Evaluation of Novel Quorum Sensing Inhibitors Targeting Auto-Inducer 2 (AI-2) for the Control of Avian Pathogenic Escherichia coli Infections in Chickens.

Avian pathogenic Escherichia coli (APEC) associated with colibacillosis results in high morbidity and mortality, and severe economic losses to the poultry industry. APEC is a zoonotic pathogen and can infect humans through contaminated poultry products. Vaccination and antibiotic treatment are currently used to control APEC infections; however, the limited effect of vaccines and the emergence of antibiotic-resistant strains have necessitated the development of novel therapeutics. Here, we evaluated seven quorum sensing inhibitors (QSI) identified in our previous study, in APEC-infected chickens. QSIs were administered orally (~92 to 120 µg/bird) and chickens were challenged subcutaneously with APEC. Among them, QSI-5 conferred the best protection (100% reduction in mortality, 82% to 93% reduction in lesions [airsacculitis, perihepatitis, lung congestion, pericarditis] severity, and 5.2 to 6.1 logs reduction in APEC load). QSI-5 was further tested in chickens raised on built-up floor litter using an optimized dose (1 mg/L) in drinking water. QSI-5 reduced the mortality (88.4%), lesion severity (72.2%), and APEC load (2.8 logs) in chickens, which was better than the reduction observed with currently used antibiotic sulfadimethoxine (SDM; mortality 35.9%; lesion severity up to 36.9%; and APEC load up to 2.4 logs). QSI-5 was detected in chicken's blood after 0.5 h with no residues in muscle, liver, and kidney. QSI-5 increased the body weight gain with no effect on the feed conversion ratio and cecal microbiota of the chickens. Metabolomic studies revealed reduced levels of 5'-methylthioadenosine in QSI-5-treated chicken serum. In conclusion, QSI-5 displayed promising effects in chickens and thus, represents a novel anti-APEC therapeutic. IMPORTANCE Avian pathogenic Escherichia coli (APEC), a subgroup of ExPEC, is a zoonotic pathogen with public health importance. Quorum sensing is a mechanism that regulates virulence, biofilm formation, and pathogenesis in bacteria. Here, we identified a novel quorum sensing autoinducer-2 inhibitor, QSI-5, which showed higher anti-APEC efficacy in chickens compared to the currently used antibiotic, sulfadimethoxine at a much lower dose (up to 4,500 times). QSI-5 is readily absorbed with no residues in the tissues. QSI-5 also increased the chicken's body weight gain and did not impact the cecal microbiota composition. Overall, QSI-5 represents a promising lead compound for developing novel anti-virulence therapies with significant implications for treating APEC infections in chickens as well as other ExPEC associated infections in humans. Further identification of its target(s) and understanding the mechanism of action of QSI-5 in APEC will add to the future novel drug development efforts that can overcome the antimicrobial resistance problem.

Characteristics of Staphylococcus aureus small colony variant and its parent strain isolated from chronic mastitis at a dairy farm in Beijing, China.

Staphylococcus aureus is a major pathogen associated to bovine mastitis and has the ability to form a slow-growing population termed the small colony variants (SCVs). From 20 samples of 5 chronic S. aureus cases, 1 SCV isolate (SCV102) was recovered simultaneously with 1 of 8 S. aureus isolates. SCV102 showed auxotrophy for thymidine and had a slow growth rate. Intracellular persistence in human mammary epithelial cells (HBL100cell line) monolayer revealed that SCV102 isolate had minimal cytopathological effects compared with its parent strains. SCV102 isolate and its parent strain S. aureus 101 indicate similar resistant pattern to four antibiotics. On the contrary, the minimal inhibitory concentrations values for chloramphenicol and sulfadimethoxine were much higher in SCV102 than that of S. aureus 101. To the best of our knowledge this is the first time the isolation of S. aureus SCV102 from a persistent bovine mastitis has been reported in Beijing (China). This study suggests that SCV102 isolate may be an important contributor to persistent bovine mastitis.

Outbreaks of severe enteric disease associated with Eimeria furonis infection in ferrets (Mustela putorius furo) of 3 densely populated groups.

CASE DESCRIPTION: 3 unrelated, densely populated, dynamic ferret populations with severe outbreaks of enteric coccidiosis were evaluated. CLINICAL FINDINGS: In each outbreak, morbidity rate was high, there were an appreciable number of deaths, and ferrets of all ages were affected. Affected individuals had acute onset of diarrhea, and feces often contained frank or digested blood. Other clinical signs included dehydration, weakness, lethargy, and weight loss. Fecal examinations of affected ferrets revealed sporadic and inconsistent shedding of coccidial oocysts. Necropsy findings included moderate to marked atrophic enteritis associated with numerous intraepithelial and fewer extracellular coccidial life stages. Sporulated oocysts isolated from feces were consistent with Eimeria furonis. A PCR assay was performed on formalin-fixed, paraffin-embedded sections of intestine for the gene encoding the small subunit of rRNA yielded products with sequences identical to those described for E furonis. TREATMENT AND OUTCOME: Supportive care and treatment with sulfadimethoxine over the course of these outbreaks was palliative, but long-term treatment was required and failed to completely eradicate infection as identified by the subsequent finding of oocysts in fecal samples. CLINICAL RELEVANCE: Enteric coccidiosis due to infection with E furonis has typically been reported to be subclinical rather than to cause severe gastrointestinal disease in ferrets. This report indicated that infection with E furonis may have contributed to severe enteric disease with high morbidity and mortality rates in 3 densely populated, dynamic groups of ferrets. Furthermore, long-term treatment with anti-coccidials may be required in outbreak situations, but may be ineffectual in completely eradicating infection.

Clinical and microbial efficacy of antimicrobial treatments of experimental avian colibacillosis.

The clinical and microbial efficacy of antimicrobial treatments of avian colibacillosis was studied, using an experimental model on chickens previously inoculated with multiresistant commensal Escherichia coli strains. One E. coli with pMG252 plasmid containing bla(FOX5) and qnrA1 genes and another E. coli with pMG298 plasmid containing bla(CTX-M15) and qnrB1 genes were first orally inoculated to chickens Both isolates were also resistant to chloramphenicol, sulphamethoxazole, trimethoprim, streptomycin, gentamicin, kanamycin, and tetracycline. The birds were then experimentally infected with an avian pathogenic E. coli (APEC), via the air sac. Treatments (oxytetracycline (OTC), trimethoprim-sulfadimethoxin (SXT), amoxicillin (AMX) or enrofloxacin (ENR) were then offered at the therapeutic doses. Symptoms, lesions in dead or sacrificed birds, and isolation and characterization of APEC from internal organs were studied. Results showed that OTC, SXT or ENR treatments could control the pathology. AMX worsened the disease, possibly due to endotoxin shock. All APEC re-isolated from internal organs showed the same antimicrobial susceptibility as the APEC inoculated strain, except for one APEC isolate from an infected OTC-treated bird, which acquired tetracycline resistance only, and one APEC isolate recovered from the air sacs of a chicken in the infected SXT-treated group, which acquired the pMG252 plasmid and became multi-resistant. Thus three antimicrobials could control the disease but the experimental model enabled, to our knowledge, the first observation of plasmid transfer from a bacterium of the intestinal tract to a pathogenic isolate from the respiratory tract.

Comparison of toltrazuril and sulphadimethoxine in the treatment of intestinal coccidiosis in pet rabbits.

Treatment of intestinal coccidiosis was studied in domestic pet rabbits. In 45 rabbits aged four months or more, coccidial oocysts were observed in the faeces of 35 rabbits at a mean density of 806 opg (range 50 to 6800 opg). Eimeria magna was the dominant species, with Eimeria media and Eimeria intestinalis also being common. The presence of the hepatic species Eimeria stiedae was not recorded. A single oral dose of 2.5 mg/kg or 5.0 mg/kg toltrazuril, or a single oral dose of 50 mg/kg sulphadimethoxine followed by its inclusion in drinking water at 1 g/4 l for nine days, were all found to significantly reduce the faecal oocyst count by 73 to 99 per cent. The extent of oocyst reduction in the faeces was not dependent on the dose of toltrazuril. Oocyst counts began to rise again in the days after treatment ceased.

Chronic intermittent diarrhea in a 14-month-old Abyssinian cat.

A 14-month-old intact, female Abyssinian cat was presented for chronic intermittent diarrhea and bilateral enlargement of the mammary glands. Gastrointestinal coccidiosis was diagnosed; therapy with sulfadi-methoxine was unsuccessful in the elimination of Isospora felis and clinical signs. Infection with Tritrichomonas foetus was diagnosed by fecal polymerase chain reaction (PCR) and successfully treated with ronidazole and dietary modification.

Nephrotic syndrome associated with administration of sulfadimethoxine/ormetoprim in a dobermann.

This case report describes sulphonamide-induced nephrotic syndrome in a young dobermann dog. The clinical signs and laboratory abnormalities resolved shortly after discontinuation of the sulphonamide antibiotic and with generalised supportive care. Since nephrotic syndrome typically carries a guarded prognosis in veterinary medicine and is poorly responsive to therapy, a thorough drug history should be an important part of the investigation of any animal with a protein-losing nephropathy.

Comparative efficacy of enrofloxacin, oxytetracycline, and sulfadimethoxine for the control of morbidity and mortality caused by Escherichia coli in broiler chickens.

The purpose of the present study was to compare the ability of enrofloxacin, oxytetracycline, and sulfadimethoxine to reduce morbidity and mortality caused by Escherichia coli (colibacillosis) in broiler chickens. The chickens were raised in 80 pens (20 birds per pen) with 20 pens representing each treatment group under simulated commercial conditions that produced a colibacillosis challenge scenario. Each group of 20 randomized pens (replicates) was given one of four water treatments. Chickens that received enrofloxacin had significantly less mortality (P < 0.01), lower average gross pathology (colibacillosis) scores (P < 0.01), and better feed-conversion ratios (P < 0.05) than did chickens that received either oxytetracycline or no medication. Chickens that received enrofloxacin had significantly less mortality and lower pathology scores than those that received sulfadimethoxine and numerically lower feed conversion than the sulfadimethoxine group. Results from the present study show that enrofloxacin is superior to oxytetracycline and sulfadimethoxine for the control of morbidity and mortality caused by E. coli in broiler chickens. Our findings will help veterinarians choose and prescribe the most efficacious antimicrobial when treating colibacillosis.

Acute columnaris infection in channel catfish, Ictalurus punctatus (Rafinesque): efficacy of practical treatments for warmwater aquaculture ponds.

Columnaris disease was induced in channel catfish, Ictalurus punctatus (Rafinesque), by bath exposure to four highly virulent isolates of Flavobacterium columnare. In untreated controls, mortality began 20 h after exposure and reached 100% by 48 h. Mortality in channel catfish given antibiotic treatments with oxytetracycline or a combination of sulphadimethoxine and ormetoprim in feed prior to bacterial challenge was zero with all four strains of F. columnare. Diquat (Zeneca Agricultural Products, Wilmington, DE, USA) was the most effective bath treatment; mortality with all four strains was zero. With potassium permanganate, chloramine-T, hydrogen peroxide and copper sulphate, bath treatment efficacy varied significantly among strains (P = 0.0346) and among treatments (P = 0.0033). Bath treatments with chloramine-T and potassium permanganate significantly reduced (P < 0.05) mortality from 100 to 75 and 69%, respectively, but copper sulphate and hydrogen peroxide treatments were not effective. Based on our results, oral antibiotics prevented columnaris disease but, of the bath treatments, only Diquat produced a dramatic reduction in the mortality of acutely infected fish. Diquat is labelled for aquatic use as an herbicide in the USA but in large ponds it is prohibitively expensive.

Prevention of pleuropneumonia in pigs by in-feed medication with sulphadimethoxine and sulphamethoxazole in combination with trimethoprim.

The prophylactic effect of in-feed medication of conventional pigs with sulphadimethoxine (SDM), sulphamethoxazole (SMX), and trimethoprim (TMP) was tested by using an Actinobacillus pleuropneumoniae infection model. In each of five experiments, six pigs were given medicated feed twice daily and three pigs received antibiotic-free feed and served as positive (unmedicated, infected) controls. The following drugs or drug combinations were tested (in mg per kg feed): 500 SDM + 100 TMP, 500 SMX + 100 TMP, 125 SMX + 25 TMP, 125 SMX (alone) and 25 TMP (alone). After six days of feed medication, all animals were endobronchially inoculated with A. pleuropneumoniae in a dose of 1-3.10(4) colony-forming units (CFU). The response to the challenge in all control pigs was characterized by fever, lethargy, anorexia, reduced water consumption, and laboured breathing. At autopsy all controls manifested a fibrinous haemorrhagic pleuropneumonia. In-feed medication with 500 SDM + 100 TMP, 500 SMX + 100 TMP as well as 125 SMX + 25 TMP resulted in an effective protection against the challenge in all treated animals. After consumption of feed medicated with 125 mg per kg SMX or 25 mg per kg TMP, pleuropneumonia was evident in all challenged pigs. The results of this study indicate an in vivo potentiation of SMX and TMP in pigs against this respiratory tract pathogen.

Identification of Blastomyces dermatitidis in the stool of a dog with acute pulmonary blastomycosis.

We report the identification of Blastomyces dermatitidis by microscopic examination of a direct faecal smear from a dog with pulmonary blastomycosis. A simultaneously obtained faecal culture grew Blastomyces dermatitidis. The fungus was also cultured from a transtracheal sample from this same dog. This report suggests that yeast-phase cells of B. dermatitidis may be recovered in the stool of dogs with pulmonary blastomycosis following transit through the gastrointestinal tract of swallowed infected sputum. Implications regarding the ecology of Blastomyces dermatitidis are discussed.

Haptoglobin response to clinical respiratory tract disease in feedlot cattle.

OBJECTIVE: To quantify haptoglobin response to respiratory tract disease in feedlot cattle, and to investigate its ability to predict disease outcome and response to antibiotic treatment. DESIGN: Randomized clinical trial. ANIMALS: 60 feedlot calves with clinical respiratory tract disease. PROCEDURE: Calves were randomly assigned to receive a standard antibiotic treatment regimen (TRT), or to observation pens without antibiotic treatment. Serum haptoglobin concentration was measured at initial and final examinations. Calves were examined for presence of gross pulmonary lesions at slaughter. RESULTS: Mean +/- SD serum haptoglobin concentration at initial examination was 67 +/- 108 mg/dl, with range of 0 to 508 mg/dl. Haptoglobin concentration at initial examination was similar for the TRT group and the group that did not receive antibiotic treatment, but at final examination, TRT-group calves had lower (P < 0.01) mean values. Calves receiving antibiotic treatment had haptoglobin concentration at or near zero at final examination. Calves not receiving antibiotic treatment had only slightly lower mean haptoglobin concentration at final examination, compared with initial examination. Within treatment groups, haptoglobin concentration was similar for cases with different outcomes. Calves with gross pulmonary lesions at slaughter had numerically higher, although statistically similar, haptoglobin concentrations at initial examination, compared with calves without lesions. CONCLUSIONS: Feedlot cattle with clinical respiratory tract disease have a large and variable haptoglobin response. Antibiotic treatment resulted in lower serum haptoglobin values, although low values were not required for full clinical recovery. CLINICAL RELEVANCE: Serum haptoglobin concentration may be an indicator of response to antibiotic therapy, although it appears to be unrelated to case severity or need for treatment.

Monodrug efficacies of sulfonamides in prophylaxis for Pneumocystis carinii pneumonia.

A remarkably high rate of adverse events is associated with the use of trimethoprim-sulfamethoxazole in patients with human immunodeficiency virus type 1 infection. We examined the efficacies of sulfonamides alone in the prevention of Pneumocystis carinii pneumonitis, with the assumption that at least some of the adverse events with the drug combination might be due to trimethoprim. With the immunosuppressed rat model, eight sulfonamides were studied at 100, 10, and 1.0 mg/kg/day (10 rats per dosage and drug). P. carinii infection was prevented in all animals (100%) receiving dosages of as little as 1.0 mg of sulfamethoxazole, sulfamethoxypyridazine, and sulfadimethoxine per kg per day, as little as 10 mg of sulfameter, sulfachlorpyridazine, and sulfaquinoxaline per kg per day; and 100 mg of sulfaguanidine and sulfanilamide per kg per day. These studies suggest that a sulfonamide, such as sulfamethoxazole, might provide effective prophylaxis for P. carinii pneumonitis without trimethoprim.

Pharmacokinetics of sulfadimethoxine and sulfamethoxazole in combination with trimethoprim after intravenous administration to healthy and pneumonic pigs.

The pharmacokinetics of two sulfonamide/trimethoprim combinations were investigated after intravenous administration to clinically healthy pigs and to the same pigs following a challenge with Actinobacillus pleuropneumoniae toxins. Endobronchial challenge with A. pleuropneumoniae toxins resulted in fever, increased white blood cell counts and decreased water and feed consumption. Healthy, as well as febrile, pigs were given sulfadimethoxine (SDM) or sulfamethoxazole (SMX) intravenously at a dose of 25 mg/kg b.w. in combination with 5 mg trimethoprim (TMP) per kg body weight. The pharmacokinetic parameters of the sulfonamides as well as their main metabolites (acetyl sulfonamides) were not significantly different in healthy and febrile pigs. In healthy and pneumonic pigs, the mean elimination half-lives of SDM were 12.9 h and 13.4 h, respectively, those of SMX 2.5 h and 2.7 h, respectively, and those of TMP 2.8 h and 2.6 h, respectively. Distribution volumes in healthy and febrile pigs of SDM and SMX varied between 0.2 and 0.4 L/kg, and those of TMP between 1.1 and 1.6 L/kg. The mean AUC of TMP was decreased and the volume of distribution and total body clearance of TMP were increased in febrile pigs. Protein binding of the drugs and metabolites studied were not significantly changed after toxin-induced fever. The extent of protein binding of SDM, SMX and TMP was in the range 94-99%, 45-56% and 40-50%, respectively. Based on knowledge of in vitro antimicrobial activity of the drug combinations against A. pleuropneumoniae it was concluded that after intravenous administration of the dose administered (30 mg/kg of the combination preparations) to healthy and pneumonic pigs, plasma concentrations of SMX and TMP were above the concentration required for growth inhibition of 50% of A., pleuropneumoniae strains for approximately 16 h, whereas bacteriostatic plasma concentrations of SDM were still present after TMP had been eliminated from plasma. Because of similar elimination half-lives of SMX and TMP in pigs this combination is preferred to the combination of SDM with TMP.

Activity of sulfadimethoxine against cryptosporidiosis in dairy calves.

Of 13 neonatal calves inoculated orally with 1.5 x 10(6) oocysts of Cryptosporidium parvum, 7 in group A were fed 5-g boluses of sulfadimethoxine for 21 consecutive days beginning 1 day before infection, and 6 calves in group B were untreated controls. Calves in group A had diarrhea for 6-18 days (mean = 11 days); those in group B had diarrhea for 4-14 days (mean = 8.7 days). The severity of diarrhea, based on a daily numerical scoring system, was similar for both groups. Calves in group A shed an average of 18 x 10(6) oocysts/ml of feces for 3.9 days; those in group B shed an average of 2.4 x 10(6) oocysts/ml of feces for 5.3 days. By 28 days of age, calves in group A vs. group B gained an average of 8.9 kg vs. 15.7 kg. These findings indicate that sulfadimethoxine did not significantly reduce the number of days or severity of diarrhea, or the number of oocysts or patent period, nor did it improve weight gains.

Anticryptosporidial activity is associated with specific sulfonamides in immunosuppressed rats.

Dexamethasone-immunosuppressed rats infected with Cryptosporidium parvum were used to assess 23 sulfonamides for anticryptosporidial activity. Five of the compounds administered before the animals were inoculated with C. parvum oocysts reduced the severity of cryptosporidial infections in the rat model. Two of the 5 agents with prophylactic activity, sulfadimethoxine and sulfamethazine, were effective also against an established infection, indicating that some sulfonamides may have therapeutic value in immunosuppressed patients with cryptosporidiosis. The findings also suggest that sulfonamide treatment of cryptosporidiosis in the immunocompromised host may not be successful unless the compound is administered continuously or over several weeks.

Efficacy of different anticoccidials against experimental coccidiosis in large white turkeys.

Two trials were conducted to compare the efficacy of currently approved anticoccidials for turkeys against challenge using a field isolate of mixed Eimeria species; E. adenoides, E. gallopavonis, and E. meleagrimitis. Poults in wire-floored cages were fed unmedicated diets from day-old to 3 wk of age. Diets were supplemented with either amprolium (AMP, 125 mg/kg), butynorate (BUT, 375 mg/kg), monensin (MON-60, 60 mg/kg; MON-100, 100 mg/kg), halofuginone (HAL; 3 mg/kg), zoalene (ZOA; 125 mg/kg), or sulfadimethoxine plus ormetoprim (SUL + ORM, 62.5 mg/kg and 37.5 mg/kg, respectively). After 2 days on the test diets, poults were individually weighed and inoculated with sporulated coccidial oocysts from the field isolate. Total fecal collections were obtained for Days 0 to 5 and 6 to 10 to estimate oocyst output. At 10 days postinoculation, the birds were individually weighed and killed to determine severity of intestinal lesions. The HAL and MON were most effective and AMP, ZOA, and SUL + ORM were least effective in maintaining weight and in reducing the severity of intestinal lesions. All the coccidiostats tested reduced oocyst passage, but poults fed HAL produced fewer oocysts. The results demonstrated differences in efficacy among anticoccidials with the more recently approved drugs providing the best protection against coccidiosis.