Validation of a microbiological method: the STAR protocol, a five-plate test, for the screening of antibiotic residues in milk.

The results of an in-house laboratory validation of a microbiological method for the screening of antibiotic residues in milk are presented. The sensitivity of this five-plate test, called Screening Test for Antibiotic Residues (STAR), was established by the analysis of milk samples spiked with 66 antibiotics at eight different concentrations. Ten different groups of antibiotics were studied: macrolides, aminoglycosides, cephalosporins, penicillins, quinolones, tetracyclines, sulphonamides, lincosamides, phenicolated and miscellaneous drugs. It was shown that 21 antibiotics were detected by the STAR protocol at or below the maximum residue limit (MRL), and that a further 27 drugs could be detected at levels from the MRL up to four times the MRL. The sensitivity of the STAR protocol was at or below the MRL for three macrolides, one tetracycline, two aminoglycosides, some sulphonamides, half of the beta-lactams, quinolones, lincosamides, trimethoprim and baquiloprim. Moreover, the STAR protocol was at least twice as sensitive as conventional methods for macrolides, quinolones and tetracyclines. The other antibiotics had limits of detection between four and 150 times the MRL. Each plate was preferentially sensitive for one or two families of antibacterials: the plate Bacillus cereus for tetracyclines, the plate Escherichia coli for quinolones, the plate Basillus subtilis for aminoglycosides, the plate Kocuria varians for macrolides, and the plate Bacillus stearothermophilus for sulphonamides and beta-lactams. This method has been used routinely on a day-to-day basis to direct the physicochemical confirmation towards one or two families of antibiotics. Considering the high cost of liquid chromatography coupled with tandem mass spectrometry detection analyses, the reduction of the range of antibiotics to test for confirmation is a significant gain in time and money.

Stability of penicillin antibiotic residues in meat during storage. Ampicillin.

Incurred samples from a pig treated with ampicillin, one of the most important penicillin antibiotic drugs used in food-producing animal treatments, were analyzed at the residue level of the drug in muscle tissue (approximately 100 microg kg(-1)) during their freezing storage and using three different techniques: quantitative microbiological assay, HPLC-UV and LC-MS. Two parameters have been specifically monitored: storage temperature (-20 and -75 degrees C) and storage packaging (ground meat or bulk meat). No significant decrease was observed during the first 3 months of storage monitoring at -20 and -75 degrees C. On the contrary, the sample preparation significantly affected the drug concentration in muscle from the very beginning of the storage. Grinding the meat before storage allowed to keep the drug near the higher level of concentration (approximately 100 microg kg(-1)) when bulk meat stored frozen systematically led to a decreased value (approximately 75 microg kg(-1)). After 8 months of storage at -20 degrees C, a significant decrease arose and was never observed at -75 degrees C. All the results were similarly obtained with the three different techniques used simultaneously, which allows to indicate a good correlation between the techniques.

In vitro toxicity of trichothecenes on human haematopoietic progenitors.

The culture of human haematopoietic progenitors, Colony-Forming-Unit Granulocyte and Macrophage (CFU-GM), has been performed in the presence of four trichothecenes, T-2 toxin, HT-2 toxin, diacetoxyscirpenol (DAS), and deoxynivalenol (DON). Our results showed that trichothecenes were cytotoxic for human haematopoietic progenitors. This work and the analysis of results described in the literature allowed us to propose that the haematologic lesions observed during human intoxication could be due to a destruction of haematopoietic progenitors such as granulocytic and macrophage colony-forming cells.

Experimental and natural infection of calves with Bunostomum phlebotomum.

An experiment was conducted to determine the efficiency of a method of experimental infection of weaner beef calves with Bunostomum phlebotomum and to compare such infection with that established by natural infection. Six calves, maintained on a concrete-floored pen, were inoculated with B. phlebotomum L3 by placing the larval inocuulum, in small volume, in the outer chamber of the ear while the animal was restrained for 18 min. Inoculation doses of 20, 30, 40, 50, 60, and 80 thousand L3 were used. Six other calves were grazed on pasture known to be heavily contaminated with hookworm. All animals were killed 72 days after experimental infection and 93 days after initial exposure to pasture infection. The experimental and naturally-infected calves became patent at 55 and 66 days, respectively, after exposure to L3. Red blood cell counts and hemoglobin values were markedly depressed in both groups and lowest values coincided with onset of patency. There was difference in liveweight changes, but both groups lost weight during the prepatent period of infection and gained weight with the onset of patency. The largest number of hookworms was established at the 30000 L3 inoculation level; little or no establishment was observed at the 2 highest levels. Sizeable adult hookworm burdens were established in 4 out of 6 pastured calves. Intestinal pathology was generally more severe in experimentally-infected calves, consisting of a thickened mucosa and masses of punctate, hemorrhagic foci. Pastured calves also acquired large burdens of Ostertagia ostertagi, particularly inhibited early fourth-stage larvae. Moderate to severe abomasal pathology and elevated plasma pepsinogen were associated with ostertagiasis in the pastured calves. The experimental infection method is efficient in establishing high levels of B. phlebotomum infection in calves currently or previously infected with other gastrointestinal nematodes.

Activity of fenbendazole against inhibited early fourth-stage larvae of Ostertagia ostertagi.

The efficacy of fenbendazole (Panacur, Hoechst-Roussel) against inhibited early fourth-stage larvae of Ostertagia ostertagi and other nematodes of the abomasum and intestinal tract was investigated in naturally infected, yearling cattle in April 1978. The time when peak levels of inhibited larvae occurred was determined by epizootiologic study which began in November 1977. All animals were removed from pasture and maintained free from further helminth infection until slaughter (19 to 21 days). The fenbendazole liquid suspension was administered as an oral drench at dose level of 10 mg/kg to 10 animals and then at dose level of 15 mg/kg to an additional 10 animals at 10 days after removal from pasture. Eleven animals were maintained as untreated controls. In cattle given the dose of 10 mg/kg, the following reductions were observed: O ostertagi adults--100%, developing stages--80%, and inhibited larvae--97%; other worm genera in the abomasum and nematodes of the intestinal tract--100%. In the cattle given the larger dose, the following reductions were observed: O ostertagi adults--100%, developing stages--98%, and inhibited larvae--99%; other worm genera in the abomasum and nematodes of the intestinal tract--100%.

Efficacy of albendazole against inhibited early fourth stage larvae of Ostertagia ostertagi.

Twelve untreated controls and 12 treated beef yearling steers were used in trials of albendazole (Smith Kline Animal Health Products) at 7.5 mg per kg (oral drench) against natural infections of inhibited fourth stage larvae of Ostertagia ostertagi. Albendazole had an efficacy of 83.8 per cent in removal of inhibited larvae. The mean number of inhibited larvae in untreated controls was 48.9 per cent. Efficacy against developing stages and adults of O ostertagi was 92.8 and 99.7 per cent, respectively. Efficacy against other worm genera in the abomasum, primarily thaemonchus and Cooperia adults, was 99.4 per cent. No signs of toxicity were observed following administration of albendazole. Some aspects of inhibition of O ostertagi in cattle in the USA are reviewed.

Effect of albendazole on gastrointestinal parasites of cattle.

The efficacy of albendazole in removing gastrointestinal nematodes and tapeworms was studied in 16 treated and 16 untreated (control) beef calves. The anthelmintic was administered as an oral drench at a dose level of 5 ml/45.5 kg of body weight (5 mg/kg). Percentage of efficacy against the various genera was: Trichostrongylus axei, Oesophagostomum radiatum, and Moniezia-100.0; Haemonchus placei, Cooperia spp, and Trichostrongylus colubriformis - greater than 99.0; Ostertagia ostertagi -98.3; and Bunostomum phlebotomum -96.2. Efficacy against Trichuris was 20.2. Efficacy against immature nematodes in the abomasum and small intestine was high, 91.9 and 86.4, respectively, but total numbers of nematodes were not large. Efficacy against immature stages in the cecum and remainder of the large intestine was 48.5. Signs of toxicosis were not observed in calves treated with albendazole.