A new diagnostic technique for identifying Angiostrongylus spp. larvae in intermediate snail species by examining the buccal cavity.

BACKGROUND: Angiostrongyliasis is a zoonotic parasitic disease caused by the rat lungworm Angiostrongylus cantonensis. The intermediate hosts of A. cantonensis are gastropods, and snail species such as Pomacea canaliculata play a key role in the transmission of human angiostrongyliasis. Detecting A. cantonensis infection in snails is an important component of epidemiological surveillance and the control of angiostrongyliasis. METHODS: In this study, a new method for diagnosing A. cantonensis infection in gastropods was developed by recovering larvae from the buccal cavity of three snail species. The entire buccal cavity of a snail was extracted, and the tissue was pressed between two microscope slides to observe whether A. cantonensis larvae were present. Our new method was compared with traditional pathogenic detection methods of lung microscopy, tissue homogenization, and artificial digestion. We artificially infected 160 P. canaliculata, 160 Cipangopaludina chinensis, and 160 Bellamya aeruginosa snails with A. cantonensis. Then, the four different detection methods were used to diagnose infection in each snail species at 7, 14, 21, and 28 days post exposure. RESULTS: We found no significant difference in the percentages of infected P. canaliculata snails using the four methods to detect A. cantonensis larvae. The radula pressing method had a mean detection rate of 80%, while the lung microscopy (81.3%), tissue homogenization (83.8%), and artificial digestion (85%) methods had slightly greater detection rates. Similarly, the percentages of infected C. chinensis snails that were detected using the radula pressing (80%), tissue homogenization (82.1%), and artificial digestion (83.8%) methods were not significantly different. Finally, the percentages of infected B. aeruginosa snails that were detected using the radula pressing (81.3%), tissue homogenization (81.9%), and artificial digestion (81.4%) methods were not significantly different. These results showed that the radula pressing method had a similar detection rate to traditional lung microscopy, tissue homogenization, or artificial digestion methods. CONCLUSIONS: This study demonstrates a new method for the qualitative screening of gastropods that act as intermediate hosts of A. cantonensis (and other Angiostrongylus species), provides technical support for the control of human angiostrongyliasis, and furthers research on A. cantonensis.

Differentiation of Bulinus senegalensis and Bulinus forskalii Snails in West Africa Using Morphometric Analysis.

PURPOSE: Accurate identification of medically important intermediate host and vector species is crucial for understanding disease transmission and control. Identifying Bulinus snails which act as intermediate host species for the transmission of schistosomiasis is typically undertaken using conchological and genital morphology as well as molecular methods. METHODS: Here, a landmark-based morphometric analysis of shell morphology was undertaken to determine its utility to distinguish the closely related and morphologically similar sister species Bulinus senegalensis and Bulinus forskalii. The method was developed to increase the accuracy of conchological morphology methods to identify Bulinus species in the field. Both species are found in West Africa, but only B. senegalensis is implicated in the transmission of urogenital schistosomiasis. RESULTS: We found when scaled down to the same length, 3-whorl and 4-whorl (juvenile) B. senegalensis shells had a longer spire, narrower body whorl and shorter aperture than B. forskalii. In contrast, 5-whorl (adult) B. senegalensis had a shorter spire, but still had a shorter aperture and narrower body whorl than B. forskalii. Canonical Variate Analysis (CVA) showed minimal overlap between B. senegalensis and B. forskalii for 3-whorl and 4-whorl shells, with a clear separation for 5-whorl shells. Overall, B. senegalensis had a consistently shorter aperture size and narrower body whorl than B. forskalii for all development stages. Spire length was variable depending on the stage of development, with 3-whorl and 4-whorl shells having the opposite trends of adult shells. CONCLUSIONS: Our study demonstrates the applicability of landmark-based morphometrics in distinguishing the medically important, Bulinus senegalensis from its morphologically similar sister species, Bulinus forskalii. We recommend using measurements based on spire length, penultimate whorl length, body whorl width and aperture size to differentiate B. senegalensis and B. forskalii, when used with the appropriate information for each shell's development stage.

Seasonal patterns of Schistosoma mansoni infection within Biomphalaria snails at the Ugandan shorelines of Lake Albert and Lake Victoria.

Intestinal schistosomiasis is hyperendemic in many sub-Saharan African countries. In Uganda, it is endemic at both Lake Albert (LA) and Lake Victoria (LV) and caused by S. mansoni that uses Biomphalaria snails as obligatory intermediate snail hosts. To shed light on local patterns of infection, we utilised two PCR-based methods to detect S. mansoni within Biomphalaria spp. as collected at the Ugandan shorelines of Lake Albert and Lake Victoria from 2009-2010. Overall, at our Lake Albert sites, the mean infection prevalence was 12.5% (15 of 120 snails), while at our Lake Victoria sites the prevalence was 5% (3 of 60 snails). At our Lake Albert sites, the highest infection prevalence of 13.3% (8 of 60 snails) was at Walukuba, while at our Lake Victoria sites, the highest infection prevalence of 10% (2 of 20 snails) was at Lwanika. Three species of Biomphalaria, B. pfeifferi, B. stanleyi and B. sudanica, were identified at our Lake Albert collection sites, while only a single species, B. choanomphala, was identified at our Lake Victoria collection sites. Biomphalaria stanleyi (2 of 20 snails; 15%) had the highest infection prevalence, followed by B. sudanica (5 of 60 snails; 13.3%), B. pfeifferi (4 of 40 snails; 10%) and B. choanomphala (3 of 60 snails; 5%). Of the Biomphalaria species identified, B. choanomphala had the highest haplotype (gene) diversity score, followed by B. stanleyi, B. sudanica and B. pfeifferi. Sites with a higher mean prevalence of S. mansoni infection had higher intra-species haplotype diversity scores than sites with a lower mean prevalence. The wet seasons (LA: 13.3%; LV: 8.7%) had a consistently higher mean infection prevalence of S. mansoni than the dry seasons (LA: 9.5%; LV: 5%) for all species and all sites tested at both Lake Albert (n = 480) and Lake Victoria (n = 320), though the difference was not statistically significant.

Comparing PCR techniques against conventional cercarial shedding methods for detecting Schistosoma mansoni infection in Biomphalaria snails.

The detection of Schistosoma mansoni infection in both its intermediate (snail) and definitive (human) hosts is useful in providing information on the transmission of schistosomiasis. Three pairs of previously designed PCR primers (SM1-7, SMF/R & ND5) used for the detection of S. mansoni infection were tested. We assess the utility of each of these primer sets for detecting S. mansoni infection both in artificially exposed laboratory bred Biomphalaria glabrata, and field infected African Biomphalaria sudanica and Biomphalaria pfeifferi. Two of the three primer sets (SMF/R & ND5) detected S. mansoni infection in snails, but amplification of S. mansoni DNA with SM1-7 was unreliable. For the artificially exposed laboratory bred B. glabrata snails, SMF/R and ND5 both detected infection in more snails than the cercarial shedding method. Infection detection rates were 62.4% for ND5, 57.1% for SMF/R and 50.4% using traditional cercarial shedding methods. Both SMF/R and ND5 detected S. mansoni infection in 91% of snails observed shedding cercariae, increasing to 98.5% when low stringency PCR methods were used. When comparing each of the detection methods using a Bayesian latent class analysis model, ND5 had the highest detection sensitivity and negative predictive value (NPV), while SMF/R had the highest detection specificity and positive predictive value (PPV). In field collected Biomphalaria snails, ND5 detected S. mansoni infection in 21 of 24 snails categorised as shedding S. mansoni cercariae and 4 of 24 snails categorised as shedding non-S. mansoni cercariae, while SMF/R detected infection in 18 of 24 snails categorised as shedding S. mansoni cercariae and in 3 of 24 snails categorised as shedding non-S. mansoni cercariae. All SMF/R and ND5 PCR products were shown to be S. mansoni indicating that these field snails must have been infected with both S. mansoni and cercariae from other Schistosoma species. This indicates that the two primer sets are specific for S. mansoni and will not amplify non-S. mansoni species when used at their recommended annealing temperatures. Both the SMF/R and ND5 primers effectively detected S. mansoni infection in three Biomphalaria species and have improved detection sensitivity over cercarial shedding.