Virulence factors, antimicrobial resistance and phylogeny of bovine mastitis-associated Streptococcus dysgalactiae.

We carried out a thorough genetic evaluation of Streptococcus dysgalactiae isolated from clinical bovine mastitis cases and performed a phylogenetic analysis to represent the evolutionary relationship between S. dysgalactiae sequences. A total of 35 S. dysgalactiae strains were isolated from cases of clinical mastitis identified at a large commercial dairy farm located near Ithaca, New York. Whole-genome sequencing identified twenty-six antibiotic resistance genes, four of which were acquired genes, in addition to fifty virulence genes. Multi-locus sequence typing detected three new sequence types (STs). We conclude that a high proportion of this microorganism carries multiple virulence determinants and resistance genes, and that this indicates its potential to cause mastitis. Eight different STs were identified, of which ST453 (n = 17) was the most prevalent and ST714, ST715, ST716 were novel STs.

Association between Mastitis Occurrence in Dairy Cows and Bedding Characteristics of Compost-Bedded Pack Barns.

Compost-bedded pack barns (CB) are receiving increasing attention as a housing system that can potentially improve the welfare of dairy cows. This study characterized the frequency and profile of pathogens isolated from clinical (CM) and subclinical (SCM) mastitis in dairy cows housed in CB. It evaluated the association between mastitis occurrence and bedding characteristics in CB systems. Over six months, seven dairy herds were visited monthly for milk and bedding sample collections. Milk samples from mastitis cases were submitted to microbiological identification by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF MS). Bedding samples were submitted to physical-chemical (pH, organic matter, moisture, and carbon to nitrogen ratio) and microbiological counting (total bacterial counts, coliforms, streptococci, and staphylococci) analyses. Regression analysis was used to determine the association between mastitis occurrence and CB characteristics. Our results showed that Escherichia coli and environmental streptococci were the most frequently isolated pathogens from CM cases, while Staphylococcus chromogenes and contagious pathogens (Staphylococcus aureus and Streptococcus agalactiae) were the most commonly isolated from SCM cases. Bedding moisture content was positively associated with the incidence of CM. The bedding carbon to nitrogen ratio was negatively associated with the incidence of SCM, and the bedding total bacteria counts tended to be associated with the incidence of SCM. Bedding counts of coliforms positively associated with the prevalence of SCM. Our results can support decision-makers in the dairy industry seeking strategies for bedding management and mastitis control.

Antimicrobial Resistance and Molecular Characterization of Staphylococcus aureus Recovered from Cows with Clinical Mastitis in Dairy Herds from Southeastern Brazil.

Staphylococcus aureus is a contagious pathogen frequently associated with bovine mastitis in Brazil. Molecular characterization of Staph. aureus isolated from affected mammary quarters of cows with clinical mastitis (CM) can provide data on epidemiological behavior of this pathogen and antimicrobial susceptibility (AMS) assessment at the genotypic level. This study genotypically characterized Staph. aureus isolates recovered from cows with CM and determined the association of genotypes and AMS. A total of 84 Staph. aureus strains identified from affected mammary quarters of cows with CM in 13 dairy herds from Southeastern Brazil were submitted for susceptibility testing to 10 antimicrobials using the technique of minimal inhibitory concentration. The same isolates were also genotyped using the spa-typing methodology. Results showed a high genotypic similarity between the Staph. aureus isolates within and between herds, which were categorized as resistant to most antimicrobials, especially to ß-lactam antibiotics. In addition, differences in AMS were observed among genotypic clusters, which may affect the efficacy of antimicrobials used to treat CM in different dairy herds.

Calculating clinical mastitis frequency in dairy cows: Incidence risk at cow level, incidence rate at cow level, and incidence rate at quarter level.

The lack of standardization in reporting clinical mastitis incidence limits the ability to compare results across multiple studies without additional calculations. There is both a biological and statistical rationale for evaluating the at-risk period at the quarter level. This study aimed to: (1) to outline an applied method for calculating clinical mastitis (CM) incidence rate at the quarter level using currently available software; and (2) to present the results of three different measurements: incidence risk at cow level, incidence rate at cow level, and incidence rate at quarter level. In an open population prospective cohort of eight commercial dairy farms monitored from May 15, 2016, to May 31, 2017, all CM cases (n = 7513) were identified by trained on-farm personnel, who collected all milk samples from all quarters with visibly abnormal milk. Microbiological identification was determined by culture and MALDI-TOF. All lactating quarters were at risk for CM. A quarter was at risk for a new CM case if there was at least 14 d between a previously diagnosed case and the current case in the same quarter, or if a different pathogen was isolated in the same quarter within 14 d. A total of 17,513,429 quarters days at risk (QDAR) were estimated. A statistical software macro and Structured Query Language (SQL) were used to bring all data together. The monthly incidence rate at the cow level was 16.6 cases per 10,000 cow-days, the monthly incidence rate at the quarter level was 4.4 cases per 10,000 QDAR and the monthly incidence risk at the cow level was 4.8 cases per 100 cows. Although the evaluation of QDAR requires additional computation when compared to other methods, it might allow for a more precise evaluation of the data and a more accurate evaluation of mastitis incidence. Clearly defining the methods used to report mastitis incidence will improve our ability to discuss and learn about the differences and similarities across studies, regions, and countries.

Whole-genome sequencing reveals high genetic diversity of Streptococcus uberis isolated from cows with mastitis.

BACKGROUND: Bovine mastitis is an important cause of economic loss in dairy farms. Streptococcus uberis is among the most frequently isolated bacterial species isolated from cows with mastitis. The aim of this study was to perform an in-depth genetic assessment of S. uberis strains isolated from bovine clinical mastitis (CM) and to perform a phylogenetic analysis to represent the evolutionary relationship among S. uberis sequences. RESULTS: A total of 159 isolates was genetically characterized using whole genome sequencing. According to the virulence determinants, all strains harbored the hasC, leuS, perR, purH, and purN virulence genes. Thirty-four resistance genes were identified in at least one strain. In terms of acquired genes, we observed that 152 (95.6 %) strains had a resistance gene to lincosamine (lnuD), 48 (30.2 %) to tetracycline (tetM), 4 (2.51 %) to tobramicine (ant6), and 1 to lincosamide (lsa(E)). MLST detected the Sequence Type (ST)797 (n = 23), while 85.5 % of the strains did not match to known STs. CONCLUSIONS: Then, eleven distinct ST were identified after we submitted the new alleles to assign new STs. The other prevalent STs observed were ST1215 (n = 58), ST1219 (n = 35), and ST1213 (n = 15). And it was not possible to identify the MLST of four strains. Phylogenetic lineages indicated a high genomic diversity of S. uberis in our collection, confirming that most strains isolated from bovine mastitis have different reservoirs, typical of environmental pathogens.

Association between antimicrobial use and antimicrobial resistance of Streptococcus uberis causing clinical mastitis.

It is unknown whether overuse of antimicrobials against clinical mastitis (CM) from Streptococcus uberis is associated with increased antimicrobial resistance (AMR). Therefore, the aim of this study was to evaluate the association between antimicrobial use (AMU) and AMR in relation to the Strep. uberis causing CM in dairy herds. A total of 83 Strep. uberis isolates were selected from a collection created during a previous study evaluating the epidemiology of CM in dairy herds (n = 17) of southeastern Brazil. For each case of CM identified on farm, the following information was recorded: cow's identification number, affected mammary quarter, date of CM diagnosis, antimicrobial commercial names, number of administrations, and descriptions of protocol changes during the treatment. Streptococcus uberis isolates were confirmed by conventional culture, MALDI-TOF mass spectrometry, and quantitative multiplex PCR analyses. Thus, a total of 8 antimicrobials commonly used for CM treatment were evaluated for antimicrobial activity against Strep. uberis isolates. The minimum inhibitory levels of antimicrobials were determined at the lowest concentrations able to inhibit 50 and 90%, respectively, of Strep. uberis isolates. Data related to the antibiotics used for treatment of CM was used to calculate the frequency of administered antimicrobials as the number of defined daily doses (DDD). The highest frequencies of resistant Strep. uberis were observed for erythromycin (80.7% resistant, R), tetracycline (R = 59%), and penicillin G (R = 57.8%), whereas against ceftiofur only 10.8% of Strep. uberis isolates were resistant, and only 1.2% of the Strep. uberis isolates were resistant to enrofloxacin. Regarding the evaluation of resistance for antimicrobial classes, the highest frequency was observed for macrolides (R = 80.7%; 19.3% susceptible, S). Additionally, a frequency of 18.7% of Strep. uberis isolates were resistant to cephalosporins (S = 81.3%), respectively. Further, 94% of Strep. uberis isolates were multiresistant; all these isolates presented resistance to at least 3 different antimicrobial classes. The overall monthly average of antimicrobial treatment incidence (ATI) among the 17 herds enrolled in the study was 23.7 DDD per 1,000 lactating dairy cows [standard deviation (SD) = 13.9], ranging from 5.0 to 55.4 DDD per 1,000 cows in lactation-day. Cephalosporins and penicillins were the most commonly used antimicrobial classes among the evaluated herds (n = 16; 94.1%), followed by tetracyclines (n = 15 herds; 88.2%), fluoroquinolones (n = 14; 82.3%), and sulfonamides (n = 14; 82.3%). The tetracycline class had the highest ATI mean (5.0 DDD per 1,000 lactating cow-days, SD = 5.8), followed by fluoroquinolones (4.7 DDD per 1,000 lactating cow-days, SD = 6.0) and cephalosporins (3.8 DDD per 1,000 lactating cow-days, SD = 6.0). The overall use of antimicrobials was associated with the resistance of Strep. uberis to the antimicrobial tetracycline.

Description of the Characteristics of Five Bedding Materials and Association With Bulk Tank Milk Quality on Five New York Dairy Herds.

Environmental mastitis represents a major challenge on dairy farms where contagious pathogens are controlled by improved milking procedures. Therefore, research focused on the environment is important to improve udder health programs. The objectives of this prospective and descriptive study were to (1) describe bedding bacterial counts, pH, and dry matter (DM) of five different bedding types (organic: manure solids, straw, paper fiber; inorganic: sand, recycled sand) and (2) explore the association between bedding bacterial counts with bulk tank milk quality. This study took place within five conveniently selected commercial dairy herds, each with a predominant bedding material in lactating pens. Bedding samples (used n = 237; fresh n = 53) were collected monthly from July 2018 to July 2019 following a standard operating procedure (SOP) to minimize sampling variability. Additionally, a bulk tank (BT) milk sample (n = 40) was collected on the same day unless milk had been picked up prior to arrival. Both BT and bedding samples were submitted to the laboratory for culture and bacterial identification and quantification of Streptococcus spp, coliforms, and non-coliforms as well as detection of several pathogens of mastitis importance. Somatic cell count was evaluated in BT samples. Within bedding type, the correlation between bedding characteristics and bacterial counts in bedding was evaluated using Pearson correlation. Within bedding type, the correlation between bacterial counts in bedding samples and bacterial counts in BT were determined. The Kruskal-Wallis test was used to evaluate the bacterial count by bedding type and to evaluate BT somatic cell count differences based on bedding type. In fresh bedding, bacterial counts were generally higher for manure solids for all bacterial groups compared with other materials. In used samples, organic materials had the highest levels of all bacterial groups. The proportion of samples with detectable organisms of mastitis importance varied within and among herds in both bedding and BT samples throughout the study period. In bedding samples, a higher DM content had the lowest levels of bacterial growth compared with those with lower DM content. Most bedding samples were on the alkaline side within a pH range of 8-11. No relationship between bacterial counts and pH was observed. No associations between BT bacteria counts and bedding bacterial counts were observed. No association between bulk tank somatic cell counts based on bedding type were observed. Despite using an SOP for bedding sampling in an effort to consistently collect samples, we still observed a large amount of variability both within and among bedding samples. This variability may have obscured any potential association between BT milk quality and bedding type.

Effect of metaphylactic administration of tildipirosin on the incidence of pneumonia and otitis and on the upper respiratory tract and fecal microbiome of preweaning Holstein calves.

The objectives of this study were to evaluate the effect of the metaphylactic use of a semi-synthetic long-acting macrolide (tildipirosin) on the prevention of pneumonia and otitis in preweaning Holstein calves, as well as its effects on the microbiome of their upper respiratory tract (URT) and feces. Newborn healthy Holstein heifers, collectively housed, were randomly allocated to 1 of 2 treatment groups: treatment (TRT; n = 932) or control (CTR; n = 927). Calves in the TRT group received a single subcutaneous injection of 4 mg/kg tildipirosin (Zuprevo, Merck Animal Health) at 7 ± 7 d of life. Calves in the CTR group received no drug injection. All enrolled calves were evaluated from 1 to 63 ± 3 d of life (weaning age) and monitored daily for any adverse health events during this period. Daily physical examination was performed to diagnose pneumonia and otitis, and body weight was measured weekly in all animals. From a randomly selected subset of 217 calves, blood samples for biochemical variables analysis and swabs were collected weekly from the URT and rectum for analysis of the nasal and fecal microbiome, respectively, via next-generation sequencing of the 16S rRNA gene. Total bacterial load was evaluated using quantitative PCR. In addition, another subset of 26 calves was randomly selected and fecal swabs were collected in a more intensive sampling to investigate the short-term effect of tildipirosin administration on the fecal microbiome. We performed general mixed linear models and logistic regression to analyze continuous and binary outcomes, respectively. Tildipirosin metaphylaxis reduced the incidence of otitis (CTR = 47.03%; TRT = 37.55%) and tended to reduce the incidence of pneumonia (CTR = 20.71%; TRT = 17.38%) and the overall mortality risk (CTR = 6.69%; TRT = 4.94%). We observed no significant differences between groups for mortality due to pneumonia (CTR = 0.86%; TRT = 0.97%) or mortality due to otitis (CTR = 2.05%; TRT = 1.39%). Calves in the TRT group had a higher average daily gain than calves in the CTR group. Furthermore, metaphylaxis had no significant effects on the total bacterial load, genus, or phylum analysis of the fecal microbiome from the 2 subset groups. However, for the URT microbiota, we observed a significant decrease in total bacterial load for the TRT group compared to the CTR group 1 week after metaphylactic injection. Tildipirosin metaphylaxis decreased the mean relative abundance of the genera Mannheimia, Moraxella, and Pasteurella but significantly increased the mean relative abundance of Mycoplasma. Although tildipirosin had no positive effect on Mycoplasma, it reduced the mean relative abundance of important pathogenic bacteria in the URT and had positive effects for the control of otitis. The metaphylactic use of tildipirosin can be a suitable strategy for the control of otitis on farms with a high prevalence of this disease.

Internal Teat Sealant Administered at Drying off Reduces Intramammary Infections during the Dry and Early Lactation Periods of Dairy Cows.

The effect of an internal teat sealant (ITS) on subsequent infection of the mammary gland was evaluated on the following mammary gland health indicators: (a) bacteriological cure of preexisting intramammary infections at drying off, (b) risk of postpartum new intramammary infections (NIMI), (c) cure and risk of new cases of subclinical mastitis (SCM), and (d) risk of postpartum clinical mastitis (CM). A total of 553 cows during late gestation were randomly assigned into two treatment protocols at drying off: (a) Dry cow therapy with 0.25 g of intramammary anhydrous cefalonium (ADCT; Cepravin®, MSD Animal Health); or (b) ADCT combined with ITS (SDCT; 4 g bismuth subnitrate; Masti-Seal®, MSD Animal Health, São Paulo, Brazil). Mammary quarter (MQ) milk samples were collected for microbiological culture and somatic cell count (SCC) at drying off and early lactation, and data from 1756 MQ were used in the multivariate logistic regression. There was no effect on the risk of bacteriological cure, SCM cure, and new cases of postpartum SCM. Still, SDCT reduced the risk of CM up to 60 days postpartum (DPP), overall NIMI risk, and the NIMI caused by major pathogens compared to ADCT. Thus, the DCT combined with ITS at drying off is effective for preventing NIMI during the dry period and CM up to 60 DPP.

Antimicrobial use for treatment of clinical mastitis in dairy herds from Brazil and its association with herd-level descriptors.

The aims of this prospective and descriptive study were to: (a) characterize treatment profile and quantify antimicrobial consumption for treatment of clinical mastitis (CM) in dairy herds of Brazil; and, (b) determine the association of antimicrobial use (AMU) for treatment of CM and herd-level descriptors, such as herd size, average milk yield, bulk milk somatic cell count (BMSCC), bulk milk total bacterial count (BMTBC), season and housing type. Data on treatment practices were obtained from 19 of 20 dairy herds selected for the study for a period of 12 months per herd. Treatment protocols were recorded in each case of CM by the farm personnel using a form that included information at the cow- and treatment-level. The frequency of antimicrobial consumption for treatment of CM was determined monthly in units of defined daily dose (DDD) and expressed as antimicrobial treatment incidence (ATI; the number of DDD per 1,000 lactating cow-days). Mixed linear regression models were used to determine the association between log-transformed ATI and herd level descriptors. The overall monthly mean ATI was 21.9 DDD per 1,000 lactating cow-days (15.4 DDD per 1,000 lactating cow-days for intramammary compounds, and 6.4 DDD per 1,000 lactating cow-days for systemically administered antimicrobials). Among intramammary drugs, aminoglycosides had the highest ATI (11.7 DDD per 1,000 lactating cow-days), followed by a treatment with a combination of tetracycline, aminoglycoside and polypeptide (10.3 DDD per 1,000 lactating cow-days). For systemically administrated antimicrobials, fluoroquinolones (6.1 DDD per 1,000 lactating cow-days), penicillin combinations (3.9 DDD per 1,000 lactating cow-days), and the combination of sulfonamide and pyrimidine (3.6 DDD per 1,000 cows per day) were the most frequently used antimicrobials. The use of combination therapy (i.e., association of intramammary and systemically administered antimicrobials) was reported for 64.3 % of treatments at the cow-level. The AMU tended to be higher in herds with highest BMSCC. In addition, a higher AMU for treatment of CM was observed during the rainy season compared to the dry season in Brazil. This seasonal effect was mostly characteristic in herds housing cows in outdoor housing systems (i.e., paddocks).

Genotyping and antimicrobial resistance of Streptococcus uberis isolated from bovine clinical mastitis.

A genotypic characterization of Streptococcus uberis isolated from clinical mastitis (CM) in dairy cows, and the association of Strep. uberis genotypes and antimicrobial susceptibility (AMS) was performed. A total of 89 isolates identified as Strep. uberis from 86 dairy cows with CM in 17 dairy herds of Southeastern Brazil were genotyped using random amplified polymorphic DNA (RAPD) analysis. After genotyping, two clusters (I and II) were created according to RAPD types. A commercial broth microdilution test was used to determine the susceptibility of Strep. uberis isolates to 8 antimicrobials (ampicillin, ceftiofur, cephalothin, erythromycin, penicillin, penicillin+novobiocin, pirlimycin and tetracycline). For each antimicrobial, we determined the minimal inhibitory concentrations that inhibit 50% (MIC50) and 90% (MIC90) of Strep. uberis strains. Differences in AMS among genotypic clusters were evaluated using mixed regression models. Overall, a great polymorphism (56 RAPD-types) was found among Strep. uberis isolates, although a higher genetic similarity (based on the PCR bands features) was observed within herds after genotypic clustering. No differences in AMS were observed among clusters. Strep. uberis isolated from bovine CM were resistant to most antimicrobials, with the exception of cephalothin and penicillin+novobiocin.

Association of herd-level risk factors and incidence rate of clinical mastitis in 20 Brazilian dairy herds.

The objectives of this study were to characterize the pathogen frequency and severity of clinical mastitis (CM) in 20 dairy herds of southeastern Brazil; and to determine the incidence rate of clinical mastitis (IRCM; overall and based on specific-pathogen groups) based on quarter time at risk and its association with risk factors at the herd-level. Data were recorded in each herd for a period of 8 to 15 months. The association between herd-level risk factors and IRCM were determined by two groups of mixed regression models: one based on the overall IRCM, and five based on the following specific-pathogen groups: contagious, other Gram-positive, Gram-negative, other, and negative culture. The following herd-level risk factors were evaluated: herd size, housing system, average daily milk yield per cow, bulk milk somatic cell count (BMSCC), and bulk milk total bacterial count (BMTBC). A total of 5957 quarter-cases of CM were recorded from 2637 cows, but only 4212 cases had milk samples collected for culture. The most frequently isolated pathogens were Escherichia coli (6.6% of total cultures), Streptococcus uberis (6.1%), and Streptococcus agalactiae (5.9%). The majority of CM cases were mild (60.3%), while 34.1% were moderate and 5.6% severe. The frequency of severe CM cases was lower for those with a Gram-positive result (4.6%) compared to a Gram-negative result (11.4%). Overall, monthly mean IRCM was 9.7 cases per 10,000 quarter-days at risk (QDAR). Herds with a geometric mean BMSCC ≥ 601 × 103 cell/mL had higher overall IRCM (16/10,000 QDAR) than those with BMSCC ≤ 600 × 103 cell/mL (≤7.7/10,000 QDAR). When the specific-pathogen groups were evaluated, for contagious pathogens, variables housing (free-stalls or compost-bedded pack barns), BMSCC (≥601 × 103 cells/mL), and average daily milk yield per cow (21 and 25 Kg/d) presented the highest IRCM. Furthermore, in Gram-negative group, herds with BMTBC ≥ 31 × 103 cfu/mL had higher IRCM compared with herds with BMTBC ≤ 30 × 103 cfu/mL. Although environmental pathogens were the most common cause of CM in this study, contagious pathogens (e.g., Strep. agalactiae and Staph. aureus) are still a concern in dairy herds of Brazil. Additionally, as there were some herd-level risk factors associated with the IRCM, there may be opportunity for management strategies aiming to improve the control of CM in dairy herds.

Molecular characterization and antimicrobial susceptibility pattern of Streptococcus agalactiae isolated from clinical mastitis in dairy cattle.

The objectives of this study were to: (a) genotypically characterize Streptococcus agalactiae isolates recovered from clinical mastitis (CM) cases in dairy cows and, (b) determine the association of antimicrobial susceptibility (AMS) and genotypes of Strep. agalactiae clustered according to the genetic similarity. A total of 89 Strep. agalactiae isolates recovered from bovine CM were genotyped using random amplified polymorphic DNA (RAPD) analysis. In addition, the AMS of the isolates was determined using a commercial broth microdilution test composed of 10 antimicrobials (penicillin, ampicillin, oxacillin, cephalothin, ceftiofur, penicillin/novobiocin, erythromycin, pirlimycin, tetracycline, and sulfadimethoxine). Descriptive analysis was used to report the frequency of RAPD-types and genotypic clusters within herd, housing system, season and CM severity scores. The minimal antimicrobial concentrations that inhibited 50% (MIC50) and 90% (MIC90) of the isolates were calculated and survival analysis was completed to verify the differences of AMS among genotypic clusters. Results of RAPD showed a great genotypic diversity of Strep. agalactiae (45 RAPD-types) and three clusters (Ia, Ib and II) were created based on the genetic similarity among genotypes. After clustering, a high genetic similarity was observed within and between herds. Overall, Strep. agalactiae showed high susceptibility to most antimicrobials, except to tetracycline and erythromycin. Differences in the AMS among clusters were observed for ampicillin, ceftiofur, erythromycin, pirlimycin, sulfadimethoxine and tetracycline. In conclusion, Strep. agalactiae is still highly susceptible to most antimicrobials, although differences in susceptibility to certain antimicrobials were observed among genotypic clusters.

Rapid identification of bovine mastitis pathogens by MALDI-TOF Mass Spectrometry / Rápida identificação de agentes causadores de mastite por espectrometria de massas MALDI-TOF

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been shown to be an alternative method for identification of bacteria via their protein profile spectra, being able to identify bacteria at the genus, species and even at subspecies level. With the aim of large-scale identification of pathogens causing mastitis by this platform, a total of 305 isolates of bacteria identified from cows with subclinical mastitis were analyzed by conventional microbiological culture (MC) as well as by MALDI-TOF MS coupled with Biotyper data processing. Approximately 89% of the identifications performed by MALDI-TOF MS were consistent with results obtained by MC. From the remaining isolates (11%), 6.3% of isolates were classified as misidentified (discordance for both genus and species level), and 4.7% showed identification agreement at the genus level but not at the species level, being classified as unidentified at species level. The disagreement results were mostly associated with identification of Streptococcus and Enterococcus species probably due to the narrow phenotypic similarity between these two genera. These disagreement results suggest that biochemical assays might be prone to identification errors and, MALDI-TOF MS therefore may be an alternative to overcome incorrect species-specific identification. Standard microbiological methods for bovine mastitis diagnosis are time consuming, laborious and prone to errors for some bacteria genera. In our study, we showed that MALDI-TOF MS coupled with Biotyper may be an alternative method for large-scale identification of bacteria isolated from milk samples compared to classical microbiological routine protocols.(AU)

A espectrometria de massas (MALDI-TOF MS) tem mostrado ser um método alternativo para a identificação de bactérias, sendo capaz de identificar as bactérias causadoras de mastite em gênero, espécie ou até mesmo subespécie. Com o objetivo de identificar os patógenos causadores de mastite em grande-escala por esta plataforma, um total de 305 isolados bacterianos oriundos de vacas com mastite subclínica foram analisados pela cultura microbiológica convencional (CM) e pela MALDI-TOF MS acoplada ao software Biotyper. Aproximadamente 89% das identificações realizadas pela MALDI-TOF MS foram consistentes com os resultados obtidos pela CM. Do restante de isolados bacterianos (11%), 6,3% foram classificados como identificação errônea (discordância de gênero e espécie), e 4,7% apresentaram concordância de gênero, mas discordância da espécie. Os resultados que apresentaram divergência estavam mais associados com a identificação das espécies de Streptococcus spp. e Enterococcus spp. devido à similaridade fenotípica entre os dois gêneros. Estes resultados divergentes sugerem que os ensaios bioquímicos podem ser propensos a erros de identificação, por isso a MALDI-TOF MS pode ser considerada um método alternativo para superar os erros de identificação da CM. A cultura microbiológica padrão e os ensaios bioquímicos utilizados na identificação de agentes causadores de mastite são demorados, trabalhosos e propensos a erros quando utilizados na identificação em nível de espécie. No presente estudo, demonstramos que a MALDI-TOF MS acoplada ao software Biotyper pode ser considerada um método alternativo de identificação de bactérias causadoras de mastite em grande-escala quando comparado com a cultura microbiológica convencional.(AU)

Randomized clinical trial comparing ceftiofur hydrochloride with a positive control protocol for intramammary treatment of nonsevere clinical mastitis in dairy cows.

The objective of this study was to compare ceftiofur hydrochloride with a positive control protocol for intramammary treatment of nonsevere clinical mastitis in dairy cows. A total of 264 clinical mastitis cases on 11 commercial dairy farms were treated with intramammary infusions, once a day for 4 d using 1 of 2 treatments: (1) ceftiofur hydrochloride 125mg; or (2) control: tetracycline 200mg + neomycin 250mg + bacitracin 28mg + prednisolone 10mg. Streptococcus agalactiae was the most frequently isolated gram-positive pathogen from clinical mastitis, followed by Staphylococcus aureus. Klebsiella spp. and Escherichia coli were the most isolated gram-negative bacteria from clinical mastitis. No significant differences were observed between treatments regarding the overall clinical cure, bacteriological cure, and new infection. No effect of treatment regimen was observed when the bacterial group (gram-positive vs. gram-negative) was evaluated. The overall clinical cure was 0.79 for ceftiofur-treated cows and 0.74 for control-treated cows, whereas the overall bacteriological cure was 0.79 for ceftiofur-treated cows and 0.76 for control-treated cows. Furthermore, the new intramammary infection was 0.10 for cows treated with ceftiofur and 0.11 for cows treated with control. In conclusion, the use of intramammary ceftiofur hydrochloride for treatment of nonsevere clinical mastitis has similar efficacy as a treatment regimen with a combination of antimicrobial agents (tetracycline + neomycin + bacitracin).

Effects of bovine subclinical mastitis caused by Corynebacterium spp. on somatic cell count, milk yield and composition by comparing contralateral quarters.

Subclinical mastitis caused by Corynebacterium spp. (as a group and at the species level) was investigated by evaluating contralateral (healthy and infected) mammary quarters for somatic cell count (SCC), milk yield and composition. Selection of cows with subclinical mastitis caused by Corynebacterium spp. was performed by microbiological culture of composite samples collected from 1242 dairy cows from 21 dairy herds. For each of the selected cows, milk yield was measured and milk samples were collected at the mammary quarter level (i.e., 1140 mammary samples collected from 285 cows) for analysis of milk composition and SCC. The identification of Corynebacterium spp. isolates was performed by 16S rRNA gene sequencing. One hundred and eighty Corynebacterium spp. isolates were identified, of which 167 (92.77%) were C.bovis and eight (4.44%) non-C.bovis; for five of the Corynebacterium spp. isolates (2.77%), sequencing of 16S rRNA genes did not allow identification at the species level. Mammary quarters infected with Corynebacterium spp. as a group had a higher geometric mean SCC (197,900 cells/mL) than healthy contralateral mammary quarters (85,800 cells/mL). Species of Corynebacterium non-C.bovis were infrequently isolated and did not change SCC, milk yield or milk solid contents when evaluated at the contralateral quarter level. Although C.bovis infection showed no effect on milk yield, fat, protein, casein or total solids in milk, it increased SCC and decreased lactose and milk solids non-fat content.

Effect of the ensiling time of hydrated ground corn on silage composition and in situ starch degradability / Efeito do tempo de ensilagem do milho moído hidratado sobre a composição da silagem e degradabilidade in situ do amido

The aim of this study was to evaluate the effect of time of ensiling on chemical composition and in situ degradability of starch of hydrated ground corn (HGC) with medium grain vitreousness. Corn grains harvested at 83% of dry matter (DM) and vitreosity content of 67% ± 3, were dried to 87% DM. Grains were milled into a device with 2 mm sieve, reconstituted to reach 67% DM, and ensiled (density of 880 Kg/m³) for up to 330 days. One HGC sample was collected monthly for in situ determination of composition, fermentation end products and for corn starch degradability. Ensiling time did not affect the DM and crude protein (CP) content of the HGC. However, starch concentration was reduced by 2.4 percentage points at 330 days compared to 3 days of ensiling. Increased concentrations of NH3-N (8.5 times), lactic acid (3.45 times), acetic acid (4.1 times), propionic acid (1.7 times), butyric acid (2.8 times) and alcohol (2.4 times) were observed during the ensiling period. The rapidly degradable fraction (fraction A) and the rate of degradation of the slowly degradable fraction (fraction C) of HGC starch were increased 3.51 and 2.21 times, respectively, during the ensiling period. Conversely, the slowly degradable fraction (fraction B) of the HGC starch was decreased 1.93 times during the ensiling period. The effective degradability of the starch ofHGC increased for passage rates by 0.02/h (79.9% vs. 94.5%); 0.05/h (65.9% vs 90.01%) and 0.08/h (56.98 vs. 86.52%) when it was evaluated at 3 vs 330 days of ensiling, respectively. In conclusion, ensiling time affected the chemical composition and increases rumen starch degradability of HGC with medium vitreousness of the grain endosperm...

O objetivo deste estudo foi avaliar o efeito do tempo de ensilagem sobre a composição química e a degradabilidade in situ do amido do milho moído hidratado (MMH) em grãos de média vitreosidade. Os grãos de milho foram colhidos com 83% de matéria seca (MS) e vitreosidade de 67% ± 3, e foram secos até atingirem 87% de MS. Os grãos foram moídos a dois milímetros, sendo posteriormente reconstituídos, 67% MS, e ensilados (densidade de 880 kg / m³) para até 330 dias. Uma amostra MMH foi coletado mensalmente para a determinação da composição, produtos finais da fermentação e para degradabilidade in situ do amido de milho. O tempo de ensilagem não afetou o teor de MS e proteína bruta (PB). No entanto, a concentração de amido foi reduzido em 2,4 pontos percentuais em comparação de 3 com 330 dias de ensilagem. Foram observados o aumento das concentrações de N-NH3 (8,5 vezes), ácidos láctico (3,45 vezes), acético (4,1 vezes), propionico (1,7 vezes), butírico (2,8 vezes) e álcool (2,4 vezes), durante o período de ensilagem. A fracção rapidamente degradável (fração A) e a taxa de degradação da fracção lentamente degradável (fração C) do amido do MMH foram aumentadas 3,51 e 2,21 vezes, respectivamente, durante o período de ensilagem. Por outro lado, a fração lentamente degradável (fração B) do amido do MMH foi diminuída em 1,93 vezes durante o período de ensilagem. A degradabilidade efetiva do amido do MMH foi aumentado para as taxas de passagem de 0,02 / h (79,9% vs. 94,5%); 0,05 / h (65,9% vs 90,01%) e de 0,08/h (56,98% vs. 86,52%)quando foi comparada o período de 3 vs 330 dias de ensilagem, respectivamente. Em conclusão, o tempo de ensilagem afetou acomposição química e aumentou a degradabilidade ruminal do amido do MMH de grãos com média vitreosidade...

Identification of Corynebacterium spp. isolated from bovine intramammary infections by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Corynebacterium species (spp.) are among the most frequently isolated pathogens associated with subclinical mastitis in dairy cows. However, simple, fast, and reliable methods for the identification of species of the genus Corynebacterium are not currently available. This study aimed to evaluate the usefulness of matrix-assisted laser desorption ionization/mass spectrometry (MALDI-TOF MS) for identifying Corynebacterium spp. isolated from the mammary glands of dairy cows. Corynebacterium spp. were isolated from milk samples via microbiological culture (n=180) and were analyzed by MALDI-TOF MS and 16S rRNA gene sequencing. Using MALDI-TOF MS methodology, 161 Corynebacterium spp. isolates (89.4%) were correctly identified at the species level, whereas 12 isolates (6.7%) were identified at the genus level. Most isolates that were identified at the species level with 16 S rRNA gene sequencing were identified as Corynebacterium bovis (n=156; 86.7%) were also identified as C. bovis with MALDI-TOF MS. Five Corynebacterium spp. isolates (2.8%) were not correctly identified at the species level with MALDI-TOF MS and 2 isolates (1.1%) were considered unidentified because despite having MALDI-TOF MS scores >2, only the genus level was correctly identified. Therefore, MALDI-TOF MS could serve as an alternative method for species-level diagnoses of bovine intramammary infections caused by Corynebacterium spp.

Identification of coagulase-negative staphylococci from bovine intramammary infection by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Coagulase-negative staphylococci (CoNS) are among the main pathogens causing bovine intramammary infection (IMI) in many countries. However, one of the limitations related to the specific diagnosis of CoNS is the lack of an accurate, rapid, and convenient method that can differentiate the bacterial species comprising this group. The aim of this study was to evaluate the ability of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to accurately identify CoNS species in dairy cow IMI. In addition, the study aimed to determine the frequency of CoNS species causing bovine IMI. A total of 108 bacterial isolates were diagnosed as CoNS by microbiological cultures from two milk samples collected from 21 dairy herds; the first sample was collected at the cow level (i.e., 1,242 composite samples from all quarters), while the second sample was collected at the mammary quarter level (i.e., 1,140 mammary samples collected from 285 cows). After CoNS isolation was confirmed by microbiological culture for both samples, all CoNS isolates (n=108) were genotypically differentiated by PCR restriction fragment length polymorphism (RFLP) analysis of a partial groEL gene sequence and subjected to the MALDI-TOF MS identification procedure. MALDI-TOF MS correctly identified 103 (95.4%) of the CoNS isolates identified by PCR-RFLP at the species level. Eleven CoNS species isolated from bovine IMI were identified by PCR-RFLP, and the most prevalent species was Staphylococcus chromogenes (n=80; 74.1%). In conclusion, MALDI-TOF MS may be a reliable alternative method for differentiating CoNS species causing bovine IMI.

Morphological characteristics of maize plants in estimate the silage chemical composition / Características morfológicas de plantas de milho estimam a composição bromatológica da silagem

The aim of this study was to estimate the chemical composition of maize silage based on the morphological characteristics of maize plants and to evaluate the effect of nitrogen fertilization and the inclusion of a microbial inoculant during the ensiling process on the production of maize silage and its morphological, qualitative and fermentative characteristics. The experimental treatments consisted of four levels of nitrogen fertilization with urea (0, 100, 200 and 300 kg ha-1) and the inclusion or exclusion of the microbial inoculants during the ensiling process. A completely randomized design was used in a 4 × 2 factorial arrangement of treatments. The maize silage chemical composition was estimated by evaluating the plant height (PH) and ear characteristics (NRE= number of rows per ear; NKE= number of kernels per ear; ELS= ear length with straw; EL= ear length without straw) using the following equations:CP= -12.44 + 5.871 × PH + 0.01814 × NRE² (R²= 0.89; P < 0.0001); NDF= 587.93-0.78×NKE-11.67×ELS-0.47×EL+0.0000007×NKE³+0.006× EL³ (R²=0.92; P = 0.003); ADF= 41.48 -0.046 × NRE2 (R2 = 0.42; P = 0.02);TDN= 57.81 - 0.0319 × NRE2 (R2 = 0.42; P = 0.02);EDDM= 56.58 + 0.035 × NRE2 (R2 = 0.42; P = 0.02) andNEL= 1.31 + 0.000757 × NRE2 (R2 = 0.41; P = 0.02). In conclusion, nitrogen fertilization increases the silage energy and protein content; while the inclusion of microbial inoculants during the ensiling process does not alter the chemical and fermentative characteristics of the maize silage.

O objetivo do presente estudo foi estimar a composição bromatológica da silagem por meio de características morfoló- gicas das plantas de milho, e avaliar o efeito de níveis de adubação nitrogenada e da inclusão de inoculante microbiano na ensilagem sobre as características produtivas, morfológicas, bromatológicas e fermentativas de silagem de milho. Os tratamentos consistiram em quatro níveis de adubações com ureia: 0; 100; 200 e 300 Kg ha-1, e da inclusão ou não de inoculante microbiano. Foi utilizado um delineamento inteiramente casualizado, com arranjo fatorial de tratamentos 4 × 2. A composição bromatológica da silagem foi estimada pela altura das plantas (AP) de milho e pelas características da espiga (NLE = número de linhas de grãos por espiga; NGE = Número de grãos por espiga; CCP = comprimento da espiga com palha; CSP = comprimento da espiga sem palha) por meio das seguintes equações: PB = -12,44 + 5,871 × AP + 0,01814 × NLE² (R² = 0,89; P < 0,0001); FDN = 587,93 – 0,78 × NGE – 11,67 × CCP – 0,47 × CSP + 0,0000007 × NGE³ + 0,006 × CCP³ (R² = 0,92; P = 0,003); FDA = 41,48 - 0,046 × NLE2 (R2 = 0,42; P = 0,02); NDT = 57,81 – 0,0319 × NLE2 (R2 = 0,42; P = 0,02); DMSE = 56,58 + 0,035 × NLE2 (R2 = 0,42; P = 0,02) e ELL = 1,31 + 0,000757 × NLE2 (R2 = 0,41; P = 0,02). Pode-se concluir que a adubação nitrogenada aumenta o teor de energia e de proteína da silagem; enquanto a inclusão de inoculante microbiano não altera as características bromatológicas e fermentativas da silagem de milho.