Piyanan Taweethavonsawat

Rapid detection of Dirofilaria immitis in mosquito vectors and dogs using a real-time fluorescence resonance energy transfer PCR and melting curve analysis

A real-time fluorescence resonance energy transfer (FRET) PCR supplemented with melting curve analysis for the rapid molecular detection of Dirofilaria immitis in mosquito vectors and dog blood samples was developed. This real-time FRET PCR was based on the fluorescence melting curve analysis of a hybrid between an amplicon generated from the D. immitis ribosomal RNA gene sequence and specific fluorophore-labeled probes. The sensitivity, specificity, accuracy, and positive and negative predictive values of this method were all 100%. Besides being highly sensitive and specific, this PCR is fast and offers a high throughput. Therefore it is a suitable and powerful tool for the diagnosis and for epidemiological surveys of canine dirofilariasis as well as for molecular xenomonitoring of D. immitis in mosquito vectors.

Efficacy of a spot on combination containing imidacloprid 10% and moxidectin 1% (Advocate(®)/Advantage(®) Multi, Bayer Animal Health) against Ancylostoma ceylanicum in cats.

Ancylostoma ceylanicum is a common zoonotic hookworm of dogs and cats, especially in the Asia-Pacific region. The objective of this study was to determine the efficacy of a spot on combination product containing imidacloprid 10% and moxidectin 1% (Advocate(®)/Advantage(®) Multi, Bayer Animal Health) against A. ceylanicum in experimentally infected cats. Sixteen kittens were each subcutaneously injected with 100 infective third-stage larvae of A. ceylanicum. Kittens were stratified by egg count and randomly allocated into control and treatment groups. The kittens in the treatment group were each treated with a spot on combination of 10% (w/v) imidacloprid and 1% (w/v) moxidectin, administered topically at recommended label dose rates. The kittens in the control group were not treated. Egg counts were performed daily until the end of the study period and compared for the treated and control groups. No eggs were detected in the treated group of kittens within 4 days of treatment and faecal samples from this group remained negative throughout the rest of the study, resulting in a treatment efficacy (egg reduction) of 100% (P<0.0001). The egg counts remained high (993 ± 666 epg) in the untreated control group for the rest of the study period. This study demonstrated that based on faecal egg count reduction, the spot on combination containing imidacloprid 10% (w/v) and moxidectin 1% (w/v) (Advocate(®)/Advantage(®) Multi, Bayer Animal Health) given at the recommended dose is highly effective against infection with A. ceylanicum in cats.

Rapid Detection and Identification of Wuchereria bancrofti, Brugia malayi, B. pahangi, and Dirofilaria immitis in Mosquito Vectors and Blood Samples by High Resolution Melting Real-Time PCR

A simple, rapid, and high-throughput method for detection and identification of Wuchereria bancrofti, Brugia malayi, Brugia pahangi, and Dirofilaria immitis in mosquito vectors and blood samples was developed using a real-time PCR combined with high-resolution melting (HRM) analysis. Amplicons of the 4 filarial species were generated from 5S rRNA and spliced leader sequences by the real-time PCR and their melting temperatures were determined by the HRM method. Melting of amplicons from W. bancrofti, B. malayi, D. immitis, and B. pahangi peaked at 81.5±0.2℃, 79.0±0.3℃, 76.8±0.1℃, and 79.9±0.1℃, respectively. This assay is relatively cheap since it does not require synthesis of hybridization probes. Its sensitivity and specificity were 100%. It is a rapid and technically simple approach, and an important tool for population surveys as well as molecular xenomonitoring of parasites in vectors.

Comparative efficacy of a spot-on formulation containing emodepside and praziquantel (Profender ®, Bayer) and praziquantel and pyrantel oral tablets (Drontal ® for Cats) against experimental Ancylostoma ceylanicum infections in cats.

Ancylostoma ceylanicum is a common zoonotic hookworm of dogs and cats throughout Asia and has also been reported to occur within the Australasian region. The aim of this study to was to determine the efficacy of a spot-on formulation containing emodepside and praziquantel (Profender(®), Bayer) and praziquantel and pyrantel oral tablets (Drontal(®) for Cats, Bayer) against experimental A. ceylanicum infections in cats. Twenty-four kittens were each subcutaneously injected with 100 infective third-stage larvae of A. ceylanicum. Kittens were stratified by egg count and randomly allocated equally into control and two treatment groups. The first group were treated with emodepside 2.1%/praziquantel 8.6% (Profender®, Bayer) at the recommended label dose. The second group was treated with 80 mg pyrantel and 20mg praziquantel (Drontal(®) for Cats, Bayer) at the recommended label dose. The kittens in the control group were not treated. Egg counts were performed daily until the end of the study period and compared for the treated and control groups. No eggs were detected in the treated group of kittens within 4 days of treatment and faecal samples from this group remained negative throughout the rest of the study, resulting in a treatment efficacy (egg reduction) of 100% (P<0.0001). The egg counts remained high (993 ± 666 epg) in the untreated control group for the rest of the study period. This study demonstrated that both combination products containing topical emodepside/praziquantel (Profender(®), Bayer) and praziquantel/pyrantel oral tablets (Drontal(®) for Cats, Bayer) given at the recommended dose is highly effective against infection with A. ceylanicum in cats.

Detection of Babesia canis vogeli and Hepatozoon canis in canine blood by a single-tube real-time fluorescence resonance energy transfer polymerase chain reaction assay and melting curve analysis

A real-time fluorescence resonance energy transfer polymerase chain reaction (qFRET PCR) coupled with melting curve analysis was developed for detection of Babesia canis vogeli and Hepatozoon canis infections in canine blood samples in a single tube assay. The target of the assay was a region within the 18S ribosomal RNA gene amplified in either species by a single pair of primers. Following amplification from the DNA of infected dog blood, a fluorescence melting curve analysis was done. The 2 species, B. canis vogeli and H. canis, could be detected and differentiated in infected dog blood samples (n = 37) with high sensitivity (100%). The detection limit for B. canis vogeli was 15 copies of a positive control plasmid, and for H. canis, it was 150 copies of a positive control plasmid. The assay could simultaneously distinguish the DNA of both parasites from the DNA of controls. Blood samples from 5 noninfected dogs were negative, indicating high specificity. Several samples can be run at the same time. The assay can reduce misdiagnosis and the time associated with microscopic examination, and is not prone to the carryover contamination associated with the agarose gel electrophoresis step of conventional PCR. In addition, this qFRET PCR method would be useful to accurately determine the range of endemic areas or to discover those areas where the 2 parasites co-circulate.

Successful treatment of Brugia pahangi in naturally infected cats with ivermectin.

Lymphatic filariasis is a common parasitic disease of cats in tropical regions including Thailand. The objective of this study was to determine the efficacy of ivermectin against microfilariae of Brugia pahangi in naturally infected cats. Eight cats naturally infected with B. pahangi were divided into control (untreated) and treated groups. Cats in the latter group were given ivermectin injection at 400 µg/kg weekly for 2 months. Microfilariae were counted every week until 48 weeks. Microfilaremia was significantly decreased in the treated group 4 weeks after starting the treatment and become zero at week 9 and afterwards. On the other hand, cats in the control group had high microfilaremia throughout the study. It was successful to treat and control B. pahangi infection in naturally infected cats using ivermectin.