Comparative epidemiology and pathophysiology of patent and latent babesiosis caused by Babesia bigemina in buffaloes and cattle from different agroclimatic zones of Punjab State, India.

To conduct comparative epidemiology of parasitologically positive (patent) and polymerase chain reaction positive (latent) cases of bovine babesiosis in Bet Region (low-lying areas adjoining Sutlej, Beas, Ravi, and Ghaggar rivers of Punjab) of diverse agroclimatic zones of Punjab state in relation to haematobiochemical parameters as patho-physiological markers, blood samples from 783 dairy animals (487 buffaloes and 296 cattle) were analysed parasitologically by Giemsa-stained blood smears (GSBS) and by molecular-based polymerase chain reaction (PCR) targeting SpeI-AvaI restriction fragment of Babesia bigemina. We ruled out the endemicity of the disease with 2.17% patent and 3.96% latent cases of B. bigemina with significantly higher prevalence (P < 0.01) in cattle than buffaloes. The spatial distribution for a guideline to local veterinary practitioners and policy-makers indicated highest number of patent and latent cases in western zone and undulating plain zone, respectively. District wise highest prevalence of patent as well as latent infection observed in SBS Nagar of undulating plain zone showed substantial agreement (Kappa value: 0.70) between the two techniques. Haematology revealed marked microcytic hyperchromic anaemia in patent animals of group I (GSBS positive; n = 17) and latent animals of group II (PCR positive; n = 14) as compared to disease-free controls (group III; n = 10). Blood urea nitrogen (BUN), creatinine and gamma-glutamyltransferase (GGT) levels were significantly increased (P < 0.05) in group I in comparison to group II and group III indicated comparative pathogenic effect of babesiosis in patent cases. Though patent cases showed higher pathogenicity of babesiosis, diagnosis of latent infection is significant as it may act as source of infection for spread to other highly prone bovines.

Tagging of Weakly Expressed Toxoplasma gondii Calcium-Related Genes with High-Affinity Tags.

Calcium ions regulate a diversity of cellular functions in all eukaryotes. The cytosolic Ca2+ concentration is tightly regulated at the physiological cytosolic concentration of 50-100 nm. The Toxoplasma gondii genome predicts the presence of several genes encoding potential Ca2+ channels, pumps, and transporters. Many of these genes are weakly expressed and likely tightly regulated due to their potential impact to the physiology of the cell. Endogenous tagging has been widely used to localize proteins in T. gondii but low level of expression of many of them makes visualization of tags difficult and sometimes impossible. The use of high-performance tags for labeling proteins expressed at low level is ideal for investigating the localization of these gene products. We designed a Carboxy-terminus tagging plasmid containing the previously characterized "spaghetti monster-HA" (smHA) or "spaghetti monster-MYC" (smMYC) tags. These tags consist of 10 copies of a single epitope (HA or MYC) inserted into a darkened green fluorescence protein scaffold. We localized six proteins of various levels of expression. Clonal lines were isolated and validated by PCR, western blot, and immunofluorescence analyses. Some gene products were only visible when tagged with smHA and in one case the smHA revealed a novel localization previously undetected.

The paternal ancestry of Uttarakhand does not imitate the classical caste system of India.

Although, there have been rigorous research on the Indian caste system by several disciplines, it is still one of the most controversial socioscientific topic. Previous genetic studies on the subcontinent have supported a classical hierarchal sharing of genetic component by various castes of India. In the present study, we have used high-resolution mtDNA and Y chromosomal markers to characterize the genetic structuring of the Uttarakhand populations in the context of neighboring regions. Furthermore, we have tested whether the genetic structuring of caste populations at different social levels of this region, follow the classical chaturvarna system. Interestingly, we found that this region showed a high level of variation for East Eurasian ancestry in both maternal and paternal lines of descent. Moreover, the intrapopulation comparison showed a high level of heterogeneity, likely because of different caste hierarchy, interpolated on asymmetric admixture of populations inhabiting on both sides of the Himalayas.

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.

PCR and ELISA vis-à-vis microscopy for detection of bovine anaplasmosis: a study on associated risk of an upcoming problem in North India.

This investigation demonstrates the status of bovine anaplasmosis caused by A. marginale in bovines from Submountain and Undulating Zone of Punjab. Out of 184 suspected animals, 25 (19.51%), 47 (31.71%), and 78 (68.75%) were positive by microscopy, indirect ELISA, and PCR assay, respectively. The microscopy showed 29% sensitivity and 99% specificity, while ELISA showed 32% sensitivity and 79% specificity in concordance with PCR assay. Five false negative samples by msp1ß PCR were reconfirmed for Anaplasma spp. targeting 16S rRNA gene. The sequence analysis showed the presence for A. marginale specific restriction site, indicating variation in the local strains of the organism resulting in no amplification with msp1ß gene primers. Of 82 samples positive by PCR, 57 were negative by ELISA indicating lower efficacy of ELISA to detect early anaplasmosis. The assessment of risk factor with results of PCR technique indicated that cattle (Odds ratio = 2.884), particularly those of age > 1 years (Odds ratio = 2.204) of district Pathankot (Odds ratio = 3.182) of Submountain Zone (Odds ratio = 2.086), were at high risk of anaplasmosis. All three districts of Submountain Zone are at higher risk indicating the impact of biotic and abiotic factors on the incidence of disease.

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.

Diagnostic Utility of Fine-Needle Aspiration Cytology (FNAC) and Frozen Section Against Histopathology in Evaluating Benign and Malignant Breast Lesions.

INTRODUCTION: Breast lesions, particularly lumps, pose concerns for females, varying between benign and malignant conditions. Accurate differentiation solely through clinical assessment is challenging, necessitating a definitive diagnostic strategy. Fine-needle aspiration cytology (FNAC) is integral in the "triple approach" to breast evaluation, offering simplicity, reliability, and cost-effectiveness. However, FNAC has limitations, occasionally failing to yield definitive diagnoses due to inherent constraints. Contrarily, frozen-section analysis, a long-standing intraoperative diagnostic method, plays a crucial role in swift diagnosis during surgeries. Despite technological advancements, frozen sections serve specific diagnostic purposes, confirming carcinoma when FNAC is inconclusive and evaluating resected margins. However, freezing artifacts may affect tissue assessment, emphasizing the continued reliance on histopathology for guiding treatment decisions. OBJECTIVES: This study was conducted at KVG Medical College and Hospital, Sullia, Karnataka, India. It aimed to analyze the morphological characteristics of benign and malignant breast lesions using FNAC, frozen section, and histopathology and evaluate the sensitivity, specificity, and predictive values of FNAC and frozen section against histopathology as the reference standard. METHODS: A cross-sectional investigation was carried out at a tertiary care hospital's Department of Pathology, on 60 female patients who presented with palpable breast masses over a span of two and a half years. FNAC was conducted, and the observations were classified into five categories as per the International Academy of Cytology guidelines. In addition, intraoperative frozen-section analysis was undertaken. A comparative analysis was conducted between the FNAC and intraoperative frozen-section findings, juxtaposed with the subsequent histopathological diagnoses. RESULTS: FNAC revealed 51.7% malignant, 45% benign, and 3.3% inadequate cases; the frozen-section analysis indicated 51.6% malignant, 45% benign, and 3.3% deferred cases; histopathology showed 53.3% malignant, 45% benign, and 1.6% borderline cases. FNAC demonstrated 93.9% sensitivity, 100% specificity, 100% positive predictive value (PPV), 93.1% negative predictive value (NPV), and 96.7% accuracy. The frozen-section analysis exhibited 96.9% sensitivity, 100% specificity, 100% PPV, 96.4% NPV, and 98.3% accuracy. CONCLUSION: Intraoperative frozen-section analysis displays superior diagnostic utility compared to preoperative FNAC. However, histopathology remains the definitive gold standard. Integrating all three diagnostic modalities is crucial for precise diagnosis and effective patient management.

Chemical Optimization of CBL0137 for Human African Trypanosomiasis Lead Drug Discovery.

The carbazole CBL0137 (1) is a lead for drug development against human African trypanosomiasis (HAT), a disease caused by Trypanosoma brucei. To advance 1 as a candidate drug, we synthesized new analogs that were evaluated for the physicochemical properties, antitrypanosome potency, selectivity against human cells, metabolism in microsomes or hepatocytes, and efflux ratios. Structure-activity/property analyses of analogs revealed eight new compounds with higher or equivalent selectivity indices (5j, 5t, 5v, 5w, 5y, 8d, 13i, and 22e). Based on the overall compound profiles, compounds 5v and 5w were selected for assessment in a mouse model of HAT; while 5v demonstrated a lead-like profile for HAT drug development, 5w showed a lack of efficacy. Lessons from these studies will inform further optimization of carbazoles for HAT and other indications.

Hypothesis-generating proteome perturbation to identify NEU-4438 and acoziborole modes of action in the African Trypanosome.

NEU-4438 is a lead for the development of drugs against Trypanosoma brucei, which causes human African trypanosomiasis. Optimized with phenotypic screening, targets of NEU-4438 are unknown. Herein, we present a cell perturbome workflow that compares NEU-4438's molecular modes of action to those of SCYX-7158 (acoziborole). Following a 6 h perturbation of trypanosomes, NEU-4438 and acoziborole reduced steady-state amounts of 68 and 92 unique proteins, respectively. After analysis of proteomes, hypotheses formulated for modes of action were tested: Acoziborole and NEU-4438 have different modes of action. Whereas NEU-4438 prevented DNA biosynthesis and basal body maturation, acoziborole destabilized CPSF3 and other proteins, inhibited polypeptide translation, and reduced endocytosis of haptoglobin-hemoglobin. These data point to CPSF3-independent modes of action for acoziborole. In case of polypharmacology, the cell-perturbome workflow elucidates modes of action because it is target-agnostic. Finally, the workflow can be used in any cell that is amenable to proteomic and molecular biology experiments.

First record of Toxoplasma gondii antibodiesin Royal Bengal tigers (Panthera tigris tigris) and Asiatic lions (Panthera leo persica) in India.

The purpose of this study was to detect the antibodies against Toxoplasma gondii in Royal Bengal tigers (Panthera tigris tigris), Asiatic lions (Panthera leo persica), leopards (Panthera pardus), and elephants (Elephas maximus indicus) residing in the Mahendra Chaudhury Zoological Park, in Chhatbir, Punjab (India) during winter and monsoon seasons. Using  indirect ELISA, 20 serum samples were analysed during the winter season. Results indicated that 1 lion (5%) tested seropositive, and 3 tigers and 1 lion (20%) were considered suspect. During the monsoon, 4 individuals (2 tigers and 2 lions, 20%) were seropositive, whereas only 1 tiger (5%) gave suspected results. Significantly higher globulin, creatinine, blood urea nitrogen, phosphorus, and creatine kinase values were recorded in seropositive and suspected groups. Levels of albumin, glucose, calcium, sodium, and iron decreased significantly in the seronegative group. Results from sero-testing 40 rodents trapped in and around the park depicted the presence of antibodies against Toxoplasma gondii in 1 individual. This study reveals the haemato-biochemical alterations in both seropositive and suspected wild felids for toxoplasmosis. Moreover, it provides the first serological evidence of T. gondii exposure in wild felids, notably Royal Bengal tigers and Asiatic lions, in India.

Spatial seroepidemiology, risk assessment and haemato-biochemical implications of bovine trypanosomiasis in low lying areas of Punjab, India.

A cross-sectional study was carried out on 594 bovines (341 buffalo adults, 31 buffalo calves, 163 cattle adults, and 59 cattle calves) to assess the exposure of native bovine population to T. evansi elicited trypanosomiasis in the low-lying areas of Punjab (India). We ruled out the endemicity of the disease with 10.77% (95%CI = 8.53-13.52) sero-positive and 23.56% (95%CI = 20.33-27.15) suspected cases by card agglutination assay. We have presented the spatial distribution of these cases as a guideline to local veterinary practitioners and policy-makers. The categorical assessment of risk factors revealed buffalo adults are the most susceptible group in the state despite insignificant differences in farm management practices. A significant increase in the WBC, platelet, AST and serum iron, and decrease in haemoglobin, haematocrit volume, and serum glucose was recorded in both T. evansi positive and suspected animals.

Nitrogen-dependent induction of atrazine degradation pathway in Pseudomonas sp. strain AKN5.

Soil isolate Pseudomonas sp. strain AKN5 degrades atrazine as the sole source of nitrogen. The strain showed expeditious growth on medium containing citrate as the carbon source and ammonium chloride as the nitrogen source as compared to citrate plus atrazine or cyanuric acid. Biochemical and nitrogen-source-dependent enzyme induction studies revealed that atrazine is metabolized through hydrolytic pathway and has two segments: the upper segment converts atrazine into cyanuric acid while the lower segment metabolizes cyanuric acid to CO2 and ammonia. Bioinformatics and co-transcriptional analyses suggest that atzA, atzB and atzC were transcribed as three independent transcripts while atzDEF were found to be transcribed as a single polycistronic mRNA indicating operonic arrangement. Transcriptional analysis showed inducible expression of atzA/B/C/DEF from atrazine grown cells while cyanuric acid grown cells showed significantly higher expression of atzDEF. Interestingly, growth profiles and enzyme activity measurements suggests that strain utilizes a simple nitrogen source (ammonium chloride) over the complex (atrazine or cyanuric acid) when grown on dual nitrogen source. These results suggest that atrazine degradation genes were up-regulated in the presence of atrazine but repressed in the presence of simple nitrogen source like ammonium chloride.

Kinetoplast Division Factors in a Trypanosome.

Inheritance of the single mitochondrial nucleoid (kinetoplast) in the trypanosome requires numerous proteins, many of whose precise roles are unclear. By considering kinetoplast DNA (kDNA) as a template for cleavage into two equal-size networks, we predicted sets of mutant kinetoplasts associated with defects in each of the five steps in the kinetoplast cycle. Comparison of these kinetoplasts with those obtained after gene knockdowns enabled assignment of proteins to five classes - kDNA synthesis, site of scission selection, scission, separation, and partitioning. These studies highlight how analysis of mutant kinetoplast phenotypes may be used to predict functional categories of proteins involved in the biogenesis of kinetoplasts.

Improvement of Aqueous Solubility of Lapatinib-Derived Analogues: Identification of a Quinolinimine Lead for Human African Trypanosomiasis Drug Development.

Lapatinib, an approved epidermal growth factor receptor inhibitor, was explored as a starting point for the synthesis of new hits against Trypanosoma brucei, the causative agent of human African trypanosomiasis (HAT). Previous work culminated in 1 (NEU-1953), which was part of a series typically associated with poor aqueous solubility. In this report, we present various medicinal chemistry strategies that were used to increase the aqueous solubility and improve the physicochemical profile without sacrificing antitrypanosomal potency. To rank trypanocidal hits, a new assay (summarized in a cytocidal effective concentration (CEC50)) was established, as part of the lead selection process. Increasing the sp3 carbon content of 1 resulted in 10e (0.19 µM EC50 against T. brucei and 990 µM aqueous solubility). Further chemical exploration of 10e yielded 22a, a trypanocidal quinolinimine (EC50: 0.013 µM; aqueous solubility: 880 µM; and CEC50: 0.18 µM). Compound 22a reduced parasitemia 109 fold in trypanosome-infected mice; it is an advanced lead for HAT drug development.

Anilinoquinoline based inhibitors of trypanosomatid proliferation.

We recently reported the medicinal chemistry re-optimization of a series of compounds derived from the human tyrosine kinase inhibitor, lapatinib, for activity against Plasmodium falciparum. From this same library of compounds, we now report potent compounds against Trypanosoma brucei brucei (which causes human African trypanosomiasis), T. cruzi (the pathogen that causes Chagas disease), and Leishmania spp. (which cause leishmaniasis). In addition, sub-micromolar compounds were identified that inhibit proliferation of the parasites that cause African animal trypanosomiasis, T. congolense and T. vivax. We have found that this set of compounds display acceptable physicochemical properties and represent progress towards identification of lead compounds to combat several neglected tropical diseases.

Series of Alkynyl-Substituted Thienopyrimidines as Inhibitors of Protozoan Parasite Proliferation.

Discovery of new chemotherapeutic lead agents can be accelerated by optimizing chemotypes proven to be effective in other diseases to act against parasites. One such medicinal chemistry campaign has focused on optimizing the anilinoquinazoline drug lapatinib (1) and the alkynyl thieno[3,2-d]pyrimidine hit GW837016X (NEU-391, 3) into leads for antitrypanosome drugs. We now report the structure-activity relationship studies of 3 and its analogs against Trypanosoma brucei, which causes human African trypanosomiasis (HAT). The series was also tested against Trypanosoma cruzi, Leishmania major, and Plasmodium falciparum. In each case, potent antiparasitic hits with acceptable toxicity margins over mammalian HepG2 and NIH3T3 cell lines were identified. In a mouse model of HAT, 3 extended life of treated mice by 50%, compared to untreated controls. At the cellular level, 3 inhibited mitosis and cytokinesis in T. brucei. Thus, the alkynylthieno[3,2-d]pyrimidine chemotype is an advanced hit worthy of further optimization as a potential chemotherapeutic agent for HAT.

Molecular survey in relation to risk factors and haemato-biochemical alteration in Theileria equi infection of equines in Punjab Province, India.

Equine piroplasmosis caused by Theileria equi, an apicomplexan intracellular haemoprotozoan parasite effects equids throughout the world. Current investigation is the first detailed epidemiological survey report based on conventional (blood smear) and primary polymerase chain reaction (PCR) for the detection of T. equi on 464 equids (426 horses and 38 donkeys/mules) in Punjab province of India. PCR targeting 18S rRNA gene of T. equi produced high fidelity 709bp amplification products with 100% concordance with blood films. The prevalence of infection was proportional (P≤0.01) to temperature and aridness of the regions, which turned out to be the most important physical factor pertaining to T. equi infection. Spatial distribution analysis revealed an increasing trend of disease prevalence from north-eastern to south-western region of Punjab by both the techniques. Haemato-biochemical analysis revealed significant decrease in total erythrocyte count and haemoglobin; and increase in mean corpuscular haemoglobin, serum globulin, glucose, creatinine, aspartate aminotransferase and bilirubin levels (P≤0.05). This study divulges the endemicity of equine theileriosis in Punjab with the prominence of various odds of infection, emphasising the pathophysiological manifestation associated with latent infection of T. equi detectable by PCR.

Clinicopatho-Biochemical Alterations Associated with Subclinical Babesiosis in Dairy Animals.

BACKGROUND: Present investigation is based on the clinicopatho-biochemical alteration related to natural tick borne bovine babesiosis in Punjab state. METHODS: Blood samples from jugular vein of 542 bovines (cattle 466 and buffaloes 76) having history of tick infestation, fever, hemoglobinurea or anemia were collected and tested for Babesia bigemina by blood smear examination and PCR targeting 18S rRNA gene to distinguish clinically and subclinically infected groups. Further the hemato-biochemical parameters were correlated with the status of infection. RESULTS: Overall, of the 542 samples tested 16.42 % were positive by PCR while only 1.66 % by blood smear examination. The trend of molecular prevalence was found to decrease from north-eastern towards western Punjab. Analysis of the hematobiochemical alterations showed significant decrease in the levels of RBC, Hb, PCV, and MCV with significant increase in TBIL, MCH and MCHC levels. CONCLUSION: As the transmission of B. bigemina is transovarian, presence of even few infected Rhipicephalus (Boophilus) microplus ticks on a subclinically infection can be the nidus of infection for whole herd, causing severe economic losses, at the same time significantly affecting the physiology of carrier animal.

Spatial distribution, risk factors and haemato-biochemical alterations associated with Theileria equi infected equids of Punjab (India) diagnosed by indirect ELISA and nested PCR.

Equine piroplasmosis is a febrile, tick-borne disease of equids predominately caused by obligatory intra-erythrocytic protozoa Theileria equi in the Indian sub-continent. A cross-sectional study was carried out on 464 equids (426 horses and 38 donkeys/mules) in Punjab, India to assess the level of exposure to equine piroplasmosis by 18S rRNA gene nested polymerase chain reaction (nPCR) and equine merozoite antigen-2 (EMA2) indirect-ELISA (enzyme linked immunosorbent assay), to investigate risk factors and haemato-biochemical alterations associated with the infection. The endemicity of the disease was confirmed by positive PCR amplification in 21.77% and positive antibody titers in 49.78% equid samples. There was a fair agreement between these two diagnostic techniques (Kappa coefficient=0.326). The spatial distribution analysis revealed an increasing trend of T. equi prevalence from north-eastern to south-western region of Punjab by both the techniques correspondingly, which proffered a direct relation with temperature and inverse with humidity variables. The relatively prominent risk factor associated with sero-positivity was the presence of other domestic animals in the herd, while the propensity of finding a positive PCR amplification was higher in donkeys/mules, animal kept at unorganised farm or those used for commercial purposes as compared to their counterparts. There was a significant increase in globulins, gamma glutamyl-transferase, total bilirubin, direct bilirubin, indirect bilirubin, glucose levels and decrease in total erythrocyte count, haemoglobin, packed cell volume by animals, which were revealed positive by nPCR (may or may not positive by indirect-ELISA) and increase in creatinine, total bilirubin, direct bilirubin, glucose and decrease in total erythrocytes count by animals, which were revealed positive by indirect-ELISA (alone). To our knowledge, this study, for the first time, brings out a comprehensive report on the status on spatial distribution of T. equi in Punjab (India) state, thoroughly investigated by molecular and serological techniques, evaluating various environmental and demographic risk factors along with the haemato-biochemical alterations in the exposed animals.