Multiple point mutations in cytochrome b gene of Babesia gibsoni - A possible cause for buparvaquone resistance.

The development and rise in drug-resistant Babesia gibsoni strain is a serious concern among veterinary practitioners. Of several therapeutic strategies followed, buparvaquone-azithromycin combination therapy is widely used to treat small Babesia infections in Asia. The present study focused on buparvaquone-induced mutations in B. gibsoni by analyzing its cytochrome b gene sequence. Among the 12 dogs that were administered with buparvaquone-azithromycin combination therapy, 8 of them were unresponsive to drug-resistant B. gibsoni infection. Hematological parameters were recorded before and after 10 day treatment plan and an improvement in these values was observed. Eight samples with persistent parasitemia after 10 day treatment plan was subjected to cytochrome b gene amplification and analyzed for mutations. On analysis, out of the 25 mutations only 9 were non-synonymous in nature; T15N, S48T, P152L, V167I, A217T, F258Y, M311I, S336G, A337S. Mutation P152L was seen near to Q01 (130-148) binding region and F258Y within the drug binding region Q02 (244-266).

Acute insulin-responsive hyperglycemia and hypocalcemia in Theileria spp. infected goat.

Theileriosis can be manifested in appreciably variable clinical forms among domestic ruminants and may often become life-threatening. The present report narrates, the quick remarkable clinical recovery of a lactating goat infected with Theileria spp., exhibiting acute insulin-responsive hyperglycemia and hypocalcemia, by providing intensive therapy. A four year old doe was presented with the complaint of acute manifestation of weakness, ventroflexion of neck with flaccid muscles, recumbency, hypersalivation, severe abdominal breathing, anorexia and polyuria since last eighteen hours. The animal kidded three kids one month before, out of which one was mummified. Clinical examination revealed severe depression, dehydration, dyspnoea, congested mucous membrane, sluggish rumen motility and reduced pupillary light reflex. Laboratory investigation revealed severe granulocytopenia, thrombocytopenia, hypocalcemia, hyperglycemia and Theileria spp. infection. The animal showed significant improvement within a few minutes of initiating the evidence-based stabilization therapy to correct hydration status, cellular glucose uptake, calcium levels and Theileria spp. infection. This case indicates the significance of investigating the metabolic status of animals suffering from theileriosis for achieving better clinical responses. Also, future studies may focus on the endocrinological perspectives of metabolic impact of Theileria spp. infection in goats.

Molecular characterization of 5S ribosomal spacer sequences of Dirofilaria repens microfilariae in dogs in Kerala, India.

The present study reports the characteristics of the 5S rRNA spacer sequences of Dirofilaria repens microfilariae isolated from dogs. The 22 nucleotide long spliced leader 1 sequences located in the 5S rRNA spacer region are completely conserved in all nematodes. There is variation in the spliced leader 1 sequences and associated sites in the 5S rRNA spacer region of D. repens. Absence of canonical SL 1 sequences distinguishes D. repens from other filarial species.

Evaluation of oxidative stress in caprine anaplasmosis and effect of vitamin E-selenium in monitoring oxidative stress.

Caprine anaplasmosis is an economically important tick-borne rickettsial disease that affects goats all over the world. Microscopic examination of stained blood smears from 162 animals revealed inclusion bodies of Anaplasma spp. in 24 cases. Genus specific PCR for Anaplasma spp. yielded positive results in 22 cases. All the diseased animals showed a significant fall in the mean values of antioxidants such as reduced glutathione, superoxide dismutase and catalase and a significant increase in the level of lipid peroxidation. Out of the 22 animals positive for anaplasmosis both in blood smear and PCR, 16 female non pregnant goats selected for study were divided into two groups consisting of eight animals each. Animals belonging to groups I and II were treated with oxytetracycline dihydrate. In addition, animals of group II were supplemented with vitamin E-selenium combination. Oxidative stress parameters were rechecked on the 10th day of treatment. At the end of the study period, a significant reduction in malondialdehyde level and a significant increase in mean value of superoxide dismutase were detected in group II. While there was a significant reduction in lipid peroxidation and a significant increase in superoxide dismutase and catalase values within both the groups after treatment, reduced glutathione showed no significant difference within the group.

Canine Brugia malayi microfilarial excretory/secretory protein-based antibody assay for the diagnosis of brugian filariasis in dogs.

Brugian filariasis is reported in dogs in Kerala, India. Antibody detection kits are not available worldwide, for detection of Brugian filariasis in dogs. A study was carried out to develop Indirect plate ELISA using excretory secretory antigen isolated from canine brugian microfilariae and compare the sensitivity and specificity with that of blood smear examination. Identification of microfilariae was done by acid phosphatase staining using Naphthol AS-TR method and Polymerase Chain Reaction for Hha 1 repeat sequence. The microfilariae were identified as Brugia malayi. Isolation of brugian microfilariae from canine blood was done by gradient centrifugation method. The isolated microfilariae were maintained in RPMI-1640 media. The pooled media was then concentrated to obtain excretory secretory protein (ESP). This ESP was used to develop Indirect ELISA. The sensitivity and specificity of the plate ELISA developed was 84 and 100 per cent respectively when compared with blood smear examination. This is the first report of successful isolation of ESP from Brugia malayi microfilariae from dogs and standardization of plate ELISA using the antigen.

Comparison of efficacy of ivermectin and diethylcarbamazine against naturally infected Brugia malayi microfilaria in dogs.

Filarial parasites like Brugia pahangi and Brugia malayi can infect dogs. Adults of Brugia genus resides in the lymphatic system and microfilariae, in blood. There are increasing reports of detection of B. malayi microfilariae in dogs. A study was undertaken to compare the efficacy of repeated oral dosing of ivermectin (IVT) and diethylcarbamazine (DEC), individually and in combination against naturally infected B. malayi microfilariae in dogs. The species of the microfilariae was confirmed by acid phosphatase staining and polymerase chain reaction. The three treatment groups were 200 mcg/kg body weight IVT daily for 14 days (I), 6.6 mg/kg body weight DEC daily for 14 days (II) and IVT and DEC together in the same dose for a period of 5 days (III). Microfilarial status of the peripheral blood was assessed on the 0th, 7th, 14th and 21st day. Haematological parameters were measured on day zero and on the 21st day. Though, all the three treatment groups showed a reduction in the microfilarial concentration through the study period, complete absence of detectable microfilaremia was not noticed in any of the three groups by 21st day. Among the haematological parameters, statistically significant difference was observed in the post-treatment means of haemoglobin levels of group III when compared with group II. Since group III regime (IVT + DEC) was shorter and just as effective as the longer ones, it is considered superior to the other two.

Diagnosis of paratuberculosis in goats by cell mediated immune response, conventional and molecular diagnostic techniques.

In the present study efficacy of single intradermal Johnin test, acid fast staining of faecal smear and IS 900 faecal polymerase chain reaction tests was evaluated in 200 goats for detection of Mycobacterium avium subsp paratuberculosis. Two hundred goats comprising 150 goats from an organised farm in Trichur district and 50 goats reared under field condition at farmers premise from Malappuram district of Kerala state formed the study population. Faecal smear from all the 200 goats was stained by Ziehl-Neelsen acid fast stain and faecal polymerase chain reaction (PCR) specific for M. avium subsp paratuberculosis (MAP); IS 900 was performed on all samples. All the animals were subjected to single intradermal Johnin test. Out of 200 goats screened for paratuberculosis, six goats (3%), 11 goats (5.5%) and 42 goats (21%) were found positive by Ziehl-Neelsen acid fast staining of faecal smear, single intradermal Johnin test and IS 900 PCR respectively. Results of the present study indicate that amplification of IS 900 insertion element was the most specific and sensitive diagnostic detection method. Single intradermal Johnin test and Ziehl-Neelsen acid fast staining did not show any significant difference.

Detection of human filarial parasite Brugia malayi in dogs by histochemical staining and molecular techniques.

Human filariasis caused by Brugia malayi is still a public health problem in many countries of Asia including India, Indonesia, Malaysia and Thailand. The World Health Organization (WHO) has targeted to eliminate filariasis by the year 2020 by Mass annual single dose Diethylcarbamazine Administration (MDA). Results of the MDA programme after the first phase was less satisfactory than expected. Malayan filariasis caused by B. malayi is endemic in the south of Thailand where domestic cat serves as the major reservoir host. There is no report about the occurrence of B. malayi in dogs. The present work was carried out to find out the incidence of microfilariasis in dogs and also to detect the presence of human filarial infection in dogs, if any. One hundred dogs above 6 months of age presented to the veterinary college Hospital, Mannuthy, Kerala, with clinical signs suggestive of microfilariasis - fever, anorexia, conjunctivitis, limb and scrotal oedema - were screened for microfilariae by wet film examination. Positive cases were subjected to Giemsa staining, histochemical staining and molecular techniques. Results of the study showed that 80% of dogs had microfilariasis; out of which 20% had sheathed microfilaria. Giemsa and histochemical staining character, PCR and sequencing confirmed it as B. malayi. High prevalence of B. malayi in dogs in this study emphasized the possible role of dogs in transmission of human filariasis.

Phytase supplemented poultry diets affect soluble phosphorus and nitrogen in manure and manure-amended soil.

Understanding P and N dynamics in manure-amended soil is essential for estimating the environmental impact of manure utilization in land applications. A laboratory incubation study was conducted to assess, (i) the effect of feeding a standard Australian commercial diet, and diets modified with phytase supplementation and reduced nonphytase phosphorus (NPP), on the concentrations of P and N (total and soluble) in the manure derived from layer hens (Gallus domesticus L.), and (ii) the change in water-soluble phoshorus (P(WSP)) and mineral N (NH(4)-N and NO(3)-N) when used as a soil amendment, applied at rates equivalent to 200 kg ha(-1) (200N) and 400 kg ha(-1) (400N). Phytase supplementation increased %P(WSP) by 8 to 12% in the manures, regardless of the levels of NPP in the diets, and in the manure-amended soils by 27 to 30% at the 200N application rate, and up to 54% at the 400N rate. Phytase significantly (P < 0.05) reduced total nitrogen (TN) content (by 12-31%) of the manures but generally produced greater nitrate accumulation in the manure-amended soils. Net nitrification, which commenced 4 wk after incubation, was accompanied by a simultaneous decrease in soil pH (by one pH unit) and a concomitant decline in %P(WSP). The decline in %P(WSP) was primarily attributed to P retention by the soil as it became more acidic. This study suggests that phytase addition not only reduces manure total N content, and increases water-soluble P, but its effects on manure total phosphorus (TP) and 2 mol L(-1) KCl extractable mineral N is influenced by the NPP level in the diet.

In vitro and in vivo profile of 5-[(4'-trifluoromethyl-biphenyl-2-carbonyl)-amino]-1H-indole-2-carboxylic acid benzylmethyl carbamoylamide (dirlotapide), a novel potent MTP inhibitor for obesity.

The synthesis of a novel gut selective MTP inhibitor, 5-[(4'-trifluoromethyl-biphenyl-2-carbonyl)-amino]-1H-indole-2-carboxylic acid benzylmethyl carbamoylamide (dirlotapide), and its in vitro and in vivo profile are described. Dirlotapide (3) demonstrated excellent potency against MTP enzyme in HepG2 cells and canine hepatocytes. This novel MTP inhibitor also showed excellent efficacy when tested in a canine food intake model.

Activation of nuclear transcription factor-kappa B is associated with the induction of inhibitory kappa B kinase-beta and involves differential activation of protein kinase C and protein tyrosine kinases during fatal murine cerebral malaria.

The levels of nuclear transcription factor-kappa B (NF-kappaB) subunits p65 and p50 and its associated kinase, inhibitory kappa B kinase (IKK) alpha and beta were monitored in cytosolic and nuclear fraction of mice cerebral cortex and cerebellum using an experimental model of fatal murine cerebral malaria (FMCM). Since protein kinase C (PKC) and protein tyrosine kinases (PTK) are known to collaborately regulate the NF-kappaB activation, we also studied the activity of these two kinases in cytosol and membrane fraction. In parallel, the levels of two PKC isoforms (alpha and delta) and tyrosine phosphorylated proteins were monitored to correlate the observed changes in the activity. Our results underscore the involvement of IKK-beta as an essential mediator of NF-kappaB activation as evinced by the nuclear translocation of p65 and p50 during CM pathology. Additional findings confirm altered activity and levels of PKC and enhanced activation of PTK and tyrosine phosphorylation of proteins during CM pathology. These signaling intricacies involving an interplay between rel family (NF-kappaB) of transcription factors, PKC and PTK may serve as an important cue in understanding the possible continuation of the post receptor signaling events associated with tumor necrosis factor-alpha induction during FMCM pathology.

Bacterially expressed and refolded receptor binding domain of Plasmodium falciparum EBA-175 elicits invasion inhibitory antibodies.

Malaria parasites make specific receptor-ligand interactions to invade erythrocytes. A 175 kDa Plasmodium falciparum erythrocyte binding antigen (EBA-175) binds sialic acid residues on glycophorin A during invasion of human erythrocytes. The receptor-binding domain of EBA-175 lies in a conserved, amino-terminal, cysteine-rich region, region F2 of EBA-175 (PfF2), that is homologous to the binding domains of other erythrocyte binding proteins such as Plasmodium vivax Duffy binding protein. We have developed methods to produce recombinant PfF2 in its functional form. Recombinant PfF2 was expressed in Escherichia coli, purified from inclusion bodies, renatured by oxidative refolding and purified to homogeneity by ion-exchange and gel filtration chromatography. Refolded PfF2 has been characterized using biochemical and biophysical methods and shown to be pure, homogenous and functional in that it binds human erythrocytes with specificity. Immunization with refolded PfF2 yields high titre antibodies that efficiently inhibit P. falciparum invasion of erythrocytes in vitro. Importantly, antibodies raised against PfF2 block invasion by a P. falciparum field isolate that invades erythrocytes using multiple pathways. These observations support the development of recombinant PfF2 as a vaccine candidate for P. falciparum malaria.