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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.
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.
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.
Streptococcus dysgalactiae is among the most important pathogens causing bovine mastitis. Unfortunately, there is presently a lack of clear knowledge about the mode of transmission — contagious or environmental — of this pathogen. To obtain more information on this, knowledge of the genetic diversity of the isolated microorganisms at the farm level can be useful. To observe the strain variety in different herds of cattle, isolates of Strep. dysgalactiae were collected from clinical mastitis samples at different farms, and the strains were typed using the pulsed-field gel electrophoresis (PFGE) method. Overall, we performed strain typing on 93 isolates from 16 farms in Germany and used an index to describe the degree of contagiosity of Strep. dysgalactiae at each farm. This index (CI) represents the number of isolates divided by the number of strains found in mastitis milk of clinical cases within a period of 14 months. The results differed between the farms. In one farm, all six Strep. dysgalactiae cases that occurred during the study period were caused by a single strain (CI = 6), while in another farm the six cases that occurred were caused by five different strains (CI = 1.2). All other farms fell between these two extremes. This indicates that Strep. dysgalactiae infections can occur via several routes of transmission. At the farm level, strain comparisons are necessary to determine the routes of transmission. Two strains were able to survive on the farm for a minimum of 14 months.
During machine milking, pathogenic microorganisms can be transmitted from cow to cow through liners. Therefore, in Germany, a spray method for the intermediate disinfection of the milking cluster is often used for prevention. This method of cluster disinfection is easy to perform, requires little time and no extra materials, and the disinfection solution is safe from outside contamination in the spray bottle. Since no data on a systematic efficacy trial are available, the aim of this study was to determine the microbial reduction effect of intermediate disinfection. Therefore, laboratory and field trials were conducted. In both trials, two sprays of 0.85 mL per burst of different disinfectant solutions were sprayed into the contaminated liners. For sampling, a quantitative swabbing method using a modified wet–dry swab (WDS) technique based on DIN 10113-1: 1997-07 was applied. Thus, the effectiveness of disinfectants based on Peracetic Acid, Hydrogen Peroxide and Plasma-Activated Buffered Solution (PABS) was compared. In the laboratory trial, the inner surfaces of liners were contaminated with pure cultures of Escherichia (E.) coli, Staphylococcus (S.) aureus, Streptococcus (Sc.) uberis and Sc. agalactiae. The disinfection of the contaminated liners with the disinfectants resulted in a significant reduction in bacteria with values averaging 1 log for E. coli, 0.7 log for S. aureus, 0.7 log for Sc. uberis and 0.8 log for Sc. agalactiae. The highest reduction was obtained for contamination with E. coli (1.3 log) and Sc. uberis (0.8 log) when PABS was applied and for contamination with S. aureus (1.1 log) and Sc. agalactiae (1 log) when Peracetic Acid Solution (PAS) was used. Treatment with sterile water only led to an average reduction of 0.4 log. In the field trial, after the milking of 575 cows, the liners were disinfected and the total microorganism count from the liner surface was performed. The reduction was measured against an untreated liner within the cluster. Although a reduction in microorganisms was achieved in the field trial, it was not significant. When using PAS, a log reduction of 0.3 was achieved; when using PABS, a log reduction of 0.2 was obtained. The difference between the two disinfection methods was also not significant. Treatment with sterile water only led to a reduction of 0.1 log. The results show that spray disinfection under these circumstances does result in a reduction in the bacteria on the milking liner surface, but for effective disinfection a higher reduction would be preferred.
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 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 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.
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.
Corynebacterium spp. are frequently detected in bovine quarter milk samples, yet their impact on udder health has not been determined completely. In this longitudinal study, we collected quarter milk samples from a dairy herd of approximately 200 cows, ten times at 14 d intervals. Bacteriologically, Catalase-positive and Gram-positive rods were detected in 22.7% of the samples. For further species diagnosis, colonies were analyzed by MALDITOF MS. Corynebacterium bovis, C. amycolatum, C. xerosis and 10 other Corynebacterium spp. were detected. The three aforementioned species accounted for 88.4%, 8.65% and 0.94% of all cultured Corynebacterium spp., respectively. For further evaluation of infection dynamics, the following three infection definitions were applied: A (2/3 consecutive samples positive for the same species), B (≥1000 cfu/mL in one sample), C (isolated from a clinical mastitis case). Infections according to definition B occurred most frequently and clinical mastitis with Corynebacterium spp. occurred once during sampling. Life tables were used to determine the duration of infection. According to infection definition A, infection durations of 111 d and 98 d were obtained for C. bovis and C. amycolatum, respectively. Exemplarily, longer lasting infections were examined for their strain diversity by RAPD PCR. A low strain diversity was found in the individual quarters that indicates a longer colonization of the udder parenchyma by C. bovis and C. amycolatum.