A new diagnostic technique for identifying Angiostrongylus cantonensis larvae in the odontophores (radula knobs) of different intermediate snail species
Authors: Zhao Yongbo , Jiang Ling , Fang Wen , Chen Shaorong , Liu Yuhua , Zhao Shenhua , Andrus Peter , Li Tianmei , Guo Yunhai 
Published: February 2024

Abstract
Background
Angiostrongyliasis is a zoonotic parasitic disease cause by the Rat lungworm, Angiostrongylus cantonensis. The intermediate hosts of A. cantonensis are gastropods, with specific snails species such as Pomacea canaliculata playing a key role in the transmission of human angiostrongyliasis. The detection of A. cantonensis infection in snails is an important part of the epidemiological surveillance and control of angiostrongyliasis. Currently, the detection methods of A. cantonensis infection in snails are divided into pathogenic detection techniques and molecular biology detection techniques, with the pathogenic detection techniques being the gold standard for detection.
Methods
In this study, a new method of diagnosing A. cantonensis infection in gastropods was developed by testing whether larvae could be recovered from the buccal cavity of three different snail species. This was done by extracting the entire buccal cavity of a snail (e.g. the jaws, radula and the radula knobs) and pressing the tissue between two microscope slides to view whether A. cantonensis larvae were present or not. To test the effectiveness of our detection method, we compared it against other traditional pathogenic detection techniques such as lung-microscopy, tissue homogenization and artificial digestion. This was done by artificially infecting 160 P. canaliculata, 160 Cipangopaludina chinensis and 160 Bellamya aeruginosa snails with A. cantonensis. Afterwards, four different detection methods (the radula pressing method, lung-microscopy, tissue homogenization, artificial digestion) were used to diagnose infection each of snail species at 7, 14, 21, and 28 days post-infection.
Results
When using each of the four methods to detect A. cantonensis larvae in P. canaliculata snails, we found no significant difference in the percentage of infected snails detected when any of the four detection methods. The radula pressing method showed a mean detection rate of 80%, while the lung-microscopy method (81.25%), tissue homogenization (83.75%) and the artificial digestion (85%) methods showed a slightly higher rate of detection (χ2 = 1.791, P = 0.617). Similarly, the rate of detection in Bellamya aeruginosa snails by the radula pressing method (81.25%), tissue homogenization (81.875%) and artificial digestion (81.375%) also showed no significant difference in the number of infected snails detected (χ2 = 0.606, P = 0.739). Lastly, the rate of detection for the Cipangopaludina chinensis snails by the radula pressing method (80%), tissue homogenization (82.125%) and artificial digestion (83.75%) also showed no significant difference in the number of infected snails detected (χ2 = 1.6, P = 0.449). These results showed that using the radula pressing method to detect the presence of A. cantonensis larvae in intermediate snails hosts has a similar rate of detection to that of other traditional Angiostrongylus detection methods such as lung-microscopy, tissue homogenization or artificial digestion. However, our method has several advantages such as it can be used to detect A. cantonensis larvae in all gastropod hosts (unlike lung-microscopy), and is suitable for large-scale, on-site detection surveys. Secondly, the method is simple and faster to perform compared other detection methods (like tissue homogenization or artificial digestion) and can be used for quick qualitative on-site screening during newly reported outbreaks.
Conclusions
This study provides a new method for the qualitative screening of gastropod hosts that act as the intermediate hosts of A. cantonensis, and provides technical support for the control of human Angiostrongyliasis and aids in the research of A. cantonensis.