Antimicrobial susceptibility of mastitis pathogens isolated from North American dairy cattle, 2011-2022.

A total of 10,890 bacterial isolates of Streptococcus dysgalactiae, Streptococcus uberis, Staphylococcus aureus and Escherichia coli isolated as etiological agents from dairy cows with mastitis by 29 veterinary laboratories across North America between 2011 and 2022 were tested for in vitro antimicrobial susceptibility by broth microdilution to ampicillin, cefoperazone, ceftiofur, cephalothin, erythromycin, oxacillin, penicillin-novobiocin and pirlimycin according to CLSI standards. Using available clinical breakpoints, antimicrobial resistance among S. dysgalactiae (n = 2406) was low for penicillin-novobiocin (0% resistance), ceftiofur (0.1%), erythromycin (3.2%) and pirlimycin (4.6%). Among S. uberis (n = 2398), resistance was low for ampicillin (0%) and ceftiofur (0.2%) and moderate for erythromycin (11.9%) and pirlimycin (18.4%). For S. aureus (n = 3194), resistance was low for penicillin-novobiocin (0%), ceftiofur (0.1%), oxacillin (0.2%), erythromycin (0.7%), cefoperazone (1.2%) and pirlimycin (2.8%). For E. coli (n = 2892), resistance was low for ceftiofur (2.8%) and cefoperazone (3.4%) and moderate for ampicillin (9.2%). Overall, the results indicate that mastitis pathogens in the United States and Canada have not shown any substantial changes in the in vitro susceptibility to antimicrobial drugs over the 12 years of the study, or among that of the proceeding survey from 2002-2010. The data support the conclusion that resistance to common antimicrobial drugs among mastitis pathogens, even to drugs that have been used in dairies for mastitis management for many years, continues to remain low.

Synergistic effects of Clostridium perfringens enterotoxin and beta toxin in rabbit small intestinal loops.

The ability of Clostridium perfringens type C to cause human enteritis necroticans (EN) is attributed to beta toxin (CPB). However, many EN strains also express C. perfringens enterotoxin (CPE), suggesting that CPE could be another contributor to EN. Supporting this possibility, lysate supernatants from modified Duncan-Strong sporulation (MDS) medium cultures of three CPE-positive type C EN strains caused enteropathogenic effects in rabbit small intestinal loops, which is significant since CPE is produced only during sporulation and since C. perfringens can sporulate in the intestines. Consequently, CPE and CPB contributions to the enteropathogenic effects of MDS lysate supernatants of CPE-positive type C EN strain CN3758 were evaluated using isogenic cpb and cpe null mutants. While supernatants of wild-type CN3758 MDS lysates induced significant hemorrhagic lesions and luminal fluid accumulation, MDS lysate supernatants of the cpb and cpe mutants caused neither significant damage nor fluid accumulation. This attenuation was attributable to inactivating these toxin genes since complementing the cpe mutant or reversing the cpb mutation restored the enteropathogenic effects of MDS lysate supernatants. Confirming that both CPB and CPE are needed for the enteropathogenic effects of CN3758 MDS lysate supernatants, purified CPB and CPE at the same concentrations found in CN3758 MDS lysates also acted together synergistically in rabbit small intestinal loops; however, only higher doses of either purified toxin independently caused enteropathogenic effects. These findings provide the first evidence for potential synergistic toxin interactions during C. perfringens intestinal infections and support a possible role for CPE, as well as CPB, in some EN cases.

Transcriptomic and genomic evidence for Streptococcus agalactiae adaptation to the bovine environment.

BACKGROUND: Streptococcus agalactiae is a major cause of bovine mastitis, which is the dominant health disorder affecting milk production within the dairy industry and is responsible for substantial financial losses to the industry worldwide. However, there is considerable evidence for host adaptation (ecotypes) within S. agalactiae, with both bovine and human sourced isolates showing a high degree of distinctiveness, suggesting differing ability to cause mastitis. Here, we (i) generate RNAseq data from three S. agalactiae isolates (two putative bovine adapted and one human) and (ii) compare publicly available whole genome shotgun sequence data from an additional 202 isolates, obtained from six host species, to elucidate possible genetic factors/adaptations likely important for S. agalactiae growth and survival in the bovine mammary gland. RESULTS: Tests for differential expression showed distinct expression profiles for the three isolates when grown in bovine milk. A key finding for the two putatively bovine adapted isolates was the up regulation of a lactose metabolism operon (Lac.2) that was strongly correlated with the bovine environment (all 36 bovine sourced isolates on GenBank possessed the operon, in contrast to only 8/151 human sourced isolates). Multi locus sequence typing of all genome sequences and phylogenetic analysis using conserved operon genes from 44 S. agalactiae isolates and 16 additional Streptococcus species provided strong evidence for acquisition of the operon via multiple lateral gene transfer events, with all Streptococcus species known to be major causes of mastitis, identified as possible donors. Furthermore, lactose fermentation tests were only positive for isolates possessing Lac.2. Combined, these findings suggest that lactose metabolism is likely an important adaptation to the bovine environment. Additional up regulation in the bovine adapted isolates included genes involved in copper homeostasis, metabolism of purine, pyrimidine, glycerol and glucose, and possibly aminoglycoside antibiotic resistance. CONCLUSION: We detected several genetic factors likely important in S. agalactiae's adaptation to the bovine environment, in particular lactose metabolism. Of concern is the up regulation of a putative antibiotic resistance gene (GCN5-related N-acetyltransferase) that might reflect an adaptation to the use of aminoglycoside antibiotics within this environment.

Evaluation of an alternative dosing regimen of a J-5 mastitis vaccine against intramammary Escherichia coli challenge in nonlactating late-gestation dairy cows.

The objective of the study was to evaluate the efficacy of an alternative vaccination regimen of a J-5 bacterin against intramammary Escherichia coli challenge in nonlactating late-gestation dairy cows. The parameters analyzed to assess the effect of vaccination were milk yield, milk conductivity, somatic cell count, J-5-specific serum IgG titers, and clinical signs. Twenty multiparous Holstein cows from the Cornell teaching and research dairy herd were paired by days in milk and were randomly selected to receive either the alternative off-label regimen of commercial J-5 bacterin or act as nonvaccinated controls. Cows received a first dose of bacterin 15 d before dry off, a second dose with the same product at the day of dry off, and the third dose 2 wk after dry off. The cows in both groups were challenged 10 d before the expected calving date. Serum IgG (total, IgG1 and IgG2) levels were higher in vaccinates compared with control cows. Eighty-five percent of challenged quarters became infected between both groups of animals. Eight of the 10 vaccinated and 9 of the 10 control cows showed signs of clinical mastitis postfreshening. A non-severe clinical mastitis was observed 24 to 48 h postparturition, characterized by flakes or clots in milk and mild swelling or pain. Off-label vaccination did reduce the clinical severity of clinical mastitis in the vaccinated group of cows as evidenced by reduced California mastitis test score, fewer flakes and lower overall clinical mastitis score. No significant differences between vaccinated and control groups were detected for rectal temperature. In conclusion, the alternative off-label vaccination scheme used in our study and evaluated in a novel E. coli challenge model did not prevent new intramammary infections but reduced clinical severity of experimentally induced E. coli mastitis.

Prednisolone and cefapirin act synergistically in resolving experimental Escherichia coli mastitis.

Mastitis in dairy cows is typically treated with intramammary antibiotics. The combination of antibiotics with corticosteroids tends to have a large market share where these products are registered. Our objective was to investigate the effect of prednisolone in combination with cefapirin on the inflammatory response of experimentally induced Escherichia coli mastitis. Six midlactating Holstein-Friesian cows were challenged in 3 quarters with E. coli and treated at 4, 12, 24, and 36 h postinfection with 300 mg of cefapirin in 1 quarter and a combination of 300 mg of cefapirin and 20mg of prednisolone in another quarter. At 24h (n=3) or 48 h (n=3) postinfection cows were euthanized for tissue sampling. Clinical scores, somatic cell count, and California mastitis test scores, as well as IL-1ß, IFN-γ, IL-4, and IL-10 levels and bacterial growth in milk, were measured every 6h. Experimental inoculation caused a moderate clinical mastitis in all cows in challenged, untreated quarters. The E. coli challenge strain was recovered from all infected quarters and confirmed by PCR-based fingerprinting. Challenged, untreated control quarters showed increased concentrations of all measured cytokines together with recruitment of polymorphonuclear neutrophilic leukocytes at 24 and 48 h postchallenge. Both treatments reduced udder swelling and sensitivity with no statistically significant difference between treatment groups. Administration of cefapirin alone or in combination with prednisolone resulted in significantly lower concentrations of IFN-γ, IL-1ß, and IL-10 compared with challenged, untreated quarters. Treated quarters did show IL-4 production, but concentrations were significantly decreased compared with untreated, challenged quarters. Quarters treated with the combination of cefapirin and prednisolone showed a significantly lower concentration of IL-4 compared with cefapirin-only treatment. At both 24 and 48 h postinoculation, the level of polymorphonuclear neutrophilic leukocyte recruitment was lowest in challenged quarters treated with a combination of cefapirin and prednisolone, followed by cefapirin alone. Taken together, treatment with cefapirin alone inhibited bacterial growth in milk and reduced the host inflammatory responses. Addition of prednisolone to cefapirin had a synergistic effect, resulting in a lower density of leukocytes in tissue and milk and a quicker restoration of milk quality.

Molecular diagnostics applied to mastitis problems on dairy farms.

Mastitis in dairy cows is among the most important diseases of dairy cattle worldwide. Mastitis is most often the response of the host to an intramammary infection. Accurate and cost-effective methods of identifying mastitis pathogens are important for the diagnosis, surveillance, and control of this economically important disease. Rapid identification methods have the potential to be extremely specific and can also discriminate among closely related organisms. A wide range of phenotyping and genotyping methods have been developed or implemented to study mastitis-causing bacteria of dairy cattle at the species and subspecies level. This article provides the basis for evaluating molecular diagnostic technologies as a routine tool in diagnosing mastitis pathogens.

The "other" gram-negative bacteria in mastitis: Klebsiella, serratia, and more.

Mastitis caused by gram-negative infections is of increasing importance on modern and well-managed dairy farms. Without a doubt, E coli tends to be the most important cause of these gram-negative infections when the data are tallied across farms.1 However, more precise investigation of individual farms often reveals a farm-specific infection pattern where a single gram-negative bacterial species predominates. Several farms with a predominance of "other" gram-negative IMIs may be observed. We have shown the presence of outbreaks on individual dairy farms with K pneumoniae, S marcescens, and Enterobacter cloacae. On farms with a predominance of these "other" gram-negative infections, a detailed epidemiologic investigation may reveal the source of these infections. It is quite surprising to identify the difference in host immune response pattern and the associated clinical and subclinical presentations of IMIs due to the different gram-negative organisms. Experimental and field observations would suggest that among the gram-negative bacterial causes of mastitis, Klebsiella spp are causing the most severe cases, closely followed by E coli and then much less clinical severity is observed in Serratia spp and Enterobacter spp cases. The precise mechanisms that would explain the difference in clinical severity are not known, but the most likely explanation appears to be the structure of the lipid A fraction of the LPS of the bacterial species. Important differences in the lipid A fraction of LPS between and within bacterial species are observed. The prevention of IMIs with gram-negative bacteria has components that are generic across species and components that are species specific. Generic prevention may be obtained by improving hygiene and reducing exposure of teat ends to environmental contamination. Also the use of a J5 bacterin is expected to provide some reduction in severity of gram-negative IMIs across bacterial species. Specific prevention programs will depend on the actual transmission behavior of the dominant species causing IMIs in the herd. Several clonal outbreaks of gram-negative bacterial species have been described. In such situations, optimal milking procedures, segregation and culling of infected animals, and targeted treatment would be advisable. Even more specific are the prevention procedures associated with S marcescens outbreaks, where resistance against specific biocides will lead to transmission of infection through teat disinfectants. Removal of these biocides from the cow environment is than essential. Antimicrobial treatment of gram-negative bacteria has often considered to be of limited value and treatment should be more targeted toward cow survival and reduction of clinical symptoms. More recently, extended treatment with a third-generation cephalosporin was reported to be efficacious in the treatment of E coli and Klebsiella spp but not of E cloacae. Further investigations in effective treatment protocols for gram-negative IMIs are warranted.

Characterization of toxin plasmids in Clostridium perfringens type C isolates.

Clostridium perfringens type C isolates cause enteritis necroticans in humans or necrotizing enteritis and enterotoxemia in domestic animals. Type C isolates always produce alpha toxin and beta toxin but often produce additional toxins, e.g., beta2 toxin or enterotoxin. Since plasmid carriage of toxin-encoding genes has not been systematically investigated for type C isolates, the current study used Southern blot hybridization of pulsed-field gels to test whether several toxin genes are plasmid borne among a collection of type C isolates. Those analyses revealed that the surveyed type C isolates carry their beta toxin-encoding gene (cpb) on plasmids ranging in size from ∼65 to ∼110 kb. When present in these type C isolates, the beta2 toxin gene localized to plasmids distinct from the cpb plasmid. However, some enterotoxin-positive type C isolates appeared to carry their enterotoxin-encoding cpe gene on a cpb plasmid. The tpeL gene encoding the large clostridial cytotoxin was localized to the cpb plasmids of some cpe-negative type C isolates. The cpb plasmids in most surveyed isolates were found to carry both IS1151 sequences and the tcp genes, which can mediate conjugative C. perfringens plasmid transfer. A dcm gene, which is often present near C. perfringens plasmid-borne toxin genes, was identified upstream of the cpb gene in many type C isolates. Overlapping PCR analyses suggested that the toxin-encoding plasmids of the surveyed type C isolates differ from the cpe plasmids of type A isolates. These findings provide new insight into plasmids of proven or potential importance for type C virulence.

Expression, crystallization and preliminary X-ray diffraction studies of recombinant Clostridium perfringens beta 2-toxin.

Clostridium perfringens is a Gram-positive sporulating anaerobic bacterium that is responsible for a wide spectrum of diseases in animals, birds and humans. The virulence of C. perfringens is associated with the production of several enterotoxins and exotoxins. beta2-toxin is a 28 kDa exotoxin produced by C. perfringens. It is implicated in necrotic enteritis in animals and humans, a disease characterized by a sudden acute onset with lethal hemorrhagic mucosal ulceration. The recombinant expression, purification and crystallization of beta2-toxin using the batch-under-oil technique are reported here. Native X-ray diffraction data were obtained to 2.9 A resolution on a synchrotron beamline at the F2 station at Cornell High Energy Synchrotron Source (CHESS) using an ADSC Quantum-210 CCD detector. The crystals belong to space group R3, with a dimer in the asymmetric unit; the unit-cell parameters are a = b = 103.71, c = 193.48 A, alpha = beta = 90, gamma = 120 degrees using the hexagonal axis setting. A self-rotation function shows that the two molecules are related by a noncrystallographic twofold axis with polar angles omega = 90.0, phi = 210.3 degrees.