Herbal Essential Oils Along with Its Amelioration with Silver Nanoparticles for Curing the Canine Demodicosis

Demodicosis, also named as demodectic mange, red mange or follicular mange (Shrestha et al., 2015). In dogs, Demodex canis is acquired from the dam during the first few hours of life, probably during suckling (Greve and Gaafar, 1966). Demodicosis can be defined on the basis of two forms localized and generalized (Shipstone, 2000) with juvenile or adult onset. The ideal confirmation of diagnosis of demodicosis were established by the laboratory analysis of the cutaneous skin scrapings Various drugs have been used for treating canine demodicosis. The bad effects of amitraz therapy include depression, sleepiness sometimes ataxia and polyphagia/polydipsia. Herbal medicine (also known as traditional, folk as well as alternative medicine) comprises of medical knowledge systems that were developed over generations within various societies before the era of modern medicine. In this approach nanotechnology played a great role in herbal medicine and more specifically in the field of drug delivery is onset to spread fastly. Though skin diseases are very important disease ending a very few works has been carried out in north-east region including Mizoram. Till date no research work has been done on herbal nano medicine against demodicosis in dogs especially in Mizoram. Keeping these points in view the present study was made to formulate the  herbal nanomedicine against demodicosis in dogs.

The study was conducted in Teaching Veterinary Clinical Complex (T.V.C.C), College of Veterinary Sciences and Animal Husbandry, Central Agricultural University, Selesih, Aizawl, Mizoram and State Veterinary Hospital, Khatla, Aizawl, Mizoram.

Molecular diagnosis (PCR)
 
PCR protocol/DNA amplification
 
The PCR assay was optimized targeting a portion of the 18S rRNA gene to amplify Demodex species.
 
DNA sequencing
 
Sequencing of target partial length was done from DNA sequencing facility, Molbiogen Organisation, Beltola, Guwahati-Assam DNA sequences were compared for similarity with sequences available in GenBank®, using the BLAST program.
 
Detection of Demodex spp. by molecular technique
 
By PCR assay, it was confirmed that total 35 cases were positive (including 20 positive cases confirmed by microscope). An approximately 483 bp PCR product were amplified in gel electrophoresis from the skin scraping of 35 dogs which were further confirmed by sequencing. The BLAST analysis confirmed 33 samples were 99.8% identical to available sequences with D. canis isolated from dogs of China (MG372354), one sample was 98.8% similarity of the sequences with Demodex canis isolated from dogs of China (KU253790) and another one samples was 96.8% similarity of the sequences with Demodex canis isolated from dogs of China (MG372359). The sequence of PCR product were annotated and submitted to NCBI GenBank for accession number. Accession number obtained from GenBank was MK177513.
 

 

 
Phylogenetic analysis
 
The phylogenetic analysis performed on the basis of the 18S rRNA gene sequence which revealed that species of the genus Demodex were clustered with China isolates (MG372354, KU253790 and MG372359). The phylogenetic analysis supported that the both genera shared a common ancestor (Fig 1).
 

 
Measurement of silver nitrate nano particle size and herbo- nano particle size by transmission electron microscope
 
Twenty microlitre of the ultrasonicated liquid sample was placed on carbon coated 200 mesh size copper grid. The grid was allowed to air dry for overnight. The images were taken by transmission electron microscope (Hitachi H-7650 model TEM) at 80 KV acceleration voltage (Fig 3 A,B,C).
 

 

Biosynthesis of silver nanoparticles
 
Synthesis of AgNPs was observed when herb formulation (Neem oil + Lemon oil + Garlic oil) was incubated with aqueous solution of silver nitrate, a gradual change of colour was observed after 15 minutes (Fig 4B). The colour of silver nitrate treated formulation turned to milky white (Fig 4C).
 

 
Transmission electron microscopy (TEM)
 
TEM image of AgNPs indicated the formation of spherical nanoparticles with a few agglomerations (Fig 4 and 5).
An average size of these nanoparticles at shaking conditions was 287.5 nm287.55±0.76 and without adding herbal formulation of AgNO­ was 144.44±0.56 (Table 3). The particle size of herbal nano formulation ranged from 200-500 nm (Fig 5) and without adding herbo formulation of AgNO3 was 100-200 nm (Fig 6).
 



 

 
Biosynthesis of silver nanoparticles
 
Biosynthesis of Ag NPs using essential herbal oil In vitro under controlled condition were carried out for the first time in this study. This study confirmed essential oils have capability for the biosynthesis of Ag NPs. The plant oil was mixed with the AgNO­ solution, a colour change from pale yellow to dark yellow and finally colloidal brown was observed within few minute. After few hours (hrs), there was no further change in the colour of the solution indicating that the entire silver salt were present in the solution had been reduced. The data obtained from transmission electron-micrograph showed distinct shape and size of nanoparticles. The particles shape were in shape with mean of 13.88±4.11 nm. AgNPs uniformly distributed with some agglomeration which revealed pattern similar to the biosynthesized AgNPs by Jain (2011). Now generally definition of a size ≤100 nm (The Royal Society and Royal Academy of Engineering 2004) as nanoparticles.
 
Therapeutic evaluation of herbal nano medicine against amitraz in demodicosis infected dogs
 
Chemotherapeutic agents used for curing the treatment of canine demodicosis are amitraz and ivermectin. Amitraz has been most commonly utilized for the treatment D. canis infection. Although this medicine is effective against D. canis, it sometimes induces side effects such as high blood sugar level, vomition, lower body temperature, diarrhea, unsteadiness (ataxia) and slow heart rate. Amitraz may cause toxicity after its oral intake in sufficient concentration. To combat these resistance problems, the present study was evaluated the acaricidal effect of Azadirachta indica oil, Citrus lemon oil and Allium sativum oil with nano particles against canine demodicosis. It was selected based on their wide pharmacological and medicinal uses and also by its various previous acaricidal activity. Neem oil contains many potent bioactive components that are present in different crude extracts. In general, the pesticidal activity of neem oil is thought to be due to azadirachtin (Isman, 1990). Neem oil are preferred for their economic benefits, low environmental persistence, lack of resistance in insects and relative non-toxicity to mammals (Jacobson, 1995). Garlic contains Sulphur compounds which mites dislike (Yadav et al., 2018). Diluted garlic oil can be used topically on the affected areas. Since garlic is antibacterial as well as applying garlic oil on the affected areas will have the benefit of minimizing lower bacterial infection. Citrus essential oils have larvicidal activity against mosquito species (Amer and Mehlhorn 2006; Lee 2006; Michaelakis et al., 2009). Lee (2006) worked on efficacy of lemon oil against sarcoptic mange and treatment  with 20% lemon oil achieved much faster recovery and rapid skin cure; this might be due the keratolytic effect of lemon. The study demonstrated herbal nano medicine to be an effective treatment for demodicosis without concurrent side effects. Therefore, the purpose and strategy of demodicosis therapy, rather than to quickly and completely eliminate the mite from hosts, have been to suppress the proliferation of mites without any adverse side effects. Based upon the results of this study, herbal nano medicine appears to be effective therapeutic regiment against demodicosis.
Conflict of interest
 
The authors declared that there is no conflict of interest.

Herbal nano medicine were found to be effective therapeutic measures and it can be used as an alternative of Amitraz against demodicosis. Further research is necessary to explicit the role of nanoparticles in canine demodicosis.

The authors are highly thankful to Vice-Chancellor of Central Agricultural University, for carrying out the research work.