Cloning, expression and purification of Eimeria maxima gametocyte antigen-EmGam56 for control of poultry coccidiosis.

Poultry coccidiosis is an important devitalizing enteric protozoan disease caused by a group of obligatory intracellular apicomplexan parasites of the Genus Eimeria contributing to major economic loss in commercial poultry worldwide. As the current method of chemotherapeutic control using ionophores in feed had led to development of drug resistant isolates, the need for development of prophylactic vaccines is the most viable alternate and eco-friendly control strategy as on date. Of the several candidate vaccines, the EmGam 56 is one of the most promising candidates which protect the birds against E. maxima, E. tenella and E. acervulina, the three most pathogenic coccidian species infecting commercial chicken. EmGam56 is a major wall forming component of macrogametocyte of E. maxima and a candidate with high immunogenicity and low virulence. The present study was planned and carried out for the generation of E.coli expressed recombinant gametocyte antigen-EmGam56 using pET 28(a+) as cloning vector and BL21 DE3 (pLysS) as prokaryotic expression system in a Bio-fermentor (New Brunswick™ Scientific BioFlo 310). The recombinant protein was purified by conventional (Ammonium sulphate precipitation) and by automatic purification system (AKTA prime) in Ni-NTA column for a planned immunization trial with experimental chickens.

Perception of Ticks and Tick-Borne Diseases Worldwide.

In this comprehensive review study, we addressed the challenge posed by ticks and tick-borne diseases (TBDs) with growing incidence affecting human and animal health worldwide. Data and perspectives were collected from different countries and regions worldwide, including America, Europe, Africa, Asia, and Oceania. The results updated the current situation with ticks and TBD and how it is perceived by society with information bias and gaps. The study reinforces the importance of multidisciplinary and international collaborations to advance in the surveillance, communication and proposed future directions to address these challenges.

In vitro efficacy of plumbagin and thymol against Theileria annulata.

Phytochemical compounds, plumbagin and thymol were evaluated for their efficacy against Theileria annulata using MTT cell viability assay. Plumbagin and thymol were found to be effective in preventing the proliferation of Theileria annulata infected bovine lymphocytes. The IC50 values of plumbagin and thymol were 0.019 µM and 0.009 µM, respectively. Plumbagin and thymol were found to be non-cytotoxic to the bovine peripheral blood mononuclear cells. However, both the compounds were found to have inhibitory effect on vero cell proliferation. Plumbagin had primarily anti-theilerial activity but thymol had primarily anti-mitotic activity. The in vitro efficacy and cell toxicity studies indicate the potential application of plumbagin, purified from Plumbago indica as a lead therapeutic molecule against T. annulata infection in cattle. Supplementary Information: The online version contains supplementary material available at 10.1007/s12639-022-01550-x.

Assessment of consistency of minimum post-mortem intervals estimated by thermal summation-based methods in medico-legal cases associated with blowflies.

BACKGROUND: This study is based on blow fly samples collected from 8 medico-legal cases in Tamil Nadu, India. The fly life stages were identified and the consistency of minimum post-mortem intervals (PMImin) estimated by different thermal summation-based methods was assessed. METHODS: PMImin of 8 medico-legal cases was estimated using six different thermal summation constants and lower developmental temperatures that are based on C. megacephala and C. rufifacies developmental data. Limits of agreement (LoA), intra class correlation coefficient (ICC) between PMImin values and margin of error of mean of difference between PMImin values were calculated. RESULTS: Intra-class correlation between the PMImin values estimated using different thermal summation constants based on C. megacephala ranged between 0.89 and 0.98 and coefficient of determination ranged between 0.93 and 0.98. Intra-class correlation between the PMImin values estimated using different thermal summation constants based on C. rufifacies ranged between 0.91 and 0.99 and coefficient of determination ranged between 0.95 and 0.99. The mean difference of PMImin values estimated using different thermal summation methods based on C. megacephala ranged between 1.8 hr and 6.6hr and margin of error ranged between 2.51 and 6.93hr. The mean difference of PMImin values estimated using different thermal summation methods based on C. rufifacies ranged between 3.33 and 31.33hr and margin of error ranged between 4.66 and 32hr. CONCLUSION: Consistency of PMImin values estimated by different thermal summation methods was good to excellent. Thermal summation constants useful in estimation of PMImin with lowest mean difference and margin of error were described.

Site-specific delivery of green tea coated aluminium magnesium silicate beads and studies on their effect against chicken coccidiosis.

In this report, the site-specific co-delivery of green tea/aluminium magnesium silicate (AMS) was reported and the specific target delivery was achieved orally. The new co-precipitation process was developed to synthesis the green tea/AMS hybrid complex and using energy-dispersive X-ray spectroscopy, Fourier-transform infrared spectroscopy and Raman confirmed its successful synthesis. The blood biocompatibility of the green tea/AMS was tested using chicken blood, and the compound is safe up to 500 mg/ml. After mixed with hydroxypropyl methylcellulose phthalate, the oral beads were synthesised using a linking agent. The oral beads underwent different pH-based dissolution studies and the results indicated that the beads specifically dissolved in gastric pH (6.5). The pharmaco kinetic studies were performed to estimate the delivery kinetics. The results revealed that the beads underwent as per the Higuchi model. The anticoccidial effects of the beads were tested using chicken. The animal studies were performed in two different modes such as prophylactic treatment and active treatment after Eimeria species challenge. The results indicated that the prophylactic treatment with beads 100% protected the chicken and the active treatment with beads after the Eimeria challenge significantly protected against the intestinal damage and it also enhanced the anticoccidial effect.

A transgenic male-only strain of the New World screwworm for an improved control program using the sterile insect technique.

BACKGROUND: The New World screwworm, Cochliomyia hominivorax, is a devastating pest of livestock endemic to subtropical and tropical regions of the Western hemisphere. The larvae of this species feed on the tissue of living animals, including man, and can cause death if untreated. Over 60 years ago, the sterile insect technique (SIT) was developed with the aim of eradicating this pest, initially from Florida but subsequently from all of North and Central America. From the outset it was appreciated that SIT would be more efficient if only sterile males were released in the field, but this was not possible until now. RESULTS: Here, we report on the development and evaluation of the first sexing strains of C. hominivorax that produce only males when raised on diet without tetracycline. Transgenic lines have been developed that possess a tetracycline repressible female-lethal genetic system. Ten of these lines show high female lethality at the late larval/pupal stages and three of them present dominant female lethality. Most of the lines were comparable to the wild type parental strain in several fitness parameters that are relevant to mass rearing in a production facility. Further, three lines performed well in male mating success and male competition assays, suggesting they would be sexually competitive in the field. Consequently, one transgenic line has been selected by the New World Screwworm Program for evaluation under mass rearing conditions. CONCLUSIONS: We conclude that the promising characteristics of the selected sexing strains may contribute to reduce production costs for the existing eradication program and provide more efficient population suppression, which should make a genetic control program more economical in regions were C. hominivorax remains endemic.

Use of electroporation as an option to transform the horn fly, Haematobia irritans: a species recalcitrant to microinjection.

The horn fly, Haematobia irritans, is a serious pest of cattle in North America. The control of horn flies has primarily relied on insecticides. However, the heavy use of insecticides has led to the development of insecticide resistance in horn flies. Novel methods to control horn flies are greatly needed. Transgenic technology is an effective tool to genetically modify insects and may lead to novel methods of pest control based on genomic approaches. Here we report a piggyBac-mediated transformation of the horn fly via electroporation. Transformation with a DsRed fluorescent marker protein coding region was verified by PCR analysis of individual fly bodies and pupal cases and sequencing of PCR products. However, Southern blot analysis failed to indicate the DsRed gene was integrated into the horn fly genome. Thus, the electroporation protocol may have caused the DsRed gene to be integrated into bacterial symbionts of the horn fly.

Post-integration silencing of piggyBac transposable elements in Aedes aegypti.

The piggyBac transposon, originating in the genome of the Lepidoptera Trichoplusia ni, has a broad host range, making it useful for the development of a number of transposon-based functional genomic technologies including gene vectors, enhancer-, gene- and protein-traps. While capable of being used as a vector for the creation of transgenic insects and insect cell lines, piggyBac has very limited mobility once integrated into the genome of the yellow fever mosquito, Aedes aegypti. A transgenic Aedes aegypti cell line (AagPB8) was created containing three integrated piggyBac elements and the remobilization potential of the elements was tested. The integrated piggyBac elements in AagPB8 were transpositionally silent in the presence of functional transposase, which was shown to be capable of catalyzing the movement of plasmid-borne piggyBac elements in the same cells. The structural integrity of one of the integrated elements along with the quality of element-flanking DNA, which is known to influence transposition rates, were tested in D. melanogaster. The element was found to be structurally intact, capable of transposition and excision in the soma and germ-line of Drosophila melanogaster, and in a DNA sequence context highly conducive to element movement in Drosophila melanogaster. These data show that transpositional silencing of integrated piggyBac elements in the genome of Aedes aegypti appears to be a function of higher scale genome organization or perhaps epigenetic factors, and not due to structural defects or suboptimal integration sites.

Pyrosequencing-based analysis of the microbiome associated with the horn fly, Haematobia irritans.

The horn fly, Haematobia irritans, is one of the most economically important pests of cattle. Insecticides have been a major element of horn fly management programs. Growing concerns with insecticide resistance, insecticide residues on farm products, and non-availability of new generation insecticides, are serious issues for the livestock industry. Alternative horn fly control methods offer the promise to decrease the use of insecticides and reduce the amount of insecticide residues on livestock products and give an impetus to the organic livestock farming segment. The horn fly, an obligatory blood feeder, requires the help of microflora to supply additional nutrients and metabolize the blood meal. Recent advancements in DNA sequencing methodologies enable researchers to examine the microflora diversity independent of culture methods. We used the bacterial 16S tag-encoded FLX-titanium amplicon pyrosequencing (bTEFAP) method to carry out the classification analysis of bacterial flora in adult female and male horn flies and horn fly eggs. The bTEFAP method identified 16S rDNA sequences in our samples which allowed the identification of various prokaryotic taxa associated with the life stage examined. This is the first comprehensive report of bacterial flora associated with the horn fly using a culture-independent method. Several rumen, environmental, symbiotic and pathogenic bacteria associated with the horn fly were identified and quantified. This is the first report of the presence of Wolbachia in horn flies of USA origin and is the first report of the presence of Rikenella in an obligatory blood feeding insect.

Intrinsic characteristics of neighboring DNA modulate transposable element activity in Drosophila melanogaster.

Identifying factors influencing transposable element activity is essential for understanding how these elements impact genomes and their evolution as well as for fully exploiting them as functional genomics tools and gene-therapy vectors. Using a genetics-based approach, the influence of genomic position on piggyBac mobility in Drosophila melanogaster was assessed while controlling for element structure, genetic background, and transposase concentration. The mobility of piggyBac elements varied over more than two orders of magnitude solely as a result of their locations within the genome. The influence of genomic position on element activities was independent of factors resulting in position-dependent transgene expression ("position effects"). Elements could be relocated to new genomic locations without altering their activity if ≥ 500 bp of genomic DNA originally flanking the element was also relocated. Local intrinsic factors within the neighboring DNA that determined the activity of piggyBac elements were portable not only within the genome but also when elements were moved to plasmids. The predicted bendability of the first 50 bp flanking the 5' and 3' termini of piggyBac elements could account for 60% of the variance in position-dependent activity observed among elements. These results are significant because positional influences on transposable element activities will impact patterns of accumulation of elements within genomes. Manipulating and controlling the local sequence context of piggyBac elements could be a powerful, novel way of optimizing gene vector activity.