Karber Method Research Articles

In vitro inactivation of SARS-CoV-2 by ozonated water via novel hand hygiene device.

The COVID-19 pandemic has heightened awareness of the need for novel surface disinfectants and hand-hygiene modalities. Ozone gas is an effective surface disinfectant, but toxicity limits its use in human applications. Ozonated water is a safer means to use ozone for disinfection, especially for human antisepsis. However, there are little data available regarding the effectiveness of ozonated water in eliminating severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). This study utilizes a novel hand hygiene device that produces a stable ozone concentration of 0.5 +/-0.1ppm in water and applies it using a proprietary spray that controls droplet size, velocity, and direction. The Device was used to apply ozonated water to a known quantity of SARS-CoV-2 Delta Variant viral particles on a non-porous surface (glass) for seven seconds. Post-exposure growth was compared to the unexposed matched control utilizing the Spearman-Karber method. Compared to control, ozonated water decreased SARS-CoV-2 viral growth by a mean log10 reduction of 4.33, or>99.99%reduction. These results suggest that the ozonated water, when applied by a spray hand hygiene device, is highly effective at surface disinfection of SARS-CoV-2.

Combined Silver Sulfadiazine Nanosuspension with Thermosensitive Hydrogel: An Effective Antibacterial Treatment for Wound Healing in an Animal Model.

Silver sulfadiazine (AgSD) is widely used in burn wound treatment due to its broad-spectrum antibacterial activity. However, its application in wound healing is greatly hindered by the low solubility of AgSD particles and their cellular cytotoxicity. Herein, we studied the safety and in vivo efficacy of nano-sized silver sulfadiazine loaded in poloxamer thermosensitive hydrogel (NS/Gel). In NS/Gel, silver sulfadiazine was prepared into silver sulfadiazine nanosuspension (NS) to improve the solubility and enhance its antibacterial activity, whereas the poloxamer thermosensitive hydrogel was selected as a drug carrier of NS to achieve slow drug release and reduced cytotoxicity. The acute toxicity of silver sulfadiazine nanosuspension was first evaluated in healthy mice, and its median lethal dose (LD50) was calculated by the modified Karber method. Furthermore, in vivo antibacterial effect and wound healing property of NS/Gel were evaluated on the infected deep second-degree burn wound mice model. The mortality ratio of mice was concentration-dependent, and the LD50 for silver sulfadiazine nanosuspension was estimated to be 252.1 mg/kg (230.8 to 275.4 mg/kg, 95% confidence limit). The in vivo dosages used for burn wound treatment (40-50 mg/kg) were far below LD50 (252.1 mg/kg). NS/Gel significantly accelerated wound healing in the deep second wound infection mice model, achieving > 85% wound contraction on day 14. Staphylococcus aureus in the wound region was eradicated after 7 days in NS/Gel group, while the bacterial colony count was still measurable in the control group. Histological analysis and cytokines measurement confirmed that the mice treated with NS/Gel exhibited well-organized epithelium and multiple keratinized cell layers compared to control groups with the modulated expression of IL-6, VEGF, and TGF-β. The combination of silver sulfadiazine nanosuspension and thermo-responsive hydrogel has great potential in clinical burn wound treatment.

Inflammatory and pathological changes in Escherichia coli infected mice

PurposeUnderstanding the inflammation and histopathological changes in vivo caused by Escherichia coli infection is of great significance for scientific research and clinical diagnosis. MethodsMice were randomly divided into 6 groups (N = 10) after adaptive feeding, and it challenged by intraperitoneal injection with different concentrations of E. coli ATCC25922. The survival situation within 7 days was recorded, and the half-lethal dose (LD50) was calculated by Karber's method. After the end, the blood, heart, liver, spleen, lung, and kidney of the mice were collected. We detected the concentration of inflammatory cytokines (IL-6, IL-β, and TNF-α) and inducible nitric oxide synthase (iNOS) in serum by ELSIA. Organs were observed by histopathological staining and electron microscope observation. ResultsThe LD50 of mice infected with E. coli was 1.371∗106 CFU/kg. The concentrations of IL-6, IL-β, and TNF-α increased with time after infection in mice, reaching the highest concentration on the 7th day. iNOS was significantly increased on the 1st day of infection, and then decreased over time (P < 0.01). Within a week after infection, the colony counts of the heart, liver, spleen, lung and kidney showed a first decrease, and then reached a surge on the 7th day. Pathological results showed that a small amount of mitochondrial swelling and autophagy were seen in the spleen, lung and kidney tissues of the infected group; and a small amount of secondary lysosomes and autophagy were also seen; but no pathological changes were found in the liver and heart. ConclusionEscherichia coli can cause inflammation and oxidative stress in mice, causing different degrees of damage to the spleen, lung, and kidney tissues, which provides theoretical support for inflammatory and pathological changes caused by Escherichia coli infection in vivo.

Improved up-and-down procedure for acute toxicity measurement with reliable LD50 verified by typical toxic alkaloids and modified Karber method

BackgroundUp-and-down procedure (UDP) was recommended to replace traditional acute toxicity methods. However, it was limited due to the long experimental period (20–42 days). To improve UDP, an improved UDP method (iUDP) was developed by shortening observation time between sequence dosages. The aim of this study was to test the reliability of iUDP to provide a reliable method for the acute toxicity measurement of valuable or minor amount compounds.MethodsOral median lethal dose (LD50) of nicotine, sinomenine hydrochloride and berberine hydrochloride were measured both by iUDP and modified Karber method (mKM).ResultsLD50 of the three alkaloids measured by iUDP with 23 mice were 32.71 ± 7.46, 453.54 ± 104.59, 2954.93 ± 794.88 mg/kg, respectively. LD50 of the three alkaloids measured by mKM with 240 mice were 22.99 ± 3.01, 456.56 ± 53.38, 2825.53 ± 1212.92 mg/kg, respectively. The average time consumed by the two methods were 22 days and 14 days respectively. Total grams of the alkaloids used by the two methods were 0.0082 and 0.0673 (nicotine), 0.114 and 1.24 (sinomenine hydrochloride), 1.9 and 12.7 (berberine hydrochloride).ConclusioniUDP could replace mKM to detect acute toxicity of substances with comparable and reliable result. And it is suitable for valuable or minor amount substances.

Acute Toxicity (LC50) Of Cyhalofop Butyl on Gambusia Holbrooki

Cyhalofob butyl is a synthetic herbist. This herbicide is a systemic herbicide and is used in weed control especially on celtic agriculture. In this study, the acute toxicity (LC50) of this herbist (Chillinger 200 EC 200, cyhalofob butyl) was investigated, which is currently used intensely in the fight against herbists in Thrace region, including Cyhalofob butyl. Acute toxicity (LC50 value) for this herbicide has been investigated in the common mosquito fish (Gambusia holbrooki) in Thrace Region. Lethal Dose 50 experiments were performed in 3 replicates in static test runs (water temperature 27.70 ±0.56 C, water pH 8.88± 0.37, and conductivity 718.25±21.113 µhos). The experiment results obtained for Chillinger 200 ec during the experiments were evaluated by Trimmed Spearman–Karber method. 96-hour LC50 value for the mosquito fish Gambusia holbrooki was calculated as 4.140 ppm.

Comparison of Time and Sequence of Permanent Tooth Eruptions in Chinese and Arabic Ethnic Groups

Introduction. Tooth eruption is a process of moving teeth into the oral cavity through the soft tissue and mucosa that lines the jaw until it comes into contact with the antagonistic teeth and functions in mastication. The timing and sequence of tooth eruptions for each individual varies, which is influenced by several factors, one of which is race and ethnicity. Indonesia is known as a multi-ethnic country, including Chinese and Arabic ethnic groups. The purpose of the study was to compare the timing and sequence of eruption of permanent teeth in ethnic Chinese and Arabs.Methods. This type of research is an observational study with a cross-sectional design. The sample of this study was 327 children consisting of 201 Chinese and 126 Arab ethnicities with an age range of 6-13 years. The eruption of all permanent teeth was examined except for the third molar. The mean time to eruption of permanent teeth was calculated using the Karber method. The data were analyzed statistically using the Mann-Whitney test.Results. The first permanent teeth erupted in the two ethnic groups were the mandibular central incisors around the age of 7 years and the last eruption was the maxillary second molar around the age of 11 years. Significant differences in the eruption time of the permanent teeth between ethnicities were shown in the maxillary central and second molars and lateral incisors and mandibular first molars (p &lt;0.05). The eruption sequence pattern in both ethnicities was almost the same except for the canines and second premolars in the upper and lower jaws.Conclusion. There are differences in the timing and sequence of eruption of permanent teeth between ethnic Chinese and Arabs.

Nitrate acute toxicity to post larvae and juveniles of Macrobrachium amazonicum (Heller, 1862).

The objective was to determine the median lethal concentration (LC50) of nitrate for post-larvae and juveniles of Macrobrachium amazonicum exposed for 96 h, and to identify histopathological alterations in the gills of juveniles exposed to nitrate. Post-larvae and juveniles of M.amazonicum were exposed to seven different concentrations of nitrate (0, 50, 100, 500, 1000, 1500 and 2000 mg.L-1) with four replicates (n = 10 prawn/experimental unit). The degree of damage to the branchial structure of juveniles was evaluated using standard histological processing with hematoxylin and eosin (H&E), subjected to the Organ Index (Iorg). LC50 values at 24, 48, 72 were estimated by the Trimmed Spearman Karber Method software and were respectively 1574, 638, 237, and 194 mg.L-1 for post-larvae and 1070, 286, 185 and 155 mg.L-1 for juveniles, respectively. From these results, the safety level of nitrate was estimated at 157, 64, 24 and 19 mg.L-1 for post-larvae and 107; 27; 18 and 16 mg.L-1 for juveniles, respectively for 24 h, 48 h, 72 h and 96 h. Damage to the gills in treatments with 100% mortality for nitrate corresponded to the high occurrence of progressive, regressive, circulatory and inflammatory damages. The other treatments, which caused lower mortality, mainly resulted in inflammation and regressive damage, whose occurrence increased with increasing concentration of nitrate. However, in closed systems, the use of anaerobic denitrifying filters or aquatic macrophytes is necessary to provide suitable water quality for the best productive performance, avoiding the negative influence of the accumulated nitrate.

Dr. PI POLYPHENOL CONTENT, PHYTOCHEMISTRY COMPOSITIONS AND ANTIOXIDANT ACTIVITY OF DIFFERENT EXTRACTS FROM MARINE SPONGE AAPTOS SUBERITOIDES GROWN IN NHATRANG BAY, VIETNAM

Objective: To investigate the content and antioxidant activities of polyphenol, the correlation between polyphenol content and their antioxidant activities, and phytochemistry compositions of different extracts from marine sponge Aaptos suberitoides commonly found growing in Nhatrang bay, Vietnam orienting application into functional food and pharmacy.&#x0D; Methods: Evaluating the toxicity of antioxidant polyphenol powder preparing from the initial concentrated extract was by the adjusted Behrens Karber method and a correlation between polyphenol content and antioxidant activities basing on the Pearson coefficient in Excel. Separating antioxidant polyphenol content was base on solvents polarization of n-hexane, chloroform, ethanol, ethyl acetate and n-butanol which the quantification of polyphenol content and antioxidant activities, and preliminary phytochemical compositions qualitative.&#x0D; Results: Antioxidant polyphenol powder did not affect mice weight during the assay time of 28 d. Polyphenol content and antioxidant activities got the highest value at chloroform extract in comparison to other extracts, a significant difference (p&lt;0.05) and strong correlation (R2&gt;0.9). Polyphenol content (122.682 mg gallic acid equivalent ml-1), total antioxidant activity (368.183 mg ascorbic acid equivalent ml-1), reducing power activity (24.08 mg FeSO4 equivalent ml-1) and DPPH scavenging (72.48±1.54 %) were the highest values. Alkaloids, flavonoids, steroids, tannins and triterpenoids existed in initial methanol extract. Weakly polarized polyphenol content was 70.27% in comparison to initial methanol extract.&#x0D; Conclusion: Antioxidant polyphenol of sponge Aaptos suberitoides has the potential for application into the field of functional food and pharmaceuticals.

Acute oral toxicity test and assessment of combined toxicity of cadmium and aflatoxin B1 in kunming mice

Cadmium and aflatoxin B1 (AFB1) are both common and widespread pollutants in food and feed. There are several reports on toxicity induced by Cadmium or AFB1 alone, but few address the toxicity caused by co-exposure to the two substances. In this study, 42 female and 42 male Kunming (KM) mice were divided into seven groups to test the acute oral toxicity of CdCl2 and AFB1, using Karber's method. The combined toxicity was assessed using the Keplinger evaluation system. Acute toxicity symptoms, deaths, and body and organ weights were evaluated, and hematological, blood biochemical, and histopathological analyses were conducted. The results revealed the following median lethal doses (LD50): LD50(Female KM mice) = 62.56 mg/kg; LD50(Male KM mice) = 48.79 mg/kg; LD50(KM mice)=55.27 mg/kg. The combined toxicity of AFB1 and CdCl2 showed an additive effect in mice, and an increase in the mixed dose of AFB1 and CdCl2 resulted in greater toxicity. These results demonstrated that the combined toxicity of AFB1 and CdCl2 was greater than the toxicities of the individual components in mice; thus, this may cause particular challenges when addressing these hazards in food and feed and the associated risk to human and animal health.

Effect and mechanism of citral against methicillin-resistant Staphylococcus aureus in vivo.

Citral is an active component of many plant extracts, and it is a safe additive used in food and cosmetics. A previous study showed that citral has a good antibacterial effect against methicillin-resistant Staphylococcus aureus (MRSA) in vitro, but its in vivo anti-infective activity has not been studied. Anti-MRSA activity and the preliminary mechanism of citral against MRSA were investigated in MRSA-infected KM mice. The ED50 was calculated using Karber's method. Groups were selected for inflammatory and oxidative stress level tests, and lung and liver tissues were counterstained with HE for detection of pathological changes. Cytokines and oxidative factors were evaluated using the ELISA method (one-way ANOVA computed using SPSS 19.0.). With the increase in the concentration of citral, the survival rate of MRSA-infected mice increased accordingly. The ED50 values of citral for intramuscular injection and intragastric administration were 0.09 and 0.26 g kg-1 respectively. Citral significantly reduced cytokines (IL-1β, IL-6, TNF-α) and oxidative factors (malondialdehyde and hydroxyl radicals) of MRSA-infected mice, whereas it increased gluthtione and superoxide dismutase levels. Citral can reduce the lung inflammatory infiltrates infected by MRSA. Citral exerted a dose-dependent anti-MRSA effect and ameliorated MRSA-induced abnormal changes in inflammation and oxidative stress. This indicates that citral has the potential for development as a new anti-MRSA drug. © 2019 Society of Chemical Industry.

Acute and Cumulative Effects of Unmodified 50-nm Nano-ZnO on Mice.

Nanometer zinc oxide (nano-ZnO) is widely used in diverse industrial and agricultural fields. Due to the extensive contact humans have with these particles, it is crucial to understand the potential effects that nano-ZnO have on human health. Currently, information related to the toxicity and mechanisms of nano-ZnO is limited. The aim of the present study was to investigate acute and cumulative toxic effects of 50-nm unmodified ZnO in mice. This investigation will seek to establish median lethal dose (LD50), a cumulative coefficient, and target organs. The acute and cumulative toxicity was investigated by Karber's method and via a dose-increasing method, respectively. During the experiment, clinical signs, mortality, body weights, hematology, serum biochemistry, gross pathology, organ weight, and histopathology were examined. The LD50 was 5177-mg/kg·bw; the 95% confidence limits for the LD50 were 5116-5238-mg/kg·bw. It could be concluded that the liver, kidney, lung, and gastrointestinal tract were target organs for the 50-nm nano-ZnO acute oral treatment. The cumulative coefficient (K) was 1.9 which indicated that the cumulative toxicity was apparent. The results also indicated that the liver, kidney, lung, and pancrea were target organs for 50-nm nano-ZnO cumulative oral exposure and might be target organs for subchronic and chronic toxicity of oral administered 50-nm ZnO.

Effect of Large Flow CO2 on Indexes of Water Quality and LD50 of Crucian Carp

CCS (Carbon dioxide capture and storage) is an effective way to reduce carbon dioxide levels of atmosphere in the near future. However, due to the geological problems in storage areas and the technical problems of CO2 injection, there is still a risk of CO2 leakage during the implementation of CCS projects. Once the stored CO2 leaks through water body, the water quality, aquatic ecosystem and fisheries in the leakage areas will be affected. Therefore, the research on impacts of CO2 on water quality and aquatic ecosystem is significant for the promotion of CCS project. The effects of different CO2 flow rates on indexes of water quality and crucian carp mortality were investigated by injecting CO2 into aquarium. In the impact experiment of CO2 to water quality, three indicators which included pH, DO (dissolved oxygen) and DCO2 (dissolved carbon dioxide) were chosen, and four types of experiments were designed in the research which included blank water, fish-farming water, water with injecting CO2 and fish-farming water with injecting CO2. The acute toxicity test included preliminary experiment and formal experiment. The 24-hour all-dead flow rate (LD100) and the 96-hour non-dead flow rate of CO2 (LD0) were determined by preliminary experiment. The equal logarithmic interval method was used to set the groups of CO2 flow rates based on the above flow rates. The survival number and mortality of experimental fish in each group were recorded and calculated at 24h, 48h, 72h and 96h. At last, the LD50 of CO2 flow rate at each node of time was calculated according to the improved Karber method. The main conclusions were obtained as follows: (1) The average background values of pH, DO and DCO2 in blank water were 7.69, 8.58mg/L and 10.05mg/L respectively. The average values of pH, DO and DCO2 in fish-farming water were 7.26, 7.31mg/L and 43.78mg/L respectively. Comparing the above conclusion, pH and DO in blank water were slightly higher than that in fish-farming water, while the DCO2 was lower obviously. (2) When CO2 was injected into the non-fish farming water, pH and DO decreased and DCO2 increased with time, and all indexes balanced quickly. The larger the CO2 flow rate, the shorter the equilibrium time. The equilibrium values of pH and DO decreased with CO2 flow rate, whereas the equilibrium concentration of DCO2 increased with CO2 flow rate. When CO2 was injected into the fish farming water, all indexes in water had the similar rule with the non-fish farming water. Comparing with the indexes of water quality in non-fish-farming water with injected CO2, pH and DCO2 in fish-farming water with injecting CO2 were higher, while the DO was lower. The reason may be the dissolution of CO2, fish's respiratory consumption, and neutralization of ammonium ions formed by manure decomposition. (3) According to preliminary experiment of acute toxicity, the 24-hour all-dead flow rate of CO2 (LD100) was 800 mL/min, and the 96-hour non-dead flow rate of CO2 (LD0) was 100 mL/min. The acute toxicity test showed that the mortality of crucian carp increased with time and CO2 flow rates. According to the semi-lethal experiment of the modified Karber method, the LD50 of CO2 to crucian carp at 24h, 48h, 72h and 96h was 1490±326 mL/m3﹒min, 799±181 mL/m3﹒min, 503±66 mL/m3﹒min, 427±0 mL/m3﹒min, respectively. (4) According to above results, the CO2 leakage of the CCS project can be evaluated by monitoring of surface water quality and crucian carp's mortality rate in the future.

Toxicity of Peracetic Acid to Fish: Variation among Species and Impact of Water Chemistry

AbstractThere has been strong interest in the use of peracetic acid (PAA) in aquaculture as it can be used to disinfect water and hard surfaces and thereby eliminate or lower the burden of fish pathogens. Unfortunately, there has been little research on the toxicity of PAA to fish. Twelve species of fingerling fish that are important to aquaculture were exposed to PAA for 24 h in static toxicity bioassays in well water. These fish were: fathead minnow, Pimephales promelas; black‐nose crappie, Pomoxis nigromaculatus; bluegill, Lepomis macrochirus; blue tilapia, Oreochromis aureus; channel catfish, Ictalurus punctatus; golden shiner, Notemigonus crysoleucas; goldfish, Carassius auratus; grass carp, Ctenopharyngodon idella; largemouth bass, Micropterus salmoides; rainbow trout, Oncorhynchus mykiss; sunshine bass, Morone chrysops × M. saxatilis; and walleye, Sander vitreus. Median lethal concentration (LC50) values were estimated with the trimmed Spearman–Karber method using nominal PAA concentrations. The mean 24‐h LC50 values ranged from 2.8 to 9.3 mg/L PAA. Fathead minnow were very sensitive and blue tilapia were very tolerant to PAA exposure; LC50 values of other species tested were within the range of 4.1–6.2 mg/L PAA. More importantly, the 24‐h no‐observed‐effect concentration (NOEC) ranged from 1.9 to 5.8 mg/L PAA; the NOEC would be considered as the safe range for culturists to investigate the use of PAA. Decreased alkalinity/hardness increased the toxicity of PAA, while a small increase of dissolved organic content had no effect on PAA toxicity. Results of the present study are important information on the safe application of PAA for the aquaculture industry.

Toxicidade aguda da amônia em juvenis de sardinha-verdadeira Sardinella brasiliensis

A sardinha-verdadeira (Sardinella brasiliensis) é o principal recurso pesqueiro do Brasil, sustentando diretamente a indústria de enlatados, como matéria prima, e indiretamente a pesca dos tuní­­deos, pois juvenis são utilizados como isca-viva. A UFSC, através do Projeto Isca-Viva, vem desenvolvendo técnicas de produção de sardinha-verdadeira em cativeiro com vistas ao uso sustentável do recurso, entretanto não existem informações sobre a toxicidade dos compostos nitrogenados no cultivo desta espécie. O objetivo deste estudo foi estimar a toxicidade aguda de amônia (NH3-N) em juvenis de sardinha-verdadeira. Grupos de cinco peixes (1,04 ± 0,20 g) foram expostos a cinco concentrações de NH3-N (0,60 a 1,47 mg L-1) durante 96 h, mais um grupo controle em triplicata. Os testes foram conduzidos em um sistema semiestático com renovação diária total dos meios experimentais. Os resultados foram apresentados como dados de mortalidade, registrados nas diferentes concentrações testadas, utilizando o software Trimmed Spearman Karber Method. A concentração letal (LC50) após 96 h de exposição a NH3-N foi estimada em 0,74 (0,61-0,90) mg L-1. Os resultados indicam que juvenis de sardinha-verdadeira são relativamente sensí­­veis í­Â amônia em relação a outros peixes marinhos e que atenção especial deve ser dada a este composto em sistemas de cultivo.

Establishment and evaluation of mouse models of Staphylococcus aureus and Klebsiella pneumoniae bloodstream infections

Objective To establish the reliable models for early diagnosis of bloodstream infections by common clinical bacteria and to provide reliable experimental models for screening and validation of biomarkers in the early stage of bacterial bloodstream infections. Methods Strains of Staphylococcus aureus (S.aureus) ATCC25923 and Klebsiella pneumoniae (K.pneumoniae) ATCC700603 were used as experimental strains. Different concentrations of bacterial suspensions were injected into mice through tail vein. LD50 (median lethal dose) was calculated by Karber method and the concentration of 1/2LD50 was used for injection. The established mouse models were evaluated based on the changes in physical signs, body weights, white blood cells (WBC), platelets and IL-6 as well as the results of blood culture, PCR and HE staining. Results LD50 of S. aureus and K. pneumoniae to ICR mice were 8.1×108 CFU/ml and 1.11×109 CFU/ml, respectively. Body weights of mice decreased significantly at the time point of 24 h after infection. WBC counts in normal mice were about (1.42±0.66)×109/L, but increased in model groups at 3 h after bacteria challenge. Compared with the control group, WBC counts in model groups increased significantly at 6 h (P<0.05), followed by decreases. Two days after infection, WBC counts in model groups increased again and remained at high levels till the 7th day. However, PLT counts in the model groups dropped dramatically within 2 days as compared with that in control group (P<0.05). Increase of IL-6 was detected in the early K. pneumoniae infection and the extent of that was larger than that in S. aureus infection. Results of the whole blood culture were all positive within 2 days after infection. PCR analysis confirmed that the bloodstream infection pathogens were S. aureus and K. pneumoniae. Alveolar exudation, edema and leukocyte infiltration were observed in lung and liver tissues with HE staining. Conclusion The mouse models of S. aureus and K. pneumoniae infections for early detection of bloodstream infections are successfully established. This study provides reliable animal models for further researches on early diagnosis of different pathogen-induced bloodstream infections. Key words: Staphylococcus aureus; Klebsiella pneumonia; Virulence factor; Bloodstream infection model

0176 Environmental persistence of porcine epidemic diarrhea virus, porcine delta corona virus, and transmissible gastroenteritis in feed ingredients

Porcine epidemic diarrhea virus (PEDV), porcine delta corona virus (PDCoV), and transmissible gastroenteritis (TGEV) are major threats to swine production. Investigations of recent outbreaks confirmed that contaminated feed plays a role in virus transmission. This risk makes it necessary to evaluate the survival of such viruses in various feed ingredients. The objective of our experiment was to characterize the inactivation of PEDV, PDCoV, and TGEV in various feed and ingredient matrices. To determine differences in virus survival, 5-g samples of complete feed, spray-dried porcine plasma, meat meal, meat and bone meal, blood meal, corn, soybean meal, and low, medium, and high oil dried distillers grains with solubles were weighed into separate scintillation vials. These samples were inoculated with 1 mL of PEDV, PDCoV, or TGEV and incubated at room temperature for up to 56 d. At each time point, surviving virus was eluted and the supernatant was inoculated into vero-81 cells for PEDV, or swine testicular cells for PDCoV and TGEV. Cells were observed daily for 10 d for cytopathic effects, and this information was used to calculate a median tissue culture infectious dose (TCID50) using the Karber method. Inactivation kinetics were determined using the Weibull model. A delta value was estimated from the model, indicating the time necessary to reduce virus concentration by 1 log. This delta value was then compared across ingredients using the mixed procedure of SAS, and correlations between ingredient proximate analysis data and delta values were determined. Results showed that soybean meal had the greatest delta value (7.50 d) for PEDV compared with other ingredients (P < 0.06). Likewise, PDCoV (42.04 d) and TGEV (42.00 d) delta values were highest in soybean meal (P < 0.001). There was a moderate positive correlation between moisture and the delta value for PDCoV (r = 0.49, P = 0.01) and TGEV (r = 0.41, P = 0.02). There was also a moderate negative correlation between lipid content and the delta value for TGEV (r = −0.51, P = 0.01), suggesting that TGEV is less stable in ingredients with greater lipid content compared with ingredients with less lipid content. In conclusion, these results indicate that the first log reduction of PDCoV and TGEV takes the greatest amount of time in soybean meal and it appears to be the result of greater moisture content.

Protection of Litopenaeus vannamei against white spot syndrome virus by electron-irradiated inactivated vaccine and prebiotic immunogen

WSSV11White Spot Syndrome Virus. is an infectious agent of shrimp and other crustaceans. Neither effective vaccines nor treatments are currently available for this disease. Although the immune system of shrimps is not comparable to that of vertebrates, shrimps can acquire protection against pathogenic challenge by building up immunity. This study for the production of radio vaccines against WSSV used a radiation facility providing 10-Mev electron beams and a 2-mA current. In this study WSSV was isolated from infected shrimp samples and multiplied in Astacus leptodactylus crayfish hemolymph and called WSSV/IRN/1/2010. Titration of WSSV was obtained in post-larvae by Karber method as 105.4 LD5022Lethal Dose50./mL and the virus was inactivated by the electron beam irradiation, the D10 value and optimum dose of electron beam were obtained at 1.85 and 13kGy, respectively. Electron beam-irradiated WSSV (EBI33Electron Beam Irradiated.-WSSV) was used as a radio vaccine to immunize L. vannamei by intramuscular injection and bath-immersion routes. Prebiotic Immunogen as a diet additive was used to improve immune responses. PD5044Protective Dose50. was calculated at 5.62 and 6.02 for the shrimp groups vaccinated with EBI–WSSV vaccine and EBI-WSSV vaccine + Prebiotic Immunogen, respectively. Calculated RPS values were 75%, 91% and 25% for the EBI-WSSV vaccine alone, EBI-WSSV + Prebiotic Immunogen and Prebiotic Immunogen alone (PID55Probiotic Immunogen Diet.) groups vaccinated by immersion route, and 73%, 82% and 18.5% by injection route, respectively. Significant difference in cumulative mortalities was observed between both vaccination groups (EBI-WSSV and EBI-WSSV+ immunogen), and the control groups (P<0.05). No significant difference in cumulative mortalities was observed between the two vaccination groups (P>0.05). Therefore, EBI-WSSV vaccine induced immunity responses in shrimp against WSSV infection and Prebiotic Immunogen enhanced this response.

Toxicological evaluation of ferrous N-carbamylglycinate chelate: Acute, Sub-acute toxicity and mutagenicity

Iron is an essential trace element that is vital important in various biological process. A deficiency in iron could induce public health problem e.g. anaemia, while an overload could induce ROS production, lipid peroxidation and DNA bases modifications. In the present study, a new iron fortifier was synthesized, and its acute/sub-acute toxicity was investigated. According to the improved Karber's method, the median lethal dose (LD50) of the ferrous N-carbamylglycinate in SD rat was 3.02 g/kg and the 95% confidence intervals were between 2.78 and 3.31 g/kg. No biologically significant or test substance-related differences were observed in body weights, feed consumption, clinical signs, organ weights, histopathology, ophthalmology, hematology, and clinical chemistry parameters in any of the treatment groups of ferrous N-carbamylglycinate at target concentrations corresponding to 150, 300, and 600 mg/kg/day for 28 days. The no observed adverse effect level (NOAEL) for ferrous N-carbamylglycinate was at least 600 mg/kg b.w. day in rats. In addition, no evidence of mutagenicity was found, either in vitro in bacterial reverse mutation assay or in vivo in mice bone marrow micronucleus assay and sperm shape abnormality assay. On the basis of our findings, we conclude that ferrous N-carbamylglycinate is a low-toxic substance with no genotoxicity.

Comparative Susceptibility of Freshwater Fish Species to Metals from Ultramafic Soils

Soils derived from ultramafic rocks have a mineralogy rich in metals like Co, Cr, Fe and Ni, and the process of extracting these metals from soil generates highly metallic waste. When disposed near of water bodies these residues pose a risk on the aquatic environment. Two fish species are used in ecotoxicity tests worldwide: Danio rerio (zebrafish), an Asian species, and Oreochromis niloticus (tilapia), an African one. However, Hyphessobrycon eques (mato grosso), a native of South America and found in Brazilian rivers, is as yet little used in ecotoxicity tests. The aims of this study were to evaluate the acute toxicity of the metals Co, Cr, Fe and Ni to the fish D. rerio, O. niloticus and H. eques, and compare its susceptibility. Tests were conducted following the recommendations of the Brazilian Association for Technical Standardization (ABNT). Stock solutions were prepared with the analytical reagents CoCl2.6H2O; K2Cr2O7; FeCl3.6H2O e NiCl2.6H2O. The quantification of metals was carried out in the stock solutions by ICP-AES. The dilution water used was synthetic softwater pH 7.3±0.1, hardness 43 mg L-1; DO 95% and temperature 25±1°C. To perform static tests 10 fish from each species were exposed for 96 hours, in duplicate, to different concentrations of the metals tested. The measured 96-hr initial lethal concentrations LC(I)50 of metals Co, Cr, Fe and Ni, calculated by the Trimmed Spearman Karber method, were 35.7, 35.9, 5.6 and 15.0 mg L-1 for D. rerio, 79.8, 32.6, 8.8 and 49.7 mg L-1 for O. niloticus and 104.1, 23.7, 8.5 and 34.5 mg L-1 for H. eques, respectively. The data showed that D. rerio has greater susceptibility to the metals tested, which justifies its increased use as a test species, except concerning Cr, for which H. eques was the most sensitive species.

Acute and subacute oral toxicity of polychlorinated diphenyl sulfides in mice: Determining LD50 and assessing the status of hepatic oxidative stress

Polychlorinated diphenyl sulfides (PCDPSs), a series of dioxin-like compounds, have been detected in various environmental samples. However, information on the toxicity of these compounds is limited. In the present study, the toxic effects of PCDPSs were assessed after acute and subacute exposure in mice. Relationships between acute toxicity, number, and position of substituted Cl atoms were assessed. In the acute study, 11 types of PCDPSs were administered to female Kunming mice by gavage, and median lethal doses (LD50s) were determined by the Karber method. Results indicated that the LD50s of lower substituted PCDPSs were smaller than higher substituted PCDPSs. Substituted positions also influenced the LD50 of PCDPSs. Terminal necropsy showed increased relative liver weights and decreased relative kidney weights. Histological examination of livers demonstrated swollen cells, inflammation, vacuolization, and necrosis. In the 28-d subacute exposure tests, 11 types of PCDPSs were dissolved in corn oil and administered to mice at doses of 1, 10, and 100 mg/kg. Superoxide dismutase (SOD) activity and malondialdehyde (MDA) levels in mouse liver were determined after the exposure period. Results suggested that lower substituted PCDPSs decreased SOD activity in the high-dose groups compared with controls, and MDA level in the 100-mg/kg dose group was significantly increased. In addition, acute toxicity of PCDPSs partly corresponded to the hepatic oxidative damage observed.