Mastitis In Animals Research Articles

Development and mouse model evaluation of a new phage cocktail intended as an alternative to antibiotics for treatment of Staphylococcus aureus-induced bovine mastitis

Bovine mastitis (BM) is a prevalent infectious disease in dairy herds worldwide, resulting in substantial economic losses. Staphylococcus aureus is a major cause of mastitis in animals, and its antibiotic resistance poses challenges for treatment. Recently, there has been a renewed interest in the development of alternative methods to antibiotic therapy, including bacteriophages (phages), for controlling bacterial infections. In this study, 2 lytic phages (designated as JDYN for vB_SauM_JDYN and JDF86 for vB_SauM_JDF86) were isolated from the cattle sewage effluent samples collected from dairy farms in Shanghai. The 2 phages have a broad bactericidal spectrum against Staphylococcus of various origins. Genomic and morphological analyses revealed that the 2 phages belonged to the Myoviridae family. Moreover, JDYN and JDF86 remained stable under a wide range of temperatures or pH and were almost unaffected in chloroform. In this study, we prepared a phage cocktail designated "PHC-1" which consisted of a 1:1:1 ratio of JDYN, JDF86 and SLPW (a previously characterized phage). PHC-1 showed the strongest bacteriolytic effect and the lowest frequency of emergence of bacteriophage insensitive mutants compared with monophages. The bovine mammary epithelial cells (MAC-T cells) and lactating mice mastitis model were used to evaluate the effectiveness of PHC-1 in vitro and in vivo, respectively. The results demonstrated that PHC-1 treatment significantly reduced bacterial load, alleviated inflammatory response, and improved mastitis pathology. Altogether, these results suggest that PHC-1 has the potential to treat S. aureus-induced bovine mastitis and that phage cocktails can combat antibiotic-resistant S. aureus infections.

ANALYSIS OF PHYSICOCHEMICAL AND NUTRITIONAL PARAMETERS OF RAW MILK FROM COMMERCIAL DAIRY FARMS – IMPLICATIONS OF ADULTERATION FOR PUBLIC HEALTH

Objective: Globally, milk adulteration is an important public health issue but more so in developing countries, for small financial gains. This study was designed to assess the physicochemical and nutritional parameters of raw milk from commercial dairy farms from a public health perspective. Material and Methods: In this study 29 bulk samples of raw cattle milk were selected, one from each dairy farm in Peshawar, using simple random sampling and analyzed using by ultrasonic milk analyser. The main parameters tested for adulteration of milk were urea, starch, hydrogen peroxide, detergent, sorbitol, quaternary ammonium compounds, boric acid, cane sugar, sodium chloride, carbonate, formalin, hypochlorite, freezing point, added water, salt percentages and electrical conductivity (EC). Additionally, nutritional quality was assessed by fats, solids-non-fats (SNF), lactose, and protein. The results were statistically analyzed by t-test using SPSS software. Results: Results showed that more than 90% of the samples had water adulteration of more than 5% due to which 55% had significantly reduced nutritional parameters but still overall nutritional quality was acceptable in 24% of the samples. 21% of samples had high electrical conductivity reflecting mastitis in animals and consequently considered unsuitable for consumption due to the resultant transmission of either antibiotics or microorganisms into the human body. Furthermore, chemical adulterants were not detected in any sample. t-test showed that most of the parameters were significantly different (p=0.05) than the population standard means. Conclusion: Milk samples had compromised quality due to water addition in half of them and raised EC values in about a quarter of the samples. Despite water adulteration, nutrients like fat and SNF remained within ranges, indicating a cow-buffalo milk blend. Most tested parameters were lower than national averages but above minimum values. No chemical adulterants were detected. There’s a need for continuous milk testing to maintain public health standards. key words: mild adulteration, commecial dairy farms

Genomic Features and Pathophysiological Impact of a Multidrug-Resistant Staphylococcus warneri Variant in Murine Mastitis

Non-aureus staphylococci (NAS) represent a major etiological factor in dairy animal mastitis, yet their role and impact remain insufficiently studied. This study aimed to elucidate the genomic characteristics of a newly identified multidrug-resistant NAS strain, specifically Staphylococcus warneri G1M1F, isolated from murine feces in an experimental mastitis model. Surprisingly, NAS species accounted for 54.35% of murine mastitis cases, with S. warneri being the most prevalent at 40.0%. S. warneri G1M1F exhibited resistance to 10 major antibiotics. Whole-genome sequencing established a genetic connection between G1M1F and S. warneri strains isolated previously from various sources including mastitis milk in dairy animals, human feces and blood across diverse geographical regions. Genomic analysis of S. warneri G1M1F unveiled 34 antimicrobial resistance genes (ARGs), 30 virulence factor genes (VFGs), and 278 metabolic features. A significant portion of identified ARGs (64%) conferred resistance through antibiotic efflux pumps, while VFGs primarily related to bacterial adherence and biofilm formation. Inoculation with G1M1F in mice resulted in pronounced inflammatory lesions in mammary and colon tissues, indicating pathogenic potential. Our findings highlight distinctive genomic traits in S. warneri G1M1F, signifying the emergence of a novel multidrug-resistant NAS variant. These insights contribute to understanding NAS-related mastitis pathophysiology and inform strategies for effective treatment in dairy animals.

Evaluating wound-healing effect of silicon-zinc-boron-containing glycerohydrogel and its effect on mammary glands of high producing dairy cows

Cow’s milk quality, which may deteriorate due to inflammatory processes in the mammary glands, remains one of the important problems of dairy farming and requires effective, safe and affordable therapeutic agents. Nanocomposite silicon-zinc-boron-containing glycerohydrogel (Si-Zn-B-gel) may become a very good solution to the issue. The paper demonstrates wound-healing effect of the glycerohydrogel and confirms its effectiveness for teat hyperkeratosis treatment indairy cows. Results of a rat burn model – based experiment suggest that Si-Zn-B-gel is a promising wound healing agent for topical use. Thus, on Day 9 complete re-epithelialization of the burn surface was observed, with fibrous structures prevailing in the granulation tissue of the dermal layer, on Day 19 a mature scar was formed with a longitudinal alignment of collagen fibers. The production tests conducted inhigh producing dairy cows have demonstrated good therapeutic effect of the Si-Zn-B gel for teat-end hyperkeratosis and confirmed its long-term effect that helps to longer maintain the results achieved during treatment. After a 7-day treatment physiological structure of up to 27.8% teats improved, on Day 14 of the experiment no severe hyperkeratotic lesions were observed and the number of teats that correspond to the physiological norm was 72.2%. Analysis of the data collected shows that the Si-Zn-B-gel is effective for teat-end hyperkeratosis treatment, thus, it prevents mastitis in animals and improves the milk quality.

Biomedical aspects of the use of bacteriocins and glycerolates – possibility of use for the treatment of mastitis in cows

Domestic and foreign data on bacteriocin nisin's biomedical and veterinary use are analysed. The mechanism of action of Nisin is based on damage to the structures of the bacterial cell, which leads to the subsequent death of the target cell and makes it possible to reduce the development of microbial resistance. Like most bacteriocins, Nisin has high biological activity due to its effectiveness in the nanomolar range and is a lowtoxic substance. Unlike antibiotics, bacteriocin nisin is completely degraded in the body of humans and animals. An analysis of the sources revealed the safe and effective use of Nisin in clinical practice for treating respiratory, gastrointestinal and skin infections and inflammatory processes in the human oral cavity due to an antimicrobial effect against several microorganisms. It has been established that antimicrobial peptides exhibit synergistic and cytotoxic effects. The effective action of nisin against a wide range of pathogens of animal mastitis has been determined. A study of literary sources on using silicon-boron-containing glycerolates in medical and veterinary practice was carried out. The effective use of glycerolates in treating inflammatory diseases in humans and animals has been established due to their reparative and regenerative effects and high transcutaneous conductivity. The data analysis confirms the feasibility of using bacteriocin nisin with silicon glycerolates and boron bisglycerolates to develop pharmaceutical compositions.

The effectiveness of the use of mastitis vaccine in breeding farms

The article presents data showing the effectiveness of the use of anti-mastitis vaccine in breeding farms of the Sverdlovsk region, based on the study of the level of diagnosed mastitis and the indicator of somatic cells in milk. Studies have shown that the use of animal immunization against mastitis has a positive effect, so 6 months after the first vaccination of animals in the breeding reproducer and plant, the number of detected mastitis decreased by 16,6% and 7,3%, respectively, and the level of somatic cells in the milk of highly productive cows by 26,5% and 10,7%, respectively. 12 months after the first vaccination scheme was applied, the total number of animals with signs of mastitis in the breeding reproducer and plant was 13,3% and 7,5%, respectively. The level of somatic cells in the nucleus amounted to 290 thousand. /ml., and the stud farm of 450 thousand/ml 24 months after first use of vaccination schemes, the level of mastitis was identified at 6,0% and 5,7% of the studied population of high yielding cows breeding farm and breeding farm, respectively. The average number of somatic cells found in milk of animals of a farm decreased by 80 thousand/ml, the farm – to 79 thousand/ml, in comparison with the previous study period. The study revealed a significant decrease in diagnosed mastitis in animals after 32 months due to the use of the STARTVAK antimastitic vaccine, the total number of diseases in the breeding reproducer decreased by 30,6% compared to the period of the beginning of vaccination, and in the breeding plant – by 26,2%. As a result, the dynamics of reduction of subclinical and clinical mastitis in the breeder compared to the period at the time of immunization was 19,5% and 11,1%, respectively. And in the stud farm, the level of subclinical and clinical mastitis decreased by 16,9% and 9,0%, respectively. The number of somatic cells in the milk of cows contained in the breeding reproducer was 152 thousand/ml, which is 83,9% lower compared to the initial study period. The level of somatic cells in the studied samples of milk of animals kept in the breeding plant was 263 thousand/ml, which is 62,2% lower than this indicator before the application of the animal vaccination program against mastitis.

Удосконалення профілактичних протимаститних заходів на молочних фермах

Goal. To study the spread of mastitis in heifers in dairy farms in the Ternopil region to improve measures for the prevention and treatment of this disease. Methods. Diagnostics of subclinical mastitis in animals was carried out using clinical studies of the mammary gland (examination, palpation, test milking of the secretion with visual assessment), mastidine test, and bacteriological studies (isolation of the pathogenic agent). Results. In the 8th month of pregnancy, when the heifer’s mammary gland begins to develop, pathogenic microorganisms can penetrate it and cause the development of an inflammatory process — it was diagnosed in 17.6% of animals. In the 9th month of pregnancy, 2 weeks before labor, the number of subclinical mastitis in heifers was diagnosed 1.7 times (p≤0.001) more often than 4 weeks before labor. This means that every third to fourth firstborn has inflammatory processes in the mammary gland even before lactation begins. Bacteria of the genera Staphylococcus and Streptococcus were mainly isolated from the udder secretions of animals suffering from subclinical mastitis. Thus, 4 weeks before calving, S. agalactiae microorganisms were recorded in 44.4% of disease cases, and S. uberis — 2 times less often (p≤0.001). 2 weeks before labor, the number of cases of mastitis caused by S. agalactiae was 35.7%, and that one caused by S. dysgalactiae and S. uberis was 2.5 times (p≤0.001) and 1.7 times (p≤ 0.01) smaller. It was also established that pathogenic microorganisms S.uberis in heifers caused subclinical mastitis 2.0–2.1 times (p≤0.001) more often than in first-born heifers during the first month of lactation. The number of cases of subclinical mastitis caused by S. aureus microorganisms in heifers and firstborns in the 1st month of lactation ranged from 28.6 to 33.4%. Conclusions. Preventive anti-mastitis measures on dairy farms should primarily consist in the diagnostics of mastitis in heifers at the 8-9th month of pregnancy and its treatment before labor.

The pharmacokinetics and pharmacodynamics of cefquinome against Streptococcus agalactiae in a murine mastitis model.

Cefquinome is a new generation cephalosporin that is effective in the treatment of mastitis in animals. In this study, we evaluated the associations between the specific pharmacokinetics and pharmacodynamics (PK/PD) of cefquinome and its antibacterial activity against Streptococcus agalactiae in a mouse model of mastitis. After a single intramammary dose of cefquinome (30, 60, 120, and 240 μg/mammary gland), the concentration of cefquinome in plasma was analysed by liquid chromatography with tandem mass spectrometry (HPLC/MS-MS). The PK parameters were calculated using a one-compartment first-order absorption model. Antibacterial activity was defined as the maximum change in the S. agalactiae population after each dose. An inhibitory sigmoid Emax model was used to evaluate the relationships between the PK/PD index values and antibacterial effects. The duration for which the concentration of the antibiotic (%T) remained above the minimum inhibitory concentration (MIC) was defined as the optimal PK/PD index for assessing antibacterial activity. The values of %T > MIC to reach 0.5-log10CFU/MG, 1-log10 CFU/MG and 2-log10 CFU/MG reductions were 31, 47, and 81%, respectively. When the PK/PD index %T > MIC of cefquinome was >81% in vivo, the density of the Streptococcus agalactiae was reduced by 2-log10. These findings provide a valuable understanding to optimise the dose regimens of cefquinome in the treatment of S. agalactiae infections.

Molecular Identification of Some Bacterial Species Causing Animal Mastitis in Basrah Province, Iraq

Molecular Identification of Some Bacterial Species Causing Animal Mastitis in Basrah Province, Iraq

Mesenchymal Stem Cells Overexpressing ACE2 Favorably Ameliorate LPS-Induced Inflammatory Injury in Mammary Epithelial Cells.

Mesenchymal stem cells (MSCs) are capable of homing injury sites to exert anti-inflammatory as well as anti-damage effects and can be used as a vehicle for gene therapy. Angiotensin-converting enzyme 2 (ACE2) plays an important role in numerous inflammatory diseases, but fewer studies have been reported in animal mastitis. We hypothesized that MSCs overexpressing ACE2 is more effective in ameliorating lipopolysaccharide (LPS)-induced inflammatory injury in mammary epithelial cells compared to MSCs alone. The results showed that MSC-ACE2 inhibited the LPS induction by upregulation of TNF-α, IL-Iβ, IL-6, and iNOS mRNA expression levels in EpH4-Ev cells compared with MSCs. Furthermore, results showed that both MSC and MSC-ACE2 were significantly activated IL-10/STAT3/SOCS3 signaling pathway as well as inhibited TLR4/NF-κB and MAPK signaling pathways, but MSC-ACE2 had more significant effects. Meanwhile, MSC-ACE2 promoted the expression of proliferation-associated proteins and inhibited the expression of the apoptosis-associated proteins in EpH4-Ev cells. In addition, MSC and MSC-ACE2 reversed the LPS-induced downregulation expression levels of the tight junction proteins in mammary epithelial cells, indicating that both MSC as well as MSC-ACE2 could promote blood-milk barrier repair, and MSC-ACE2 was more effective. These results suggested that MSCs overexpressing ACE2 were more anti-inflammatory as well as anti-injurious action into LPS-induced inflammatory injury in the EpH4-Ev cells. Thus, MSCs overexpressing ACE2 is expected to serve as a potential strategy for mastitis treatment.

Biotechnology of the New Al-Test - an Indicator for the Diagnosis of Subclinical Mastitis and an Indication of the Effectiveness of the Bactericidal Drug Dioxinor for Serous Mastitis in Lactating Sheep

The article is devoted to the study of the effectiveness of the Al-test indicator for rapid diagnosis of mastitis in ewes. In laboratory studies using the Al-test diagnosticum, the main criterion for the presence of subclinical mastitis in animals was the formation of a well-formed purple jelly-like clot (moderate to dense) of in the milk samples, which was partially (+) or entirely (++) of thrown of out the from the hole in plate with stirring. On the plate, with a negative reaction (-), the mixture of milk and the indicator remained homogeneous, pale raspberry colored, with a dubious (+) - it contained traces of the formation of the color of raspberry jelly. To detect latent mastitis in sheep, we proposed the rapid test of the A1 test, which is an aqueous solution of sulfanol and cresol red. Unlike a 2% mastidine solution, it is more sensitive. We also recommend for the treatment of serous mastitis in lactating sheep, intramuscularly administer Dioxinor at a dose of 0.1 ml / kg body weight, in twice a day until complete recovery and oxytocin, at a dose of 5 units in once a day on of the first 2 days of treatment, and also of carry out arrogant novocaine blockade according to D.D. Logvinov, twice 0.25% novocaine of solution, at a dose of 0.5 ml / kg body weight. In the experimental group, where the complex antimicrobial preparation Dioxinor was used, the effectiveness of treating serous mastitis in sheep was 96,3%.

PK/PD Analysis by Nonlinear Mixed-Effects Modeling of a Marbofloxacin Dose Regimen for Treatment of Goat Mastitis Produced by Coagulase-Negative Staphylococci

Simple SummaryCoagulase-negative staphylococci are main pathogens that produce goat mastitis. Marbofloxacin is a third-generation fluoroquinolone approved to treat mastitis in animals. Since the efficacy of an antimicrobial is related with its concentration in the site of infection, and the latter depends of dose and biological processes that determine the distribution of the antimicrobial in different tissues and secretions, the objectives of this study were to evaluate the efficacy of a dose regimen of marbofloxacin (10 mg/kg/24 h) administered intramuscularly for five days in goats with mastitis induced by coagulase-negative staphylococci, by an evaluation of the concentrations of marbofloxacin achieved in blood and milk over time (called pharmacokinetics), and characterizing the concentration–effect relationship of marbofloxacin against coagulase-negative staphylococci in Mueller Hinton broth and goat milk, by time kill assays, in order to determine the concentrations of marbofloxacin related with an adequate bacterial count reduction (measured by efficacy index AUC/MIC). The proposed dose regimen was adequate for the treatment of goat mastitis produced by coagulase-negative staphylococci, resulting in a microbiological and clinical cure of all animals. The animal model used in this study provided important pharmacokinetic information about the effect of the infection on the pharmacokinetics of marbofloxacin. Pharmacodynamic modeling showed that marbofloxacin concentrations needed for antimicrobial efficacy were higher in goat milk compared with Mueller Hinton broth. Bacterial resistance to antimicrobials is a serious problem, since marbofloxacin is considered a critically important antimicrobial, and its rational and prudent use could extend its utility over time.Coagulase-negative staphylococci are main pathogens that produce goat mastitis. Marbofloxacin is a third-generation fluoroquinolone approved for treat mastitis in animals. The objectives of this study were: (i) to determine the pharmacokinetics of marbofloxacin (10 mg/kg/24 h) in serum and milk administered intramuscularly for five days in goats with mastitis induced by coagulase-negative staphylococci; (ii) to characterize the concentration–effect relationship of marbofloxacin against coagulase-negative staphylococci in Mueller Hinton broth and goat milk; (iii) to determine AUC/MIC cutoff values of marbofloxacin, and (iv) to perform a PK/PD analysis to evaluate the efficacy of the dose regimen for the treatment of goat mastitis produced by coagulase-negative staphylococci. Marbofloxacin presented context-sensitive pharmacokinetics, influenced by the evolution of the disease, which decreased marbofloxacin disposition in serum and milk. Marbofloxacin showed a median (95% CI) fAUC/MIC values for MIC of 0.4 and 0.8 µg/mL of 26.66 (22.26–36.64) and 32.28 (26.57–48.35) related with −2 log10CFU/mL reduction; and 32.26 (24.81–81.50) and 41.39 (29.38–128.01) for −3 log10CFU/mL reduction in Mueller Hinton broth. For milk, −2 log10CFU/mL reduction was achieved with 41.48 (35.29–58.73) and 51.91 (39.09–131.63), and −3 log10CFU/mL reduction with 51.04 (41.6–82.1) and 65.65 (46.68–210.16). The proposed dose regimen was adequate for the treatment of goat mastitis produced by coagulase-negative staphylococci, resulting in microbiological and clinical cure of all animals. The animal model used in this study provided important pharmacokinetic information about the effect of the infection on the pharmacokinetics of marbofloxacin. Pharmacodynamic modeling showed that fAUC/MIC cutoff values were higher in goat milk compared with Mueller Hinton broth.

JUSTIFICATION OF CONSTRUCTIVE-REGIME PARAMETERS OF VARIABLE RUBBER OF VARIABLE SECTION

Domestic and foreign industries do not produce milking machines that would fully comply with the physiology of animals. Full milking of the udder and reduction of cows with ma-stit significantly depends on the operation of the milking machine. The quality of serial milking cups does not fully meet modern zootechnical requirements. In the process of their work, especially at the beginning and at the end of milking cows, "idle" milking can take place at nominal vacuum. Milking at high vacuum often irritates the teat sphincter, which often causes mastitis in animals, followed by cancer. Also, along with the growth of milk production, its quality is extremely important. The main technical solutions for milking machines with controlled change of vacuum pressure are analyzed. Some theoretical and experimental studies of the milking machine, which provides a reduction of the vacuum load on the udder and a rigid vibration effect on the teat sphincter at nominal vacuum in the process of machine milking cows. Despite the many known solutions to improve the milking machine, the task of creating a design that combines high productivity and stimulation of the reflex remains relevant. Designs of milking machines are now being improved mainly in two directions, namely - the development of milking machines that allow you to adjust the operating parameters depending on the level of milk yield and design improvements of milking machines that stimulate the milk reflex without causing the animal negative effects. Theoretical studies of the working process of machine milking allowed to systematize the method of engineering calculation of the milking machine with controlled mode of vacuum pressure on the teats of the cow and to determine the optimal design parameters of the executive mechanisms. The obtained analytical expressions characterize the influence of their parameters and mode of operation on the dynamics of milk production during machine milking of cows.

Occurrence of Beta-Lactamases Genes in Beta-Lactam Resistant Bacteria Isolated from Milk of Goats with Sub-Clinical Mastitis in Thika Sub-County, Kenya

For decades, beta-lactam antibiotics have played a critical role in the control of mastitis in animals. However, the emergence of beta-lactam-resistant bacteria poses a challenge to both human and veterinary medicine. In this regard, bacterial isolates from milk samples collected from dairy goats with sub-clinical mastitis from Thika Sub-county, Kenya, were used in the current study to interrogate the occurrence of beta-lactamases genes in bacterial isolates (Klebsiella pneumoniae, Staphylococcus aureus, and Acinetobacter spp., and Coagulase-negative Staphylococci) with known phenotypic resistance profiles to penicillin G, cephalexin, cefoxitin, and cefotaxime. Four target genes, blaTEM, blaSHV, blaCTX, blaOXA, and blaKPC were amplified using a polymerase chain reaction, and compared with Escherichia coli American Type Culture Collection 35218 and non-standard Klebsiella pneumonia positive controls. Out of the 46 samples, 44 samples (95.7%) harbored blaTEM with two samples of the 44 bacterial isolates, also possessing the blaSHV gene. Only one isolate of Klebsiella pneumonia and Acinetobacter spp had a combination of blaTEM and blaSHV. None of the bacteria had blaCTX, blaOXA, and blaKPC genes. The data indicated that sub-clinical mastitis in dairy goats in Thika Sub-county is associated with the bacteria carrying beta-lactamases genes, suggesting that the use of beta-lactam antibiotics for the treatment of sub-clinical mastitis may result in the treatment failure and potential transfer of the infectious bacteria to humans and other animals. The current study recommends the use of an alternative class of antibiotics for the management of beta-lactam-resistant bacteria.

Unprocessed milk as a source of multidrug-resistant Staphylococcus aureus strains

Staphylococcus aureus is the most relevant pathogen of animal mastitis and milk-related intoxications. Its presence in directly sold milk is rather not to be expected if strict udder health management and regular microbiological control of raw milk are performed. In this one-year survey, we present the results of monthly microbiological testing of milk from vending machines in Croatia for S. aureus and its multi-drug resistance. Staphylococcus aureus was detected in 27.58% of the samples. Among 60 tested isolates from 10 farmers, a total of 41 isolates were resistant to at least one antimicrobial agent (68.33%). A Multiple Antibiotic Resistance (MAR) index of 0.2 or higher had 48.8% of the resistant isolates, which is considered a high-risk potential for the spread of antimicrobial resistance. The majority of the isolates were resistant to penicillin and ampicillin followed by ciprofloxacin, ceftazidime, and kanamycin. The results impose the need for improving the control measures in the raw milk distribution chain focused on MAR risk reduction.

Isolation, Molecular Identification and Multidrug Resistance Profiling of Bacteria Causing Clinical Mastitis in Cows

Background: Mastitis, the inflammation of parenchyma of mammary gland is frequently considered to be costliest and complex disease prevalent in India. Mastitis is caused by pathogens like Staphylococcus spp., Streptococcus spp., Mycoplasma bovis, E. coli, Klebsiella spp., Citrobacter spp., Enterobacter spp. and Entercoccus. The treatment of mastitis in animals is carried out using antibiotics. Treatment failure in mastitis is due to increased antibiotic resistance of mastitis pathogens and also due to indiscriminate use of antibiotics without testing in vitro antibiotic sensitivity test against causal organisms. In comparison to cultural method, PCR assays takes less time for detection of bacteria from the mastitis milk samples. Present research work was carried out regarding isolation, identification and multiple drug resistance profile of clinical bovine mastitis associated pathogens using conventional as well as molecular approach. Methods: In the present study, 73 mastitis milk samples were collected from Anand and Panchmahal district of Gujarat. The milk samples were subjected for cultural isolation and DNA extraction for identification of bacteria by cultural and PCR method. Antimicrobial sensitivity pattern of the isolates were carried by disc diffusion method and isolates were categorized in multiple drug resistant. Result: In the present study, Out of 73 mastitis milk samples collected from cows 48 (65.75%) cows were positive for bacterial isolation and S. aureus was the most predominant bacterial species. PCR from the mastitis milk additionally detected bacteria in culturally negative milk samples. Most sensitive drug was gentamicin and most of the isolates (90.19%) showed the multiple drug resistance for the two to nine drugs with 0.1 to 0.6 multiple antibiotic resistance index.

Immunoprotective evaluation of <I>Escherichia coli</I> outer membrane protein A against the main pathogens of animal mastitis

Purpose: To evaluate prokaryotic expression of the Escherichia coli (E. coli) outer membrane protein A (OmpA) and its immunoprotective function against the main pathogens of animal mastitis.Methods: A molecular cloning method was used to develop a prokaryotic strain expressing OmpA protein, which was purified by Ni-affinity chromatography. Polyclonal antiserum was generated in mice immunized with OmpA protein. Enzyme-linked immunosorbent assay (ELISA) and western blotting were used to determine the titer and verify anti-OmpA serum specificity, respectively. Interaction between OmpA antiserum and main pathogens of animal mastitis was verified by ELISA and a pull-down method. The immune protective function of OmpA protein was evaluated in mice challenged with pathogens of animal mastitis. Optimal fermentation conditions to produce OmpA protein were determined by the L9(34) orthogonal test.Results: A prokaryotic strain expressing OmpA protein was developed, and purified OmpA was used to develop a mouse polyclonal antibody. The anti-OmpA serum exhibited high specificity and a titer of 1:1600. Anti-OmpA serum directly interacted with E. coli and Staphylococcus aureus (S. aureus). OmpA demonstrated a significant immune protective function of 58.33 % against E. coli and 46.15 % against S. aureus. The optimal conditions for expressing fermentation OmpA were a strain absorbance of 0.5 at a wavelength of 600 nm, IPTG final concentration of 0.3 mmol/L, induction time of 12 h, and induction temperature of 28 °C.Conclusion: OmpA possesses selective immunogenicity and a significant immune protective effect against the main pathogens of animal mastitis. The results suggest that OmpA may potentially be used as a vaccine for animal mastitis.
 Keywords: E. coli, OmpA protein, Immunoprotection, Animal mastitis, Protein fermentation

Vaccination of cows against mastitis-an alternative to antibiotics application

Data demonstrating the effectiveness of the antimastitic vaccine in bred livestock farms in the Sverdlovsk region by examining the level of diagnosed mastitis and the index of somatic cells (SCC) in milk are given. Application of animal immunization against mastitis revealed that 6 months after the first vaccination of animals in the pedigree breeding unit and plant, the number of identified mastitis reduced by 16.6% and 7.3%, respectively, and the level of SCC in the milk of highly productive cows by 26.5% and 10.7%, respectively. 12 months after the first vaccination protocol was applied. The overall number of animals with evidence of mastitis in the pedigree breeding unit and plant was 13.3% and 7.5%, respectively. The SCC level in 1 organization was 290 thousand/ml, and in 2 - 450 thousand / ml. The research found a significant reduction in the diagnosed mastitis in animals after 32 months due to the application of the antimastitis vaccine. The overall number of diseases reduced by 30.6% in organization 1 and 26.2% in organization 2 comparing to the period of the beginning of vaccination. Results: the dynamics of decrease in asymptomatic and clinical mastitis in organization 2 comparing to the period at the time of immunization was 19.5% and 11.1%, respectively. And in organization 1, the level of asymptomatic and clinical mastitis decreased by 16.9% and 9.0%, respectively. The quantity of SCC in the milk of cows kept in the research organization 1 had a value of 152 thousand/ml, which is 83.9% lower comparing to the first period of the study. The level of SCC in the tested samples of milk of animals kept in organization 2 was 263 thousand/ml, which is 62.2% lower than this index before the application of the animal vaccination program against mastitis.

Management Practices for Rearing of Gangatiri Cattle in Native Tract

The present study was purposively conducted in native tract of Gangatiri cattle in Uttar Pradesh. The information were collected from 108 respondents, who were rearing Gangatiri cattle for their livelihood. It was observed that most (53.70%) of the respondents practicing 6-10 hrs grazing of their Gangatiri herd. All the farmers were providing natural service to cows in estrus, maximum number of them (54.63%) between 12 to 16 hrs after detection of estrus and 53.70 per cent of them with bulls available in village surroundings. Maximum number of respondents (35.20%) initially used indigenous knowledge for disease treatment and after that consulted with veterinary doctor/ stockman. Major diseases prevelant in the area include FMD, HS, BQ and mastitis. Gangatiri cows were found to be more resistance to the diseases and heat tolerant as compared to crossbred cattle. Majority (81.48%) of respondents kept their cattle on kachcha floor in good sanitary conditions. About 70.37% respondents made cattle shed as a part of their houses. Majority (59.25 %) of the cattle owners were using knuckling method of milking, which may be a cause of mastitis in animals. It can be concluded that there is need of awareness programmes on scientific cattle rearing practices and interventions like supply of semen of bulls of high genetic merit under AI programme or bulls of good genetic merit for natural breeding, adequate grazing land and inputs for health management practices are needed to make the Gangatiri cattle rearing more beneficial.

Sodium houttuyfonate inhibits LPS‑induced mastitis in mice via the NF‑κB signalling pathway.

Sodium houttuyfonate (SH) has been indicated to play an important anti‑inflammatory role. Previous studies have confirmed that SH can inhibit the NF‑κB pathway in lipopolysaccharide (LPS)‑induced mastitis in bovine mammary epithelial cells. However, the effects of SH on LPS‑induced mastitis in animals should be verified to further evaluate its actual value. In the present study, the anti‑inflammatory effects of SH were investigated in mouse models and a mouse mammary epithelial cell line. Hematoxylin and eosin staining (H&E) showed that SH therapy significantly alleviated the pathological changes in mammary glands. Myeloperoxidase (MPO) activity analysis demonstrated that SH substantially decreased MPO activity invivo. RT‑qPCR results showed that SH reduced the expression of interleukin (IL)‑1, IL‑6 and tumor necrosis factor α both invivo and invitro. In addition, western blot results indicated that SH suppressed the phosphorylation of nuclear factor kappa‑light‑chain‑enhancer of activated B‑cells (NF‑κB) p65 protein and reduced the degradation of inhibitor of kappa light polypeptide gene enhancer in B‑cells alpha protein invivo and invitro. These results demonstrated that SH ameliorates LPS‑induced mastitis by inhibiting the NF‑κB pathway.