Dr Dina Resume.docx

Resume Cat flea, currently a member of Ctenocephalides genus, is considered as the most successful ectoparasite on Earth. Cat flea is also known as “pinjal” in Indonesian. Ctenocephalides sp. (Siphanoptera: Pulicidae) (Bouché) is the most common species found in cats and dogs all around the world. The parasite that broadens from insects, in mammals, is closely associated with human. As pets, dogs and cats have a significant potential for disease transmissions caused by pathogenic microbes. (Lawrence, et al., 2014). Many debates in morphologic characteristics and status of Ctenocephalides sp. (Lawrence, et. al., 2014) exist. Until today, there is no scholarly study of the status of this species originated from Manado City. Ctenocephalides felis is a competent vector for zoonotic pathogens, such as Rickettsia felis and Bartonella sp. Increasing knowledge of variety and phylogenetic of the flea is important in understanding pathogenic transmission cycle that “supported” by the fleas (Hii et.al., 2015). Cat flea causes not only health problem in animals, also in human. As animal that interacts with human very much, C. felis cat flea is very potential as disease vector. Microbial analyses that correlate well to the body surface and digestive tract of C. felis by conventional methods have many limitations. Nowadays studies in microbiology field report that 99% of microbes cannot be cultured in the artificial growth media in the laboratory. Thus, there are still many pathogenic microbes that are associated with vector insects but not yet known because they cannot be isolated and cultured in the laboratory growth media. Based on data from Manado Husbandry Office, cat population in Manado City is relatively high. Cat population is divided into domestic cats and cats that were initially nurtured but live wild or were poorly nurtured. Cat population is mostly found in the region of Karombasan, Malalayang, Paal 2, and Ranotana. Infestation of C. felis in domesticated cats and wildly live cats is different. However, wild cats can be the infestation media of C. felis to domesticated cats. This study is focused on characteristics study of morphology, molecular barcoding and metagenomics of C. felis isolated from cat fleas in Manado city. The study aims to (1) analyse morphological and morphometric characteristics of C. felis cat fleas in Manado city, (2) analyse the genetic variety of C. felis cat fleas in Manado, and (3) analyse the characteristics of bacteria in C. felis cat fleas in Manado. In each location 10 cats were taken, then C. felis cat fleas were taken from certain body parts: head, neck, chest, crease, and back, ten of each, with no distinguishing sexes. Study of morphological and morphometric characteristics of C. felis cat fleas used 3D Digital Microscope Hirox KH-8700. The morphological characteristics study of cat fleas were carried out according to the method of Balvin et al., (2012), and Khan and Rahman (2012). The observed parameters were eye colour, abdomen colour, wing colour, and hair intensity in the body organ. Then the morphometric characters are body length (PT), femur length of the back leg (PFTB), femur width of the back leg (LFTB), hind limb tibia length (PTTB), tibia width of the back leg (LTTB), ctenidium length, and eye diameter (LM). The genetic variety of C. felis cat flea is observed by using Gen CO1 with Polymerase Chain Reaction (PCR) 1

method. Sequencing data results were analysed with software Geneious 9.0 (Kearse et al., 2012) and programme Mega 6 (Tamura et al., 2013). The obtained sequences were then analysed with homology analysis using BLAST (Basic Local Alignment Search Tool) (Astchul et al., 1990) which was online connected with the NCBI GenBank (National Center of Biotechnology Information). Phylogenetic tree reconstruction was made by using neighbour-joining method and minimum evolution as the comparator. Calculation of matrices of genetic distance with the Kimura-2 parameter model is implemented in the pairwise distance calculation with Bootstrap 1000 replications in the MEGA (Molecular Evolutionary Genetics Analysis) software version 6.0 (Tamura et al., 2013). Sample sequences were compared with the NCBI GenBank collections. Pathogenic bacteria characteristics in C. felis cat flea used 16S rRNA. Metagenome data analysis was uploaded to MG-RAST server (https://metagenomics.anl-gov (Meyer et al., 2008). Taxonomy task was used SEED database and metabolic tasks were used the Subsystems database. In both tasks, we used e-value maximum of 1e-5, minimum identity of 60% and alignment length maximum of 15bp. The metagenome accession numbers of the MG-RAST (http://metagenomics.anl.gov/metagenomics.cgi?page=MetagenomeProject&project=8742) are: 4565458.3, 4565459.3, 4565460.3, 4565461.3, 4565462.3, and 4565463.3. The Rarefaction curve for each sample reached into the taxonomic depth. For the taxonomical analysis purpose, OTU table from MG-RAST server was retrieved and was analysed in QIIME v.1.9.1 (Caporaso et al., 2010). In standardised sequence numbers among samples, they are rarely used into 289.800 sequencing. Study results showed that 1. Based on morphologic study, C. felis cat fleas in Manado have same characteristics. Morphometric results of cat fleas from Ranotana showed body size and other segments are longer than ones from Karombasan, Paal 2, and Malalayang. 2. C. felis cat fleas in Manado have a 99% similarity. Molecular barcoding profile of Gen CO1 of Manado C.felis cat fleas is not recorded in an international molecular barcoding system. 3. Study results showed that there was 15 families and 19 genera of bacteria in the body and digestive tract of C. felis cat fleas. There are three bacteria in the cat fleas, i.e. Staphylacoccus, Corynobacterium, and family Plannacoccaceae, that are pathogenic to humans, and not recorded yet in the NCBI GenBank. Study results showed that C. felis adults in Manado have same morphologic characteristics. Nevertheless, based on morphometric data, C. felis isolated from cats of Ranotana has a bigger average morphometric size of body organ than isolated C. felis from cats in Karombasan, Malalayang, and Paal 2. Based on cytochrome c oxidase subunit 1 (CO1), fleas that were isolated from cat in Manado have 99% similarity with gene CO1 sequence of C. felis in the NCBI GenBank. However, profile of molecular barcoding of the gene CO1 is not recorded in international molecular barcoding system. In metagenomics analysis result, is found 15 families and 19 genera of bacteria that exist in the body and digestive tract of C. felis. There are some genera of bacteria which never reported found from C. felis, but are pathogenic to humans.

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