Tropical Biomedicine 34(4): 804–814 (2017)

Identification of Brugia malayi in dogs in Kerala, India

Chirayath, D.1*, Alex, P.C.2, Pillai, U.N.3, George, S.4, Ajithkumar, S.5 and Panicker, V.P.6 1,2,3,5Department of Clinical Veterinary Medicine, College of Veterinary and Animal Sciences, Mannuthy, Kerala Veterinary and Animal Sciences University, Pookode, India 4,6Department of Veterinary Biochemistry, College of Veterinary and Animal Sciences, Mannuthy, Kerala Veterinary and Animal Sciences University, Pookode, India *Corresponding author e-mail: [email protected] Received 2 June 2017; received in revised form 12 August 2017; accepted 13 August 2017

Abstract. Identification of filarial species in dogs is clinically important because of zoonotic concerns and therapeutic implications. The present study was carried out to identify the filarial parasites causing microfilaraemia in dogs in Thrissur District, Kerala- an endemic area for human Brugian filariasis. Out of the 1600 dogs screened by wet blood film examination, 130 were positive for microfilariasis. Giemsa staining of blood smears revealed that 90 out of 130 dogs had unsheathed microfilariae, 24 had sheathed microfilariae and 16 had combined infection of sheathed and unsheathed microfilariae. Results of micrometry and histochemical staining of the sheathed microfilariae were in conformity with that of Brugia malayi. The DNA isolated from the sheathed microfilariae amplified the primers specific for the Hha 1 repeats of the B. malayi. Cloning and sequencing revealed that the amplified fragment corresponded to the 140-292 base pairs of the 320 base pair Hha1 repeat of Brugia malayi. The amplified DNA fragment also contained restriction sites for Alu 1 and Rsa 1which confirmed that the present isolate is Brugia malayi. The present study confirmed the presence of B. malayi in dogs in Kerala, India.

INTRODUCTION the potential to infect humans and hence significant from a public health perspective Identification of filarial species in dogs is (MegatAbd Rani et al., 2010). clinically important because of zoonotic Dogs were not considered as reservoir concerns and therapeutic implications hosts for B. malayi. Recently B. malayi (Rishniw et al., 2006). Dirofilariasis in dogs microfilariae similar to that causing filariasis is usually diagnosed by identification of in human beings have been identified in microfilariae in blood. Filariae such as dogs in Kerala, India (Ambily et al., 2011). Dirofilaria immitis, Acanthocheilonema Contradicting the above finding, Ravindran reconditum, Acanthocheilonema dracun- et al., 2014, reported that sheathed micro- culoides, Dirofilaria repens, Brugia filariae from dogs in Alappuzha district, malayi, Brugia pahangi, and Brugia Kerala, were B. pahangi and B. malayi like ceylonensis can cause canine microfilaremia microfilariae. and differentiating these microfilariae is Detection of Brugia malayi in domestic clinically important. Infection with A. dogs, in a highly endemic area for lymphatic reconditum and A. dracunculoides have filariasis, poses great threat to the control few clinical consequences, while D. repens and eradication of human lymphatic infection has been associated with filariasis. Brugian filariasis accounts for subcutaneous granulomas and pruritus approximately 5% of lymphatic filariasis in dogs. Moreover dogs infected with cases in India where over 40 million people microfilariae of D. repens can act as a carrier are estimated to be infected with either B. for human infection. All canine filariae have malayi or Wuchereria bancrofti. The role of

804 dogs (and cats) as reservoirs of Brugian used for micrometry of microfilariae were filariasis has important implications for calibrated using the standard method parasite control strategies. If canine and (Garcia, 2001). Measurements of micro- feline reservoir hosts exist in these areas, a filariae were taken using the calibrated more inter-sectorial approach in control microscope and compared with the standard strategies may be required in addition to the measurements of the parasites (Soulsby, traditional use of Mass Drug Administration 2005). (MDA) programs advocated by the World Health Organization. Therefore, accurate 2.3 Acid Phosphatase Staining differentiation of canine microfilariasis and Approximately 5 ml of blood was drawn successful treatment and elimination of from the cephalic vein of each dog and was microfilariae from canine host is very allowed to clot. The clot was loosened with important. an applicator stick and was washed with 5 The present work was carried out to ml of distilled water. The water and serum clarify the doubts on the speciation of were then poured into a test tube and sheathed microfilariae detected in dogs in centrifuged for 5 minutes at 1000 x g. The Kerala. supernatant fluid was discarded leaving a drop in the bottom of the tube. A drop of this fluid containing the resuspended sediment MATERIALS AND METHODS was placed on a glass slide and examined for microfilariae. Smears were prepared from 1 Selection of cases the sediment were air dried, fixed in absolute A total of 1600 dogs above six months of age acetone at 4ºC for one minute and stained for presented to the Medicine Unit of Veterinary the demonstration of acid phosphatase Hospital Mannuthy and Kokkalai (Thrissur activity. District, Kerala State, India) with various The naphthol AS-TR- phosphate method complaints, checkup and for vaccinations of Barka was used to demonstrate acid were screened by wet blood film examination phosphatase activity (Chalifoux and Hunt, during morning hours between 8.30 am to 12 1971). noon. Wet film positive dogs were selected for the study. 3 Molecular methods 3.1 Microfilarial DNA Extraction 2 Parasitological Methods Microfilarial DNA was extracted using Blood 2.1 Giemsa staining Genomic DNA Miniprep Purification Spin Kit Peripheral blood smears were air dried and from Himedia (HiPurA, MB504) with slight fixed by dipping the film briefly in a coplin modification of manufacturer’s instruction. jar containing absolute methyl alcohol. The According to the manufacturer’s instruction, film was air dried and stained with diluted proteinase K digestion at 56ºC need be carried Giemsa stain (1:20, vol/vol) for 20 minutes. out only for 10 minutes. It required 16 hours The slide was washed in buffered water, air of proteinase K digestion at 56ºC for dried and examined under oil immersion extraction of DNA of sheathed microfilariae. objective of the microscope. Identification of microfilariae was done based on 3.2 Specific PCR for B. malayi morphological peculiarities (Soulsby, 2005). DNA isolated from all the samples positive for sheathed microfilariae were subjected 2.2 Micrometry to species specific PCR for B. malayi. The An ocular micrometer disk with 50 divisions presence of B. malayi was confirmed using was compared with a calibrated stage the primers specific for the Hha I repetitive micrometer having a scale of 0.1 and 0.01 sequence of B. malayi (Thanchomnang et mm divisions. Low power, high power and oil al., 2008). The primer sequences were immersion objectives of the microscope

805 BM-F (5' TCA TTA GAC AAG GAT ATT GGT and sheathed microfilariae. Occurrence of TC 3') microfilariasis in dogs was 8.1%. Occurrence BM R (5' TTT AAA CTA TAA AAT GAC AAC of unsheathed microfilariae was 6.6% and ACA 3') sheathed microfilariae was 1.8%.

The PCR reaction contents included: 2.5 µl Parasitological Methods of 10X reaction buffer, 2.5 µl of 2mM dNTP In Giemsa stained smears, sheathed mixture, 20 pmoles of each primer, 2 µl microfilariae had a bluish violet coloured template DNA solution 1 unit of Taq body and a pink coloured sheath extending polymerase and double distilled H2O added beyond both the anterior and posterior ends. to a total volume of 25 µl. The PCR cycling Microfilariae were smaller in size compared conditions used were: 95ºC, 2 minute for to the unsheathed microfilariae and had initial denaturation and then 31 cycles of multiple kinks over the body. The anterior 95ºC for one minute, 54.3ºC one minute and end was blunt and there was a clear area 72ºC for 2 minutes followed by 72ºC for 5 devoid of nuclei called head space or minutes. Ten microlitres of PCR products cephalic space. Cephalic space was twice were analysed by using 1.5% agarose gel long compared to the width and devoid of any electrophoresis, stained by ethidium bromide nuclei. Nuclear column was darkly stained and visualized under the transilluminator. and tightly packed throughout the body preventing the visualization of individual 3.3 Cloning and Sequencing of PCR nucleus. The tail was pointed with a Products and Sequence Data Analysis discontinuous row of nuclei. There were two B. malayi specific, 153-bp PCR amplicon was terminal nuclei which were widely separated cloned and sequenced at Bioserve, Priority at the tip of the tail (Fig. 1). The nerve ring Life Sciences, Hyderabad, India. The vector appeared as an oblique clear space devoid used was pGEM-T Easy (Promega). The of nuclei, traversing the entire width of the sequences were analysed using BLAST microfilariae. Excretory pore was seen as an and pair wise alignment with reference oval clear space opening to one side of the sequences of B. malayi (Accession No. body. Anal pore also appeared as an oval M12691.1). Restriction site analysis for clear space opening to one side of the body. Alu I and Rsa I was done for the cloned Presence of sheath and two distinctly sequences, reference sequences of Brugia separated terminal nuclei confirmed it as malayi (No. M12691.1), Brugia pahangi microfilariae of genus Brugia. The size of (M12692.1), Brugia timori (No. AF499118) the microfilariae and proportion of cephalic and Hha1sequences of human and canine space suggested that the microfilariae were microfilarial isolates (JN413104, JN601135, similar to human filarial parasite B. malayi. JN601136 and JN601137) submitted from The microfilarial length varied from Alappuzha, Kerala (Ravindran, et al., about 190 to 220 micrometer (µm) with a 2014) using restriction site analysis tool mean of 215.0 µm and width of 4.35 to 6.52 ‘Webcutter 2.0’. µm with a mean of 5.0 µm. The cephalic space ranged from 8.6 to 10 micrometer with a mean of 9.50 µm. The nerve ring appeared as an RESULTS oblique clear space devoid of nuclei, traversing the entire width of the microfilariae Screening of dogs for microfilariae at around 45.35 to 48.50 micrometer with Out of the 1600 dogs screened by wet blood a mean of 46.0 µm from the anterior end. film examination, 130 were positive for Excretory pore was seen as an oval clear microfilariae. On Giemsa staining of blood space opening to one side of the body at smears, 90 out of 130 were having unsheathed about 61.88 to 78.50 micrometer with a mean microfilariae, 24 were having sheathed of 72.5 µm from anterior end. Anal pore also microfilariae and 16 were having unsheathed appeared as an oval clear space opening to

806 Figure 1. Giemsa staining of microfilariae. one side of the body at about 171.88 to 184.50 acid phosphatase activity in these four areas micrometer with a mean of 180.00 µm from (Fig. 2). The four regions were amphids, the anterior end. excretory pore, anal pore and phasmid. Acid Histochemical staining of sheathed phosphatase activity at excretory pore and microfilariae revealed two staining patterns. anal pore appeared as oval red spots opening In one type, four points in the microfilariae to one side of the body of microfilariae. Acid have taken bright red colour, indicating the phosphatase activity at amphid and phasmid

807 Figure 2. Acid phasphatase staining of sheathed microfilariae. appeared as two small red lines resembling Molecular methods the sign. Second type showed staining at Primers specific for B. malayi amplified a two points, viz., excretory pore and anal fragment having a size between 100 and pore as red spots. Among the 24 cases with 200 bp from all the 30 samples containing sheathed microfilariae alone, 21 (87.5%) sheathed microfilariae (Fig. 3). revealed four point staining pattern and 3 Sequencing the cloned fragment revealed (12.5%) revealed two point staining pattern. the presence of 153 bp and the sequences The histochemical staining pattern suggested were analysed using NCBI BLAST. Analysis that the microfilariae were similar to human showed 97% identity with B. malayi isolate filarial parasite B. malayi. 1 Alappuzha (Accession No. JN413104.1)

808 Figure 3. Gel electrophoresis of filarial PCR products using B. malayi specific primers.

Hha I repeat region (which was isolated Restriction Analysis from human samples in Alappuzaha district Restriction endonuclease analysis is of Kerala) and 96% identity with B. malayi presented in Fig. 5. There were three ex. canine Kadakkarappally 2 (Accession restriction endonuclease (RE) sites, two for No. JN601137.1) Hha I repeat region (which Alu I and one for Rsa I in the 322 bp Hha I was isolated from canine samples in repeat region of B. malayi reference Kadakkarapally region of Alappuzha district). sequence (Accession No. M12691.1). The 153 With 100% query coverage, sequences of bp Hha I repeat sequence of the present the present isolate showed 93% identity isolate (Mannuthy) also showed one site each with the reference sequences of B. malayi for these two enzymes, which is about 38 bp (Accession No. M12691.1). apart, similar to that in the reference Pair wise sequence alignment of the sequence. Absence of the second Alu I site is 153 base pair sequences with reference due to a transversion mutation (Fig. 4). sequence of B. malayi (Accession No. Compared to the present isolate, Hha I M12691.1), presented in Fig. 4, revealed repeat region of B. malayi isolate from 93% sequence identity. The sequences human beings of Alappuzha (Accession No. revealed a deletion of 7 bases at position JN 413104) also contained one site each for 65 to 71 and a transversion mutation at Alu I and Rsa I but at a different region of the positions 101 and 102 base pairs. sequence (Fig. 5). Restriction sites of the two

809 Figure 4. Pair wise sequence alignment of B. malayi Hha 1 repeat region with reference sequences.

Figure 5. Restriction map of Brugia isolates (Alu I and Rsa I). enzymes in B. timori also showed a similar Analysing the B. pahangi Hha I repeat location to that of the Alappuzha isolate. sequence of reference isolate (Accession No. Two RE sites each for Alu I and Rsa I were M12692.1), revealed a total absence of RE observed in B. malayi isolate from canine sites for both Alu I or Rsa I. This difference in (Kadakkarappally 1) whereas the other B. the cleavage sites of REs, Alu I and Rsa I in malayi isolate from canine (Kadakkarapally the repetitive DNA, allow to distinguish B. 2) contained only one site for Alu I alone in malayi and B. pahangi. But in the repeat the region similar to that of the present isolate DNA of Brugia isolated from Pattanakkad (B. malayi Mannuthy). (Alappuzha) [Accession No. JN601135]

810 named as B. pahangi, has two RE sites each, (Ufomadu, 1990; Kanjanopas et al., 2001; for Alu I and Rsa I, in the region somewhat Kobosa et al., 2004 and Ambily, 2009). Histo- similar to that of B. malayi (Alappuzha) and chemical staining pattern of B. pahangi is B. timori. different from that of B. malayi. Brugia The difference in the cleavage sites of pahangi microfilariae show heavy and REs, Alu I and Rsa I in the repetitive Hha 1, diffused acid phosphatase activity along the allowed to distinguish B. malayi and B. entire body (Yen and Mak, 1978, Kobosa et pahangi and confirmed that the canine al., 2004 and Nuchprayoon et al., 2006). sheathed microfilariae detected in the Amplification with B. mlalayi specific present study were Brugia malayi itself. primers (Thanchomnang et al., 2008) Parasitological studies and molecular produced a fragment between 100 to 200 bp. studies confirmed that the sheathed Sequencing revealed it as a 153 bp fragment microfilariae detected in dogs are that of and its analysis helped to distinguish B. B. malayi. The presence of microfilariae, malayi from B. pahangi. McReynolds et al. in peripheral blood in morning hours between (1986) analysed the 320 bp repeat DNA 8.30 am to 12 noon indicates that they are sequences from B. malayi and B. pahangi subperiodic strain of B. malayi. and showed that the differences between the Hha I repeated sequences of B. malayi and B. pahangi could be used to distinguish DISCUSSION the two species by restriction site poly- morphisms and by differences in specific In the present study, the occurrence of regions of the DNA sequence. The differences microfilariasis in dogs in Thrissur district, in DNA sequences between B. malayi and Kerala was found to be 8.1%. The reported B. pahangi were not random, but are occurrence of microfilariasis in dogs in clustered in two regions, between 140-154 Thrissur was 7.5% (Radhika, 1997) and 7% bp and 231-275 bp. Comparison of these two (Sabu et al., 2005). The present study also regions between the species showed only agrees with the above findings. Therefore 60–65% homology. Although the Hha I screening for microfilaria must be made repeats of B. malayi and B. pahangi have mandatory for all canine cases. an average homology of 89%, half the Presence of sheath and two distinctly differences are clustered in a region of 66 separated terminal nuclei in Giemsa stained nucleotides that has a homology of only 72%. smears of microfilariae indicated that they Similar observations were also reported by belong to the genus Brugia. The size of the Williams et al. (1988). The 153 bp fragment microfilariae and proportion of cephalic obtained in this study encompasses the space led to the conclusion that the two clustered region of Hha I repeat DNA, microfilariae were similar to sub periodic harboring species specific differences form of the human filarial parasite B. malayi between B. malayi and B. pahangi. rather than the canine filarial worm Brugia In the present study, analysis of the 153 pahangi (Schacher, 1962; Chansiri et al., bp fragment revealed 97% and 96% similarity 2002; Soulsby, 2005 and Ambily et al., 2011). with Hha I repeat region of B. malayi isolate Histochemical staining of sheathed micro- from human blood sample and canine blood filariae revealed two staining patterns. In one sample respectively of Alappuzha region, type, four points in the microfilariae took Kerala. With human B. malayi reference bright red colour, indicating acid phosphatase sequences (Accession No. M12691.1) and activity in these four areas. The four regions B. timori, 93% identity was observed. Pair were amphids, excretory pore, anal pore and wise sequence alignment with reference to phasmid. The second type showed staining B. malayi Hha I repeat region of 322 bp at two points, viz., excretory pore and anal revealed that the 153 bp correspond to a pore as red spots. Both these types of staining region of 140-292 bp. McReynolds et al. patterns were reported for B. malayi (1986) showed that the differences in DNA

811 sequences between B. malayi and B. it as B. pahangi (Ravindran et al., 2014) is pahangi are clustered in two regions, to be re-examined. Xie et al. (1994) reported between 140-154 bp and 231-275 bp of a that the Hha I repeat DNA of B. malayi 322 bp Hha I repeat. Thus, the amplified and B. timori were to be virtually identical. product in the present study encompassed These two species are morphologically and the most divergent regions of Hha I repeats molecularly very closely related among the of the two species. The primers used here is Brugia species. species specific for B. malayi and hence, All the above findings such as Giemsa the amplification was specific to the Hha I staining morphology, acid phosphatase repeats of B. malayi. Sequence alignment staining pattern,species specific amplifi- also showed a deletion of 7 bases at positions cation of a 153 bp fragment and RE sites for 65 to 71, which might have contributed to Alu I and Rsa I and their similar location the adaptation of the parasite to the new to that of reference B. malayi sequences host (dogs). clearly identified the present isolate as B. Further, restriction endonuclease malayi. Even though, the natural host for B. analysis of the 153 bp Hha I repeat region malayi is human beings, sequence variations obtained in the present study revealed RE such as deletion of seven base pairs and a sites for Alu I and Rsa I separated by a transversion mutation, which deleted an Alu distance of 38 bp in the positions exactly same I site might have influenced it’s adaptation as that of the reference B. malayi sequence. in dogs. One Alu I site in the present isolate is deleted B. malayi microfilariae are of three due to a transverse mutation. This might physiological forms, nocturnally periodic, also play a role in the adaptation of the nocturnally sub periodic or even diurnally parasite in the new host. McReynolds et al. sub periodic. In nocturnally periodic form (1986) reported that RE cleavage sites in microfilariae appear in peripheral blood in the repetitive DNA allow to distinguish B. peak levels at midnight, in nocturnally sub malayi and B. pahangi. They showed that B. periodic, microfilariae present in peripheral malayi repetitive DNA sequence is restricted blood throughout the day but in high by Alu I and Rsa I but B. pahangi sequence is concentration from the afternoon onwards. not. Hence, it is suggested that the 153 bp In diurnally sub periodic, microfilariae fragment obtained in the present study is present in peripheral blood throughout the a part of Hha I repeat DNA of B. malayi day but in high concentration in the morning microfilariae and not that of B. pahangi. hours (Denham and McGreevy, 1977). From Comparison of RE sites of the two enzymes India, only nocturnally periodic form of in the Hha I repeat DNA of the present isolate B. malayi has been reported in human and published B. malayi and B. timori beings (Sabesan et al., 2010). In Mastomys isolates revealed the presence of RE sites natalensis, B. malayi exhibited diurnal at similar positions in the B. malayi isolate subperiodicity indicating that in a different from canine, Kadakkarapally 1. Whereas, host one form of microfilariae can change the RE site of B. malayi from human beings its periodicity (Reddy et al., 1984). Sub (Alappuzha) were at a different region of periodic form of B. malayi were detected the Hha I repeat DNA, similar to that of B. in animals such as leaf monkeys (Nutman, timori, there was a total absence of RE sites 2001). In the present study also, B. malayi for these enzymes in B. pahangi (Reference microfilariae identified in dogs were of Sequence). However, the presence of RE subperiodic form. The study confirmed the sites for these enzymes (two sites for each presence of B. malayi microfilariae in dogs enzyme) in this region of Brugia isolate from in Kerala, India and documented detailed Pattanakkad (Alapuzha) in a region similar description of morphological and molecular to that of B. malayi (Alappuzha) and B. features of the microfilariae. timori (Reference Sequence) and naming

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