Acaricidal activity of paste of methanolic extract of leaf of Azadirachta indica, Eucalyptus hybrid, Saraca asoca and Murraya koenigii against the bovine ticks in in-vitro condition.

The objective of this study was to evaluate the acaricidal activity of a paste made from freeze-dried methanolic extracts of air-dried leaf powders of Azadirachta indica, Eucalyptus hybrid, Saraca asoca, and Murraya koenigii against Rhipicephalus (Boophilus) microplus on cellulose paper. The extracts were tested in both single form (100% and 50% concentration) and dual combination (prepared by mixing equal proportions of the extracts in 100% concentration). The results showed a direct proportional relationship (p < 0.05) between the concentration of the extracts and the mortality percentage at 72 h post-treatment, as well as the inhibition of oviposition (I.O.) percentage. The highest mortality rate of 98.75 ± 1.25% and I.O. of 44.47 ± 0.87% was observed with the A. indica extract at 100% concentration, followed by E. hybrid, S. asoca, and M. koenigii. The combination of A. indica and E. hybrid extracts had a mortality rate of 87.5 ± 5.59% and I.O. of 42.91 ± 0.44%, followed by the combinations of S. asoca: E. hybrid, A. indica: S. asoca, E. hybrid: M. koenigii, and A. indica: M. koenigii. The extracts of A. indica and E. hybrid demonstrated the highest mortality and inhibition of oviposition percentages compared to the other extracts in both single and dual combinations. These extracts required 72 h to reach their maximum mortality.

Loop-mediated isothermal amplification assay for the rapid detection of Anaplasma marginale in cattle based on major surface protein 5 gene.

BACKGROUND & OBJECTIVES: Timely intervention is needed to minimize the economic losses of vector-borne bovine anaplasmosis which can be possible by the isothermal amplification assay. METHODS: Anaplasma marginale in the cattle of south Gujarat, India was detected in the PCR and LAMP by amplifying the fragment of msp5 gene. The PCR product was digested with EcoRI, and sequenced to confirm its pathogen specific detection. RESULTS: Species specific PCR observed a band of 457 bp of msp5 DNA following 1% agarose gel electrophoresis. Positive LAMP reaction turned into yellow colour while negative sample depicted original pink colour. A detection limit of PCR and LAMP was up to 10-6 and 10-8 of the original genomic DNA of A. marginale, respectively. A single cut site of EcoRI was observed in the PCR product. Current msp5 DNA sequences of A. marginale (MW538962 and MW538961) showed 100% homology with the published sequences. Monophyletic lineage type relationship was observed with high bootstrap proportion among the msp5 DNA sequences of A. marginale in the phylogram. Prevalence rate of A. marginale was significantly higher (p<0.05) in the PCR [43/280 (15.36%)] and LAMP [62/280 (22.14%)] than the microscopic technique [17/280 (6.07%)]. Diagnostic sensitivity, specificity, positive and negative predictive values at 95% CI for LAMP assay with respect to PCR were 93.02%, 90.72%, 64.52% and 98.62%, respectively. INTERPRETATION & CONCLUSION: Thus LAMP can be a practical alternative to the PCR for the diagnosis of A. marginale infection in the cattle even in field condition.

Dry preservation of Toxocara vitulorum by plastination technique.

BACKGROUND AND AIM: The most widely adopted technique to preserve the gross specimen of the parasite is immersions and storage in liquid preservatives. The present study aimed to describe the dry method of the preservation of Toxocara vitulorum using plastination technique. MATERIALS AND METHODS: Acetone dehydrated parasites were incubated at -20°C for 1 month in five different plastination solutions, prepared by mixing melamine and turpentine oil with clove oil (MTCl)/chloroform (MTC)/isopropanol (MTI)/benzene (MTB)/xylene (MTX) in 1:1:1 ratio to infiltrate the polymer. Technical personnel was asked to assign weekly score for dryness, stickiness, shrinkage, glossiness, flexibility, and odor of the prepared model on a 5-point scale. RESULTS: Overall, the plastinated parasites were dry, non-sticky, glossy, odorless, chemical-free, harmless, to some extent flexible, with detectable morphological structure including natural form but lost their natural color, and cuticle became translucent. A varying level of shrinkage was noted in all types of plastinated model, but it was least in MTCl model. One month post-plastination, the mean evaluation score for glossiness was maximum in the parasite plastinated in MTCl solution (4.50±0.17), followed by MTC (3.72±0.32), MTX (3.56±0.38), MTB (2.83±0.37), and MTI (2.31±0.33). Likewise, for flexibility, the score was maximum in the parasite plastinated in MTCl solution (4.36±0.16), followed by MTB (3.11±0.14), MTC (2.94±0.41), MTX (2.75±0.41), and MTI (1.97±0.28). The degree of dryness, stickiness, and odor of the prepared model varies non-significantly (p>0.05) with the polymer mixtures. Maximum shrinkage percentage in terms of length and width was 4.24% and 50%, respectively, in the parasites plastinated in MTB solution. Shrinkage percentage was minimal (1.81% in length and 25% in width) in the MTCl plastinated parasites. Shrinkage percentage in terms of dimension was statistically non-significant among the different polymer solutions. Plastinated models withstand the process of microbial decomposition. There were 5 and 11 odd points in favor of plastination and formalin preservation technique, respectively. CONCLUSION: The prepared T. vitulorum model in MTCl can be used as an adjunct to the parasite preserve in 10% formalin solution. The plastination technique can be used as an alternative method of liquid preservation.

Molecular Assessment of Anaplasma marginale in Bovine and Rhipicephalus (Boophilus) microplus Tick of Endemic Tribal Belt of Coastal South Gujarat, India.

BACKGROUND AND METHODS: This study described the detection, prevalence and phylogeny of Anaplasma marginale in the bovine (cattle and buffaloes) and Rhipicephalus (Boophilus) microplus tick belonged to the tribal area of coastal South Gujarat, India, by amplifying 576 bp of major surface protein (msp) 5 gene using custom designed primers in the polymerase chain reaction (PCR). RESULTS: The PCR detection limit was up to 20 parasites/µl of blood in sensitivity experiment, and observed 100% specificity against Trypanosoma evansi, Babesia bigemina and Theileria annulata. Prevalence rate of the A. marginale in the bovine (n = 211)) was 18.48% and 6.64% (p < 0.05) as per the PCR and Giemsa stained blood smear, respectively. Febrile animals (35%) observed significantly (p < 0.05) higher incidence rate than the non-febrile (14.62%). The amplified msp5 had single cut site for the EcoR1 enzyme, upon digestion yielded two fragments of 365 and 211 bp on 1.0% agarose gel. The current sequence (KC811329) showed 100% homology and 1064 total score with the published nucleotide sequences of msp5 of A. marginale in the NCBI-BLAST study. Monophyletic relationship was observed with high bootstrap proportion (> 76% in Neighbor-Joining/ Maximum Likelihood) between the current and published nucleotide sequences in the phylogeny. Twenty out of 39 A. marginale infected bovine recorded R. (B.) microplus on their body surface, out of which 18 had detected the infection. The rickettsia was in 55%, 65% and 25% of anterior half, posterior half and egg of tick, respectively. CONCLUSION: The test detected A. marginale in a carrier, pre-symptomatic and symptomatic vertebrate hosts (cattle and buffalo) and different body parts of the starved R. (B.) microplus including its egg. The current genotype could be an explanation for the frequent outbreaks of bovine anaplasmosis in the targeted areas.

Plastination of macroparasites: An eco-friendly method of long-term preservation.

AIM: Preservation of macroparasites by infiltrating the polymer in the tissues can defy the inherited shortcoming of classical wet preservation method. MATERIALS AND METHODS: Preservation was done by infiltrating the melamine alone or with xylene (MX)/chloroform (MC)/turpentine oil (MT) in 1:1 and hardener (MH) in 9:1 ratio in the tissues of the gross specimen of the animal parasites. RESULTS: The plastinated models withstand the process of microbial decomposition, and remain intact in the environmental conditions. The polymer mixture resists the entry of the water molecule, and model dried just after taking out it from the water tank. Overall, the plastinated parasites were dry, non-sticky, glossy, odorless, chemical free, and harmless, to some extent flexible, with detectable morphological structure, and retain their natural form but lost their natural color. Full marks were assigned to the degree of dryness, non-stickiness, and odorlessness to the model plastinated in different solutions on a five-point scale. For flexibility, the score was 1.2, 2.2, and 2.4 for the plastinated model in melamine/MH, MX/MC, and MT solutions, respectively. The average score of glossiness was 4.6 and 5 for the specimen plastinated in melamine/MH and MX/MC/MT solutions, respectively. The degree of dryness, glossiness, stickiness, and flexibility varies non-significantly, with the polymer mixtures used. CONCLUSION: The prepared model can be used to educate the students/general mass population.

Sero-detection of Toxocara canis infection in human with T.canis recombinant arginine kinase, cathepsin L-1 and TES-26 antigens.

Three recombinant antigens viz. arginine kinase, cathepsin L-1 and TES-26 of Toxocara canis were expressed in Escherichia coli and evaluated for their potential in the detection of T. canis larval infection in human in immunoglobulin G-enzyme linked immunosorbent assay (IgG-ELISA). Results of the IgG-ELISA with the above recombinant antigens were confirmed with commercially available IgG detection kit for T. canis infection used as a standard test. All three recombinant antigens were 100% sensitive in the detection of positive cases (n = 6) of T. canis infection in human and were screened for their cross-reactivity in human patients with history of Toxoplasma gondii, Plasmodium vivax, Entamoeba histolytica, hydatid and hookworm infections. The recombinant TES-26 antigen showed higher specificity and cross-reacted with T. gondii infection sera only. However, arginine kinase and cathepsin L-1 recombinant antigens showed cross-reactions with sera of patients infected with T. gondii, P. vivax and E. histolytica but not with the patient sera infected with hydatid and hookworm. These results show that recombinant TES-26 is a potential diagnostic candidate antigen for human toxocarosis caused by migrating T. canis larvae.

Physio-biochemical parameters: a potential tool for target-selective treatment of haemonchosis in the small ruminants.

This study aims to evaluate the conjunctiva colour-based FAMACHA score (FS) coupled with a body condition score (BCS), haemogram and stressor hormone level estimation, in identifying post-mortem (PM)/coproscopically proven individuals wanting therapy for economically important gastrointestinal (GI) helminths, Haemonchus contortus, in the small ruminants. The incidence of haemonchosis was significantly (p < 0.05) higher (60.81%) in the ruminants with FS = 3. The H. contortus count in the animals with FS 2, 3 and 4 was 23.2 ± 0.37, 62 ± 2.5 and 74 ± 3.2 (p < 0.05) [positive correlation (r = 0.841 in goats; r = 0.828 in sheep, p < 0.05)], respectively, with corresponding 2.8 ± 0.15, 2 ± 0.3 and 2 ± 0.16 BCS (negative correlation, p > 0.05). The infected animals of FS 2, 3 and 4 measured 8.2 ± 0.0, 7.5 ± 0.23 and 6.7 ± 0.34 g/dl Hb (r = -0.452, p = 0.01) in goats/9.3 ± 0.8, 8.6 ± 0.5 and 7.6 ± 0.3 g/dl Hb (r = -0.511, p = 0.05) in sheep with 21.2, 19.8 ± 1.8 and 17.8 ± 0.2% PCV (r = -0.369, p = 0.05) in goats/26.7 ± 1.2, 22.2 ± 0.2 and 20.9 ± 0.6% PCV (r = -0.251, p = 0.03) in sheep, respectively. The FS 2, 3 and 4 infected goats/sheep measured 6.1 ± 0, 7.9 ± 1.0 and 9.5 ± 0.9 (p < 0.05)/5.8 ± 2.3, 6.9 ± 1.2 and 7.8 ± 0.2% (p < 0.05) mid-granulocyte [(r = 0.928 (goats)/0.834 (sheep), p < 0.05], while the cortisol level was 15.6, 23 ± 4.5 and 42 ± 2.3 (p = 0.23)/12.1 ± 0, 15.9 ± 1.2 and 24 ± 3.4 (p = 0.29) µg/dl, respectively. The infected ruminants recorded low (p < 0.05) level of Hb/PCV while high level of mid-granulocytes/cortisol. Specificity of FAMACHA test was maximized (100%) when FS = 4 was considered anaemic, but sensitivity was low (35.29% in goats; 25% in sheep). The false negatives was 5.9 (goat)/12.5 (sheep)% when FS ≥ 3 was considered anaemic. The small ruminants with FS ≥ 3, BCS ≤ 2.5, Hb ≤ 7.5 g/dl (goats)/8.6 g/dl (sheep), PCV ≤ 19.8% (goats)/22.2% (sheep) and mid-granulocyte ≥7.9% (goats)/6.9 ± 1.2% (sheep) can be subjected to target-selective treatment for haemonchosis in the field simultaneously maximizing the economic benefit to the farmers.

Major sites of cancer occurrence among men and women in gandhinagar district, India.

BACKGROUND: This study examines major cancer sites among the population of Gandhinagar district, India during the year 2009-2011. OBJECTIVE: To study leading cancer incidents and mortality and their age distribution in both sexes in Gandhinagar district. MATERIALS AND METHODS: Primary data were collected from various sources and entered in computer and analyzed. Quality checks were done, and duplicate cases were eliminated. For mortality data, death registration units were contacted. RESULTS: Total 2360 incident cases (1374 males and 986 females) and 736 mortality cases (464 males and 272 females) were recorded during the year 2009-2011 in Gandhinagar district. Among males, the leadings sites were mouth, tongue, lung, esophagus, hypopharynx, and larynx, whereas in females they were breast, cervix, ovary, mouth, tongue and myeloid leukemia. Majority of cases were found in the age group of 35-64 years and the proportion in male and female in this age group was 62.51% and 71.05%, respectively. CONCLUSION: The study helps to understand the possible cancer patterns in Gandhinagar district. Foremost causes of cancer in leading sites in males were tobacco related, and the proportion of cancers associated with tobacco was 53% in our study. It highlights the possibility of easy and early detection of cancers, especially by oral cancer screening in the population. Further, the findings highlight the need of cancer cervix and breast screening among the women at regular intervals through camp approach in the community, as these are the most common sites (40% of female cancers).