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Staphylococcus aureus is recognized worldwide as one of the major agents of dairy cow intra-mammary infections. This microorganism can express a wide spectrum of pathogenic factors used to attach, colonize, invade and infect the host. The present study evaluated 120 isolates from eight different countries that were genotyped by RS-PCR and investigated for 26 different virulence factors to increase the knowledge on the circulating genetic lineages among the cow population with mastitis. New genotypes were observed for South African strains while for all the other countries new variants of existing genotypes were detected. For each country, a specific genotypic pattern was found. Among the virulence factors, fmtB, cna, clfA and leucocidins genes were the most frequent. The sea and sei genes were present in seven out of eight countries; seh showed high frequency in South American countries (Brazil, Colombia, Argentina), while sel was harboured especially in one Mediterranean country (Tunisia). The etb, seb and see genes were not detected in any of the isolates, while only two isolates were MRSA (Germany and Italy) confirming the low diffusion of methicillin resistance microorganism among bovine mastitis isolates. This work demonstrated the wide variety of S. aureus genotypes found in dairy cattle worldwide. This condition suggests that considering the region of interest might help to formulate strategies for reducing the infection spreading.
Mastitis poses a considerable threat to productivity and to animal welfare on modern dairy farms. However, the common way of antibiotic treatment does not always lead to a cure. Unsuccessful cures can, among other reasons, occur due to biofilm formation of the causative agent. This has attracted interest from researchers to introduce promising alternative therapeutic approaches, such as the use of beneficial lactic acid bacteria (LAB). In fact, using LAB for treating mastitis probably requires the formation of a beneficial biofilm by the probiotic bacteria. The present study investigated the ability of five LAB strains, selected on the basis of results from previous studies, to remove and to replace pathogenic biofilms in vitro. For this purpose, Staphylococcus (S.) aureus ATCC 12,600 and two strains—S. xylosus (35/07) and S. epidermidis (575/08)—belonging to the group of coagulase negative staphylococci (CNS) were allowed to form biofilms in a 96-well plate. Subsequently, the LAB were added to the well. The biofilm challenge was evaluated by scraping off and suspending the biofilm cells, followed by a plate count of serial dilutions using selective media. All the LAB strains successfully removed the staphylococcal biofilms. However, only Lactobacillus (L.) rhamnosus ATCC 7469 and L. plantarum 2/37 formed biofilms of their own to replace the pathogenic ones.
In order to reduce antimicrobial treatment and prevent environmental mastitis, the aim of the present study was to investigate associations between herd level factors and microbial load on teat ends with environmental mastitis pathogens. Quarterly farm visits of 31 dairy farms over a one-year period were used for statistical analysis. During each farm visit, teat-skin swabs, bedding and air samples were taken and management practices and herd parameters were documented. Total mesophilic bacteria, esculin-positive streptococci and coliform bacteria were examined in the laboratory procedures from teat skin and environmental samples. Esculin-positive streptococci and coliform bacteria on teat ends increased with high temperature–humidity indices (THI) in the barn during the spring and summer. Significantly more coliform bacteria on teat ends were found in herds with an increased percentage of normal or slightly rough teat ends. Cleaning cubicles more frequently, pre-cleaning teats before milking as well as post-dipping them after milking had a decreasing effect of teat-skin load with total mesophilic and coliform bacteria at the herd level. To conclude, teat-skin bacterial load with environmental pathogens is subject to fluctuations and can be influenced by aspects of farm hygiene.
To reduce the negative effects of mastitis in dairy heifers in early lactation on the future milking performance, the aim of this study was to define the time-related period of intramammary infections and to relate this to risk factors at heifer and quarter level for intramammary infections and subclinical mastitis. In total, 279 German Holstein Frisian heifers in three farms in Northern and Eastern Germany were included in this study. Quarter milk samples for cytomicrobiological examination were collected 3 +- 1 days after calving and 17 +- 3 days after calving, and risk factors
at heifer and quarter level associated with intramammary infections and clinical mastitis were recorded during the trial period. Data were analyzed using logistic regression procedures and odds ratios were calculated. Calving at older ages increased the odds of intramammary infections with non-aureus staphylococci (NAS) and coryneforms 17 +- 3 days after calving compared to heifers calving at a younger age. Detaching of milking cups during milking is a risk factor for new infections between day 3 +- 1 and 17 +- 3 postpartum. The milk yield after calving is associated with a decrease in intramammary infections with environmental pathogens and clinical mastitis. A high milk yield assists the development of udder edema with an increased risk of intramammary infections with NAS and coryneforms. An increased somatic cell count (SCC) after calving increased the odds of intramammary infections with contagious pathogens 17 +- 3 days postpartum. The early lactation has an important influence on udder health and intramammary infections postpartum in dairy heifers. Udder quarters eliminated pathogens during early lactation by 6.9% for cases in
this study. New infections manifest themselves up until 17 +- 3 days postpartum, especially with Corynebacterium spp. and NAS. In total, 82.9% of the infected quarters showed new infections with another pathogen species 17 +- 3 days postpartum than 3 +- 1 days postpartum. In conclusion, the early lactation has an important influence on udder health and intramammary infections postpartum in heifers with the possibility that udder quarters eliminate pathogens, but also the danger that new infections manifest themselves. Thus, the prevention of new infections by minimizing the associated risk factors is of great importance.
The lytic efficacy of bacteriophages against Staphylococcus aureus isolates from bovine milk was investigated in vitro, regarding possible applications in the therapy of udder inflammation caused by bacterial infections (mastitis). The host range of sequenced, lytic bacteriophages was determined against a collection of 92 Staphylococcus (S.) aureus isolates. The isolates originated from quarter foremilk samples of clinical and subclinical mastitis cases. A spot test and a subsequent plaque assay were used to determine the phage host range. According to their host range, propagation and storage properties, three phages, STA1.ST29, EB1.ST11, and EB1.ST27, were selected for preparing a bacteriophage mixture (1:1:1), which was examined for its lytic activity against S. aureus in pasteurized and raw milk. It was found that almost two thirds of the isolates could be lysed by at least one of the tested phages. The bacteriophage mixture was able to reduce the S. aureus germ density in pasteurized milk and its reduction ability was maintained in raw milk, with only a moderate decrease compared to the results in pasteurized milk. The significant reduction ability of the phage mixture in raw milk promotes further in vivo investigation.
The present research study investigated the susceptibility of common mastitis pathogens—obtained from clinical mastitis cases on 58 Northern German dairy farms—to routinely used antimicrobials. The broth microdilution method was used for detecting the Minimal Inhibitory Concentration (MIC) of Streptococcus agalactiae (n = 51), Streptococcus dysgalactiae (n = 54), Streptococcus uberis (n = 50), Staphylococcus aureus (n = 85), non-aureus staphylococci (n = 88), Escherichia coli (n = 54) and Klebsiella species (n = 52). Streptococci and staphylococci were tested against cefquinome, cefoperazone, cephapirin, penicillin, oxacillin, cloxacillin, amoxicillin/clavulanic acid and cefalexin/kanamycin. Besides cefquinome and amoxicillin/clavulanic acid, Gram-negative pathogens were examined for their susceptibility to marbofloxacin and sulfamethoxazole/trimethoprim. The examined S. dysgalactiae isolates exhibited the comparatively lowest MICs. S. uberis and S. agalactiae were inhibited at higher amoxicillin/clavulanic acid and cephapirin concentration levels, whereas S. uberis isolates additionally exhibited elevated cefquinome MICs. Most Gram-positive mastitis pathogens were inhibited at higher cloxacillin than oxacillin concentrations. The MICs of Gram-negative pathogens were higher than previously reported, whereby 7.4%, 5.6% and 11.1% of E. coli isolates had MICs above the highest concentrations tested for cefquinome, marbofloxacin and sulfamethoxazole/trimethoprim, respectively. Individual isolates showed MICs at comparatively higher concentrations, leading to the hypothesis that a certain amount of mastitis pathogens on German dairy farms might be resistant to frequently used antimicrobials.
The aim of the current study was to investigate the effects of the prepartum external treatment of teats with a combination of four lactic acid bacteria strains viz. Lactobacillus (Lb.) rhamnosus ATCC 7469, Lactococcus lactis subsp. lactis ATCC 11454, Lb. paracasei 78/37 (DSM 26911), and Lb. plantarum 118/37 (DSM 26912) on the postcalving udder health of dairy heifers. The study used a split-udder design. Two weeks before the expected calving date, one of two contralateral teats of a teat pair was dipped with an aqueous suspension of lactic acid bacteria (final bacterial counts 8.40–8.47 log10-transformed CFU/mL) once in a week until calving; the other teat of the pair was not treated. After calving, quarter foremilk samples were taken and investigated cyto-microbiologically. In total, 629 teat pairs of 319 heifers were included. There was an association between the treatment and intramammary infections caused by the major udder-pathogenic bacteria Staphylococcus aureus, Streptococcus dysgalactiae, and enterococci, as well as clinical mastitis in the first 100 days after calving. The present study indicates that intramammary infections with major pathogens and clinical mastitis may be prevented by regular prepartum external application of lactic acid bacteria in dairy heifers.
The antimicrobial activity of a phagemixture and a lactic acid bacteriumagainst Staphylococcus aureus isolates from bovine origin was investigated in vitro with regard to possible applications in the therapy of udder inflammation (mastitis) caused by bacterial infections. The S. aureus isolates used for inoculation derived from quarter foremilk samples of mastitis cases. For the examination of the antimicrobial activity, the reduction of the S. aureus germ density was determined [log10 cfu/mL]. The phage mixture consisted of the three obligatory lytic and S. aureus-specific phages STA1.ST29, EB1.ST11 and EB1.ST27 (1:1:1). The selected Lactobacillus plantarum strain with proven antimicrobial properties and the phage mixture were tested against S. aureus in milk, both alone and in combination. The application of the lactic acid bacterium showed only a low reduction ability for a 24 h incubation period. The bacteriophage mixture as well as its combination with the lactic acid bacterium showed high antimicrobial activity against S. aureus for a 24 h incubation period at 37 C, with only the phage mixture showing significance.
The control of clinical mastitis on dairy farms is an essential part of animal health management. Knowledge of the causative microorganisms, the cure rates achievable in the field and essential associated factors are crucial for proper control. The objectives of the present study were to characterize clinical mastitis cases in Germany and to analyze factors influencing cure rates and the recurrence rate. Milk samples of every clinical mastitis case occurring on 12 participating farms were examined cytomicrobiologically. Post-treatment quarter samples were taken after 14 and 21 days. Treatments were performed according to existing farm protocols. Of 2883 clinical mastitis cases, the most prevalent pathogens were Streptococcus (S.) uberis (20.2%) and coliforms (11.6%). In 35% of the milk samples, no bacteriological growth was detected. The overall bacteriological cure rate was 73.3%, while the cytological cure rate was 22.3%, the full cure rate 21.4% and the recurrence rate 18.8%. Regarding the pathogen distribution of severe mastitis, coliform bacteria were detected in 30.5% of the cases, whereas S. uberis was detected in 26.5% thereof. The results show that severe mastitis is caused almost as frequently by Gram-positive as by Gram-negative microorganisms. The low cytological cure rates show that the therapy needs to be further developed with regard to calming the inflammation. The obtained data can be very helpful in assessing internal mastitis scenarios and the effect of measures and therapies.
The objective of this study was to investigate the occurrence of bacteremia in dairy cows with severe mastitis. Milk samples were collected from affected udder quarters, and corresponding blood samples were collected from dairy cows with severe mastitis at the time of diagnosis before any therapeutic measures were undertaken. The cultural detection of pathogens in blood classified a bacteremia. Further diagnostic tests were performed to provide evidence of bacteremia. This was realized by PCR with regard to S. aureus, E. coli and S. uberis and the Limulus test. Detection of culturable pathogens in the blood of cows with severe clinical mastitis was rare and occurred in only one of 70 (1.4%) cases. Overall, bacterial growth was detected in 53 of 70 (75.7%) milk samples. S. uberis (22/70), E. coli (12/70) and S. aureus (4/70) were the most frequently isolated pathogens from milk of cows with severe mastitis. PCR was performed in 38 of 70 (54.3%) blood samples. PCR was positive in eight of 38 cases. S. uberis was found most frequently in six blood samples (8.6%). E. coli was found on PCR in one blood sample (1.4%). S. aureus was identified in one blood sample (1.4%). When Coliforms were detected in the quarter milk sample, a Limulus test was performed in the corresponding blood sample. In three of 15 cases, the Limulus test was positive (4.3% of samples). Further studies are needed to investigate the occurrence of bacteremia in cows with severe mastitis in a higher population size.
The aim of this study was to define the time-related period of intramammary infections and its relation to risk factors for intramammary infections and clinical mastitis at cow and quarter levels. In total, 269 German Holstein Frisian dairy cows on three farms in Northern and Eastern Germany were included in this study. Quarter milk samples were collected at dry-off, 3 ± 1 days after calving and 17 ± 3 days after calving, for cytomicrobiological examination. Risk factors at quarter- and cow-level associated with intramammary infections and clinical mastitis were recorded during the trial period. Data were analyzed using logistic regression procedures and odds ratios were calculated. Calving for the second time increased the odds of clinical mastitis during the first 100 days of lactation compared to cows calving for the third time or more. A high milk yield after calving was a risk factor for new infections, with environmental pathogens 17 ± 3 days postpartum. A body condition score after calving less than 3.5 was associated with a decreased risk of having an intra-mammary infection (IMI) with non-aureus staphylococci and coryneforms 3 ± 1 days postpartum and consistent body condition between dry-off and early lactation decreased the risk of intramammary infections after calving. The absence of a ring of hyperkeratosis at the teat apex shown at dry-off was associated with a lower risk of intramammary infections with environmental pathogens 17 ± 3 days postpartum. This study shows the important influence of the dry period and early lactation on intramammary infections and clinical mastitis postpartum in dairy cows. Udder quarters may have eliminated pathogens during the dry period in 43.6% of cases in this study. Additionally, new infections occurred during early lactation, so 5.1% more quarters were infected 17 ± 3 days compared to 3 ± 1 days postpartum. New infections can be traced to non-aureus staphylococci and Staphylococcus aureus from dry-off up until 3 ± 1 days postpartum, and to non-aureus staphylococci, Staphylococcus aureus and Streptococcus uberis, after calving. In total, 88.7% of the infected quarters showed new infections with another pathogen species 3 ± 1 days postpartum than at dry-off, and 89.2% of the quarters 17 ± 3 days postpartum than 3 ± 1 days postpartum. In conclusion, the early lactation has just as important an influence on intramammary infections postpartum in dairy cows as the dry period. There is the possibility that udder quarters eliminate pathogens during the early lactation, especially during the dry period. However, there is also the danger that new infections manifest, with a large proportion of new infections occurring after calving. Thus, additional control strategies are of great importance to prevent new infections occurring during early lactation as well as during the dry period to reduce negative effects on milk yield and culling hazards in dairy cows by minimizing the associated risk factors
To reduce ineffective antimicrobial usage in the treatment of non-severe clinical mastitis (CM) in cows with long-lasting udder diseases, non-antibiotic therapy with a non-steroidal anti-inflammatory drug (NSAID) was conducted and evaluated in a non-blinded, positively controlled, non-inferiority trial. Therefore, three-time systemic ketoprofen treatment at intervals of 24 h was evaluated in comparison with the reference treatment of solely antibiotic therapy in a field study on nine free-stall dairy farms located in Northern Germany. Cows with previous CM cases in current lactation and/or with long-lasting high somatic cell counts in preceding dairy herd improvement test days were randomly allocated to one of the two treatment groups in cases of mild to moderate CM. Quarter foremilk samples of the affected quarters were taken for cyto-bacteriological investigation before treatment as well as ~14 and 21 d after termination of therapy. Both treatment groups were compared regarding the bacteriological cure (BC) as the primary outcome. Clinical cure (CC) and no CM relapse within 60 d after the end of treatment (no R60) were chosen as secondary outcomes. The study resulted in the following outcomes: Streptococcus uberis was most frequently identified in microbiological culture from pre-treatment samples, followed by Staphylococcus aureus and Escherichia coli and other coliforms. No significant differences between the NSAID treatment and the reference treatment were detected regarding CC and CM recurrence (no R60). Although the sole ketoprofen therapy resulted in a numerically lower likelihood of BC, there were no significant differences to the reference treatment. Considering the selection criteria in this study, the results indicate that in mild to moderate CM cases exclusive treatment with ketoprofen may constitute an alternative to antimicrobial intramammary therapy, providing an opportunity for reduction of antibiotic usage. However, non-inferiority evaluations were inconclusive. Further investigations with a larger sample size are required to confirm the results and to make a distinct statement on non-inferiority.
In this species differentiation study of Corynebacterium spp. (C. spp.), quarter foremilk samples from 48 farms were included. These were obtained from both clinically healthy cows and those with clinical mastitis. First, all samples were examined cyto-microbiologically and all catalase-positive rods were differentiated using the direct transfer method in MALDI-TOF MS. C. bovis, C. amycolatum, C. xerosis, and five other species were identified with proportions of 90.1%, 7.7%, and 0.8% for the named species, respectively, and 1.4% for the remaining unnamed species. In addition, somatic cell count (SCC) was determined by flow cytometry. Based on this, the isolates were classified into four udder health groups: “latent infection”, “subclinical mastitis”, “clinical mastitis” and “others”. Approximately 90% of isolates of C. bovis and C. amycolatum were from latently and subclinically infected quarters. Of the C. bovis isolates, 5.8% were obtained from milk samples from clinical mastitis, whereas C. amycolatum was not present in clinical mastitis. The distribution of groups in these two species differed significantly. The geometric mean SCC of all species combined was 76,000 SCC/mL, almost the same as the SCC of C. bovis. With 50,000 SCC/mL, the SCC of C. amycolatum was slightly below the SCC of C. bovis. Through the species-level detection and consideration of SCC performed here, it is apparent that individual species differ in terms of their pathogenicity. Overall, their classification as minor pathogens with an SCC increase is confirmed.
To optimise udder health at the herd level, identifying incurable mastitis cases as well as providing an adequate therapy and culling strategy are necessary. Cows with clinical mastitis should be administered antibiotic medication if it is most likely to improve mammary cure. The somatic cell count (SCC) in milk of the monthly implemented Dairy Herd Improvement (DHI) test represents the most important tool to decide whether a cow has a promising mammary cure rate. Differential cell count (DCC) facilitates the specification of the immunological ability of defence, for example by characterising leukocyte subpopulations or cell viability. The aim of this study was to assess the DCC and cell viability in DHI milk samples regarding the cytological (CC) and bacteriological cure (BC) of the udder within a longitudinal study, thereby gaining a predictive evaluation of whether a clinical mastitis benefits from an antibiotic treatment or not. The cows enrolled in this study had an SCC above 200,000 cells/mL in the previous DHI test. Study 1 assessed the CC by reference to the SCC of two consecutive DHI tests and included 1010 milk samples: 28.4% of the mammary glands were classified as cytologically cured and 71.6% as uncured. The final mixed logistic regression model identified the total number of non-vital cells as a significant factor associated with CC. An increasing amount of non-vital cells was related to a lower individual ability for CC. Cows which were in the first or second lactation possessed a higher probability of CC than cows having a lactation number above two. If animals developed a clinical mastitis after flow cytometric investigation, the BC was examined in study 2 by analysing quarter foremilk samples microbiologically. Taking 48 milk samples, 81.3% of the mammary glands were classified as bacteriologically cured and 18.7% as uncured. The percentage of total non-vital cells tended to be lower for cows which were cured, but no significance could be observed. This study revealed that the investigation of the proportion of non-vital cells in DHI milk samples can enhance the prognosis of whether an antibiotic treatment of clinical mastitis might be promising or not. Prospectively, this tool may be integrated in the DHI tests to facilitate the decision between therapy or culling.
Severe mastitis can lead to considerable disturbances in the cows’ general condition and even to septicemia and death. The aim of this cross-sectional study was to identify factors associated with the severity of the clinical expression of mastitis. Streptococcus (Str.) uberis (29.9%) was the most frequently isolated pathogen, followed by coliform bacteria (22.3%). The majority of all mastitis cases (n = 854) in this study were either mild or moderate, but 21.1% were severe. It can be deduced that the combination of coliform pathogens and increasing pathogen shedding of these showed associations with severe mastitis. Furthermore, animal-related factors associated with severe disease progression were stages of lactation, and previous diseases in the period prior to the mastitis episode. Cows in early lactation had more severe mastitis. Ketosis and uterine diseases in temporal relation to the mastitis were associated with more severe mastitis in the diseased cows. Hypocalcemia was significantly associated with milder mastitis. As another factor, treatment with corticosteroids within two weeks before mastitis was associated with higher severity of mastitis. Knowledge of these risk factors may provide the basis for randomized controlled trials of the exact influence of these on the severity of mastitis.
As part of the European Network for Optimization of Veterinary Antimicrobial Treatment (ENOVAT), a webinar on the topic “Mastitis Treatment in Lactation” was held, in which eight mastitis experts from different European countries (Spain, The Netherlands, Estonia, Ireland, Poland, Finland, Germany, and Italy) presented their treatment approaches for clinical mastitis in lactation. The aim of this study was to compare the therapeutic approaches to identify commonalities and differences. In all eight participating countries, the decision to start treatment is usually made by the veterinarians, while the farm personnel are responsible for treatment administration. Antibiotic treatment is then typically administered intramammarily. The treatment duration often depends on the label instructions and is frequently extended if Staphylococcus aureus or Streptococcus uberis is involved. Administering supportive therapy, especially non-steroidal anti-inflammatory drugs (NSAIDs) is an established practice in all countries. Penicillin is the first-choice drug for the treatment of mastitis in an increasing number of countries. The use of critically important antimicrobials (CIAs) such as quinolones and third- and fourth-generation cephalosporins is at a low level in Finland and The Netherlands. In Estonia, Germany, Italy, and Spain, the use of CIAs is declining and is only allowed if milk samples are analyzed in advance following the legal framework. Systems for monitoring antibiotic use are being introduced in more and more countries. This exchange of different views will help the European countries to move towards a common high standard of antimicrobial stewardship in veterinary medicine.
Subclinical mastitis in heifers during early lactation affects udder health, future milk production and, therefore, the risk of premature culling. The aim of this cross-sectional study was to identify pre- and post-partum risk factors associated with a high heifer mastitis rate (HMR), and to find out which period (either pre- or post-partum) contains more risk factors and consequently should be the focus of mastitis control in heifers. A total of 77 herds were included in this study and the potential animal- and farm-related risk factors were recorded during a one-time farm visit. The HMR was provided by the dairy herd improvement test (DHI) as the annual average of the past 11 DHIs. For this study, data were analyzed in two models using generalized linear models. Each model examined the association between possible risk factors and HMR, one including only prepartum risk factors and the other one only post-partum risk factors. One identified pre-partum risk factor was the proportion of udder-healthy cows in the herd. Post-partum risk factors were the type of teat cleaning procedure before milking, teat disinfection, treatment of mastitis in heifers, a body condition score (BCS) of >3.0 in fresh heifers, and the combination of a teat cleaning procedure with a teat disinfectant. The results show the importance of the period shortly after calving for udder health in heifers, as four of the five significant risk factors were identified in this period and three of them were related to the milking process. However, further research with a higher number of herds is needed to minimize individual herd effects.
Although Corynebacterium spp. can be regularly associated with subclinical and clinical mastitis cases in dairy cows, knowledge on their reservoirs in dairy farms is sparse. Therefore, samples were collected at 10 visits with 14 day intervals from bedding material (n = 50), drinking troughs (n = 20), different walking areas (n = 60), cow brushes (n = 8), fly traps (n = 4), the passage to pasture (n = 9) as well as milking liners (n = 80) and milker gloves (n = 20) in one dairy cow farm. Additionally, quarter foremilk samples from all lactating cows (approximately 200) were collected at each visit. All samples underwent microbiological examination and cultured isolates were identified using MALDI-TOF MS. Most Corynebacterium spp. that were cultivated from milk were also isolated from the housing environment and milking-related niches (C. amycolatum, C. confusum, C. stationis, C. variabile, C. xerosis) or from milking-related niches only (C. frankenforstense, C. pilosum, C. suicordis). C. bovis was not cultivated from any environmental niche, while being the dominant species in milk samples. This study demonstrates that many Corynebacterium spp. present in milk samples can also be isolated from the cows’ environment. For C. bovis, the most relevant Corynebacterium species with regard to intramammary infections, it indicates that environmental reservoirs are of little relevance.
The aim of this cross-sectional study was to investigate the occurrence of bacteremia in severe mastitis cases of dairy cows. Milk and corresponding blood samples of 77 cases of severe mastitis were bacteriologically examined. All samples (milk and blood) were incubated aerobically and anaerobically to also investigate the role of obligate anaerobic microorganisms in addition to aerobic microorganisms in severe mastitis. Bacteremia occurred if identical bacterial strains were isolated from milk and blood samples of the same case. In addition, pathogen shedding was examined, and the data of animals and weather were collected to determine associated factors for the occurrence of bacteremia in severe mastitis. If Gram-negative bacteria were detected in milk samples, a Limulus test (detection of endotoxins) was also performed for corresponding blood samples without the growth of Gram-negative bacteria. In 74 cases (96.1%), microbial growth was detected in aerobically incubated milk samples. The most-frequently isolated bacteria in milk samples were Escherichia (E.) coli (48.9%), Streptococcus (S.) spp. (18.1%), and Klebsiella (K.) spp. (16%). Obligatory anaerobic microorganisms were not isolated. In 72 cases (93.5%) of the aerobically examined blood samples, microbial growth was detected. The most-frequently isolated pathogens in blood samples were non-aureus Staphylococci (NaS) (40.6%) and Bacillus spp. (12.3%). The Limulus test was positive for 60.5% of cases, which means a detection of endotoxins in most blood samples without the growth of Gram-negative bacteria. Bacteremia was confirmed in 12 cases (15.5%) for K. pneumoniae (5/12), E. coli (4/12), S. dysgalactiae (2/12), and S. uberis (1/12). The mortality rate (deceased or culled) was 66.6% for cases with bacteremia and 34.1% for cases without bacteremia. High pathogen shedding and high humidity were associated with the occurrence of bacteremia in severe mastitis.
Antimicrobials are widely used to cure intramammary infections (IMI) in dairy cows during the dry period (DP). Nevertheless, the IMI cure is influenced by many factors and not all quarters benefit from antimicrobial dry cow treatment (DCT). To evaluate the true effect of antibiotic DCT compared to self-cure and the role of causative pathogens on the IMI cure, a retrospective cross-sectional study was performed. The analysis included 2987 quarters infected at dry-off (DO). Information on DCT, causative pathogens, somatic cell count, milk yield, amount of lactation, Body Condition Score, and season and year of DO were combined into categorical variables. A generalized linear mixed model with a random cow, farm and year effect and the binary outcome of bacteriological cure of IMI during the DP was conducted. In the final model, a significant effect (p < 0.05) on DP cure was seen for the DO season and the category of causative pathogens (categories being: Staphylococcus aureus, non-aureus staphylococci, streptococci, coliforms, ‘other Gram-negative bacteria’, ‘other Gram positive bacteria’, non-bacterial infections and mixed infections), while antibiotic DCT (vs. non-antibiotic DCT) only showed a significant effect in combination with the pathogen categories streptococci and ‘other Gram-positive bacteria’.
To effectively prevent and control bovine mastitis, farmers and their advisors need to take infection pathways and durations into account. Still, studies exploring both aspects through molecular epidemiology with sampling of entire dairy cow herds over longer periods are scarce. Therefore, quarter foremilk samples were collected at 14-d intervals from all lactating dairy cows (n = 263) over 18 wk in one commercial dairy herd. Quarters were considered infected with Staphylococcus aureus, Streptococcus uberis, or Streptococcus dysgalactiae when ≥100 cfu/mL of the respective pathogen was detected, or with Staphylococcus epidermidis or Staphylococcus haemolyticus when ≥500 cfu/mL of the respective pathogen was detected. All isolates of the mentioned species underwent randomly amplified polymorphic DNA (RAPD)-PCR to explore strain diversity and to distinguish ongoing from new infections. Survival analysis was used to estimate infection durations. Five different strains of Staph. aureus were isolated, and the most prevalent strain caused more than 80% of all Staph. aureus infections (n = 46). In contrast, 46 Staph. epidermidis and 69 Staph. haemolyticus strains were isolated, and none of these caused infections in more than 2 different quarters. The 3 most dominant strains of Strep. dysgalactiae (7 strains) and Strep. uberis (18 strains) caused 81% of 33 and 49% of 37 infections in total, respectively. The estimated median infection duration for Staph. aureus was 80 d, and that for Staph. epidermidis and Staph. haemolyticus was 28 and 22 d, respectively. The probability of remaining infected with Strep. dysgalactiae or Strep. uberis for more than 84 and 70 d was 58.7 and 53.5%, respectively. Staphylococcus epidermidis and Staph. haemolyticus were not transmitted contagiously and the average infection durations were short, which brings into question whether antimicrobial treatment of intramammary infections with these organisms is justified. In contrast, infections with the other 3 pathogens lasted longer and largely originated from contagious transmission.
To design cost-effective prevention strategies against mastitis in dairy cow farms, knowledge about infection pathways of causative pathogens is necessary. Therefore, we investigated the reservoirs of bacterial strains causing intramammary infections in one dairy cow herd. Quarter foremilk samples (n = 8056) and milking- and housing-related samples (n = 251; from drinking troughs, bedding material, walking areas, cow brushes, fly traps, milking liners, and milker gloves), were collected and examined using culture-based methods. Species were identified with MALDI-TOF MS, and selected Staphylococcus and Streptococcus spp. typed with randomly amplified polymorphic DNA-PCR. Staphylococci were isolated from all and streptococci from most investigated locations. However, only for Staphylococcus aureus, matching strain types (n = 2) were isolated from milk and milking-related samples (milking liners and milker gloves). Staphylococcus epidermidis and Staphylococcus haemolyticus showed a large genetic diversity without any matches of strain types from milk and other samples. Streptococcus uberis was the only Streptococcus spp. isolated from milk and milking- or housing-related samples. However, no matching strains were found. This study underlines the importance of measures preventing the spread of Staphylococcus aureus between quarters during milking.
The aim of this cross-sectional study was to investigate associated factors of the severity of clinical mastitis (CM). Milk samples of 249 cases of CM were microbiologically examined, of which 27.2% were mild, 38.5% moderate, and 34.3% severe mastitis. The samples were incubated aerobically and anaerobically to investigate the role of aerobic and anaerobic microorganisms. In addition, the pathogen shedding was quantitatively examined, and animal individual data, outside temperature and relative humidity, were collected to determine associated factors for the severity of CM. The pathogen isolated the most was Escherichia coli (35.2%), followed by Streptococcus spp. (16.4%). Non-aureus staphylococci (NaS) (15.4%) and other pathogens (e.g., Staphylococcus aureus, coryneforms) (15.4%) were the pathogens that were isolated the most for mild mastitis. Moderate mastitis was mostly caused by E. coli (38%). E. coli was also the most common pathogen in severe mastitis (50.6%), followed by Streptococcus spp. (16.4%), and Klebsiella spp. (10.3%). Obligate anaerobes (Clostridium spp.) were isolated in one case (0.4%) of moderate mastitis. The mortality rate (deceased or culled due to the mastitis in the following two weeks) was 34.5% for severe mastitis, 21.7% for moderate mastitis, and 4.4% for mild mastitis. The overall mortality rate of CM was 21.1%. The pathogen shedding (back logarithmized) was highest for severe mastitis (55,000 cfu/mL) and E. coli (91,200 cfu/mL). High pathogen shedding, low previous somatic cell count (SCC) before mastitis, high outside temperature, and high humidity were associated with severe courses of mastitis.