Prevalence of gastro-intestinal parasites in buffalo calves from different agro-climatic zones of Punjab.

Faecal samples from 1,582 buffalo calves of up to 6 months of age from 13 districts falling under four major agro-climatic zones of Punjab state, India were examined for gastrointestinal (GI) parasites for a period of one year (May 2008 to April 2009). The results revealed prevalence of GI parasitic infections as 73.58 per cent calves and Eimeria sp. (54.55 %) was the most prevalent GI parasite. Significant differences (P < 0.01) were found in overall prevalence of Toxocara vitulorum, strongyles and Strongyloides papillosus in buffalo calves of all four major agro-climatic zones of Punjab. Highest prevalence of T. vitulorum was recorded in undulating plain region (12.43 %) while lowest infection was recorded from western region (3.55 %). Further, the highest prevalence of strongyle infection was recorded in western region (32.26 %) and lowest in undulating plain region (19.46 %) thus showing a negative correlation in prevalence of T. vitulorum and strongyles. Highest and lowest infection of S. papillosus was recorded from central plain region (32.33 %) and western plain region (20.86 %), respectively whereas, the prevalence of Eimeria sp. and Moniezia expansa was comparable in all four major agro-climatic zones. The variation in prevalence recorded in the different agro-climatic zones is due to the climatic variation and thus the data generated could be of immense help in formulation of effective strategies for GI parasite control in different agro-climatic zones.

Comparison of nested PCR and microscopy for the detection of cryptosporidiosis in bovine calves.

The comparative efficacies of different conventional parasitological methods and nested PCR for diagnosis of bovine cryptosporidiosis in faecal samples were evaluated. Among the 100 samples collected from calves in and around Ludhiana Direct faecal smear staining technique revealed 25.0 % positivity for the oocysts of Cryptosporidium spp. with sensitivity and specificity of 68.12 and 92.98 %, respectively. Zinc sulphate solution floatation and saturated sugar solution floatation staining techniques showed sensitivity and specificity of 83.92 and 96.36; 81.03 and 98.14 %, respectively. Products of the primary PCR of Cryptosporidium spp. directed against small subunit (18S) ribosomal RNA when employed as template in nested PCR produced the amplicons of desired size (834 bp) in 47.0 % of the samples. Amplification of 834 bp fragment was also observed in positive control, while no amplification was observed in negative control. Results indicated PCR assays as highly sensitive and specific techniques for the screening of the samples for Cryptosporidium spp. but in developing countries and under field conditions where limited resources do not allow the application of PCR assays, concentration staining methods are recommended.

Coprological investigation on neonatal bovine cryptosporidiosis in Ludhiana, Punjab.

An overall prevalence of 38.90 % was recorded for bovine cryptosporidiosis on coprological examination of 144 faecal samples collected from neonatal cattle calves from organised dairy farms in and around Ludhiana, Punjab by modified Ziehl-Neelsen staining. Further, a gradual decline in the percent prevalence was seen with increase in the age of the host from <1 month (64.1 %) to 4-5 months (12.5 %). The highest prevalence was recorded during the monsoon season (47.06 %) followed by summer (37.73 %) and winter (30.0 %) season and the seasonal variation was statistically significant (p ≤ 0.05). Female calves showed higher prevalence (44.32 %) than their male counterparts (27.66 %). Further, prevalence of cryptosporidiosis was significantly higher (p ≤ 0.05) in the diarrhoeic calves (52.70 %) as compared to the non-diarrhoeic (24.28 %) thus indicating a relatively higher risk (1.75 times) of the disease in diarrhoeic than normal calves.

Comparative evaluation of real time PCR assay with conventional parasitological techniques for diagnosis of Trypanosoma evansi in cattle and buffaloes.

For comparative evaluation, a real time PCR assay was standardized by using TaqMan primer and probe targeting the internal transcribed spacer 1 (ITS-1) region of rRNA for Trypanosoma evansi and sensitivity was evaluated by using DNA, extracted from diethyleamino ethane cellulose purified trypanosomes and trypanosomes infected whole blood of mice. The minimum detection limit for purified trypanosomal DNA was 0.01 ng (≈ 0.33 genomic DNA of T. evansi) whereas for whole blood the minimum detection limit was 0.1 ng (≈ 6.12 genomic DNA). T. evansi infected mice blood samples were collected at different interval post infection and were analysed by conventional parasitological methods (CPT) viz. wet blood smear, thin blood smear, thick blood smear, quantitative buffy coat and real time PCR and found that TaqMan assay was two fold sensitive than CPT in case of in vivo infectivity in mice and gave positive signal at 36 h post infection where as QBC and blood smear examination was able to detect at 60 h and 72 h post infection respectively. A total 109 (80 cattle and 29 buffaloes) blood samples were collected from in and around Ludhiana district and analysed by CPT and real time PCR. The overall prevalence of T. evansi by CPT in cattle and buffaloes was 2.75 per cent. The prevalence rate was 2.5 per cent in cattle and 3.45 per cent in buffaloes. By real time PCR overall prevalence was 12.84 per cent in cattle and buffaloes, with a prevalence rate of 12.50 per cent in cattle and 13.79 per cent in buffaloes.

Pathological studies on experimental Trypanosoma evansi infection in Swiss albino mice.

The pathology of Trypanosoma evansi infection was studied in Swiss albino mice using cattle isolate of the parasite. Sixteen Swiss albino mice were used in the experiment and were divided into two groups viz. infected group (I) and uninfected healthy control group (II) comprising 12 and four mice, respectively. Twelve mice from group I were infected with 1 × 10(5) purified trypanosomes. Systematic necropsy examination specifically of the infected mice (group I) as well as of healthy control (group II) was performed and pathological changes were recorded. The different tissue samples were collected in 10 % neutral buffered formal saline and were used to study the histopathological changes. Gross post-mortem examination revealed enlargement of spleen, petechial haemorrhages in liver in the terminal stages of disease. Tissue sections revealed presence of numerous trypanosomes in blood vessels of liver, spleen, brain and kidneys. Microscopically, liver revealed lesions varying from vacuolar degeneration, coagulative necrosis along with congestion and haemorrhages. Spleen showed extensive haemorrhages in red pulp area, haemosiderosis and aggregation of histiocytes resulting in multinuclear giant cell formation. Lungs revealed oedema, congestion and mild inflammatory changes. Brain revealed mild degenerative changes along with congestion of meningeal blood vessels. Kidneys showed tubular degeneration, congestion and cellular infiltration. Heart revealed mild degenerative changes along with interstitial oedema. All changes were consistent with trypanosome infection and were confirmed by presence of trypanosomes in most of the tissue sections examined.

Drug combination therapy in control of cryptosporidiosis in Ludhiana district of Punjab.

The present report describes outbreak of cryptosporidiosis in neonatal cross bred cattle calves ageing 1-2 months in an organized dairy farm. The protozoan infection was confirmed by identifying bright red oocysts of Cryptosporidium spp. in the faecal samples after staining with modified acid Fast Zeihl-Neelsen stain. Metronidazole and furazolidone combination was able to induce clinically and parasitological recovery. This is believed to be the first report on the successful use of this drug combination against cryptosporidiosis.

Serodetection of Ehrlichia canis infection in dogs from Ludhiana district of Punjab, India.

Dot-ELISA (enzyme-linked immunosorbent assay) Immunocomb(®) assay was conducted to detect the presence of antibodies against Ehrlichia canis in blood samples of 60 privately owned dogs suspected to be infected with E. canis from the Small Animal Clinics, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab (India). Antibodies reactive to E. canis were detected in plasma in 48 samples out of 60 samples by Immunocomb(®) Dot-ELISA. Out of these 39.58% samples were low positive (Titre 1:20-1:40), 31.25% were medium positive (Titre 1:80-1:640) and 29.16% were high positive (Titre >1,280), for the infection. When examined by microscopy, only two samples revealed typical E. canis morulae. Haemato-cellular examination revealed thrombocytopenia along with anaemia and leucopenia. Results suggest that E. canis infection circulates in dogs in India in low non-detectable numbers by microscopy and is transmitted by the brown dog tick Rhipicephalus sanguineus.

Parasitic zoonoses in India: an overview.

Parasitic zoonotic diseases are prevalent throughout India at varying rates. First reports of zoonotic parasites and new emerging diseases have been recorded in both the human and animal populations in recent decades. The prevalence of zoonotic parasites is likely to be an underestimate, owing to the lack of proper surveillance and the shortage of information about the existence of asymptomatic animal carriers. Emergence of diseases such as human echinococcosis/hydatidosis, neurocysticercosis, cryptosporidiosis and toxoplasmosis in those with acquired immune deficiency syndrome, together with the re-emergence of cutaneous leishmaniosis, poses a serious threat in India and the prevention and control of these parasitic zoonoses, and others, is a great challenge.

Immunoaffinity chromatographic analysis for purification of specific diagnostic antigens of Paramphistomum epiclitum.

Polypeptide profile of somatic antigen of Paramphistomum epiclitum (PSAg) and Gastrothylax crumenifer (GSAg) was studied by SDS-PAGE. PSAg and GSAg showed 14 and 19 polypeptides in the range of 14.9-95.5 and 13.7-129.6 kDa with six common polypeptides of mol wt 16.8, 21.8, 23.7, 35.5, 43.4 and 70.8 kDa. P. epiclitum experimentally infected sheep sera were used for identification of specific immuno-dominant peptide in the range of 37-40 kDa against P. epiclitum by western blotting. Hyperimmune sera (HIS) was raised in rabbit against the identified polypeptide, IgG was separated from HIS and an immunoaffinity column was constructed with a binding percentage of 83.74 of IgG with CNBr activated Sepharose 4B. Purification of somatic antigen (PSAg) was done with immunoaffinity chromatography and 37-40 kDa protein antigen was isolated in pure form with recovery percentage of 2.97%. This purified fraction of somatic antigen can be used as a candidate antigen for development of serological assay for early diagnosis of paramphistomosis among livestock.

Standardization and application of indirect plate ELISA for immunodiagnosis of paramphistomosis in ruminants.

Indirect plate enzyme-linked immunosorbent assay was standardized and evaluated for its effectiveness in immunodiagnosis of paramphistomosis in experimental and clinical cases in sheep, goat, cattle and buffaloes by using somatic whole adult antigen of Paramphistomum epiclitum and Gastrothylax crumenifer. Plate enzymelinked immunosorbent assay (ELISA) was standardized using 2 µg/ml of antigen concentration with 1:200 and 1:1,000 of sera and conjugate dilution. Indirect Plate ELISA was able to demonstrate the antibody titre at different weeks postinfection in experimental sheep. Immune response at weekly interval varied in all the four experimental sheep. A paired t-test between two types of somatic antigens (P. epiclitum and G. crumenifer) showed that experimental sheep sera showed more affinity for homologous antigen as compared to heterologous antigen. A comparison of plate ELISA on suspected field sera and fecal samples examination by sedimentation method revealed that 77 samples were found to be positive by ELISA but only seven by fecal examination. Sensitivity of plate ELISA was found to be 85.71%, whereas specificity was 23.65% indicating that this test is quite sensitive for clinical cases; an early diagnosis, however, lacks specificity. In comparison to ELISA test the sensitivity and specificity of fecal examination were 7.79 and 88%, respectively.

Therapeutic trial of ivermectin against Notoedres cati var. cuniculi infection in rabbits.

Effect of 400 micrograms/kg of ivermectin against natural infection of Notoedres cati var. cuniculi mange in rabbits was evaluated in twenty rabbits which showed typical symptoms of notoedric mange and were randomly divided into two groups. Group I consisted of 15 rabbits treated with single s/c injection of ivermectin (Ivomec, Dynamic Pharmacals, Bombay). In group II, five infected untreated rabbits were kept as control. Daily observations of clinical improvement and on the basis of examination of skin scrapings on days 0, 3, 6, 9, 16, 23 and 30 post treatment were done. Complete visual shedding of lesions was seen on day 6 after treatment and skin scrapping were found negative for mites after 7th day of treatment till the end of experiment. Histopathologically mites were present in stratum corneum and many were noticed in the burrows in epidermis. Marked hyperkeratosis, hyperplasia and acantholysis along with ballooning degeneration of epithelial cells of epidermis were seen.

Sarcocystis and sarcocystosis in India.

Sarcocystosis, in recent times has been recognized as a disease in animals and man. With the attention of scientists on this problem all over the world, work on prevalence, morphology, life cycle, transmission, pathogenesis, immunology, biochemistry and prophylaxis of this parasite has been initiated in domestic animals in India.

Toxicity of cyst extract of Sarcocystis fusiformis from buffalo in rabbits and mice.

The toxic effect of cysts of Sarcocystis fusiformis (of buffalo origin) in rabbits and mice have been studied. It is shown that inoculation of a 2 mg ml-1 concentration of protein of the macrosarcocyst extract (ME) into rabbits and mice was lethal. The toxic substance was thermolabile at greater than or equal to 60 degrees C for 30 min. Protease and trypsin caused inactivation, while papain did not affect the toxicity. ME was found to be non-dialysable, but precipitable. There was also no effect of a pH of 6-9, while pH 4-5 altered the toxicity and pH 10 only partially affected the toxic effect.

Effect of temperature on the infectivity of Sarcocystis miescheriana cysts in pork.

Sarcocysts of Sarcocystis miescheriana in the thigh muscles of pigs became non-infective to pups after heating infected pork in minute pieces at 60 degrees C for 20 min, 70 degrees C for 15 min and 100 degrees C for 5 min. Similar pieces of infected muscle tissues, when exposed to -4 degrees C for 2 days or -20 degrees C for 1 day, became non-infective to pups. The experiment suggests that pork containing sarcocysts of S. miescheriana, and possibly of S. suihominis, requires cooking at a minimum of 70 degrees C for 15 min or freezing at -4 degrees C for 2 days or -20 degrees C for 1 day for making it safe for consumption.

Spirometra sp. in a domestic cat in India.

Adult Spirometra sp. were found in an experimental cat. This is the first report from northern India. It is suggested that this infection may have resulted from the feeding of raw fish offal.

Host resistance and faecal sporocyst excretion in dogs exposed to repeated infection with Sarcocystis levinei.

A marked reduction in the faecal excretion of sporocysts was observed in experimental pups, following the repeated oral administration to them of buffalo cardiac muscle infected with Sarcocystis levinei. Sporocysts excreted from days 9 to 25 post-infection (pi) exhibited a gradual reduction in the quantum. Maximum intensity of excretion of sporocysts was recorded between days 9 and 16 pi, becoming moderate after day 16, light after day 21 and completely absent after day 36. After the subsequent feeding to pups of S. levinei infected buffalo cardiac tissues at 40 day intervals the quantity of sporocysts shed was less, the prepatent period was prolonged and the patent period was considerably shortened. The peak period of excretion varied depending upon the number of exposures of the pups to the infected S. levinei tissues from buffaloes (Bubalus bubalis).

Heavy sarcocystosis in the ocular musculature of cattle and buffaloes.

A high prevalence of 71.5 per cent and 69.7 per cent of sarcocystosis was observed in the ocular musculature of cattle and buffaloes respectively, in Bihar, India. The concentration of cysts in the eye muscle was also usually heavy. Ocular musculature appears to be a preferred site for the development of Sarcocystis in these intermediate hosts, second only to the heart muscle. The species of Sarcocystis involved in the present study were morphologically indistinguishable from S. cruzi in cattle and S. levinei in buffaloes. This appears to be the first report on the occurrence of S. cruzi and S. levinei in ocular musculature.