erroneous results with the analysis of environmental samples. The purpose of this study was to compare cultural, molecular, and combined assays for the most effective method of virus detection in variable environmental samples. Using ICC/PCR, stock enterovirus inocula of [Formula: see text]10 PFU were PCR positive in at least 4/5 replicate flasks after only 5 h of incubation in cell culture, and in all flasks after [Formula: see text]10 h. An inoculum of one PFU was detected by PCR after 20 h of cell culture incubation while for concentrations of virus below one PFU, 25 h of incubation was sufficient. Similarly, hepatitis A virus (HAV) inocula of 100 MPN/flask, produced indeterminate CPE in cell culture, but were clearly detected by ICC/PCR following 48 h of incubation. Lower levels of HAV, 1 and 10 MPN, were detected by ICC/PCR after 96 to 72 h of incubation, respectively. Cell culture lysates from 11 environmental sample concentrates of sewage, marine water, and surface drinking water sources, were positive for enteroviruses by ICC/PCR compared to 3 positive by direct RT-PCR alone. Results from ICC/PCR eventually agreed with cell culture but required [Formula: see text]48 h of incubation, compared to as long as 3 weeks for CPE following incubation with BGM and FRhK cells.Key words: RT-PCR, cell culture, ICC/PCR, enterovirus, hepatitis A virus.

Genetic structure of an introduced pest, grape phylloxera (Daktulosphaira vitifoliae Fitch), in Europe

A model for the genetic structure of grape phylloxera populations in Europe was developed using hierarchical sampling techniques and AFLP-PCR (amplified fragment length polymorphism - polymerase chain reaction) methodology. One-hundred three European and 6 North American phylloxera populations were studied. An additional European sampling set comprising 60 samples was analyzed to study regional subdivision. The populations grouped into two clusters loosely correlated with collection site location. Phylloxera populations collected from northern (above lat 43°) geographic regions were significantly different from southern (below 43°) populations. The northern cluster was more heterogeneous than the southern cluster, possibly reflecting holocyclic versus anholocyclic reproduction. Microgeographic scales of phylloxera genetic structure displayed as much variation within as among host plants. The host plant did not affect the genetic structure of European phylloxera as revealed in two independent experiments.Key words: Daktulosphaira vitifoliae, aphid, genetic structure, AFLP-PCR.