Susceptibility evaluation and PK/PD integration of tulathromycin against Actinobacillus pleuropneumoniae during the mutant selection window.

Introduction: Actinobacillus pleuropneumoniae (APP) is a serious pathogen that affects the development of livestock breeding. Due to excessive use of antimicrobial drugs, many multidrug-resistant bacteria have emerged and spread, which have threatened the livestock industry. Therefore, we established a peristaltic pump infection model (PPIM) to evaluate the susceptibility change and pharmacokinetic/pharmacodynamic (PK/PD) integration of tulathromycin against APP during the mutant selection window (MSW) for preventing the emergence of mutant-resistant bacteria. Methods: The 99% minimum inhibitory concentration (MIC99) and mutant prevention concentration (MPC) of tulathromycin against APP were measured using the agar-plate method. After the model of dynamic infection had been established based on tulathromycin data in lungs, different dosages were administered to make the drug concentrations located in different parts of the MSW. The population and sensitivity of APP were monitored. Tulathromycin concentrations were measured by high-performance liquid chromatography-tandem mass spectrometry. Finally, a sigmoid Emax model was used to analyze the relationships between PK/PD parameters and antibacterial effects. Results and discussion: The values of MIC, MIC99, and MPC of tulathromycin against APP were 2, 1.4, and 44.8 µg/mL, respectively. The PPIM was stable. An elimination effect without regrowth was observed at 5.6 to 44.8 µg/mL (-4.48 to -7.05 Log10 CFU/mL, respectively). The MIC of APP increased 32-fold at 8 MIC99. AUC168 h/MIC99 had the best fit with the antibacterial effect (R 2 = 0.9867). The AUC168 h/MIC99 required to achieve bacteriostatic, bactericidal, and clearance effects were 1.80, 87.42, and 198 h, respectively. Our results could provide guidance for the clinical application of tulathromycin to treat APP infection and avoid the generation of drug-resistant bacteria.

Evaluation of antimicrobial and non-steroidal anti-inflammatory treatments for BRD on health and welfare in fattening bulls: a cross-sectional study.

Our study aimed to evaluate the effect of different treatments for BRD on health and welfare in fattening bulls. A total of 264 bulls were enrolled. Welfare was assessed on day 2 (T0) and day 15 (T1) after arrival. A decrease in the welfare level was observed from T0 to T1. All bulls were inspected clinically at T0 and T1 revealing an increase of skin lesions and lameness in T1. In both periods, a high incidence of respiratory disease was observed. A prevalence of 79.55% and 95.45% of Mycoplasma bovis using RT-PCR and culture at T0 and T1 respectively was observed. Blood samples were collected for haematology at T0 and T1. At T0, 36 animals were individually treated for BRD with an antimicrobial (IT), 54 received a metaphylactic treatment with tulathromycin (M), 150 received a metaphylactic treatment with tulathromycin plus a second antimicrobial (M + IT) whereas 24 were considered healthy and therefore not treated (NT). Additionally, 128 were treated with a non-steroid anti-inflammatory (NSAID). Neutrophils of M + IT were significantly higher than groups NT and M and the lymphocytes of M + IT were significantly lower than that of IT. White blood cells, neutrophils and N/L ratio of animals treated with an NSAID was significantly higher than that not treated. Lung inspection of 172 bulls at the abattoir indicated that 92.43% presented at least one lung lesion. A statistically significant effect of the NSAID treatment on the lung lesions was observed. Our findings indicate that BRD was a major welfare and health concern and evidence the difficulties of antimicrobial treatment of M. bovis.

Evaluation of Biological Equivalence for Generic Tulathromycin Injections in Cattle.

The recent expiration of patents for the antibiotic tulathromycin has led to a significant increase in the number of generic tulathromycin products (GTPs) available. This study aims to evaluate the bioequivalence of four GTPs, which experienced a rapid increase in market share. The bioequivalence was evaluated by performing pharmacokinetic assessments. The four selected GTPs (Tulaject, Tulagen, Toulashot, and T-raxxin) were compared with the reference product, Draxxin. A dose of 2.5 mg/kg.bw/day was administered via subcutaneous injection, and blood samples were collected 460 times from 20 Holstein cattle. Plasma concentrations of tulathromycin were measured over time using LC-MS/MS analysis. Bioequivalence was evaluated using a statistical program for pharmacokinetic parameters, including the area under the concentration time curve (AUC) and the maximum plasma concentration (Cmax). The bioequivalence was considered proven if the difference between the test and reference products was within 20% for both AUC and Cmax. The results showed that the confidence interval (CI, 90%) for both AUC and Cmax values was within the 80~120% range, demonstrating the bioequivalence of the four GTPs compared to Draxxin. This study provides evidence for the bioequivalence of the selected GTPs, contributing to their validation for use as effective antibiotics.

Tulathromycin and Diclazuril Lack Efficacy against Theileria haneyi, but Tulathromycin Is Not Associated with Adverse Clinical Effects in Six Treated Adult Horses.

Equine theileriosis, caused by Theileria haneyi and Theileria equi, leads to anemia, exercise intolerance, and occasionally, death. Theileriosis-free countries prohibit the importation of infected horses, resulting in significant costs for the equine industry. Imidocarb dipropionate is the only treatment for T. equi in the United States, but lacks efficacy against T. haneyi. The goal of this study was to assess the in vivo efficacy of tulathromycin and diclazuril against T. haneyi. Fourteen T. haneyi-infected horses were utilized. Six were treated with eight weekly 2.5 mg/kg doses of tulathromycin. Three were treated daily for eight weeks with 2.5 mg/kg diclazuril. Three were pre-treated with 0.5 mg/kg diclazuril daily for one month to determine whether low-dose diclazuril prevents infection. Following infection, the dose was increased to 2.5 mg/kg for eight weeks. Two infected horses remained untreated as controls. The horses were assessed via nested PCR, physical exams, complete blood counts, serum chemistry panels, and cytology. Tulathromycin and diclazuril failed to clear T. haneyi and the treated and control groups exhibited similar parasitemia and packed cell volume declines. To obtain additional safety data on tulathromycin use in adult horses, necropsy and histopathology were performed on tulathromycin-treated horses. No significant lesions were detected.

A Single Intranasal Dose of Bacterial Therapeutics to Calves Confers Longitudinal Modulation of the Nasopharyngeal Microbiota: a Pilot Study.

To address the emergence of antimicrobial-resistant pathogens in livestock, microbiome-based strategies are increasingly being sought to reduce antimicrobial use. Here, we describe the effects of intranasal application of bacterial therapeutics (BTs) on the bovine respiratory microbiota and used structural equation modeling to investigate the causal networks after BT application. Beef cattle received (i) an intranasal cocktail of previously characterized BT strains, (ii) an injection of metaphylactic antimicrobial (tulathromycin), or (iii) intranasal saline. Despite being transient colonizers, inoculated BT strains induced longitudinal modulation of the nasopharyngeal bacterial microbiota while showing no adverse effect on animal health. The BT-mediated changes in bacteria included reduced diversity and richness and strengthened cooperative and competitive interactions. In contrast, tulathromycin increased bacterial diversity and antibiotic resistance and disrupted bacterial interactions. Overall, a single intranasal dose of BTs can modulate the bovine respiratory microbiota, highlighting that microbiome-based strategies have potential in being utilized to mitigate bovine respiratory disease in feedlot cattle. IMPORTANCE Bovine respiratory disease (BRD) remains the most significant health challenge affecting the North American beef cattle industry and results in $3 billion in economic losses yearly. Current BRD control strategies mainly rely on antibiotics, with metaphylaxis commonly employed to mitigate BRD incidence in commercial feedlots. However, the emergence of multidrug-resistant BRD pathogens threatens to reduce the efficacy of antimicrobials. Here, we investigated the potential use of novel bacterial therapeutics (BTs) to modulate the nasopharyngeal microbiota in beef calves, which are commonly administered metaphylactic antibiotics to mitigate BRD when sourced from auction markets. By direct comparison of the BTs with an antibiotic commonly used for BRD metaphylaxis in feedlots, this study conveyed the potential use of the BTs to modulate respiratory microbiome and thereby improve resistance against BRD in feedlot cattle.

Association between Tulathromycin Treatment for Bovine Respiratory Disease and Antimicrobial Resistance Profiles among Gut Commensals and Foodborne Bacterial Pathogens Isolated from Feces of Beef Steers.

ABSTRACT: This study was conducted to evaluate the association between a therapeutic dose of tulathromycin for bovine respiratory disease in beef steers and the antimicrobial and multidrug resistance profiles of the gastrointestinal tract commensals Escherichia coli and Enterococcus spp. and the foodborne pathogens Salmonella enterica and Campylobacter spp. isolated from fecal samples. Individual fecal samples were collected on days 0, 14, and 28 from 70 beef steers that were housed in a single pen and had been treated or not treated with tulathromycin. Samples were cultured for bacterial isolation, and isolates were tested for antimicrobial susceptibility with the broth microdilution method to determine the MICs of clinically relevant antimicrobials used in both human and veterinary medicine. Generalized linear mixed effects models were fitted to estimate the prevalence of the bacterial species and the prevalence of resistant isolates over time and between treated and nontreated cattle and of multidrug-resistant isolates. Model-adjusted mean prevalences of E. coli, Enterococcus spp., S. enterica, and Campylobacter spp. were 99.5, 85.9, 1.5, and 17.7%, respectively. The prevalence of erythromycin-resistant Enterococcus spp. was significantly higher on day 14 (59.7%) than on day 28 (22.2%). A higher prevalence of erythromycin-resistant Enterococcus spp. was found in samples from treated (59.3%) than in samples from nontreated (27.6%) animals. Multidrug resistance (three or more antimicrobial classes) was observed in 8.4% of E. coli isolates and 62.7% of Enterococcus isolates. The administration of tulathromycin was significantly associated with an increased prevalence of erythromycin-resistant Enterococcus spp. isolates.

Pharmacokinetic/Pharmacodynamic Relationships of Tulathromycin Against Actinobacillus pleuropneumoniae in a Porcine Tissue Cage Infection Model.

Tulathromycin is a semi-synthetic macrolide antibiotic that is highly effective in treating respiratory tract bacterial infections. We evaluated the in vivo antibacterial activity of tulathromycin against Actinobacillus pleuropneumoniae in piglets and determined its pharmacokinetic/pharmacodynamic (PK/PD) relationships using a tissue cage infection model. A. pleuropneumoniae (108 CFU/ml) was exposed to tulathromycin via intramuscular injection followed by a collection of cage tissue fluids at various intervals. The percentage of time the drug concentration remained above the minimum inhibitory concentration (MIC) divided by the dosing interval (%T > MIC) was the best PK/PD index to describe the antibacterial efficacy of tulathromycin (R 2 = 0.9421). The %T > MIC values required to achieve 1 - log10CFU/ml reductions and bactericidal activity (3 - log10CFU/ml reduction) were 50.8 and 96.38%, respectively. These results demonstrated that maintaining %T > MIC above 96.38% achieved bactericidal activity and thereby optimized the clinical dosage.

A single LC-MS/MS validated method for tulathromycin quantification in plasma, seminal plasma, and urine to be applied in a pharmacokinetic study in bull.

Tulathromycin is a macrolide antibiotic generally used for the treatment of respiratory diseases in cattle and swine. This work proposes an improvement of a previously published LC-MS/MS method for tulathromycin determination in pig serum, here validated in three different bull matrices: plasma, seminal plasma, and urine. The approach is based on a quick protein precipitation with acetonitrile, filtration, and sample dilution before injection, allowing to rapidly process large batches of samples. Analytes separation was obtained using a BEH C18 (50 × 2.1 mm, 1.7 µm) column, maintained at 40°C with a chromatographic run of 5 min. The method was fully validated over concentration ranges suitable for field levels of tulathromycin found in each matrix (0.01-1 µg/ml for plasma, 0.05-5 µg/ml for seminal plasma, and 0.1-10 µg/ml for urine), showing good linearity during each day of testing (R2 always >0.99). Accuracy and precision were within ±15% at all QC concentrations in all the three matrices. Furthermore, the use of tulathromycine-d7 as internal standard mitigated the potential impacts of matrix effect. The validated technique was successfully applied to samples collected during a pharmacokinetic study in bulls, allowing to monitor tulathromycin concentrations over time in the three matrices. To our knowledge, this is the first validated approach for LC-MS/MS quantification of tulathromycin in seminal plasma and urine.

Emergence and Mechanism of Resistance of Tulathromycin Against Mycoplasma hyopneumoniae in a PK/PD Model and the Fitness Costs of 23S rRNA Mutants.

Macrolides are widely used in diseases caused by Mycoplasma spp. The new semi-synthetic macrolide antibiotic tulathromycin is currently in wide use for the treatment of respiratory diseases of livestock. The objective of this study was to evaluate the antibacterial effect of tulathromycin against Mycoplasma hyopneumoniae using an in vitro pharmacokinetic/pharmacodynamic (PK/PD) model to reveal mechanisms of antibiotic resistance and to evaluate the fitness of drug-resistant strains. In this study, high performance liquid chromatography-tandem mass spectrometry was used to determine drug concentrations for the in vitro model after dosing. The peak concentrations were in the range 0.3125-20 µg/mL (1 × MIC-64 × MIC). The ratio of the area under the concentration-time curve (AUC) over 72 h divided by the MIC (AUC72h/MIC) had the highest correlation with the antibacterial effect of tulathromycin against M. hyopneumoniae. Tulathromycin also showed concentration-dependent antimicrobial effects and promoted the emergence of drug-resistant bacteria after being cultured for 168 h and most were mutations in 23S rRNA at site A2058G (E.coli numbering) and only a single isolate was an A2058T (E.coli numbering) mutant. In the presence of reserpine, we determined the MIC of tulathromycin, tilmicosin, tiamulin and tylosin against these drug-resistant bacteria and the strains with efflux pump mechanisms were found among the strains resistant to tilmicosin. Gene expression analysis indicated that the ABC and MATE transporter efflux pump genes RS01935, RS02670, RS01115, RS01970, RS02395 and RS03540 (MATE family efflux transporter) were up-regulated in the three strains (P < 0.05 or P < 0.01). These investigations provide guidance for clinical administration of tulathromycin and elucidate the mechanism and fitness cost of drug resistance in M. hyopneumoniae.

Pharmacokinetics of tulathromycin on plasma and semen of beef bulls.

The objective of this investigation was to evaluate the pharmacokinetic parameters of tulathromycin in plasma and semen of beef bulls after administering a single sc dose at two different sites in the neck. Four Simmental bulls with excellent temperament received a comprehensive physical exam that included breeding soundness examination. In addition, blood was collected and analyzed for CBC and chemical panel in order to rule out any subclinical liver or kidney disease. All bulls were diagnosed as healthy and satisfactory potential breeders. The mean plasma levels of tulathromycin for the two neck sites of sc administration were not different between posterior aspect of the ear where it attaches to the head (RP; regio parotidea; 77.9 ± 43.3 ng/mL; X ± SD) and to the middle of the neck (RC; regio collis lateralis; 73.7 ± 39.7 ng/mL; P = 0.84). The mean seminal plasma levels of tulathromycin after administration in the RP was 608 ± 374 ng/mL and for RC was 867 ± 599 ng/mL without differences between both sites (P = 0.29). The mean level of tulathromycin in plasma was 75.8 ± 40.2 ng/mL, which was lower than mean seminal plasma levels of 781 ± 482 ng/mL (P = 0.001). The plasma peak tulathromycin concentration (Cmax) was 160 ± 27 ng/mL at 21 ± 6 h (Tmax) post-administration. The seminal plasma Cmax was 1539 ± 44.4 ng/mL at 33.00 ± 18.00 h (Tmax) post-administration. The Cmax between plasma and seminal plasma were different (P = 0.008) without any differences in Tmax between plasma and seminal plasma (P = 0.35). The terminal half-life for plasma tulathromycin (81.4 ± 27.6 h) showed a tendency to be shorter than in seminal plasma (114.7 ± 21.7; P = 0.10). The plasma area under the curve concentration time from the first to the last sample (AUC0-last) was 15,440 ± 1717 ng/mL/h, which was significatively smaller compared with 171,071 ± 58,556 ng/mL/h for seminal plasma AUC0-last (P = 0.01). The plasma means residence time from the first to the last sample (MRT0-last) was 89.3 ± 5.1 h and it was shorter than for seminal plasma of 96.6 ± 5.0 h (P = 0.05). From the present investigation, it was concluded that tulathromycin is a suitable antibiotic based in its pharmacokinetic properties that could be used for treatment of bull genital infections when its application is indicated.

In vivo Pharmacokinetic/Pharmacodynamic (PK/PD) Profiles of Tulathromycin in an Experimental Intraperitoneal Haemophilus parasuis Infection Model in Neutropenic Guinea Pigs.

Tulathromycin is a semi-synthetic macrolide antimicrobial that has an important role in veterinary medicine for respiratory disease. The objective of the study was to develop a pharmacokinetic/pharmacodynamic (PK/PD) model to examine the efficacy and determine an optimal dosage of tulathromycin intramuscular (IM) treatment against Haemophilus parasuis infection induced after intraperitoneal inoculation in neutropenic guinea pigs. The PKs of tulathromycin in serum and lung tissue after intramuscular administration at doses of 1, 10, and 20 mg/kg in H. parasuis-infected neutropenic guinea pigs were evaluated by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The tulathromycin minimum inhibitory concentration (MIC) against H. parasuis was ~16 times lower in guinea pig serum (0.03 µg/mL) than in cation-adjusted Mueller-Hinton broth (CAMHB) (0.5 µg/mL). The ratio of the 168-h area under the concentration-time curve (AUC) to MIC (AUC168h/MIC) positively correlated with the in vivo antibacterial effectiveness of tulathromycin (R 2 = 0.9878 in serum and R 2 = 0.9911 in lung tissue). The computed doses to achieve a reduction of 2-log10 CFU/lung from the ratios of AUC72h/MIC were 5.7 mg/kg for serum and 2.5 mg/kg for lung tissue, which lower than the values of 13.2 mg/kg for serum and 8.9 mg/kg for lung tissue with AUC168h/MIC. In addition, using as objective a 2-log10 reduction and an AUC0-72h as the value of the PK/PD index could be more realistic. The results of this study could provide a solid foundation for the application of PK/PD models in research on macrolide antibiotics used to treat respiratory diseases.

Effect of antibiotic treatment on Mycoplasma hyopneumoniae detection and infectious potential.

Mycoplasma hyopneumoniae (M. hyopneumoniae) causes significant economic losses in the swine industry. Antibiotics with activity against Mycoplasma spp. are employed for disease mitigation and pathogen elimination. However, veterinarians are often challenged with the detection of M. hyopneumoniae by PCR after antibiotic treatment, thus raising the question whether the bacterium is still infectious. The objective of this study was to evaluate the effect of tulathromycin treatment on M. hyopneumoniae detection and infectious potential during the acute and chronic phases of infection. For each infection phase, one age-matched naïve gilt was placed in contact with one M. hyopneumoniae infected gilt that was either treated with tulathromycin, treated and vaccinated, or non-treated, for 14 days. Four replicates per treatment group were performed for each infection phase. A numerical reduction in relative bacterial load was observed in acutely treated gilts compared to non-treated gilts. The rate at which naïve gilts became infected with M. hyopneumoniae was numerically reduced when co-housed with treated, acutely infected gilts compared to those housed with non-treated, infected gilts. During the chronic infection phase, M. hyopneumoniae was detected by PCR in more than 50 % of treated infected gilts and persisted for up to three months post-treatment. Transmission was not detected in all treatment groups however, the possibility that the pathogen was infectious could not be completely ruled out. Further research focused on assessing M. hyopneumoniae detection and viability post-treatment is necessary to guide control and elimination efforts.

The Anti-Nociceptive Potential of Tulathromycin against Chemically and Thermally Induced Pain in Mice.

The present study was conducted to evaluate the analgesic potential of the new triamilide macrolide antibiotic, tulathromycin, at 20 and 40 mg/kg of body weight (BW), subcutaneously against acute pain in mice. Acute pain was induced either chemically (using acetic acid-induced writhing and formalin-induced pain tests) or thermally (using hot-plate, and tail-flick tests). In the acetic acid-induced writhing test, tulathromycin induced a dose-dependent and significant decrease in the number of writhes compared with the control group. In the late phase of the formalin test, a significant decline in hind paw licking time compared with the control group was observed. In the hot-plate and tail-flick tests, tulathromycin caused a dose-dependent and significant prolongation of latency of nociceptive response to heat stimuli, compared with the control group. These findings may indicate that tulathromycin possesses significant peripheral and central analgesic potentials that may be valuable in symptomatic relief of pain, in addition to its well-established antibacterial effect.

Metagenomic Analysis of the Fecal Archaeome in Suckling Piglets Following Perinatal Tulathromycin Metaphylaxis.

The gastrointestinal microbiome plays an important role in swine health and wellbeing, but the gut archaeome structure and function in swine remain largely unexplored. To date, no metagenomics-based analysis has been done to assess the impact of an early life antimicrobials intervention on the gut archaeome. The aim of this study was to investigate the effects of perinatal tulathromycin (TUL) administration on the fecal archaeome composition and diversity in suckling piglets using metagenomic sequencing analysis. Sixteen litters were administered one of two treatments (TUL; 2.5 mg/kg IM and control (CONT); saline 1cc IM) soon after birth. Deep fecal swabs were collected from all piglets on days 0 (prior to treatment), 5, and 20 post intervention. Each piglet's fecal archaeome was composed of rich and diverse communities that showed significant changes over time during the suckling period. At the phylum level, 98.24% of the fecal archaeome across all samples belonged to Euryarchaeota. At the genus level, the predominant archaeal genera across all samples were Methanobrevibacter (43.31%), Methanosarcina (10.84%), Methanococcus (6.51%), and Methanocorpusculum (6.01%). The composition and diversity of the fecal archaeome between the TUL and CONT groups at the same time points were statistically insignificant. Our findings indicate that perinatal TUL metaphylaxis seems to have a minimal effect on the gut archaeome composition and diversity in sucking piglets. This study improves our current understanding of the fecal archaeome structure in sucking piglets and provides a rationale for future studies to decipher its role in and impact on host robustness during this critical phase of production.

Effects of rifampicin on plasma pharmacokinetics of tulathromycin in goats.

We investigated the pharmacokinetic profile of co-administration of tulathromycin with rifampicin. Healthy male goats were allocated to three groups (n = 8) as Group A (single dose 2.5 mg/kg tulathromycin s.c.), B (10 mg kg-1  day-1 rifampicin p.o. daily for 7 days and single dose 2.5 mg/kg tulathromycin s.c. on 8th day), and C (10 mg kg-1  day-1 rifampicin p.o. daily for 21 days and single dose 2.5 mg/kg tulathromycin s.c. on 8th day). Blood samples were collected from jugular veins. Plasma samples were analyzed for tulathromycin by liquid chromatography-mass spectrometry (LC-MS/MS). Peak plasma concentration (Cmax ) values of tulathromycin were 1,390 ± 173, 958 ± 106, and 807 ± 116 ng/ml in groups A, B, and C, respectively. Cmax value of group A was greater than other groups (p < .05). Mean residence time based on time zero to last sample time (MRTlast ) values were 52 ± 1, 56 ± 4 and 66 ± 4 hr in A, B, and C groups, respectively whereas mean residence time based on time zero extrapolated to infinity (MRTINF_obs ) values were 69 ± 4, 85 ± 5, and 86 ± 4 hr, respectively. MRTlast and MRTINF_obs values were greater in B and C groups than group A (p < .05). These findings suggest that rifampicin administration may change several pharmacokinetic parameters of tulathromycin in goats.

Effect of anti-inflammatory compounds or antibiotic administration on receiving performance and physiological responses of transported heifers.

The objectives of this experiment was to evaluate the effects of products with anti-inflammatory properties (yeast product [YEA; 20 g/heifer daily] or astragalus polysaccharide [APS; 20 g/heifer daily]) or an antibiotic (TUL, tulathromycin; 0.025 mL/kg body weight [BW]) on receiving performance and stress responses of transported heifers. Angus heifers (n = 80) were ranked by BW (315 ± 6 kg) and assigned to one of four treatments (five pens per treatment, four heifers per pen) 7 d before shipping 1,400 km (day -7): 1) fed a basal diet of ad libitum hay and concentrate supplement (CON) from day -7 to day 29; 2) YEA in supplemental concentrate from day -7 to day 7 (YEA); 3) APS in supplemental concentrate from day -7 to day 7 (APS); 4) administration of TUL at loading for shipping (day 0; TUL). Upon arrival at the receiving facility (day 1), heifers within each treatment were ranked by BW and assigned to 20 feedlot pens in the same manner as pre-transport. Daily dry matter intake (DMI) was recorded from day 1 to day 28. Full BW was recorded on days -7, -1, 0, 1, 28, and 29. Blood samples were collected on days -7, -1, 1, 4, 7, 14, and 28. Over the receiving period, average daily gain (ADG) and gain: feed did not differ (P ≥ 0.19) for YEA, APS, and TUL, which were greater (P ≤ 0.01) than CON. Average daily gain was also lower (P < 0.01) for CON vs. YEA, APS, and TUL from day -7 to day 28. During the first week of receiving, hay, concentrate, and total DMI were lower (P < 0.01) in CON than the YEA, APS, and TUL, but did not differ (P ≥ 0.13) among these three groups. Hay and total DMI were still lower (P < 0.01) in CON vs. TUL in the second week. Total DMI was greater (P = 0.01) for TUL vs. YEA, and greater (P < 0.01) for YEA vs. CON. Serum nonesterified fatty acid concentrations were greater (P ≤ 0.05) for CON and TUL vs. YEA and APS on day 1. Plasma cortisol concentrations were greater (P ≤ 0.05) for YEA and CON vs. APS and TUL on day 1. Serum tumor necrosis factor-α concentrations were lower (P ≤ 0.05) for APS vs. CON, YEA, and TUL on days 1 and 4. Plasma haptoglobin concentrations were greater (P ≤ 0.05) for CON vs. YEA, APS, and TUL on days 1 and 4, greater (P ≤ 0.05) for YEA, APS vs. TUL on day 1, and greater (P = 0.03) for YEA vs. TUL on day 4. Plasma ceruloplasmin concentrations were greater (P ≤ 0.05) for CON vs. YEA, APS and TUL vs. APS on days 1, 4, and 7. In conclusion, YEA, APS, and TUL modulated the physiological stress responses and alleviated the performance losses caused by long-distance transportation.

Influence of early use of antimicrobial on the health and performance of Holstein calves in the first month of life / Influência do uso precoce de antimicrobiano na sanidade de bezerras holandesas no primeiro mês de vida

The early use of antimicrobial therapy has been introduced in many farms to prevent diarrhea and respiratory disease in young calves; however, there is controversy about whether this practice has a beneficial effect on the health of these animals. This study evaluated the influence of the early use of antimicrobials on the health and performance of neonatal Holstein calves. Twenty-six Holstein calves were screened and divided into two groups, according to the administration (ATB+), or not (ATB-) of tulathromycin (2.5mg/kg, subcutaneously) within the first 12 hours of life. Calves were evaluated by general clinical examination, fecal score, respiratory score, and external palpation of the umbilical region, besides fecal output of dry matter. Anemia was determined by using an automatic system and, also, using a commercial kit for iron dosage. Diarrhea was diagnosed by a centrifuge-flotation technique using a sugar solution (Cryptosporidium) and multiplex semi-nested RT-PCR (rotavirus/coronavirus). The performance of the calves was estimated by Daily Weight Gain (DWG). The young dairy calves were evaluated within 12 hours of birth (≤12h) and at 3-5th (D3-5), 7-9th (D7-9), 13-15th (D13-15), 20-23rd (D20-23), and 27-30th (D27-30) days of life. No difference was noted between the ATB+ and ATB- groups concerning heart rate, respiratory frequency, and rectal temperature. Erythrogram showed a higher frequency of anemia in ATB- group (P=0.016) at the D3-5 check-up; lower values of serum iron were also observed simultaneously (P=0.051). Thirteen cases of respiratory disease were detected during this study; however, no significant difference was observed between the groups in this regard. The frequency of diarrhea (fecal score 2-3) was high in both groups, peaking at D13-D15. No differences were noted between the groups regarding the frequency of diarrhea when considering the dry fecal matter. The predominant etiological agent for diarrhea was Cryptosporidium spp.. The DWG was similar between groups, with maximum weight reduction on D13-15. The administration of tulathromycin in prophylactic dose (2.5mg/kg) at birth decreased the frequency of anemia but did not influence weight gain or the prevalence of diarrhea.(AU)

O uso precoce de antimicrobianos tem sido adotado em muitas fazendas para profilaxia das diarreias e doença respiratória em bezerras, no entanto existem controvérsias sobre os beneficios desta prática na saúde desses animais. Esta pesquisa avaliou a influência do uso precoce de antimicrobiano na sanidade e desempenho de bezerras holandesas recém-nascidas. Para tanto foram selecionadas 26 bezerras Holandesas distribuídas de acordo com a aplicação (ATB+) ou não (ATB-) de tulatromicina (2,5mg/Kg) por via subcutânea até 12h de vida. As bezerras foram examinadas por meio de exame clínico geral, escore fecal, escore respiratório e palpação externa da região umbilical, além da matéria seca fecal. A presença de anemias foi determinada pelo eritrograma utilizando sistema automático e além da dosagem de ferro utilizando kit comercial. O diagnóstico etiológico das diarreias foi investigado por meio da técnica de flutuação em solução saturada de sacarose (Cryptosporidium) e multiplex semi-nested RT-PCR (rotavírus/coronavírus). O desempenho das bezerras foi estimado pelo ganho de peso. As bezerras foram avaliadas até doze horas após o nascimento (≤12h); 3-5º (D3-5); 7-9º (D7-9); 13-15º (D13-15); 20-23º (D20-23); e 27-30º dias de vida (D27-30). Não foram encontradas diferenças entre os grupos ATB+ e ATB- em relação à frequência cardíaca, frequência respiratória e temperatura retal. O eritrograma revelou maior frequência de anemias no grupo ATB- (P=0,016) no D3-5. Neste momento também foram observados menores valores de ferro sérico (P=0,051). Foram detectados treze casos de doença respiratória durante o estudo, no entanto não foi possível detectar diferença entre os grupos. A frequência de diarreias (escore fecal 2 e 3) foi alta em ambos os grupos, observando-se pico no D13-15 (ATB+=92,3%; ATB-=92,3%). Não observamos diferenças entre os grupos em relação a frequência de diarreia considerando-se a matéria seca fecal. O agente etiológico predominante nas diarreias foi o Cryptosporidium. O ganho de peso diário foi igual entre grupos, com intensa redução no GPD no D13-15. A administração de tulatromicina na dose profilática (2,5mg/Kg) ao nascimento diminuiu a frequência de anemias e não influenciou no ganho de peso e prevalência de diarreias.(AU)

Evaluation of new generation macrolides for the treatment and metaphylaxis of contagious bovine pleuropneumonia (CBPP) in cattle experimentally infected with Mycoplasma mycoides subspecies mycoides.

BACKGROUND: Contagious bovine pleuropneumonia (CBPP) caused by Mycoplasma mycoides subspecies mycoides (Mmm) is an important disease of cattle that causes serious economic losses. With the known effectiveness of new generation macrolides, tulathromycin and gamithromycin were assessed in comparison with oxytetracycline as a positive control and saline as a negative control for effectiveness in inhibiting lung lesion development, promoting resolution, preventing spread and bacteriological clearance in susceptible local cattle breeds in two separate studies in Kenya and Zambia. Animals were monitored for clinical signs, sero-conversion as well as detailed post-mortem examination for CBPP lesions. RESULTS: Using the Hudson and Turner score for lesion type and size, tulathromycin protected 90%, gamithromycin 80%, and oxytetracycline 88% of treated animals in Kenya. In Zambia, all animals (100%) treated with macrolides were free of lung lesions, while oxytetracycline protected 77.5%. Using the mean adapted Hudson and Turner score, which includes clinical signs, post-mortem findings and serology, tulathromycin protected 82%, gamithromycin 56% and oxytetracycline 80% of the animals in Kenya whereas in Zambia, tulathromycin protected 98%, gamithromycin 94% and oxytetracycline 80%. The saline-treated groups had 93 and 92% lesions in Kenya and Zambia respectively, with Mmm recovered from 5/14 in Kenya and 10/13 animals in Zambia. Whereas the groups treated with macrolides were free from lesions in Zambia, in Kenya 5/15 tulathromycin-treated animals and 6/15 gamithromycin-treated animals showed lesions. Oxytetracycline-treated animals showed similarities with 3/14 and 4/15 showing lesions in Zambia and Kenya respectively and Mmm recovery from one animal in Kenya and six in Zambia. In both studies, lesion scores of saline-treated groups were significantly higher than those of the antibiotic treated groups (p < 0.001). In sentinel animals, CBPP lesions were detected and Mmm recovered from one and two animals mixed with the saline-treated groups in Kenya and Zambia respectively. CONCLUSIONS: This study demonstrated that tulathromycin, a mycoplasmacidal, can achieve metaphylactic protection of up to 80%, while non-recovery of Mmm from sentinels suggests macrolides effectiveness in preventing spread of Mmm. It is recommended that further studies are conducted to evaluate strategies comparing vaccination alone or combining vaccination and antibiotics to control or eradicate CBPP.

Genetic characterization of susceptible and multi-drug resistant Mannheimia haemolytica isolated from high-risk stocker calves prior to and after antimicrobial metaphylaxis.

Bovine Respiratory Disease (BRD) is a major threat to animal health and welfare in the cattle industry. Strains of Mannheimia haemolytica (Mh) that are resistant to multiple classes of antimicrobials are becoming a major concern in the beef industry, as the frequency of isolation of these strains has been increasing. Mobile genetic elements, such as integrative conjugative elements (ICE), are frequently implicated in this rapid increase in multi-drug resistance. The objectives of the current study were to determine the genetic relationship between the isolates collected at arrival before metaphylaxis and at revaccination after metaphylaxis, to identify which resistance genes might be present in these isolates, and to determine if they were carried on an ICE. Twenty calves culture positive for Mh at arrival and revaccination were identified, and a total of 48 isolates with unique susceptibility profiles (26 from arrival, and 22 from revaccination) were submitted for whole-genome sequencing (WGS). A phylogenetic tree was constructed, showing the arrival isolates falling into four clades, and all revaccination isolates within one clade. All revaccination isolates, and one arrival isolate, were positive for the presence of an ICE. Three different ICEs with resistance gene modules were identified. The resistance genes aphA1, strA, strB, sul2, floR, erm42, tetH/R, aadB, aadA25, blaOXA-2, msrE, mphE were all located within an ICE. The gene bla-ROB1 was also present in the isolates, but was not located within an ICE.

Tissue residue depletion and estimation of extralabel meat withdrawal intervals for tulathromycin in calves after pneumatic dart administration.

The objectives of this study were to evaluate the injection site pathology and determine tissue residue depletion of tulathromycin in calves following pneumatic dart administration and to calculate the associated extralabel withdrawal interval (WDI). Castrated male Holstein calves were injected with ~2.6 mg/kg tulathromycin via pneumatic dart administration. At 1 (n = 2), 6, 12, 18, and 24 d after drug injection (n = 3/time point), calves were euthanized, and muscle, liver, kidney, fat, and injection site samples were harvested and analyzed for tulathromycin concentrations using a LC-MS/MS method. Gross pathology and histopathology evaluations on the injection site samples were also performed. Pneumatic dart administration of tulathromycin caused severe localized lesions of hemorrhage and edema on days 1 and 6, as well as severe pathological reactions in the subcutaneous muscle on days 1, 6, and 12. Slight to moderate reactions were still observed in the majority of the skin or subcutaneous/muscle samples on day 24. Measured tulathromycin concentrations were converted to calculate the concentrations of the marker residue CP-60,300 by dividing a conversion factor of 1.4. The data were used to calculate extralabel WDIs based on the guidelines from U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA). The results showed that tulathromycin concentrations were the highest in the liver (4,877.84 ± 65.33 µg/kg), kidney (5,819.52 ± 1,087.00 µg/kg), muscle (1,717.04 ± 140.35 µg/kg), injection site (51,884.05 ± 7,529.34 µg/kg), and fat (161.69 ± 36.48 µg/kg) at 6, 1, 1, 1, and 1 d, respectively, after treatment. Tulathromycin concentrations remained above the limit of quantification of 5 µg/kg in all tissues at 24 d. The calculated WDIs based on kidney data were 26 d using EMA method, 36 d using FDA method based on CP-60,300 data, and 45 d using FDA method based on tulathromycin data. These results suggest that pneumatic dart administration of tulathromycin causes injection site reactions in calves and an extended WDI is needed. One limitation of this study was the small sample size of 3 that did not meet FDA guideline requirement. Therefore, the calculated WDIs should be considered as preliminary and additional studies that use a larger number of animals and directly measure the concentrations of the marker residue CP-60,300 are needed to make a more conclusive recommendation on the extralabel WDI.