Cell Differentiation of Bovine Milk Control Samples to Improve Prognosis of Mastitis Cure.

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.

Prevention of Intramammary Infections by Prepartum External Application of a Teat Dip Containing Lactic Acid Bacteria with Antimicrobial Properties in Dairy Heifers.

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.

Biofilm Challenge: Lactic Acid Bacteria Isolated from Bovine Udders versus Staphylococci.

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.

Efficacy of a commercial glutaraldehyde footbath product for the control of bovine digital dermatitis.

OBJECTIVE: To evaluate the relative efficacy of a novel, commercially available disinfecting agent containing ≥ 15 to < 20 % glutaraldehyde in a hydro-colloid matrix ("EIMÜ Klauen Sprint®"; Eimermacher, Nordwalde, Germany, Biozid-Desinfektionsmittel, BAuA Nr. N-57213) as a walkthrough footbath in order to treat digital dermatitis of dairy cattle. MATERIAL AND METHODS: The study was conducted in a dairy farm located in northern Germany from June 1st, 2015 to September 5th, 2015. Eighty-eight cows were enrolled in this study. In both trials of the study, a walkthrough footbath was applied on five consecutive milking times per week. Trial 1 was a blinded, randomised and controlled intervention study. The therapy group (n = 40) received the novel glutaraldehyde product in a 2 % solution while the control group (n = 40) was led through a footbath containing pure water. Trial 2 was a longitudinal study to investigate the development of claw health over a longer time period regarding the use of the new footbath solution. In trial 2 the disinfecting footbath was applied to all trial cows (therapy and control group) of trial 1. The development of claw health and lameness was systematically monitored in the two groups on days 0, 9, 17, 29, 35 (trial 1) and days 42, 68, 94 (trial 2), respectively. The adjusted odds of digital dermatitis lesions (DDL) throughout the study were analysed by mixed logistic regression models. RESULTS: The odds of DDL were 0.15 for the glutaraldehyde foot-bath group compared to the control group. Hence, the disinfecting footbath significantly reduced the DDL prevalence and increased the cure rate of DDL. However, no preventive effect on new digital dermatitis (DD) cases could be identified. CONCLUSION: The efficacy of the novel glutaraldehyde footbath was evaluated and it was concluded that "EIMÜ Klauen Sprint®" was well tolerated over a longer period of time and might therefore be an effective tool to control DD. CLINICAL RELEVANCE: In future, "EIMÜ Klauen Sprint®" could be used within a Mortellaro sanitation concept and thereby might help to solve a major problem of dairy cattle farms.

Biofilm formation and adhesion to bovine udder epithelium of potentially probiotic lactic acid bacteria.

Mastitis is one of the most important diseases threatening modern dairy herds. The idea of fighting the disease through colonising the udder with lactic acid bacteria (LAB), thereby building a beneficial biofilm, is the base for a probiotic approach towards mastitis control. The purpose of this study was to screen 13 LAB strains (eleven wild strains, two ATCC strains) inhibitory to the growth of mastitis-causing pathogens for their in vitro ability to form a biofilm and to adhere to bovine glandular mammary epithelium in order to assess their probiotic potential. Furthermore, we aimed to gain knowledge about the chemical nature of the adhesins involved by subjecting the bacteria to various chemical and enzymatical pre-treatments. The biofilms were grown on hydrophilic glass and on hydrophobic polypropylene in de Man, Rogosa and Sharpe (MRS) broth and afterwards quantified with a crystal violet assay. Biofilm formation was observed in all strains. However, the extent strongly depended on the strain, surface charge and medium. The adhesion assay also revealed a strong strain dependency, but this trait was also present in all of the investigated LAB isolates. Depending on the strain, chemical or enzymatical pre-treatment revealed carbohydrate molecules as well as proteins and lipids to be crucial for the adhesion of LAB to epithelial cells. The seven strains showing the strongest biofilm formation and/or adhesion represent promising candidates for further investigation in order to develop a probiotic remedy for the treatment of mastitis. Still, their safety for consumers and patients as well as their capability to colonise the udder remain to be investigated in in vivo studies.

In vitro ability of mastitis causing pathogens to form biofilms.

This Research Communication describes the study of in vitro biofilm formation of mastitis causing pathogens. Biofilms are communities of bacteria that are attached to a surface and to each other and are embedded in a self-produced matrix of extracellular polymeric substances. Biofilm formation is an important virulence factor that may result in recurrent or persistent udder infections and treatment failure through increased resistance to antibiotics and protection against host defences. In the present study 252 bacterial isolates from milk samples from bovine udder quarters with intramammary infections were examined with Congo Red agar (CRA) method and tube method (TM) for their ability to form biofilms. Both tests revealed a high number of biofilm-positive strains. Literature reports that the cure rates for Staphylococcus aureus infected udders are lower (27%) in comparison to cure rates of Streptococcus uberis (64-81%) or coagulase-negative staphylococci (CNS) mastitis (80-90%). The findings of the present study suggest that biofilm formation is not the main factor for the differences in cure rates of the various bacteria genera, because all tested pathogen groups showed a similarly high proportion of biofilm formation. Further research is needed to detect microbial biofilms on bovine udder epithelia.

A field study evaluation of Petrifilm™ plates as a 24-h rapid diagnostic test for clinical mastitis on a dairy farm.

Clinical mastitis is one of the most common and expensive diseases of dairy cattle. To make an informed treatment decision, it is important to know the causative pathogen. However, no detection of bacterial growth can be made in approximately 30% of all clinical cases of mastitis. Before selecting the treatment regimen, it is important to know whether the mastitis-causing pathogen (MCP) is Gram-positive or Gram-negative. The aim of this field study was to investigate whether using two 3M Petrifilm™ products on-farm (which conveys a higher degree of sample freshness but also bears a higher risk for contamination than working in a lab) as 24-h rapid diagnostic of clinical mastitis achieved results that were comparable to the conventional microbiological diagnostic method. AerobicCount (AC)-Petrifilm™ and ColiformCount (CC)-Petrifilm™ were used to identify the total bacterial counts and Gram-negative bacteria in samples from clinical mastitis cases, respectively. Missing growth on both plates was classified as no bacterial detection. Growth only on the AC-Petrifilm™ was assessed as Gram-positive, and growth on both Petrifilm™ plates was assessed as Gram-negative bacterial growth. Additionally, milk samples were analysed by conventional microbiological diagnostic method on aesculin blood agar as a reference method. Overall, 616 samples from clinical mastitis cases were analysed. Using the reference method, Gram-positive and Gram-negative bacteria, mixed bacterial growth, contaminated samples and yeast were determined in 32.6%, 20.0%, 2.5%, 14.1% and 1.1% of the samples, respectively. In 29.7% of the samples, microbiological growth could not be identified. Using the Petrifilm™ concept, bacterial growth was detected in 59% of the culture-negative samples. The sensitivity of the Petrifilm™ for Gram-positive and Gram-negative MCP was 85.2% and 89.9%, respectively. The specificity was 75.4% for Gram-positive and 88.4% for Gram-negative MCP. For the culture-negative samples, sensitivity was 41.0% and specificity was 91.0%. The results indicate that the Petrifilm™ concept is suitable for therapeutic decision-making at the farm level or in veterinary practice. As this concept does not allow any statement about the genus or species of microorganisms, relevant MCP should be assessed periodically at the herd level with conventional microbiological diagnostics.

[Cure rates of chronic subclinical Staphylococcus aureus mastitis in lactating dairy cows after antibiotic therapy]. / Heilungsraten chronischer subklinischer Staphylococcus aureus-Mastitiden nach antibiotischer Therapie bei laktierenden Milchkühen.

Staphylococcus (S.) aureus is an important mastitis causing pathogen in dairy cows worldwide. The aim of this controlled and randomized study was to analyze the effects of an antibiotic treatment on chronic subclinical S.aureus mastitis during lactation.The study was conducted between July 2011 and December 2011 in Northern Germany including 134 udder quarters (i. e. 103 dairy cows) infected with S. aureus. The animals were randomly divided into two groups (control and treatment group). Quarter foremilk duplicate samples were taken on days 0, 7, 32 and 39 from each infected udder quarter for microbiological analysis and somatic cell count determination. Treatment consisted of cephalexin (200 mg intramammarily 5 times every 12 h) plus marbofloxacine (2 mg/kg BM subcutaneously 3 times every 24 h). "Pathogen elimination" was assessed as the status, when no S. aureus was isolated from the quarter samples of days 32 and 39. "Cure" was defined as the status, when in addition to pathogen elimination the somatic cell count of the quarter in both milk samples was below 100 000/ml. Animals of the treatment group showed a pathogen elimination rate of 35.9% and a cure rate of 21.9%. The rates for the control group were 21.4% and 8.6%, resp. The differences between groups were statistically significant. These results indicate that pathogen elimination and cure rates of chronic subclinical S. aureus mastitis are low after an intramammary cephalexin and subcutaneous marbofloxacine treatment, but still significantly better than without any antibiotic treatment.

The association between bedding material and the bacterial counts of Staphylococcus aureus, Streptococcus uberis and coliform bacteria on teat skin and in teat canals in lactating dairy cattle.

Several mastitis-causing pathogens are able to colonize the bovine teat canal. The objective of this study was to investigate the association between the treatment of sawdust bedding with a commercial alkaline conditioner and the bacterial counts on teat skin and in the teat canal. The study used a crossover design. Ten lactating Holstein cows that were free of udder infections and mastitis were included in the study. The animals were bedded on either untreated sawdust or sawdust that had been treated with a hydrated lime-based conditioner. Once a day, fresh bedding material was added. After 3 weeks, the bedding material was removed from the cubicles, fresh bedding material was provided, and the cows were rotated between the two bedding material groups. Teat skin and teat canals were sampled using the wet and dry swab technique after weeks 1, 2, 3, 4, 5 and 6. Staphylococcus aureus, Streptococcus uberis, Escherichia coli and other coliform bacteria were detected in the resulting agar plate cultures. The treatment of the bedding material was associated with the teat skin bacterial counts of Str. uberis, Esch. coli and other coliform bacteria. An association was also found between the bedding material and the teat canal bacterial counts of coliform bacteria other than Esch. coli. For Staph. aureus, no associations with the bedding material were found. In general, the addition of a hydrated lime-based conditioner to sawdust reduces the population sizes of environmental pathogens on teat skin and in teat canals.

The association between teat end hyperkeratosis and teat canal microbial load in lactating dairy cattle.

Most pathogens that cause bovine mastitis invade the udder lumen through the teat canal. Amino acids and intercellular lipids may support microbial colonisation of the teat canal epithelium by pathogenic microorganisms. The aim of this study was to investigate the association between teat end hyperkeratosis, which is induced by machine milking, and teat canal microbial load. Contralateral teats, which differed in teat end hyperkeratosis scores, were identified in a split-udder experiment. The teat canal's microbial load was evaluated using the wet and dry swab technique. Staphylococcus (S.) aureus, Streptococcus (Sc.) uberis, Escherichia (E.) coli and other coliforms were detected by agar plate cultures. The positive detection of E. coli and the log(10)-transformed E. coli load of a teat canal were significantly associated with the teat end hyperkeratosis score (P<0.05). There were significant differences with respect to positive findings for E. coli, as well as the microbial load of E. coli and Sc. uberis, between the less-calloused and the more-calloused teat of a pair. For S. aureus, no significant associations between hyperkeratosis score and teat canal microbial load were detected. In general, a teat with a highly calloused teat end had an increased teat canal microbial load compared with the contralateral teat, characterised by a lower callosity. The results of the present study indicate that the environmental pathogen load is associated with teat end hyperkeratosis. Further research is needed to identify factors that may affect teat canal microbial load in lactating dairy cattle.

[Microbiological colonisation of bovine teat canal--significance and influencing factors]. / Die mikrobielle Besiedlung des Zitzenkanals bei Milchkühen--Bedeutung und Einflussfaktoren.

The teat canal of lactating dairy cattle is of particular importance for the defence of facultative pathogenic and pathogenic microorganisms (e.g., staphylococci) invading the bovine udder. Furthermore the canal is usually colonized with microorganisms, too. In addition to microorganisms inhibiting mastitis pathogens the teat canal is colonized by staphylococci. The microbial colonization can be influenced by the environment of the animals, the care and disinfection of the teat skin and indirectly by the effects of forces being associated with machine milking. Because of vacuum fluctuations occurring under the teat tip microorganisms, which colonize the teat canal, can invade the bovine mammary gland and cause infections there. This paper gives a review of the microbial colonization of the bovine teat duct and of influencing factors on the microbial populations as well as of the significance of the teat canal colonization for the development of mastitis.

[Effects of an additional nonsteroidal anti-inflammatory therapy with carprofen (Rimadyl Rind) in cases of severe mastitis of high yielding cows]. / Effekte einer zusätzlichen entzündungshemmenden Therapie mit Carprofen (Rimadyl Rind) bei schweren Mastitiden hochleistender Milchkühe.

This field study focuses on the possible effects of a combination of nonsteroidal anti-inflammatory treatment (carprofen) and a local and parenteral antibiotic on cure rates, survival rate and return to milk production of severe clinical mastitis cases. 69 cows in 3 herds (blocked by parity) with severe clinical mastitis during the first 120 d of lactation (median = 28 d) were treated with antibiotics and one-half of these cows were treated with 1.4 mg/kg bodyweight carprofen (Rimadyl Rind, Pfizer GmbH Tiergesundheit, Germany). Double milk samples for bacteriology were collected from clinically affected udder quarters before treatment and at 14 (+/- 3) and 21 (+/- 3) days after commencement of treatment for cytomicro-temperature, clinical, bacteriological, cytobacteriological cure rate and in the number of cows that were defined as treatment failures (i.e., died, re-treated, relapse). Six (22.2%) vs. seven (19.4%) cows in the carprofen and control groups failed, respectively. The milk yield was significantly higher in the carprofen-treated group compared with the control group after treatment. The present work gives first indications that treatment of cows with severe clinical mastitis with a combination of carprofen and antibiotics could result in a faster return to milk production compared to treatment with antibiotics alone. If this effect can be affiliated to the administration of carprofen alone has to be examined in further studies.

[Efficacy of extended intramammary therapy to treat moderate and severe clinical mastitis in lactating dairy cows]. / Wirksamkeit einer verlängerten Arzneimittelapplikation zur Behandlung mittel- und hochgradiger klinischer Mastitiden bei Milchkühen.

Streptococcus uberis and Staphylococcus aureus are important mastitis pathogens in dairy cows in the entire world. Recent publications showed that an extended therapy can be more efficient in combating such intramammary infections. The objective of this study was to evaluate the effect of an extended intramammary therapy to treat moderate and severe mastitis cases in lactating dairy cows under field conditions in northern Germany. From December 2005 to August 2007, a total of 157 clinical mastitis cases on 10 farms in northern Germany were enrolled in the study and randomly assigned (blocked by parity and body temperature) to one of two treatment groups (intrammammary lincomycin/neomycin 1.5-d (ALK) or 5-d (ALL)). Clinical, microbiological and cytomicrobiological cure rates were evaluated. Treatments were initiated before culture results. Cows were observed and evaluated on d 1 to 6, 19 and 26. Six cases in 157 (3.8%) resulted in a therapy change in between 48 h after mastitis detection. Overall, treatments were not significantly different to controls regarding clinical cure rate. However, when the microbiological cure rate was evaluated, differences were observed. ALL appeared form infections. We conclude that in farms with Streptococcus uberis mastitis, the 5-d extended lincomycin/neomycin treatment regimen was significantly more efficient in microbiological cure than the standard 1.5-d treatment.

Evaluation of increased milking frequency as an additional treatment for cows with clinical mastitis.

This field study focused on the possible effects of increased milking frequency (milking four times a day in comparison with milking twice a day) on clinical and bacteriological cure rates of clinical, antibiotically treated mastitis cases. Parameters tested were clinical, microbiological and full (cytomicrobiological) cure as well as the development of milk yield after the clinical mastitis episode. Cows from a large dairy herd meeting the study criteria (n=93) were assigned to two treatment groups by a systematic randomization scheme (blocked by body temperature <=or >39.5 degrees C). Both groups were randomly divided by experimental treatments: a) antibiotic intramammary treatment and milking 2-times a day; b) antibiotic intramammary treatment and milking 4-times a day. Treatments were initiated before the culture results were known. Cows were surveyed and evaluated on days 1-6, 24 and 31. No significant differences between treatment and control groups regarding clinical cure, microbiological cure, full cure and milk production could be established. Applying a 4-times a day milking regime did not lead to any significant effect, either positive or negative. Therefore, the results suggest that milking 4-times a day as a supporting therapy for mild, moderate and severe antimicrobially treated mastitis cases cannot be recommended.