Effect of Excess Fluoride Exposure on Radiographic and Histopathological Changes in Long Bones of Rabbit.

The present study aimed to investigate the effects of excess fluoride exposure on long bones in young rabbits (Oryctolagus cuniculus). New Zealand White rabbits (n = 30) were randomly divided into five equal groups and were provided drinking water containing 0, 50, 100, 200, and 400 µg added fluoride per ml ad lib for a period of 90 days. Blood samples were collected on days 0, 45, and 90 of the experiment, and femur samples were collected for fluoride estimation on day 90 after radiography of long bone before sacrifice. Study revealed significant increase in serum fluoride concentration following oral intake of excess fluoride. Alterations in activities of alkaline phosphatase, aspartate transaminase, alanine transaminase, and concentrations of creatinine and urea nitrogen in blood plasma were also recorded in animals receiving excess fluoride, though changes revealed inconsistent pattern. Radiographic changes in long bones in fluoride exposed rabbits included widening of metaphysis, thinning of cortical region, and a variety of osteopenic changes like osteoporosis and osteomalacia that were more prominent in animals receiving 200 ppm or more than 200 ppm fluoride in drinking water. Important changes in histomorphology of growth plate in long bones recorded in excess fluoride (> 100 ppm) exposed rabbits included irregular thickening of epiphyseal growth plate with haphazard orientation of chondrocytes forming nodular protrusion into metaphysis. Fluoride exposure induced both osteogenesis and osteoporosis to a degree varying with dose of fluoride exposure.

Toxic effect of sodium fluoride on hydroxyproline level and expression of collagen-1 gene in rat bone and its amelioration by Tamrindus indica L. fruit pulp extract.

Excessive fluoride intoxication plays an important role in the development of dental, skeletal and non-skeletal fluorosis. The aim of this study was to ascertain the toxic effect of excessive fluoride ingestion on the level of hydroxyproline and expression of type 1 collagen gene in rat bone and its amelioration by supplementation with Tamarindus indica fruit pulp extract. Forty albino rats were randomly assigned to four groups. The first group served as control and received only tap water. The second group received sodium fluoride (200 ppm) through drinking water. The third group received T. indica fruit pulp extract (200 mg/kg body weight) alone and the fourth group received the T. indica fruit pulp extract (200 mg/kg body weight) along with fluorinated drinking water (200 ppm) daily by gavage for a period of 90 days. The level of hydroxyproline and expression of type 1 collagen gene using quantitative real time PCR in the tibia bone decreased significantly with continuous exposure to sodium fluoride. Co-administration of T. indica fruit pulp extract during exposure to fluoride through drinking water restored the level of calcium, phosphorus and alkaline phosphatase in serum and the concentration of hydroxyproline in urine. It increased the level of hydroxyproline and expression of type 1 collagen gene in the tibia as compared to untreated fluoride-exposed rats. It is concluded that T. indica fruit pulp extract has an ameliorative potential to protect the bone from fluoride induced collagen damage.

Psoroptes cuniculi induced oxidative imbalance in rabbits and its alleviation by using vitamins A, D3, E, and H as adjunctive remedial.

The oxidant/antioxidant balance of rabbits naturally infected with Psoroptes cuniculi and treated with ivermectin +/- vitamins A, D(3), E, and H supplementation was investigated. Two groups of seven mixed ♂ and ♀, 6-to-8 month-old New Zealand White rabbits, diagnosed Psoroptes mites-positive by skin scraping examination and seven clinically healthy control rabbits were examined. Blood samples were obtained on day 0 and at 28 days post-therapy to determine oxidative stress indices. On day 0, the levels of lipid peroxides were significantly higher (P ≤ 0.01) in the Psoroptes-infected rabbits compared with the healthy controls while those of reduced glutathione and the activities of the antioxidant enzymes glutathione peroxidase, glutathione-S-transferase, catalase, and superoxide dismutase were significantly lower (P ≤ 0.01). Vitamin supplementation of the ivermectin-treated rabbits revealed both faster clinical (14 days) and parasitological (10 days) recovery. It was concluded that significant alteration of oxidant/antioxidant balance is a factor in the pathogenesis of P. cuniculi infestation of rabbits, and recovery can be enhanced by combining ivermectin treatment with vitamin A, D(3,) E, and H supplementation.

Detecting diclofenac in livestock carcasses in India with an ELISA: a tool to prevent widespread vulture poisoning.

Diclofenac, a non-steroidal anti-inflammatory drug (NSAID), has caused catastrophic vulture declines across the Indian sub-continent. Here, an indirect ELISA is used to detect and quantify diclofenac in 1251 liver samples from livestock carcasses collected across India between August 2007 and June 2008, one to two years after a ban on diclofenac manufacture and distribution for veterinary use was implemented. The ELISAs applicability was authenticated with independent data obtained using LC-ESI/MS. Of 1251 samples, 1150 (91.9%) were negative for diclofenac using both methods, and 60 (4.8%) were positive at 10-4348 and 10-4441 µg kg(-1) when analysed by ELISA and LC-ESI/MS, respectively. The residue level relationship in the 60 positive samples was highly significant (p < 0.001, r(2) = 0.644). Data suggest that this immunological assay could be used not only for cost effective sample screening, but also for residue level semi-quantification.

The role of apoptosis in immunosuppression of dogs with demodicosis.

The aim of the present study was to evaluate the status of apoptosis in peripheral blood leukocytes of dogs with demodicosis. A total of 26 dogs suffering from demodicosis, and positive for Demodex canis mites by skin scraping, participated in the study, 13 with localized demodicosis (LD) and 13 with generalized demodicosis (GD). A further 13 clinically healthy dogs, all of whom were negative for mites upon skin scraping, were used as controls. The dogs with GD revealed significantly higher (P ≤ 0.0001) percentage of leukocytes with externalization of phosphatidylserine (PS) and depolarized mitochondrial membrane potentials (ΔΨm) as compared with the dogs with LD and healthy controls. These dogs also revealed significantly lower values (P ≤ 0.0001) of hematological parameters viz. hemoglobin, total erythrocytes count total leukocytes count, lymphocytes, monocytes and neutrophils. Significantly higher (P ≤ 0.0001) percentages of leukocytes with externalization of PS and depolarized ΔΨm were also found in dogs with LD as compared with the healthy controls. These dogs also revealed significantly lower values of Hb (P ≤ 0.0001), TEC (P=0.025), TLC (P ≤ 0.0001), lymphocytes (P=0.008), monocytes (P ≤ 0.0001) and neutrophils (P=0.03). It is concluded that premature apoptosis of PBL may be implicated in the immunosuppression of the dogs with demodicosis.

Effectiveness of action in India to reduce exposure of Gyps vultures to the toxic veterinary drug diclofenac.

Contamination of their carrion food supply with the non-steroidal anti-inflammatory drug diclofenac has caused rapid population declines across the Indian subcontinent of three species of Gyps vultures endemic to South Asia. The governments of India, Pakistan and Nepal took action in 2006 to prevent the veterinary use of diclofenac on domesticated livestock, the route by which contamination occurs. We analyse data from three surveys of the prevalence and concentration of diclofenac residues in carcasses of domesticated ungulates in India, carried out before and after the implementation of a ban on veterinary use. There was little change in the prevalence and concentration of diclofenac between a survey before the ban and one conducted soon after its implementation, with the percentage of carcasses containing diclofenac in these surveys estimated at 10.8 and 10.7%, respectively. However, both the prevalence and concentration of diclofenac had fallen markedly 7-31 months after the implementation of the ban, with the true prevalence in this third survey estimated at 6.5%. Modelling of the impact of this reduction in diclofenac on the expected rate of decline of the oriental white-backed vulture (Gyps bengalensis) in India indicates that the decline rate has decreased to 40% of the rate before the ban, but is still likely to be rapid (about 18% year(-1)). Hence, further efforts to remove diclofenac from vulture food are still needed if the future recovery or successful reintroduction of vultures is to be feasible.

Interferon Alpha Characterization and Its Comparative Expression in PBM Cells of Capra hircus and Antelope cervicapra Cultured in the Presence of TLR9 Agonist.

TLR9 plays pivotal role in innate immune responses through upregulation of costimulatory molecules and induction of proinflammatory cytokines like type I interferons including interferon alpha (IFNA). The present study characterized IFNA cDNA and predicted protein sequences in goat and black buck. Response of the PBM cells to TLR9 agonist CpG ODN C and Phorbol Myristate Acetate (PMA) was evaluated by realtime PCR. IFNA coding sequences were amplified from leukocyte cDNA and cloned in pGEMT-easy vector for nucleotide sequencing. Sequence analysis revealed 570 bp, IFNA ORF encoding 189 amino acids in goat and black buck. Black buck and goat IFNA has 92.1% to 94.7% and 93% to 95.6% similarity at nucleotide level, 86.3% to 89.5% and 70.9% to 91.6% identity at amino acid level with other ruminants, respectively. Nonsynonymous substitutions exceeding synonymous substitutions indicated IFNA evolved through positive selection among ruminants. In spite of lower total leukocyte count, the innate immune cells like monocytes and neutrophils were more in black buck compared to goat. In addition, CpG ODN C-stimulated PBM cells revealed raised IFNA transcript in black buck than goat. These findings indicate sturdy genetically governed immune system in wild antelope black buck compared to domestic ruminant goat.

Sequence characterization and polymerase chain reaction-restriction fragment length polymorphism of the mitochondrial DNA 12S rRNA gene provides a method for species identification of Indian deer.

Characterization of species-specific molecular markers and development of a method for identification of Indian deer species is necessary to monitor illegal trade of parts and products for better conservation and management of the endangered species. In this investigation, we characterized the 12S rRNA gene sequence for differentiation of Indian deer species and developed a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)-based method for their identification. Universal primers were used for the amplification of the mitochondrial 12S rRNA gene from genomic DNA of chital or spotted deer, hog deer, barking deer, sika deer, musk deer and sambar. PCR products of chital, hog deer and Himalayan musk deer were cloned and sequenced for the first time. Among the Indian deer species, more than 90% similarity was observed in the mitochondrial 12S rRNA gene. The sequences of the above deer species were restriction mapped with the help of Lasergene (DNAstar Inc., Madison, WI, USA). PCR amplicon of these deer species were subjected to restriction digestion with Rsa1, Dde1, Bsr1 and BstSF1 endonucleases that showed a species-specific RFLP pattern. This technique provides a reliable and efficient tool for identification of deer species using a variety of biomaterials.

Molecular characterization of coding sequences and analysis of Toll-like receptor 3 mRNA expression in water buffalo (Bubalus bubalis) and nilgai (Boselaphus tragocamelus).

Toll-like receptor 3 (TLR3), an antiviral innate immunity receptor recognizes double-stranded RNA, preferably of viral origin and induces type I interferon production, which causes maturation of phagocytes and subsequent release of chemical mediators from phagocytes against some viral infections. The present study has characterized TLR3 complementary DNA (cDNA) in buffalo (Bubalus bubalis) and nilgai (Boselaphus tragocamelus). TLR3 coding sequences of both buffalo and nilgai were amplified from cultured dendritic cell cDNA and cloned in pGEMT-easy vector for characterization by restriction endonucleases and nucleotide sequencing. Sequence analysis reveals that 2,715-bp-long TLR3 open reading frame encoding 904 amino acids in buffalo as well as nilgai is similar to that of cattle. Buffalo TLR3 has 98.6 and 97.9% identity at nucleotide level with nilgai and cattle, respectively. Likewise, buffalo TLR3 amino acids share 96.7% identity with cattle and 97.8% with nilgai. Non-synonymous substitutions exceeding synonymous substitutions indicate evolution of this receptor through positive selection among these three ruminant species. Buffalo and nilgai appear to have diverged from a common ancestor in phylogenetic analysis. Predicted protein structures of buffalo and nilgai TLR3 from deduced amino acid sequences indicate that the buffalo and nilgai TLR3 ectodomain may be more efficient in ligand binding than that of cattle. Furthermore, TLR3 messenger RNA expression in tissues as quantified by real-time PCR was found higher in nilgai than buffalo.

Removing the threat of diclofenac to critically endangered Asian vultures.

Veterinary use of the nonsteroidal anti-inflammatory (NSAID) drug diclofenac in South Asia has resulted in the collapse of populations of three vulture species of the genus Gyps to the most severe category of global extinction risk. Vultures are exposed to diclofenac when scavenging on livestock treated with the drug shortly before death. Diclofenac causes kidney damage, increased serum uric acid concentrations, visceral gout, and death. Concern about this issue led the Indian Government to announce its intention to ban the veterinary use of diclofenac by September 2005. Implementation of a ban is still in progress late in 2005, and to facilitate this we sought potential alternative NSAIDs by obtaining information from captive bird collections worldwide. We found that the NSAID meloxicam had been administered to 35 captive Gyps vultures with no apparent ill effects. We then undertook a phased programme of safety testing of meloxicam on the African white-backed vulture Gyps africanus, which we had previously established to be as susceptible to diclofenac poisoning as the endangered Asian Gyps vultures. We estimated the likely maximum level of exposure (MLE) of wild vultures and dosed birds by gavage (oral administration) with increasing quantities of the drug until the likely MLE was exceeded in a sample of 40 G. africanus. Subsequently, six G. africanus were fed tissues from cattle which had been treated with a higher than standard veterinary course of meloxicam prior to death. In the final phase, ten Asian vultures of two of the endangered species (Gyps bengalensis, Gyps indicus) were dosed with meloxicam by gavage; five of them at more than the likely MLE dosage. All meloxicam-treated birds survived all treatments, and none suffered any obvious clinical effects. Serum uric acid concentrations remained within the normal limits throughout, and were significantly lower than those from birds treated with diclofenac in other studies. We conclude that meloxicam is of low toxicity to Gyps vultures and that its use in place of diclofenac would reduce vulture mortality substantially in the Indian subcontinent. Meloxicam is already available for veterinary use in India.

Molecular cloning and evolution of the gene encoding the precursor of Nilgai (Boselaphus tragocamelus) interleukin 2.

Interleukin 2 is a key immunoregulatory cytokine that is involved in T helper differentiation and is known to induce interferon gamma (IFN-gamma) in natural killer (NK) and T-cell. The gene for interleukin-2 (IL-2) was amplified from cDNA pool prepared from ConAstimulated peripheral blood mononuclear cells (PBM cells) isolated from Nilgai reared in semicaptivity. The amplified IL-2 gene was cloned and nucleotide sequences were determined. The sequencing result showed that Nilgai IL-2 cDNA contains 468 bp long ORF encoding 155 amino acids (Genbank Accession No DQ017831). Homology comparison of nucleotide revealed that Nilgai IL-2 sequence is evolutionary more related to buffalo than cattle IL-2 sequence.