Duplex real-time PCR methods for molecular detection and characterization of canine tick-borne haemoparasites from Punjab state, India.

BACKGROUND: Microscopy is a routinely used technique for the diagnosis of canine tick-borne haemoparasitic diseases in various clinical laboratories worldwide. In an attempt to provide better diagnostic assay to the clients for effective management of these diseases duplex real-time PCR assays were applied. METHODS AND RESULTS: Blood samples (n = 338) aseptically collected from suspected dogs of Central Plain Zone of Punjab state, India were subjected to SYBR Green based real-time duplex PCR assays for simultaneous detection of B. vogeli & E. canis and B. gibsoni & H. canis. Results revealed an overall prevalence rate of canine tick-borne haemoparasites as 54.1%, amongst which H. canis was the predominant (25.4%), followed by B. gibsoni (16.3%), E. canis (10.7%) and B. vogeli (1.8%). Sensitivity and specificity of the duplex assays ranged from 59.04 to 100.0% and 58.12 to 92.52%, respectively and their strength of agreement was ″fair″ with kappa value statistics. A significant (p < 0.05) association between prevalence of B. gibsoni, H. canis and E. canis infection with risk factors like sex, breed, season and location was recorded. The ancestral background of the field isolates of haemoparasites was also studied by phylogenetic analysis of their nucleotide sequences. CONCLUSIONS: SYBR Green dye based duplex real-time PCR assays proved to be highly sensitive, specific, rapid and affordable diagnostic tests for use by clinicians to save the life of pets.

Comparative View of Lung Vascular Endothelium of Cattle, Horses, and Water Buffalo.

Endothelium plays an important role in maintaining the vascular barrier and physiological homeostasis. Endothelium also is fundamental to the initiation and regulation of inflammation. Endothelium demonstrates phenotypic and functional heterogeneity not only among various organs but also within an organ. One of the striking examples would be the pulmonary endothelium that participates in creating blood-air barrier. Endothelium in large pulmonary blood vessels is distinct in structure and function from that lining of the pulmonary capillaries. This chapter focuses on the comparative aspects of pulmonary endothelium and highlight unique differences such as the presence of pulmonary intravascular macrophages among select species.

Pulmonary innate inflammatory responses to agricultural occupational contaminants.

Agricultural workers are exposed to many contaminants and suffer from respiratory and other symptoms. Dusts, gases, microbial products and pesticide residues from farms have been linked to effects on the health of agricultural workers. Growing sets of data from in vitro and in vivo models demonstrate the role of the innate immune system, especially Toll-like receptor 4 (TLR4) and TLR9, in lung inflammation induced following exposure to contaminants in agricultural environments. Interestingly, inflammation and lung function changes appear to be discordant indicating the complexity of inflammatory responses to exposures. Whereas the recent development of rodent models and exposure systems have yielded valuable data, we need new systems to examine the combined effects of multiple contaminants in order to increase our understanding of farm-exposure-induced negative health effects.

A composite polyphenol-rich extract improved growth performance, ruminal fermentation and immunity, while decreasing methanogenesis and excretion of nitrogen and phosphorus in growing buffaloes.

The effects of a composite polyphenolic-rich extract (CPRE) on ruminal fermentation, nutrient utilisation, growth performance, excretion of nitrogen and phosphorus and methane emission were studied in growing buffaloes. Four herbal dry extracts prepared from Acacia arabica (babul; bark), Acacia catechu (cutch; bark), Punica granatum (pomegranate; peel) and Eugenia jambolana (Indian blackberry; seeds) were mixed in an equal proportion (1:1:1:1) to prepare the CPRE that contained mainly phenolic compounds (146 g/kg), flavonoids (41.7 g/kg) and saponins (40.5 g/kg). First, in vitro tests were performed for ruminal fermentation and feed degradability using ruminal fluid as inocula and CPRE at 0 to 40 g/kg substrate to decide an optimal dose of CPRE for an in vivo study on buffaloes. In the animal study, 20 buffaloes were randomly assigned to two groups (n = 10)-a control diet and a CPRE diet (control diet added with extra 20 g/kg of CPRE). The in vitro tests suggested that addition of CPRE at 20 g/kg substrate increased degradability of substrate, short-chain fatty acid concentration and propionate proportion, and reduced methane production, acetate proportion, acetate:propionate ratio and ammonia concentration in fermentation media, which were also noted in the rumen of buffaloes. Feeding CRPE to buffaloes did not affect feed intake, but increased daily body weight gain, dry matter and crude protein digestibility and nitrogen and phosphorus retention in the body. Total bacteria, methanogens and protozoal numbers were similar between two groups, but Fibrobacter succinogenes increased in the rumen of buffaloes fed CPRE. Concentrations of total, essential, non-essential and glucogenic amino acids were greater in the plasma of CPRE-fed buffaloes. Cell-mediated immune response improved in the CPRE-fed buffaloes compared with the control group. Estimated methane production and excretion of nitrogen and phosphorus per unit of body weight gain decreased in the CPRE group. The comprehensive results of this study clearly suggested that the composite polyphenol-rich feed additive at 20 g/kg diet improved growth performance, ruminal fermentation, immunity and plasma amino acids profile, whereas it reduced indicators of environmental impacts of buffalo production.

Fipronil induces lung inflammation in vivo and cell death in vitro.

BACKGROUND: Fipronil is an insecticide that acts at the gamma-aminobutyric acid receptor and glutamate-gated chloride channels in the central nervous systems of target organisms. The use of fipronil is increasing across the globe. Presently, very little data exist on the potential impact of exposure to fipronil on the lungs. METHODS: We studied effects of intranasal (N = 8) and oral (N = 8) treatment with fipronil (10 mg/kg) on lungs of mice. Control mice were given groundnut oil orally (N = 7) or ethanol intranasally (N = 7) as these were the vehicles for respective treatments. RESULTS: Hematoxylin-eosin stained lung sections showed normal histology in the control lungs compared to the thickened alveolar septa, disruption of the airways epithelium and damage to vascular endothelium in the intranasal and the oral groups. Mice exposed to fipronil either orally or intranasally showed increased von Willebrand factor staining in the endothelium and septal capillaries. Compared to the control mice, TLR4 expression in airway epithelium was increased in mice treated intranasally but not orally with fipronil. Oral fipronil reduced TLR9 staining in the airway epithelium but intranasal exposure caused intense staining in the alveolar septa and airway epithelium. There were higher numbers of TLR4 positive cells in alveolar septa in lungs of mice treated intranasally (P = 0.010) compared to the respective control and orally treated mice but no significant differences between treatments for TLR9 positive stained cells (P = 0.226). The U937 macrophage cells exposed to fipronil at concentrations of 0.29 µm to 5.72 µm/ml over 3- or 24-hour showed significant increase in cell death at higher concentrations of fipronil (P < 0.0001). Western blots revealed no effect of fipronil on TLR4 (P = 0.49) or TLR9 (P = 0.94) expression on macrophage cell line. CONCLUSION: While both oral or intranasal fipronil treatments induced signs of lung inflammation, the number TLR4-positive septal cells was increased only following intranasal treatment. Fipronil causes macrophage cell death without altering TLR4 and TLR9 expression in vitro.

Indoxacarb interaction alters immunotoxic and genotoxic potential of endotoxin.

Indoxacarb is commonly used to effectively control pests, cockroaches, termites, fleas, and houseflies. Although the toxicological profile of indoxacarb had already been well characterized, we examined the possible toxicological interaction with indoxacarb and endotoxin. Male Swiss albino mice aged 8-10 weeks were orally administered indoxacarb dissolved in groundnut oil at 4 mg/kg/day and 2 mg/kg/day for 90 days. On day 91, five animals from each group were challenged with lipopolysaccharides (LPS) at 80 µg/mouse, administered intranasally. Indoxacarb at 4 mg/kg significantly decreased Total leukocyte count, lymphocytopenia, and neutrophilia. Both doses of indoxacarb combined with LPS resulted in significant lymphocytopenia. Indoxacarb did not produce DNA damage in comet assay, but when combined with LPS, it resulted in a significant increase in tail length, tail moment, and olive moment. The data indicate that indoxacarb at 4 mg/kg administered orally for 90 days induced immune-response change. Further, both doses of indoxacarb, when combined with LPS, accelerate immunotoxicity and endotoxin-induced DNA damage.