Microscopic and molecular investigation of vector borne haemoprotozoan diseases in dromedary camel of North Gujarat, India.

BACKGROUND OBJECTIVES: Vector-borne haemoprotozoan diseases comprise diverse group of single celled organism transmitted by haematophagus invertebrates. The current study was aimed at the identification of major haemoprotozoan (Babesia, Theileria and Trypanosoma) in dromedary camel of North Gujarat region in India using microscopy and Polymerase Chain Reaction (PCR). METHODS: A total of 234 blood samples were screened by the microscopic and molecular detection assays. Molecular prevalence studies of Theileria, Trypanosoma spp and Babesia was undertaken using 18s ribosomal DNA, RoTat 1.2 and SS rRNA gene respectively. The data relating to microscopic and molecular prevalence along with associated risk factors were analysed by statistical methods. RESULTS: The overall prevalence of hamoprotozoan disease based on microscopic and molecular investigation was 23.50%. The sensitivity and specificity (95% Confidence Interval) of PCR assay was 100% in comparison to microscopy (45.45 % sensitive and 100 % specific). The kappa coefficient between PCR and microscopy indicated good level of agreement with a value of 0.704 and SE of 0.159. INTERPRETATION CONCLUSION: Despite holding much significance to the animal sector, little work has been undertaken in regional parts of India regarding camel parasites. The present study offers first preliminary research data investigating haemoprotozoan disease using parasitological and molecular methods in camels in the region.

Microscopic and molecular investigation of vector borne haemoprotozoan diseases in dromedary camel of North Gujarat.

BACKGROUND OBJECTIVES: Vector borne haemoprotozoan diseases comprise diverse group of single celled organism transmitted by haematophagus invertebrates. The current study was aimed at identification of major haemoprotozoan (Babesia, Theileria and Trypanosoma) in dromedary camel of North Gujarat region using microscopy and Polymerase Chain Reaction (PCR). METHODS: A total of 234 blood samples were screened by the microscopic and molecular detection assays. Molecular prevalence studies of Theileria, Trypanosoma spp and Babesia was undertaken using 18s ribosomal DNA, RoTat 1.2 and SS rRNA gene respectively. The data relating to microscopic and molecular prevalence along with associated risk factors were analysed by statistical methods. RESULTS: The overall prevalence of hamoprotozoan disease based on microscopic and molecular investigation was 23.50%. The sensitivity and specificity (95% Confidence Interval) of PCR assay was 100% in comparison to microscopy (45.45% and 100%). The kappa coefficient between PCR and microscopy indicated good level of agreement with a value of 0.704 and SE of 0.159. INTERPRETATION CONCLUSION: Despite holding much significance to the animal sector, little work has been undertaken in regional part of India regarding camel parasites. The present paper offers the first preliminary research data investigating haemoprotozoan disease using parasitological and molecular methods in camels in the region.

Molecular identification and genetic characterization of Theileria and Anaplasma infection in sheep and goat of North Gujarat, India.

Hemoprotozoal diseases are significant health concerns in small ruminants. The present study was conducted to identify and characterize the species of Theileria and Anaplasma in sheep and goats located in different districts of North Gujarat, India. A total of 226 (Banaskantha = 175, Patan = 26, and Bhuj = 25) blood samples were collected from sheep (n = 78) and goats (n = 148), and 46 ticks were collected and identified from sheep and goats. PCR assays were carried out using genus and species-specific primers for Theileria targeting 18S rRNA locus and for Anaplasma targeting the msp5 gene. Overall, 37.2% sheep (29/78) and 10.8% of goats (16/148) were positive for Theileria by PCR, whereas 15.4% of sheep (12/78) and 25.7% goats (38/148) were positive for Anaplasma infection. Moreover, mixed infection was found in 4.4% (10/226) of sheep and goats by PCR. Sanger sequencing of Theileria and Anaplasma positives revealed a high similarity to T. ovis and A. ovis using NCBI blast, respectively. Phylogenetic analyses revealed that the Anaplasma spp. DNA sequences belonged to the A. ovis group and closely associated with the A. ovis nucleotide sequence strain Haibei isolated in China from sheep (GQ483471). The phylogenetic analysis based on the SSU rRNA locus revealed that the Theileria ovis DNA sequences belonged to the T. ovis group and closely related to MW440586 isolated in Kerala, India, from a goat. The majority of ticks (91.3%) were identified as Hyalomma. In conclusion, Theileria ovis and Anaplasma ovis were commonly identified species in sheep and goats and transmitted mainly by Hyalomma ticks in North Gujarat, India, which is important baseline data for future research and control strategies. This is the first report on Theileria and Anaplasma co-infections in sheep and goats from North Gujarat, India.

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.

Morphological and molecular characterization of Paramphistomum epiclitum of small ruminants.

Morphological and molecular identification can pave the way to design the most effective control measures against the Paramphistomum epiclitum in small ruminants. Morphology of the flukes had described the features of Paramphistomum genus. Body was conical with concave ventral and convex dorsal surface, tegumental spines all around the body in the immature stage, terminal funnel shape oral sucker, sub-terminal acetabulum, blind caeca with a serpentine course touching the anterior level of the acetabulum. Vitelline glands were at the lateral margins of the body extended from the pharynx to the posterior sucker. Testes were lobed and tandem, wavy post-testicular uterus and genital pore behind intestinal bifurcation. Sequence analyses of internal transcribed spacer (ITS)-2+ (PCR products of approximately 500 bp) of 10 flukes yielded 2 genotypes, Navsari isolate 1 and 2. In BLAST analysis, ITS-2+ genotypes were 97.3-99% similar with published sequences (KF564870, JF834888, KF642983 and JX678254) of P. epiclitum of Paramphistomatidae. Two genotypes depicted 4 single nucleotide polymorphisms (NPs) in the form of transitions (C-T at 10 and 18; G-A at 255; A-G at 367 locus), 1 triple NPs (CGT-GAA between 21-23 loci) and missing A base at codon 40 in the genotype 1. Average AT and GC content was 49.61% and 50.38%, respectively. Trees topology inferred by Neighbor Joining and Maximum Likelihood methods of ITS2+ of trematodes were similar, with small difference of bootstrap values. Navsari genotypes formed a tight cluster with the P. epiclitum, originated from different location with high bootstrap value and 0.004-0.011 estimated evolutionary divergence.

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.

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.

Staining of Platyhelminthes by herbal dyes: An eco-friendly technique for the taxonomist.

AIM: An environment compatible technique to stain Platyhelminthes, Fasciola gigantica, Gastrothylax crumenifer, Taenia solium, and Moniezia expansa using aqueous and alcoholic extract of sugar beet (Beta vulgaris), China rose (Hibiscus rosa-sinensis), and red rose (Rosa hybrida) were described to minimized the deleterious effects of the synthetic dyes. MATERIALS AND METHODS: Aqueous/ethanolic extracts of roses were extracted from the flowers while red beet was extracted from the roots. RESULTS: Stained helminthes acquired a comparable level of pigmentation with the distinction of their internal structure in these natural dyes. The flukes (liver and rumen) internal structure, oral and ventral/posterior sucker, cirrus sac, gravid uterus, testes, ovary, and vitallaria were appeared pink color in aqueous and alcoholic extract of either China or red rose and yellow to brown color in sugar beet stain. The interior of the proglottid of T. solium and M. expansa took yellow to brown color with good contrast in sugar beet stain and of pink to pink-red in China and red rose stain. CONCLUSION: The extract of roses (red rose followed by China rose) followed by red beet possess the potential to replace the conventional stains in the taxonomic study of Platyhelminthes parasites.