6penuliar.pmd C. B. T. Garcia and others 49 SCIENCE DILIMAN (JULY-DECEMBER 2014) 26:2, 49-60 Seroprevalence and Risk Factors Associated with Seropositivity to Toxoplasma gond ii among Stray and Domestic Cats (Fel is silvestris catus) in Metro Manila Christel Bohn T. Garcia Ma. Jill ian P. Talavera Gil M. Penul iar* University of the Philippines Diliman _______________ *Corresponding Author ISSN 0115-7809 Print / ISSN 2012-0818 Online ABSTRACT Toxoplasma gond ii is a protozoan parasite that causes toxoplasmosis. It is widespread in the environment and infects a variety of warm-blooded animals, causing miscarriages and birth problems. Previous studies in the Philippines have determined the seropositivity of T. gondii in humans. H ow ev e r, t h e s e r o p r e v a l e n ce o f t h e p a r a s i t e a m o n g h o u s e h o l d p e t s , par t i c u l a r l y i t s fe l i n e d e f i n i t i ve h o s t , r e m a i n s i n s u f f i c i e n t . T h i s s t u d y aimed to: (1) determine the seroprevalence of T. gond ii antibodies among domestic and stray cats in the Philippines; and, (2) to analyze the risk factors associated with seropositivity. Blood samples from 59 domestic and stray cats were collected and tested for T. gond ii seropositivity using a c o m m e r c i a l l y a v a i l a b l e i n d i r e c t E L I S A k i t , w h i l e p e t o w n e r s a n d handlers were given questionnaires about their cats. Thirteen or 22.03% o f t h e c a t s w e r e s e r o p o s i t i v e to T. g o n d i i , a n d r i s k f a c t o r a n a l y s i s revealed a signif icant difference betw een domestic and stray cats with regard to diet (p = 0.026, OR = 8.333, c = 0.299) and domestication (p = 0.039, OR = 5.000, c = 0.276). Cats fed with table food tested 31.43% seropositive compared to the 4.35% of those fed with cat food, whereas 33.33% of the stray cats were seropositive compare d to 7.69% for domestic cats. O d d s r a t i o t e s t s h o w e d t h a t t h e r i s k f a c t o r s s t u d i e d w e r e a s s o c i a t e d with higher likelihood of T. gond ii seropositivity. These results implicate diet and environment in the transmission dynamics of T. gond ii among cats. Keywords: Toxoplasma gond ii, seroprevalence, risk factor analysis, indirect ELISA φ  φ  Seroprevalence and Risk Factors Associated with Seropositivity 50 INTRODUCTION Toxoplasma gondii is an intracellular parasite that infects a wide range of warm- blooded animals, including cats, dogs, and humans. It is present worldwide and is of medical and veterinary importance due to its ability to cause miscarriages and birth defects in intermediate hosts (Garcia and others 2012). Infection by this zoonotic parasite causes toxoplasmosis, occurring through the ingestion of cysts in undercooked and raw meat or the accidental ingestion of oocysts from the environment (Duan and others 2012). T. gondii only undergoes sexual reproduction and gametogenesis within its def initive host, the members of Family Felidae; this serves as the only method for the production of oocysts (Webster 2007). Oocysts are environmentally stable and are shed in the cat’s feces. These remain infectious for approximately two years, causing widespread contamination and providing a source of infection to humans and other intermediate hosts (Yan and others 2012). Previous studies on T. gondii in the Philippines have covered toxoplasmosis in rats, cats, pigs, and humans (Advincula and others 2010). An earlier study on the seroprevalence of T. gondii among Filipinos in the Philippines revealed an overall seropositivity of approximately 11.1% in the Metro Manila area, which is a relatively low seropositivity compared to rural areas like Leyte (30.1%) and Mindoro (61.2%) (Kawashima 2000). Domestic cats (Felis silvestris catus) are household pets that are exposed to similar environments as humans but are less cautious of the cleanliness of their immediate environment. This increases their exposure to T. gondii and should provide a more accurate estimate of the prevalence of the parasite in the environment. As the parasite’s def initive host, cats are crucial for the parasite to reach maturity and complete its life cycle. Consequently, T. gondii is able to manipulate the behavior of its intermediate host to enhance transmission to the def initive host (Webster 2007). Domestic cats, therefore, represent a major source of contamination and infection for humans and other potential hosts. T. gond ii infection can be detected through several diagnostic methods. One of these is the indirect enzyme-linked immunosorbent assay (ELISA), a serological test that is one of the most widely used methods for the diagnosis of toxoplasmosis. Another technique is the polymerase chain reaction (PCR), a highly specif ic and C. B. T. Garcia and others 51 sensitive molecular test that enables detection of the parasite DNA (Castillo-Morales and others 2012). This method, however, requires the use of costly reagents. This study aimed to (1) determine the seroprevalence of T. gondii antibodies among domestic and stray cats in the Philippines using indirect ELISA and (2) to analyze the risk factors associated with seropositivity. Findings from this study will be signif icant in monitoring and controlling the possible infection of intermediate hosts, particularly humans, because of their interaction with cats. MATERIALS AND METHODS Consent Forms An introductory letter indicating the purpose of the study and the assistance needed had been sent to pet owners, shelter administrators, and veterinarians from different sampling sites in Metro Manila for approval. A consent form explaining the purpose of the study and rights regarding participation had also been provided. Questionnaire Prior to blood extraction, information regarding the age, sex, breed, diet, location, presence of other cats, contact with the outdoor environment (for domestic cats), health condition, medical history, litter information, and domestication was obtained through questionnaires f illed out by the owners, shelter administrators, and veterinarians. Sampl ing Sites and Blood Collection Blood samples from domestic cats were collected from the Makati Dog and Cat Hospital, Riverside Village, and Companion Animal Veterinary Clinic, whereas samples from stray cats were collected from the Philippine Society for the Prevention of Cruelty to Animals and the Marikina City Vet Off ice. One milliliter of whole blood sample was collected by licensed veterinarians via venipuncture of the cephalic veins (McCurnin and Bassert 2002). Proper animal restraint was accomplished before venipuncture. The elbow of the cats was kept extended and pressure was applied such that the vein remains f illed with blood. After the venipuncture, f irm pressure was applied to the puncture site for 60 s to prevent hematoma formation (McCurnin and Bassert 2002). Fifty-nine cat blood samples were successfully obtained. The blood was stored in red-topped vacutainer tubes Seroprevalence and Risk Factors Associated with Seropositivity 52 (contains no anticoagulant), labeled, and left in a slanting position for 10 min at room temperature (RT ) to facilitate clotting. The tubes were then kept inside a Styrofoam box with ice (Stevens and others 2007) to keep the temperature low. The blood samples were then transported to the Medical Microbiology Laboratory in UP Diliman, where they were either stored in the refrigerator or immediately processed. Serum Preparation The vacutainer tubes were taken out of storage and left undisturbed at RT for 10 min, to aid in clotting (www.invitrogen.com). Three hundred microliters of serum was then taken from the tubes, and transferred into sterile 1.5 mL microcentrifuge tubes. The serum was centrifuged at 1000 x g for 10 min, and immediately transferred to a sterile 1.5 mL microcentrifuge tube in aliquots of 250 μL. The tubes were stored in the freezer (www.invitrogen.com) until a suff icient number of samples were obtained. Serological Assay IgG antibodies against T. gondii in the serum samples were detected through indirect ELISA using a commercially available kit (ID Screen® Toxoplasmosis Indirect Multi- species from ID.vet Innovative Diagnostics, Montpellier, France) following the manufacturer’s instructions. Samples were prepared in a 96-well microplate coated with P30 antigen of T. gond ii. To each well, 90 μL Dilution Buffer 2 was added, followed by 10 μL of the Negative Control in wells A1 and B1, and 10 μL of the Positive Control in wells C1 and D1. Serum samples were thawed and 10 μL were dispensed into the remaining wells. Microplates were then incubated for 45 min at RT. The wells were washed thrice with 300 μL Wash Solution, then 100 μL of Conjugate was added, followed by incubation for 30 min at RT. The wells were washed thrice with 300 μL Wash Solution, then 100 μL Substrate Solution was added, followed by incubation in the dark for 15 min at RT. The reaction was stopped by adding 100 μL Stop Solution. The optical density (OD) of the samples and controls were measured at 450 nm and recorded using a microplate reader. This assay was performed twice. Interpretation and Val idation of Results The OD values were tested for validity using the readings of the positive and negative controls. The test was considered valid if the mean OD values of the C. B. T. Garcia and others 53 (1)S/P = OD sample –OD NC OD PC –OD NC × 100 positive control was greater than 0.350 (OD PC >0.350), and if the ratio of the OD values of the positive and negative controls was greater than 3.5 (OD PC /OD NC >3.5). The Sample/Positive (S/P) percentage was computed for each sample using Equation 1. The results for each sample were labeled as either negative (S/P < 40%), doubtful (40% < S/P < 50%), or positive (S/P > 50%). Analysis of Data Statistical analysis of the prevalence of T. gondii across potential risk factors for infection was performed using Chi-square test in SPSS software (Release 20.0 standard version, SPSS Inc. , Armonk, New York). Risk factors that were analyzed included sex, diet, domestication (domestic versus stray), and location (from veterinary clinics versus from animal shelters). P values less than 0.05 were considered to be statistically signif icant (Duan and others 2012). Otherwise, the null hypothesis was accepted, and the risk factor in question was concluded to play no role in T. gondii infection. Phi and Cramer’s V values were also reported using Cramer’s Phi test to determine the strength of association between T. gondii infection and the risk factors. Phi values from 0–0.30 indicated absence of relationship to weak relationship, 0.31–0.70 implied a moderate relationship, and 0.71–1.0 suggested a strong relationship (Release 20.0 standard version, SPSS Inc. , Armonk, New York). Odds ratios (ORs) were also noted to compare the relative odds of the occurrence of T. gondii infection across the risk factors involved. Odds ratio equal to 1 indicated that the risk factor does not affect the likelihood of infection, ORs greater than 1 indicated that the risk factor is associated with higher likelihood of infection, and ORs less than 1 indicated that the risk factor is associated with lower likelihood of infection (Szumilas 2010). Waste Disposal Potentially infectious materials were decontaminated using an autoclave prior to disposal (CDC 2009). Seroprevalence and Risk Factors Associated with Seropositivity 54 RESULTS Val id ity of the Assay Two separate assays were performed and both were deemed valid as each set met the conditions for validity. The ratios of the OD values of the positive and negative controls for the f irst and second batches were 3.51 and 3.53, respectively. Seroprevalence Out of the 59 blood samples collected, 22.03% were seropositive to T. gondii and 72.88% were negative for infection. Three samples exhibited a doubtful result. The seroprevalence of T. gondii in cats from Marikina, Manila, and Makati were 27.78%, 40%, and 14.29%, respectively (Table 1). Table 1. Seroprevalence of T. gond ii in cats (Fel is catus) based on geographic location in the Phil ippines Makati City 2 10 2 14 14.29 Manila City 6 9 0 15 40 Marikina City 5 12 1 18 27.78 Pasig City 0 2 0 2 - Antipolo City 0 10 0 10 - Total 13 43 3 59 Prevalence of T. gond ii(%) Positive Negative Doubtful Total Table 2. Seroprevalence of T. gond ii infection in cats (Fel is catus) across each risk factor Total 13 43 3 59 Stray 11 21 1 33 59 Data Domestic 2 22 2 26 completed In Clinics 2 20 2 24 57 2 from In Shelters 11 21 1 33 private homes Male 6 15 2 23 56 3 without Female 7 25 1 33 information Table food 11 23 1 35 58 1 without Cat food 1 20 2 23 information Positive Negative Doubtful Total Sum Details C. B. T. Garcia and others 55 Table 3. Summary of statistical analysis for potential risk factors associated with T. gond ii infection * OR=1 means that the risk factor does not affect the odds of infection, OR<1 means that the risk factor is associated with lower odds of infection, and OR>1 means that the risk factor is associated with higher odds of infection. * * P-values<0.05 are considered statistically significant. * * * Phi values from 0–0.30 indicate absence of relationship to weak relationship, 0.31–0.70 with a moderate relationship, and 0.71–1.0 with a strong relationship. Risk Factor Odds ratio(OR) * p-value** Phi value( c)*** Sex 1.733 0.403 0.115 Diet 8.333 0.026 0.299 Domestication 5.000 0.039 0.276 Location 5.000 0.054 0.262 φ  The seroprevalence across risk factors are summarized in Table 2. Each risk factor was observed to indicate at least one cat to be positive. Those shown to be seropositive to T. gondii were 33.33% (11/33) in stray cats, 7.69% (2/26) in domestic cats, 8.33% (2/24) in cats kept in clinics, 33.33% (11/33) in cats from shelters, 26.09% (6/23) in male cats, 21.21% (7/33) in female cats, 31.43% (11/ 35) in cats fed with table food, and 4.35% (1/23) in cats fed with cat food. Risk Factor Analysis A statistically signif icant association with seropositivity was found with domestication and diet (p<0.05). The proportion of seropositive cats was found to be higher in stray cats and in cats fed with table food. Cramer’s Phi, however, indicated that the correlation is not strong, but the OR test suggested that all risk factors are associated with higher odds of T. gondii infection (OR>1, Table 3). DISCUSSION Cats are considered reservoirs of zoonotic diseases such as toxoplasmosis. These animals that live in close contact with humans increase the risk of transfer of potential infections, especially to immuno-compromised individuals (Grøndalen and others 2004). Whether domestic or stray, cats are exposed to the same environment as humans and may be used as sentinels that reflect the spread of T. gondii in the environment. A high seropositivity in cats would indicate a high risk of infection in the community. The seropositivity obtained in this study was 22.03% (13/59). This rate of T. gondii infection is rather low relative to those reported in other countries, which can be as high as 80% of tested cats (Alvarado-Esquivel and others 2007). Seroprevalence and Risk Factors Associated with Seropositivity 56 A third of the stray cats (33.33%) were seropositive compared to 7.69% in domestic cats. This difference in seropositivity was based on statistical tests (p<0.05). Studies conducted in other countries similarly reveal that stray cats generally have higher seropositivity rates and are more prone to infection (Meireles and others 2004, Miró and others 2004, Raeghi and others 2011). The differences in the lifestyles of stray cats and domestic cats affect their daily encounters with the potential parasite, which subsequently affect their health. Domestic cats are kept indoors, and are often personally carried or kept in cages when brought outside the house. They are generally exposed to clean, controlled environments free of T. gondii contamination. They may, however, get infected by the ingestion of oocysts that have been left by other cats, or the ingestion of cysts in raw or uncooked contaminated meat. Stray cats, on the other hand, are exposed to environments that are not maintained to the same degree of sanitation compared to the inside of a house. The outside environment contains numerous possible sources of contamination, such as raw or uncooked food scraps from domestic garbage, and droppings from intermediate hosts like mice, birds, and reptiles, which may contain cysts. Interaction with other free-roaming stray cats also hastens the spread of infections. This is supported by the ORs for domestication, which suggested the risk factor is likely to increase the odds of seropostivity to T. gond ii in cats (OR=5.000, Table 3). A difference in seropositivity values was also seen in cats according to their diets. A higher percentage of positive result was seen in cats fed with table food or leftovers (31.43%), relative to cats fed with cat food (4.35%). The odds ratio test also indicated that diet has the highest OR value (8.333), corresponding to the increased chances of infection in cats fed with table food (Table 3). These results are similar to those obtained in studies conducted in Brazil and Mexico by Lucas and others (1999), and Alvarado-Esquivel and others (2007), respectively. Table food, when fed to cats, is often mixed with uncooked or undercooked meat and by-products that are easily contaminated. Cat food, on the other hand, is factory-processed and is less exposed to the environment prior to feeding, as it is kept in containers or packages. Companies that manufacture animal food undergo regular monitoring procedures to ensure the safety of their products. Protocols in these companies involve the application high temperatures (>100 °C) in the production of commercial cat food. This ensures the elimination of viable tissue cysts, as T. gondii becomes nonviable at temperatures above 66 °C (Meireles and others 2004). In addition, cats fed with cat food are most likely domestic, which as previously mentioned, are less prone to infection. C. B. T. Garcia and others 57 A larger percentage of males (26.09%) were seropositive to T. gondii compared to females (21.21%). The difference, however, was not statistically signif icant (p>0.05). This was also seen in studies conducted by Alvarado-Esquivel and others (2007), Hooshyar and others (2007), and Wu and others (2011) in Mexico, Iran, and China, respectively. This may be attributed to the territoriality and roaming behaviors associated with male cats. These allow them to cover a wider range of areas, which increases their chances of coming into contact with oocysts in contaminated meat and environments, and with other cats, particularly females (Reyes and others 2013). A study by Maruyama and others (1998) suggested that male cats may have increased chances of infection through aggressive encounters during estrus period. The OR test, however, confirmed that sex is a risk factor involved with higher likelihood of T. gondii infection (OR=1.733, Table 3). Seropositivity was also higher in cats being cared for in animal shelters (33.33%) compared to those kept in veterinary clinics (8.33%). OR test conf irmed that location as a risk factor can possibly increase the likelihood of T. gondii infection (OR=5.000). Generally, the cats from animal shelters were usually stray cats, whereas cats from veterinary clinics were usually domestic cats. In this study, the same holds true. The main difference was the sample size, with the risk factor for location having fewer samples due to incomplete data supplied by the participants. As it is, the higher seropositivity in cats from animal shelters may be explained by the same reasons justifying the higher seropositivity among stray cats. In conclusion, the seroprevalence of T. gondii among cats in Metro Manila is relatively low compared to other countries, which may indicate that the parasite is not as widespread in the areas covered by the study. Results from the risk factor analysis also emphasize the role of diet and environment in the transmission dynamics of T. gondii among cats. ACKNOWLEDGMENTS The results of this paper were presented as poster during the 43rd Annual Convention and Scientif ic Meeting of the Philippine Society for Microbiology, Cebu City, 2014. We thank Juvy Ann C. Palma for technical assistance. This work was supported by the Off ice of the Vice Chancellor for Research & Development of the University of the Philippines Diliman, through the Ph.D. Incentive Awards (Project No. 121218 PhDIA). Seroprevalence and Risk Factors Associated with Seropositivity 58 REFERENCES Advincula J.K.d.C. , Iewida S.Y.P. , Salibay C.C. 2010. Serologic detection of Toxoplasma gondii infection in stray and household cats and its hematologic evaluation. Scientia Medica (Porto Alegre) 20: 76-82. Alvarado-Esquivel C. , Liesenfeld O. , Herrera-Flores R.G. , Sanchez-Ramirez B.E. , Gonzalez- Herrera A. , Matrinez-Garcia S.A. , Dubey J.P. 2007. Seroprevalence of Toxoplasma gond ii Antibodies in Cats From Durango City, Mexico. J. Parasitol 93(5): 1214-1216. Castillo-Morales V.J. , V iana K.Y.A , Ed. Guzmán-Marín S. , Jiménez-Coello M.J. , Segura- Correa J.C. , Aguilar-Caballero A.J. , Ortega-Pacheco A. 2012. Prevalence and Risk Factors of Toxoplasma gond ii Infection in Domesticated Cats from the Tropics of Mexico Using Serological and Molecular Tests. Interdisciplinary Perspectives on Infectious Diseases 2012. Center for Disease Control and Prevention (CDC), 2009. Biosafety in Microbiological and Biomedical Laboratories, 5 th Edition. Available from http: //www.cdc.gov/. Accessed 24 May 2013. D u a n G . , T i a n Y. M . , L i B . F . , Ya n g J . F . , L i u Z . L . , Yu a n F . Z . , Z h u X . Q. , Zo u F . C . 2 0 1 2 . Seroprevalence of Toxoplasma gond ii infection in pet dogs in K unming, Southwest China. Parasites & Vectors 5:18. Garcia G. , Sotomaior C. , Nascimento A .J. , Navarro I.T. , Soccol V.T. 2012. Toxoplasma gond ii in goats from Curitiba, Paraná, Brazil: risks factors and epidemiology. Rev. Bras. Parasitol. Vet . , Jaboticabal 21: 42-47. Grøndalen J. , Sævik B. , Sørum H. 2008. Companion animals as reservoir for zoonotic diseases. European Journal of Companion Animal Practice 18: 213-22. Hooshyar H. , Rostamkhani P. , Talari S. , Arbabi M. 2007. Toxoplasma gond ii infection in stray cats. Iranian Journal of Parasitology 2(1): 18-22. IBM Corp. Released 2011. IBM SPSS Statistics for Windows, Version 20.0. Armonk, NY: IBM Corp. Kawashima T. , Khin-Sane-W in, Kawabata M. , Barzaga N. , Matsuda H. , Konishi E. 2000. Prevalence of antibodies to Toxoplasma gondii among urban and rural residents in the Philippines. Southeast Asian J Trop Med Public Health 4: 742-6. L i f e Te c h n o l o g i e s T M P r o t o c o l s . i n v i v o R N A i P r o t o c o l s . Av a i l a b l e f r o m h t t p : / / www.invitrogen.com/. Accessed 30 April 2013. Lucas S.R.R. , Hagiwara M.K. , Loureiro Vd.S. , Ikesaki J.Y.H. , Birgel E.H. 1999. Toxoplasma gond ii infection in Brazilian outpatient cats. Rev. Inst. Med. Trop. S. Paulo 41(4): 221-224. C. B. T. Garcia and others 59 Maruyama S. , Hiraga S. , Yokoyama E. , Naoi M. , Tsuruoka Y. , Ogura Y. , Tamura K. , Namba, S. , Kameyama Y. , Nakamura S. , Katsube Y. 1998. Seroprevalence o f Bartonella henselae and Toxoplasma gondii infections among pet cats in Kanagawa and Saitama Prefectures. Journal of Veterinary Medical Science 60(9): 997-1000. McCurnin D.M. , Basser t J.M. 2002. Diagnostic Sampling and Treatment Techniques. In: Clinical Textbook for Veterinary Technicians, 5th edition. Philadelphia, USA: Elsevier Inc. Available from http: //www.catnmore.com/animals/pdfs/VenipunctureHow To.pdf. Accessed 30 April 2013. Meireles L.R. , Galist eo A .J. J r. , Pompeu E. , Andrade H.F. J r. 2004. Toxoplasma gond i i spreading in an urban area evaluated by seroprevalence in free-living cats and dogs. Tropical Medicine and International Health 9(8): 876-881. Miró G. , Montoya A. , Jiménez S. , Frisuelos C. , Mateo M. , Fuentes I. 2004. Prevalence of antibodies to Toxoplasma gond ii and intestinal parasites in stray, farm and household cats in Spain. Veterinary Parasitology 126: 249-255. Raeghi S. , Sedeghi S. , Sedeghi S. 2011. Prevalence of Toxoplasma gond ii antibodies in cats in Urmia, northwest of Iran. The Journal of Animal & Plant Sciences 21(2): 132- 134. Rey e s M . F . , G u ev a r a V. G . , S a n Ro q u e D. G . D. G . , F l o r e s M . L . S . , L a s t i c a E . A . 2 0 1 3 . Seroprevalence o f Toxoplasma gond ii antibodies in domestic shor t-haired cats ( Fel is ca t u s ) in a wildlife facility in Manila. Philippine Journal of Ve terinary and Animal Sciences 39(1): 99-106. S t ev e n s V. L . , Pa te l A .V. , F e i g e l s o n H . S . , Ro d r i q u e z C . , T h u s M . J . , Ca l l e E . E . 2 0 0 7. C r y o p r e s e r v a t i o n o f W h o l e B l o o d S a m p l e s C o l l e c t e d i n t h e F i e l d f o r a L a r g e Epidemiologic Study. Cancer Epidemiol Biomarkers Prev 16: 2160-2163. Szumilas M. 2010. Explaining Odds Ratios. J Can Acad Child Adolesc Psychiatry 19(3): 227-229. Webster J.P. 2007. The effect of Toxoplasma gond ii on animal behaviour: playing cat and mouse. Schizophrenia Bulletin 33: 752-6. Wu S.M. , Zhu X.Q. , Zhou D.H. , Fu B.Q. , Chen J. , Yang J.F. , Song H.Q. , Went Y.B. , Ye D.H. 2011. Seroprevalence of Toxoplasma gond ii infection in household and stray cats in Lanzhou, northwest China. Parasites & Vectors 4: 214. Yan C. , Fu L.L. , Yue C.L. , Tang R.X. , Liu Y.S. , Lv L. , Shi N. , Zeng P. , Zhang P. , Wang D.H. , Zhou D.H. , Zhu X.Q. , Zheng K.Y. 2012. Stray dogs as indicators of Toxoplasma gond ii distribut ed in the environment: the f irst repor t across an urban-rural gradient in China. Parasites & Vectors 5: 5. Seroprevalence and Risk Factors Associated with Seropositivity 60 _______________ Christel Bohn T. Garcia has a BS Biology degree from the Institute of Biology, UP Diliman, and is an active member of the UP Association of Biology Majors and UP Pre-Medical Society. Ma. Jill ian P. Talavera is a fourth year BS Biology student of the Institute of Biology, UP Diliman. She is currently conducting her undergraduate thesis on microbiology under the Medical Microbiology Laboratory and plans to take up graduate studies on microbiology as well. Gil M. Penul iar is an Assistant Professor of the Institute of Biology, UP Diliman. He has a Doctorate degree in Medical Science from Gunma University Graduate School Medicine, and a Master’s degree in Microbiology from the UP Diliman. His f ield of specialization is systems microbiology.