Molecular expression and characterization of Taenia solium TS14 gene for sensitive detection of porcine cysticercosis

In the present study, molecular expression and characterization of TS14 gene of Taenia solium (Indian isolate) was carried out for sero-prevalence study of porcine cysticercosis. The complete open reading frame (ORF) of TS14 gene was amplified by RT-PCR from mRNA isolated from T. solium cysticerci (Indian isolate). The amplicon was cloned into the pET-32a(+) expression vector and used to transform Escherichia coli BL21 codon(+) cells to produce TS14 antigen. The nucleotide and deduced amino acid sequences of the TS14 were aligned against the related sequences of T. solium available in public domain for in silico analysis by DNA STAR and MEGA version 4.0 softwares. The nucleotide sequence revealed that the TS14 gene of T. solium (Indian isolate) encodes 85 amino acids and its nucleotide sequence had 98.8% to 99.5% sequence homology with that of China, Peru and Mexico isolates. A high-level expression of the recombinant protein was observed in the molecular range (Mr) of 29 kDa. The rTS14 was confirmed by its specific immunoreactivity against hyper-immune and known reference positive pig sera (8). The protein was serologically non-reactive to known cysticercus negative pig sera samples and further lacks cross-reactivity against hydatid cyst positive pig sera. The present preliminary investigation revealed the good potential of rTS14 for serodiagnosis of T. solium cysticercosis

Prevalence of porcine cysticercosis in Bareilly, Northern India

Aim: The objective of this study was to investigate the prevalence of Taenia solium cysticercosis in pigs slaughtered at makeshift houses in Bareilly, Northern India. Materials and Methods: Local makeshift slaughter houses were visited weekly in Bareilly to explore the prevalence of the porcine cysticercosis in this area. 175 pigs were screened for cysticercosis and prevalence was correlated to age, sex and breed of pigs. Results: A total of 175 pigs were examined for cysticercosis out of which 9 (5.14%) were found positive for porcine cysticercosis. Sex-wise prevalence of this infection in male and female was recorded as 4.82% (4/83) and 5.43% (5/92), respectively. The infection was higher (5.34%) in the young age group of 1-12 months as compared to the older stocks of 13-24 months of age group (4.54%). Prevalence of porcine cysticercosis was relatively higher in cross bred pigs (5.88%, 6/102) than in the non-descript local breed of pigs (4.11%, 3/73). Conclusion: The present study reveals that T. solium cysticerci infection is prevalent in swine population of Bareilly. Keeping in view the zoonotic importance, strict hygienic measures need to be undertaken for prevention of human infection

LEPTOTROMBIDIUM DELIENSE INFESTATION IN DOMESTIC DOGS FROM INDIA, A VECTOR OF SCRUB TYPHUS: A CASE REPORT

Scrub typhus is a vector-borne, zoonotic disease caused by Orientia tsutsugamushi. Several members of the genus Leptotrombidium have gained importance due to their potential role as vectors as well as reservoirs for O. tsutsugamushi. The larvae of Leptotrombidium species are primary parasites of ground-dwelling rodents. However, changes in climate, and host specificity makes them adapt to other animals and play a role in the perpetuation of various (re)- emerging pathogens between animals and humans. Two male mongrel dogs aged six months were presented to the College of Veterinary Sciences and Animal Husbandry, Central Agricultural University, Mizoram, India with a history of skin lesions and intense pruritus. Routine skin scraping examination of samples revealed the presence of Leptotrombidium deliense larvae. Considering the public health importance of L. deliense infestation, an attempt was made to screen the dogs for O. tsutsugamushi and other haemoprotozoans. Microscopic and molecular tests were negative for haemoprotozoan parasites and O. tsutsugamushi, respectively. Both the dogs were successfully treated with parenteral ivermectin and topical fipronil spra

Co-immunization efficacy of recombinant antigens against Rhipicephalus microplus and Hyalomma anatolicum tick infestations

This article belongs to the Collection Advances in Tick Research.The immunoprophylactic management of ticks is the most effective option to control tick infestations and counter spread the acaricide resistance problem worldwide. Several researchers reported an inconsistent efficacy of the single antigen-based immunization of hosts against different tick species. In the present study, to develop a multi-target immunization protocol, proteins from Rhipicephalus microplus BM86 and Hyalomma anatolicum subolesin (SUB) and tropomyosin (TPM) were targeted to evaluate the cross-protective potential. The sequence identities of the BM86, SUB, and TPM coding genes amongst Indian tick isolates of targeted species were 95.6–99.8%, 98.7–99.6%, and 98.9–99.9%, respectively, while at the predicted amino acid level, the identities were 93.2 to 99.5, 97.6 to 99.4, and 98.2 to 99.3%. The targeted genes were expressed in the eukaryotic expression system, pKLAC2-Kluyveromyces lactis, and 100 µg each of purified recombinant protein (Bm86-89 kDa, SUB-21 kDa, and TPM-36 kDa) mixed with adjuvant was injected individually through the intramuscular route at different sites of the body on days 0, 30, and 60 to immunize cross-bred cattle. Post-immunization, a statistically significant (p < 0.001) antibody response (IgG, IgG1, and IgG2) in comparison to the control, starting from 15 to 140 days, against each antigen was recorded. Following multi-antigen immunization, the animals were challenged twice with the larvae of R. microplus and H. anatolicum and theadults of H. anatolicum, and a significant vaccine efficacy of 87.2% and 86.2% against H. anatolicum larvae and adults, respectively, and 86.7% against R. microplus was obtained. The current study provides significant support to develop a multi-antigen vaccine against cattle tick species.The authors are grateful to the Indian Council of Agricultural Research (ICAR), New Delhi, India for funding through the National Agricultural Science Fund (Grant number NASF/ABA-6015/2016-17/357 and NFBSFARA/BSA-4004/2013-14. The APC is funded by authors.Peer reviewe

Functional characterization of candidate antigens of Hyalomma anatolicum and evaluation of its cross-protective efficacy against Rhipicephalus microplus

Hyalomma anatolicum and Rhipicephalus microplus seriously affect dairy animals and immunization of host is considered as a sustainable option for the management of the tick species. Identification and validation of protective molecules are the major challenges in developing a cross-protective vaccine. The subolesin (SUB), calreticulin (CRT) and cathepsin L-like cysteine proteinase (CathL) genes of H. anatolicum were cloned, sequenced and analysed for sequence homology. Both Ha-SUB and Ha-CRT genes showed very high level of homogeneity within the species (97.6–99.4% and 98.2–99.7%) and among the tick species (77.3–99.3% and 85.1–99.7%) while for Ha-CathL the homogeneity was lower among ticks (57.5–89.5%). Besides tick species, both Ha-SUB and Ha- CRT genes showed high level of homogeneity with dipterans (47.2–53.4% and 72.0–74.4%) and nematodes (64.0% by CRT). The level of expression of the conserved genes in different stages of the tick species was studied. The differences in fold change of expression (FCE) of the targeted genes in life stages of tick were not statistically significant except Ha-SUB in eggs and in frustrated females, Ha-CRT in fed male and Ha-CathL in unfed and frustrated females where highest FCE was recorded. The functional properties of the genes were studied by RNAi technology and a significant level of gene suppression (p < 0.05) resulted in very low percentage of engorgement of treated ticks viz., 3.7%, 11.1% and 30.0% in Ha-SUB, Ha-CRT and Ha-CathL respectively, in comparison to control was recorded. The recombinant proteins rHa-SUB, rHa-CRT and rHa-CathL encoded by the genes were expressed in prokaryotic expression system. They were evaluated for cross-protective efficacy and found to be respectively, 65.4%, 41.3% and 30.2% protective against H. anatolicum and 54.0%, 37.6% and 22.2%, against R. microplus infestations.Authors are thankful to World Bank funded National Agricultural Innovation Project [NAIP/Comp-4/C2066/2008-09] and National Fund for Basic Strategic and Frontier Application Research in Agriculture Project no. [NFBSFARA/BSA-4004/2013-14 and NASF/ABA-6015/2016-17/357] for establishing the laboratory facilities for conducting the experiments.Peer Reviewe

Analysis of genetic diversity in Indian isolates of Rhipicephalus microplus based on Bm86 gene sequence

This article belongs to the Section Veterinary Vaccines.The control of cattle tick, Rhipicephalus microplus, is focused on repeated use of acaricides. However, due to growing acaricide resistance and residues problem, immunization of animals along with limited use of effective acaricides is considered a suitable option for the control of tick infestations. To date, more than fifty vaccine candidates have been identified and tested worldwide, but two vaccines were developed using the extensively studied candidate, Bm86. The main reason for limited vaccine commercialization in other countries is genetic diversity in the Bm86 gene leading to considerable variation in vaccine efficacy. India, with 193.46 million cattle population distributed in 28 states and 9 union territories, is suffering from multiple tick infestation dominated by R. microplus. As R. microplus has developed multi-acaricide resistance, an efficacious vaccine may provide a sustainable intervention for tick control. Preliminary experiments revealed that the presently available commercial vaccine based on the BM86 gene is not efficacious against Indian strain. In concert with the principle of reverse vaccinology, genetic polymorphism of the Bm86 gene within Indian isolates of R. microplus was studied. A 578 bp conserved nucleotide sequences of Bm86 from 65 R. microplus isolates collected from 9 Indian states was sequenced and revealed 95.6–99.8% and 93.2–99.5% identity in nucleotides and amino acids sequences, respectively. The identities of nucleotides and deduced amino acids were 94.7–99.8% and 91.8–99.5%, respectively, between full-length sequence (orf) of the Bm86 gene of IVRI-I strain and published sequences of vaccine strains. Six nucleotides deletion were observed in Indian Bm86 sequences. Four B-cell epitopes (D519-K554, H563-Q587, C598-T606, T609-K623), which are present in the conserved region of the IVRI-I Bm86 sequence, were selected. The results confirm that the use of available commercial Bm86 vaccines is not a suitable option against Indian isolates of R. microplus. A country-specific multi-epitope Bm86 vaccine consisting of four specific B-cell epitopes along with candidate molecules, subolesin and tropomyosin in chimeric/co-immunization format may provide a sustainable option for implementation in an integrated tick management system.The authors are grateful to the Indian Council of Agricultural Research, New Delhi, for funding through the National Agricultural Science Fund (Grant numberNASF/ABA-6015/2016-17/357 and NFBSFARA/BSA-4004/2013-14 and also wish to thank The University Grant Commission (UGC) for providing Junior Research Fellowship (JRF). The APC is funded by the Indian Veterinary Research Institute.Peer reviewe

Genetic Diversity of <i>Rhipicephalus (Boophilus) microplus</i> for a Global Scenario: A Comprehensive Review

Rhipicephalus microplus poses a substantial threat to livestock health and agricultural economies worldwide. Its remarkable adaptability to diverse environments and hosts is a testament to its extensive genetic diversity. This review delves into the genetic diversity of R. microplus, employing three pivotal genetic markers: the cytochrome c oxidase I (COX1) gene, ribosomal genes, and microsatellites. The COX1 gene, a crucial tool for genetic characterization and phylogenetic clustering, provides insights into the adaptability of ticks. Ribosomal genes, such as internal transcribed spacer regions (ITS-1 and2) as well as 18S and 28S, are routinely utilized for species differentiation. However, their use is limited due to indels (insertions and deletions). Microsatellites and minisatellites, known for their high polymorphism, have been successfully employed to study populations and genetic diversity across various tick species. Despite their effectiveness, challenges such as null alleles and marker variations warrant careful consideration. Bm86, a well-studied vaccine candidate, exhibits substantial genetic diversity. This diversity directly influences vaccine efficacy, posing challenges for developing a universally effective Bm86-based vaccine. Moreover, the review emphasizes the prevalence of genes associated with synthetic pyrethroid resistance. Identifying single nucleotide polymorphisms in the acaricide-resistant genes of R. microplus has facilitated the development of molecular markers for detecting and monitoring resistance against synthetic pyrethroids. However, mutations in sodium channels, the target site for synthetic pyrethroid, correlate well with the resistance status of R. microplus, which is not the case with other acaricide target genes. This study underscores the importance of understanding genetic diversity in developing effective tick management strategies. The choice of genetic marker should be tailored based on the level of taxonomic resolution and the group of ticks under investigation. A holistic approach combining multiple markers and integrating additional molecular and morphological data may offer a more comprehensive understanding of tick diversity and relationships. This research has far-reaching implications in formulating breeding programs and the development of vaccine against ticks and tick-borne diseases (TTBDs) as well as strategies for the management of resistant ticks

Identification and characterization of vaccine candidates against Hyalomma anatolicum—Vector of Crimean‐Congo haemorrhagic fever virus

Crimean-Congo haemorrhagic fever (CCHF) is a tick borne viral disease reported from different parts of the world. The distribution of the CCHF cases are linked with the distribution of the principal vector, Hyalomma anatolicum in the ecosystem. Presently, vector control is mainly dependent on repeated application of acaricides, results in partial efficacy and generated acaricide resistant tick strains. Amongst the different components of integrated management programme, immunization of hosts is considered as one of the sustainable component. To restrict CCHF virus spreading, use of anti-Hyalomma vaccines appears as a viable solution. Accordingly, present study was under taken to characterize and evaluate vaccine potential of two conserved molecules, ferritin2 (FER2) and tropomyosin (TPM). Silencing of the genes conferred a cumulative reduction (rejection + unable to engorge) of 61.3% in FER2 and 70.2% in TPM respectively. Furthermore, 44.2% and 72.7% reduction in engorgement weight, 63.6% and 94.9% reduction in egg masses in FER2 and TPM silenced ticks in comparison to LUC-control group was recorded. The recombinant protein, rHaFER2 was characterized as 35 kDa protein with pI of 5.84 and possesses iron binding domains. While rHaTPM is a 51kDa protein with pI of 4.94 having calcium binding domains. Immunization of cross-bred calves by rHaFER2 conferred 51.7% and 51.2% protection against larvae and adults of H. anatolicum challenge infestations. While rHaTPM conferred 63.7% and 66.4% protection against larvae and adults infestations, respectively. The results were comparable with the data generated by RNAi and it clearly showed the possibility for the development of anti-hyalomma vaccine to manage CCHF virus and Theileria annulata infection in human and animals.The work is supported by the infrastructure developed through funding from National Agricultural Science Fund of ICAR (NFBSFARA/BSA‐4004/2013‐14). The senior author is highly thankful to the DST, Government of India for providing INSPIRE fellowship for doctoral programme and to publication committee of ICMR—NIRTH, Jabalpur (ICMR‐NIRTH/PSC/09/2018)