C. Locatelli

Cefquinome sulfate behavior after intramammary administration in healthy and infected cows

Maintenance of adequate drug concentration at the site of infection is an important problem in mastitis antibiotic therapy, and the efficacy of intramammary β-lactams can be optimized by maintaining the drug concentration at the site of infection above the minimum inhibitory concentration (MIC) as long as possible. The most important pharmacokinetic and pharmacodynamic parameter for efficacy evaluation is time during which drug concentrations exceed the MIC (t>MIC). In this study, we assessed the pharmacokinetic profile of cefquinome (CFQ) after repeated intramammary administration in healthy cows and cows subclinically infected with Staphylococcus aureus as well as the MIC of Staph. aureus field strains. In addition, the degree of drug passage was investigated from udder to bloodstream by measuring systemic drug absorption in healthy and infected animals. Cefquinome concentrations were quantified by HPLC (UV-visible detection) in milk samples collected from quarters and from blood serum samples. The systemic drug absorption was negligible in healthy and subclinically infected animals (maximum concentration 0.09±0.02 and 0.1±0.01 μg/mL in healthy and subclinically infected animals, respectively). The MIC(90) value for CFQ in Staph. aureus field strains (n=20) was 0.24 μg/mL. The pharmacokinetic and pharmacodynamic evaluation, determined by t>MIC, showed an equal persistence of CFQ in all quarters, indicating an equivalent activity of the drug regardless of the pathological status of the udder. Moreover, with literature data regarding CFQ MIC, the t>MIC has been calculated for other bacterial species.

Genetic analysis of small ruminant lentiviruses following lactogenic transmission

Lactogenic transmission plays an important role in the biology of lentiviruses such as HIV and SIV or the small ruminant lentiviruses (SRLV). In this work we analyzed the characteristics of viruses that goats, naturally infected with two strains of SRLV, transmitted to their kids. The spectrum of viral genotypes transmitted was broader and the efficiency of transmission greater compared to their human and simian counterparts. The newly described A10 subgroup of SRLV was more efficiently transmitted than the B1 genotype. The analysis of a particular stretch of the envelope glycoprotein encompassing a potential neutralizing epitope revealed that, as in SIV, the transmitted viruses were positively charged in this region, but, in contrast to SIV, they tended to lack a glycosylation site that might protect against antibody neutralization. We conclude that the physiology of the ruminant neonatal intestine, which permits the adsorption of infected maternal cells, shaped the evolution of these particular lentiviruses that represent a valid model of lactogenic lentivirus transmission.

Efficacy of vaccination on Staphylococcus aureus and coagulase-negative staphylococci intramammary infection dynamics in 2 dairy herds.

The aim of this study was to evaluate vaccine efficacy of a commercial vaccine (Startvac, Hipra Spain) aimed at reducing intramammary infections (IMI) with Staphylococcus aureus and coagulase-negative staphylococci under field conditions. During the 21-mo duration of the study, 1,156 lactations from 809 cows were enrolled in 2 herds. During the first phase of the trial, all cows that were due to calve were vaccinated until approximately 50% of cows in the milking herd were vaccinated (at ~6mo). At that point, when 50% vaccination coverage was reached, cows that were due to calve were randomly assigned to be vaccinated or left as negative controls. Cure rate, rate of new infection, prevalence, and duration of infections were analyzed. Vaccination resulted in a moderate reduction in incidence of new staphylococcal IMI and a more pronounced reduction in duration of IMI associated with reduction of the basic reproduction ratio of Staph. aureus by approximately 45% and of coagulase-negative staphylococci by approximately 35%. The utilization of vaccine in combination with other infection-control procedures, such as excellent milking procedures, treatment, segregation, and culling of known infected cattle, will result in an important reduction in incidence and duration of intramammary staphylococcal infections.

Cefoperazone sodium preparation behavior after intramammary administration in healthy and infected cows

Selection of the antimicrobial agent and maintenance of adequate drug concentrations at the site of infection are the most relevant problems in mastitis antibiotic therapy. Intramammary drug efficacy can be maximized by keeping drug concentrations at the site of infection above the minimum inhibitory concentration (MIC) as long as possible; the most important pharmacokinetic and pharmacodynamic (PK/PD) measure for efficacy evaluation is time during which drug concentrations exceed the MIC (t>MIC). To evaluate this measure, the PK profile of cefoperazone (CFP) after single intramammary administration in healthy and subclinical infected Staphylococcus aureus cows and the MIC of Staph. aureus field strains were assessed. In addition, the degree of drug passage from udder to bloodstream was investigated by measuring systemic drug absorption in healthy and infected animals. Cefoperazone concentrations were quantified by HPLC in quarter milk samples and blood serum samples. Systemic drug absorption was negligible in healthy animals (0.020+/-0.006 microg/mL serum at 4 h), whereas it was higher in infected animals (0.102+/-0.079 microg/mL at 4h and 0.025 microg/mL at 24 h), probably due to the damage of epithelial cell junctions caused by subclinical infections. The MIC90 value for CFP in Staph. aureus field strains (n=24) was 0.64 microg/mL. The PK/PD evaluation, determined by t>MIC, showed a longer persistence of CFP in infected quarters than in healthy ones (mean residence time was 8.37+/-1.51 vs. 11.42+/-5.74 h in September and 2.07+/-0.43 vs. 3.31+/-0.91 h in October), with a t>MIC of 45+/-6 h for infected quarters versus 38+/-5 h for healthy quarters different only in October. This could mean a prolonged time in which microorganisms are exposed to drug activity and thus, a greater efficacy of the drug.

CTX-M1 ESBL-Producing Klebsiella pneumoniae subsp. pneumoniae Isolated from Cases of Bovine Mastitis

Escherichia coli and Klebsiella spp., followed by Serratia spp. and Enterobacter spp., are the most frequent Gram-negative pathogens isolated from bovine clinical mastitis (8). The incidence of mastitis due to such bacteria has increased in recent years (7). Klebsiella is usually referred as particularly aggressive and prone to cause severe clinical mastitis, which responds poorly to treatment and is likely to have a fatal outcome (6, 13). Klebsiellae are defined as germs that easily produce enzymes like extended-spectrum β-lactamases (ESBL), thanks to their ability to survive longer than other Gram-negative rods in the environment and on the skin and, in particular, to allow ESBL genes to evolve (9). At the onset of clinical mastitis, it is impossible to identify the causative agent; thus, it is necessary to use broad-spectrum intramammary preparations which contain narrow- to extended-spectrum cephalosporins, alone or in combination with other antibiotics (1). The broad-spectrum cephalosporin ceftiofur is used as well in supportive systemic therapy (6). Few recent works have studied Klebsiella isolation and ESBL detection in veterinary medicine (2, 16), and no data are available describing ESBL-mediated resistance in Klebsiella spp. isolated from bovine mastitis. In this research, the antimicrobial susceptibility and ESBL production were evaluated in Klebsiella pneumoniae isolated from animals with bovine clinical mastitis and selected for their resistance to ceftiofur, a broad-spectrum cephalosporin. Susceptibility to a range of antimicrobials, including those approved for both human and animal use (Table ​(Table1),1), was determined by disk diffusion assay according to Clinical and Laboratory Standards Institute interpretative criteria (3). Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 were used as quality control standards. TABLE 1. Comparison of resistance profiles of the nine isolates From March 2008 to March 2009, 140 klebsiellae were isolated from milk samples, representing 26.6% of the total number of Gram-negative isolates obtained from bovine clinical mastitis. The isolates were subcultured on MacConkey agar supplemented with 8 μg/ml of ceftiofur (5). Nine isolates, identified as Klebsiella pneumoniae, were able to grow in the presence of 8 μg of ceftiofur. All Klebsiella pneumoniae isolates were screened against the panel in Table ​Table11 by the disk diffusion method and for ESBLs by the double-disk diffusion assay. A strain was considered an ESBL producer when it showed the expansion of an inhibition zone between a disk containing amoxicillin-clavulanate (20 and 10 μg) and disks containing, respectively, ceftazidime (30 μg) and cefotaxime (30 μg) placed 25 mm apart (10). All Klebsiella pneumoniae isolates were tested by PCR targeting the Ambler class A β-lactamase genes blaSHV, blaTEM, and blaCTX, identified in Klebsiella pneumoniae animal isolates (16), using previously published primer sets (14, 15). Sequencing of amplified genes completed the study. The complete resistance profiles and the PCR results are summarized in Table ​Table1.1. Only isolate 205, identified as Klebsiella pneumoniae subsp. pneumoniae, had a positive result to the double-disk test and was resistant to all the β-lactams, including cefotaxime and ceftriaxone, and sensitive to amoxicillin-clavulanate. The amplification of a CTX-M class enzyme-encoding gene confirmed such a phenotype, and sequencing of the gene identified the blaCTX-M1 group. To our knowledge, it is the first report about a CTX-M1 enzyme produced by a Klebsiella pneumoniae subsp. pneumoniae isolate in a case of bovine clinical mastitis. Isolate 205 also displayed a positive PCR result for genes blaSHV and blaTEM, and so did isolates 204 and 251. A previous randomized search detected only ESBL-producing E. coli and no Klebsiella spp. (11). The generally low level of in vitro resistance and the low prevalence of ESBL-producing strains (0.7%) should guarantee a good outcome of drug treatment for Klebsiella involved in mastitis, but antimicrobial resistance is not the only factor affecting therapy efficacy (1, 4). However, the described isolation must encourage that attention be paid to a phenomenon which could evolve and be enforced under the pressure of antimicrobial therapy, as the use of extended-spectrum cephalosporins in veterinary medicine may select ESBL producers (12). It is recommended to screen klebsiellae from cases of bovine mastitis in order to keep the prevalence of ESBL producers under control.

Effects of intramammary infections on somatic cell score and milk yield in Sarda sheep

Abstract AIM: To evaluate the effects of intramammary infections (IMI) on somatic cell score (SCS) and milk yield in dairy ewes. METHODS: Monthly milk samples were collected from a flock of 202 Sarda sheep, over a period of 7 months, for bacteriological culture and measurement of somatic cell counts (SCC). During the same period, milk yield was measured daily using electronic milk meters connected to each half-udder cluster of the milking machine. SCC was transformed to SCS using a base-2 log transformation. One SCS is equivalent to a SCC of 25,000 cells/ml, and each increase of 1 in SCS is associated with doubling of the SCC. IMI was defined by the presence of five or more colonies of similar morphology isolated from a milk sample (≥500 cfu/ml). The effect of IMI on SCS and milk yield was assessed using a generalised estimating equation (GEE). RESULTS: There were 1,186 udder halves with IMI from 2,828 milk samples, a prevalence of 41.9%. The distribution of bacterial species within the 1,186 culture-positive samples was comprised of 476 (40.1%) Staphylococcus epidermidis, 172 (14.5%) Staph. chromogenes, 38 (3.2%) Staph. caprae, 134 (11.3%) Staph. simulans, 114 (9.6%) Streptococcus uberis, 123 (10.4%) Strep. dysgalactiae, and 129 (10.9%) Strep. equi subsp. zooepidemicus. SCS was greater in udder halves with IMI (mean 7.71; SD 0.82) than in udder halves without IMI (mean 5.53; SD 1.02) (p<0.01). IMI due to streptococcal species were associated with greater SCS (mean 8.24; SD 0.62) than those due to staphylococcal species (mean 7.48; SD 0.79) (p<0.01). Milk yield from udder halves with IMI was lower (mean 439 (SD 162) ml/half udder/day) than from udder halves without IMI (mean 602 (SD 170) ml/half udder/day) (p<0.01). IMI due to staphylococcal species was associated with a lower milk yield (mean 399 (SD 167) ml/half udder/day) than IMI due to streptococcal species (mean 427 (SD 156) ml/half udder/day) (p<0.01). CONCLUSIONS: These findings provide sheep milk producers with information on the losses associated with subclinical mastitis, which can be used to evaluate the economics of prevention and treatment protocols concerning udder health in ovine dairy flocks.

Milk hygiene and udder health in the periurban area of Hamdallaye, Niger

The prevalence of intra-mammary infections in dairy herds was studied in Hamdallaye, Niger. A total of 956 milk samples were collected in 2007 from 239 lactating cows of four local breeds in eight traditional herds; the first sampling was undertaken in the dry season at morning milking, and the second in the rainy season at evening milking. Staphylococcus aureus, Coagulase-Negative Staphylococci (CNS) and environmental microorganisms were detected in significantly (p < 0.05) more samples in the rainy season, 55.2%, than in the dry season, 27.1%. Statistically significant (P < 0.05) differences in prevalence were observed among herds and according to lactation number. Infections were assigned to four classes, according to the major pathogen, and the respective mean somatic cell counts during the dry season were: S. aureus, 775 × 103 cells/ml; CNS, 447 × 103 cells/ml; environmental microorganisms, 407 × 103 cells/ml; and non-infected, 262 × 103 cells/ml. Most of the tested strains were sensitive to antibiotics, and selected strains of S. aureus (n = 15) were negative to the multiplex PCR tests for production of enterotoxins.

Short communication: Epidemiology and genotyping of Candida rugosa strains responsible for persistent intramammary infections in dairy cows

The present study was undertaken during an outbreak of clinical and subclinical mastitis in 14 dairy cows caused by Candida rugosa, in which high somatic cell counts were seen and cases did not respond to antibiotic treatment. Intramammary infection cured spontaneously in 10 cows, whereas 4 cows were culled as a result of persistent infections. Repeated sampling of these cows and biomolecular analysis of the isolates showed that the infections were caused by the same genotype, even over a period of 2 lactations. Random amplification of the genome of C. rugosa milk isolates gave 3 different DNA banding patterns (genotypes G1, G2, and G3). Viable cells of C. rugosa were also isolated from various environmental sources and were present in high concentrations in total mixed ration samples, which could be considered the primary source of diffusion of viable yeast cells in the environment, as demonstrated by genotyping. The proven capacity of these microorganisms to survive in the environment of the cow, such as the total mixed ration, bedding, water, and cow skin, and to cause persistent intramammary infections highlights the importance of mycotic spread in dairy herds.

Clonal diversity, virulence-associated genes and antimicrobial resistance profile of Staphylococcus aureus isolates from nasal cavities and soft tissue infections in wild ruminants in Italian Alps

Staphylococcus aureus is a commensal and a pathogenic bacterium that causes a wide variety of diseases in humans and animals with a high impact on public health and the livestock industry. S. aureus virulence pattern, antimicrobial resistance profile and host specialization are of great concern both in livestock and in companion animals. Concerning wild animals, S. aureus carriage and antimicrobial resistance profile has been recently investigated in free-ranging species both in aquatic and terrestrial environment. Here we report genotyping (spa typing, Multilocus Sequence Typing and SCCmec typing), virulence and antimicrobial resistance profile of four S. aureus isolated in Alpine chamois (Rupicapra r. rupicapra) and roe deer (Capreolus capreolus), euthanized due to walking impairment and signs of disorientation. S. aureus was isolated from nasal cavities in both wild ruminant species and in soft tissue infections in chamois. A marked S. aureus genetic heterogeneity was detected: spa type t1523, sequence type 45 (Clonal Complex 45), and spa type t1328, ST22 (CC22) from the nasal cavities and the liver of a chamois kid respectively, t1773, ST700 (CC130) from an adult chamois abscess, and a new sequence type, ST2712, belonging to CC97 from the roe deer nasal cavities. One of the main findings was the confirmation that the t1328, ST22 isolate, obtained from the liver of the chamois kid, was a methicillin-resistant S. aureus (MRSA) harbouring a SCCmec cassette type IV. The set of virulence marker and toxin genes investigated showed profiles characteristic of the S. aureus lineages detected, including those of the human adapted ST (CC) 22 and ST (CC) 45 isolates.

Pseudomonas aeruginosa in Dairy Goats: Genotypic and Phenotypic Comparison of Intramammary and Environmental Isolates

Following the identification of a case of severe clinical mastitis in a Saanen dairy goat (goat A), an average of 26 lactating goats in the herd was monitored over a period of 11 months. Milk microbiological analysis revealed the presence of Pseudomonas aeruginosa in 7 of the goats. Among these 7 does, only goat A showed clinical signs of mastitis. The 7 P. aeruginosa isolates from the goat milk and 26 P. aeruginosa isolates from environmental samples were clustered by RAPD-PCR and PFGE analyses in 3 genotypes (G1, G2, G3) and 4 clusters (A, B, C, D), respectively. PFGE clusters A and B correlated with the G1 genotype and included the 7 milk isolates. Although it was not possible to identify the infection source, these results strongly suggest a spreading of the infection from goat A. Clusters C and D overlapped with genotypes G2 and G3, respectively, and included only environmental isolates. The outcome of the antimicrobial susceptibility test performed on the isolates revealed 2 main patterns of multiple resistance to beta-lactam antibiotics and macrolides. Virulence related phenotypes were analyzed, such as swarming and swimming motility, production of biofilm and production of secreted virulence factors. The isolates had distinct phenotypic profiles, corresponding to genotypes G1, G2 and G3. Overall, correlation analysis showed a strong correlation between sampling source, RAPD genotype, PFGE clusters, and phenotypic clusters. The comparison of the levels of virulence related phenotypes did not indicate a higher pathogenic potential in the milk isolates as compared to the environmental isolates.