Tularemia - a re-emerging disease with growing concern.

Tularemia caused by Gram-negative, coccobacillus bacterium, Francisella tularensis, is a highly infectious zoonotic disease. Human cases have been reported mainly from the United States, Nordic countries like Sweden and Finland, and some European and Asian countries. Naturally, the disease occurs in several vertebrates, particularly lagomorphs. Type A (subspecies tularensis) is more virulent and causes disease mainly in North America; type B (subspecies holarctica) is widespread, while subspecies mediasiatica is present in central Asia. F. tularensis is a possible bioweapon due to its lethality, low infectious dosage, and aerosol transmission. Small mammals like rabbits, hares, and muskrats are primary sources of human infections, but true reservoir of F. tularensis is unknown. Vector-borne tularemia primarily involves ticks and mosquitoes. The bacterial subspecies involved and mode of transmission determine the clinical picture. Early signs are flu-like illnesses that may evolve into different clinical forms of tularemia that may or may not include lymphadenopathy. Ulcero-glandular and glandular forms are acquired by arthropod bite or handling of infected animals, oculo-glandular form as a result of conjunctival infection, and oro-pharyngeal form by intake of contaminated food or water. Pulmonary form appears after inhalation of bacteria. Typhoidal form may occur after infection via different routes. Human-to-human transmission has not been known. Diagnosis can be achieved by serology, bacterial culture, and molecular methods. Treatment for tularemia typically entails use of quinolones, tetracyclines, or aminoglycosides. Preventive measures are necessary to avoid infection although difficult to implement. Research is underway for the development of effective live attenuated and subunit vaccines.

Molecular detection of Coxiella-like endosymbionts in Rhipicephalus microplus from north India.

Apart from the tick-borne pathogens affecting human and animal health, ticks also harbor various non-pathogenic endosymbionts with dynamic ecological interactions. These endosymbionts are unexplored from the Indian ticks; hence this pilot study was conducted. Seventy-nine ticks were collected from Nainital district of Uttarakhand state of north India and were identified as Rhipicephalus microplus morphologically and by molecular analysis. PCR and sequence analysis were carried out to detect the presence of Rickettsia-like, Coxiella-like and Francisella-like endosymbionts in these ticks. Based on the partial 16S rRNA gene sequence, Coxiella-like endosymbiont (CLE) was detected in the adult and other life-cycle stages of ticks with 96.6-97.7% nucleotide sequence identity with the published CLE sequences from GenBank. The phylogenetic analysis revealed that the CLE from R. microplus were clustered with the CLE from other Rhipicephalus species. All these CLE formed distinct clades from the pathogenic Coxiella burnetii. None of the tick samples was found positive for Rickettsia-like and Francisella-like endosymbionts in the present study. We also demonstrated the vertical transmission of CLE from surface sterilized and laboratory reared fully engorged adult females to the eggs and the larvae. However, large scale studies are to be conducted to detect various endosymbionts and endosymbiont-tick associations in the Indian tick species and to explore these associations for tick and tick-borne disease control.

Serodiagnosis of Fasciola gigantica Infection in Buffaloes with Native Cathepsin-L Proteases and Recombinant Cathepsin L1-D.

AIM: Serodiagnosis of Fasciola gigantica natural infection in buffaloes with recombinant cathepsin L1-D and native cathepsin-L protease antigens. METHODS: The recombinant cat L1-D antigen was expressed in prokaryotic expression system and native cathepsin-L proteases were purified by alcoholic fractionation from adult F. gigantica flukes. Buffaloes (n  = 325) were screened for anti-Fasciola antibodies with the above antigens in immunoglobulin-G-enzyme linked immunosorbent assay (IgG-ELISA). RESULTS: The recombinant cat L1-D antigen showed positive reactivity with 101/122 necropsy positive animals but 21/122 necropsy confirmed positive animals were negative in this ELISA (sensitivity 82.8%). However, 30/203 (14.8%) necropsy negative animals for Fasciola were seropositive with specificity of 85.2%. With native cat-L protease, 104/122 necropsy confirmed positive animals were ELISA positive but 18/122 necropsy positive animals were seronegative, thereby depicting the sensitivity of 85.2%. But ELISA with this antigen showed 27/203 (13.3%) necropsy negative animals as positive (specificity 86.7%). CONCLUSIONS: Comparative evaluation of both the antigens showed that they are suitable for serodiagnosis of F. gigantica infection in buffalo herds.

Evaluation of Echinococcus granulosus recombinant EgAgB8/1, EgAgB8/2 and EPC1 antigens in the diagnosis of cystic echinococcosis in buffaloes.

Three recombinant proteins of Echinococcus granulosus including two antigen B sub-units EgAgB8/1 and EgAgB8/2 and Echinococcus protoscolex calcium binding protein 1 (EPC1) were expressed in prokaryotic expression vectors. The diagnostic potential of these three recombinant proteins was evaluated in the detection of cystic echinococcosis in buffaloes in IgG-ELISA. The EgAgB8/1 and EgAgB8/2 recombinant proteins reacted fairly with the hydatid infected buffaloes with sensitivity of 75.0% and 78.6%, respectively and specificity of 75.8% while EPC1 recombinant protein showed higher sensitivity (89.3%) but lower specificity (51.5%). Cross-reactivity of these three antigens was assayed with buffalo sera naturally infected with Explanatum explanatum, Paramphistomum epiclitum, Gastrothylax spp., Fasciola gigantica and Sarcocystis spp. EgAgB8/1 and EPC1 antigens cross-reacted with all these sera while EgAgB8/2 showed no cross-reaction with Sarcocystis spp. and reacted with some of the E. explanatum infected buffalo sera. This study explores the potential of three hydatid antigens viz. EgAgB8/1, EgAgB8/2 and EPC1 for their diagnostic potential in buffaloes positive for cystic echinococcosis.

RNA interference in Fasciola gigantica: Establishing and optimization of experimental RNAi in the newly excysted juveniles of the fluke.

Fasciolosis caused by Fasciola gigantica is a neglected tropical disease but a constraint on the growth and productivity of cattle, buffaloes and sheep in the tropical countries of Asia and Africa. Resistance to commonly used anthelmintics in Fasciola has increased the need to search for alternative therapeutic targets. RNA interference is the current tool of choice in the search for such targets in Fasciola. The susceptibility of juvenile Fasciola hepatica to double stranded (ds) RNA induced RNAi has been established but in F. gigantica a single preliminary report on RNAi induced mRNA transcript knockdown is available. Here we optimized conditions for RNAi in the liver fluke F.gigantica targeting six genes including superoxide dismutase (SOD), σ class of glutathione-s-transferase (GST), cathepsin (Cat) L1-D, Cat B1, Cat B2 and Cat B3 that showed robust transcriptional silencing of the targets following exposure of the newly excysted juveniles (NEJs) to long (170-223 nt) dsRNA. Knockdown was shown to be concentration dependent with significant mRNA transcript suppression occurring at 5 ng / µl that showed further suppression with the increase in the dsRNA concentration. The dsRNA induced persistent silencing of the mRNA transcript of SOD and σGST up to 15 days of observation. Delivery of the long dsRNA and siRNA to the newly excysted juveniles by soaking method was found to be efficient by tracking the uptake and diffusion of Cy3 labelled siRNA and long dsRNA in the flukes. Off-target effects of dsRNA trigger on some of the non-target genes were detected in the present investigation on RNAi in F. gigantica. The dsRNA induced superoxide dismutase protein suppression while impact of RNAi on other target proteins was not studied. There is no in vitro culture system for prolonged survival of the F. gigantica and in the present study in vitro maintenance of the NEJs is reported for a period of 3 weeks. The present study is the first attempt on optimization of RNAi protocols in F. gigantica where long dsRNA allowed for an efficient and persistent gene silencing, opening prospects for functional validation of putative vaccine and therapeutic targets in this neglected parasite.

Sero-detection of Toxocara canis infection in human with T.canis recombinant arginine kinase, cathepsin L-1 and TES-26 antigens.

Three recombinant antigens viz. arginine kinase, cathepsin L-1 and TES-26 of Toxocara canis were expressed in Escherichia coli and evaluated for their potential in the detection of T. canis larval infection in human in immunoglobulin G-enzyme linked immunosorbent assay (IgG-ELISA). Results of the IgG-ELISA with the above recombinant antigens were confirmed with commercially available IgG detection kit for T. canis infection used as a standard test. All three recombinant antigens were 100% sensitive in the detection of positive cases (n = 6) of T. canis infection in human and were screened for their cross-reactivity in human patients with history of Toxoplasma gondii, Plasmodium vivax, Entamoeba histolytica, hydatid and hookworm infections. The recombinant TES-26 antigen showed higher specificity and cross-reacted with T. gondii infection sera only. However, arginine kinase and cathepsin L-1 recombinant antigens showed cross-reactions with sera of patients infected with T. gondii, P. vivax and E. histolytica but not with the patient sera infected with hydatid and hookworm. These results show that recombinant TES-26 is a potential diagnostic candidate antigen for human toxocarosis caused by migrating T. canis larvae.

Molecular detection and genetic diversity of Babesia gibsoni in dogs in India.

Babesia gibsoni is a tick borne intraerythrocytic protozoan parasite causing piroplasmosis in dogs and has been predominantly reported in Asian countries, including Japan, Korea, Taiwan, Malaysia, Bangladesh and India. The present communication is the first evidence on the genetic diversity of B. gibsoni of dogs in India. Blood samples were collected from 164 dogs in north and northeast states of India and 13 dogs (7.9%) were found positive for B. gibsoni infection by microscopic examination of blood smears. Molecular confirmation of these microscopic positive cases for B. gibsoni was carried out by 18S rRNA nested-PCR, followed by sequencing. Nested-PCR for the 18S rRNA gene was also carried out on microscopically B. gibsoni negative samples that detected a higher percentage of dogs (28.6%) infected with B. gibsoni. Genetic diversity in B. gibsoni in India was determined by studying B. gibsoni thrombospondin-related adhesive protein (BgTRAP) gene fragments (855bp) in 19 isolates from four north and northeast states of India. Phylogenetic analysis of the BgTRAP gene revealed that B. gibsoni parasite in India and Bangladesh formed a distinct cluster away from other Asian B. gibsoni isolates available from Japan, Taiwan and Korea. In addition, tandem repeat analysis of the BgTRAP gene clearly showed considerable genetic variation among Indian isolates that was shared by B. gibsoni isolates of Bangladesh. These results suggested that B. gibsoni parasites in a different genetic clade are endemic in dogs in India and Bangladesh. Further studies are required for better understanding of the genetic diversity of B. gibsoni prevalent in India and in its neighbouring countries.

Detection of benzimidazole resistance in gastrointestinal nematodes of sheep and goats of sub-Himalyan region of northern India using different tests.

The present investigation was planned with the objective of studying the status of benzimidazole (BZ) resistance in gastrointestinal nematodes (GIN) of sheep and goats of different agro-climatic zones of sub-Himalyan region of northern India using in vivo faecal egg count reduction test (FECRT) and in vitro tests namely egg hatch assay (EHA) and larval development assay (LDA). Out of fourteen flocks, FECRT detected resistance in eight flocks (two sheep flocks and six goat flocks) with FECR% ranging from 54.95 to 90.86. Pre treatment coproculture contained predominantly Haemonchus contortus, followed by Trichostrongylus spp., Oesophagostomum and Strongyloides, while post treatment coproculture results showed that only H. contortus survived fenbendazole (FBZ) (in FECRT) or thiabendazole (TBZ) (in LDA) treatment except in three flocks of Tarai region {one sheep flock (Us1), and two goat flocks (Ug1 and Ug5)} where BZ resistant Trichostrongylus were also detected. The GIN of those eight farms which were found resistant by FECRT were also detected resistant by EHA. Arithmetic mean and range of ED50 value of susceptible group was found to be 0.059 µg/ml and 0.037-0.096 µg/ml, respectively, and the same for the resistant group were found to be 0.119 µg/ml and 0.101-0.147 µg/ml, respectively. With LDA, the arithmetic mean and range of LC50 value of susceptible group was found 0.0030 µg/ml and 0.001-0.005 µg/ml, respectively, and those of resistant group was found 0.0105 µg/ml and 0.009-0.012 µg/ml, respectively. The values of Spearman rank correlation coefficient indicated that negative correlation was found between FECR% and ED50 and between FECR% and LC50 while positive correlation existed between ED50 and LC50 value and the p-values indicated that these correlations were statistically highly significant. In the present study, FECRT and EHA gave comparable results with regard to detection of BZ resistance in GIN in sheep and goats. Although with LDA, the threshold LC50 value could not be established as for EHA but LDA indicated the presence of low level of resistance in GIN of both sheep and goats. For effective worm control, regular monitoring for anthelmintic resistance is important to know the status of anthelmintic efficacy in a particular agro-climatic zone. The baseline information thus generated will enable timely management of benzimidazoles resistance in GIN.