vulnerability and poor health conditions. Royal Belum,

vulnerability and poor health conditions. Royal Belum,Kelantan state of Malaysia, bordered by Thailand on the north are inhabited bya group indigenous community known as Jahai tribe is the example ofcircumstances stated.

The potential risk of zoonosis to the Jahai Community arethe land concessions for plantation and logging activities, resourcesoverexploitation, tourism, unbalanced diets and poor health care facilities(United Nations Human Rights Council, 2016). The public health concern isfurther raised in this group of people when a recent disease outbreak occurredin Sungai Kejar of Royal Belum where 50% of Jahai children were wiped out (TheStar, 2015).           Thepresence of unknown origin of feral dogs and cats in the Jahai village furtherpossess a threat of spreading zoonotic diseases to the population. Sharing thecommon source of untreated water among the community and the feral animals is arisk factor of transmitting water-borne zoonoses such as leptospirosis. Close borderingto Thailand where leptospirosis is endemic possessedanother risk to the Jahai community. In year 2001, 2002 and 2003, Thailand reported a total case of 10217, 6864, and 4958cases respectively (Tangkanakul et al.,2005).

Living at close proximity to other wildlife such as rats, squirrelsand bats also facilitate the possible transmission of leptospire to the community. Bats and rats are popular forharbouring leptospiral serovars in their bodies and transmit to other speciesincluding humans (Roth, 1964; Faine et al., 1999; Richardson and Gauthier,2003; Matthias et al.

, 2005; Vashi et al., 2010).                     Moreresearch need to be done to evaluate the current zoonosis status in theindigenous area. Necessary measures need to be taken to enhance the healthcare, access to health facilities, food subsidy, clean and treated water,population control of the feral dogs and cats and reduce exploitation of landuse to prevent further zoonotic outbreak among the indigenous people in thefuture.               3.

0 MATERIALS AND METHODS 3.1 Sample Collection          Prior to sample collection,consent was obtained from Jahai community, Belum, Gerik District, Perak. Thisstudy was conducted by obtaining approval from Institutional Animal Care andUse Committee (IACUC) (UPM/IACUC/AUP-R067/2016).         Total of fortyanimals (37 dogs and 3 cats) were selected to represent the isolatedpopulations in the indigenous village, Belum. The animals were manuallyrestrained by the handlers for venipuncture blood collection. Jugular vein wasselected as blood collection site and approximately 2mL of blood were withdrawnfrom each animal and place into plain tubes by veterinarians. Information fromeach animal such as species, sex, age and colour were recorded. 3.

2Transportation and Storage of Samples          Blood tubes containing thesamples were stored in a polystyrene ice box with ice packs (temperature ~4°C). The blood samples were then sent to Bacteriology Laboratory in Faculty ofVeterinary Medicine, Universiti Putra Malaysia. Centrifugation of the bloodsamples was performed at 4000rpm for 5 minutes, followed by transferring theblood sera (supernatant) into 1.5 mL Eppendorf tubes. All the blood sera werethen stored at -20°C for further analysis using Microscopic Agglutination Test(MAT). 3.3Microscopic Agglutination Test (MAT)          Total of twelveserovars of Leptospira were selectedto be tested using Microscopic Agglutination Test (MAT) which include Canicola, Pomona, Icterohaemorrhagiae,Grippotyphosa, Australis, Pyrogenes, Lai, Celledoni,Bataviae, Javanica, Hardjo and Copenhageni. Prior to testing, 1000µL ofeach Leptospira serovar (liveantigen) was sub-cultured in Ellinhausen-McCullough-Johnson-Harris (EMJH)medium and incubation at 30°C for 7 – 10 days.

After the incubation period, thesub-cultured live antigens were examined under dark field microscopy andestimated using 0.5 MacFarland standards (1.5 x 108 CFU/mL) beforebeing used for MAT.          Sterile 96-wellsmicrotiter plate consist of 8 rows and 12 columns were used.

The plate wasdivided into half to load 14 samples with 1 positive and 1 negative control. Atotal of 3 microtiter plates were required for 40 samples in each serovar. Theserial dilution of the plates is up to 1:1600 and were prepared as the stepsfollow:1.  Allwells of the microtiter plates were filled with 50µL phosphate buffer saline (PBS)of       pH7.2. 2.

 Additional 46µL of PBS were added to all wells of column 1 and column 7.3.  Next,4µL of serum samples were added to wells of column 1 and column 7.4. Serial dilution was done for each serum sample by pipetting 50µL ofmixtures starting      fromwells of column 1 to column 6. The last 50µL of mixtures was discarded.

5.  Step4 was repeated for sera samples in column 7 and starting from wells of column 7to      column12.6.

  Forthe positive control well, step 1 and 2 were repeated followed by adding 10µLof      hyperimmuneserum. Serial dilution was performed as in step 4.7.  Fornegative control, step 1 was repeated.8.  50µLof live antigens were added to all wells including wells of positive andnegative      control.9.

  Themicrotiter plated were then covered and mixed in incubator shaker for 5minutes.10. All the microtiter plates were incubated at37°C for 2 hours.

11. After incubation, one loop of the mixturewas taken out using inoculation loop from the       positiveand negative control wells were placed onto glass side for dark fieldmicroscopic       examination.12. Step 11 was repeated for all other wells toexamine for any antibody-antigen         agglutinationunder dark field microscopy. The cut-off point was set at 1:100 (OIE       TerrestrialManual 2008) in which the sample was recorded as positive if at least 50%        agglutinationoccurs and the endpoint dilution is determined.13. All the results were recorded. 1:50   1:100   1:200   1:400   1:800   1:1600      1:50   1:100   1:200   1:400   1:800   1:1600                                                                                                                   Figure 1: Sterile96-Wells Microtiter Plate Containing Serum Sample, Positive Control andNegative Control               4.

0RESULTS          In this study,forty (n=40) animals (37 dogs and 3 cats) were selected randomly from theisolated population of dogs and cats in indigenous village, Belum. All theselected animals were not known for their origin and exposure to leptospira.The dogs and cats were not claim (no ownership) by any indigenous people in thevillage and feed on scraps and sometimes fish offered by the villagers.

Theinformation of the animals was tabulated as follow:TABLE 4 : DATAOF ANIMALS (DOGS AND CATS) COLLECTED Number Species Gender Age Colour 1 Dog F Adult Creamy 2 Dog M Adult Creamy 3 Cat F Pregnant Orange 4 Dog M Adult Creamy 5 Dog M Adult Creamy 6 Dog M Adult Mixed 7 Dog M Adult Creamy 8 Dog M Adult Brown 9 Dog M Puppy Brown 10 Dog M Adult Mixed 11 Dog F Adult Creamy 12 Dog M Adult Creamy 13 Dog M Adult Brown 14 Dog M Adult Creamy 15 Dog M Adult Brown 16 Cat M Adult Orange 17 Dog M Adult Creamy 18 Dog M Adult Creamy 19 Dog M Puppy Brown 20 Dog M Adult Brown 21 Dog M Adult Creamy 22 Cat F Adult Mixed 23 Dog M Adult Brown 24 Dog M Adult Creamy 25 Dog M Adult Brown 26 Dog F Puppy Creamy 27 Dog F Puppy Creamy 28 Dog M Puppy Creamy 29 Dog M Adult Brown 30 Dog M Adult Creamy 31 Dog M Puppy Mixed 32 Dog F Adult Creamy 33 Dog M Adult Creamy 34 Dog M Adult Creamy 35 Dog M Puppy Brown 36 Dog F Puppy Creamy 37 Dog F Puppy Creamy 38 Dog M Puppy Brown 39 Dog M Adult Brown 40 Dog F Adult Creamy            The animals are categorized into dogs (37/40) and cats (3/40),adult and young and male and female. In the dog category, 73% (27/37) are adultand 27% (10/37) are puppy whilst in cat category, all the 3 cats are adult and1 of them was pregnant. Of the 37 dogs, 78% (29/37) are male and the remaining22% (8/37) are female whereas out of 3 cats, 33.33% (1/3) is male and 66.66%(2/3) are female.Figure 2 : Species of sampled animals (dogs and cats)Figure 3 : Age group of sampled animalsFigure 4 : Gender group of sampled animals4.1Microscopic Agglutination Test (MAT) Results          Out of 37 dog’s serumsamples, the seroprevalence of Leptospirosis was 8.1 % (3/37) where 3 of thesamples showed positive results at a cut-off titer of 1:100.

Of the 3 samples(number 6, 10 and 40), sample number 6 reacted with serovar Celledoni with atiter of 1:200; sample number 10 reacted to both serovars Celledoni and Laiwith titers of 1:400 and 1:100 respectively; sample number 40 was seropositivefor serovar Australis with a titer of 1:200. All the seropositive dogs wereadult with 67% were male (2/3) and 33% was female (1/3). Besides theseropositive dogs, some samples (number 12, 29 and 32) also showed low titer(1:50) of MAT which were insufficient to qualify as seropositive. Both samplenumber 12 and 29 showed low titer toward serovar Icterohaemorrhagiae; number 32showed low titer to both serovars Celledoniand Copenhageni.          For cat’s serumsamples, the seroprevalence of Leptospirosis was 33% (1/3) where 1 of thesamples showed positive titer of 1:400 against serovar Lai. The seropositivecat was female adult (pregnant).

2.7%   2.7%   5.4%     Figure 5 : Seroprevalence of Leptospirosis of dog’sserum samples collectedFigure 6 :Percentage of Seropositive Dogs against Gender   Figure 7 : Seroprevalence of Leptospirosis of cat’sserum samples collectedFigure 8 :Percentage of Seropositive Cats against Gender            5.0DISCUSSION          In thestudy, the seroprevalence of the isolated population of dogs in Belum villagewas 8.1%.

The positive cut-off point was set at 1:100 by following the standardset by OIE. The predominant serovars detected include Cellodoni (5.4%), Lai (2.7%) and Australis (2.7%). Out of the 3 seropositive samples, one of thesample showed seropositive toward 2 serovars which are Cellodoni and Lai.

Therewere also low titer (1:50) reported in the study which include serovars Icterohaemorrhagiae, Celledoni and Copenhageni in 3 other samples. The finding shared a common serovarof Australis that also found in othercanine leptospirosis studies done in Malaysia (Lau et al., 2016; Wong, 2016; Bo, 2017). Majority of the seropositivesamples showed titers of 1:100 or 1:200 which may indicate the early stage ofleptospirosis, previous infection and vaccination (Picardeau, 2013). Butvaccination is not applicable in this group of isolated population. The lowtiter detected in 3 other samples cannot be neglected in the study as it canindicate an early infection or case of recovery from previous infection.

One ofthe positive samples recorded a high titer of 1:400 against serovar Celledoni and this might indicate activeinfection of leptospirosis. The dog was poor in body condition score butapparently healthy without any clinical presentation of leptospirosis. All thedogs were scattered around the Belum village with majority living at thelakeside and known to be feral as none of the dogs was claimed by any of theindigenous people. Generally, the dogs were emaciated probably due tounbalanced diets. They are fed on scraps and sometimes fish offered by thevillagers. The dogs were not known of their origin, immune and health statusdue to the isolated location in Belum. The temperament of the dogs was hardlyapproachable and fierce probably due to less domesticated.

          On the other hand,the seroprevalence of cats reported in the study was 33% and the predominantserovar detected was Lai. The onlyseropositive cat in the study was pregnant. To date, there is no felineleptospirosis prevalence study done in Malaysia and hence the result isincomparable. But according to Jamshidi etal (2009), the prevalence of feline leptospirosis was 27% with predominantserovars of Canicola, Hardjo and Icterohaemorrhagiae in Iran.  The cats were living closer to the villagersand mainly staying underneath their raised-floor houses. The seropositive catrecorded a surprisingly high titer of 1:400 against serovar Lai. This can be due to previousexposure to leptospiral antigens or active infection. The cat was apparentlyhealthy and show no clinical signs of leptospirosis.

Cats are known to beinfected with leptospirosis but rarely presented with clinical signs (Dickeson& Love, 1993; Agunloye & Nash, 1996). The pathogenesis of felineleptospirosis is similar to canine infection, but even with that, clinicalsigns are rarely developed in cats despite the development of histologicallesions in the kidneys and liver (Jamshidi, 2009). However, the infected catsare still able to shed the leptospiresthrough their urine up to 3 months based on an experiment (Willoughby, 2004).This possessed a zoonotic risk of spreading leptospiresto the indigenous people.          In the study, ithighlighted that uncommon serovars such as Celledoniand Lai can be found in dogs. Thisfinding is contrast with other canine leptospirosis studies reported inMalaysia where Canicola, Icterohaemorrhagiae, Bataviae, Australis, Pomona andPyrogenes are detected.

         The sample size of the study may be inadequate which possibly lead tooverestimate or underestimate the seroprevalence. As mentioned, the feral dogswere unapproachable and fierce, and due to lack of manpower and restrainingtools, sampling was unrewarding during the study. Feral cat was limited andscattered around the village which lead to the least number of samples.Seroprevalence can also be underestimated due to false negative results of MAT.This happened if the infecting serovars are not included in the testing paneland highly possible in our study due to unknown origin of the feral dogs andcats.                                             Figure 9 : Map of Royal Belum State Perak (source from WWF Malaysia)                          Figure10: Sungai Tiang, Royal Belum (source from http://saifudin-mtb.blospot.

com)          Theindigenous village of our study located at Tiang River, Belum. The only humaninhabitant in the village is Jahai community. Under the Perak State ParksCorporation Enactment 2001, Royal Belum was gazetted as a protected area on 3May 2007 and the area encompasses a total of 117 500 hectare in the mostnorthern part of Perak, Malaysia (coloured in green on the map). Royal Belumstate is bordered with Thailand on the north, Kelantan state on the east andGadong River on the west. The landscape of Royal Belum consists of forest,small areas of grassland, abandoned agricultural plots, and a large man-madelake, Temenggor Lake. The Lake was built to generate electricity and watersupply to the town. Due to the presence of lake, flood is a common incidence inthe area especially during rainy season. A flood incidence had hit the RoyalBelum Park in early year of 2015 and caused damage to the wooden houses of theindigenous people (New Straits Times, 2015).

According to Centers for DiseaseControl and Prevention (CDC) 2017, exposure of flood water carriers thepotential of leptospirosis infection and outbreak are usually followed byflood. Other than that, the water from the lake was shared among the indigenouspeople and the feral dogs and cats in the area. The indigenous people statedthat they prefer to stay near to river as it ease them from getting domesticusage and catching fish (New Straits Times, 2016). Domestic usage such aswashing clothes, bathing and cleaning by the indigenous people were observedduring our study in the area.  Besides,the water also acts as a medium of transportation for the villagers to get tothe other location by boat. The feral dogs and cats use the water as a sourceof drinking water.

Another risk of transmission of leptospirosis between thepeople and the feral are proposed due the reason.          According to Suut et al(2016), a local study done in rural communities in Sarawak reported that aseroprevalence of human leptospirosis was 37.4%. The seroprevalence is higherthan other studies done in Malaysia which only ranged from 8.4% to 25.75%(Rafizah et al.

, 2013; Shafei et al., 2012; Karim et al., 2003). The proposed reason of the high prevalence is due tothe demographics of the rural communities live in settlement built along theriver and engaged in economic activities such as fishing, collecting jungleproduce and farming.

The situation is akin to the Jahai community in Sungai Tiang,Belum.           On theother hands, wildlife can also act a potential carrier of leptospire (Bengis, 2004). Belum state is notable for its wildlifediversity and majority of the species are characteristic of the tropicalrainforest whilst minority of the species are from seasonal tropical forest ofThailand. A study conducted locally by Thayaparan et al (2013) on seroprevalence of leptospiral infection in wildliferevealed high seropositive among monkey, rats, bats, squirrel and mongoosespecies. Rats from Royal Belum rainforest was tested positive for carryingpathogenic leptospires(Mohamed-Hassan et al.

, 2012). TheBelum area provide high risk for transmitting leptospires from the reservoir animals. The risk of transmission ofleptospirosis from the wildlife to the feral dogs and cats is highly possiblein the Belum region and this further increase the zoonotic burden to theindigenous people.

            Another risk factor of leptospirosis spreading in Belum state is theclose bordering with Thailand where leptospirosis is endemic. Thailand hadreported a total case of 10217, 6864, and 4958 cases of leptospirosis in theyear 2001, 2002 and 2003 respectively (Tangkanakul et al., 2005). Sharing the same rainforest between Belum state andThailand facilitate the movement of wildlife in the area.           Withall the potential risk factors mentioned, leptospirosis outbreak is not an astonishingevent that could happen in near future. Hence, thorough prevalence study whichinclude other species (human, rats and other wildlife) is necessary to have aninsight of leptospirosis status in this area.

Intervention by the government inpreventive measures are needed to curb and prevent future outbreak of leptospirosisin this isolated population. Lack of access to medical facilities, balanceddiets, clean and treated water and public education are the major problems tothe Jahai population.  6.0CONCLUSION          In conclusion, theoverall seroprevalence of canine leptospirosis was 8.1% in total of 37 dogswhilst feline leptospirosis recorded 33% in total of 3 cats studied. Thepredominant serovars reported were Celledoni,Lai and Australis in dogs and Laiin cats.

These findings showed that leptospiresare existing in the environment of Belum state. 7.0RECOMMENDATIONS          Infuture study, more area of the Belum state should be included to have a largersample size and a more precise estimation of seroprevalence of leptospirosis.

Sampling methods need to be revise as the animals in the area are lessdomesticated and require proper restraining tools, restraining techniques andwell-trained personnel to reduce stress and injuries to both the handlers andanimals. 

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