Persistent Intramammary Infections Research Articles

Employment of mastoparan-like peptides to prevent Staphylococcus aureus associated with bovine mastitis.

Bovine mastitis is a frequent infection in lactating cattle, causing great economic losses. Staphylococcus aureus represents the main etiological agent, which causes recurrent and persistent intramammary infections because conventional antibiotics are ineffective against it. Mastoparan-like peptides are multifunctional molecules with broad antimicrobial potential, constituting an attractive alternative. Nevertheless, their toxicity to host cells has hindered their therapeutic application. Previously, our group engineered three mastoparan-L analogs, namely mastoparan-MO, mastoparan-R1, and [I5, R8] MP, to improve cell selectivity and potential. Here, we were interested in comparing the antibacterial efficacy of mastoparan-L and its analogs against bovine mastitis isolates of S. aureus strains, making a correlation with the physicochemical properties and structural arrangement changes promoted by the sequence modifications. As a result, the analog's hemolytic and/or antimicrobial activity was balanced. All the peptides displayed α-helical folding in hydrophobic and membrane-mimetic environments, as determined by circular dichroism. The peptide [I5, R8] MP stood out for its enhanced selectivity and antibacterial features related to mastoparan-L and the other derivatives. Biophysical approaches revealed that [I5, R8] MP rapidly depolarizes the bacterial membrane of S. aureus, causing cell death by subsequent membrane disruption. Our results demonstrated that the [I5, R8] MP peptide could be a starting point for the development of peptide-based drugs for the treatment of bovine mastitis, with the advantage of no residue in milk, which would help reduce the use of classical antibiotics.IMPORTANCEStaphylococcus aureus is a leading cause of mastitis, the world's most important dairy cattle disease. The multidrug resistance and zoonotic potential of S. aureus, besides the likelihood of antibiotic residues in milk, are of critical concern to public and animal health. Antimicrobial peptides offer a novel antimicrobial strategy. Here, we demonstrate that [I5, R8] MP is a potent and selective peptide, which acts on S. aureus by targeting the bacterial membrane. Therefore, understanding the physicochemical determinants and the modes of action of this class of antimicrobials opens novel prospects for peptide development with enhanced activities in the bovine mastitis context.

Periparturient blood T-lymphocyte PD-1 and CTLA-4 expression as potential predictors of new intramammary infections in dairy cows during early lactation (short communication)

BackgroundThe periparturient period in dairy cows is marked by immunosuppression which increases the likelihood of infectious disorders, particularly also mastitis. An in-depth understanding of peripartum leukocyte biology is vital for the implementation of highly successful post-partum disease prevention measures. Immune checkpoint molecules, such as programmed death 1 (PD-1) and cytotoxic T-lymphocyte antigen 4 (CTLA-4), are critical inhibitory receptors expressed on immune cells, particularly T cells, that drive immunosuppressive signaling pathways. However, the potential role of immune checkpoint molecules expression in T-cells on udder health has never been explored. Thus, the association between the occurrence of new postpartum intramammary infections (IMIs) and the expression of programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) on blood T-cells during the peripartum period was investigated.ResultsIn this study, the incidence of IMIs by any pathogen in early lactation was not associated with a higher expression of PD-1 and CTLA-4 in the periparturient period. However, the incidence of IMIs by major pathogens throughout the first month of lactation was significantly associated with higher expression of PD-1 at 14 days before calving (P = 0.03) and CTLA-4 at parturition (P = 0.03) by blood T-cells. Also, the expression of CTLA-4 at D0 (P = 0.012) by T-cells was associated with the occurrence of persistent IMIs during the first month of lactation.ConclusionsTo our knowledge, this is the first report to investigate the expression of PD-1 and CTLA-4 by blood T-lymphocytes during the periparturient period in dairy cows and to explore their relationship with the incidence of new IMIs in the postpartum period. Thus, a comprehensive understanding of leukocyte biology during peripartum would appear to be a prerequisite for the identification of resilient dairy cows or targets innovative (immunological) non-antibiotic approaches in the transition period.

Phenotypic and genotypic assessment of iron acquisition in diverse bovine-associated non-aureus staphylococcal strains

Although the role of iron in bacterial infections has been well described for Staphylococcus (S.) aureus, iron acquisition in (bovine-associated) non-aureus staphylococci and mammaliicocci (NASM) remains insufficiently mapped. This study aimed at elucidating differences between four diverse bovine NASM field strains from two species, namely S. chromogenes and S. equorum, in regards to iron uptake (with ferritin and lactoferrin as an iron source) and siderophore production (staphyloferrin A and staphyloferrin B) by investigating the relationship between the genetic basis of iron acquisition through whole genome sequencing (WGS) with their observed phenotypic behavior. The four field strains were isolated in a previous study from composite cow milk (CCM) and bulk tank milk (BTM) in a Flemish dairy herd. Additionally, two well-studied S. chromogenes isolates originating from a persistent intramammary infection and from a teat apex were included for comparative purpose in all assays. Significant differences between species and strains were identified. In our phenotypical iron acquisition assay, while lactoferrin had no effect on growth recovery for all strains in iron deficient media, we found that ferritin served as an effective source for growth recovery in iron-deficient media for S. chromogenes CCM and BTM strains. This finding was further corroborated by analyzing potential ferritin iron acquisition genes using whole-genome sequencing data, which showed that all S. chromogenes strains contained hits for all three proposed ferritin reductive pathway genes. Furthermore, a qualitative assay indicated siderophore production by all strains, except for S. equorum. This lack of siderophore production in S. equorum was supported by a quantitative assay, which revealed significantly lower or negligible siderophore amounts compared to S. aureus and S. chromogenes. The WGS analysis showed that all tested strains, except for S. equorum, possessed complete staphyloferrin A (SA)-synthesis and export operons, which likely explains the phenotypic absence of siderophore production in S. equorum strains. While analyzing the staphyloferrin A and staphyloferrin B operon landscapes for all strains, we noticed some differences in the proteins responsible for iron acquisition between different species. However, within strains of the same species, the siderophore-related proteins remained conserved. Our findings contribute valuable insights into the genetic elements associated with bovine NASM pathogenesis.

Genotypic characterization of Staphylococcus chromogenes and Staphylococcus simulans from Swedish cases of bovine subclinical mastitis

Staphylococcus chromogenes and Staphylococcus simulans are commonly found in intramammary infections (IMI) associated with bovine subclinical mastitis, but little is known about genotypic variation and relatedness within species. This includes knowledge about genes encoding antimicrobial resistance (AMR) and potential virulence factors (pVF). The aim of this study was therefore to investigate these aspects by whole-genome sequencing of milk isolates from Swedish dairy cows with subclinical mastitis in an observational study. We also wanted to study if specific genotypes were associated with persistent IMI and the inflammatory response at udder quarter level. In total, 105 and 118 isolates of S. chromogenes and S. simulans, respectively, were included. Isolates were characterized using a 7-locus multilocus sequence typing (7-MLST), core genome analysis and in-silico analysis of AMR and pVF genes. Forty-seven sequence types (ST) and 7 core genome clusters of S. chromogenes were identified, and the most common ST were ST-6 and ST-109, both belonging to cluster VII. A 7-locus MLST scheme for S. simulans was not available, but 3 core genome clusters and 5 subclusters were described. Overall, substantial variation in ST and clusters among cows and herds were found in both species. Some ST of S. chromogenes were found in several herds, indicating spread between herds. Moreover, within-herd spread of the same genotype was observed for both species. Only a few AMR genes (blaZ, strpS194, vga(A)) were detected in a limited number of isolates, with the exception of blaZ coding for β-lactamase, which was identified in 22% of the isolates of S. chromogenes with ST-19, ST-102, and ST-103 more commonly carrying this gene compared with other STs. However, the blaZ gene was not identified in S. simulans. The average total number of pVF detected per isolate was similar in S. chromogenes (n = 30) and S. simulans (n = 33), but some variation in total numbers and presence of specific pVF or functional groups of pVF, was shown between ST/clusters within species. Differences in inflammatory response and potentially in persistent IMI at udder quarter level were found between S. chromogenes subtypes but not between S. simulans subtypes. In conclusion, the results from the present study generates new insight into the epidemiology of bovine S. chromogenes and S. simulans IMI, which can have implications for future prevention and antimicrobial treatment of infections related to these species.

Bovine-associated staphylococci and mammaliicocci trigger T-lymphocyte proliferative response and cytokine production differently

We examined whether distinct staphylococcal and mammaliicoccal species and strains trigger B- and T-lymphocyte proliferation and interleukin (IL)-17A and interferon (IFN)-γ production by peripheral blood mononuclear cells in nulliparous, primiparous, and multiparous dairy cows. Flow cytometry was used to measure lymphocyte proliferation with the Ki67 antibody, and specific monoclonal antibodies were used to identify CD3, CD4, and CD8 T lymphocyte and CD21 B lymphocyte populations. The supernatant of the peripheral blood mononuclear cell culture was used to measure IL-17A and IFN-γ production. Two distinct, inactivated strains of bovine-associated Staphylococcus aureus [one causing a persistent intramammary infection (IMI) and the other from the nose], 2 inactivated Staphylococcus chromogenes strains [one causing an IMI and the other from a teat apex), as well as an inactivated Mammaliicoccus fleurettii strain originating from sawdust from a dairy farm, and the mitogens concanavalin A and phytohemagglutinin M-form (both specifically to measure lymphocyte proliferation) were studied. In contrast to the "commensal" Staph. aureus strain originating from the nose, the Staph. aureus strain causing a persistent IMI triggered proliferation of CD4+ and CD8+ subpopulations of T lymphocytes. The M. fleurettii strain and the 2 Staph. chromogenes strains had no effect on T- or B-cell proliferation. Furthermore, both Staph. aureus and Staph. chromogenes strains causing persistent IMI significantly increased IL-17A and IFN-γ production by peripheral blood mononuclear cells. Overall, multiparous cows tended to have a higher B-lymphocyte and a lower T-lymphocyte proliferative response than primiparous and nulliparous cows. Peripheral blood mononuclear cells of multiparous cows also produced significantly more IL-17A and IFN-γ. In contrast to concanavalin A, phytohemagglutinin M-form selectively stimulated T-cell proliferation.

Genetic and Antimicrobial Resistance Profiles of Mammary Pathogenic E. coli (MPEC) Isolates from Bovine Clinical Mastitis.

Mammary pathogenic E. coli (MPEC) is one of the main pathogens of environmental origin responsible for causing clinical mastitis worldwide. Even though E. coli are strongly associated with transient or persistent mastitis and the economic impacts of this disease, the virulence factors involved in the pathogenesis of MPEC remain unknown. Our aim was to characterize 110 MPEC isolates obtained from the milk of cows with clinical mastitis, regarding the virulence factor-encoding genes present, adherence patterns on HeLa cells, and antimicrobial resistance profile. The MPEC isolates were classified mainly in phylogroups A (50.9%) and B1 (38.2%). None of the isolates harbored genes used for diarrheagenic E. coli classification, but 26 (23.6%) and 4 (3.6%) isolates produced the aggregative or diffuse adherence pattern, respectively. Among the 22 genes investigated, encoding virulence factors associated with extraintestinal pathogenic E. coli pathogenesis, fimH (93.6%) was the most frequent, followed by traT (77.3%) and ompT (68.2%). Pulsed-field gel electrophoresis analysis revealed six pulse-types with isolates obtained over time, thus indicating persistent intramammary infections. The genes encoding beta-lactamases detected were as follows: blaTEM (35/31.8%); blaCTX-M-2/blaCTX-M-8 (2/1.8%); blaCTX-M-15 and blaCMY-2 (1/0.9%); five isolates were classified as extended spectrum beta-lactamase (ESBL) producers. As far as we know, papA, shf, ireA, sat and blaCTX-M-8 were detected for the first time in MPEC. In summary, the genetic profile of the MPEC studied was highly heterogeneous, making it impossible to establish a common genetic profile useful for molecular MPEC classification. Moreover, the detection of ESBL-producing isolates is a serious public health concern.

Differential immune response to two Staphylococcus aureus strains with distinct adaptation genotypes after experimental intramammary infection of dairy cows

The aim of this study was to evaluate and compare the ability of two S. aureus strains with different adaptation genotypes (low and high) to the bovine mammary gland (MG) to establish an intramammary infection (IMI) and induce an immune response after an experimental challenge in lactating cows. Two isolates (designated 806 and 5011) from bovine IMI with different genotypic profiles, harboring genes involved in adherence and biofilm production, belonging to different capsular polysaccharide (CP) type, accessory gene regulator (agr) group, pulsotype (PT) and sequence type/clonal complex (ST/CC) were selected. Strains 806 and 5011 were associated with low (nonpersistent-NP) and high (persistent-P) adaptation to the MG, respectively. Strain 806 (NP) was characterized as agr group II, cap5 positive and ST350; strain 5011 (P) agr group I, cap8 positive and CC188. Three groups of clinically healthy cows, 4 cows/treatment group, were inoculated by the intramammary route with strain 806 (NP), strain 5011 (P) and pyrogen-free saline solution. All mammary quarters challenged with strain 806 (NP) developed mild clinical mastitis between 1 and 7 d post inoculation (pi). Quarters challenged with strain 5011 (P) developed a persistent IMI; bacteria were recovered from milk from d 7 pi and up to d 56 pi. In quarters inoculated with strain 806 (NP) the inflammatory response induced was greater and earlier than the one induced by strain 5011 (P), since a somatic cell count (SCC) peak was observed at d 2 pi, while in quarters inoculated with strain 5011 (P) no variations in SCC were observed until d 4 pi reaching the maximum values at d 14 pi; indicating a lower and delayed initial inflammatory response. The highest levels of nitric oxide (NO) and lactoferrin (Lf) detected in milk from quarters inoculated with both S. aureus strains coincided with the highest SCC at the same time periods, indicating an association with the magnitude of inflammation. The high levels of IL-1β induced by strain 806 (NP) were associated with the highest SCC detected (d 2 pi); while quarters inoculated with strain 5011 (P) showed similar IL-1β levels to those found in control quarters. In quarters inoculated with strain 806 (NP) two peaks of IL-6 levels on d 2 and 14 pi were observed; while in quarters inoculated with strain 5011 (P) IL-6 levels were similar to those found in control quarters. The strain 806 (NP) induced a higher total IgG and IgG1 response; while strain 5011 (P) generated a higher IgG2 response (even against the heterologous strain). The present study demonstrated that S. aureus strains with different genotype and adaptability to bovine MG influence the local host immune response and the course and severity of the infectious process.

Comparison of Virulence Patterns Between Streptococcus uberis Causing Transient and Persistent Intramammary Infection.

The objectives of this study were determined by two experiments including Experiment 1 (EXP1) using Streptococcus uberis obtained from a weekly longitudinal study to compare virulence patterns between transient and persistent intramammary infection (IMI), and Experiment 2 (EXP2) using a stored-known-appearance PFGE strain of a contagious S. uberis to determine a change of virulence patterns after long-term transmission. For EXP1, quarter milk samples from 31 milking cows were aseptically and longitudinally collected once a week for 10 weeks. A total of 14 S. uberis isolates from quarters with 1 and >4 weeks of duration of IMI were categorized as transient and persistent IMI, respectively. For EXP2, 11 isolates of a stored-known-appearance PFGE strain of S. uberis from our previous study (1) were randomly selected, including 5 from transient IMI (1 month) and 6 from persistent IMI (>1 month). The virulence profiles of all isolates were investigated, including sua, hasAB, hasC, gapC, pauA, and CAMP factor or cfu, using PCR. The Kaplan–Meier estimates were used to calculate the duration of IMI in EXP1. Approximately 50% of field S. uberis IMI was spontaneously cured within 1 week, while 25% was not cured within 10 weeks. From EXP1, 4 virulence patterns were found in 14 isolates. The majority of patterns for transient S. uberis did not include hasAB (63.6%), the gene relating to capsule formation. Regardless of transient or persistent IMI, a high similarity of the virulence pattern within a PFGE strain was found in EXP2. Few changes of virulence pattern within a PFGE strain were found or were related to its subsequently changing to transient IMI.

Molecular Typing and Antimicrobial Susceptibility Profile of Staphylococcus aureus Isolates Recovered from Bovine Mastitis and Nasal Samples.

Simple SummaryStaphylococcus aureus is a major, prevalent mastitis pathogen, representing a real issue for bovine udder health, with unquestionable importance in human and veterinary medicine. The present study thus aimed to determine the antimicrobial resistance and the diversity of S. aureus recovered from transient and persistent intramammary infections and from extramammary niches (e.g., nares/muzzles) in dairy cows. We found that a large proportion of S. aureus strains exhibited multidrug resistance to antimicrobials, including resistance to antimicrobials that are critically important to human health. S. aureus isolates from transient and persistent IMIs did not differ, suggesting that the persistence of bovine intramammary infections (IMIs) was mainly determined by host factors, although S. aureus isolated from extramammary niches are not an important source of S. aureus intramammary infections. Furthermore, a discrepancy in antimicrobial resistance between S. aureus strains isolated from nares/muzzles and intramammary infections was observed.In the present study, we aimed to determine the antimicrobial resistance and molecular typing of Staphylococcus aureus recovered from transient and persistent intramammary infections and nares/muzzles in dairy cows. We investigated the antimicrobial resistance of 189 S. aureus strains using a broad antimicrobial susceptibility profile. Furthermore, 107 S. aureus isolates were strain-typed using staphylococcal protein-A (spa) typing. A large proportion of strains exhibited multidrug resistance to antimicrobials, including resistance to critically important antimicrobials, although no methicillin-resistant S. aureus strains were found. Our study did not strengthen the idea that extramammary niches (i.e., nares/muzzles) are an important source of S. aureus for bovine mastitis. A discrepancy in the antimicrobial resistance between S. aureus strains isolated from nares/muzzles and milk samples was observed. Furthermore, S. aureus isolates from transient and persistent intramammary infections (IMIs) did not differ by spa typing, suggesting that the persistence of bovine IMIs was determined by cow factors. Thus, the high level of multidrug-resistant S. aureus found in the two herds, considered together with the predominance of a well udder-adapted S. aureus strain, may contribute to our knowledge of the history of the high prevalence of mastitis caused by S. aureus, which is of great concern for animal and public health.

Longitudinal study on the effects of intramammary infection with non-aureus staphylococci on udder health and milk production in dairy heifers

We conducted a longitudinal study to evaluate the effect of non-aureus staphylococci (NAS) causing subclinical intramammary infections (IMI) on quarter milk somatic cell count (qSCC) and quarter milk yield (qMY). In total, 324 quarters of 82 Holstein Friesian heifers were followed from calving to 130 d in milk (DIM) and were sampled 10 times each at 14-d intervals. The IMI status of each quarter was determined based on bacterial culture results at the current and previous or next sampling day, or both. The qSCC was determined on each sampling day and the average qMY on sampling day was available through stored daily milk weight data in the management program of the automatic milking system. A transient IMI (tIMI) was defined as a case where a specific pathogen was isolated from a quarter on only one sampling day and not on the previous or next sampling day. When the same bacterial strain, as defined by random amplification of polymorphic DNA-PCR, was isolated from the same quarter on multiple sampling days, it was defined as a persistent IMI (pIMI) status on those sampling days; a pIMI episode was defined as the combination of multiple consecutive pIMI statuses with the same bacterial strain on different sampling days. During this study, 142 subclinical IMI with NAS occurred in 116 different quarters from 64 animals, yielding in total 304 NAS isolates belonging to 17 different species. The prevalence of NAS was highest in the first 4 DIM. Overall, the predominant species was Staphylococcus chromogenes (52% of the isolates), followed by S. epidermidis (9.2%), S. xylosus (8.2%), and S. equorum (5.9%). Staphylococcus chromogenes was the only species for which an effect on qSCC and qMY could be analyzed separately; the other NAS species were considered as a group because of their low prevalence. Eighteen out of 40 IMI (45%) caused by S. chromogenes persisted over at least 2 sampling days, whereas only 10 of 102 (9.8%) IMI caused by other NAS species persisted for at least 2 sampling days. The average duration of pIMI episodes was 110.4 d for S. chromogenes and 70 d for the other NAS species. Remarkably, 17 of the 18 pIMI episodes with S. chromogenes started within the first 18 DIM. The qSCC was highest in quarters having a pIMI with a major pathogen, followed by quarters having a pIMI with S. chromogenes, and a pIMI with other NAS. Transient IMI with other NAS or with a major pathogen caused a small but significantly higher qSCC, whereas the qSCC in quarters having a tIMI with S. chromogenes was not statistically different compared with noninfected quarters. No significant differences in qMY were observed between quarters having a pIMI or tIMI with S. chromogenes or with the other NAS species compared with noninfected quarters, despite the higher qSCC. Quarters having a pIMI with major pathogens showed significantly lower daily milk production. Surprisingly, quarters that cured from an IMI with S. chromogenes had a significantly lower qMY than noninfected quarters.

The effect of intramammary infection in early lactation with non-aureus staphylococci in general and Staphylococcus chromogenes specifically on quarter milk somatic cell count and quarter milk yield

This longitudinal study aimed to evaluate the impact of subclinical intramammary infection (IMI) with non-aureus staphylococcal (NAS) species in the first 18 d in milk (DIM) on the quarter milk somatic cell count (qSCC) and quarter milk yield (qMY) during the first 4 mo of lactation in Holstein Friesian heifers. Quarter milk samples were collected from 82 heifers from 1 to 4 DIM until 130 DIM on a biweekly (14 d) basis for determination of the qSCC; qMY data were available through the automatic milking systems. The quarter samples collected on the first (1-4 DIM) and second (15-18 DIM) sampling days were used for bacteriological culturing to determine the IMI status. In this study, 324 quarters from 82 heifers were enrolled, of which 68 were NAS-infected at the first sampling day. Only 16 (23.5%) of these quarters were still NAS-infected at the second sampling day, demonstrating the high spontaneous cure rate of these infections shortly after calving; 9 of these 16 cases were infected with the same NAS species. Interestingly, none of the NAS-infected quarters at the first sampling day acquired a new infection with a major pathogen at the second sampling day, whereas 2.3% of the noninfected quarters did. All 102 isolates phenotypically identified as NAS were further identified to the species level. Staphylococcus chromogenes was the most prevalent species on the first (29.4% of all NAS) and second (52.9%) sampling days. Quarters infected with Staph. chromogenes at the first sampling day had a significantly higher qSCC in later lactation than noninfected quarters, whereas this was not true for quarters infected with all other NAS species (i.e., as a group of species). The average daily qMY in the first 4 mo of lactation did not differ between noninfected quarters and quarters infected with Staph. chromogenes or all other NAS species at the first sampling day. Persistently NAS species-infected quarters in the first 18 DIM (i.e., infected with the same NAS species on the first and second sampling days) had the highest qSCC later in lactation, followed by quarters with a new NAS IMI (i.e., noninfected at the first sampling day and infected with NAS at the second sampling day). The qSCC from transiently NAS species-infected quarters (i.e., not infected with the same NAS species at the second sampling day) was not significantly higher in later lactation compared with that in noninfected quarters. The IMI status of quarters in the first 18 DIM, combining culture results at 1 to 4 and 15 to 18 DIM (new, persistent, and transient IMI), was not significantly associated with daily qMY in the first 4 mo after calving. In general, NAS should be considered minor pathogens with no adverse effect on daily qMY in quarters of heifers infected in the first 18 DIM and with a high spontaneous cure rate. Staphylococcus chromogenes was the most prevalent species, causing an increase in qSCC comparable to the level of quarters infected with a major pathogen; Staph. chromogenes caused most infections that persisted through at least the first 18 DIM.

Staphylococcal intramammary infection dynamics and the relationship with milk quality parameters in dairy goats over the dry period

The objectives of this study were (1) to report the rates of new intramammary infection (IMI) and spontaneous IMI cure over the dry period in 3 dairy goat herds; (2) to evaluate the factors predicting infection dynamics over the dry period; and (3) to define milk quality parameter thresholds that predict infection dynamics over the dry period. Two consecutive udder-half milk samples were collected 10 to 14 d apart before dry-off from 288 goats in 3 herds, and 2 consecutive udder-half samples were collected 7 to 14 d apart in the following lactation, with the first sample being collected ≤10 d in milk, from 200 of the same goats. In 2 of the herds, udder-half milk samples were also collected at the same time points (n = 312 halves; 157 goats) for measurement of milk quality parameters. Standard aerobic culture of milk samples was performed for the detection of mastitis pathogens. To rule out the presence of Mycoplasma spp. IMI, milk samples were also cultured on modified Hayflick medium. Non-Mycoplasma isolates were speciated using MALDI-TOF mass spectrometry. Staphylococcal isolates, when not identified by MALDI-TOF, were speciated using partial gene sequence analysis of rpoB or tuf. When >1 sample from an udder half yielded the same species, available isolates from the first and last positive samples for that species were strain-typed using pulsed-field gel electrophoresis. Incidence of new IMI and cure rate were computed. Generalized linear mixed regression models were built to evaluate the associations between new IMI and pre-dry somatic cell score (SCS), between IMI persistence and half-level SCS, and between IMI persistence and pre-dry IMI species. Thresholds for pre-dry SCS and lactose concentration were computed to predict IMI persistence. Overall, 12.6% (48/380) of halves had a persistent IMI. Cumulative incidence of new IMI over the dry period was 13.2%, and cure rate was 52.0%. Pre-dry SCS was not associated with odds of new IMI or IMI persistence. Pre-dry IMI species was not associated with odds of persistence. Lactose concentration was not associated with odds of persistence. Regardless of culture data, the optimal pre-dry SCS threshold to detect IMI that would persist into the next lactation was 8.7, with sensitivity and specificity of 50 and 73.8%, respectively. Further studies on the effect of control measures on species-specific incidence and cure rates during the dry period are warranted.

Intramammary infections with different non-aureus staphylococci in dairy cows

Subclinical mastitis causes an increase in milk somatic cell count (SCC) and can lead to reduced milk production and early culling. In many countries, non-aureus staphylococci (NAS) is the most common bacterial finding in subclinical mastitis of dairy cows. New methodology makes it possible to identify NAS species, but knowledge about the epidemiology is limited. The objective of this project was to improve advisory services for mastitis control by investigating associations between NAS and SCC, milk production, and persistence of intramammary infections (IMI). Farmers who had sent milk samples to the Swedish National Veterinary Institute (Uppsala, Sweden) were asked to participate if NAS was identified in the samples. Participating farmers were asked to resample all udder quarters of the cow once within 1 mo. Regression models were used to investigate associations between NAS and cow factors, udder quarter California mastitis test and SCC, and persistence of IMI. Associations with cow composite milk yield and SCC were also investigated. In total, 671 cows from 201 herds were enrolled in the study, and 19 NAS species were identified, of which the 4 most common were Staphylococcus epidermidis, Staphylococcus simulans, Staphylococcus chromogenes, and Staphylococcus haemolyticus. Persistent IMI was more common in udder quarters with Staphylococcus hyicus and S. simulans and less common in those with Staphylococcus saprophyticus IMI. β-Lactamase production by the different NAS species varied from 0 to 100%. There was a significant association between NAS species and California mastitis test and SCC of udder quarters, and this varied depending on parity. The cow composite milk SCC at the test milking before the initial sample was taken differed significantly with NAS species, but not at the subsequent test milking. Milk yield-at the test milking before or after the initial sample-did not differ significantly for NAS species. There were no significant associations between milk yield or SCC and persistent NAS IMI. In conclusion, the NAS species affects SCC and persistent IMI differently but not milk yield.

Transmission dynamics of intramammary infections caused by Corynebacterium species

The development of reliable models for transmission of intramammary infections (IMI) is the subject of extensive research. Such models are useful to enhance the identification and understanding of factors that affect pathogen-specific IMI dynamics. Longitudinal transmission models are valuable for predicting infection outbreak risks, quantifying the effectiveness of response tactics, and performing response planning. In this work, we focused on modeling Corynebacterium spp. by using a compartmental model. Previous investigations have considered modeling the transmission dynamics of several bacterial pathogens, but not Corynebacterium spp. We established a Corynebacterium spp. Susceptible-Infectious-Susceptible (SIS) model. We simulated the model numerically by using parameters that we estimated by a generalized linear model approach, using month of study as the time variable. The data, from which the parameters of the model were estimated, were obtained in a field trial conducted in 2 US dairy herds. Altogether, 786 cows were sampled at least once during the 13-mo study period. The total number of quarter milk cultures and cases of IMI caused by Corynebacterium spp. were 11,744 and 556, respectively, in farm A; the corresponding figures for farm B were 11,804 and 179. Our modeling study included only transmission from persistent IMI caused by Corynebacterium spp. within the lactation pens. The rate of new infections was significantly related to preexisting IMI in both farms, underscoring the importance of preexisting Corynebacterium spp. IMI for the transmission of Corynebacterium spp. within lactation pens. The estimated basic reproduction numbers (R0) in the 2 farms were 1.18 and 0.98, respectively. The nonsignificant disparity in R0 was associated with significant differences in cure rates between farms.

Epidemiological and partial budget analysis for treatment of subclinical Staphylococcus aureus intramammary infections considering microbiological and cytological scenarios

An innovative method was investigated to aid in the elimination of Staphylococcus aureus (S. aureus) intramammary infections (IMI) from dairy herds. A stochastic model explore the economic benefit of three-day or eight-day treatment of subclinical IMI in all S. aureus infected cows or in only those with a somatic cell count (SCC) exceeding 200,000 cells/ml. An epidemiological model was developed to run parallel to the economic model that would predict the S. aureus IMI likely to persist, develop new infections and clinical mastitis.In the economic model a first algorithm was used to consider the low prevalence (LP) scenario and made use of S. aureus prevalence information provided by retrospective analysis of microbiological and cytological results in South Africa (2008–2012). The data used considered Staphylococcus aureus prevalence from [1.495; 1.595]95% to [6.72; 6.95]95% for SCC≤200,000 and SCC>200,000 cells/ml respectively. A second algorithm considered the high prevalence (HP) scenario to evaluate a simulated situation with a 5[U1] [R12] to 25% prevalence. Scenarios of low or high transmission ratio (TR) were included in the model according to the hygiene management on the farm. Probabilities and costs were calculated over 255days. The economic models predicted average cost indices for low S. aureus IMI and low TR to vary from −3179 ZAR (South African Rands) when subclinical cases with SCC higher than 200,000 cell/ml were treated for eight days, to −3663 ZAR when all subclinical S. aureus IMI were treated for three days. With a HP and high TR of S. aureus the average cost indices changed from −18,042 ZAR when none to −5433 ZAR per 255days when all S. aureus IMI were treated for eight days.The epidemiological model in this study predicted substantial benefit of treatment mainly in high TR scenarios. New IMI decreased up to77% in the three-day and up to 91% in the eight-day treatment scenarios. In the HP scenarios, persistent IMI were reduced by 94%. The number of clinical cases predicted with no treatment for subclinical infections was higher than the total number of clinical and subclinical cases in scenarios where cows were treated three or eight days.Initial prudent treatment of subclinical IMI resulted in less overall treatments and less new, persistent and clinical cases. Combined results of economic and epidemiological models indicated that the option that cost the least did not always have the best epidemiological outcome. Models may assist in optimising and balancing decisions relating to financial and IMI.

Antimicrobial resistance profiles of Staphylococcus aureus clusters on small dairy farms in southern Brazil

ABSTRACT: In intensive dairy farming, persistent intramammary infection has been associated with specific Staphylococcus (S.) aureus strains, and these strains may be resistant to antimicrobials. The objective of this study was to evaluate the antimicrobial resistance phenotypes of S. aureus isolates and to assess the distribution and the persistence of clonal groups in small dairy herds of southern Brazil. Milk samples were collected from all lactating cows from 21 dairy farms over a two-year period, totaling 1,060 samples. S. aureus isolates were tested for susceptibility to thirteen antimicrobials using the disk diffusion method. The total DNA of the isolates was subjected to SmaI digestion followed by pulsed-field gel electrophoresis (PFGE). Banding patterns differing by ≤4 bands were considered members of a single PFGE cluster. The frequency of S. aureus isolation ranged from 3.45% to 70.59% among the 17 S. aureus-positive herds. Most S. aureus isolates (87.1%) were susceptible to all antimicrobials; resistance to penicillin (18.2%) was the most frequently observed. The 122 isolates subjected to macrorestriction analysis were classified into 30 PFGE-clusters. Among them, only 10 clusters were intermittent or persistent over the two-year period. The majority (93.6%) of isolates belonging to persistent and intermittent clusters were susceptible to all tested antimicrobials. S. aureus intramammary colonization in small dairy farms of southern Brazil is most frequently caused by sporadic PFGE clusters, although some persistent clusters can arise over time. Both sporadic and persistent isolates were highly susceptible to antimicrobials.

Local host response following an intramammary challenge with Staphylococcus fleurettii and different strains of Staphylococcus chromogenes in dairy heifers.

Coagulase-negative staphylococci (CNS) are a common cause of subclinical mastitis in dairy cattle. The CNS inhabit various ecological habitats, ranging between the environment and the host. In order to obtain a better insight into the host response, an experimental infection was carried out in eight healthy heifers in mid-lactation with three different CNS strains: a Staphylococcus fleurettii strain originating from sawdust bedding, an intramammary Staphylococcus chromogenes strain originating from a persistent intramammary infection (S. chromogenes IM) and a S. chromogenes strain isolated from a heifer’s teat apex (S. chromogenes TA). Each heifer was inoculated in the mammary gland with 1.0 × 106 colony forming units of each bacterial strain (one strain per udder quarter), whereas the remaining quarter was infused with phosphate-buffered saline. Overall, the CNS evoked a mild local host response. The somatic cell count increased in all S. fleurettii-inoculated quarters, although the strain was eliminated within 12 h. The two S. chromogenes strains were shed in larger numbers for a longer period. Bacterial and somatic cell counts, as well as neutrophil responses, were higher after inoculation with S. chromogenes IM than with S. chromogenes TA. In conclusion, these results suggest that S. chromogenes might be better adapted to the mammary gland than S. fleurettii. Furthermore, not all S. chromogenes strains induce the same local host response.

Genomic and Phenomic Study of Mammary Pathogenic Escherichia coli.

Escherichia coli is a major etiological agent of intra-mammary infections (IMI) in cows, leading to acute mastitis and causing great economic losses in dairy production worldwide. Particular strains cause persistent IMI, leading to recurrent mastitis. Virulence factors of mammary pathogenic E. coli (MPEC) involved pathogenesis of mastitis as well as those differentiating strains causing acute or persistent mastitis are largely unknown. This study aimed to identify virulence markers in MPEC through whole genome and phenome comparative analysis. MPEC strains causing acute (VL2874 and P4) or persistent (VL2732) mastitis were compared to an environmental strain (K71) and to the genomes of strains representing different E. coli pathotypes. Intra-mammary challenge in mice confirmed experimentally that the strains studied here have different pathogenic potential, and that the environmental strain K71 is non-pathogenic in the mammary gland. Analysis of whole genome sequences and predicted proteomes revealed high similarity among MPEC, whereas MPEC significantly differed from the non-mammary pathogenic strain K71, and from E. coli genomes from other pathotypes. Functional features identified in MPEC genomes and lacking in the non-mammary pathogenic strain were associated with synthesis of lipopolysaccharide and other membrane antigens, ferric-dicitrate iron acquisition and sugars metabolism. Features associated with cytotoxicity or intra-cellular survival were found specifically in the genomes of strains from severe and acute (VL2874) or persistent (VL2732) mastitis, respectively. MPEC genomes were relatively similar to strain K-12, which was subsequently shown here to be possibly pathogenic in the mammary gland. Phenome analysis showed that the persistent MPEC was the most versatile in terms of nutrients metabolized and acute MPEC the least. Among phenotypes unique to MPEC compared to the non-mammary pathogenic strain were uric acid and D-serine metabolism. This study reveals virulence factors and phenotypic characteristics of MPEC that may play a role in pathogenesis of E. coli mastitis.

A C-type lectin from Bothrops jararacussu venom disrupts Staphylococcal biofilms.

Bovine mastitis is a major threat to animal health and the dairy industry. Staphylococcus aureus is a contagious pathogen that is usually associated with persistent intramammary infections, and biofilm formation is a relevant aspect of the outcome of these infections. Several biological activities have been described for snake venoms, which led us to screen secretions of Bothrops jararacussu for antibiofilm activity against S. aureus NRS155. Crude venom was fractionated by size-exclusion chromatography, and the fractions were tested against S. aureus. Biofilm growth, but not bacterial growth, was affected by several fractions. Two fractions (15 and 16) showed the best activities and were also assayed against S. epidermidis NRS101. Fraction 15 was identified by TripleTOF mass spectrometry as a galactose-binding C-type lectin with a molecular weight of 15 kDa. The lectin was purified from the crude venom by D-galactose affinity chromatography, and only one peak was observed. This pure lectin was able to inhibit 75% and 80% of S. aureus and S. epidermidis biofilms, respectively, without affecting bacterial cell viability. The lectin also exhibited a dose-dependent inhibitory effect on both bacterial biofilms. The antibiofilm activity was confirmed using scanning electron microscopy. A pre-formed S. epidermidis biofilm was significantly disrupted by the C-type lectin in a time-dependent manner. Additionally, the lectin demonstrated the ability to inhibit biofilm formation by several mastitis pathogens, including different field strains of S. aureus, S. hyicus, S. chromogenes, Streptococcus agalactiae, and Escherichia coli. These findings reveal a new activity for C-type lectins. Studies are underway to evaluate the biological activity of these lectins in a mouse mastitis model.

Characterization of persistent and transient Escherichia coli isolates recovered from clinical mastitis episodes in dairy cows

Escherichia coli usually cause transient intramammary infections in dairy cows, but persistent intramammary infections have been observed. The objective of the study was to compare antimicrobial resistance and virulence genes found in persistent and transient E. coli isolated from clinical mastitis cases in a cohort of 91 Canadian dairy herds monitored over a 2-year period. Antimicrobial susceptibility was determined by broth microdilution and the presence of 27 virulence genes associated with extra-intestinal E. coli infections was determined by colony hybridization. Proportion of resistance in persistent E. coli ranged from 0.0% (enrofloxacin) to 27.8% (ampicillin and tetracycline). Proportion of resistance in transient E. coli ranged from 0.0% (enrofloxacin) to 16.8% (tetracycline). Odds of being classified as a persistent isolate increased by a factor of 1.6 (95% CI: 1.1, 2.4) for each aditional resistance observed (e.g. isolates resistant to four antimicrobial agents had 1.6 times higher odds of belonging to the persistent groups compared to isolates demonstrating resistance to three agents). Persistency was associated with higher odds of resistance to ampicillin (OR: 9.8, P<0.01) or cephalothin (OR: 7.6, P=0.02). Persistent isolates had 5.4 times higher odds (95% CI: 1.2, 24.0) of harboring virulence gene iroN. Similarly, persistent isolates had 8.6 times higher odds (95% CI: 2.8, 27.1) of possessing the virulence gene sitA. In conclusion, this study confirmed that persistency of intramammary E. coli isolates is associated with certain traits. Findings concerning iron-acquisition shed new light on the mechanisms of intramammary survival.