Novel polymorphisms in the octopamine receptor gene of amitraz resistant population of Rhipicephalus sanguineus sensu lato, in south India.

The brown dog tick or Rhipicephalus sanguineus sensu lato is an ixodid tick, responsible for the dissemination of pathogens that cause canine infectious diseases besides inflicting the direct effects of tick bite. The hot humid climate of Kerala, a south Indian state, is favorable for propagation of tick vectors and acaricides are the main stay of tick control. Though the resistance against synthetic pyrethroids is reported among these species, the status of amitraz resistance in R. sanguineus s. l. in the country is uncertain due to the lack of molecular characterisation data and scarce literature reports. Hence the present study was focused on the phenotypic detection and preliminary genotypic characterisation of amitraz resistance in the R. sanguineus s. l. A modified larval packet test (LPT) on a susceptible isolate was performed to determine the discriminating dose (DD). Further LPT-DD on 35 tick isolates was carried out to detect amitraz resistance robustly, along with that full dose response bioassays on the resistant isolates were performed. The results indicated that amitraz resistance is prevalent with 49 per cent of the samples being resistant. Amplification of exon 3 of octopamine receptor gene from both the susceptible and resistant larval isolates was carried out. Amplicons of ten pooled amitraz susceptible and ten pooled amitraz resistant representative samples were sequenced and analysed, unveiling a total of three novel non-synonymous mutations in the partial coding region at positions V32A, N41D and V58I in phenotypically resistant larval DNA samples. In silico analysis by homology modelling and molecular docking of the mutated and unmutated receptors showed that these mutations had reduced the binding affinity to amitraz. However, lack of mutations in the octopamine receptor gene in three of the pooled low order resistant R. sanguineus s. l. larval samples could be suggestive of other mechanisms associated with amitraz resistance in the region. Hence, further association studies should be carried out to confirm the association of these mutations with target insensitivity in R. sanguineus s. l. ticks, along with exploring the status of metabolic resistance and other mechanisms of resistance.

A Novel PCR-RFLP to Detect and Differentiate Schistosoma spindale and S. indicum, the Pathogenic Schistosomes in Indian Cattle.

PURPOSE: Visceral schistosomosis is an economically important trematode infection caused by Schistosoma spindale and S. indicum in among ruminants. The lack of sensitive diagnostic tools has often led to underestimation of the prevalence in live animals. A sensitive copro-PCR targeting partial mitochondrial gene was developed to detect Schistosoma spp. However, this protocol could not differentiate between the two species. This study was conducted to explore the possibility of species differentiation using restriction fragment length polymorphism of PCR products (PCR- RFLP). METHODS: Polymerase chain reaction was carried out to amplify mitochondrial gene of adult S. spindale and S. indicum. Copro PCR was done with schistosome-positive faecal samples. A novel PCR-RFLP was designed targeting the Hpy166II recognition sequence in the mitochondrial gene sequence of S. indicum. RESULT: The PCR using primers targeting the mitochondrial gene of S. spindale and S. indicum amplified a distinct product of approximately 454 bp with adult fluke as well as faecal DNA, which upon RFLP with Hpy166II yielded 330 bp and 124 bp products with S. indicum amplicons alone. CONCLUSION: The novel PCR-RFLP possesses the potential to be used in epidemiological surveys among bovines and in snail intermediate hosts to screen for S. spindale and S. indicum infection.

Candidatus Dirofilaria Hongkongensis Infections in Humans During 2005 to 2020, in Kerala, India.

We tried to determine the epidemiology and species of human dirofilariasis observed at two tertiary care hospitals in Kerala. We searched the hospital database to identify cases of dirofilariosis from January 2005 to March 2020. Along with human isolates, one dog Dirofilaria isolate was also subjected to PCR and sequencing of pan filarial primers cytochrome oxidase subunits 1 and 12S rDNA. We documented 78 cases of human dirofilariosis. The orbit, eyelid, and conjunctiva were the most commonly affected sites. Molecular characterization identified one dog and five human isolates as Candidatus Dirofilaria Hongkongensis. A rare case of subconjunctival infestation by B. malayi was also documented. Human dirofilariosis is a public health problem in the state of Kerala in India, and it is mostly caused by Candidatus Dirofilaria Hongkongensis. We propose that all diroifilaria isolates are subjected to sequencing for identification.

Bacillus thuringiensis strains from Western Ghats of India possess nematocidal property against Haemonchus contortus larvae of goats.

Nematocidal properties of spore crystal mixtures of six Bacillus thuringiensis (Bt) strains (KAU 49, 50, 52, 61, 99 and 424) collected from Western Ghats, a biodiversity hot spot of India, were analysed against Haemonchus contortus larvae isolated from goats. One dose nematocidal assay dose response to lyophilised spore-crystal mixtures (SCM) of the six Bt strains were determined by adding 200 µg/mL of each SCMs to culture plate wells containing aqueous suspension of H. contortus larvae. Out of the strains screened, KAU 50 and 424 were found to possess nematocidal properties. Maximum nematocidal properties were exhibited 7 days post-inoculation of the lyophilised SCMs. The 50 per cent lethal concentrations deduced by log probit analysis for KAU 50 was found to be 130.59 µg/mL, whereas that of KAU 424 was found to be 144.536 µg/mL at 95 per cent confidence level. This is the first report on the nematocidal propery of Bt strains against Haemonchus contortus larvae isolated from goats. Further studies are needed for identification and characterisation of the toxin.

Canine Brugia malayi microfilarial excretory/secretory protein-based antibody assay for the diagnosis of brugian filariasis in dogs.

Brugian filariasis is reported in dogs in Kerala, India. Antibody detection kits are not available worldwide, for detection of Brugian filariasis in dogs. A study was carried out to develop Indirect plate ELISA using excretory secretory antigen isolated from canine brugian microfilariae and compare the sensitivity and specificity with that of blood smear examination. Identification of microfilariae was done by acid phosphatase staining using Naphthol AS-TR method and Polymerase Chain Reaction for Hha 1 repeat sequence. The microfilariae were identified as Brugia malayi. Isolation of brugian microfilariae from canine blood was done by gradient centrifugation method. The isolated microfilariae were maintained in RPMI-1640 media. The pooled media was then concentrated to obtain excretory secretory protein (ESP). This ESP was used to develop Indirect ELISA. The sensitivity and specificity of the plate ELISA developed was 84 and 100 per cent respectively when compared with blood smear examination. This is the first report of successful isolation of ESP from Brugia malayi microfilariae from dogs and standardization of plate ELISA using the antigen.

Comparison of efficacy of ivermectin and diethylcarbamazine against naturally infected Brugia malayi microfilaria in dogs.

Filarial parasites like Brugia pahangi and Brugia malayi can infect dogs. Adults of Brugia genus resides in the lymphatic system and microfilariae, in blood. There are increasing reports of detection of B. malayi microfilariae in dogs. A study was undertaken to compare the efficacy of repeated oral dosing of ivermectin (IVT) and diethylcarbamazine (DEC), individually and in combination against naturally infected B. malayi microfilariae in dogs. The species of the microfilariae was confirmed by acid phosphatase staining and polymerase chain reaction. The three treatment groups were 200 mcg/kg body weight IVT daily for 14 days (I), 6.6 mg/kg body weight DEC daily for 14 days (II) and IVT and DEC together in the same dose for a period of 5 days (III). Microfilarial status of the peripheral blood was assessed on the 0th, 7th, 14th and 21st day. Haematological parameters were measured on day zero and on the 21st day. Though, all the three treatment groups showed a reduction in the microfilarial concentration through the study period, complete absence of detectable microfilaremia was not noticed in any of the three groups by 21st day. Among the haematological parameters, statistically significant difference was observed in the post-treatment means of haemoglobin levels of group III when compared with group II. Since group III regime (IVT + DEC) was shorter and just as effective as the longer ones, it is considered superior to the other two.

Seroprevalence of bovine intestinal schistosomosis in different agro- ecological zones of south India using excretory-secretory antigen based ELISA.

Intestinal schistosmosis caused by Schistosoma spindale and S. indicum is an important snail borne trematode infection that adversely affects the production and productivity of bovines in India. The present communication reports the high seroprevalence of infection among dairy cattle and buffaloes, under field conditions, utilizing a sensitive and specific excretory-secretory antigen based ELISA. Comparison of ELISA with copro-PCR, microscopy and post-mortem mesentery examination revealed the diagnostic superiority of ELISA. Seroprevalence of infection was mapped for the first time in India with special emphasis to agro- ecological zones. Anti-schistosome antibodies were detected in 34.96% of dairy cattle and buffaloes in the state with the evidence of significant influence of topography on the prevalence of infection. The study also highlighted the need to trace endemic pockets of infection in the country through efficient ante-mortem surveillance and to initiate anti-schistosome therapy prior to animal transport.

Molecular detection of Hepatozoon canis in dogs from Kerala.

India has a wide range of agro-climatic zones which is highly conducive for a diverse range of vectors and canines are continuously exposed to the risk of spectrum of tick borne protozoan diseases. The brown dog tick, Rhipicephalus sanguineus is widely prevalent among dogs in Kerala and there is a high prevalence of this tick transmitted Babesia and Ehrlichia spp. infection. However, the incidence of Hepatozoon canis transmitted by the same tick species had not been reported in the state since 2004. Preliminary screening of client owned dogs revealed six dogs to be positive for typical gelatin capsule shaped gamonts of H. canis within neutrophils in blood smear by microscopic examination. A PCR assay was standardized to amplify a specific 737 bp fragment of 18S rRNA gene of H. canis. Phylogenetic analysis revealed closest relationship with West Indies isolate deposited at GenBank database. The present study records the molecular detection of this haemoparasite in the state, for the first time.

Molecular evidence of haemoparasites in ixodid ticks of dogs- first report in India.

The distribution of ixodid ticks of the genera Rhipicephalus and Haemaphysalis spp. in tropical regions of India contributes to many serious tick-borne parasitic and rickettsial infections in domestic and wild canines. A preliminary molecular survey of the most prevalent haemoparasites in ixodid ticks of carnivores in Kerala, South India was undertaken using multiplex PCR. Babesia vogeli, B. gibsoni and Ehrlichia canis could be detected in R. sanguineus ticks, while H. bispinosa harboured B. gibsoni alone. Future investigations including transmission trials are to be undertaken to prove the vector potentiality of these ticks in this geo-climatic zone.

High prevalence of small Babesia species in canines of Kerala, South India.

AIM: Canine babesiosis is an important vector-borne hemoparasitic disease caused by Babesia canis vogeli and Babesia gibsoni, in India. The communication places on record the salient findings of the study directed to detect and characterize the pathogenic B. gibsoni isolates of Kerala state. MATERIALS AND METHODS: A total of 150 dogs were examined for the presence of hemoparasites by light microscopy as well as by PCR targeting the 18S rRNA gene of B. gibsoni. Hematological parameters were also analysed. Phylogenetic tree was constructed based on Tamura kei model adopting ML method. RESULTS: A sensitive and specific polymerase chain reaction assay was developed with newly designed primer pair BAGI-F/BAGI-R for the amplification of 488 bp fragment of 18S rRNA gene of B. gibsoni. Out of the 150 dogs examined, molecular evidence of B. gibsoni was recorded in 47.3% animals, while light microscopy detected the infection in 26.67% cases. The phylogenetic analyses revealed that B. gibsoni, Kerala, isolate was closest and occurred together with Bareilly isolate. Anemia and thrombocytopenia were the significant hematological alterations in chronic B. gibsoni infection. CONCLUSION: A high prevalence of natural infection of B. gibsoni was detected among the study population. The affected animals showed anaemia and thrombocytopenia. Phylogenetic analysis of this pathogenic isolate from south India revealed the closest similarity with Bareilly isolates.

Molecular identification of Babesia spp. in naturally infected dogs of Kerala, South India.

Canine babesiosis is a tick borne haemoprotozoan disease caused by large and small intraerythrocytic apicomplexan piroplasms of the genus Babesia spp. The clinical manifestations of the disease vary from inapparent subclinical form to hyperacute shock related haemolytic crisis. Microscopic examination of blood smears from suspected dogs revealed Babesia canis and B. gibsoni in 3.45 and 25.86 % of samples respectively. A seminested PCR based on previously published species specific primers targeting the 18S rRNA gene was utilized to identify the Babesia species infecting dogs of Kerala at the sub species level. The study revealed 57.5 % prevalence of Babesia spp. among dogs. This report also presents the first molecular evidence of Babesia canis vogeli and B. gibsoni among naturally infected dogs in Kerala, South India. Molecular survey revealed a high prevalence of B. gibsoni infections when compared with B. canis vogeli infections among canines of the state. Preliminary survey of the tick population revealed the presence of Rhipicephalus sanguineus, R. haemaphysaloides and Haemaphysalis bispinosa in infected animals. Further studies need be directed towards utilizing the PCR protocol for confirming the vectors of these species in the region.

Molecular characterization of human Dirofilaria isolates from Kerala.

Background & objectives: Human dirofilariosis is a well-recognized zoonosis caused by several species of the genus Dirofilaria. The disease is prevalent among canines and human beings in Kerala. The objective of the present study was to confirm the human Dirofilaria isolates by molecular characterization. Methods: The worms or segments obtained from human sources were subjected to diagnostic polymerase chain reaction (PCR) targeting the cytochrome oxidase subunit 1 (COI) and 5S ribosomal RNA (rRNA) genes of Dirofilaria repens. The amplicons were sequenced and analyzed. Results: The filariid nematodes recovered from ocular as well as subcutaneous tissue of human origin were identified as D. repens based on PCR targeting COI as well as 5S rRNA genes. The phylogenetic analysis of the COI gene nucleotide sequence obtained in the present study showed that D. repens shared the closest evolutionary relationship with D. honkongensis. Interpretation & conclusions: Molecular identification of D. repens isolated from human source assumes significance from the point of zoonotic threat of this mosquito-borne nematode. Phylogenetic analysis revealed a close evolutionary relationship with Asian isolate of D. honkongensis. Timely detection and treatment of infection in dogs, together with mosquito control, should be an integral part of the control strategy of this disease.

Efficacy of Dot-ELISA using different antigens in detecting anti-schistosome antibodies among bovines in field conditions.

Schistosomosis has been recognised as one of the major parasitic diseases of livestock and human beings. Schistosoma spindale is the major cause of visceral schistosomosis among bovines of Kerala State. Besides pathology in animals, it has been long known that cercariae of S. spindale are a common cause of dermatitis in human beings in Asia. However, detection of this disease based on coprology has underestimated the prevalence of this economically important disease among cattle of the State. An efficient diagnostic tool providing unequivocal evidence of infection in living animals is perhaps, the key to formulate and deliver control measures to the target population. It is also crucial for an enhanced understanding of parasite epidemiology. The utility of excretory-secretory proteins as diagnostic and vaccine candidates for schistosomosis has been a focus of medical research since long. There exists a paucity of information with regard to analysis of ES proteins of S. spindale and their incorporation to develop sensitive and specific serodiagnostic tool. Hence a study was designed to evaluate the efficacy of Dot-ELISA incorporating different antigens of S. spindale and to validate the test under field conditions.

Occurrence of human sparganosis in Kerala.

Human sparganosis is a rare zoonotic disease caused by the larval stages of Spirometra sp. A seven cm worm recovered from a painful swelling in the right inguinal region of a 9 year old boy from Kunnamkulam in Thrissur District, Kerala was identified as sparganum based on morphology. This is the first case report of the disease from Kerala state. Possible mode of infection and control measures are suggested. Increased public awareness about risks associated with drinking contaminated water is important to prevent further incidence in the locality.

Canine filarial infections in a human Brugia malayi endemic area of India.

A very high prevalence of microfilaremia of 42.68 per cent out of 164 canine blood samples examined was observed in Cherthala (of Alappuzha district of Kerala state), a known human Brugia malayi endemic area of south India. The species of canine microfilariae were identified as Dirofilaria repens, Brugia malayi, and Acanthocheilonema reconditum. D. repens was the most commonly detected species followed by B. pahangi. D. immitis was not detected in any of the samples examined. Based on molecular techniques, microfilariae with histochemical staining pattern of "local staining at anal pore and diffuse staining at central body" was identified as D. repens in addition to those showing acid phosphatase activity only at the anal pore. Even though B. malayi like acid phosphatase activity was observed in few dogs examined, they were identified as genetically closer to B. pahangi. Hence, the possibility of dogs acting as reservoirs of human B. malayi in this area was ruled out.

Molecular characterization of Theileria orientalis causing fatal infection in crossbred adult bovines of South India.

The disease condition attributed to have been caused by Theileria orientalis is generally benign. However, it is also thought that the parasite, at least some strains of it, can cause fatal disease. The present communication deals with the clinical signs, postmortem lesions and diagnosis of a fatal disease due to T. orientalis which caused mortality in crossbred adult bovines of South India. High body temperature, lacrimation, nasal discharge, swollen lymph nodes and haemoglobinuria were the symptoms observed. The postmortem lesions observed were punched out ulcers in abomasum, enlargement of spleen, massive pulmonary oedema, frothy exudates in trachea, epicardial and endocardial haemorrhage and haemorrhagic duodenitis. Peripheral blood smear examination revealed rod shaped Theileria sp. organisms. Polymerase chain reaction that amplify the T. orientalis specific P(32/33) gene, followed by cloning and sequencing, revealed maximum homology with Narathiwat (Thailand) and Jingole -1 (Indonesia) isolates which were positioned as isolate type 7 of T. orientalis.