Potential of phage EF-N13 as an alternative treatment strategy for mastitis infections caused by multidrug-resistant Enterococcus faecalis.

Bovine mastitis is the most common and costly disease affecting dairy cattle throughout the world. Enterococcus faecalis is one of the environmental origin mastitis-causing pathogens. The treatment of bovine mastitis is primarily based on antibiotics. Due to the negative impact of developing antibiotic resistance and adverse effects on soil and water environments, the trend toward use of nonantibiotic treatments is increasing. Phages may represent a promising alternative treatment strategy. However, it is unknown whether phages have therapeutic effects on E. faecalis-induced mastitis. Thus, the objective of this study was to investigate the degree of protection conferred by a phage during murine mastitis caused by multidrug-resistant E. faecalis. Enterococcus faecalis was isolated from the milk of dairy cows with mastitis, and a phage was isolated using the E. faecalis isolates as hosts. The bactericidal ability of the phage against E. faecalis and the ability to prevent biofilm formation were determined in vitro. The therapeutic potential of the phage on murine mastitis was evaluated in vivo. We isolated 14 strains of E. faecalis from the milk of cows with mastitis, all of which exhibited multidrug resistance, and most (10/14) could form strong biofilms. Subsequently, a new phage (EF-N13) was isolated using the multidrug-resistant E. faecalis N13 (isolated from mastitic milk) as the host. The phage EF-N13 belongs to the family Myoviridae, which has short latent periods (5 min) and high bursts (284 pfu/cell). The genome of EF-N13 lacked bacterial virulence-, antibiotic resistance-, and lysogenesis-related genes. Furthermore, bacterial loading in the raw milk medium was significantly reduced by EF-N13 and was unaffected by potential IgG antibodies. In fact, EF-N13 could effectively prevent the formation of biofilm by multidrug-resistant E. faecalis. All of these characteristics suggest that EF-N13 has potential as mastitis therapy. In vivo, 1 × 105 cfu/gland of multidrug-resistant E. faecalis N13 resulted in mastitis development within 24 h. A single dose of phage EF-N13 (1 × 104, 1 × 105, or 1 × 106 pfu/gland) could significantly decrease bacterial counts in the mammary gland at 24 h postinfection. Histopathological observations demonstrated that treatment with phage EF-N13 effectively alleviated mammary gland inflammation and damage. This effect was confirmed by the lower levels of proinflammatory cytokines IL-6, IL-1ß, and tumor necrosis factor-α in the mammary gland treated with phage EF-N13 compared with those treated with phosphate-buffered saline. Overall, the data underscored the potential of phage EF-N13 as an alternative therapy for bovine mastitis caused by multidrug-resistant E. faecalis.

Natural photosensitizer-loaded in micellar copolymer to prevent bovine mastitis: A new post-dipping protocol on milking.

Good management practices such as post-dipping applications (post-milking immersion bath) contribute to the dairy cattle health during lactation and minimize the appearance of mastitis (an infection in the mammary gland). The post-dipping procedure is performed conventionally using iodine-based solutions. The search for therapeutic modalities that are not invasive and do not cause resistance to the microorganisms that cause bovine mastitis instigates the interest of the scientific community. In this regard, antimicrobial Photodynamic Therapy (aPDT) is highlighted. The aPDT is based on combining a photosensitizer (PS) compound, light of adequate wavelength, and molecular oxygen (3O2), which triggers a series of photophysical processes and photochemical reactions that generate reactive oxygen species (ROS) responsible for the inactivation of microorganisms. The present investigation explored the photodynamic efficiency of two natural PS: Chlorophyll-rich spinach extract (CHL) and Curcumin (CUR), both incorporated into the Pluronic® F127 micellar copolymer. They were applied in post-dipping procedures in two different experiments. The photoactivity of formulations mediated through aPDT was conducted against Staphylococcus aureus, and obtained a minimum inhibitory concentration (MIC) of 6.8 mg mL-1 for CHL-F127 and 0.25 mg mL-1 for CUR-F127. Only CUR-F127 inhibited Escherichia coli growth with MIC 0.50 mg mL-1. Concerning the count of microorganisms during the days of the application, a significant difference was observed between the treatments and control (Iodine) when the teat surface of cows was evaluated. For CHL-F127 there was a difference for Coliform and Staphylococcus (p < 0.05). For CUR-F127 there was a difference for aerobic mesophilic and Staphylococcus (p < 0.05). Such application decreased bacterial load and maintained the milk quality, being evaluated via total microorganism count, physical-chemical composition, and somatic cell count (SCC).

Ozone use in the treatment of subclinical mastitis in dairy cows.

This research communication paper addresses the hypothesis that the use of therapeutic alternatives for mastitis, such as intramammary ozone, can cure the disease with lower costs and without harmful residues for human consumption and without formation of microbial resistance like the ones caused by indiscriminate use of antibiotics in dairy farms. The study was performed in 36 mammary quarters from 12 dairy cows with subclinical mastitis grade three. The experimental units were randomly assigned into four groups and each group received a treatment. Treatments comprised (a) 20 µg/ml ozone gas; (b) 40 µg/ml ozone gas; (c) negative control treatment of 12.5 µg/ml ozonated saline and (d) positive control treatment of 100 mg of cephalexin + 100 mg of neomycin + 10 mg of prednisolone, all by intramammary injection. In all quarters, milk was collected before and after the application of treatments for California mastitis test and evaluation of milk composition, somatic cell count, and bacterial cultures. The results indicated that the use of intramammary ozone did have a therapeutic effect, and whilst this was less than that of antibiotics, ozone does confer some advantages. Treated milk had a good composition, the treatment cost was low, milk withdrawal may not be necessary and there is no risk of antibiotic resistance.

Isolation and evaluation of the efficacy of bacteriophages against multidrug-resistant (MDR), methicillin-resistant (MRSA) and biofilm-producing strains of Staphylococcus aureus recovered from bovine mastitis.

BACKGROUND: Staphylococcus aureus (S. aureus) is one of the major causes of bovine mastitis with significant economic losses around the worldwide. The emergence of multidrug-resistant (MDR), methicillin-resistant (MRSA) and biofilm-producing strains of S. aureus challenges the treatment strategies based on the antibiotic application. Today, alternative or combinational treatment options such as bacteriophage application has received much attention. The goal of the present study was to focus on isolation and evaluation of the efficacy of bacteriophages with specific lytic activity against S. aureus strains with low cure rates (MDR, MRSA and biofilm-producing strains). RESULTS: In the present study, two phages belonging to the Podoviridae family with specific lytic activity against S. aureus were isolated from the sewage of dairy farms and designated as Staphylococcus phage M8 and Staphylococcus phage B4. Latent period and burst size for Staphylococcus phage M8 (70 min, 72 PFU/cell) and Staphylococcus phage B4 (30 min, 447 PFU/cell) were also defined. Our results revealed the susceptibility of MDR (4/20; 20%), MRSA (4/13; 30.8%) and biofilm-producing (1/10; 10%) strains to Staphylococcus phage M8. Moreover, one biofilm-producing strain (1/10; 10%) was susceptible to Staphylococcus phage B4. Furthermore, both phages kept their lytic activity in milk. They reduced the S. aureus population by about 3 logs in cultured milk after 8 h of incubation. CONCLUSION: In conclusion, it seems that both phages had the potential to serve as biological control agents alone or in combination with other agents such as antibiotics against infections induced by S. aureus. However, further studies are needed to investigate the efficacy of these phages in vivo.

Persistence of Staphylococcus spp. in milk from cows undergoing homeopathy to control subclinical mastitis.

BACKGROUND: Mastitis is one of the major diseases in dairy cattle, as it causes great economic losses to producers due to the reduction of milk production and changes in the quality of the product. The disease is mainly caused by bacteria of the genus Staphylococcus spp., these microorganisms can express various virulence factors, such as biofilms for example. In herds with organic management, producers and technicians use unconventional ways to treat and control the disease, such as homeopathy. However, it is not known if this type of treatment is able to control pathogenic bacteria such as those of the genus Staphylococcus, of relevance to animal and human health. Thus, the objective of this study was to investigate the production of biofilm in vitro and its genes by Staphylococcus spp. isolated in the milk of cows treated with homeopathy, as well as the persistence of microorganisms in animals. METHODS: Ninety-nine isolates of Staphylococcus spp. from cows treated and not treated with homeopathy were identified by internal transcribed space-polymerase chain reaction and investigated for the presence of the icaABCD, bap, aap, atlE, and bhp genes and in vitro biofilm production using the adhesion method on polystyrene plates. The enzyme restriction profile was determined by Pulsed-Field Gel Electrophoresis. Clusters of S. aureus and S. epidermidis with three or more isolates had an isolate selected for Multilocus Sequence Typing. RESULTS: The frequency of S. aureus isolations was similar in treated and untreated cows, while 71.4% of the coagulase-negative identified were isolated in cows treated with homeopathy. The distribution of the operon ica genes was similar in animals with and without treatment, except for the icaD gene, more frequent in treated cows. Production of biofilm was associated with presence of one or more genes from the icaADBC operon. S. aureus revealed a greater diversity and greater dissemination in cows treated and not treated with homeopathy. Sequence Types ST1, ST5, and ST126 were identified in S. aureus. CONCLUSIONS: The presence of biofilm-associated genes and the in vitro production of biofilms, combined with the persistence of clonal profiles of Staphylococcus spp. demonstrate other forms of control for bovine mastitis should be researched for organic production herds.

Efficacy Assessment of Phage Therapy in Treating Staphylococcus aureus-Induced Mastitis in Mice.

The primary aim of this study was to evaluate the efficacy of phage against mastitis induced by drug-resistant S. aureus in a mouse model. In this study, five S. aureus phages-4086-1, 4086-2, 4086-3, 4086-4, and 4086-6-were isolated from milk samples secreted by mastitis cows. Transmission electron microscopy showed that all the five phages had icosahedral heads and short non-contractile tails, which are typical characteristics of the family Podoviridae. All these phages were species-specific against S. aureus. The one-step growth curve showed a short latency period (10-20 min) and high burst size (up to 400 PFU/infected cell). To evaluate the effectiveness of the phage 4086-1 in the treatment against mastitis, a mouse model of mastitis was challenged with drug-resistant S. aureus. The results showed the proliferation of S. aureus in the mammary glands was significantly inhibited after treating by phage 4086-1. The concentrations of TNF-α and IL-6 decreased significantly, which demonstrated the phages could effectively alleviate the inflammatory responses. Furthermore, the histopathological analysis showed that inflammatory infiltration in the mammary glands was significantly reduced. These results demonstrate that phage may be a promising alternative therapy against mastitis caused by drug-resistant S. aureus.

Intramammary treatment using allogeneic pure platelet-rich plasma in cows with subclinical mastitis caused by Gram-positive bacteria.

The aims of the study were (1) to compare the cure risk of intramammary treatment of pure platelet rich plasma (P-PRP) or cefquinome sulfate (CS) in cows with subclinical mastitis (SCM) caused by Gram-positive bacteria, evaluated via somatic cell count (SCC) and the microbiological analysis of milk; (2) to compare the inflammatory/anti-inflammatory response of mammary gland to both treatments through the analyses of interleukins (IL), interferon gamma (IFN-γ), and tumour necrosis factor alpha (TNF-α) in milk. A non-inferiority randomized clinical trial was conducted. The null hypothesis was that cure risk in the experimental group (P-PRP) was inferior to the reference group (CS). A total of 103 cows were selected according to SCC and presence of Gram-positive bacteria, 49 cows were treated with CS and 54 cows were treated with P-PRP after determination of its cellular and molecular quality control. Cure was assessed by milk analyses at day 21 and 22 after treatment. Cows that remained with SCM were retreated at day 26, and cure assessed at day 47 and 48. Overall, bacteriological cure was observed in 16 cows (30%) of the P-PRP group, and 35 cows (71%) in CS group. Staphylococcus aureus cure risk was higher in CS group, but inconclusive for Streptococcus spp. The mean SCC increased in relation to time only in the P-PRP group. A direct relation between time and treatment for IL-1, IL-2, and IL-6 was observed, while no differences were observed for IL-4. Furthermore, IL-1 and IL-2 increased in cows treated twice in both groups. IL-8, IFN-γ, and TNF-α showed a significant interaction between time and treatment. IFN-γ concentration was lower in the P-PRP group compared to the CS on days 0 and 22. Leukocyte counts were lower in P-PRP when compared to whole blood. TGF-ß1 and PF4 concentrations were higher in platelet lysates in comparison to P-PRGS and plasma. Moreover, PDGF-BB concentration was significantly higher in platelet lysates in comparison to plasma. Results obtained in this study demonstrate that SCM treated with PRP showed a lower rate of bacteriologic cure when compared to animals treated with CS.

Outbreak of Pseudomonas aeruginosa mastitis in a dairy cow herd in northern Greece and its control with an autogenous vaccine.

In a dairy cow herd consisted of herd of 200 lactating Holstein-Friesian cows and heifers, clinical signs of mastitis in 40 out of 170 animals were observed. Treatments with antibiotics were proved ineffective. Milk bacterial cultures from 15 affected animals revealed Pseudomonas aeruginosa. An autogenous vaccine was administered subcutaneously, twice in a month period, to all adults. Cases of clinical mastitis declined significantly (p⟨0.0001) during next 3 months.

Laboratory-based evaluation of a simplified point-of-care test intended to support treatment decisions in non-severe bovine clinical mastitis.

To limit the use of antimicrobials in dairy cattle, farmers are increasingly encouraged to adopt targeted treatment decisions based on knowledge of the pathogens causing clinical mastitis (CM), whereby treatment of non-severe CM is generally recommended for gram-positive mastitis but not for gram-negative or culture-negative mastitis. The objectives of this study were to conduct a laboratory-based evaluation of the performance of a simplified slide test as a tool to differentiate gram-positive CM from other cases of CM, and to compare its performance against a commercially available on-farm test that is commonly used in our area (VétoRapid). Test outcomes after 24-48 h incubation were compared to results from bacteriological culture and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS). Milk samples (n = 156) were obtained from cases of severe and non-severe CM on seven farms and collected by farm personnel. After removal of small numbers of contaminated samples and organisms with unknown species identity, the simplified slide test showed high sensitivity and accuracy (>80%), similar to the comparator test. For most outcomes of interest (culture positive, Escherichia coli, or gram-positive growth), the specificity of the slide test was higher than the specificity of the comparator test. When considering non-severe cases of CM only, and interpreting detection of gram-positive organisms as indicative of the need for antimicrobial treatment, the simplified test had higher specificity (77.4% v. 60.4%) and higher positive predictive value (79.7% v. 70.0%) than the comparator test and similar sensitivity (83.9% v. 87.5%). The proportion of sampled CM cases, contaminated samples and gram-positive mastitis cases - which affects the positive and negative predictive value, the economic value of diagnostic testing and its potential to reduce antimicrobial use - differed between farms. The simplicity and accuracy of the slide test could make it an attractive tool for farmers to target antimicrobial treatment of non-severe clinical mastitis.

Casein hydrolyzate for drying-off lactating mammary quarters in cows with chronic mastitis.

In this research communication we address the hypothesis that a single intramammary infusion of casein hydrolyzate (CH) would have a similar effect to three intramammary infusions of CH for drying-off quarters with chronic mastitis (CM) during lactation. Sixty cows with CM were selected and randomly distributed into two treatment groups: (a) three intramammary CH infusions (100 mg, 50 ml per infusion, with 24-h intervals) or (b) single intramammary CH infusion (300 mg, 50 ml). Milk samples from the treated and untreated quarters were collected for microbiological culture and somatic cell count (SCC) before and after CH infusions. Milk yield was recorded and a manual pressure index measurement was used to evaluate cessation of lactation. Of the 60 quarters selected, 43 (71.67%) had positive microbiological culture. The quarters treated with three intramammary CH infusions had higher udder pressure index than those treated with single CH infusion. However, the average milk yield and composite SCC of three functional quarters were not different among treatments. Therefore, a single infusion of CH has the potential to be used as an alternative method for drying-off mammary quarters with CM during lactation.

[Literature review on phyto-therapeutics for the treatment of bovine mastitis]. / Literaturübersicht zum Thema ­Phytotherapie zur Behandlung von ­Mastitis beim Rind.

INTRODUCTION: Literature review on phyto-therapeutics for the treatment of bovine mastitis.

INTRODUCTION: Afin de trouver une alternative aux traitements antibiotiques conventionnels des mammites bovines, une recherche bibliographique a été menée en 2015 sur les agents phytothérapeutiques qui ont été étudiés in vitro et utilisés in vivo (sur des patients) dans le monde entier. Une recherche bibliographique est l'une des premières étapes du développement d'un agent phytothérapeutique peroral contre les mammites bovines en utilisant la méthode de «pharmacologie inverse¼. Des phytothérapies citées dans le monde entier dans tous les types d'administration ont été compilées et comptées. Un total de 935 citations pertinentes différentes ont été trouvées dans 195 publications, qui ont été séparées et comptées en fonction de leur type d'appli­cation et de l'espèce cible. La liste de toutes les plantes et les citations peuvent être téléchargés à partir de http://vets.ch/desktop/liste-des-plantes_fuchs_def_sat.pdf . On a répertorié dans la présente étude 106 plantes pour l'application perorale, 45 plantes pour l'application intra mammaire et 24 huiles essentielles (perorale, intra mammaire ou topique) pour le traitement des mammites bovines. La condition préalable à cette sélection était que les plantes soient mentionnées dans la littérature comme agent thérapeutique. En outre, il a été pris en compte si des études in vitro ou in vivo avaient déjà été menées. D'une part, cette liste devrait servir de preuve de l'utilisation traditionnelle des plantes dans le traitement des mammites bovines et, d'autre part, constituer une base pour de nouvelles recherches.

Advances in therapeutic and managemental approaches of bovine mastitis: a comprehensive review.

Mastitis (intramammary inflammation) caused by infectious pathogens is still considered a devastating condition of dairy animals affecting animal welfare as well as economically incurring huge losses to the dairy industry by means of decreased production performance and increased culling rates. Bovine mastitis is the inflammation of the mammary glands/udder of bovines, caused by bacterial pathogens, in most cases. Routine diagnosis is based on clinical and subclinical forms of the disease. This underlines the significance of early and rapid identification/detection of etiological agents at the farm level, for which several diagnostic techniques have been developed. Therapeutic regimens such as antibiotics, immunotherapy, bacteriocins, bacteriophages, antimicrobial peptides, probiotics, stem cell therapy, native secretory factors, nutritional, dry cow and lactation therapy, genetic selection, herbs, and nanoparticle technology-based therapy have been evaluated for their efficacy in the treatment of mastitis. Even though several strategies have been developed over the years for the purpose of managing both clinical and subclinical forms of mastitis, all of them lacked the efficacy to eliminate the associated etiological agent when used as a monotherapy. Further, research has to be directed towards the development of new therapeutic agents/techniques that can both replace conventional techniques and also solve the problem of emerging antibiotic resistance. The objective of the present review is to describe the etiological agents, pathogenesis, and diagnosis in brief along with an extensive discussion on the advances in the treatment and management of mastitis, which would help safeguard the health of dairy animals.

A potential treatment approach for subclinical mastitis in dairy cows: auriculotherapy of the auricular branch of the vagus nerve.

This research aims to test the hypothesis that neural therapy (NT) on the auricular branch of the vagus nerve (ABVN) in dairy cows diagnosed with subclinical mastitis (SCM) results in a reduction of the somatic cell count. Therapeutic options for SCM are mostly based on use of antibiotics and often lead to unsatisfactory results. An alternative therapy targeting the anti-inflammatory properties of the vagus nerve showing good efficacy, economic viability and without major side effects would be of considerable interest. Auriculotherapy (AT) was performed using three repeated infiltrations of 8.0 mg (0.4 ml) procaine hydrochloride (2%) at the location of the ear tag associated with the auricular acupuncture point (AAP) of the udder. Some 85 clinically healthy cows from nine dairy farms were sampled for evaluation of quarter somatic cell count (QSCC) on four days (d0, d2, d4 and d6). Quarters with a QSCC > 100 000 cells/ml on d0 were included in the analysis. Over the study period, a total of 784 quarters were analysed, 385 control (CON) quarters from 40 cows and 399 treated (TRE) quarters from 45 cows. Results showed that AT of the ABNV resulted in a significant reduction of the QSCC after three treatments. The effect was independent of bacteriological culture results of the quarter milk samples. The bacteriological cure rate, however, was not influenced by AT. To our knowledge this is the first report of AT reducing QSCC in dairy cows with SCM. Before AT can be regarded as an alternative therapeutic approach, further research should focus on possible long-term effects of AT on the reduction of SCC, any bacterial elimination and the neural pathways of AT in dairy cows with SCM.

Bacteriophage has beneficial effects in a murine model of Klebsiella pneumoniae mastitis.

Bovine mastitis caused by Klebsiella pneumoniae is usually treated with antibiotics, thereby potentially increasing antimicrobial resistance. The objective of this study was to evaluate efficacy of a bacteriophage, isolated from dairy farm wastewater, as a treatment for a murine model of K. pneumoniae mastitis. A lytic bacteriophage CM8-1 was isolated, morphological and biological characteristics were assessed with transmission electron microscopy and double-layer plate, and its genome was sequenced and analyzed. Furthermore, effectiveness of this bacteriophage for treatment of a murine model of K. pneumoniae mastitis was evaluated based on the following mammary gland characteristics: morphological changes; number of K. pneumoniae; and mRNA and protein expression of pro-inflammatory factors TNF-α, IL-1ß, IL-6, and IL-8. Bacteriophage CM8-1 had an incubation period of 30 min and a burst time of 20 min. Its viability and adsorption were stable at 30 to 50°C, but decreased significantly at >60°C, with no significant change in viability or infectivity at pH 6 to 10. In a murine model of K. pneumoniae mastitis, injecting bacteriophage CM8-1 into the mammary gland 2 h after inoculation with K. pneumoniae resulted in reductions in bacterial counts in the murine mammary gland, improvements in mammary gland tissue morphology, and reductions in mRNA and protein expression of pro-inflammatory factors. Bacteriophage CM8-1 had stable biological characteristics and suppressed K. pneumoniae mastitis when injected into the mammary gland 2 h latera in mice bacterial inoculation.

[Bovine teat lesions - Retrospective data evaluation of 116 German Holstein cows]. / Zitzenverletzungen ­ retrospektive Datenauswertung von 116 Deutsch-Holstein-Kühen.

OBJECTIVE: The management and treatment of teat injuries represent major challenges in modern dairy farming. The injury per se, its treatment and the handling of the cow during the healing process interfere with the principal purpose of the cow, which is the production of saleable milk. The study presents treatment and post-operative management based on the evaluation of a large data set. MATERIALS AND METHODS: The data from patients of the Clinic of Ruminants and Swine of the University Leipzig from May 2012 to July 2017 were analysed in a retrospective study. The location and type of injury, initial and post-operative medication, and treatment and outcome were analyzed. RESULTS: In total, 120 teat injuries in 116 cows were treated. The teat canal was the most common location of injuries (n = 85; 70.8 %), and closed injuries (without an external wound) were the most frequent type (n = 73; 60.8 %). Of all teat canal injuries, 65 (54.2 %) were internal and did not involve the skin. Most of these injuries were treated under theloscopic guidance directly after initial examination irrespective of concomitant disorders, such as thelitis (except severe acute), cysternitis and mastitis. The majority of treated teats (n = 98; 87.4 %) had functional healing and could be milked, 8 (7.2 %) of treated teats were amputated and in the remaining functional healing was achieved after a second surgery. According to an owner survey at least 700 days post-treatment, 23 (21.5 %) of the surgically treated cows and 50 % (n = 2) of the non-surgically treated cows were slaughtered in consequence of complications associated with the treated teat or the corresponding quarter. CONCLUSION AND CLINICAL RELEVANCE: Teat injuries are commonly seen in dairy practice. A successful outcome is obtainable when surgical or medical treatment is administered promptly and consequently in addition to an appropriate post-operative management and monitoring.

Intramammary antibiotics with complementary acupuncture decreases milk serum N-acetyl-beta-D-glucosaminidase concentrations in dairy cattle with subclinical mastitis.

The objective of this research is to determine whether intramammary antibiotics with complementary acupuncture can reduce bovine mammary inflammation due to subclinical mastitis. Lactating cows were selected based on milk with a somatic cell count (SCC) greater than 500,000 cells/ml. Pre- and post-treatment milk samples were collected to determine SCC, aerobic bacterial content, milk ion conductivity, total protein, lactate dehydrogenase (LDH) and N-acetyl-beta-D-glucosaminidase (NAGase) concentrations. Milk serum was prepared from milk samples by double centrifugation. Concentrations of LDH and NAGase were determined using commercial enzyme-linked immunosorbent assays. Cows being treated with intramammary antibiotics were separated by random assignment to the acupuncture group (n = 10) and a no-acupuncture (control) group (n = 9). Both the acupuncture and control group were restrained for 30 min in a head catch 12 hr apart for a total of four times. For front quarters affected by subclinical mastitis, the acupuncture points used were spleen (SP) 12, SP 17, SP 18, SP 21, stomach (ST) 18 and conception vessel (CV) 12. For rear quarters affected by subclinical mastitis, the acupuncture points used were bladder (BL) 30, BL 30-1, BL 49, kidney (KI) 10, conception vessel (CV) 2 and CV 3. All parameters were compared using a Student t test. Significance was defined as p < .05. Compared to control cows, complementary acupuncture treatment reduced NAGase enzymatic activity in quarters of cows with subclinical mastitis. The reduction in NAGase suggests that complementary acupuncture treatment may be associated with healing of the damaged mammary epithelial cells, which are the primary source of NAGase activity in milk serum.

Therapeutic effects of conditioned - DPBS from amniotic stem cells on lactating cow mastitis.

OBJECTIVE: Bovine mastitis results in economic loss due to decrease in milk production. Antibiotic ointments are commonly used for treating. However, residue and anti-microbial resistance warranted attention progressively. Fortunately, stem cell anti-inflammatory properties and paracrine expression of cytokines accelerates wound healing and suppresses inflammatory reactions in mastitis. The objective of this study is to use the conditioned-Dulbecco's pluripotent stem cells (DPBS) from amniotic membrane stem cells (AMSCs) in treating bovine mastitis. MATERIALS AND METHODS: The cows with mastitis were divided into two groups. In antibiotic control group, the cows were given tetraneomycin ointment. In conditioned-DPBS of AMSCs treatment group, amniotic membrane was collected for AMSCs after delivery. With expression of surface antigen and potential of tri-linage differentiation, AMSCs were injected into mammary glands. Then, milk was sampled every three days to monitor the effect of both treatments. The quality of milk was measured with pH, titratable acidity, free calcium ions and somatic cell count. RESULTS: Our results demonstrated the Bovine AMSCs expressed CD44, low levels of CD4 and no CD105. Bovine AMSCs demonstrated the differentiation capability in the tri-cell lineages. Mastitis treatment with conditioned-DPBS from AMSCs (experimental group) and conventional antibiotics (control group) showed insignificant difference in pH value and titratable acidity. The level of ionic calcium concentration in the conditioned-DPBS group decreased from 3rd day to 12th day, while the level in the antibiotic group decreased from 0 day to 12th day. The somatic cell number was similar in both groups, which meet the standard of Taiwan milk collection. CONCLUSION: In conclusion, conditioned-DPBS from bovine AMSCs has the therapeutic potential to treat bovine mastitis and may replace antibiotics therapy in the future.

In vitro and in vivo assessment of phage therapy against Staphylococcus aureus causing bovine mastitis.

OBJECTIVE: The aim of this study was to assess the efficacy of lytic bacteriophages on Staphylococcus aureus causing bovine mastitis, by in vitro and in vivo assays using Galleria mellonella and murine mastitis models. METHODS: Between May and December 2016, ten S. aureus (five methicillin-resistant and five methicillin-sensitive) isolates were isolated from milk samples of cattle with mastitis in Belgium and Norway. The isolates were assessed in vitro for their susceptibility to four lytic bacteriophages (Romulus, Remus, ISP and DSM105264) and subsequently in vivo in G. mellonella larvae and in murine mastitis model. RESULTS: Romulus, Remus and ISP showed a lytic activity against the S. aureus isolates in vitro. A larvae survival rate below 50% was observed at 4 days post-inoculation (DPI) in the groups infected with a methicillin-sensitive S. aureus isolate and treated with these three phages in vivo. An incomplete recovery of the mouse mastitis was observed at 48h post-inoculation (HPI) in the groups infected and treated with the ISP phage in vivo. CONCLUSIONS: The observations are much more pronounced statistically between the infected- phosphate buffered saline (PBS)-treated and infected-phage-treated groups in G. mellonella and the murine mastitis model demonstrating an effect of the phages against S. aureus associated with bovine mastitis.

Effects of intramammary infusion of Bifidobacterium breve on mastitis pathogens and somatic cell response in quarters from dairy cows with chronic subclinical mastitis.

The present study assessed the effects of intramammary infusion of Bifidobacterium breve (B. breve) on mastitis-causing pathogens and on the somatic cell counts (SCC) in lactating cows with chronic subclinical mastitis. The bacteriological cure rates of 42 quarters from 42 cows infected with Staphylococcus aureus, Corynebacterium bovis, coagulase-negative staphylococci, and environmental streptococci were 18.2% (2/11), 14.3% (1/7), 58.8% (10/17), and 28.6% (2/7), respectively, on day 14 after B. breve infusion. In a second trial, B. breve was infused into 18 quarters from 18 cows with chronic subclinical mastitis from which pathogens had not been isolated; the rates of quarters showing SCC > 50 × 104 cells/ml prior to B. breve infusion that decreased to < 30 × 104 cells/ml after infusion were significantly (p < .01) increased to 61.1% (11/18) on day 14 compared to that prior to infusion (0/18). The intramammary infusion of B. breve appears to be a non-antibiotic approach for elimination of minor pathogens and decreasing SCC in quarters with chronic subclinical mastitis in dairy cows.

Bacteriophages and associated endolysins in therapy and prevention of mastitis and metritis in cows: Current knowledge.

Bacteriophages and the associated endolysins have been proposed as an alternative to antibiotic treatment of mastitis and metritis in cows. Many bacteriophages have been isolated and characterized with a large amount of lytic potential against the bacteria causing mastitis and metritis in cows. Several endolysins with marked lytic activity against mastitis pathogens in vitro were also produced from staphylococcal and streptococcal bacteriophages. In the few clinical studies, however, there has been marginal efficacy of bacteriophages in the therapy of mastitis caused by Staphylococcus aureus. Similarly, lytic bacteriophages have marked antimicrobial activity in vitro against E. coli strains from the uteri of postpartum dairy cows. In clinical studies, however, neither administration of bacteriophages early postpartum nor prepartum was effective in the prevention of metritis in cows. More clinical studies on the effectiveness of bacteriophages and the associated endolysins in the prevention and therapy of mastitis and metritis in cows, therefore, are needed.