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Turkiye Parazitolojii Dergisi Jun 2024() is an obligate intracellular, zoonotic protozoan parasite of interest to physicians and veterinarians with its highly complex structure. It is known to infect about... (Review)
Review
() is an obligate intracellular, zoonotic protozoan parasite of interest to physicians and veterinarians with its highly complex structure. It is known to infect about one-third of the world's population. Since it is a zoonotic disease, it is necessary to keep the animal population under control in order to prevent human exposure. Many studies have been conducted on the detection of and it has been determined that there are three clonal groups consisting of types 1, 2, 3. Developments in molecular studies have led to changes in the taxonomy and new developments in parasitic diseases. It has helped in diagnosis, treatment, development of antiparasitic drugs and research on resistance. They also provided research on vaccine studies, genetic typing and phylogenetics of parasitic diseases. Conventional polymerase chain reaction (PCR), real-time PCR and genotyping studies conducted today increase our knowledge about . Methods such as genes and 18S rRNAs are mostly used in PCR, and methods such as MS, MLST, PCR-RFLP, RAPD-PCR and HRM are used in genotyping. Toxoplasmosis is a disease that is within the framework of the concept of one health and must attract attention, has not yet been eradicated in the world and needs joint studies for humans, animals and ecosystems to be eradicated. This can only be possible by establishing interdisciplinary groups, conducting surveys and training.
Topics: Toxoplasma; Animals; Humans; Toxoplasmosis; Toxoplasmosis, Animal; Zoonoses; Genotype
PubMed: 38958491
DOI: 10.4274/tpd.galenos.2024.70298 -
Turkiye Parazitolojii Dergisi Jun 2024and are enteric protozoa that can cause a variety of gastrointestinal diseases, especially in vulnerable people like children, the elderly, and those with impaired...
OBJECTIVE
and are enteric protozoa that can cause a variety of gastrointestinal diseases, especially in vulnerable people like children, the elderly, and those with impaired immune systems. In order to ascertain the microbiological quality of the recreational water from Araromi Beach in Ilaje Local Government Area, Ondo State, Nigeria. This risk assessment is of great significance to human health protection against waterborne diseases. The aim of this study was to determine the microbial quality of recreational water from Araromi Beach in Ilaje Local Government Area, Ondo State, Nigeria.
METHODS
Microscopic examination of and oocysts were done.
RESULTS
Results revealed maximum occurrence of (20 oocysts/100 mL) of water sample in the month of April and maximum occurrence of (300 cysts/100 mL) of water sample in the month of June. Additionally, according to Kolmogorov-Smirnov tests for normalcy Ho =0.05, and were not regularly distributed in the water samples collected from the beach throughout the study period. The average likelihood of contracting and infections after consuming 100 mL of beach water was 0.96 and 0.35, respectively. The risks of infection associated with was lower than those associated with in water from the beach, but were both above the acceptable risk limit of 10-4.
CONCLUSION
The results of this study indicate that and may represent serious health hazards to people who engage in aquatic activities. Adopting a comprehensive strategy that includes regular inspections, enhanced detection techniques, and the prevention of aquatic environment pollution may provide clean and safe recreational water for all, thereby safeguarding the public's health.
Topics: Cryptosporidium parvum; Giardia lamblia; Nigeria; Humans; Seawater; Risk Assessment; Water Microbiology; Giardiasis; Cryptosporidiosis; Recreation; Oocysts
PubMed: 38958402
DOI: 10.4274/tpd.galenos.2024.69733 -
Memorias Do Instituto Oswaldo Cruz 2024Chagas disease is a tropical neglected disease that affects millions of people worldwide, still demanding a more effective and safer therapy, especially in its chronic... (Review)
Review
Chagas disease is a tropical neglected disease that affects millions of people worldwide, still demanding a more effective and safer therapy, especially in its chronic phase which lacks a treatment that promotes substantial parasitological cure. The technical note of Romanha and collaborators published in 2010 aimed establish a guideline with the set of minimum criteria and decision gates for the development of new agents against Trypanosoma cruzi with the focus on developing new antichagasic drugs. In this sense, the present review aims to update this technical note, bringing the state of the art and new advances on this topic in recent years.
Topics: Chagas Disease; Trypanocidal Agents; Animals; Trypanosoma cruzi; Drug Evaluation, Preclinical; Disease Models, Animal; Humans; Drug Development
PubMed: 38958341
DOI: 10.1590/0074-02760240057 -
Ethiopian Journal of Health Sciences Jan 2024
Topics: Humans; Malaria, Vivax; Antimalarials; Plasmodium vivax; Aminoquinolines
PubMed: 38957344
DOI: 10.4314/ejhs.v34i1.1 -
Parasites & Vectors Jul 2024Toxoplasma gondii infection affects a significant portion of the global population, leading to severe toxoplasmosis and, in immunocompromised patients, even death....
BACKGROUND
Toxoplasma gondii infection affects a significant portion of the global population, leading to severe toxoplasmosis and, in immunocompromised patients, even death. During T. gondii infection, disruption of gut microbiota further exacerbates the damage to intestinal and brain barriers. Therefore, identifying imbalanced probiotics during infection and restoring their equilibrium can regulate the balance of gut microbiota metabolites, thereby alleviating tissue damage.
METHODS
Vimentin gene knockout (vim-/-) mice were employed as an immunocompromised model to evaluate the influence of host immune responses on gut microbiota balance during T. gondii infection. Behavioral experiments were performed to assess changes in cognitive levels and depressive tendencies between chronically infected vim-/- and wild-type (WT) mice. Fecal samples were subjected to 16S ribosomal RNA (rRNA) sequencing, and serum metabolites were analyzed to identify potential gut probiotics and their metabolites for the treatment of T. gondii infection.
RESULTS
Compared to the immunocompetent WT sv129 mice, the immunocompromised mice exhibited lower levels of neuronal apoptosis and fewer neurobehavioral abnormalities during chronic infection. 16S rRNA sequencing revealed a significant decrease in the abundance of probiotics, including several species of Lactobacillus, in WT mice. Restoring this balance through the administration of Lactobacillus murinus and Lactobacillus gasseri significantly suppressed the T. gondii burden in the intestine, liver, and brain. Moreover, transplantation of these two Lactobacillus spp. significantly improved intestinal barrier damage and alleviated inflammation and neuronal apoptosis in the central nervous system. Metabolite detection studies revealed that the levels of various Lactobacillus-related metabolites, including indole-3-lactic acid (ILA) in serum, decreased significantly after T. gondii infection. We confirmed that L. gasseri secreted much more ILA than L. murinus. Notably, ILA can activate the aromatic hydrocarbon receptor signaling pathway in intestinal epithelial cells, promoting the activation of CD8 T cells and the secretion of interferon-gamma.
CONCLUSION
Our study revealed that host immune responses against T. gondii infection severely disrupted the balance of gut microbiota, resulting in intestinal and brain damage. Lactobacillus spp. play a crucial role in immune regulation, and the metabolite ILA is a promising therapeutic compound for efficient and safe treatment of T. gondii infection.
Topics: Animals; Gastrointestinal Microbiome; Mice; Toxoplasma; Mice, Knockout; Brain Injuries; Probiotics; Brain; Lactobacillus; Disease Models, Animal; Immunocompromised Host; Toxoplasmosis; RNA, Ribosomal, 16S; Male; Intestines
PubMed: 38956725
DOI: 10.1186/s13071-024-06349-8 -
Zhongguo Xue Xi Chong Bing Fang Zhi Za... Jun 2024To investigate the development and dynamic changes of cysts in the brain of mice following infection with different forms of , so as to provide insights into for...
OBJECTIVE
To investigate the development and dynamic changes of cysts in the brain of mice following infection with different forms of , so as to provide insights into for toxoplasmosis prevention and control.
METHODS
ICR mice at ages of 6 to 8 weeks, each weighing 20 to 25 g, were intraperitoneally injected with tachyzoites of the PRU strain at a dose of 1 × 10 tachyzoites per mouse, orally administered with cysts at a dose of 20 oocysts per mouse or oocysts at a dose of 200 oocysts per mouse for modeling chronic infection in mice, and the clinical symptoms and survival of mice were observed post-infection. Mice were orally infected with cysts at doses of 10 (low-dose group), 20 (medium-dose group), 40 cysts per mouse (high-dose group), and the effect of different doses of infections on the number of cysts was examined in the mouse brain. Mice were orally administered with cysts at a dose of 20 cysts per mouse, and grouped according to gender (female and male) and time points of infections (20, 30, 60, 90, 120, 150, 180 days post-infection), and the effects of gender and time points of infections on the number of cysts was examined in the mouse brain. In addition, mice were divided into the tachyzoite group (Group T), the first-generation cyst group (Group C1), the second-generation cyst group (Group C2), the third-generation cyst (Group C3) and the fourth-generation cyst group (Group C4). Mice in the Group T were intraperitoneally injected with tachyzoites at a dose of 1 × 10 tachyzoites per mouse, and the cysts were collected from the mouse brain tissues 30 days post-infection, while mice in the Group C1 were orally infected with the collected cysts at a dose of 30 cysts per mouse. Continuous passage was performed by oral administration with cysts produced by the previous generation in mice, and the effect of continuous passage on the number of cysts was examined in the mouse brain.
RESULTS
Following infection with tachyzoites, cysts and oocysts in mice, obvious clinical symptoms were observed on days 6 to 13 and mice frequently died on days 7 to 12. The survival rates of mice were 67.0%, 87.0% and 53.0%, and the mean numbers of cysts were (516.0 ± 257.2), (1 203.0 ± 502.0) and (581.0 ± 183.1) in the mouse brain ( = 11.94, < 0.01) on day 30 post-infection with tachyzoites, cysts and oocysts, respectively, and the numbers of cysts in the brain tissues were significantly lower in mice infected with tachyzoites and oocysts than in those infected with cysts (all values < 0.01). The survival rates of mice were 87.0%, 87.0% and 60.0%, and the mean numbers of cysts were (953.0 ± 355.5), (1 084.0 ± 474.3) and (1 113.0 ± 546.0) in the mouse brain in the low-, medium- and high-dose groups on day 30 post-infection, respectively ( = 0.42, > 0.05). The survival rates of male and female mice were 73.0% and 80.0%, and the mean numbers of cysts were (946.4 ± 411.4) and (932.1 ± 322.4) in the brain tissues of male and female mice, respectively ( = 1.63, > 0.05). Following continuous passage, the mean numbers of cysts were (516.0 ± 257.2), (1 203.0 ± 502.0), (896.8 ± 332.3), (782.5 ± 423.9) and (829.2 ± 306.0) in the brain tissues of mice in the T, C1, C2, C3 and C4 groups, respectively ( = 4.82, < 0.01), and the number of cysts was higher in the mouse brain in Group 1 than in Group T ( < 0.01). Following oral administration of 20 cysts in mice, cysts were found in the moues brain for the first time on day 20 post-infection, and the number of cysts gradually increased over time, peaked on days 30 and 90 post-infection and then gradually decreased; however, the cysts were still found in the mouse brain on day 180 post-infection.
CONCLUSIONS
There is a higher possibility of developing chronic infection in mice following infection with cysts than with oocysts or tachyzoites and the most severe chronic infection is seen following infection with cysts. The number of cysts does not correlate with the severity of chronic infection, and the number of cysts peaks in the mouse brain on days 30 and 90 post-infection.
Topics: Animals; Mice; Female; Male; Mice, Inbred ICR; Brain; Chronic Disease; Toxoplasmosis, Animal; Toxoplasma; Toxoplasmosis; Disease Models, Animal
PubMed: 38952318
DOI: 10.16250/j.32.1374.2024044 -
Zhongguo Xue Xi Chong Bing Fang Zhi Za... Jun 2024To investigate the involvement of the high mobility group box protein B1 (HMGB1)-Toll-like receptor 2 (TLR2)/TLR4-nuclear factor κB (NF-κB) pathway in the intestinal...
OBJECTIVE
To investigate the involvement of the high mobility group box protein B1 (HMGB1)-Toll-like receptor 2 (TLR2)/TLR4-nuclear factor κB (NF-κB) pathway in the intestinal mucosal injury induced by infection, and to examine the effect of oxymatrine (OMT) on in mice.
METHODS
Forty SPF 4-week-old BALB/c mice were randomly divided into four groups, including the control group, infection group, glycyrrhizin (GA) group and OMT group. Each mouse was orally administered with 1 × 10 oocysts one week in the infection, GA and OMT groups following dexamethasone-induced immunosuppression to model intestinal infections in mice. Upon successful modeling, mice in the GA group were intraperitoneally injected with GA at a daily dose of 25.9 mL/kg for successive two weeks, and animals in the OMT group were orally administered OMT at a daily dose of 50 mg/kg for successive two weeks, while mice in the control group were given normal food and water. All mice were sacrificed two weeks post-treatment, and proximal jejunal tissues were sampled. The pathological changes of mouse intestinal mucosal specimens were observed using hematoxylin-eosin (HE) staining, and the mouse intestinal villous height, intestinal crypt depth and the ratio of intestinal villous height to intestinal crypt depth were measured. The occludin and zonula occludens protein 1 (ZO1) expression was determined in mouse intestinal epithelial cells using immunohistochemistry, and the relative expression of , , , myeloid differentiation primary response gene 88 () and was quantified in mouse jejunal tissues using quantitative real-time PCR (qPCR) assay.
RESULTS
HE staining showed that the mouse intestinal villi were obviously atrophic, shortened, and detached, and the submucosal layer of the mouse intestine was edematous in the infection group as compared with the control group, while the mouse intestinal villi tended to be structurally intact and neatly arranged in the GA and OMT groups. There were significant differences among the four groups in terms of the mouse intestinal villous height ( = 6.207, = 0.000 5), intestinal crypt depth ( = 6.903, = 0.000 3) and the ratio of intestinal villous height to intestinal crypt depth ( = 37.190, < 0.000 1). The mouse intestinal villous height was lower in the infection group than in the control group [(321.9 ± 41.1) μm vs. (399.5 ± 30.9) μm; = 4.178, < 0.01] and the GA group [(321.9 ± 41.1) μm vs. (383.7 ± 42.7) μm; = 3.130, < 0.01], and the mouse intestinal crypt depth was greater in the infection group [(185.0 ± 35.9) μm] than in the control group [(128.4 ± 23.6) μm] ( = 3.877, < 0.01) and GA group [(143.3 ± 24.7) μm] ( = 2.710, < 0.05). The mouse intestinal villous height was greater in the OMT group [(375.3 ± 22.9) μm] than in the infection group ( = 3.888, < 0.01), and there was no significant difference in mouse intestinal villous height between the OMT group and the control group ( = 1.989, > 0.05). The mouse intestinal crypt depth was significantly lower in the OMT group [(121.5 ± 27.3) μm] than in the infection group ( = 4.133, < 0.01), and there was no significant difference in mouse intestinal crypt depth between the OMT group and the control group ( = 0.575, > 0.05). The ratio of the mouse intestinal villous height to intestinal crypt depth was significantly lower in the infection group (1.8 ± 0.2) than in the control group (3.1 ± 0.3) ( = 10.540, < 0.01) and the GA group (2.7 ± 0.3) ( = 7.370, < 0.01), and the ratio of the mouse intestinal villous height to intestinal crypt depth was significantly higher in the OMT group (3.1 ± 0.2) than in the infection group ( = 15.020, < 0.01); however, there was no significant difference in the ratio of the mouse intestinal villous height to intestinal crypt depth between the OMT group and the control group ( = 0.404, > 0.05). Immunohistochemical staining showed significant differences among the four groups in terms of occludin ( = 28.031, < 0.000 1) and ZO1 expression ( = 14.122, < 0.000 1) in mouse intestinal epithelial cells. The proportion of positive occluding expression was significantly lower in mouse intestinal epithelial cells in the infection group than in the control group [(14.3 ± 4.5)% vs. (28.3 ± 0.5)%; = 3.810, < 0.01], and the proportions of positive occluding expression were significantly higher in mouse intestinal epithelial cells in the GA group [(30.3 ± 1.3)%] and OMT group [(25.8 ± 1.5)%] than in the infection group ( = 7.620 and 5.391, both values < 0.01); however, there was no significant differences in the proportion of positive occluding expression in mouse intestinal epithelial cells between the GA or OMT groups and the control group ( = 1.791 and 2.033, both values > 0.05). The proportion of positive ZO1 expression was significantly lower in mouse intestinal epithelial cells in the infection group than in the control group [(14.4 ± 1.8)% vs. (24.2 ± 2.8)%; = 4.485, < 0.01], and the proportions of positive ZO1 expression were significantly higher in mouse intestinal epithelial cells in the GA group [(24.1 ± 2.3)%] ( = 5.159, < 0.01) and OMT group than in the infection group [(22.5 ± 1.9)%] ( = 4.441, < 0.05); however, there were no significant differences in the proportion of positive ZO1 expression in mouse intestinal epithelial cells between the GA or OMT groups and the control group ( = 0.037 and 0.742, both values > 0.05). qPCR assay showed significant differences among the four groups in terms of ( = 21.980, < 0.000 1), ( = 20.630, < 0.000 1), ( = 17.000, = 0.000 6), ( = 8.907, = 0.000 5) and expression in mouse jejunal tissues ( = 8.889, = 0.000 7). The relative expression of [(5.97 ± 1.07) vs. (1.05 ± 0.07); = 6.482, < 0.05] 、 [(5.92 ± 1.29) vs. (1.10 ± 0.14); = 5.272, < 0.05] 、 [(5.96 ± 1.50) vs. (1.02 ± 0.03); = 4.644, < 0.05] 、 [(3.00 ± 1.26) vs. (1.02 ± 0.05); = 2.734, < 0.05] and [(2.33 ± 0.72) vs. (1.04 ± 0.06); = 2.665, < 0.05] was all significantly higher in mouse jejunal tissues in the infection group than in the control group. A significant reduction was detected in the relative expression of (0.63 ± 0.01), (0.42 ± 0.10), (0.35 ± 0.07), (0.70 ± 0.11) and (0.75 ± 0.01) in mouse jejunal tissues in the GA group relative to the control group ( = 8.629, 5.830, 11.500, 4.729 and 6.898, all values < 0.05), and the relative expression of , , , and significantly reduced in mouse jejunal tissues in the GA group as compared to the infection group ( = 7.052, 6.035, 4.084, 3.165 and 3.274, all values < 0.05). In addition, the relative expression of (1.14 ± 0.60), (1.00 ± 0.24), (1.14 ± 0.07), (0.96 ± 0.25) and N (1.12 ± 0.17) was significantly lower in mouse jejunal tissues in the OMT group than in the infection group ( = 7.059, 5.320, 3.510, 3.466 and 3.273, all values < 0.05); however, there were no significant differences between the OMT and control groups in terms of relative expression of , , , or in mouse jejunal tissues ( = 0.239, 0.518, 1.887, 0.427 and 0.641, all values > 0.05).
CONCLUSIONS
infection causes intestinal inflammatory responses and destruction of intestinal mucosal barrier through up-regulating of the HMGB1-TLR2/TLR4-NF-κB pathway. OMT may suppress the intestinal inflammation and repair the intestinal mucosal barrier through inhibiting the activity of the HMGB1-TLR2/TLR4-NF-κB pathway.
Topics: Animals; Cryptosporidiosis; Quinolizines; Mice, Inbred BALB C; Cryptosporidium parvum; Toll-Like Receptor 4; Mice; Toll-Like Receptor 2; NF-kappa B; Alkaloids; HMGB1 Protein; Signal Transduction; Male; Intestinal Mucosa; Matrines
PubMed: 38952315
DOI: 10.16250/j.32.1374.2024019 -
Zhongguo Xue Xi Chong Bing Fang Zhi Za... Jun 2024To prepare and characterize the mouse polyclonal antibody against the dense granule protein 24 (GRA24) of , and explore its preliminary applications.
OBJECTIVE
To prepare and characterize the mouse polyclonal antibody against the dense granule protein 24 (GRA24) of , and explore its preliminary applications.
METHODS
The GRA24 coding sequences of different strains were aligned using the MEGA-X software, and the dominant peptide of the GRA24 protein was analyzed with the Protean software. The base sequence encoding this peptide was amplified using PCR assay and ligated into the pET-28a vector, and the generated GRA24 truncated protein was transformed into BL21. After induction by isopropyl-beta-D-thiogalactopyranoside (IPTG), the expression and purification of the recombinant GRA24 protein was analyzed using sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE). BALB/c mice were immunized by subcutaneous injection with the purified recombinant GRA24 truncated protein to generate the polyclonal antibody, and the titer of the polyclonal antibody was measured using enzyme linked immunosorbent assay (ELISA). The specificity of the polyclonal antibody was tested using Western blotting, and the intracellular localization of the polyclonal antibody was investigated using immunofluorescence assay (IFA).
RESULTS
SDS-PAGE showed successful construction of the recombinant expression plasmid, and Coomassie brilliant blue staining showed the generation of the high-purity recombinant GRA24 truncated protein. ELISA measured that the titer of the polyclonal antibody against the GRA24 truncated protein was higher than 1:208 400, and Western blotting showed that the polyclonal antibody was effective to recognize the endogenous GRA24 proteins of different strains and specifically recognize the recombinant GRA24 truncated protein. Indirect IFA showed that the GRA24 protein secreted 16 hour following invasion in host cells.
CONCLUSIONS
The polyclonal antibody against the GRA24 protein has been successfully prepared, which has a widespread applicability, high titers and a high specificity. This polyclonal antibody is available for Western blotting and IFA, which provides the basis for investigating the function of the GRA24 protein.
Topics: Animals; Toxoplasma; Protozoan Proteins; Mice, Inbred BALB C; Mice; Antibodies, Protozoan; Female; Recombinant Proteins; Antibody Specificity; Antigens, Protozoan
PubMed: 38952314
DOI: 10.16250/j.32.1374.2024083 -
Zhongguo Xue Xi Chong Bing Fang Zhi Za... May 2024The global malaria epidemic is still severe. Because of simple procedures, rapid detection and accuracy results, rapid diagnostic test (RDT) has become the most... (Review)
Review
The global malaria epidemic is still severe. Because of simple procedures, rapid detection and accuracy results, rapid diagnostic test (RDT) has become the most important and the most widely used diagnostic tool for malaria prevention and control. However, deletions in the RDT target histidine-rich protein 2/3 () genes may cause false-negative results of RDT, which has been included as one of the four biological threats to global malaria elimination. This article reviews the applications of RDT in the global malaria diagnosis, analyzes the threats and challenges caused by gene deletion, proposes methods for monitoring gene deletion, and summarizes the causes and countermeasures of negative RDT detections, so as to provide insights into consolidation of malaria elimination achievements in China and contributions to global malaria elimination.
Topics: Protozoan Proteins; Humans; Antigens, Protozoan; Plasmodium falciparum; Malaria, Falciparum; Gene Deletion; Diagnostic Tests, Routine; China; Rapid Diagnostic Tests
PubMed: 38952308
DOI: 10.16250/j.32.1374.2024089 -
Veterinary Medicine and Science Jul 2024Sarcocystis is a food-borne zoonotic protozoan whose final hosts are humans, dogs, cats, and other carnivores and intermediate hosts are birds and mammals, especially...
BACKGROUND
Sarcocystis is a food-borne zoonotic protozoan whose final hosts are humans, dogs, cats, and other carnivores and intermediate hosts are birds and mammals, especially humans and herbivores. Humans become infected by eating raw and undercooked meat contaminated with bradyzoites or by consuming water or food contaminated with the sporocyst stage of the parasite.
OBJECTIVES
The aim of this study was to investigate the effects of gamma radiation and electron beam on the survival rate of Sarcocystis bradyzoites in infected beef and to determine the effective dose.
METHODS
Three replicates of 100 g of infected meat were treated with different doses (0.5, 1, 1.5 and 2 kGy). As a control, 20 g of contaminated meat was stored separately at 4°C. The viability of the bradyzoites after digestion in pepsin solution was assessed, stained (trypan blue) and unstained, under a stereomicroscope. To assess survival of the bradyzoites, the irradiated meat samples were fed to 30 dogs. After 10 days, faecal samples were examined for sporocysts.
RESULTS
The results showed that the highest and lowest mortality rate of Sarcocystis bradyzoites in infected organs using electron beam at a dose of 2 kGy were 92.5% and 100%, respectively, and the lowest mortality rate at a dose of 0.5 kGy were 2.5% and 7.89%, respectively.
CONCLUSION
The results of statistical analysis showed that the mortality rate of Sarcocystis bradyzoites was significant between different doses of gamma ray and electron beam, so that gamma rays were better compared to electron beam in destroying Sarcocystis bradyzoites.
Topics: Sarcocystis; Animals; Cattle; Sarcocystosis; Red Meat; Gamma Rays; Dogs; Food Irradiation; Dose-Response Relationship, Radiation; Cattle Diseases; Electrons
PubMed: 38952247
DOI: 10.1002/vms3.1519