Sequence and functional variability of Toll-like receptor 9 gene in equines.

Significant structural differences in the extracellular domain of toll-like receptor 9 (TLR9) account for species-specific recognition of its ligand CpG-ODN sequences. TLR9 is extensively studied in human, mice and some domestic animals. The recognition ability appears to be utilized differently by various species and breeds, but so far no comprehensive study exists about the equine TLR9 gene. We characterized TLR9 sequences of Marwari and Zanskari breeds of horses and Poitu donkey. We sequenced and identified the protein coding regions of equine TLR9 and compared with other animals and human beings. Furthermore, we also analyzed the amino acid substitutions and their likely implications on functions. The analysis revealed 14% evolutionary divergence between equine and human TLR9, while it was 1% between the Equus caballus and Equus asinus and less than 1% within Equus caballus. In phylogenetic analysis of predicted amino acids, the indigenous equines grouped with thoroughbred Equus caballus, while human, cattle, dog, sheep, mice, and buffalo formed separate clades. Furthermore, we also analyzed the amino acid substitutions and their likely implications on functions by sorting intolerant from tolerant (SIFT) analysis and predicted two substitutions of amino acids (D80N and S822P) in Marwari horses in leucine rich repeat 1 (LRR1) without any functional effects. The substitutions (V214A and Y579C) in LRR 3 and LRR11 in Zanskari horses were predicted to have functional consequences. Out of overall 8 substitutions, three substitutions (I420V, S970R and R1001C) were found in Equus asinus in LRR7, LRR 13, and toll interleukin receptor (TIR) domains, while the substitution G649S is observed in Poitu donkey only. We report for the first time that despite the conserved residues, the striking effect of substitutions, found within the TLR9 genes of different equine breeds/species may have possible implications.

Sodium alginate and gum acacia hydrogels of zinc oxide nanoparticles reduce hemolytic and oxidative stress inflicted by zinc oxide nanoparticles on mammalian cells.

Zinc oxide nanoparticles are important nanomaterials currently under research due to their applicability in nanomedicine. Toxicity of ZnO NPs has been extensively studied and has been shown to affect various cell types and animal systems. In this study, we investigated hemolytic potential and oxidative stress inflicted by ZnO NPs and ZnO NPs-loaded-sodium alginate-gum acacia hydrogels on horse erythrocytes and African green monkey kidney (Vero) cells. Our study provides a better understanding of the hemolytic and oxidative effects of interaction of ZnO NPs and ZnO NPs released from polymeric hydrogels with the biological system. Remarkable aggregation of erythrocytes was noted in the higher concentration of ZnO NPs treated erythrocytes as compared to erythrocytes treated with ZnO NPs-loaded hydrogels. ZnO NPs-loaded hydrogels treated Vero cells significantly reduced oxidative stress as evidenced by less malondialdehyde production as compared to that of ZnO NPs treated cells. Normal horse erythrocytes when treated with ZnO NPs in in vitro condition undergo oxidative damage, and contribute in augmenting the toxicity. We demonstrated that polymeric ZnO NPs reduced the undesirable effects provoked by ZnO NPs on mammalian cells.

Sodium alginate and gum acacia hydrogels of ZnO nanoparticles show wound healing effect on fibroblast cells.

An ideal biomaterial for wound dressing applications should possess antibacterial and anti-inflammatory properties without any toxicity to the host cells while providing the maximum healing activity. Zinc oxide nanoparticles (ZnONPs) possess antimicrobial activity and enhance wound healing, but the questions regarding their safety arise before application to the biological systems. We synthesized ZnONPs-loaded-sodium alginate-gum acacia hydrogels (SAGA-ZnONPs) by cross linking hydroxyl groups of the polymers sodium alginate and gum acacia with the aldehyde group of gluteradehyde. Here, we report the wound healing properties of sodium alginate/gum acacia/ZnONPs, circumventing the toxicity of ZnONPs simultaneously. We demonstrated the concentration-dependent zones of inhibition in treated cultures of Pseudomonas aerigunosa and Bacillus cereus and biocompatability on peripheral blood mononuclear/fibroblast cells. SAGA-ZnONPs hydrogels showed a healing effect at a low concentration of ZnONPs using sheep fibroblast cells. Our findings suggest that high concentrations of ZnONPs were toxic to cells but SAGA-ZnONPs hydrogels significantly reduced the toxicity and preserved the beneficial antibacterial and healing effect.

Synthesis and evaluation of isometamidium-alginate nanoparticles on equine mononuclear and red blood cells.

Isometamidium hydrochloride (ISMM) is an effective drug for the treatment of trypanosomosis, but it causes local and systemic toxicity. Isometamidium hydrochloride has limited therapeutic index and exhibit considerable variation in their prophylactic activities. We developed a trypanocidal nanoformulation using ISMM and polymers sodium alginate/gum acacia to enhance the efficacy of the drug at lower doses, while minimizing undesirable side effects. It was characterized by transmission electron microscopy and infrared spectroscopy for evaluation of size, morphology, functional groups, etc. In vitro cytotoxicity studies were performed by metabolic resazurin assay at different concentrations of isometamidium-loaded alginate/gum acacia nanoparticles using equine peripheral blood mononuclear cells. Hemolytic assay revealed significantly less toxicity compared to the conventional drug. The results demonstrate that the developed drug delivery module can be evaluated in suitable animal models to evaluate its potency.

Diversity of interferon inducible Mx gene in horses and association of variations with susceptibility vis-à-vis resistance against equine influenza infection.

Equine influenza (EI) is primarily an infection of the upper respiratory tract and is one of the major infectious respiratory diseases of economic importance in equines. Re-emergence of the disease, species jumping by H3N8 virus in canines and possible threat of human pandemic due to the unpredictable nature of the virus have necessitated research on devising strategies for preventing the disease. The myxovirus resistance protein (Mx) has been reported to confer resistance to Orthomyxo virus infection by modifying cellular functions needed along the viral replication pathway. Polymorphisms and differential antiviral activities of Mx gene have been reported in pigs and chicken. Here we report the diversity of Mx gene, its expression in response to stimulation with interferon (IFN) α/ß and their association with EI resistance and susceptibility in Marwari horses. Blood samples were collected from horses declared positive for equine influenza and in contact animals with a history of no clinical signs. Mx gene was amplified by reverse transcription from total RNA isolated from peripheral blood mononuclear cells (PBMCs) stimulated with IFN α/ß using gene specific primers. The amplified gene products from representative samples were cloned and sequenced. Nucleotide sequences and deduced amino acid sequences were analyzed. Out of a total 24 amino acids substitutions sorting intolerant from tolerant (SIFT) analysis predicted 13 substitutions with functional consequences. Five substitutions (V67A, W123L, E346Y, N347Y, S689N) were observed only in resistant animals. Evolutionary distances based on nucleotide sequences with in equines ranged between 0.3-2.0% and 20-24% with other species. On phylogenetic analysis all equine sequences clustered together while other species formed separate clades.

Quinapyramine sulfate-loaded sodium alginate nanoparticles show enhanced trypanocidal activity.

AIM: To reduce the dose, toxic effects and to ensure sustained release of quinapyramine sulfate (QS), a highly effective drug against Trypanosoma evansi. MATERIALS & METHODS: QS-loaded sodium alginate nanoparticles (QS-NPs) were formed by emulsion-crosslinking technology using dioctyl-sodium-sulfosuccinate and sodium alginate. The formulation was characterized for size, stability, morphology and functional groups by a zetasizer, scanning electron microscopy, atomic force microscopy, transmission electron microscopy and Fourier transform infrared spectroscopy. In vitro safety and toxicity studies were performed by metabolic assay in Vero cell lines, and in vivo efficacy was evaluated in mice. RESULTS: QS-NPs were <60 nm with 96.48% entrapment efficiency and 3.70% drug loading. The formulation showed an initial burst effect followed by slow drug release in accordance with quasi-Fickian Higuchi diffusion mechanism. QS-NPs were much less toxic and able to clear the parasite at a much lower concentration than QS. CONCLUSION: The QS-NPs synthesized are safe, less toxic and highly effective compared with QS.

Globalization and Livestock Biosecurity.

Globalization has resulted in enhanced trade in livestock and livestock products leading to increased risk of diseases to livestock and human beings. The emergence of highly contagious viral diseases of livestock and poultry such as foot-and-mouth disease, peste des petits ruminants, African swine fever (ASF), Newcastle disease, avian influenza and zoonotic diseases caused by viruses like Ebola, West Nile, Nipah, Hendra and swine influenza (H1N1) have necessitated the formulation of policies and regulatory frameworks for preventing ingress of exotic diseases and controlling dissemination of endemic diseases within the country. Biosecurity measures are important to maintain and improve animal health and reduce the risks. In this review, the risks of introduction of infections through livestock and livestock products, routes of transmission and general biosecurity measures to reduce these risks have been discussed.

Immunotherapeutic potential of CpG oligodeoxynucleotides in veterinary species.

Innate immunity plays a critical role in host defense against infectious diseases by discriminating between self and infectious non-self. The recognition of infectious non-self involves germ-line encoded pattern recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs). The PAMPs are the components of pathogenic microbes which include not only the cell wall constituents but also the unmethylated 2'-deoxy-ribo-cytosine-phosphate-guanosine (CpG) motifs. These CpG motifs present within bacterial and viral DNA are recognized by toll-like receptor 9 (TLR9), and signaling by this receptor triggers a proinflammatory cytokine response which, in turn, influences both innate and adaptive immune responses. The activation of TLR9 with synthetic CpG oligodeoxynucleotides (ODNs) induces powerful Th1-like immune responses. It has been shown to provide protection against infectious diseases, allergy and cancer in laboratory animal models and some domestic animal species. With better understanding of the basic biology and immune mechanisms, it would be possible to exploit the potential of CpG motifs for animal welfare. The research developments in the area of CpG and TLR9 and the potential applications in animal health have been reviewed in this article.

A novel genomic constellation (G10P[3]) of group A rotavirus detected from buffalo calves in northern India.

Group A bovine rotaviruses cause gastroenteritis and calf mortality leading to significant economic losses to dairy farmers in India. Due to segmented nature of the RNA genome and wide host range, vast genetic and antigenic diversity exists among different isolates of rotavirus. Molecular characterization of locally prevalent group A rotavirus strains in buffalo population in north India was undertaken. Out of a total of 455 faecal samples, 21 samples (4.61%) were positive for bovine rota virus (BRV) as determined by PAGE and ELISA, whereas of these only 15 isolates yielded specific products for VP4 and VP7 genes by RT-PCR. Genotyping by nested PCR typed G6, G10 and P[11] genotypes but VP4 genes of 11 isolates remained untyped. The phylogenetic and evolutionary analysis of nucleotide and predicted amino acid sequences of the cloned products of VP4 and VP7 genes confirmed typing results obtained by nested PCR for G6, G10 and P[11] and classified the untyped isolates as P[3] genotypes. In this study, it was observed that G6P[11] (26.66%) and G10P[3] (73.34%) group A rotaviruses are circulating in buffalo herds of organized farms in north India. Unusual reassortants G10P[3] of group A rotaviruses isolated from buffalo calves show novel genomic constellations indicative of interspecies reassortment.