BACKGROUND:Fasciola gigantica (F. gigantica) is a widespread zoonotic trematode that poses a substantial burden on livestock and public health in endemic regions. Elucidating host-parasite interactions relies largely on proteomic data. Excretory-Secretory proteins (ESPs) can play central roles in the host-parasite interactions, immune evasion, and parasite pathogenicity; however, proteomic information on F. gigantica infecting buffalo remains scarce, particularly from Pakistan. OBJECTIVES:In this study, we present the first comprehensive proteomic characterization of adult F. gigantica recovered from naturally infected buffaloes in Punjab, Pakistan, with a specific focus on putative excretory-secretory proteins. METHODOLOGY:Somatic protein extracts from adult flukes were initially assessed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, confirming a high abundance and broad molecular weight distribution of parasite proteins. Subsequent liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis generated the largest proteomic dataset for F. gigantica to date, a total of 4,777 proteins. In‒silico prediction using DeepLoc 2.0, SignalP, and transmembrane helices (TMHMM), enabled the identification of a substantial repertoire of putative ESPs, including both classical secretory proteins possessing N-terminal signal peptides and non-classical secreted proteins lacking transmembrane helices. Functional annotation and Gene Ontology (GO) enrichment analysis revealed that these proteins were predominantly associated with catalytic, hydrolase, and oxidoreductase activities, highlighting their roles in tissue invasion and nutrient acquisition. CONCLUSIONS:Among 4,777 proteins, 95 proteins associated with molecular functions, 77 in biological processes, and 65 in cellular components were linked to immune regulation, stress response, metabolic adaptation, and parasite survival. Pathway enrichment further highlighted their involvement in protein processing, signal transduction, and mechanisms facilitating host invasion and chronic infection. Collectively, these findings emphasize the vital role of proteins (somatic/putative secreted) in orchestrating host-parasite interactions and sustaining chronic infection.

Fasciolosis caused by Fasciola gigantica is a parasitic disease affecting livestock and occasionally humans in tropical and subtropical regions of Africa and Asia. Increasing reports of triclabendazole resistance have emphasized the need for alternative control strategies and vaccine development. Calcium-binding proteins are promising antigenic targets involved in host-parasite interactions. This study aimed to clone, express, and characterize tegumental calcium-binding protein-4 (TCBP-4) of F. gigantica, and to evaluate its immunomodulatory effects on host peripheral blood mononuclear cells (PBMCs). The TCBP-4 gene was amplified and cloned into the pET-28a(+) expression vector in Escherichia coli BL21(DE3). The recombinant protein (rFg-TCBP-4) was purified, and verified using SDS-PAGE and Western blotting. Bioinformatic analyses predicted physicochemical and structural properties. Interaction of rFg-TCBP-4 with PBMCs was examined by immunofluorescence. The immunomodulatory effects of rFg-TCBP-4 were evaluated by measuring cytokine production using enzyme-linked immunosorbent assay (ELISA), along with nitric oxide production, and PBMC proliferation and migration assays. The TCBP-4 gene (∼576 bp) was expressed as a recombinant protein with a molecular weight of ∼26.2 kDa. Bioinformatic analyses revealed conserved EF-hand calcium-binding motifs and predicted cytoplasmic localization. rFg-TCBP-4 specifically interacted with PBMCs and modulated cytokine production in a dose-dependent manner, characterized by decreased levels of pro-inflammatory cytokines (IFN-γ, IL-17, and TNF-α) and increased levels of regulatory cytokines (IL-4, IL-10, and TGF-β). In addition, rFg-TCBP-4 enhanced nitric oxide production, PBMC proliferation, and migration. rFg-TCBP-4 possesses immunomodulatory properties and may contribute to host immune regulation during F. gigantica infection. This protein represents a promising antigen for further investigation in vaccine development against fasciolosis.

Echinococcus granulosus sensu lato (s.l.), the causative agent of cystic echinococcosis (CE) cause enormous health, production, and economic impacts on livestock and human population. Buffaloes play a central role in Pakistan's livestock economy, and often serve as key intermediate hosts in the transmission of E. granulosus s.l. However, little is known about CE in buffaloes and associated productive and financial risks in southern Punjab, Pakistan. Out of 1689 buffaloes (male-678, female-1011), 256 were found infected (95%CI = 13.45-16.87), with a 15.16% prevalence (X2 = 30.30). Samples primarily the liver, lungs, heart, spleen, and kidneys were collected from three cities of Bahawalpur division demonstrated maximum prevalence (18.1%) in Bahawalpur (95%CI = 15.35-20.85). The prevalence was higher in females (19.09%) (95%CI = 16.67-21.33) than males (9.29%), (95%CI = 7.10-11.48). The highest age-wise prevalence (21.72%) was found in older buffaloes >7 years (95%CI = 19.03-24.41), followed by 11.34% in >5-≤7 years (95%CI = 8.35-14.33) and 3.09% in >3-≤5 years (95%CI = 1.30-4.90). The most favorable organ for cyst development was the liver (11.78%), followed by the lungs (3.37%, X2 = 85.22). The partial mitochondrial cytochrome oxidase 1 (COX1) sequence was used for molecular characterization of randomly selected five buffalo isolates of E. granulosus sensu stricto (s.s). Sequence analysis showed that five E. granulosuss.l. isolates obtained from buffaloes were 96.4-99.15% similar to E. granulosus s.s. but differed from other reference sequences by significant nucleotide variations, such as G1/G3 GenBank sequences BE1(G1/98.10%; G3/98.88%), BE2(G1/97.83%; G3/98.60%), BE3(G1/97.61%; G3/97.13%), BE4(G1/96.40%; G3/97.12%), and BE5 (G1/97.20%; G3/99.15%). The presence of notable micronucleotide variations suggests local strain adaptation, which may influence transmission dynamics and host compatibility. These findings demonstrate that buffaloes, particularly older females play a critical role in sustaining CE transmission in the study area. Their infection status, combined with close interactions among livestock, Canidae, and humans, creates a high-risk interface for environmental contamination and spill-over of CE into human populations. Targeted control measures focusing on slaughter hygiene, dog access to infected offal, and surveillance of high-risk livestock groups are therefore essential for reducing zoonotic transmission.

BACKGROUND:The identification of B-cell epitopes (BCEs) is fundamental to advancing epitope-based vaccine design, therapeutic antibody development, and diagnostics, such as in neglected tropical diseases caused by parasitic pathogens. However, the structural complexity of parasite antigens and the high cost of experimental validation present certain challenges. Advances in Artificial Intelligence (AI)-driven protein engineering, particularly through machine learning and deep learning, offer efficient solutions to enhance prediction accuracy and reduce experimental costs. METHODOLOGY/PRINCIPAL FINDINGS:Here, we present deepBCE-Parasite, a Transformer-based deep learning model designed to predict linear BCEs from peptide sequences. By leveraging a state-of-the-art self-attention mechanism, the model achieved remarkable predictive performance, achieving an accuracy of approximately 81% and an AUC of 0.90 in both 10-fold cross-validation and independent testing. Comparative analyses against 12 handcrafted features and four conventional machine learning algorithms (GNB, SVM, RF, and LGBM) highlighted the superior predictive power of the model. As a case study, deepBCE-Parasite predicted eight BCEs from the leucine aminopeptidase (LAP) protein in Fasciola hepatica proteomic data. Dot-blot immunoassays confirmed the specific binding of seven synthetic peptides to positive sera, validating their IgG reactivity and demonstrating the model's efficacy in BCE prediction. CONCLUSIONS/SIGNIFICANCE:deepBCE-Parasite demonstrates excellent performance in predicting BCEs across diverse parasitic pathogens, offering a valuable tool for advancing the design of epitope-based vaccines, antibodies, and diagnostic applications in parasitology.

The lumpy skin disease virus (LSDV) is a member of the Capripox genus of the Poxviridae family. It is the causative agent of lumpy skin, a highly contagious disease of cattle, water buffalo, sheep, and goats. In 2022, several outbreaks of LSD were reported in the Cholistan region of Pakistan, which has a large population of livestock living in arid conditions. A total of 230 blood/serum and scab samples were collected from three LSD outbreak locations. Clinically, affected cattle showed acute clinical signs characterized by skin nodules, fever, enlarged lymph nodes, emaciation, and lower leg edema. Hematological findings revealed non-significant changes in red blood cell and white blood cell counts (some animals had leukocytosis while others were leukopenic) whereas, hemoglobin level were significantly low. Platelet count, MPV, PCT, , P-LRC, and P-LCC were elevated. Granulocytes were significantly low in LSD affected cattle while lymphocyte counts were significantly high. Serological findings revealed elevated protein levels, along with high creatinine and ALT concentrations. Amplification of DNA-dependent RNA polymerase 30 kDa subunit gene (RPO30) confirmed the presence of LSD virus in all suspected samples. Phylogenetic analysis showed that all Pakistani isolates clustered closely with isolates from neighboring countries. The SNPs differences were less than 20 among these isolates, indicating their close resemblance with each other. It can, therefore, be inferred that our LSD strains might be originated from neighboring Asian countries, that were affected by LSD in previous years.

Haemonchus contortus poses a global challenge as a parasite affecting small ruminants, yet the problem of absence of an effective vaccine against H. contortus infection still exists. This investigation sought to appraise the immunological reaction induced by recombinant H. contortus excretory/secretory-24 (rHcES-24) in combination with complete Freund’s adjuvant (CFA) and bio-polymeric nanoparticles (NPs) within a murine model. In this study, rHcES-24 was encapsulated in poly(d, l-lactide-co-glycolide) (PLGA) and chitosan (CS) NPs, administered subcutaneously to mice. Researchers analyzed the NPs using scanning electron microscope (SEM) and assessed lymphocyte proliferation, specific antibodies, cytokines, T cell proliferation (CD3e+CD4+, CD3e+CD8a+), and phenotypic alteration in splenocytes (CD11c+CD83+, CD11c+CD86+) through flow cytometry to understand the immune response. The results demonstrated that the administration of nanovaccines (NVs) prompted immune responses towards Th1 pathway. This was indicated by notable enhancements in the production of specific antibodies, heightened cytokine levels, and a robust proliferation of lymphocytes observed in mice that received the NVs compared to control groups. Remarkably, mice vaccinated with the antigen-loaded NPs formulations exhibited considerably higher proportions of splenic dendritic cells (DCs) and T cells in comparison to those receiving the traditional adjuvant or the control groups. Incorporating HcES-24 protein into NPs effectively conferred immunity against H. contortus, paving the way for developing a targeted and commercial vaccine.

Enterotoxaemia is a severe disease caused by Clostridium perfringens and render high mortality and huge economic losses in livestock. However, scanty information and only few cases are reported about the presence and patho-physiology of enterotoxaemia in camels. The bacterium induces per-acute death in animals due to rapid production of different lethal toxins. The necropsy of camels (per-acute = 15, acute = 3) was conducted at 18 outbreaks of enterotoxaemia in camels in the desert area of Bahawalpur region. At necropsy, the serosal surfaces of visceral organs in the abdominal, peritoneal and thoracic cavities were found to have petechiation with severe congestion. Moreover, both the cut-sections of different visceral organs and the histo-pathological analysis revealed the pathological lesions in heart, lungs, kidneys, spleen, small and large intestines. Grossly, the kidneys were severely congested, hyperemic, swollen and softer in consistency. Under the microscope, different sections of kidneys indicated that the convulated and straight tubules were studded with erythrocytes. In the intestines, there were stunting fusion of crypts and villi. Similarly, various histo-pathological ailments were also observed in the heart, lungs and spleen. At blood agar, the collected samples showed beta hemolytic colonies of C. perfringens that appeared as medium sized rods microscopically and stained positively on Gram staining. Multiplex PCR revealed C. perfringens type A (α and β2 genes) and D (epsilon gene) and the deaths were found to be significantly higher due to C. perfringens type D compared to those by C. perfringens type A. Hence, it has been concluded that enterotoxaemia in camel affects multiple organs and becomes fatal, if occurred due to C. perfringens type D.

Background: Haemonchus contortus (H. contortus), a nematode with global prevalence, poses a major threat to the gastrointestinal health of sheep and goats. In an effort to combat this parasite, a nanovaccine was created using a recombinant ADP-ribosylation factor 1 (ARF1) antigen encapsulated within poly lactic-co-glycolic acid (PLGA). This study aimed to assess the effectiveness of this nanovaccine in providing protection against H. contortus infection. Methods: Fifteen goats were randomly divided into three groups. The experimental group received two doses of the PLGA encapsulated rHcARF1 (rHcARF1-PLGA) nanovaccine on days 0 and 14. Fourteen days after the second immunization, both the experimental and positive control groups were challenged with 8000 infective larvae (L3) of H. contortus, while the negative control group remained unvaccinated and unchallenged. At the end of the experiment on the 63rd day, all animals were humanly euthanized. Results: The results showed that the experimental group had significantly higher levels of sera IgG, IgA, and IgE antibodies, as well as increased concentrations of cytokines, such as IL-4, IL-9, IL-17, and TGF-β, compared to the negative control group after immunization. Following the L3 challenge, the experimental group exhibited a 47.5% reduction in mean eggs per gram of feces (EPG) and a 55.7% reduction in worm burden as compared to the positive control group. Conclusions: These findings indicate that the nanovaccine expressing rHcARF1 offers significant protective efficacy against H. contortus infection in goats. The results also suggest the need for more precise optimization of the antigen dose or a reassessment of the vaccination regimen. Additionally, the small sample size limits the statistical rigor and the broader applicability of the findings.

Legumains belonging to C_13 peptidase family of proteins, and are ubiquitously disseminated among all vertebrate and invertebrate organisms, and have been implicated in innumerable biological and cellular functionality. Herein, we characterized and evaluated immunoregulatory characteristics of Legumain-1 from Fasciola gigantica (Fg-LGMN-1) during its interaction with host immune cells. The isopropyl-ß-d-thiogalactopyranoside (IPTG) stimulated RFg-LGMN-1 protein was positively detected by rat serum containing anti-RFg-LGMN-1 polyclonal antibodies. Furthermore, the uptake of RFg-LGMN-1 by goat monocytes was successfully confirmed using Immunofluorescence Assay (IFA). The immunohistochemical analysis revealed the native localization of LGMN-1 protein on the periphery and internal structures such as suckers, pharynx, and genital pore of the adult parasite, thereby validating its presence in excretory-secretory (ES) products of F. gigantica. The RFg-LGMN-1 co-incubated with concanavalin-A (Con-A) stimulated the increase of interleukin 2 (IL-2), IL-10, and IL-17 in monocytes derived from peripheral blood mononuclear cells (PBMCs) in the concentration-dependent manner. However, the IL-4 cytokine in response to the RFg-LGMN-1 protein declined. These results illuminated the role of LGMN-1 during the parasite-host interface. Our findings elaborated additional evidence that Legumain protein play a role in the manipulating host immune responses during parasite infections. However, further evaluation of RFg-LGMN-1 protein in context of its immunomodulatory roles should be conducted to enhance our understandings of the mechanisms employed by F. gigantica to evade host immune responses.

Haemonchus contortus (H. contortus), a globally distributed nematode, is recognized as a significant pathogenic agent affecting the gastrointestinal health of both sheep and goats. In the context of addressing this parasitic threat, a nanovaccine was developed, comprising a recombinant ARF1 antigen encapsulated within the biopolymer PLGA (poly (lactic-co-glycolic acid) nanomaterial. The aim of this investigation was to evaluate the effectiveness of this nanovaccine in conferring defense against H. contortus infection, with fifteen goats evenly divided into three groups. The experimental group received two immunizations with the nanovaccine (rHcARF1-PLGA) on days 0 and 14. Subsequently, after a 14-day interval following the second immunization, both the experimental and positive control groups were subjected to a challenge with 8000 infective larvae (L3) of H. contortus. The negative control group remained unvaccinated and was not exposed to L3 challenge. The findings revealed a significant increase in sera IgG, IgA and IgE antibody concentrations, and heightened levels of cytokines, including IL-4, IL-9, IL-17, and TGF-β in the experimental group post-immunization compared to the negative control. Following the L3 challenge, the experimental group exhibited a remarkable reduction of 47.5% and 55.7% in mean eggs per gram of feces (EPG) and worm burdens, respectively. In conclusion, this investigation demonstrates the partial protective potential of the nanovaccine expressing rHcARF1 against H. contortus infection in goats.

Theileria annulata (T. annulata) is intra-erythrocytic protozoan parasite which is more prevalent in tropical and sub-tropical countries. It has a significant economic impact on the productivity of the dairy industry, and buparvaquone is used to treat infected animals in the prevalent regions of the world. Systematically, buparvaquone targets the cyto-b gene to break the electron transport chain (ETC) and Theileria annulata peptidyl-prolyl isomerase 1 (TaPIN1) gene to destabilize transcription factor JUN (c-JUN) to inhibit proliferation of infected cells, which ultimately leads to the death of T. annulata. The reported studies on drug resistance is due to inappropriate drug application, evolutionary characteristics of the cytochrome b (cyto-b) gene and oncogenic signaling pathways gene (TaPIN1) make the parasite resistant against buparvaquone. Hence, this systematic review was designed to find out non-synonymous mutation in genes (cyto-b and TaPIN1) responsible for drug resistance reported from Tunisia, Turkey, Egypt, Sudan, Iran, Pakistan, China and Germany with reference to the T. annulata Ankara strain of cyto-b (accession no. XM_949625.1) and TaPIN1 (accession no. TA18945) wild type genes. Non-synonymous point mutations were found in cyto-b (Q01 at 130-148 and Q02 at 253-262 regions) and TaPIN1 (A53P and A53T) genes. These point mutations are responsible for developing buparvaquone resistance against T. annulata infection. These genes can be used as biomarkers for the identification of drug resistance in any endemic area. To avoid the complication of drug resistance, development of genetically resistant cattle breeds, potent vaccines and anti-theilerial drugs (Trifloxystrobin and anti-cancerous) are currently required to control proliferating economically important T. annulata parasites.

Haemonchus contortus (H. contortus) is a gastrointestinal parasite affecting small ruminants, leading to a significant decline in animal productivity. In this study, we developed a nanovaccine by encapsulating the recombinant protein rHcES-15, derived from the excretory/secretory products of H. contortus, within biodegradable poly (D, L-lactide-co-glycolide) (PLGA) nanoparticles (NPs). To construct the nanovaccine, PLGA NPs were prepared using a modified double emulsion solvent evaporation technique. Scanning electron microscopy (SEM) illustrated successful encapsulation of rHcES-15 within PLGA NPs, with a size ranging between 350-400 nm. The encapsulation efficiency (EE) of the antigen in the nanovaccine was determined to be 72%. A total of forty experimental mice were divided into five groups, receiving the nanovaccine on day 0 and being humanely sacrificed at the end of the 14-day trial. The stimulation index (SI) from mice vaccinated with the nanovaccine indicated an amplified lymphocyte proliferation and a significant increase in anti-inflammatory cytokines (IL-4, IL-10, and IL-17). Furthermore, the percentages of T-cells (CD4+, CD8+) and dendritic cell phenotypes (CD83+, CD86+) were substantially upregulated in mice immunized with the nanovaccine compared to control groups and the rHcES-15 group. Similarly, higher levels of antigen-specific serum immunoglobulins (IgG1, IgG2a, IgM) were observed in response to the nanovaccine compared to both the antigenic (rHcES-15) and control groups. In conclusion, the data strongly supports the notion that encapsulation of rHcES-15 within PLGA NPs effectively stimulates immune cells in vivo, ultimately augmenting antigen-specific adaptive immune responses against H. contortus. This discovery highlights the promising potential of the nanovaccine, justifying additional investigations to ascertain its efficacy finally.

The present study was designed to investigate the prevalence, epizootological profile, and haematological and serum biochemistry changes in coccidiosis infected pet dogs of Rawalpindi-Islamabad, Pakistan. For this purpose, 150 blood and faecal samples were collected from dogs of various breeds. Data regarding the epizootolgical risk factors were collected through a questionnaire at the time of sample collection. The faecal examination illustrated a 16.67% prevalence of coccidiosis in dogs. Data analysis of associated risk factors revealed that locality (P = 0.001), domestication (P = 0.000), and sanitary conditions (P = 0.000) have a significant impact. Whereas there was a nonsignificant association among age, gender, breed and faecal consistency in coccidiosis infected animals. Various hematological parameters were compared and revealed the non-significant difference in coccidiosis positive samples compared to the control group. The biochemical parameters showed significant differences in serum total protein (P = 0.001) and serum albumin (P = 0.003) among infected and control animal samples. This study findings provide baseline data and valuable information to the owners, public health authorities, researchers and the veterinary community on the risk factors, complications and probable transmission of canine coccidiosis.

Haemonchus contortus is a gastrointestinal parasite that adversely impacts small ruminants, resulting in a notable reduction in animal productivity. In the current investigation, we developed a nanovaccine by encapsulating the recombinant protein rHcES-15, sourced from the excretory/secretory products of H. contortus, within biodegradable poly (D, L-lactide-co-glycolide) (PLGA) nanoparticles (NPs). The development of this nanovaccine involved the formulation of PLGA NPs using a modified double emulsion solvent evaporation technique. Scanning electron microscopy (SEM)verified the successful encapsulation of rHcES-15 within PLGA NPs, exhibiting a size range of 350-400 nm. The encapsulation efficiency (EE) of the antigen in the nanovaccine was determined to be 72%. A total of forty experimental mice were allocated into five groups, with the nanovaccine administered on day 0 and the mice euthanized at the end of the 14-day trial. The stimulation index (SI) from the mice subjected to the nanovaccine indicated heightened lymphocyte proliferation (*** p < 0.001) and a noteworthy increase in anti-inflammatory cytokines (IL-4, IL-10, and IL-17). Additionally, the percentages of T-cells (CD4(+), CD8(+)) and dendritic cell phenotypes (CD83(+), CD86(+)) were significantly elevated (** p < 0.01, *** p < 0.001) in mice inoculated with the nanovaccine compared to control groups and the rHcES-15 group. Correspondingly, higher levels of antigen-specific serum immunoglobulins (IgG1, IgG2a, IgM) were observed in response to the nanovaccine in comparison to both the antigenic (rHcES-15) and control groups (* p < 0.05, ** p < 0.01). In conclusion, the data strongly supports the proposal that the encapsulation of rHcES-15 within PLGA NPs effectively triggers immune cells in vivo, ultimately enhancing the antigen-specific adaptive immune responses against H. contortus. This finding underscores the promising potential of the nanovaccine, justifying further investigations to definitively ascertain its efficacy.

Eimeria maxima (E. maxima) are an intracellular apicomplexan protozoan that causes intestinal coccidiosis in chickens. The purpose of this research was to develop a novel delivery approach for recombinant E. maxima (rEm) 14-3-3 antigen to elicit enhanced immunogenic protection using poly (D, L-lactide-co-glycolide) (PLGA) and chitosan (CS) nanoparticles (NPs) against E. maxima challenge. The morphologies of prepared antigen-loaded NPs (PLGA/CS-rEm14-3-3 NPs) were visualized by a scanning electron microscope. The rEm14-3-3 and PLGA/CS-rEm14-3-3 NPs–immunized chicken-induced changes of serum cytokines, IgY-antibody level, and T-lymphocyte subsets and protective efficacies against E. maxima challenge were evaluated. The results revealed that encapsulated rEm14-3-3 in PLGA and CS NPs presented spherical morphology with a smooth surface. The chickens immunized with only rEm14-3-3 and PLGA/CS-rEm14-3-3 NPs elicited a significant (p<0.05) higher level of IFN-γ cytokine, stimulated the proportions of CD4+/CD3+, CD8+/CD3+ T-cells, and provoked sera IgY-antibody immune response compared to control groups (PBS, pET-32a, PLGA, and CS). Whereas, PLGA-rEm14-3-3 NP–immunized chicken provoked a higher level of IFN- γ production and IgY-antibody response rather than CS-rEm14-3-3 and bare antigen, relatively. The animal experiment results ratified that PLGA-rEm14-3-3 NP–immunized chicken significantly alleviated the relative body weight gain (%), decreased lesion score, and enhanced oocyst decrease ratio compared to CS-rEm14-3-3 NPs and only rEm14-3-3. The anti-coccidial index of the chicken vaccinated with the PLGA-rEm14-3-3 NPs was (180.1) higher than that of the Cs-rEm14-3-3 NPs (167.4) and bare antigen (165.9). Collectively, our statistics approved that PLGA NPs might be an efficient antigen carrier system (Em14-3-3) to act as a nanosubunit vaccine that can improve protective efficacies in chicken against E. maxima challenge.

Intestinal parasitic infection is one of the major challenges in obtaining optimal production and maintaining the health and welfare of all animals including cattle and buffaloes. Anti-parasitic treatments appear to be a reliable countermeasure. However, the effectiveness and selection of suitable anthelmintics require situational assessments in a given locality. In the current study, the efficacy and impact of benzimidazole (albendazole) were assessed in a total of 400 (100 each) on the performance of buffaloes, buffalo-heifer, cattle, and cattle-heifers at two commercial dairy farms in the Province of Punjab, Pakistan. Additionally, the cost-benefit ratio was calculated by assessing the inputs (medication, feed, and labor cost) and outputs (milk and weight gain). The qualitative and quantitative examination of helminth eggs in each type of animal indicated a prevalence of 73.3, 78.3, 76.6, and 85.0% in cattle, cattle-heifers, buffaloes, and buffaloes-heifers, respectively. Specifically, a highest rate (10.0-13.3%) of Haemonchus sp. infection was only observed in cattle and heifers, while Fasciola sp. infections (10.0-11.6%) were the most often found species in buffaloes and heifers. The highest anthelmintic impacts (egg per gram of feces, p < 0.001) were observed on day 14 post-medication. Until 60 days of post-anthelmintic treatment, an average increase of 0.8 and 0.7 L in milk production per day in cattle and buffaloes, respectively while a total of 11.45 and 9.45 kg body weight were noticed in cattle-heifer and buffaloes-heifer, respectively. Cumulative cost-benefit analysis indicated a positive correlation between treated and non-treated animals. These findings reiterate the importance of anthelmintic drugs in reducing the impacts of parasites on the productivity, health, and well-being of an animal under high infection challenges.

In the present study, we used an isobaric tag for relative and absolute quantitation (iTRAQ) proteomics technology to characterize the differentially expressed proteins (DEPs) in the liver, hepatic lymph nodes (hLNs), and spleen of buffaloes infected with Fasciola gigantica (F. gigantica). We also used the parallel reaction monitoring (PRM) method to verify the expression levels of the DEPs in the three infected tissues. At three days post-infection (dpi), 225, 1821, and 364 DEPs were detected in the liver, hLNs, and spleen, respectively. At 42 dpi, 384, 252, and 214 DEPs were detected in the liver, hLNs, and spleen, respectively. At 70 dpi, 125, 829, and 247 DEPs were detected in the liver, hLNs, and spleen, respectively. Downregulation of metabolism was prominent in infected livers at all time points, and upregulation of immune responses was marked in the hLNs during early infection (three dpi); however, no changes in the immune response were detected at the late stages of infection (42 and 70 dpi). Compared to the hLNs, there was no significant upregulation in the levels of immune responses in the infected spleen. All the identified DEPs were used to predict the subcellular localization of the proteins, which were related to extracellular space and membrane and were involved in host immune responses. Further PRM analysis confirmed the expression of 18 proteins. These data provide the first simultaneous proteomic profiles of multiple organs of buffaloes experimentally infected with F. gigantica.

Toxoplasmosis, caused by the intracellular protozoon Toxoplasma gondii, is a significant parasitic zoonosis with a world-wide distribution. As a main transmission route, human infection can be acquired by the ingestion of T. gondii oocysts from the environment (e.g., soil, water, fruits and vegetables). Regarding the detection of T. gondii oocysts in environmental samples, the development of a time-saving, cost-effective and highly sensitive technique is crucial for the surveillance, prevention and control of toxoplasmosis. In this study, we developed a new method by combining recombinase-aided amplification (RAA) with CRISPR-Cas12a, designated as the RAA-Cas12a-Tg system. Here, we compared this system targeting the 529 bp repeat element (529 bp-RE) with the routine PCR targeting both 529 bp-RE and ITS-1 gene, respectively, to assess its ability to detect T. gondii oocysts in soil samples. Our results indicated that the 529 bp RE-based RAA-Cas12a-Tg system was able to detect T. gondii successfully in nearly an hour at body temperature and was more sensitive than the routine PCR assay. The sensitivity of this system reached as low as 1 fM with high specificity. Thus, RAA-Cas12a-Tg system provided a rapid, sensitive and easily operable method for point-of-care detection of T. gondii oocysts in soil, which will facilitate the control of T. gondii infection in humans and animals.