Repurposing Ionophores as novel antimicrobial agents for the treatment of bovine mastitis caused by Gram-positive pathogens.

Increasing reports of multidrug-resistant bacterial infections in animals has created a need for novel antimicrobial agents that do not promote cross-resistance to critically important antimicrobial classes used in human medicine. In response to the recent emergence of antimicrobial resistance in several bovine mastitis pathogens, in vitro antimicrobial susceptibility was determined for four polyether ionophores (lasalocid, monensin, narasin and salinomycin) against Staphylococcus spp. and Streptococcus spp. isolated from clinical cases. In addition, erythrocyte haemolysis and WST-1 cell proliferation assays were used to assess in vitro mammalian cell cytotoxicity and biofilm susceptibility testing was performed using the minimum biofilm eradication concentration (MBEC™) biofilm assay. Lasalocid, monensin, narasin and salinomycin exhibited bacteriostatic antimicrobial activity against all pathogens tested, including methicillin-resistant staphylococci, with MIC90 values <16 µg/ml. Narasin and monensin displayed the least toxicity against mammalian cell lines and all compounds significantly reduced viable cell numbers in a Staphylococcus aureus biofilm. Based on in vitro characterization, all four ionophores offer potentially novel treatments against bovine mastitis but in vivo studies will be essential to determine whether acceptable safety and efficacy is present following intramammary administration.

Safety evaluation of lasalocid use in Chinese ring-necked pheasants (Phasianus colchicus).

Coccidiosis remains a significant threat to the welfare of game farm-reared pheasants in the United States. Although lasalocid has been demonstrated to be effective against pheasant specific coccidia, information regarding its safety in this species is lacking. The purpose of this study was to gather data on the safety of lasalocid when fed to Chinese ring-necked pheasants at one, two, and three times the recommended high dose of lasalocid used for prevention of coccidiosis in other poultry at three times the normal treatment period. Pheasant chicks (approximately 1 day-old; n = 160) were randomly blocked by sex into four treatment groups and given their respective diets continuously for 6 wk. No significant differences were observed in overall feed consumption, weight gain, feed conversion rates, clinical pathology measurements, or tissue gross and histopathologic evaluations between controls and treatment groups associated with lasalocid administration. Based on the results of this study it appears that lasalocid fed at the recommended rate of 125 ppm is safe in Chinese ring-necked pheasants.

Comparison effect of lasalocid, diclazuril, probiotic and symbiotic on histomorpholical changes of small intestine induced by E. tenella.

This study aimed to investigate the comparison of effect of anticoccidal drugs including lasalocid and diclazuril with probiotic and synbiotic on the growth performance and intestinal morphology in broiler chicken. One hundred eighty chickens (Ross 308, 1 day old) were randomly divided into 6 equal groups (n=30) including the negative control (basal diet), the positive control (basal diet+oral inoculation of 3×104 sporulated oocytes of E. tenella, and four treatment groups. At days of 28 and 49 of age, 9 chickens were blindly chosen from each group were scarified by decapitation and their various segments of small intestine including ileum, jejunum, and duodenum were evaluated histomorphologically. We found that the economic losses resulted from coccidial infection in the poultry industry are caused by the decreased performance of broiler chicken induced by morphological changes in the any three segments specially jejunum. The anticoccidial drugs, synbiotic and probiotic can partially prevent morphological changes in any three segments of small intestine in broiler chicken with coccidiosis. Since morphological changes in the jejunum begin earlier than in other parts and surface area of jejunal villi is important for nutrition absorbance as well as growth performance, lasolacid was found to a be more efficient treatment in this regard.

Monensin and the prevention of tryptophan-induced acute bovine pulmonary edema and emphysema.

3-Methylindole, a ruminal fermentation product of tryptophan, induces acute pulmonary edema and emphysema in cattle, and 3-methylindole is present in the ruminal fluid and blood of cows with a natually occurring form of this disease. Monensin, a polyether antibiotic and widely used feed additive for beef cattle, prevented tryptophan-induced acute bovine pulmonary edema and emphysema. Monensin acted by reducing the ruminal conversion of L-tryptophan to 3-methylindole both in vitro and in vivo. Lasalocid, also a polyether antibiotic, showed similar effects in vitro. These results provide a promising approach to prevention of this major respiratory disease of cattle.

Efficacy of lasalocid against coccidiosis in Chinese ring-necked pheasants.

Two battery efficacy studies were conducted with field isolates of Eimeria spp. from outbreaks of coccidiosis on pheasant-rearing farms in Georgia (GA) and Illinois (IL), United States. The coccidian isolates were mixed species containing E. phasiani, E duodenalis, and E colchici. Lasalocid (LAS) was tested prophylactically at 120 parts per million (ppm) in the feed. Anticoccidial activity was assessed, in comparisons between infected and uninfected control birds, by the effects on weight gain and oocyst passage. The GA isolate reduced weight gain of infected controls by 64% (P < 0.05) during the week after inoculation. LAS-treated birds also suffered some depressed gain, but average gains were significantly improved (P < 0.05) in comparison with that of the infected controls. Fecal oocyst counts of infected controls averaged 10-24 x 10(6)/day for days 5-11 postinoculation (PI), after which oocyst passage declined. Birds given LAS passed 10-20 x 10(6) oocysts on days 5-6 PI, after which counts dropped to much lower levels. Oocyst passage was significantly reduced (P < 0.05) by LAS treatment on all but days 5 and 6 PI. The IL isolate reduced weight gain by 36% in infected controls, relative to uninfected controls (P < 0.05). LAS-treated birds also suffered reduced gain, but the gain was significantly improved in comparison with the infected controls. Oocyst counts averaged 3-9 x 10(6)/day on days 6-14 PI, but were significantly reduced by LAS treatment on all but day 6 PI. Thus, LAS was effective in pheasants against the tested field isolates of Eimeria spp. and was well tolerated at 120 ppm in the feed. However, the level of control was not as high as previously observed. Possible explanations for this difference include an emergence of drug resistance from prior use of LAS and a differential efficacy of LAS against different species of Eimeria used in the studies.

Efficacy of treatments with toltrazuril 7.5% and lasalocid sodium in sheep naturally infected with Eimeria spp.

The objective of this study was to evaluate the efficacy of an experimental formulation of toltrazuril 7.5% + Trimix™ on a naturally acquired infection of Eimeria spp. in suckling lambs kept on pasture and, in another trial, evaluate the comparative efficacy between lasalocid and toltrazuril 7.5% + Trimix™ in newly weaned sheep under feedlot conditions that had been naturally infected with Eimeria spp. In the first experiment, 30 suckling lambs were divided into two groups: A - treated with toltrazuril 7.5% + Trimix™ and B- control. In experiment 2, 30 weaned sheep were divided into three groups: I - treated with toltrazuril 7.5% + Trimix™, II - treated with lasalocid and III - control. Treatment group A showed an efficacy of 90, 99.4 and 87.3% on days 5, 10 and 20, respectively. Treatment group I had an efficacy of 98.2, 92.6 and 94.5%, while group II had an efficacy of 72.7, 81.6 and 95.9% on days 7, 21 and 42, respectively. Eight Eimeria species were identified; E. ovinoidalis was the most common. Treatment with the toltrazuril 7.5% +Trimix ™ formulation was effective against Eimeria spp. in suckling lambs in field conditions and lambs weaned in under feedlot conditions.

[Cryptosporidiosis in newborn calves in Ankara region: clinical, haematological findings and treatment with Lasalocid-NA]. / Cryptosporidien-lnfektionen bei neugeborenen Kälbern aus der Umgebung von Ankara: klinische und hämatologische Untersuchungen sowie Ergebnisse der Behandlung mit Lasalocid-Na.

In the first part of the present study, a total of 109 faeces samples collected from calves suffering from diarrhoea were examined for Cryptosporidium parvum oocytes and 39 (35.8%) of them were found to be positive. On the basis of oocyte counts, 14 (36%) samples were assessed as mildly infected and 25 (64%) samples as heavily infected. The occurrence of the disease was more common in winter (56.4%) than during other seasons (autuma 0%, summer 15.4% spring 28.2%. In the present study, the ionophore polyetherantibiotic Lasalocid-Na, that is licensed as a feed additive (Bovatec, 15% Lasalocid-Na, Roche AG) in Turkey, was administered to 11 calves naturally infected with Cryptosporidium and its therapeutic effect was evaluated. Lasalocid-Na (8 mg/ kg BW) was given once daily for 3 days added to the milk. The clinical parameters of infected calves were evaluated before and 3 days after the treatment in 24 hour intervals. The oocyst counts of faeces of calves with cryptosporidiosis were between 15 x 10(6) and 96 x 10(6)/mL before treatment. No oocystes were found in faecal samples of 3 calves (27.3%) after 48 hours and 4 (40%) calves after 72 hours of treatment, respectively. The number of oocytes in the faeces of the remaining calves varied between 90 and 1.2 x 10(6)/mL during the respective period. The number of oocystes before treatment was significantly higher than the number of oocytes after treatment. One of the calves died 56 h after the first treatment despite the treatment. The pH of venous blood was decreased prior to treatment as expected. The lowest pH was 6.83, the lowest bicarbonate concentration was 3.80 mmol/l and the lowest base excess was -31.2 mmol/l. After the treatment, pH, pCO2, HCO3- and BE values of the venous blood increased significantly and reached physiological values before discharge. The differences between the values assessed before the treatment and at the 2nd, 3rd, and 4th sampling time were statistically significant. Number of leucocyte and haemoglobin concentration before the treatment were significantly higher than the values after treatment (p < 0.01). These values returned back to physiological ranges 72 hours after first treatment. Lasalocid-Na was rather well tolerated. Side effects such as the decline of the suckling reflex and intoxication symptoms in respect to present administration form and dosage were found only in one calf (9%). Treatment procedure in the present study with Lasalocid-Na was found to be suitable in combination with an adequate infusion therapy for the treatment of calves on farms with problems related to cryptosporidiosis. Although the use of Lasolacid-Na for the treatment of Cryptosporidium infection in the EU is banned, it might be used as an alternative drug outside of the EU since it has a successful effect for preventing reinfections.

Interaction of T-2 fusariotoxin with anticoccidial efficacy of lasalocid in chickens.

Two battery tests were carried out to reveal whether or not there is an interaction between T-2 fusariotoxin and lasalocid, similar to that known with some ionophorous anticoccidials. In the first experiment, an excessive dose level of toxin (6 ppm) significantly reduced the anticoccidial efficacy of lasalocid at 75 ppm and almost completely eliminated that at 37.5 ppm in cockerels which had been infected with oocysts of Eimeria tenella and E. mitis. In the second experiment, the same dose of lasalocid was tested in combination with 0.5-1.25 ppm levels of toxin, i.e. levels occurring in the field, and all but the lowest level (0.5 ppm) significantly reduced the anticoccidial efficacy of lasalocid. Thus, in cases of outbreaks of coccidiosis, there is need to keep in mind such interaction between anticoccidials and mycotoxins.

Studies on the stage of action of lasalocid against Eimeria tenella and Eimeria acervulina in the chicken.

Broiler chickens in battery pens were either fed a diet containing 100 ppm lasalocid or no drug for 24 h prior to inoculation with sporulated oocysts of Eimeria tenella or Eimeria acervulina. Different groups of birds remained on medicated feed for 24, 48, 72, 96, 120 or 144 h after inoculation. Conversely, other groups started on an unmedicated diet, were given medicated feed at different times after oocyst inoculation. Starting lasalocid medication 24 h (E. tenella) or 48 h (E. acervulina) after inoculation reduced the lesions and improved the weight gain. There was no significant difference in performance of birds after withdrawal of the drug at 48 h (E. tenella) or 72 h (E. acervulina) and thereafter. Starting lasalocid medication at 96 or 120 h did not suppress but rather reduced oocyst production.

Effect of feeding lasalocid to pregnant ewes experimentally infected with Toxoplasma gondii.

Sixteen 50 day gestational ewes were fed lasalocid at the rate of 30 g t-1 and were orally inoculated with 100 infective Toxoplasma gondii oocysts 5 days after beginning feeding of lasalocid. Seventeen control ewes were similarly inoculated with T. gondii and were not fed lasalocid. The rate of abortion and neonatal mortality in both treated and untreated ewes was similar, indicating that feeding lasalocid was not effective in preventing T. gondii abortion in sheep.

Anticoccidial evaluation of halofuginone, lasalocid, maduramicin, monensin and salinomycin.

The activities of five anticoccidials were compared against Eimeria species in/of chickens, in controlled in vivo and in vitro laboratory studies. Two more recent and potent market entries (maduramicin and halofuginone) were compared with three older polyether antibiotic anticoccidials (monensin, lasalocid and salinomycin). Halofuginone, lasalocid, maduramicin, monensin and salinomycin were evaluated at 3, 125, 5, 120 and 66 ppm, respectively, of active drug in the diets. At these levels, all five drugs demonstrated significant activity against Eimeria tenella, E. maxima, E. necatrix, E. brunetti and E. acervulina (in vivo). Monensin was least effective against E. tenella, and one of the lesser efficacious drugs against E. necatrix, maduramicin, was least effective against E. maxima. In studies of single Eimeria species infections, comparable weight gains were noted for the drugs. In the mixed Eimeria species infections, however, birds treated with maduramicin had significantly higher weight gains than did birds medicated with monensin. Unlike in vivo potencies, titration in vitro indicated that monensin was most potent (active at 10(-6) mcg ml-1), and maduramicin and lasalocid least potent (inactive at less than or equal to 10(-3) mcg ml-1).

In vitro and in vivo efficacy of lasalocid for treatment of experimental cryptosporidiosis.

In vitro viability of purified Cryptosporidium parvum oocysts, exposed for 30, 60, 90 and 120min to 0.27mg/ml lasalocid suspension was evaluated by inclusion or exclusion of two fluorogenic vital dyes and an excystation technique. Continuously, preventive and curative efficacies at different doses (9, 6.75, 5.625 and 4.5mg/kg body weight) and regimens of lasalocid against cryptosporidial infection were evaluated on an experimental neonatal mice model. In vitro assays demonstrated a decrease in the oocyst viability related to an increase in exposure time for exposure to the lasalocid suspension. The infection was eradicated when the suspension was administered with a dose of > or = 6.75mg/kg body weight. No apparent toxic effects were observed.

Acquired immunity to Eimeria tenella in lasalocid-treated chickens.

The effect of restricted medication with lasalocid sodium on the development of acquired immunity against Eimeria tenella was evaluated. The medication was allowed for all or part of 6-day test period (one day before until 4 days after infection). The parameters used for such evaluations were lesion score, caecal, bursal and splenic weights. The optimum treatment time for the drug was clearly indicated by lesion score which was very low when the medication was initiated 1 day before until 1 day after inoculation, but only partly effective if given on Day 2 post-inoculation. The challenge with higher doses on 14th day of immunizing infection revealed a reverse picture where the higher lesions were recorded by the groups where medication was started earlier than the delayed treatment groups. This indicates partial interference with the development of immunity in the earlier treatment groups. Birds treated on Day 4 p.i. were not significantly different (P less than 0.05) from the infected unmedicated control group, suggesting no interference in acquired immunity. A correlation was noticed between day of treatment, the lesion score and weight gain of the caecum as well as the spleen. After both immunizing and challenge infections, the bursa did not show any significant variation in weight, whereas the weight of the spleen did vary. The infected unmedicated group and the delayed-treatment groups had a comparatively higher splenic weight than the uninfected unmedicated group of birds.(ABSTRACT TRUNCATED AT 250 WORDS)

Residual activity of anticoccidial drugs in chickens after withdrawal of medicated feeds.

Seven anticoccidial drugs commonly used in poultry (diclazuri), monensin, salinomycin, halofuginone, nicarbazin, robenidine, amprolium, and lasalocid) were tested for residual activity after withdrawal. In each test, the products were given at the recommended level to cages of 10 broiler chickens. Oral inoculation with coccidia was given after withdrawal of medication. Birds pretreated with 1 ppm of diclazuril and inoculated with Eimeria tenella after drug withdrawal had normal weight gain and very low lesion scores. Residual activity depleted gradually over several days, as shown by higher lesion scores when medication was withdrawn for up to 3 days before inoculation. Similar results were observed when young birds were inoculated with a mixture of E. tenella, E. maxima and E. acervulina, and also when birds were given diclazuril to market weight (6 weeks of age) and inoculated with a mixture of six species of Eiméria (The above species plus E. brunetti, E. mitis, and E. necatrix) after withdrawal of medication for 2 days. In contrast, there was no evidence of residual anticoccidial activity with nicarbazin, halofuginone, lasalocid, amprolium, salinomycin or monensin. Overall, the residual activity was unique to diclazuril.

Anticoccidial drug resistance in the southeastern United States: polyether, ionophorous drugs.

Coccidia were isolated from poultry farms in the U.S. and tested for sensitivity to the polyether, ionophorous drugs. Of 52 isolates, 24 were individual; Eimeria spp. and 28 were mixed species. Eimeria tenella, E. maxima, and E. acervulina were commonly present in mixed isolates. The isolates differed considerably in response to individual drugs and to the drugs as a group. All isolates responded to some extent, so none was judged completely resistant to the ionophores, but control of some isolates was poor, with up to 47% of medicated birds dying in one instance. Based on lesion score reduction, the best indicator of effectiveness, salinomycin provided the best overall control (64%), followed by lasalocid (56%) and monensin (52%). When weight gain was used as evidence of resistance, lasalocid provided the best control (97%), followed by salinomycin (90%) and monensin (81%). Examination of testing methods revealed that the arbitrarily selected endpoint should significantly affect th number of isolates designated resistant or sensitive.

Efficacy of ionophorous anticoccidial drugs against coccidia in farm-reared pheasants (Phasianus colchicus) from Illinois.

Litter samples obtained from a ring-necked pheasant propagation farm in Illinois contained coccidia: 57.5% of the oocysts were Eimeria duodenalis, 24.9% were E. tetartooimia, 8.8% were E. phasiani, and 8.8% were E. pacifica. Ionophorous anticoccidial drugs were tested for efficacy against the pheasant coccidia. All three drugs reduced oocyst production and prevented mortality in young pheasants; unmedicated infected controls had a 40% mortality rate. Monensin at 120 ppm in the feed was coccidiocidal against E. duodenalis and E. tetartooimia, partly coccidiocidal against E. pacifica, and only partly coccidiostatic against E. phasiani. Salinomycin at 60 ppm in the feed was highly efficacious and coccidiocidal against all four species, but the salinomycin-medicated pheasants gained the least of all medicated birds. Lasalocid at 120 ppm in the feed was the most effective, with nearly complete coccidiocidal activity against all four coccidial species.

Efficacy evaluation of lasalocid plus roxarsone combination medication with different geographic field strains of Eimeria acervulina.

Performance of broiler chickens medicated with lasalocid alone (at 125 ppm) or in combination with roxarsone (at 50 ppm) was evaluated in battery and floorpen trials after challenge with geographically different field strains of coccidia containing predominately the upper intestinal species Eimeria acervulina. No significant difference in bird performance measured at 6 days postinfection (PI) was observed between lasalocid plus roxarsone-medicated (L+RM) or lasalocid-medicated (LM) birds challenged in separate battery trials with mixed-species inocula from Alabama or Georgia containing 92% or 88% E. acervulina, respectively. In contrast, L+RM birds challenged in another battery trial with a Louisiana mixed-species inoculum containing 92% E. acervulina showed significant reduction in average weight gain at 6 days PI compared with LM-challenged birds. A floorpen trial done with the same Louisiana inoculum showed significant reduction in average bird weight gain at 27 and 35 days of age (6 and 14 days PI) for L+RM-challenged birds compared with both unmedicated-nonchallenged (UMNC) control and LM-challenged birds. The LM+R groups were significantly lower in average bird weight at 27 days of age than the unmedicated-challenged controls. Feed conversions (FCs) for L+RM birds were significantly higher than those for the UMNC control birds during time of challenge (21-27 days of age) and for the 1-to-27-day-of-age time period. No significant difference in FC was seen between the UMNC and LM groups. Results of this study showed that performance of broiler birds medicated with lasalocid plus roxarsone could vary for geographically different mixed-species challenge inocula that contained predominately E. acervulina.

Sensitivity of field isolates of Eimeria species to monensin and lasalocid in the chicken.

Isolates of Eimeria species obtained from broiler or from breeder farms were compared for their sensitivity to two ionophorous anticoccidial drugs, monensin and lasalocid. All of 25 isolates from broiler farms were resistant to 100 ppm monensin or 90 ppm lasalocid, while 14 of 16 isolates were resistant to monensin and seven of 16 to lasalocid from breeder farms (replacement layer and broiler breeder).

Effect of including lasalocid or monensin singly or in combination with furazolidone on the growth and feed consumption of turkey poults.

Lasalocid and monensin are widely used to control coccidiosis in broilers, but not in turkey poults. Four feeding trials were conducted to determine the performance of turkey poults when these compounds were used singly or in combination with 100 ppm of furazolidone. Bodyweights and feed consumption were significantly depressed for five weeks after hatching by 150 ppm of lasalocid. Combining furazolidone with lasalocid ameliorated the toxic effect of lasalocid. Bodyweights were significantly depressed by 150 ppm of monensin in the fifth week after hatching, but there was no significant depression in feed consumption. Furazolidone exacerbated any toxic effects of monensin. Data indicate that monensin may be used safely at dosages greater than the recommended level of 60 to 99 ppm, but should not be used in combination with furazolidone.

Prevention of lactic acidosis in cattle by lasalocid or monensin.

Intraruminal administration of lasalocid or monensin (1.3 mg/kg body weight) effectively prevented in glucose- or corn-induced lactic acidosis in cattle. Administering the antibiotics for 7 days before experimentally inducing acidosis with corn (27.5 g/kg body weight), effectively prevented acidosis, while 2 days' were sufficient to prevent glucose-induced acidosis (12.5 g/kg body weight). The different responses observed in the two trials probably stemmed from the difference in amounts of carbohydrate used to induce acidosis. Antibiotic-treated cattle had higher rumen pH values and lower L(+) and D(-) lactate concentrations that control cattle that received no antibiotics. Ruminal VFA in control cattle decreased, while total VFA and the molar proportion of propionate increased in antibiotic-treated cattle after grain engorgement. Control cattle exhibited classic signs of acidosis, such as lowered blood pH; increased blood lactate, particularly D(-) isomer; hemoconcentration, and depleted alkali reserve with a pronounced based deficit. Antibiotic-treated cattle exhibited no signs of systemic acidosis.