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Background and Aim: Trypanosoma evansi is a blood and tissue protozoan parasite affecting domestic and wild animals. The T. evansi Thai strain, namely, T. evansi from dairy cattle number 953 (TEDC 953) strain, has been successfully isolated from dairy cattle and cultivated in vitro. The in vitro-cultivated parasite is useful for biological studies, evaluation of novel chemotherapeutic agents, and production of antigens for diagnostic tests. This study aimed to observe the histopathology and virulence of an in vitro-adapted T. evansi TEDC 953 strain in vivo. Materials and Methods: The histopathology and virulence of the TEDC 953 strain were clarified in mice. Six mice were infected with 1 × 105 trypomastigotes of TEDC 953 strain intraperitoneally, and four mice were in the negative control. Parasitemia was monitored daily, and the mice were euthanized on 30 days post-infection (DPI). Internal organs were collected for histopathological examination using hematoxylin and eosin staining. Results: Histopathological lesions were found in the liver, lung, heart, kidney, spleen, and brain of the inoculated mice. The main histopathological feature was lymphoplasmacytic inflammation in parenchyma and perivascular areas of multiple organs, and the severity of histopathological changes was related to the presence of trypomastigotes in the regional vessels. Granulomatous inflammation was seen in meninges, pleura, renal capsule, renal pelvis, and spleen of some infected mice. Four mice died at 17, 24, 26, and 27 DPI with an average parasitemia of 4.05 × 1011 trypomastigotes/mL. The average survival time was 23.5 DPI (mice = 4). Conclusion: This study confirmed that the TEDC 953 strain is infectious and pathogenic in mice after the continuously cultivated in vitro. To replace the use of experimental animals, the in vitro-cultivated parasite can be used instead in further studies.

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Background: Amidst the era of widespread resistance, there has been a renewed interest in older antibiotics such as fosfomycin, owing to its activity against certain resistant Gram-negative pathogens, including multidrug-resistant variants expressing extended spectrum ß-lactamases or carbapenemases. The goal of the study was to investigate pharmacokinetic/pharmacodynamic (PK/PD) index and PK/PD targets of fosfomycin in murine thigh and kidney infection models, employing clinical isolates of Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae). Methods: Seven isolates of E. coli (one wild-type and six clinical isolates) and five isolates of K. pneumoniae (one wild-type and four clinical isolates) were utilized for in vivo PK/PD studies. Single-dose plasma PK studies were conducted in infected mice by subcutaneous route. PD index was determined from exposure-response analysis employing 24-hr dose fractionation studies in neutropenic murine thigh infection model, while pharmacodynamic targets (PDTs) were derived from both thigh and kidney infection models. Results: Dose fractionation studies demonstrated that in vivo efficacy of fosfomycin best correlated with AUC/MIC for E. coli (R2 = 0.9227) and K. pneumoniae (R2 = 0.8693). The median AUC/MIC linked to 1 log10 kill effects were 346.2 and 745.2 in thigh infection model and 244.1 and 425.4 in kidney infection model for E. coli and K. pneumoniae, respectively. The mice plasma protein binding of fosfomycin was estimated to be 5.4%. Conclusions: The in vivo efficacy of fosfomycin against Enterobacterales was best described by AUC/MIC. The PDTs derived from this study may help define the coverage potential of fosfomycin at the clinical doses approved.

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The concentration and the isotopic composition of the redox-active essential elements Cu and Fe were investigated in blood plasma and specific brain regions (hippocampus, cortex, brain stem and cerebellum) of mice to assess potential alterations associated with sepsis-associated encephalopathy induced by lipopolysaccharide (LPS) administration. Samples were collected from young (16-22 weeks) and aged (44-65 weeks) mice after intraperitoneal injection of the LPS, an endotoxin inducing neuroinflammation, and from age- and sex-matched controls, injected with phosphate-buffered saline solution. Sector-field single-collector inductively coupled plasma-mass spectrometry was relied upon for elemental analysis and multi-collector inductively coupled plasma-mass spectrometry for isotopic analysis. Significant variations were observed for the Cu concentration and for the Cu and Fe isotope ratios in the blood plasma. Concentrations and isotope ratios of Cu and Fe also varied across the brain tissues. An age- and an inflammatory-related effect was found affecting the isotopic compositions of blood plasma Cu and cerebellum Fe, whereas a regional Cu isotopic redistribution was found within the brain tissues. These findings demonstrate that isotopic analysis of essential mineral elements picks up metabolic changes not revealed by element quantification, making the two approaches complementary.

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Combined antiretroviral therapy ushered an era of survivable HIV infection in which people living with HIV (PLH) conduct normal life activities and enjoy measurably extended lifespans. However, despite viral control, PLH often experience a variety of cognitive, emotional, and physical phenotypes that diminish their quality of life, including cognitive impairment, depression, and sleep disruption. Recently, accumulating evidence has linked persistent CNS immune activation to the overproduction of glutamate and upregulation of glutaminase (GLS) activity, particularly in microglial cells, driving glutamatergic imbalance with neurological consequences. Our lab has developed a brain-penetrant prodrug of the glutamine antagonist 6-diazo-5-oxo-L-norleucine (DON), JHU083, that potently inhibits brain GLS activity in mice following oral administration. To assess the therapeutic potential of JHU083, we infected mice with EcoHIV and characterized their neurobehavioral phenotypes. EcoHIV-infected mice exhibited decreased social interaction, suppressed sucrose preference, disrupted sleep during the early rest period, and increased sleep fragmentation, similar to what has been reported in PLH but not yet observed in murine models. At doses shown to inhibit microglial GLS, JHU083 treatment ameliorated all of the abnormal neurobehavioral phenotypes. To explore potential mechanisms underlying this effect, hippocampal microglia were isolated for RNA sequencing. The dysregulated genes and pathways in EcoHIV-infected hippocampal microglia pointed to disruptions in immune functions of these cells, which were partially restored by JHU083 treatment. These findings suggest that upregulation of microglial GLS may affect immune functions of these cells. Thus, brain-penetrable GLS inhibitors like JHU083 could act as a potential therapeutic modality for both glutamate excitotoxicity and aberrant immune activation in microglia in chronic HIV infection.

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Early detection of Schistosoma japonicum (S. japonicum) within its intermediate and definitive hosts is crucial for case finding and disease surveillance, especially in low-endemic areas. Recombinase polymerase amplification (RPA) has many advantages over traditional methods of DNA-amplification, such as polymerase chain reaction (PCR), including high sensitivity and specificity whilst being deployable in resource-poor schistosomiasis-endemic areas. Here, we evaluated the performance of a basic RPA assay targeting the 28srDNA gene fragment of S. japonicum (Sj28srDNA) using schistosome-infected Oncomelania hupensis (O. hupensis) and mouse models, compared to the traditional pathological method and a PCR assay. Overall S. japonicum infection prevalence within O. hupensis hosts by microscopic dissection, PCR and RPA was 9.29% (13/140), 32.14% (45/140) and 51.43% (72/140), respectively, presenting significant differences statistically (χ2 = 58.31, p < 0.001). It was noteworthy that infection prevalence by PCR and RPA performed was 34.44% (31/90) and 53.33% (48/90) in snails within 6 weeks post-infection, while the dissection method detected all samples as negatives. In addition, the basic RPA assay presented positive results from the fourth week post-infection and third day post-infection when detecting fecal DNA and serum DNA, respectively, which were extracted from a pooled sample from mice infected with 20 S. japonicum cercariae. This study suggests that the RPA assay has high potential for early detection of S. japonicum infection within its intermediate and definitive hosts.

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BACKGROUND AND AIM: Antimicrobial photodynamic therapy (aPDT) has shown great potential for treatment of superficial or localized multidrug-resistant (MDR) Acinetobacter baumannii infections. The purpose of this study was to investigate the cytotoxicity and in vivo safety of aloe-emodin (AE), and its photodynamic treatment efficacy against MDR A. baumannii infections. METHODS: The cytotoxicity (dark toxicity) and phototoxicity of AE to human immortalized keratinocytes and mice fibroblasts were detected by CCK-8 kit. Low and high doses of AE were intravenously injected into mice to evaluate the safety of AE in vivo. Bioluminescent MDR A. baumannii strain was employed to establish the infection model on BALB/c mice after skin scald, and infection status and therapeutic effect of AE-mediated aPDT were assessed by animal imaging system. The peripheral blood of mice was analyzed by flow cytometer. RESULTS: AE had low cytotoxicity to human immortalized keratinocytes and mice fibroblasts, and had certain phototoxicity to these cells under light irradiation. The in vivo experiments demonstrated that AE caused no obvious effects on the weight and pathological changes of mice. AE-mediated aPDT was effective in the treatment of MDR A. baumannii caused infections in mice after skin scald. CONCLUSIONS: AE has potential to be used in the photodynamic treatment of MDR A. baumannii caused superficial infections after scald.

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Leishmania major is a protozoan parasite that causes cutaneous Leishmaniasis disease in human beings and animals. The disease is prevalent in tropical and semitropical countries and has great health importance. The present study aimed to identify the histological changes in the organs infected with L. major and to provide a sophisticated diagnostic method for infection through detecting TGF-ß cytokine by immunohistochemistry technique(IHC) from October 2020 to January 2021. A total of 40 samples of paraffin blocks were used for different organs including skin, spleen, liver, kidney, and heart of male and female BALB/c mice, aged 6-8 weeks, which were previously infected subcutaneously with L. major promastigotes at a dose of 1×107 promastigotes/moues. The result indicated epidermal hyperplasia with diffuse severe lymphohistiocytic inflammatory cells infiltration in the dermis. Hyperplasia of the lymphoid follicles was observed in infected spleen and scattered polymorphonuclear cells mainly neutrophil masses with a random distribution of microgranulomas foci composed of lymphocytes and macrophages within the liver parenchyma around central veins and portal areas. The infected kidney showed aggregation of perivascular mononuclear cells (lymphocytes and macrophages) in the renal cortex. Mononuclear lymphocytes and macrophages were observed within the heart parenchyma especially around blood vessels. Additionally, evaluation of TGF-ß1 expression was highly strong for skin, spleen, relatively strong for liver, heart, and weak for the kidney. In conclusion, infection was accompanied by clinical and histological changes as well as inflammatory diseases. Furthermore, the determination of TGF-ß expression level depends on the diagnosis of infection. A clear understanding of immune mechanisms is essential for preventing, treating, and controlling strategies of this infection.

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Echinococcosis is a serious helminthic zoonosis with a great impact on human health and livestock husbandry. However, the clinically used drugs (benzimidazoles) have a low cure rate, so alternative drugs are urgently needed. Currently, drug screenings for echinococcosis are mainly phenotype-based, and the efficiency of identifying active compounds is very low. With a pharmacophore model generated from the structures of active amino alcohols, we performed a virtual screening to discover novel compounds with anti-echinococcal activity. Sixty-two compounds from the virtual screening were tested on Echinococcus multilocularis protoscoleces, and 10 of these compounds were found to be active. After further evaluation of their cytotoxicity, S6 was selected along with two active amino alcohols for in vivo pharmacodynamic and pharmacokinetic studies. At the two tested doses (50 and 25 mg/kg), S6 inhibited the growth of E. multilocularis in mice (14.43 and 9.53%), but no significant difference between the treatment groups and control group was observed. Treatment with BTB4 and HT3 was shown to be ineffective. During the 28 days of treatment, the death of mice in the mebendazole, HT3, and BTB4 groups indicated their toxicity. The plasma concentration of S6 administered by both methods was very low, with the Cmax being only 1 ng/ml after oral administration and below the detection limit after intramuscular administration. In addition, the plasma concentrations of BTB4 and HT3 in vitro did not reach high enough levels to kill the parasites. The toxicities of these two amino alcohols indicated that they are not suitable for further development as anti-echinococcal drugs. However, further attempts should be made to increase the bioavailability of S6 and modify its structure. In this study, we demonstrate that pharmacophore-based virtual screenings with high drug identification efficiency could be used to find novel drugs for treating echinococcosis.

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The incidence of mono- and multinuclear cells and their expression of pro- and antifibrotic factors were studied in cultured peritoneal macrophages from intact and BCG-infected mice. Generally, the expression of factors increased with an increase in the number of nuclei per cell. However, the expression was higher in macrophages from BCG infected mice, except the cells with 3 and more nuclei, extremely rarely expressing IL-1α in cultures from intact and BCG-infected animals. The number of macrophages with 3 and more nuclei, expressing CatD, was comparable with the number of mono- and binuclear macrophages. Presumably, this was determined by various mechanisms of formation of multinuclear (3-5 and more nuclei) macrophages, for example, by amitosis.

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Invasive fungal infections threat the life of immunocompromised patients. Chromogranin A N-46 (CGA-N46), corresponding to the 31st to 76th amino acids of the N-terminus of human chromogranin A, is an antifungal peptide. In order to elucidate the antifungal effects of CGA-N46 in vivo, we studied its effects on cell-mediated immunity in Candida krusei-infected mice. The results showed that the treatment with CGA-N46 increased the average body weight and decreased the mortality of the immunocompromised mice model infected with Candida krusei. The spleen and thymus indices of treated mice has markedly increased compared with that of the control group (P<0.05), and the immune cell levels in peripheral blood also increased significantly (P<0.05). The immuno-modulatory effect of CGA-N46 (60 mg/kg/day) was found to be comparable to that of terbinafine. Additionally, CGA-N46 could alleviate or eliminate histopathological symptoms in the liver, spleen, kidney, and lung tissues. In conclusion, the present study suggests that CGA-N46 may offer a new strategy for antifungal therapeutic option. This study is an essential step in elucidating the effect of CGA-N46 in vivo.

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Oral infection has become the most important transmission mechanism of Chagas disease in Brazil. For this study, the development of Trypanosoma cruzi infection in mice, induced by the oral and intraperitoneal (IP) routes, was compared. Four groups of Swiss mice were used to evaluate the influence of parasite genetics, number of parasites, inoculation volume and developmental stages on the development of the orally induced infection: 1 - blood trypomastigotes (BT) via oral; 2 - BT via IP; 3 - culture metacyclic trypomastigotes (MT) via oral; and 4 - culture MT via IP. Animals inoculated orally showed levels of parasitemia, as well as infectivity and mortality rates, lower than animals inoculated via IP, regardless of DTU (discrete typing unit) and inoculum. Animals infected with TcII showed higher levels of these parameters than did animals infected with TcI. The larger volume of inoculum showed a greater capacity to cause an infection when administered via the oral route. BT infection was more virulent than culture MT infection for both routes (oral and IP). However, mice inoculated orally with BT showed lower levels than via IP, while mice inoculated orally with culture MT showed similar levels of infection to those inoculated via IP. Mice inoculated with culture MT showed more histopathological changes than those inoculated with BT, regardless of the inoculation route. These results indicate that this alternative experimental model is useful for evaluating infection by T. cruzi isolates with subpatent parasitemia and low virulence, such as those belonging to the TcI and TcIV DTUs, which are prevalent in outbreaks of orally transmitted Chagas disease.

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Aromatic diamidines have been successfully used to combat a wide range of parasites that cause important human infections. Recently we reported that a N-phenyl-substituted analogue of furamidine (DB569) exerts a micromolar trypanocidal activity against Trypanosoma cruzi in vitro. Since DB569 also reduces the cardiac parasitism and increases the survival rates of T. cruzi-infected mice, our present aim was to analyze the potential protection of DB569 in the development of altered cardiac electrical conduction system during acute and chronic T. cruzi infection. In our experimental model of acute infection (Swiss mice inoculated with Y strain of T. cruzi), the prevailing disorder observed in electrocardiogram (ECG) analyses was sinus bradycardia. This ECG alteration was reverted in acutely infected mice treated with DB569. Interestingly, the DB569 treatment reduced significantly the numbers of CD8(+) T cells in the cardiac infiltration. In addition, the noticed protection of DB569 in the ECG findings of acutely-infected animals was further extended to the chronic infection. Our data suggest that the reversion to and further maintenance of normal ECG profile in the DB569-treated infected animals may be associated with the reduced cardiac CD8(+) lymphocyte infiltration and parasitism that might be ultimately contributing to their increased survival rates.