Characterization and Demonstration of the Value of a Lethal Mouse Model of Middle East Respiratory Syndrome Coronavirus Infection and Disease.

UNLABELLED: Characterized animal models are needed for studying the pathogenesis of and evaluating medical countermeasures for persisting Middle East respiratory syndrome-coronavirus (MERS-CoV) infections. Here, we further characterized a lethal transgenic mouse model of MERS-CoV infection and disease that globally expresses human CD26 (hCD26)/DPP4. The 50% infectious dose (ID50) and lethal dose (LD50) of virus were estimated to be <1 and 10 TCID50 of MERS-CoV, respectively. Neutralizing antibody developed in the surviving mice from the ID50/LD50 determinations, and all were fully immune to challenge with 100 LD50 of MERS-CoV. The tissue distribution and histopathology in mice challenged with a potential working dose of 10 LD50 of MERS-CoV were subsequently evaluated. In contrast to the overwhelming infection seen in the mice challenged with 10(5) LD50 of MERS-CoV, we were able to recover infectious virus from these mice only infrequently, although quantitative reverse transcription-PCR (qRT-PCR) tests indicated early and persistent lung infection and delayed occurrence of brain infection. Persistent inflammatory infiltrates were seen in the lungs and brain stems at day 2 and day 6 after infection, respectively. While focal infiltrates were also noted in the liver, definite pathology was not seen in other tissues. Finally, using a receptor binding domain protein vaccine and a MERS-CoV fusion inhibitor, we demonstrated the value of this model for evaluating vaccines and antivirals against MERS. As outcomes of MERS-CoV infection in patients differ greatly, ranging from asymptomatic to overwhelming disease and death, having available both an infection model and a lethal model makes this transgenic mouse model relevant for advancing MERS research. IMPORTANCE: Fully characterized animal models are essential for studying pathogenesis and for preclinical screening of vaccines and drugs against MERS-CoV infection and disease. When given a high dose of MERS-CoV, our transgenic mice expressing hCD26/DPP4 viral receptor uniformly succumbed to death within 6 days, making it difficult to evaluate host responses to infection and disease. We further characterized this model by determining both the ID50 and the LD50 of MERS-CoV in order to establish both an infection model and a lethal model for MERS and followed this by investigating the antibody responses and immunity of the mice that survived MERS-CoV infection. Using the estimated LD50 and ID50 data, we dissected the kinetics of viral tissue distribution and pathology in mice challenged with 10 LD50 of virus and utilized the model for preclinical evaluation of a vaccine and drug for treatment of MERS-CoV infection. This further-characterized transgenic mouse model will be useful for advancing MERS research.

Efficacy of cefepime, ertapenem and norfloxacin against leptospirosis and for the clearance of pathogens in a hamster model.

Animals and humans with severe leptospirosis may require empirical treatment. Although many antibiotics are active against multiple leptospira serovars in vitro, their efficacy in vivo is limited. We evaluated the efficacy of cefepime (daily dose: 2, 5, 10, and 20 mg/kg), ertapenem (daily dose: 2.5, 5, and 10 mg/kg) and norfloxacin (daily dose: 20, 40, and 80 mg/kg) for the treatment of leptospirosis and the ability to clear leptospira in target organs (liver, kidney, lung, heart, and spleen) in a lethal hamster model using Leptospira interrogans serovar Autumnalis. The histopathology of infected kidney, lung and liver was also evaluated using hematoxylin and eosin stain (H&E stain). All untreated animals, serving as a negative control, died with leptospira existing in the target organs between the 5th and 7th day after infection. All of the treated groups displayed improved survival compared to the untreated group and demonstrated a dose-dependent decrease in the presence of leptospira in the target organs. Cefepime showed survival benefit comparable to the standard treatment, doxycycline. We conclude that all of the antibiotics tested in vivo produce a statistically significant survival advantage, alleviate tissue injury and decrease the abundance of leptospira in target organs.

Deferoxamine mesylate enhances virulence of community-associated methicillin resistant Staphylococcus aureus.

Staphylococcus aureus is a leading cause of bacterial infections. Strains of community-associated methicillin-resistant S. aureus (CA-MRSA), such as USA300, display enhanced virulence and fitness. Patients suffering from iron overload diseases often undergo iron chelation therapy with deferoxamine mesylate (DFO). Here, we show that USA300 uses this drug to acquire iron. We further demonstrate that mice administered DFO I.P., versus those not administered DFO, had significantly higher bacterial burden in livers and kidneys after I.V. challenge with USA300, associated with increased abscess formation and tissue destruction. The virulence of USA300 mutants defective for DFO uptake was not affected by DFO treatment.

A murine model of coxsackievirus A16 infection for anti-viral evaluation.

Coxsackievirus A16 (CA16) is one of the main causative agents of hand, foot and mouth disease (HFMD), which is a common infectious disease in children. CA16 infection may lead to severe nervous system damage and even death in humans. However, study of the pathogenesis of CA16 infection and development of vaccines and anti-viral agents are hindered partly by the lack of an appropriate small animal model. In the present study, we developed and characterized a murine model of CA16 infection. We show that neonatal mice are susceptible to CA16 infection via intraperitoneal inoculation. One-day-old mice infected with 2×10(6)TCID50 of CA16/SZ05 strain consistently exhibited clinical signs, including reduced mobility, and limb weakness and paralysis. About 57% of the mice died within 14days after infection. Significant damage in the brainstem, limb muscles and intestines of the infected mice in the moribund state was observed by histological examination, and the presence of CA16 in neurons of the brainstem was demonstrated by immunohistochemical staining with a CA16-specific polyclonal antibody, strongly suggesting the involvement of the central nervous system in CA16 infection. Analysis of virus titers in various organs/tissues collected at 3, 6 and 9days post-infection, showed that skeletal muscle was the major site of virus replication at the early stage of infection, while the virus mainly accumulated in the brain at the late stage. In addition, susceptibility of mice to CA16 infection was found to be age dependent. Moreover, different CA16 strains could exhibit varied virulence in vivo. Importantly, we demonstrated that post-exposure treatment with an anti-CA16 monoclonal antibody fully protected mice against lethal CA16 infection. Collectively, these results indicate the successful development of a CA16 infection mouse model for anti-viral evaluation.

In vivo anthelmintic activity of Anogeissus leiocarpus Guill & Perr (Combretaceae) against nematodes in naturally infected sheep.

The identification of new anthelmintic drugs becomes a priority because of the availability of a handful of drugs, cost of treatments, and recent emergence of drug resistance. Medicinal plants are a good source of bioactive compounds for development of drugs. In this study, in vivo efficacy of Anogeissus leiocarpus was assessed in sheep naturally infected with gastrointestinal nematodes. Fecal examination, serological analyses, and necropsy were carried out to determine the egg and worm-burden reduction. The administration of ethanolic extract (single oral dose of 80 mg/kg) of A. leiocarpus induced a moderate fecal egg reduction (81 %) and adult worm-burden reduction (87 %) against Haemonchus contortus and Trichostrongylus colubriformis (82 %). The plant exhibited high efficacy against adult Strongyloïdes papillosus (100 %), Gaigeria pachyscelis (90 %), Cooperia curticei (100 %), and Oesophagostomum columbianum (95 %) but low efficacy against Trichostrongylus axei (67 %) and Trichuris globulosa (79 %). All these helminthes were sensitive to fenbendazole, except O. columbianum which showed a decrease susceptibility (17 %). The plant extract also improved certain biological parameters by increasing bodyweight from 0.7 ± 2.9 to 3.3 ± 1.9 % and improving hematocrit of 6.9 ± 1.6 % 3-week posttreatment. It emerges from the results that the plant possesses significant effectiveness on diarrhea; all treated animals gave normal feces. This study has shown that A. leiocarpus could find an application in the control of multiparasitism in small ruminants.

Acute oral toxicity of methanolic seed extract of Cassia fistula in mice.

BACKGROUND AND OBJECTIVE: Cassia fistula is widely used in traditional medicine to treat various types of ailments. The evaluation of toxic properties of C. fistula is crucial when considering public health protection because exposure to plant extracts can result in undesirable effects on consumers. Hence, in this study the acute oral toxicity of C. fistula seeds extract was investigated in mice. RESULTS: Oral administration of crude extract at the highest dose of 5000 mg/kg resulted in no mortalities or evidence of adverse effects, implying that C. fistula in nontoxic. Throughout 14 days of the treatment no changes in behavioural pattern, clinical sign and body weight of mice in both control and treatment groups. Also there were no any significant elevations observed in the biochemical analysis of the blood serum. Further, histopathological examination revealed normal architecture and no significant adverse effects observed on the kidney, heart, liver, lung and spleen. CONCLUSIONS: Overall, the results suggest that, the oral administration of C. fistula methanolic seeds extract did not produce any significant toxic effect in mice. Hence, the extract can be utilized for pharmaceutical formulations.

Immunotoxicity of zearalenone in Balb/c mice in a high subchronic dosing study counteracted by Raphanus sativus extract.

Radish (Raphanus sativus) is a cruciferous plant, rich on flavonoids, isothiocyanates, and phenolic acids. They show anti-inflammatory and immunomodulatory activity both in vitro and in vivo. Isothiocyanates and flavonoids have been reported previously to prevent low-sub-chronic dose of zearalenone (ZEN) causing immunotoxicity. The present study focuses on the amelioration of fusarotoxicosis in Balb/c mice by feeding two concentrations of radish extract. The extract at 15 and 30 mg/kg bw, was evaluated to reduce the deleterious effects in immunological parameters of high subchronic doses of 40 and 80 mg of ZEN/kg bw on modulation of lipopolysaccharide (LPS). ZEN consuming mice showed a "dose-related" decrease in weight gain and in the immune relative weights organs. Moreover, Atrophy and lymphoid depletion were seen in the histopathology of spleen. Ingestion of ZEN at either level had a significant effect on total red blood cell numbers and on their relative number of lymphocytes. Likewise, ZEN alters the production of regulatory cytokines and antibody of LPS stimulated mice. By contrast, the additions of radish extract with a low or high dose of ZEN moderately decreased the affected mice and/or the severity of lesions, and all tested parameters were normal or at least near normal levels. In addition, the radish extract alone did not produce any significant changes in all tested parameters compared with the controls. In conclusion, radish extract was effective for the protection of high dose ZEN-immunotoxication in mice and it could contribute to a solution of the ZEN immunotoxicity in humans and in farm animals.

Cytotoxic compounds generated in heated oil and assimilation of oil in Wistar rats.

We have previously suggested that gluten binds with decomposition products from thermally oxidized oil during frying, and that low-molecular-weight compounds bound to browned gluten damage the liver in rats. Ten-week-old male Wistar rats were fed for 11 weeks ad libitum a diet containing 7 wt% fresh frying oil and 0.1 wt% gluten heated in/without oil at 180 degrees C for 10 h. Feces collected weekly and serum were subjected to lipid and hematological analyses, respectively. Values obtained in the analyses did not differ from those of the control group. The results show that thermally processed gluten does not influence the digestion, absorption, metabolism, and growth of rats, regardless of the cytotoxic low-molecular-weight compounds, and that ingested fresh oil was assimilated normally. Together with the previous results, the odor of thermally processed gluten stimulated the rats' appetite, and completely assimilable fresh oil and cytotoxic low-molecular-weight compounds bound to gluten were ingested, and thus organ damage and rapid body weight increase were observed. As commercial deep-fried products are often made with repeatedly used oil with periodically added fresh oil, similar to the present experimental diet, obesity and organ damage may also occur in humans.

The prophylactic effectiveness of various antifungal agents against the progression of trichosporonosis fungemia to disseminated disease in a neutropenic mouse model.

Neutropenic mice with latent trichosporonemia were given various antifungal agents (amphotericin B, fluconazole, itraconazole) or saline to determine which antifungal agent could be useful for prophylaxis. The 3-week-survival rate was 80% in the fluconazole group, 50% in the amphotericin B group, 45% in the itraconazole group, and 30% in the saline group. Compared with the other antifungal agents, fluconazole offered superior prophylaxis against the progression of trichosporonosis fungemia to disseminated disease (P<0.05). These results suggest that clinical studies are warranted to investigate fluconazole prophylaxis of trichosporonosis progression in neutropenic patients, such as people receiving chemotherapy and patients who have received solid organ transplants.