Evaluation of an on-farm culture system for the detection of subclinical mastitis pathogens in dairy cattle.

The purpose of this observational study was to compare the performance of a novel on-farm culture (OFC) test with the reference method (RM) in identifying pathogens, and in particular Staphylococcus aureus, associated with subclinical mastitis (SCM) in dairy cattle. The OFC test (Mastatest HiSCC; Mastaplex Limited) for SCM uses a cartridge with 2 × 12 wells allowing 1 sample to be analyzed in duplicate (24 wells) or 2 samples analyzed simultaneously, each in 12 wells. Results of the milk analyses are reported hierarchically (Staph. aureus → coagulase-negative staphylococci (CNS) → other gram positive or coliform/gram negative → no bacteria present) and emailed within 24 h. Milk samples (617 quarter level from 158 cows and 70 cow level) were collected from 288 cows [individual cow somatic cell count (ICSCC) ≥150,000 cells/mL] on 9 purposefully selected farms known to have a high prevalence of clinical and subclinical Staph. aureus mastitis in Southland New Zealand. Quarter samples were analyzed individually (617 samples) and after animal-level pooling, providing 228 (158 + 70) cow-level samples. Samples were analyzed by the OFC test (in duplicate) and the RM (culture agar medium and latex test based on the recommendation by the National Mastitis Council) and identifications confirmed with MALDI-TOF mass spectrometry. Sensitivity (Se), specificity (Sp), positive predictive value (PPV), and negative predictive value (NPV) for detection of Staph. aureus were all ∼90% with tight 95% confidence limits, and Cohen's kappa (κ) for agreement between the OFC test and RM was 0.81. Kappa for agreement between the OFC test duplicates was 0.93. About 35% of cows had only one quarter infected with Staph. aureus and all these animals could still be identified when pooled cow-level milk was analyzed. Although the high prevalence of Staph. aureus in the herds used in this study does not affect the Se and Sp values, it does elevate the PPV value (and decrease the NPV) and therefore use of PPV to extrapolate to a population with lower prevalence is not appropriate. For CNS, Sp, PPV, and NPV were all >0.8, κ was ≥0.6, and Se was >0.7. Kappa for agreement between the OFC test duplicates was 0.83. A result of "no bacteria detected" was reported in 13% of the cows with 93% agreement between OFC test and RM. We conclude that the OFC test provides a reliable method for detecting Staph. aureus in pooled cow-level milk even if only one quarter is infected; in the absence of Staph. aureus in the milk, it reliably identified CNS in pooled cow-level milk; it reliably identified cows with <10 cfu/10 µL of their milk. Compared with the RM, the method was rapid with results returned in 24 h of loading the cartridge.

Selective and deferred treatment of clinical mastitis in seven New Zealand dairy herds.

Mastitis is the most frequent reason for antibiotic use in New Zealand dairy cattle and technologies reducing and targeting this use contribute to responsible product stewardship. Rapid identification of pathogen and antibiotic susceptibility facilitate targeted treatment but currently involve a minimum 24 h delay. Studies from confinement systems where Gram-negative organisms are responsible for a significant proportion of mastitis, indicate selective treatment can reduce antibiotic use without reducing clinical or bacteriological cure. However, in New Zealand's seasonal, pastoral dairy system, mastitis is dominated by Gram-positive organisms and if treatment is deferred, it is vital both short- and long-term clinical health outcomes are not compromised. Mastatest® is a diagnostic system for bovine mastitis indicating the pathogen and its antibiotic sensitivity within 24 h of sampling. This study focused on evaluating this system's ability to control antibiotic usage whilst achieving equivalent bacteriological and clinical cure rates alongside long term individual somatic cell count (ISCC) outcomes as conventional treatment choices. Mild to moderate mastitis cases in the 100 days after calving in 6467 cows from 7 farms were milk sampled and randomly allocated to a positive control group non-selective treatment or a culture-based treatment. All milk samples were processed using Mastatest®. For the positive control, the quarter was treated immediately with 3 treatments of procaine penicillin every 12 h. For the selective treatment group, treatment was delayed for 24 h and then informed by pathogen and antibiotic sensitivity from the Mastatest® result. Gram-negative and no-growth quarters were untreated. Gram-positive quarters were treated with the antibiotic for which the lowest in vitro antimicrobial sensitivity was reported. Re-sampling was carried out from affected quarter(s) approximately 21 days after initial diagnosis and cultured for bacterial identification. Clinical recurrence within 60 days and ISCC data was recorded at herd tests over the duration of the lactation. Antimicrobial usage and days of milk withhold pending clearance of antibiotic residues were also noted. There was no difference in bacteriological or clinical cure rate between the two treatment groups. Final herd test ISCC and days of milk withhold from supply did not differ between groups. Antibiotic usage was 24 % less (95 % predictive interval = 12-47 %) in the selective group. This study suggests that on farm decisions about deferred treatment of mastitis using Mastatest® to identify the intramammary pathogen can reduce the antimicrobial usage with no loss in bacterial or clinical cure and with no effect on ISCC over the lactation.

Comparison of an on-farm point-of-care diagnostic with conventional culture in analysing bovine mastitis samples.

The performance of a new point-of-care diagnostic (Mastatest), an on-farm test designed to identify bacteria and provide antibiotic sensitivity testing information from milk samples, was compared with standard microbiological culture methods. A total of 292 milk samples from clinical mastitis cases in dairy cows on New Zealand dairy farms were examined, and latent class analysis was used to estimate the performance characteristics of both tests. Two hundred and fifty-six samples (87.7%) demonstrated bacterial infection in standard culture, and 269 (92.1%) using the point-of-care diagnostic. The most common bacterial species detected was Streptococcus uberis, found in 195 samples (66.8%) using standard culture and 190 samples (65.1%) using the point-of-care diagnostic. Latent class analysis found no significant differences in test characteristics between the point-of-care diagnostic and standard culture. The estimated sensitivity and specificity of the point-of-care diagnostic against all targets combined were 94.6 and 72.1% respectively; the corresponding estimates for standard culture were 90.5 and 73.9%. Comparison of antibiotic susceptibility testing using the point-of-care diagnostic and the reference method showed similar trends and, in some cases, identical MIC50 and MIC90 values, with at most one antibiotic dilution difference.

An observational study of the effect of vibration on the caking of suspensions in oily vehicles.

An oily suspension of penethamate (PNT) that was physically stable on storage, caked solidly during road/air transport. This paper reports on the caking behaviour of PNT oily suspension formulations exposed to vibrations in a lab-based test designed to simulate road/air transport. The lab-test was used to study the effects of container type (glass v PET) and formulation (oil, surfactant type and concentration) on the physical stability of suspension under vibration. Redispersibility of the sediment was lower at longer vibrations times and at higher intensity of vibration. Caking on vibration was strongly influenced by the type of container (caking in glass but not in PET) possibly due to tribo-charging of particles. Caking on vibration was dependent on the formulation: type and concentration of surfactant; type of oil. The physical stability of oily suspensions, and the effect of vibration are two areas which have been largely neglected in the pharmaceutical literature. This paper discusses some potential mechanisms for the observations but studies using fully characterised materials are required. Finally we conclude that static testing of physical stability of oily suspensions is not sufficient and that a vibrational stress test is required.

Stability of penethamate, a benzylpenicillin ester prodrug, in oily vehicles.

Penethamate (PNT) is an ester prodrug of benzylpenicillin which is marketed as dry powder for reconstitution with aqueous vehicle prior to injection. The purpose of this paper was to investigate the chemical stability of PNT in oily formulations to provide a basis for a ready-to-use (RTU) oil-based PNT formulation. The chemical stability of PNT solutions and suspensions in light liquid paraffin (LP), medium chain triglyceride (MIG), ethyl oleate (EO) and sunflower oil (SO) was investigated at 30 °C. Solid state stability of PNT powder and stability of PNT in EO suspensions with different moisture contents were also evaluated. The solubility of PNT in the oils was in order SO > EO > MIG > LP. Degradation of PNT was rapid in oily solutions and less than 10% remained after 7-15 days. Stability of PNT decreased with increase in moisture content in ethyl oleate suspensions. PNT was stable over four weeks in the solid state. Hydrolysis, due to moisture in the oil formulation is not the only degradation mechanism. PNT stability (% drug remaining) in oily suspensions after 3.5 months was in the order LP (96.2%) > MIG (95.4%) > EO (94.1%) > SO (86%). A shelf-life of up to 5.5 years at 30 °C may be achieved for PNT suspension in these oils.

Pre-formulation and chemical stability studies of penethamate, a benzylpenicillin ester prodrug, in aqueous vehicles.

Penethamate (PNT) is a diethylaminoethyl ester prodrug of benzylpenicillin used to treat bovine mastitis via the intramuscular route. Because of its instability, PNT products must be reconstituted before administration and the reconstituted injection has a short shelf life (7 days at 2-8°C). The purpose of this paper was to investigate whether the stability of PNT can be improved in order to achieve a chemically stable ready-to-use aqueous-based PNT formulation or at least to extend the shelf life of the reconstituted suspension. A chemical stability study of PNT in aqueous-based solutions as a function of pH, buffer strength, solvent mixtures and temperature, supported by studies of its solubility in mixed solvents, allowed predictions of the shelf life of PNT solution and suspension formulations. PNT degraded in aqueous solutions by several pathways over the pH range 2.0-9.3 with a V-shaped pH-rate profile and a minimum pH of around 4.5. The stability of PNT solutions in mixed solvents was greater than in aqueous solutions. For example, in propylene glycol:citrate buffer (60:40, v/v, pH 4.5), the half-life of PNT was 4.3 days compared with 1.8 days in aqueous buffer. However, solubility of PNT in the mixed solvent was higher than that in aqueous solution and this had an adverse effect on the stability of suspensions. By judicious choosing of pH and mixed solvent, it is possible to achieve a storage life of a PNT suspension of 5.5 months at 5°C, not sufficient for a ready-to-use product but a dramatic improvement in the storage life of the reconstituted product.