Targeted drug delivery using beeswax-derived albendazole-loaded solid lipid nanoparticles in Haemonchus contortus, an albendazole-tolerant nematode.

Targeted delivery has not been achieved for anthelmintic treatment, resulting in the requirement of excess drug dose leading to side effects and therapeutic resistance. Gastrointestinal helminths take up lipid droplets from digestive fluid for energy production, egg development, and defense which inspired us to develop biocompatible and orally administrable albendazole-loaded solid lipid nanoparticles (SLN-A) that were derived from beeswax and showed drug loading efficiency of 83.3 ± 6.5 mg/g and sustained-release properties with 84.8 ± 2.5% of drug released at pH 6.4 within 24 h at 37 °C. Rhodamine B-loaded SLN showed time-dependent release and distribution of dye in-vitro in Haemonchus contortus. The sustained-release property was shown by the particles that caused enhancement of albendazole potency up to 50 folds. Therefore, this formulation has immense potential as an anthelminthic drug delivery vehicle that will be able to reduce the dose and drug-induced side effects by enhancing the bioavailability of the drug.

Faecal prevalence and histopathological evaluation of coccidiosis in bovine calves.

Bovine coccidiois, caused by Eimeria spp. is widely prevalent around the globe and responsible for huge economic losses by causing morbidity and mortality among young calves. The present study was designed to evaluate the prevalence as well as to evaluate histopathological alterations associated with it. The faecal samples were collected from 700 bovine calves upto two month of age from August 2019 to July 2021 and screened for Eimeria oocycts. The intestinal tissue samples of 37 calves were also collected which died during the study period after showing symptoms of diarrhea and examined for histological lesions. The faecal prevalence of Eimeria observed in our study was 2.29% (16/700) while in tissue samples only two out of 37 were found positive for Eimeria infection. Tissue sections revealed various stages of Eimeria gametogony, variable congestion, haemorrhage, and necrosis along with cryptic dilatation and mononuclear cell infiltration. Coccidia was not found to be associated with season, age and sex of calf. Bovine coccidiosis was found to be endemic with low prevalence but severe onset characterized by moderate to severe congestion and inflammatory reaction mainly in the ileum and caecum.

Severe Infection of Nippostrongylus brasiliensis in Bandicota bengalensis Inhabiting Commensal Areas of Punjab, India: Prevalence, Risk Factor Analysis, Molecular Identification and Phylogenesis.

PURPOSE: The present study aimed to record the prevalence, risk factors, molecular identification, and phylogeny of Nippostrongylus brasiliensis found in the small intestine of the lesser bandicoot rat, Bandicota bengalensis, a wild rodent species. METHODS: A total of 100 bandicoot rats live trapped at two commensal urban locations (50 each), i.e., a fish market and railway station, in Ludhiana, Punjab State (India), from November 2020 to October 2021, were analysed for the presence of N. brasiliensis, a nematode parasite of zoonotic importance. RESULT: Overall, the small intestine of 43.00% of the rats was found severely infected with bright red coloured adult N. brasiliensis of both sexes (total of 1439 specimens). Faecal samples contained ellipsoidal and thin-shelled eggs measuring 62.25-74.70 m in length and 33.20-37.35 m in breadth. No significant (P > 0.05) effect of host age, sex, or season was observed on the rate of infection. The parasite intensity and mean abundance ranged from 27.68-38.04 and 10.52-18.26, respectively, indicating a high risk of disease transmission. Based on the morphology, the nematode parasite was identified as Nippostrongylus sp. Molecular identification was confirmed through PCR amplification of the mitochondrial cytochrome oxidase I gene, which showed a single band of approximately 355 bp. A comparison of the present isolate with the available sequences of Nippostrongylus species across the globe showed 100% nucleotide homology with N. brasiliensis sequences available in GenBank from Japan (AP017690), the USA (U57035), and New Zealand (NC033886). CONCLUSION: The study indicates that B. bengalensis inhabiting commensal urban areas is a reservoir host for N. brasiliensis, which if transmitted to humans and animals visiting the area may pose a potential health risk. The study thus suggests proper rodent population management close to human habitations to avoid the transmission of disease-causing agents.

Evaluation and Correlation of Multiple Anthelmintic Resistances to Gastrointestinal Nematodes Using Different Fecal Egg Count Reduction Methods in Small Ruminants of Punjab, India.

PURPOSE: Concordance of multiple anthelmintic resistances for gastrointestinal nematodes in small ruminants by three average-based and two individually based fecal egg count reduction (FECR) tests was evaluated and corrected. METHODS: Sheep and goats (≥ 8 weeks) from five farms were randomly assigned to three treatment groups (I, II, III; n = 10 per group) and one untreated control group (Group IV; n = 10). Group I received fenbendazole at the dose rate of 5 and 10 mg/kg, Group II received ivermectin at the dose rate of 0.2 and 0.4 mg/kg, and Group III received levamisole at the dose rate of 8 and 12 mg/kg body weight orally for sheep and goat, respectively. Three average-based methods of FECR (FECR1, FECR2 and FECR3) and two individually based methods of FECR (iFECR1 and iFECR2) were evaluated. RESULTS: For fenbendazole resistance, Spearman correlation coefficient for FECR1 was non-significant with other formulae, but for FECR2 with FECR3, FECR3 with iFECR1 and iFECR1 with iFECR2 coincidence was significant at 1%, while for FECR2 with iFECR2 and FECR3 with iFECR2 it was significant at 5%. Spearman correlation coefficients for ivermectin resistance were significant at 1% level and for levamisole it showed significant coincidence at 1% for FECR1 with FECR2 and iFECR1, FECR2 with FECR3 and iFECR1, and iFECR1 with iFECR2, while for FECR1 with FECR3 and iFECR2 coincidence was significant at 5% level. Concordance of kappa values indicated that the coincidence of the prevalence of anthelmintic resistance (95% CI) among the five farms was non-significant. CONCLUSIONS: Concordance between the standard average-based FECR and individually based methods suggests that either method could be applied to small ruminant farms.

Theileria equi and Babesia caballi infection of equids in Punjab, India: a serological and molecular survey.

A cross-sectional study was conducted in Submountain undulating, Undulating plain, Western and Western plain agro-climatic zones of Punjab province, India, to determine the prevalence, agreement between diagnostic tests and associated related risk factors of Theileria equi and Babesia caballi infection in equids (horses, donkey, mules). An overall prevalence of 14.14 and 0.0% of T. equi and B. caballi was recorded by multiplex polymerase chain reaction targeting 18S ribosomal RNA (rRNA) for both the parasites and 75 and 1.11% by competitive enzyme-linked immunosorbent assay in a representative sample of 180 animals. Only two animals with positive antibody titre from B. caballi and none with PCR indicated T. equi as the predominant haemoprotozoan responsible for equine piroplasmosis in the study area. Among the PCR-positive animals, presence of tick vectors in farm vicinity was the most influential associated with T. equi infection (P = 0.002; odds ratio (OR) 9.30; 95% confidence interval (CI) = 3.32-27.10). For animals with higher anti-T. equi antibody titres, strong association of sero-prevalence for T. equi was recorded with age, sex, usage, tick infestation and deworming/vaccination status of host animals and farm management strategies. The study has demonstrated the possible absence of B. caballi in both conducive and non-conducive areas of Punjab and demonstrated T. equi as the potential agent of equine piroplasmosis in Punjab.

Detection and assessment of risk factors associated with natural concurrent infection of Trypanosoma evansi and Anaplasma marginale in dairy animals by duplex PCR in eastern Punjab.

Duplex PCR consisting of two primer sets within a single mixture for the simultaneous detection of Anaplasma marginale and Trypanosoma evansi was standardized and employed on 219 blood samples collected from cattle (165) and buffaloes (54) from eastern Punjab to evaluate the status of concurrent infection and associated risk factors. The reaction produced 257- and 407-bp amplification products targeting repetitive nucleotide sequence of T. evansi and msp1ß gene of A. marginale, respectively. The nucleotide sequence analysis of individual amplicons expressed the fidelity of the primer pairs used; duplex PCR was 100% sensitive and 92.66 % specific with conventional microscopy for the detection of mixed infections. Among the agro-climatic zones of interest, undulating zone was at higher risk of T. evansi infection (odds ratio (OR) = 1.75, 95% confidence interval (CI) = 0.94-3.27), and submountain zone (OR = 1.89, 95% CI = 1.11-3.33) for A. marginale. For the concurrent infection, the relative risk among the two zones was almost unity. The cross-bred cattle population was at the highest risk of infection, may it be solo infection of T. evansi (OR = ∞, 95% CI = 1.18-∞)/A. marginale (OR = 6.39, 95% CI = 1.14-125.3) or dual infection (OR = ∞, 95% CI = 0.39-∞) of both as the indigenous cattle are resistant to the infection. Cross-bred cattle were at approximately three times the risk than buffaloes. For the dual infection, the cattle calves were at about 2.5 times higher risk than buffalo calves. Results indicate the endemic status of these infections in the region and mark out the commodities at great risk and requiring better surveillance.

Molecular prevalence of Babesia bigemina and Trypanosoma evansi in dairy animals from Punjab, India, by duplex PCR: a step forward to the detection and management of concurrent latent infections.

Specific duplex polymerase chain reaction (PCR) was employed on 411 (386 cattle and 25 buffaloes) blood samples of dairy animals from 9 districts of Punjab, India, for simultaneous detection of Babesia bigemina and Trypanosoma evansi. The results were compared and correlated with conventional Giemsa stained thin blood smear (GSTBS) examination and haematological alterations to know the clinical status and pathogenicity of infections. The Bg3/Bg4 and TR3/TR4 primers were used in duplex PCR for B. bigemina and T. evansi amplified products of 689 bp and 257 bp, respectively. The overall prevalence by duplex PCR was found to be 36.49, 2.43, and 3.41% for T. evansi, B. bigemina, and dual infection, respectively. A more significant difference was observed for dual infection status (P ≤ 0.005) as compared to T. evansi (P ≤ 0.05) and B. bigemina (P ≤ 0.01) among various districts under study. A very low prevalence of T. evansi (0.73%) and B. bigemina (0.48%) was seen by GSTBS. The highly sensitive, specific, and cost-effective duplex PCR was able to detect latent T. evansi and B. bigemina infection in cattle and buffaloes. Haematological evaluation revealed marked pathology in B. bigemina infected group and in dual infected group in contrast to that infected with T. evansi alone.

Differential proteomic profiles from distinct Toxoplasma gondii strains revealed by 2D-difference gel electrophoresis.

Toxoplasma gondii is an obligate intracellular protozoan that infects mammals and birds. Human infection during pregnancy may cause severe damage to the fetus. Reactivation of latent infection in immunocompromised patients can cause life-threatening encephalitis. T. gondii strains are highly diverse but only a few lineages (Type I, II and III) are widely spread. In mouse model, Type I strains are highly virulent, whereas Type II and III strains are intermediately or non virulent. It is not clear how much quantitative difference exists in proteomic profiles among these distinct T. gondii lineages. In the present study, the proteomic profiles of T. gondii tachyzoites from these lineages were investigated by two dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mass spectrometry (MS) technologies. A total of 2321 protein spots were detected. Overall, the GT1 strain of Type I lineage and the strain PTG of Type II lineage have highly similar proteomic profiles and both are different from that of the CTG strain of Type III lineage. Eighty-four protein spots were differentially expressed by greater than 1.5-fold in relative abundance and 10 of them were identified to 7 T. gondii proteins in existing database. Investigation of the quantitative differences in proteomics among distinct T. gondii strains should facilitate our understanding of difference in biological processes and pathogenesis of distinct T. gondii genotypes, which will provide basic information to determine treatment regimen for different manifestation of toxoplasmosis.