Genome editing technologies, mechanisms and improved production of therapeutic phytochemicals: Opportunities and prospects.

Plants produce a large number of secondary metabolites, known as phytometabolites that may be employed as medicines, dyes, poisons, and insecticides in the field of medicine, agriculture, and industrial use, respectively. The rise of genome management approaches has promised a factual revolution in genetic engineering. Targeted genome editing in living entities permits the understanding of the biological systems very clearly, and also sanctions to address a wide-ranging objective in the direction of improving features of plant and their yields. The last few years have introduced a number of unique genome editing systems, including transcription activator-like effector nucleases, zinc finger nucleases, and miRNA-regulated clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9). Genome editing systems have helped in the transformation of metabolic engineering, allowing researchers to modify biosynthetic pathways of different secondary metabolites. Given the growing relevance of editing genomes in plant research, the exciting novel methods are briefly reviewed in this chapter. Also, this chapter highlights recent discoveries on the CRISPR-based modification of natural products in different medicinal plants.

Quantitative ethnoveterinary study on plant resource utilization by indigenous communities in high-altitude regions.

For millennia, ethnic knowledge has been intricately tied to local biodiversity and woven into the fabric of rural communities. Growing scientific evidence suggests that merging ethnic knowledge with new scientific findings can lead to socially acceptable and environmentally friendly approaches essential for the long-term prosperity of local communities. In the high-altitude region, where livestock raising is a key income source, and plant-based utilization for ethno-veterinary practices is widely practiced. In this context, this study was conducted with the aim of documenting the ethno-veterinary use of plant resources in different bio-geographical regions of Jammu and Kashmir's Himalayas (J & KH). Semi-structured interviews and group discussions were used to collect information. Principal component analysis (PCA) and Pearson correlation were conducted to analyze the data. We documented 148 species from 53 families that locals used for various purposes: medicine, fodder, tonic, antidote, magic, and also used to protect themselves from ectoparasite such as Pediculus humanus capitis by the local inhabitants. There were significant differences in the relative usage of plant resources across the three biogeographic regions. Comparatively, the highest number (41%) of plant species were used for ethnoveterinary in the Jammu region, while the lowest number (28%) of species were used in Kashmir. Across the regions, Kashmir and Jammu had the highest level of species similarity (17%), while Jammu and Ladakh had the lowest (1%). A cross-regional assessment of plant resources revealed that 18% of plants were shared among the regions. The reported use of Amaranthus blitum, Morus alba, Ficus palmata, Vitex negundo, Juniperus semiglobosa, Ulmus wallichiana, and Rumex nepalensis are novel for the ethno-veterinary uses of this part of the Himalayan region. The various dry unique traditional fodder preparations (gaaslov, gass khor, pan baath, kaandbaath, Lovgooad, Karb, and Phungma) from plant resources are reported for the first time from the Himalayan region and can be ascribed to the novelty of this study. Plant resources were not only a source of fodder and medicine but also presented themselves as an opportunity for livelihood generation. Therefore, our findings bridge the knowledge gap by documenting key ethnoveterinary applications of native plant species from the study region that are used to cure livestock diseases and disorders by the mountain inhabitants.

Control of tick infestations and pathogen prevalence in cattle and sheep farms vaccinated with the recombinant Subolesin-Major Surface Protein 1a chimeric antigen.

BACKGROUND: Despite the use of chemical acaricides, tick infestations continue to affect animal health and production worldwide. Tick vaccines have been proposed as a cost-effective and environmentally friendly alternative for tick control. Vaccination with the candidate tick protective antigen, Subolesin (SUB), has been shown experimentally to be effective in controlling vector infestations and pathogen infection. Furthermore, Escherichia coli membranes containing the chimeric antigen composed of SUB fused to Anaplasma marginale Major Surface Protein 1a (MSP1a) (SUB-MSP1a) were produced using a simple low-cost process and proved to be effective for the control of cattle tick, Rhipicephalus (Boophilus) microplus and R. annulatus infestations in pen trials. In this research, field trials were conducted to characterize the effect of vaccination with SUB-MSP1a on tick infestations and the prevalence of tick-borne pathogens in a randomized controlled prospective study. METHODS: Two cattle and two sheep farms with similar geographical locations and production characteristics were randomly assigned to control and vaccinated groups. Ticks were collected, counted, weighed and classified and the prevalence of tick-borne pathogens at the DNA and serological levels were followed for one year prior to and 9 months after vaccination. RESULTS: Both cattle and sheep developed antibodies against SUB in response to vaccination. The main effect of the vaccine in cattle was the 8-fold reduction in the percent of infested animals while vaccination in sheep reduced tick infestations by 63%. Female tick weight was 32-55% lower in ticks collected from both vaccinated cattle and sheep when compared to controls. The seroprevalence of Babesia bigemina was lower by 30% in vaccinated cattle, suggesting a possible role for the vaccine in decreasing the prevalence of this tick-borne pathogen. The effect of the vaccine in reducing the frequency of one A. marginale msp4 genotype probably reflected the reduction in the prevalence of a tick-transmitted strain as a result of the reduction in the percent of tick-infested cattle. CONCLUSIONS: These data provide evidence of the dual effect of a SUB-based vaccine for controlling tick infestations and pathogen infection/transmission and provide additional support for the use of the SUB-MSP1a vaccine for tick control in cattle and sheep.

Expression of immunoregulatory genes and its relationship to lead exposure and lead-mediated oxidative stress in wild ungulates from an abandoned mining area.

Lead (Pb) is a highly toxic metal that can induce oxidative stress and affect the immune system by modifying the expression of immunomodulator-related genes. The aim of the present study was to investigate the association between Pb exposure and the transcriptional profiles of some cytokines, as well as the relationship between Pb exposure and changes in oxidative stress biomarkers observed in the spleen of wild ungulates exposed to mining pollution. Red deer and wild boar from the mining area studied had higher spleen, liver, and bone Pb levels than controls, indicating a chronic exposure to Pb pollution. Such exposure caused a depletion of spleen glutathione levels in both species and disrupted the activity of antioxidant enzymes, suggesting the generation of oxidative stress conditions. Deer from the mining area also showed an induced T-helper (Th )-dependent immune response toward the Th 2 pathway, whereas boar from the mining area showed a cytokine profile suggesting an inclination of the immune response toward the Th 1 pathway. These results indicate that environmental exposure to Pb may alter immune responses in wild ungulates exposed to mining pollution. However, evidence of direct relationships between Pb-mediated oxidative stress and the changes detected in immune responses were not found. Further research is needed to evaluate the immunotoxic potential of Pb pollution, also considering the prevalence of chronic infectious diseases in wildlife in environments affected by mining activities.

Gene expression profile suggests that pigs (Sus scrofa) are susceptible to Anaplasma phagocytophilum but control infection.

BACKGROUND: Anaplasma phagocytophilum infects a wide variety of hosts and causes granulocytic anaplasmosis in humans, horses and dogs and tick-borne fever in ruminants. Infection with A. phagocytophilum results in the modification of host gene expression and immune response. The objective of this research was to characterize gene expression in pigs (Sus scrofa) naturally and experimentally infected with A. phagocytophilum trying to identify mechanisms that help to explain low infection prevalence in this species. RESULTS: For gene expression analysis in naturally infected pigs, microarray hybridization was used. The expression of differentially expressed immune response genes was analyzed by real-time RT-PCR in naturally and experimentally infected pigs. Results suggested that A. phagocytophilum infection affected cytoskeleton rearrangement and increased both innate and adaptive immune responses by up regulation of interleukin 1 receptor accessory protein-like 1 (IL1RAPL1), T-cell receptor alpha chain (TCR-alpha), thrombospondin 4 (TSP-4) and Gap junction protein alpha 1 (GJA1) genes. Higher serum levels of IL-1 beta, IL-8 and TNF-alpha in infected pigs when compared to controls supported data obtained at the mRNA level. CONCLUSIONS: These results suggested that pigs are susceptible to A. phagocytophilum but control infection, particularly through activation of innate immune responses, phagocytosis and autophagy. This fact may account for the low infection prevalence detected in pigs in some regions and thus their low or no impact as a reservoir host for this pathogen. These results advanced our understanding of the molecular mechanisms at the host-pathogen interface and suggested a role for newly reported genes in the protection of pigs against A. phagocytophilum.

Vaccination with BM86, subolesin and akirin protective antigens for the control of tick infestations in white tailed deer and red deer.

Red deer (Cervus elaphus) and white-tailed deer (Odocoileus virginianus) are hosts for different tick species and tick-borne pathogens and play a role in tick dispersal and maintenance in some regions. These factors stress the importance of controlling tick infestations in deer and several methods such as culling and acaricide treatment have been used. Tick vaccines are a cost-effective alternative for tick control that reduced cattle tick infestations and tick-borne pathogens prevalence while reducing the use of acaricides. Our hypothesis is that vaccination with vector protective antigens can be used for the control of tick infestations in deer. Herein, three experiments were conducted to characterize (1) the antibody response in red deer immunized with recombinant BM86, the antigen included in commercial tick vaccines, (2) the antibody response and control of cattle tick infestations in white-tailed deer immunized with recombinant BM86 or tick subolesin (SUB) and experimentally infested with Rhipicephalus (Boophilus) microplus, and (3) the antibody response and control of Hyalomma spp. and Rhipicephalus spp. field tick infestations in red deer immunized with mosquito akirin (AKR), the SUB ortholog and candidate protective antigen against different tick species and other ectoparasites. The results showed that deer produced an antibody response that correlated with the reduction in tick infestations and was similar to other hosts vaccinated previously with these antigens. The overall vaccine efficacy was similar between BM86 (E=76%) and SUB (E=83%) for the control of R. microplus infestations in white-tailed deer. The field trial in red deer showed a 25-33% (18-40% when only infested deer were considered) reduction in tick infestations, 14-20 weeks after the first immunization. These results demonstrated that vaccination with vector protective antigens could be used as an alternative method for the control of tick infestations in deer to reduce tick populations and dispersal in regions where deer are relevant hosts for these ectoparasites.

Targeting arthropod subolesin/akirin for the development of a universal vaccine for control of vector infestations and pathogen transmission.

Diseases caused by arthropod-borne pathogens greatly impact on human and animal health. Recent research has provided evidence that tick protective antigens can be used for development of vaccines with the dual target of controlling arthropod infestations and reducing their vector capacity for pathogens. As reviewed herein, protective antigens such as subolesin/akirin, which are highly conserved across vector species, show promise for use in development of a universal vaccine for the control of arthropod infestations and the reduction of pathogen transmission. However, further research is needed in critical areas towards achieving this goal.

Expression of recombinant Rhipicephalus (Boophilus) microplus, R. annulatus and R. decoloratus Bm86 orthologs as secreted proteins in Pichia pastoris.

BACKGROUND: Rhipicephalus (Boophilus) spp. ticks economically impact on cattle production in Africa and other tropical and subtropical regions of the world. Tick vaccines constitute a cost-effective and environmentally friendly alternative to tick control. The R. microplus Bm86 protective antigen has been produced by recombinant DNA technology and shown to protect cattle against tick infestations. RESULTS: In this study, the genes for Bm86 (R. microplus), Ba86 (R. annulatus) and Bd86 (R. decoloratus) were cloned and characterized from African or Asian tick strains and the recombinant proteins were secreted and purified from P. pastoris. The secretion of recombinant Bm86 ortholog proteins in P. pastoris allowed for a simple purification process rendering a final product with high recovery (35-42%) and purity (80-85%) and likely to result in a more reproducible conformation closely resembling the native protein. Rabbit immunization experiments with recombinant proteins showed immune cross-reactivity between Bm86 ortholog proteins. CONCLUSION: These experiments support the development and testing of vaccines containing recombinant Bm86, Ba86 and Bd86 secreted in P. pastoris for the control of tick infestations in Africa.

Mucosal immunization of sheep with a Maedi-Visna virus (MVV) env DNA vaccine protects against early MVV productive infection.

Gene gun mucosal DNA immunization of sheep with a plasmid expressing the env gene of Maedi-Visna virus (MVV) was used to examine the protection against MVV infection in sheep from a naturally infected flock. For immunization, sheep were primed with a pcDNA plasmid (pcDNA-env) encoding the Env glycoproteins of MVV and boosted with combined pcDNA-env and pCR3.1-IFN-gamma plasmid inoculations. The pcDNA plasmid used in the control group contained the lacZ coding sequences instead of the env gene. Within a month post-challenge, the viral load in the vaccinated group was lower (p < or = 0.05) and virus was only detected transiently compared with the control group. Furthermore, 2 months later, neutralizing antibodies (NtAb) were detected in all the control animals and none of the vaccinated animals (p < or = 0.01). These results demonstrated a significant early protective effect of this immunization strategy against MVV infection that restricts the virus replication following challenge in the absence of NtAb production. This vaccine protective effect against MVV infection disappeared after two years post-challenge, when active replication of MVV challenge strain was observed. Protection conferred by the vaccine could not be explained by OLA DRB1 allele or genotype differences. Most of the individuals were DRB1 heterozygous and none was totally resistant to infection.

Molecular cloning and mRNA tissue-expression of two isoforms of the ovine costimulatory molecule CD80 (B7-1).

Using RT-PCR and rapid amplification of cDNA ends (RACE), we cloned two putative alternatively spliced transcripts of the sheep CD80 (B7-1) molecule that encode both transmembrane (TM) and secreted (s) forms of CD80 protein. Comparison of the amino acid sequence of the TM form of ovine CD80 with the sequence of cattle, swine and human CD80 indicated that the deduced protein had a higher degree of similarity to cattle (87% of amino acid identity) than to pig (68%) and human sequence (53% of homology). In tissues, RT-PCR using primers for the TM and the sCD80 transcripts indicated that the expression of both CD80 transcripts was almost exclusively expressed in the hematolymphoid system, with the exception of the uterus. The sCD80 transcript was expressed in peripheral blood mononuclear cells (PBMC), uterus and lymph node, whereas the TM-CD80 transcript was very weakly detected only in PBMC cells. Our result indicates that mRNA transcripts encoding both membrane-bound and secreted CD80 proteins are expressed in sheep like in other animals. However, in contrast with the CD80 molecules from other species, the secreted form of sheep CD80 seems to be the predominant form expressed in the ovine PBMC and other tissues, suggesting that the TM-CD80 represents a rare transcript in this species. Interestingly, the expression of both forms of the CD80 molecule was not affected by treatment of sheep PBMC with Concanavalin A (ConA), as detected by RT-PCR. This is the first report describing the identification of a B7 costimulatory transcript in sheep.

Molecular cloning and structural analysis of the porcine homologue to CD97 antigen.

CD97 is a member of a novel subfamily of leukocyte proteins that are characterized by the presence of tandemly repeated extracellular epidermal growth factor (EGF)-like domains and a seven-span transmembrane region, known as EGF-TM7. We here report the cloning of cDNA encoding the pig homologue of CD97. A pig CD97 specific probe was generated by PCR amplification of pig leukocyte cDNA, using primers based on consensus regions among the known sequences of mouse and human CD97. Screening of a pig aorta smooth muscle cDNA library identified one clone containing an open reading frame (ORF) that encoded an 18 amino acid putative signal peptide, a 141 amino acid sequence consisting of three EGF domains, a mucin-like spacer region of 276 amino acid, containing a G-protein coupling motif of 52 amino acids, followed by a 250 amino acid region containing seven membrane spanning domains and a 47 amino acid cytoplasmic tail. The amino acid sequence of the clone was 75, 67 and 59% homologous to cattle, human and mouse CD97 antigen, respectively. Therefore, it was termed pig CD97. Pig CD97 antigen shares many structural features with human, cattle and mouse CD97. RT-PCR analysis of cDNA from different pig cells and tissues showed that CD97 was highly expressed in leukocytes and lymph node cells. This is the first report describing the identification of a member of the EGF-TM7 family in the pig.