Effect of Carbonyl Cyanide Chlorophenylhydrazone on Intrabacterial Concentration and Antimicrobial Activity of Amphenicols against Swine Resistant Actinobacillus pleuropneumoniae and Pasteurella multocida.

Effects and mechanism of carbonyl cyanide chlorophenylhydrazone (CCCP) on antimicrobial activity of florfenicol (FF) and thiamphenicol (TAP) were investigated against amphenicol-resistant Actinobacillus pleuropneumoniae and Pasteurella multocida isolated from diseased swine. Broth microdilution and time-kill assays indicated that CCCP dose-dependently and substantially (4-32 fold MIC reduction) improved amphenicol antimicrobial activity. When combined with CCCP at the lowest literature reported dose (2-5 µg/mL), 85% FF resistant A. pleuropneumoniae and 92% resistant P. multocida showed significantly reduced FF MICs (≥ 4-fold). In contrast, none or few of the susceptible A. pleuropneumoniae and P. multocida had FF MICs reduction ≥ 4-fold. 90% FF resistant A. pleuropneumoniae and 96% resistant P. multocida carried the floR gene, indicating strong association with the FloR efflux pump. With CCCP, the intracellular FF concentration increased by 71% in floR+ resistant A. pleuropneumoniae and 156% in floR+ resistant P. multocida strains but not the susceptible strains. The degree of reduction in TAP MICs was found consistently in parallel to FF for both bacteria. Taken together, partially attributed to blockage of drug-efflux, the combination of FF or TAP with CCCP at sub-cytotoxic concentrations was demonstrated and showed feasibility to combat amphenicol-resistant A. pleuropneumoniae and P. multocida isolated from diseased swine.

Coexistence of Fosfomycin Resistance Determinant fosA and fosA3 in Enterobacter cloacae Isolated from Pets with Urinary Tract Infection in Taiwan.

To analyze the characteristics of fosA and fosA3 in Enterobacter cloacae isolated from aspirated and catheterized urine culture specimens of companion pets in Taiwan. A total of 19 E. cloacae isolates from pets with urinary tract infection were screened for the presence of fosA, fosA3, and fosC2 and for the genetic context of them by PCR amplification and sequencing. The transferability, resistance phenotypes, plasmid replicon typing properties and genetic environments of fosA- and/or fosA3-positive strains were characterized. Five E. cloacae isolates were positive for fosA and three coharbored fosA and fosA3. No fosC determinant was detected. Transconjugants of fosA3 were successfully acquired, while the acquisition of fosA transconjugants was failed. The minimum inhibitory concentrations (MICs) of the three fosA3-positive isolates and their transconjugants were ≥256 mg/L, whereas the MICs of the five fosA-positive isolates ranged from 64 mg/L to 256 mg/L. Three plasmid replicons (InCFrepB, InCL/M, and InCHI2) were identified in fosA- and fosA3-positive E. cloacae isolates. Different genetic contexts lay in the downstream region of fosA and fosA3, respectively. Eight distinct patterns based on the similarity value of more than 80% were typed for all the 8 fosA-positive isolates. In conclusion, the fosA concomitant with fosA3 were found in E. cloacae isolates. The fosA3 not only exhibits stronger activity of inactivating fosfomycin than fosA but also possesses stronger potential to spread than fosA. Different genetic backgrounds exist in these fosA- and fosA3-positive isolates, and different mobile elements may confer the dissemination of fosA and fosA3.

Characterization of Escherichia coli Isolated from Day-old Chicken Fluff in Taiwanese Hatcheries.

Avian colibacillosis resulting from avian pathogenic Escherichia coli (APEC) seriously disrupts poultry production. Hatcheries are the main source of chickens for commercial farms. To characterize the potential pathogenicity of E. coli strains isolated from hatcheries, 2344 fluff samples from 1-day-old chickens were collected from hatching incubators between October 2016 and November 2017. Among the hatcheries, the incidence of E. coli varied from 0% to 16.9%, with an overall incidence of 2.0%. High incidences reflected inadequate sanitation in some hatcheries. We also compared 20 clinically isolated APEC strains with fluff-originated E. coli in terms of existence of 10 virulence-associated genes (VAGs) and antimicrobial-resistance genes, and antimicrobial resistance using minimum inhibitory concentration (MIC) values. Our results showed that APEC more-frequently possessed most of the assessed VAGs (papC, astA, cvaC, hlyF, fyuA, iroN, iutA, iss, and ompT), suggesting that fluff-originated E. coli is less likely to cause avian colibacillosis. However, fluff-originated E. coli more-frequently expressed the adhesion gene fimC, which could confer higher upper respiratory tract adhesion. Both APEC and fluff-originated E. coli demonstrated multidrug resistance including 100% resistance to ampicillin, amoxicillin, cephalexin, florfenicol, and trimethoprim-sulfamethoxazole. Based on median MIC values, fluff-originated E. coli was more susceptible to antibiotics. However, resistance-gene existence did not significantly differ between groups, suggesting that fluff-originated E. coli should still be a public health concern. Molecular subtyping with XbaI-digested pulsed-field gel electrophoresis revealed that only a few strains showed identical patterns, indicating that a variety of contamination sources were present within individual hatcheries. Identical strains within the same hatchery may indicate vertical transmission from parent flocks. Overall, this is the first study to characterize fluff-originated E. coli. Our results suggest that it has lower pathogenicity than APEC and that thorough sanitation should be performed to reduce the occurrence of fluff-originated E. coli in hatcheries.

Caracterización de Escherichia coli aislada de plumón de pollitos de un día en plantas de incubación en Taiwán. La colibacilosis aviar resultante de Escherichia coli patogénica aviar (APEC) puede afectar de manera severa a la producción avícola. Las plantas de incubación son la fuente principal de pollos para las granjas comerciales. Para caracterizar la patogenicidad potencial de las cepas de E. coli aisladas de las plantas de incubación, se recolectaron 2344 muestras de plumón de pollos de un día de edad de plantas de incubación entre octubre del 2016 a noviembre del 2017. Entre las plantas de incubación, la incidencia de E. coli fue del cero por ciento hasta el 16.9%, con una incidencia global del 2.0%. Las altas incidencias reflejaron una sanidad inadecuada en algunas plantas. También se compararon 20 cepas de E. coli patogénica aviar aisladas de casos clínicos con E. coli originada del plumón con relación a la presencia de 10 genes asociados a la virulencia (VAG), con genes asociados con la resistencia a los antimicrobianos y se evaluó la resistencia a los antimicrobianos mediante valores de concentración mínima inhibitoria (MIC). Los resultados mostraron que la E. coli patogénica aviar poseía con mayor frecuencia la mayoría de los genes asociados con virulencia evaluados (papC, astA, cvaC, hlyF, fyuA, iroN, iutA, iss y ompT), lo que sugiere que la E. coli originada del plumón es menos probable que cause colibacilosis aviar. Sin embargo, la E. coli originada de plumón expresó con mayor frecuencia el gene de adhesión fimC, que podría conferir una mayor adhesión en el tracto respiratorio superior. Tanto la E. coli patogénica aviar como la E. coli originada del plumón demostraron resistencia a múltiples fármacos, incluida una resistencia del 100% a la ampicilina, amoxicilina, cefalexina, florfenicol y trimetoprimsulfametoxazol. Con base la mediana de los valores de la concentración mínima inhibitoria, la E. coli originada del plumón fue más susceptible a los antibióticos. Sin embargo, la existencia del gene de resistencia no fue diferente significativamente entre los grupos, lo que sugiere que la E. coli originada del plumón puede ser un problema de salud pública. El subtipo molecular con electroforesis en gel de campo pulsado digerido con XbaI reveló que solo unas pocas cepas mostraron patrones idénticos, lo que indica que una variedad de fuentes de contaminación estaban presentes en las plantas de incubación individuales. Las cepas idénticas dentro de la misma planta de incubación pueden indicar transmisión vertical a partir de las parvadas paternas. En general, este es el primer estudio en caracterizar E. coli originada del plumón. Estos resultados sugieren que tiene una patogenicidad más baja que la E. coli patogénica aviar y que se debe realizar una limpieza y desinfección exhaustivas para reducir la aparición de E. coli originada de plumón en las plantas de incubación.

Assessment of fish iridoviruses using a novel cell line GS-1, derived from the spleen of orange-spotted grouper Epinephelus coioides (Hamilton) and susceptible to ranavirus and megalocytivirus.

A new cell line (GS-1) was developed from the spleen tissue of the orange-spotted grouper, Epinephelus coioides applied for viral infection studies of fish ranavirus and megalocytivirus. The cells proficiently multiplied in Leibovitz's L-15 medium supplemented with 10% fetal bovine serum at temperatures between 20°C and 32°C. Morphologically, the cell line comprised fibroblast-like cells, and this was confirmed by immunostaining with vimentin, fibronectin, and desmin antibodies. The optimal temperature for grouper iridovirus (GIV) and infectious spleen and kidney necrosis virus (ISKNV) proliferation in GS-1 cells was 25°C, and the highest titer of GIV was 108.4 TCID50/ml, and the highest titer of ISKNV was 105.2 TCID50/ml. Electron micrographs showed that the mean diameter of GIV virions was 180-220 nm, which was larger than ISKNV virions (160-200 nm). Negatively stained GIV particles possessed an envelope structure that was assembled by the three-layered structure with an inner electron-dense core surrounded by a lighter coat (mean diameter, 27 ± 3 nm). The highest GIV-induced mortality of groupers occurred at 25°C, whereas the highest ISKNV-induced mortality occurred at 30°C. In summary, GS-1 cell line is a valuable tool for isolating and investigating fish ranavirus and megalocytivirus in the same host system.

Antimicrobial Susceptibility Patterns of Brachyspira Species Isolated in Taiwan.

Some members of the Brachyspira genus cause diseases such as swine dysentery (SD) and porcine intestinal (or colonic) spirochetosis. Severe economic losses are caused by decreased feed intake and increased feed conversion ratio, as well as costs associated with treatment and death. A loss of clinical efficacy of some antimicrobial agents authorized for treating SD has been observed in many countries. The aim of this study was to analyze the antimicrobial susceptibility of Brachyspira isolated from Taiwan and to investigate the mechanism of decreased susceptibility to macrolides. A total of 55 Brachyspira isolates obtained from the grower-finisher period were evaluated in this study. These isolates included B. hyodysenteriae (n = 37), B. murdochii (n = 11), B. pilosicoli (n = 5), B. intermedia (n = 1), and B. innocens (n = 1). Antimicrobial susceptibility testing was performed to examine 12 selected antimicrobial agents. The results showed that the 50% and 90% minimum inhibitory concentration (MIC) values of the tested macrolides were all >256 µg/ml. The MIC50 of lincomycin, tiamulin, carbadox, olaquindox, ampicillin, amoxicillin, doxycycline, oxytetracycline, and gentamicin were 32, 1, ≤0.125, ≤0.125, 0.5, 0.25, 2, 2, and 2 µg/ml. The genetic basis of the decreased susceptibility to tylosin and lincomycin in Brachyspira spp. was investigated and the results showed a possible connection to the mutations at position A2058 and G2032 of the 23S rRNA gene. These findings demonstrated that, in Taiwan, there may be a decrease in susceptibility of Brachyspira spp. to antimicrobials commonly used for the treatment of SD.

Antimicrobial susceptibility, serotypes and genotypes of Pasteurella multocida isolates associated with swine pneumonia in Taiwan.

Pasteurella multocida (PM) can cause progressive atrophic rhinitis and suppurative bronchopneumonia in pigs. The present study performed antimicrobial susceptibility testing and serotype and genotype identification on the 62 PM strains isolated from the lungs of diseased pigs with respiratory symptoms. Antimicrobial susceptibility testing examined 13 antimicrobial agents (amoxicillin, cefazolin, doxycycline, flumequine, enrofloxacin, florfenicol, kanamycin, lincomycin, Linco-Spectin (lincomycin and spectinomycin), erythromycin, tylosin, tilmicosin and tiamulin). Antimicrobial resistance ratios were over 40% in all of the antimicrobial agents except for cefazolin. The highest levels of resistance (100%) were found for kanamycin, erythromycin and tylosin. The majority of isolated strains was serotype D:L6 (n=35) followed by A:L3 (n=17). Comparison of the antimicrobial resistance levels between the two serotypes showed that the antimicrobial resistance rates were higher in D:L6 than in A:L3 for all the tested antimicrobials except for tylosin and tilmicosin. For PM with erm(B), erm(T) or erm(42), the results showed no significant difference compared with non-resistance gene strains in phenotype. Pulsed-field gel electrophoresis genotyping using ApaI restriction digestion of the genomic DNA demonstrated that there were 17 distinct clusters with a similarity of 85% or more, and the genotyping result was similar to that of serotyping. The results of the present study demonstrated that the PM isolated from diseased pigs in Taiwan was resistant to multiple antimicrobials, and the distribution of antimicrobial resistance was associated with pulsotype and serotype.

Prevalence of plasmid-mediated quinolone resistance in Escherichia coli isolated from diseased animals in Taiwan.

Escherichia coli (E. coli) is a zoonotic pathogen that often causes diarrhea, respiratory diseases or septicemia in animals. Fluoroquinolones are antimicrobial agents used to treat pathogenic E. coli infections. In this study, 1,221 E. coli strains were isolated between March, 2011 and February, 2014. The results of the antimicrobial susceptibility testing showed a high prevalence of quinolone resistance. The antimicrobial resistance rates of these E. coli isolates to nalidixic acid (NAL) were 72.0% in swine, 81.9% in chickens, 81.0% in turkeys, 64.0% in ducks and 73.2% in geese. Among these isolates, the positive rate for the plasmid-mediated quinolone resistance (PMQR) determinant was 14.8% (181/1,221); the detection rate for qnrS1 was the highest (10.2%), followed by aac(6')-Ib-cr (4.5%) and qnrB2 (0.3%). The quinolone-resistance determining regions (QRDRs) analysis for the PMQR-positive isolates showed that the strains with mutations at codon 83 or 87 in GyrA were resistant to NAL. To the best of our knowledge, this is the first report of occurrence of qnrB2, qnrS1 and aac(6')-Ib-cr genes and high frequency (56.4%; 102/181) of mutation in gyrA or parC among PMQR-positive E. coli strains derived from diseased animals in Taiwan.

Pharmacokinetics and tissue depletion of doxycycline administered at high dosage to broiler chickens via the drinking water.

The recommended use of doxycycline (DC) to broiler chicken is 100 mg/L via the drinking water and a 7-day withdrawal time (WDT). However, study of a higher dosage is desirable because of the possible increase of antimicrobial resistance and disease spectrum. Tissue DC residues exceeding the current maximum residue levels (MRL) was our major concern. Therefore, serum concentration and tissue depletion of DC hyclate after administration of 200 mg/L of DC in the drinking water for five consecutive days were studied. The steady-state DC concentration (8.3 ± 0.9 µg/mL) was reached on the third day of medication. The elimination constant (0.05 ± 0.01 1/h), half-life (14.9 ± 1.4 h), area under concentration versus time curve (81.0 ± 9.9 h·µg/mL) and mean residence time (22.7 ± 2.5 h) were obtained using a non-compartmental pharmacokinetic model. It was determined that the current 7-day WDT regulation was still legitimate for the kidney and liver as well as for the breast and leg muscles, which were estimated by linear regression analysis of the 99% upper distribution limit. The unregulated heart and gizzard were considered safe even when the lowest MRL of muscle (100 ng/g) was applied. While at the present time the extra-label use of drugs is only allowed under specific conditions, in the future it may become necessary to increase the general dosage of DC, and the current results suggest a safe range of DC hyclate in chicken; however, skin/fat tissue residues warrant further studies.

Florfenicol As a Modulator Enhancing Antimicrobial Activity: Example Using Combination with Thiamphenicol against Pasteurella multocida.

Synergistic effects between the same class of antibiotics are rarely reported. Our previous study found synergistic-like interaction between florfenicol (FFC) and thiamphenicol (TAP) against Staphylococcus aureus. Here, the enhanced antimicrobial activity was evaluated in 97 clinical isolates of both Gram-negative and Gram-positive bacteria. Susceptible strains were initially identified by checkerboard microdilution assay (fractional inhibitory concentration index [FICI] ≤ 0.625), followed by confirmation of synergism using the time-kill methodology (≥2 log10 CFU/ml reduction). In all, 43% of Pasteurella multocida tested were susceptible to the enhanced bactericidal effect. In chicken fowl cholera models, FFC and TAP combination at much lower dosage that is correspondent to their MIC deduction provided maximum protection in vivo. Furthermore, synergistic combination of FFC with oxytetracycline (OTC) against Pseudomonas aeruginosa in vitro was also demonstrated. Based on the enhanced uptake of TAP and OTC, FFC presumably elicits enhanced antimicrobial activity in an orderly manner through alteration of bacterial membrane permeability or efflux systems and subsequent increase of intracellular concentration of the antibiotics used in combination. Results of ethidium bromide accumulation assay and RNA-seq showed little evidence for the involvement of efflux pumps in the synergy but further investigation is required. This study suggests the potentiality of a novel combination regimen involving FFC as an initiating modulator effective against both Gram-positive and Gram-negative bacteria depending on the antibiotics that are combined. The observed improvement of bacteriostatic effect to bactericidal, and the extended effectiveness against FFC-resistant bacterial strains warrant further studies.

Antimicrobial resistance of Escherichia coli isolates from canine urinary tract infections.

This study determined the antimicrobial resistance profiles of Escherichia coli isolates from dogs with a presumptive diagnosis of urinary tract infection (UTI). Urine samples from 201 dogs with UTI diagnosed through clinical examination and urinalysis were processed for isolation of Escherichia coli. Colonies from pure cultures were identified by biochemical reactions (n=114) and were tested for susceptibility to 18 antimicrobials. The two most frequent antimicrobials showing resistance in Urinary E. coli isolates were oxytetracycline and ampicillin. Among the resistant isolates, 17 resistance patterns were observed, with 12 patterns involving multidrug resistance (MDR). Of the 69 tetracycline-resistant E. coli isolates, tet(B) was the predominant resistance determinant and was detected in 50.9% of the isolates, whereas the remaining 25.5% isolates carried the tet(A) determinant. Most ampicillin and/or amoxicillin-resistant E. coli isolates carried blaTEM-1 genes. Class 1 integrons were prevalent (28.9%) and contained previously described gene cassettes that are implicated primarily in resistance to aminoglycosides and trimethoprim (dfrA1, dfrA17-aadA5). Of the 44 quinolone-resistant E. coli isolates, 38 were resistant to nalidixic acid, and 6 were resistant to nalidixic acid, ciprofloxacin and enrofloxacin. Chromosomal point mutations were found in the GyrA (Ser83Leu) and ParC (Ser80Ile) genes. Furthermore, the aminoglycoside resistance gene aacC2, the chloramphenicol resistant gene cmlA and the florfenicol resistant gene floR were also identified. This study revealed an alarming rate of antimicrobial resistance among E. coli isolates from dogs with UTIs.

Production of monoclonal antibody against ORF72 of koi herpesvirus isolated in Taiwan.

A monoclonal antibody (MAb) was generated against the capsid protein (ORF 72) of koi herpesvirus (KHV) isolated from diseased koi Cyprinus carpio in Taiwan. The clone of MAb-B2 was obtained by immunizing mice with whole virus particles and further identified using indirect enzyme-linked immunosorbent assay and Western blot assay. In addition, it detected KHV in KHV-infected cells but not in those of mock-infected cells as demonstrated by indirect immunofluorescence assay. The neutralization test showed that MAb-B2 neutralized KHV. Furthermore, we uncovered that MAb-B2 recognizes the ORF72 of KHV as revealed by liquid chromatography-tandem mass spectrometry and Western blot assays. Additionally, MAb-B2 has been used as a diagnostic tool for detection of KHV in clinical samples by immunohistochemistry. Collectively, our results indicated that MAb-B2 could be used in the development of a diagnostic kit for diagnosis of KHV infections and ORF72 protein of KHV might be a candidate for future vaccine development.

Comparison of postoperative effects between lidocaine infusion, meloxicam, and their combination in dogs undergoing ovariohysterectomy.

OBJECTIVE: To compare the postoperative analgesic effects of intravenous (IV) lidocaine, meloxicam, and their combination in dogs undergoing ovariohysterectomy. STUDY DESIGN: Prospective, randomized, double-blind, controlled clinical trial. ANIMALS: Twenty-seven dogs aged (mean ± SD) 16.1 ± 7.5 months and weighing 22.4 ± 17.9 kg scheduled for ovariohysterectomy. METHODS: Anaesthesia was induced with propofol and maintained with isoflurane. Dogs (n = 9 in each group) were allocated to receive just prior to and during surgery one of the following regimens: M group, 0.2 mg kg(-1) IV meloxicam then a continuous rate infusion (CRI) of lactated Ringer's at 10 mL kg(-1) hour(-1); L group, a bolus of lidocaine (1 mg kg(-1) IV) then a CRI of lidocaine at 0.025 mg kg(-1) minute(-1); and M + L group, both the above meloxicam and lidocaine treatments. Pain and sedation were scored, and venous samples taken for serum cortisol and glucose measurement before and at intervals for 12 hours after anaesthesia. Pain scores were assessed using a multi-parameter subjective scoring scale (cumulative scale 0-21) by three observers. The protocol stated that dogs with a total score exceeding 9 or a sub-score above 3 in any one category would receive rescue analgesia. Sedation was scored on a scale of 0-4. RESULTS: There were no significant differences in subjective pain scores, serum cortisol, and glucose concentrations between the three groups. The highest pain score at any time was 5, and no dog required rescue analgesia. None of the three regimens caused any observable side effects during or after anaesthesia. At 1 and 2 hours after extubation dogs in group L were significantly more sedated than in the other two groups. CONCLUSIONS AND CLINICAL RELEVANCE: This study suggests that, with the scoring system used, IV lidocaine and meloxicam provide similar and adequate post-operative analgesia in healthy dogs undergoing ovariohysterectomy.

Genetic analysis of fish iridoviruses isolated in Taiwan during 2001-2009.

To investigate the genetic relationships between field strains of iridoviruses gathered from various fish species in Taiwan, viruses that were collected from 2001 to 2009 were analyzed. Open reading frames encoding the viral major capsid protein (MCP) and adenosine triphosphatase (ATPase) were sequenced for phylogenetic analysis. Our results indicated that iridoviruses from Taiwan aquaculture fishes could be classified into two groups: prior to 2005, the viruses were closely related to members of the genus Ranavirus; and after 2005, they were similar to members of the genus Megalocytivirus. Based on the analysis of MCP amino acid sequences, virus isolates were divided into 4 major genotypes that were related to ISKNV, RSIV, FLIV, and GIV, respectively. Pairwise comparisons of MCP genes showed that the ranavirus was an epidemic pathogen for economically important species in the major production regions and cultured marine fish, while the megalocytivirus isolates were sensitive to host range. In addition, the distribution of synonymous and non-synonymous changes in the MCP gene revealed that the iridoviruses were evolving slowly, and most of the variations were synonymous mutations. The Ka/Ks values were lower than one, and hence, the viruses were under negative selection.

Differential heat stability of amphenicols characterized by structural degradation, mass spectrometry and antimicrobial activity.

Heat stability of amphenicols and the relationship between structural degradation and antimicrobial activity after heating has not been well investigated. Florfenicol (FF), thiamphenicol (TAP), and chloramphenicol (CAP) were heated at 100 degrees C in water, salt water, soybean sauce and chicken meat for up to 2h. Degradation and antimicrobial activity of the compounds was evaluated using capillary electrophoresis (CE) with UV-DAD spectrometry, minimum inhibitory concentration (MIC) assay, and gas chromatography with electron impact ionization mass spectrometry (GC-EI-MS). Heat stability of amphenicols in matrices was ranked as water> or =salt water>soybean sauce>meat, suggesting that heat degradation of amphenicols was accelerated in soybean sauce and was not protected in meat. Heat stability by drug and matrices was ranked as FF>TAP=CAP in water, FF=TAP>CAP in salt water, TAP> or =FF=CAP in soybean sauce, and TAP> or =FF=CAP in meat, indicating differential heat stability of amphenicols among the 3 drugs and in different matrices. In accordance with the less than 20% degradation, the MIC against Escherichia coli and Staphylococcus aureus did not change after 2h heating in water. A 5-min heating of amphenicols in water by microwave oven generated comparable percentage degradation to boiling in water bath for 30 min to 1h. Both CE and GC-MS analysis showed that heating of FF produced TAP but not FF amine as one of its breakdown products. In conclusion, despite close similarity in structure; amphenicols exhibited differential behavior toward heating degradation in solutions and protein matrices. Although higher degradations of amphenicols were observed in soybean sauce and meat, heating treatment may generate product with antimicrobial activity (FF to TAP), therefore, heating of amphenicol residues in food cannot always be assumed safe.

Cartap-induced cytotoxicity in mouse C2C12 myoblast cell line and the roles of calcium ion and oxidative stress on the toxic effects.

Our previous study has demonstrated that instead of neuromuscular blockage cartap, an organonitrogen insecticide, could cause a marked irreversible Ca2+-dependent contracture in both isolated mouse and rabbit phrenic nerve-diaphragms. We further examined the potential of direct myocytotoxicity of cartap and the possible roles of calcium ion and oxidative stress on cartap-induced muscle cell injury using the mouse myoblast cell line, C2C12. Cartap exerted a dose- and time-dependent cytotoxic effect in C2C12 cells measured by MTT colorimetric assay and trypan blue dye exclusion. The extracellular activities of both creatine kinase (CK) and lactate dehydrogenase (LDH) were elevated in the cartap-treated groups at or greater than 100 microM. The isoenzymatic profiles showed that the elevations were mainly due to CK-3, LDH-3, and LDH-4. Following the addition of 0.5-2.5mM EGTA, a Ca2+ chelator, or 30-100 microM verapamil, an L-type Ca2+ channel blocker, the cartap-induced reduction in MTT metabolic rate of C2C12 cells was significantly restored in a dose-dependent manner in both EGTA and verapamil-treated cells. Furthermore, EGTA could significantly reduce the cartap-induced elevation in the levels of total extracellular CK and LDH activities. Additionally, cartap significantly increased the level of endogenous reactive oxygen species (ROS) in C2C12 cells in a dose- and time-dependent manner. The cartap-induced ROS generation could be significantly inhibited by antioxidants, including Vitamins C and E, catalase, and superoxide dismutase, with catalase the most effective. EGTA could significantly inhibit cartap-induced ROS generation in a dose-dependent manner. The results suggested that cartap could induce ROS generation in C2C12 cells via a Ca2+-dependent mechanism resulting in subsequent cytotoxicity, at least partially, to C2C12 cells. It is speculated that both Ca2+ and Ca2+-induced ROS may also play the central role on the myogenic contracture and myofiber injury of the diaphragm leading to respiratory failure and subsequent death in rabbits exposed ocularly to cartap.

Susceptibility to cartap-induced lethal effect and diaphragmatic injury via ocular exposure in rabbits.

Cartap is extensively used to control agricultural pests. Pertinent literatures have indicated that it causes no eye irritation [D.E. Ray, Insecticides derived from plants and other organisms, in: W.J. Hayes, E.R. Laws (Eds.), Handbook of Insecticide Toxicology, Classes of Insecticides, vol. 2, Academic Press, New York, 1991, p. 611; C. Tomlin, Cartap, in: C. Tomlin (Ed.), The Insecticide Manual, 12th ed., British Crop Protection Council, Surrey, UK, 2000, p. 144]; however, the instillation of a little cartap through the eye has caused death in rabbits. The aim of this study was to determine the ocular toxicity of cartap in New Zealand White rabbits. Cartap was directly instilled into the low conjunctival sac of eyes, at doses of 0, 5, 7.5, 10 and 12.5 mg/kg body weight. The changes in the enzymes and isoenzymes of creatine kinase (CK), lactate dehydrogenase (LD), as well as pathological changes in the muscles of the heart, thigh and diaphragm were determined in the cartap-treated rabbits. Moreover, the neuromuscular effect of cartap was examined using the isolated rabbit phrenic-nerve diaphragm model. The results indicated that rabbits developed severe signs and they died within 20 min of ocular instillation. The ocular LD50 of cartap was 8.1 mg/kg body weight. Treatment with cartap increased the activities of CK and LD enzymes and their isoenzymes, CK-1, CK-2, and CK-3 in serum, and CK-3 and LD-5 in the diaphragm. Microscopically, hypercontraction bands and the rupture of myofibers of the diaphragm were observed in dead rabbits. Cartap did not affect nerve-evoked twitch but induced irreversible contracture and twitch depression on the isolated rabbit's diaphragm. These results indicate that the rabbit is susceptible to cartap toxicity; the effect of cartap caused contracture and damage to the diaphragm might play a pivotal role in respiratory paralysis and death of rabbits during intoxication.