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.