Mouse Lethality Test Research Articles

Hypercapsule is the cornerstone of Klebsiella pneumoniae in inducing pyogenic liver abscess.

To investigate the mechanisms of Klebsiella pneumoniae-induced pyogenic liver abscess (PLA). Forty-three K. pneumoniae strains from PLAs and 436 from non-PLAs were collected. Their differences were compared for virulence genes and factors, sequence types, and serotypes. Virulence genes wzi, wzy-K1, and wzi+wzy-K1 were deleted in K. pneumoniae NTUH-K2044. Various analyses, such as transmission electron microscopy, neutrophil killing tests, and mouse lethality tests, were used to confirm the consequent changes. Differences were found between K. pneumoniae strains from PLA and non-PLA samples for virulence genes and factors, including metabolism genes (allS and peg-344), capsular polysaccharide (CPS)-synthesis channel gene (wzy-K1), CPS-regulating genes (p-rmpA, p-rmpA2, and c-rmpA), and siderophore genes (iucA and iroN). When wzy-K1 was positive, the difference between PLA and non-PLA samples was only observed with c-rmpA. Δwzi, Δwzy-K1, and ΔwziΔwzy-K1 strains reverted to hypovirulence. In the Kupffer cell stimulation assay, interleukin (IL)-6, IL-12, IL-10, and transforming growth factor-β secretions were found to be equivalent in NTUH-K2044, Δwzi, Δwzy-K1, and ΔwziΔwzy-K1 groups. Lower IL-1β and higher tumor necrosis factor-α secretions were observed for Δwzi, Δwzy-K1, and ΔwziΔwzy-K1. Hypercapsule production is the cornerstone of hypervirulence, regardless of exopolysaccharides. K1 K. pneumoniae-induced PLA may decrease core inflammatory cytokines rather than increase anti-inflammatory cytokines. Exopolysaccharides could also attenuate the inflammatory response to aid in the immune escape of K. pneumoniae.

Niclosamide Modulates the Cellular and Humoral Immune Response in Balb/c Mice.

Niclosamide, a STAT3 inhibitor, is widely under investigation due to its anti-cancer properties. STAT3 also exhibits an exciting role in the immune responses. This study aimed to evaluate the impact of niclosamide on immune response of mice. Niclosamide was administered to balb/c mice. To evaluate cell-mediated immune response, a contact-hypersensitivity (CHS) test, cyclophosphamide-induced neutropenic assay, and carbon clearance test were performed, whereas a humoral immune response was evaluated by hemagglutination assay (HA) and mice lethality test. The concentration of TGF-β1 was determined by enzyme-linked immunosorbent assay (ELISA) on murine peritoneal macrophages. In the CHS test, niclosamide caused a decrease in skin thickness, significantly exhibiting a decrease in inflammation. A highly significant decrease in overall leukocyte count (lymphocytes and neutrophils) was observed before and after cyclophosphamide injection as compared with the control group. However, only a highly significant decrease in the neutrophil percentage was observed. Niclosamide has decreased the phagocytic process immensely compared with the control. In the HA titer, niclosamide was found to reduce the antibodies' titer compared with the negative control group. In the mice lethality test, the treatment groups have shown an increase in the percentage of mortality. TGF-β1 elevated in peritoneal macrophages when treated with niclosamide, in a dose-dependent manner. Niclosamide exerts potent immunomodulatory effects by significantly suppressing cell-mediated and humoral immune responses and increasing the levels of TGF-β1 in mice. Niclosamide might be added as an adjuvant to immunosuppressive drugs for the treatment of autoimmune diseases.

Phenotypic characterization, detection of virulence factors, and antibacterial susceptibility profile of clinical isolates of Klebsiella pneumoniae

Klebsiella pneumoniae has been one of the most occurring bacteria in clinical samples causing community-acquired and nosocomial infections. It has been developing resistance to most antimicrobial agents. These have increased the pathogenicity and the chance for the evolvement of more invasive K. pneumoniae. The study was aimed at the identification and detection of Klebsiella pneumoniae from clinical samples. Some selected virulence factors were detected and the antibacterial susceptibility profile of the isolates was conducted. The incubation period, infective dose, and mortality rate of K.pneumoniae were measured using Baggs albino rats (BALB/c) strain. The isolation and identification of 56 Klebsiella pneumoniae from 223 clinical samples were made. Out of 56 K. pneumoniae isolates, all manifested mucoid phenotype, 44(78.6%) capsule antigen and 13(23.2%) Siderophore. The antimicrobial test conducted identified Zinacef 51(91.1%), Rocephin 50 (89.3%), and Ampiclox 50 (91.1%) as the most resistant. Ciprofloxacin 49 (87.5%), chloramphenicol 35(62.5%), Gentamycin 32(57.1%), and Amoxicillin 28 (50%) were the most susceptible to Klebsiella pneumoniae. The mouse lethality test shows that K. pneumoniae hypermucoviscous strain can cause 41.7% lethality and 66.7% mortality in Baggs Albino rats. Meanwhile an infection dose 105cfu/ml, 107cfu/ml and 109cfu/ml produced an incubation period of 9 days, 7days and 5days. The chi-square test shows no significant association between the isolate and the gender of the patients, but there is a significant association between the samples and isolates identified p <0.05. Likewise, the association between mucoid phenotype, capsule antigen, siderophore, and the type of infection is significant (p<0.05). It is finally concluded that K. pneumoniae is the second most prevailing bacterium causing community-acquired infection the resistant pattern recorded identified the test bacteria as a multidrug-resistant strain and manifestation of the virulent factor depends on the type and site of infection caused by the K. pneumoniae.

Prevalence of Carbapenem-Resistant Hypervirulent Klebsiella pneumoniae and Hypervirulent Carbapenem-Resistant Klebsiella pneumoniae in China Determined via Mouse Lethality Tests.

ObjectiveTo investigate the epidemiology of carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-HvKP) and hypervirulent carbapenem-resistant Klebsiella pneumoniae (Hv-CRKP).MethodsTotally 436 K. pneumoniae strains were collected from 7 hospitals in mainland China between 2017.01 and 2018.02. Sequence types, serotypes, antimicrobial-resistance and virulence genes were analyzed. Additionally, string test, capsule stain, Periodic Acid Schiff stain, fitness analysis, quantitative real-time PCR and mouse lethality test were also performed. Molecular combinations were used to screen putative bla KPC(+)-HvKP and Hv-bla KPC(+)-KP, followed by the confirmation of mouse lethality test.ResultsDiverse detection rates were found for the virulence genes, ranging from c-rmpA (0.0%) to entB (100.0%). According to the molecular criteria, 127, 186, 9 and 26 strains were putatively denoted as HvKP, bla KPC(+)-KP, bla KPC(+)-HvKP and Hv-bla KPC(+)-KP. Mouse lethality test confirmed 2 bla KPC(+)-HvKP strains (JS184 and TZ20) and no Hv-bla KPC(+)-KP. JS184 showed K2 serotype, thin capsule, positive exopolysaccharid and string test. TZ20 presented K20 serotype, thin capsule, negative exopolysaccharide and string test. Compared with the positive control NTUH-K2044, equal galF expression and growth curves were confirmed for JS184 and TZ20.ConclusionsMolecular determination of CR-HvKP and Hv-CRKP brings remarkable bias compared with mouse lethality test. The exact prevalence of CR-HvKP is less than 1.0%, which of Hv-CRKP is much lower.

In-vivo Immunomodulatory Activities of Isolated Compounds from the Leaves of Amaranthus spinosus and Achyranthes aspera

The purpose of this study was to look into the Immunomodulatory action of isolated compound of Achyranthes aspera and Amaranthus spinosus in experimental model of immunity. Cellular immunity was carried out by neutrophil adhesion test and carbon clearance assay, whereas, humoral immunity was analyzed by mice lethality test and indirect haemagglutination assay. The dose was selected after acute toxicity study of isolated compound of Achyranthes aspera and Amaranthus spinosus and administered at 5 mg/kg orally. The Levamisole (0.68mg/kg, p.o) was used as standard. Isolated compound of Achyranthes aspera (IAA) and Amaranthus spinosus (IAS) at 5 mg/kg produced significant increases in adhesion of neutrophils and an increase in phagocytic index in carbon clearance assay. Both doses of IAA and IAS significantly prevented the mortality induced by bovine Pasteurella multocida in mice. Treatment of animals with IAA, IAS and Levamisole significantly increased the circulating antibody titre in indirect haemagglunation test. Among the different Isolates, (IAS) was more effective in cellular immunity models than the (IAA). However, Both the Isolates IAA and IAS exhibited similar protection in humoral immunity procedures. It is concluded that isolated compound of Achyranthes aspera(IAA) and Amaranthus spinosus(IAS)possesses potential for augmenting immune activity by cellular and humoral mediated mechanisms more at doseof (5 mg/kg).

Virulence among different types of hypervirulent Klebsiella pneumoniae with multi-locus sequence type (MLST)-11, Serotype K1 or K2 strains

BackgroundTwo different types of hypervirulent K. pneumoniae (HvKp), the MLST-11 and serotype K1/K2 strains, have been frequently described in recent studies. Although these two types of strains were described to be HvKp, their virulence was not compared. In this study, in vitro and in vivo approaches were used to assess differences in virulence.Materials and methodsA total of twenty-nine isolates, including 6 strains of each of serotype K1 and K2 isolates and 17 strains of ST11 isolates, were selected for this study. Phenotypic tests of virulence were performed by the string test and analysis of the virulent associated genes was detected by PCR. In vitro models of serum resistance and phagocytosis were used as the parameters to assess the virulence. In-frame deletion of virulence-associated genes was performed to study their contributions to virulence. The median lethal dose, i.e., the LD50, in mice was determined following IP injection.ResultsAlthough serotype K1 and K2 strains and ST11 isolates had similar virulence gene profiles, the ST11 isolates showed less serum and phagocytic resistance than the serotype K1/K2 isolates. The mouse lethality test revealed that all ST11 isolates were unable to cause lethality, even at > 107 CFU, while serotypes K1 and K2 showed an LD50 at ≤ 103 CFU. Aerobactin or capsule knockout mutants exhibited a lower LD50 than the parental strain, while capsule mutants showed a more significant decrease in LD50.ConclusionSince there was a significant difference in virulence levels between the two types of HvKp when assessed in in vitro and in vivo models, it may be better to use the designation "HvKp" for some strains based on animal studies to avoid confusion. Virulence and non-virulence could be analysed in a relative manner, especially in comparison studies.

Molecular Studies on Pasteurella multocida in Ducks

Pasteurella multocida is the common cause of Duck septicemia (Pasteurellosis) which affects ducks leading to high economic losses to duck producers. Pasteurella multocida infections are associated with severe, life-threatening systemic disease involving both hemorrhagic pneumonia and septicemia. A cross-sectional study was carried out on 220 samples collected from different breeds of ducks (Mallard, Muscovy and Baladi) from Minufiya governorate, Egypt. Only 16 cases were positive for Pasteurella multocida infection (7.3%). These isolates were confirmed microscopically, biochemically and rapidly by using Vitek2 compact system. The application of PMT-ELISA and mouse lethality test for the 16 isolates of P.multocida serotypes for differentiation between toxigenic and non toxigenic isolates. All tested of 16 isolates showed to be toxigenic by using PMT-ELISA and mouse lethality test. The application of PCR and multiplex PCR for the detection of tox A gene and capsular serotyping of toxigenic isolates of P.multocida respectively. All 16 P.multocida isolates were positive to tox A and capsular type A (100%). This study concluded that P. multocida serve as a major cause of Duck septicemia (Pasteurellosis) which affects ducks leading to high economic losses in poultry industry. Also indicated that the majority of P. multocida serotypes are toxigenic.

Evaluation of the Immunomodulatory Activity of the MethanolMethylene Chloride Extract of Parkia Biglobosa Jacq Benth. (Fabaceae) Stem Bark in Mice

The study was carried out to assess the effect of methanol/methylene chloride extract (MME) of Parkia biglobosa stem bark on specific and non-specific immune responses in rodents. Adult Swiss albino mice (20-35 g) of either sex were divided into five groups (n=5) and treated for 10 days. The effect of MME on adaptive immunity was evaluated using neutrophil adhesion test and cyclophosphamide-induced neutropenia while mice lethality test was used to evaluate its effect on non-specific immunity. Results of the neutrophil adhesion test showed a significant (P<0.05) increase in neutrophil adhesion at 250 mg/kg. However, there was decrease in neutrophil adhesion at 500mg/kg and 750 mg/kg respectively. The extract also gave no significant protection from cyclophosphamide-induced immunosuppression. Phytochemical analysis revealed the presence of alkaloids, glycosides, saponins, terpenoids, flavonoids and tannins. The oral LD50 of the extract was greater than 5000 mg/kg and considered to be relatively safe. The findings above show that the stem barks extract of the plant enhanced some aspects of specific immunity and therefore to some extent, hold promise for use as an immunostimulatory agent though it may not offer good protection from the effect of cyclophosphamide on the haematopoietic system.

Evaluation of immunomodulatory effects of lomefloxacin in mice

Lomefloxacin is a flouroquinolone antibiotic that is quite efficacious against many gram negative and gram positive pathogens. Lomefloxacin evince antibacterial effects by modifying DNA gyrase in gram negative pathogens and topoisomerase IV in gram positive pathogens. This study is designed to assess the immunomodulatory effects of lomefloxacin in male albino mice. Three doses of lomefloxacin 12.5 mg/kg, 25 mg/kg and 50 mg/kg were used and delayed type hypersensitivity assay, cyclophosphamide induced neutropenic assay, carbon clearance assay, heamagglutination assay and mice lethality test were performed to evaluate the effects of lomefloxacin on immune system of mice. DTH assay has depicted the significant immunosuppressant potential of lomefloxacin at 25 mg/kg and 50 mg/kg dose. Total leukocyte count have exhibited highly significant reduction (P<0.001) in leukocyte count after cyclophosphamide administration. Differential leukocyte count has shown significant (P<0.01) reduction in lymphocyte count, whereas, highly significant (P<0.001) increase in monocyte count and significant (P<0.05) increase neutrophil count has been observed. In carbon clearance assay, highly significant (P<0.01) increase in phagocytic index has been noted at 12.5 mg/kg and 25 mg/kg doses. Humoral immune system responses are suppressed in dose dependent manner by both heamagglutination assay (P<0.001) and mice lethality assay (P<0.001). Results clearly depict that lomefloxacin possess quite significant immunomodulatory potential.

Assessment of antimicrobial and immunomodulatory activities of termite associated fungi, Termitomyces clypeatus R. Heim (Lyophyllaceae, Basidiomycota)

BackgroundTermitomyces clypeatus (T. clypeatus) is an edible mushroom specie which grows in various areas in Cameroon. It is indicated by several healers in treatment of several infectionsand is frequently used for this purpose. However, no study has been reported on its antibacterial and immunomodulatory activities which were the aim of this study.MethodsDisc diffusion method was used to appreciate the bactericidal activity against 4 bacteria and 2 yeast species. The immunomodulatory activities were assessed in mice, where the extract was administered by gavage and as supplement in the feed. The treatment was done for 10 days. Delayed type hypersensitivity test was carried out to assess the cell mediated immune response while the effect on humoral immunity was evaluated using hemagglutination assay and mice lethality test.. The body weight was also recorded in mice used for delayed hypersensitivity.ResultsIt was found that extract of T. clypeatus highly inhibited the growth of bacteria and yeast at different ratios compared to the medium (P < 0.05). The extract of T. clypeatus has reversed the immunosuppressed effects of dexamethasone on antibody formation. In addition, it significantly decreased of the mice lethality rate in mice infected by Pasteurella multocida (P < 0.05). Administration of T. clypeatus also significantly increased the delayed type hypersensitivity response in healthy and dexamethasone immunosuppressed mice (P < 0.05), but it significantly reduced the body weight of mice after 10 days.ConclusionThe results provided basic information demonstrating the antibacterial activity and immunostimulatory activity of T. clypeatus on both cell-mediated and humoral immunity.

Evaluation of immunomodulatory activity of tenoxicam in mice

Purpose: The present study was conducted to evaluate the effect of tenoxicam on cellular and humoral immunity. Methods: Tenoxicam (2.5 - 10mg/kg) was administered at three different doses to three groups of mice and the cellular immune responses were studied using delayed hypersensitivity response (DTH) and cyclophosphamide-induced neutropenia while the humoral immune response was evaluated using hemagglutination test and mice mortality ratio. Normal saline and cyclophosphamide were used as negative and positive controls, respectively. Results: DTH assay resulted in a significant reduction in skin thickness (p 5 mg >2.5 mg> negative control group. A dose dependent reduction response was observed (p<0.05) in haemagglutination assay (HA). In mice lethality test mortality ratios of 2.5 mg, 5 mg, 10 mg tenoxicam were 60 %, 80% and 100 %, respectively, compared to 20 % and 100 % for normal saline group and cyclophosphamide, respectively Conclusion: The results suggest that tenoxicam suppresses both cellular and humoral immunity in mice. Keywords: Tenoxicam, Cellular immunity, Humoral immunity

Evaluation of the immunomodulatory effect of Eclipta prostrata whole plant extract on albino rats

The study aims to determine the immunomodulatory effect of Eclipta prostrata whole plant extract on albino rats by using various methods viz., carbon clearance test, cyclophosphamide induced neutropenia and neutrophil adhesion test. The results of Eclipta prostrata whole plant; increased the phagocytic index in carbon clearance test; prevented mortality induced by Eclipta prostrata by 17% in mice lethality test and prevented the cyclophosphamide induced neutropenia. The Eclipta prostrata also affected the humoral immunity; Both EHD ( Eclipta prostrata high dose) and ELD ( Eclipta prostrata low dose) produced a significant increase the serum immunoglobulin levels and produced a significant increase in antibody titre in indirect haemagglutination test. Eclipta prostrata low dose showed significant effect only in increasing the serum immunoglobulins level whereas it increased mortality in mice lethality test. To conclude, Eclipta prostrata bark possesses immunostimulant activity. In high dose, it stimulates both cellular and humoral arms of the immune response and the low dose stimulates only the production of antibodies.

Exploring the venom of the forest cobra snake: Toxicovenomics and antivenom profiling of Naja melanoleuca

A toxicovenomic analysis of the venom of the forest cobra, N. melanoleuca, was performed, revealing the presence of a total of 52 proteins by proteomics analysis. The most abundant proteins belong to the three-finger toxins (3FTx) (57.1wt%), which includes post-synaptically acting α-neurotoxins. Phospholipases A2 (PLA2) were the second most abundant group of proteins (12.9wt%), followed by metalloproteinases (SVMPs) (9.7wt%), cysteine-rich secretory proteins (CRISPs) (7.6wt%), and Kunitz-type serine proteinase inhibitors (3.8wt%). A number of additional protein families comprised each <3wt% of venom proteins. A toxicity screening of the fractions, using the mouse lethality test, identified toxicity in RP-HPLC peaks 3, 4, 5 and 8, all of them containing α-neurotoxins of the 3FTx family, whereas the rest of the fractions did not show toxicity at a dose of 0.53mg/kg. Three polyspecific antivenoms manufactured in South Africa and India were tested for their immunoreactivity against crude venom and fractions of N. melanoleuca. Overall, antivenoms immunorecognized all fractions in the venom, the South African antivenom showing a higher titer against the neurotoxin-containing fractions. This toxicovenomic study identified the 3FTx group of α-neurotoxins in the venom of N. melanoleuca as the relevant targets to be neutralized. Biological significanceA toxicovenomic analysis of the venom of the forest cobra, also known as black cobra, Naja melanoleuca, was performed. Envenomings by this elapid species are characterized by a progressive descending paralysis which starts with palpebral ptosis and, in severe cases, ends up with respiratory arrest and death. A total of 52 different proteins were identified in this venom. The most abundant protein family was the three-finger toxin (3FTx) family, which comprises almost 57.1wt% of the venom, followed by phospholipases A2 (PLA2) (12.9wt%). In addition, several other protein families were identified in a much lower percentage in the venom. A toxicity screening of the fractions, using the mouse lethality assay, identified four peaks as those having toxicity higher than that of the crude venom. These fractions predominantly contain α-neurotoxins of the 3FTx family. This toxicovenomic characterization agrees with the clinical and experimental manifestations of envenomings by this species, in which a strong neurotoxic effect predominates. Therefore, our findings suggest that immunotherapy against envenomings by N. melanoleuca should be directed towards the neutralization of 3FTxs; this has implications for the improvement of current antivenoms and for the development of novel antivenoms based on biotechnological approaches. A screening of the immunoreactivity of three antivenoms being distributed in sub-Saharan Africa revealed that they immunoreact with the fractions containing α-neurotoxins, although with different antibody titers.

Comparative assay of Vipera ammodytes antivenom potency

The finding of the most appropriate way to assess precisely the antivenom efficacy represents one of the major issues for antivenom standardization and success increasing of antivenom therapy. The efficacy of experimental Vipera ammodytes antivenom raised in sheep was determined using in vivo mouse lethality test, respectively, L-aminoacid oxidase, total proteinase and phospholipase A2 antienzymatic effectiveness. The values gained for the antivenom potency depend on the method of measure. So, some of the most toxic venom proteins own phospholipase A2 activity and provide the highest antivenom potency (lowest effective dose) values by antienzymatic assay method. This value is similar with total antiproteolytic antivenom potency value, but almost three times higher than value obtained by L-aminoacid oxidase (low toxic viper venom protein) antienzymatic assay method.

Therapeutic management of botulism in dairy cattle

Aim:To report the successful recovery of few dairy cattle from botulism in response to a modified therapeutic strategy.Materials and Methods:Seventy four naturally-occurring clinical cases of bovine botulism encountered during the period of 2012-2014 which were confirmed by mouse lethality test became material for this study. Affected animals were made into three groups based on the treatment modifications made during the course of study.Results and Discussion:With the modified therapeutic regimen, 17 animals recovered after 7-10 days of treatment. Clinical recovery took 2-30 days. Animals which were not given intravenous fluid and calcium recovered uneventfully. Cattle which were already treated with intravenous fluids, calcium borogluconate, and antibiotics did not recover. They were either died or slaughtered for salvage.Conclusion:In cattle with botulism, administration of Vitamin AD3E and activated charcoal aid the clinical recovery. Besides, strictly avoiding anti-clostridial antibiotics, fluid therapy, and calcium therapy may facilitate the clinical recovery. Upon fluid administration, the pulmonary congestion existed in the ailing cattle might have worsened the anoxia. Administration of antibiotics like penicillin, aminoglycosides, and tetracyclines further worsen the neuronal paralysis by increasing the availability of botulinum neurotoxin. Cattle in early botulism have fair chances of recovery with the modified therapy.

Clostridium cluster I and their pathogenic members in a full-scale operating biogas plant.

A biogas production plant operating with main and secondary digesters (MD, SD) was analysed for the diversity of bacteria from Clostridium cluster I and its pathogenic members. The plant was run in two parallel lines, both receiving silages, and one, in addition, cattle manure (CM). Quantitative PCR of 16S rRNA genes from directly extracted DNA indicated that cluster I represented 0.2 to 5.6 % of the total bacterial communities. Its prevalence was particularly low in CM and also in SD compared to MD, indicating its decline during fermentation. In contrast, another highly abundant clostridial group, i.e. the "faecal" cluster XIVa, remained quantitatively unaffected during fermentation. A total of 85.1 % of 581,934 rRNA gene sequences gathered by group-specific PCR from the silages, CM and digesters could be assigned to cluster I. All remaining sequences fell into other clostridial groups. The three most dominant operational taxonomic units (OTUs) introduced with CM were from cluster I, and they declined during fermentation. Fermentation with CM significantly increased OTUs of clostridia outside of cluster I but not within. The only OTUs related to pathogens were detected for Clostridium botulinum with 0.18 % of all cluster I sequences in maize silage and less than 0.01 % in the other substrates and digester materials. These OTUs could be assigned to all four established C. botulinum groups, thus, potentially covering all seven neurotoxins. Mouse lethality tests of samples with suspected presence of C. botulinum, however, indicated no toxigenic potential and, thus, no risk associated with the rare occurrence of these OTUs.

Phenotypic, antimicrobial susceptibility profile and virulence factors of Klebsiella pneumoniae isolated from buffalo and cow mastitic milk

Studies on the prevalence and virulence genes of Klebsiella mastitis pathogens in a buffalo population are undocumented. Also, the association of rmpA kfu, uge, magA, Aerobactin, K1 and K2 virulent factors with K. pneumoniae buffalo, and cow mastitis is unreported. The virulence of K. pneumoniae was evaluated through both phenotypic and molecular assays. In vivo virulence was assessed by the Vero cell cytotoxicity, suckling mouse assay and mice lethality test. Antimicrobial susceptibility was tested by disk diffusion method. The 45 K. pneumoniae isolates from buffalo (n = 10/232) and cow (n = 35/293) milk were isolated (45/525; 8·6%) and screened via PCR for seven virulence genes encoding uridine diphosphate galactose 4 epimerase encoding gene responsible for capsule and smooth lipopolysaccharide synthesis (uge), siderophores (kfu and aerobactin), protectines or invasins (rmpA and magA), and the capsule and hypermucoviscosity (K1 and K2). The most common virulence genes were rmpA, kfu, uge, and magA (77·8% each). Aerobactin and K1 genes were found at medium rates of 66·7% each and K2 (55·6%). The Vero cell cytotoxicity and LD (50) in mice were found in 100% of isolates. A multidrug resistance pattern was observed for 40% of the antimicrobials. The distribution of virulence profiles indicate a role of rmpA, kfu, uge, magA, Aerobactin, and K1 and K2 in pathogenicity of K. pneumoniae in udder infections and invasiveness, and constitutes a threat for vulnerable animals, even more if they are in combination with antibiotic resistance.

Characterization and neutralization of Nemopilema nomurai (Scyphozoa: Rhizostomeae) jellyfish venom using polyclonal antibody

Jellyfish stings have often caused serious health concerns for sea bathers especially in tropical waters. In the coastal areas of Korea, China and Japan, the blooming and stinging accidents of poisonous jellyfish species have recently increased, including Nemopilema nomurai. We have generated a polyclonal antibody against N. nomurai jellyfish venom (NnV) by the immunization of white rabbits with NnV antigen. In the present study, the antibody has been characterized for its neutralizing effect against NnV. At first, the presence of NnV polyclonal antibody has been confirmed from the immunized rabbit serum by Enzyme linked immunosorbent assay (ELISA). Then, the neutralizing activities of the polyclonal antibody have been investigated using cell-based toxicity test, hemolysis assay, and mice lethality test. When the polyclonal antibody was preincubated with NnV, it shows a high effectiveness in neutralizing the NnV toxicities in a concentration-dependent manner. Moreover, we explored proteomic analyses using 2-D SDS-PAGE and MALDI-TOF mass spectrometry to illustrate the molecular identities of the jellyfish venom. From this, 18 different protein families have been identified as jellyfish venom-derived proteins; the main findings of which are matrix metalloproteinase-14, astacin-like metalloprotease toxin 3 precursor. It is expected that the present results would have contributed to our understandings of the envenomation by N. nomurai, their treatment and some valuable knowledge on the pathological processes of the jellyfish stinging.

Mouse compound muscle action potential assay: An alternative method to conduct the LD50 botulinum toxin type A potency test

PurposeWe performed a prevalidation of the compound muscle action potential (CMAP) assay to determine the potency of botulinum neurotoxin type A (BoNT/A) with the aim of substituting for the mouse lethality test (LD50), which is used for quality control. MethodsPrevalidation experiments were performed to demonstrate the specificity, linearity, accuracy, precision, range, limit of quantitation (LOQ), and robustness of the assay. For specificity, toxin detection ability was determined in the presence of neutralizing antibodies (0.8 and 8 IU/mL). Linearity of this assay was determined by measuring CMAP amplitude using nine concentrations (n = 3) in the range of 1–100 U/mL (n = 3). Accuracy was assessed using five concentrations (n = 3) in the range of 4–40 U/mL. Intermediate precision was confirmed by analyzing individually prepared reagents on multiple days by one operator (n = 3). Different body weights (23–25 and 25–27 g) and measurement times (3–5 and 5–7 min) after anesthetic induction were tested to assess robustness. ResultsThis assay might have BoNT/A specificity, based on the CMAP amplitude recovery using a concentration of neutralizing antibodies. The calibration curves were linear over the range of 2–40 U/mL (R2 = 0.982). The accuracy of 14 determinations was within the range of 89.8–118.6% compared to the theoretical values among 15 determinations, except one (131.3%). Assay variability was acceptable with coefficients of variation of 4.3–14.4%. The range of quantification and the LOQ were 4–40 U/mL and 4 U/mL, respectively. Different body weights and measurement times after inducing anesthesia had no effect on CMAP amplitude. ConclusionsThese results suggest that the mouse CMAP assay is an alternative method to the standard LD50 potency test and meets the requirement of the three Rs (particularly refinement and reduction).

Effect of Stigmas of Crocus sativus L. (saffron) on Cell Mediated and Humoral Immunity

Crocus sativus stigmas (saffron) are most commonly used as spice, coloring, and flavoring agent in the preparation of various foods. The effect of saffron on the cell mediated immune system was evaluated by using different experimental animal models such as carbon clearance test; cyclophosphamide induced neutropenia, neutrophil adhesion test, whereas humoral mediated immunity was analysed by its effect on serum immunoglobulins, mice lethality test, and indirect hemagglutination test. Saffron was orally administered as suspension at doses of 50 and 100 mg/kg; Ocimum sanctum L. extract (100 mg/kg p.o) was used as a reference drug. Saffron significantly increased the level of serum immunoglobulins and circulating antibody titre, prevented the mortality in mice when challenged with lethal Pasteurella multocida toxin suggesting an increase in overall humoral mediated immunity. Also, there was a significant increase in the neutrophil adhesion to nylon fibers. Saffron was not effective in preventing cyclophosphamide-induced neutropenia. However in carbon clearance test, only the low dose was effective in producing a significant increase in the phagocytic index with the high dose failing to do so. The results from the present investigation indicate that C. sativus at low doses posses humoral and cell mediated immunity, thus substantiating its traditional claim as a potential immunostimulant and as an anticancer agent.