Cow Milk Samples Research Articles

Prevalence of Mastitis and Phenotypic Characterization of Methicillin-Resistant Staphylococcus aureus in Lactating Dairy Cows of Selected Dairy Farms in and Around Adama Town, Central Ethiopia.

Background:The emergence of Methicillin resistant Staphylococcus aureus (MRSA) poses a serious public health threat. Strains of Staphylococcus aureus resistant to β-lactam antibiotics are known as MRSA. MRSA has gained attention as community pathogen. MRSA has been increasingly reported as emerging problem in veterinary medicine. However, little is known in Ethiopia. This study was, therefore, conducted to identify MRSA, to determine its drug susceptibility patterns, and mastitis infection in dairy cattle in and around Adama town, Central Ethiopia.Methods:A cross-sectional study was conducted to estimate the occurrence of MRSA in mastitic dairy cows in and around Adama town, central Ethiopia. A total of 384 lactating cows were included from the conveniently selected dairy farms in the study area. Approximately 10 ml of milk was aseptically collected from clinical and subclinical mastitic cows into sterile universal bottles after discarding the first 3 milking streams. Then, Staphylococcus aureus was isolated using the conventional bacteriological procedure. Resistance to methicillin was detected using the Kirby-Bauer disc diffusion antibiotic susceptibility method. Oxacillin disc was used to detect methicillin resistant Staphylococcus aureus strains. Antimicrobial susceptibility test was conducted against MRSA strains using streptomycin (S, 10 µg), amoxicillin (Am, 25 µg), kanamycin (k, 30 µg), nalidixic acid (NA, 30 µg), oxytetracycline (OT, 30 µg) sulphonamide (S, 300 µg) and ceftriazole (CRO, 30 µg).Results:The study found that the prevalence of mastitis was 121(31.5%). Among this 37(30.6%) were clinical mastitis and 84 (69.4%) of them were sub-clinical mastitis. Of 121 mastitis cases, Staphylococcus aureus was isolated in 37 (30.6%) of mastitic cow milk samples. The prevalence of mastitis was significantly affected by breed, age, floor type and hygienic status of the milkers (P < .05). Moreover, 32.4% of Staphylococcus aureus isolates were resistant to oxacillin. A total of 75% percent of MRSA isolates were resistant to amoxicillin, 66.7% were resistant to oxytetracycline, and 50% were resistant to sulphonamide. However, 75% of MRSA isolates were susceptible to kanamycin, 58.3% were susceptible to streptomycin, and 50% were susceptible to nalidixic acid.Conclusion:The study revealed that relatively high number of strains are resistant to the antibiotics commonly used in the therapeutic protocol of many human and animal infections. Therefore, antimicrobial susceptibility test should be carried out at a regular basis and proper hygienic practices should be introduced at farm level. Creating public awareness about transmission, prevention and control of MRSA should also be considered.

Genes conferring anti­microbial resistance in cattle with subclinical mastitis

This study was carried out to evaluate the antimicrobial resistance (AMR) as a risk factor associated with some microorganisms isolated from subclinical mastitis (SCM) milk samples from Holstein Friesian dairy animals in Fayoum area, Egypt. The percentage of the SCM in the farm was found to be 41.18% and 63.88% at quarter and cows level respectively, with mean somatic cell count (SCC) of 8.8×105 ± 9.2×103 cells/mL and electrical conductivity (EC) 6.27 ± 0.066 mS/cm for SCM quarter milk samples. Out of the total 444 SCM cow milk samples, the most often isolated microorganisms were Staphylococcus aureus: 296 (66.6%), Enterococcus spp.: 230 (51.80%), Escherichia coli: 210 (47.29%) and Streptococcus agalactiae: 106 (23.87%). AMR was determined by disc diffusion test and the corresponding resistance genes were detected by PCR. Results of the in vitro susceptibility tests performed and the phenotypes indicated that the highest resistance to antibiotics for isolated microorganisms was against penicillin followed by amoxicillin + clavulanic acid, oxacillin and tetracycline, whereas moderate resistance was exhibited to oxytetracycline, ampicillin, sulfamethazole/trimethoprim, cefotaxime and erythromycin. However the most effective antibiotics against most isolates were nitrofurantoin and gentamicin followed by enrofloxacin, norfloxacin and cefoxitin. It was shown that the resistance to tetracyclines was due to the tetK or tetA(A) genes, the resistance to β-lactams (penicillins) – to blaZ and blaTEM genes, to macrolides (erythromycin): to ermB and ermC genes. Methicillin resistance genes were mecA, mec1 and mecC, glycopeptides (vancomycin) resistance gene was vanA, and norfloxacin resistance was attributed to norA gene.

Carbonate and silicate dissolving bacteria isolated from home-made yogurt samples.

In the current study, twenty-eight bacterial strains were isolated from home-made yogurt samples from Ağrı Province, Turkey. The bacterial strains were identified by conventional and molecular techniques. Among the twenty- eight isolates, seventeen isolates were identified according to the 16 S rDNA region and determined to belong to five different genus including Sphingomonas (8 isolates), Burkholderia (5 isolates), Lactobacillus (2 isolates), Lactococcus (1 isolate), Staphylococcus (1 isolate). In this study, the presence of Burkholderia in home-made yogurt samples were reported for the first time, whereas Sphingomonas was detected for the second time. We also investigated the carbonate (CaCO3 and MgCO3) and silicate (‎CaSiO3 and MgSiO3) dissolving potential of seventeen bacterial isolates. Among these seventeen bacterial isolates, fifteen bacterial isolates have CaCO3-dissolving and 10 bacterial isolates have MgCO3-dissolving potential. The silicates dissolution ability was relatively less than that of carbonates dissolving. We observed that six bacterial isolates have CaSiO3 and only two bacterial isolates have MgSiO3 dissolution abilities. In conclusion, this work clearly shows the diversity of bacteria existing in fermented cow milk samples in Ağrı Province, Turkey, which could be considered as valuable sources for lactic acid bacteria (LAB) isolation and further probiotic potential.

Mycoplasma isolation in milk samples from dairy herds in Chile

Mycoplasma bovine mastitis is a highly contagious disease, usually associated with clinical cases refractory to antibiotic treatment. The aim of this study was the isolation of Mycoplasma species in cattle milk samples from dairy herds in Chile. Bulk tank milk samples selected by convenience from 91 Holstein Friesian dairy herds located in Los Rios (66) and Los Lagos (25), the two most important dairy Regions in the country, were collected. Additionally, 100 individual milk samples from cows with a high incidence of clinical mastitis, refractory to antibiotic therapy, and negative bacteriological results for traditional mastitis pathogens, all from the Biobio Region and received in our diagnostic laboratory, were included. All samples were cultured for 10 days on PPLO medium. The differentiation of suspect colonies between genus Mycoplasma and Acholeplasma was performed by the digitonin test and a specific PCR. The species identification was performed by a M. bovis specific PCR and 16S rRNA sequencing. Mycoplasma was isolated from 3 (3.3%) bulk tank milk samples and 2 (2%) individual cow milk samples. All colonies were identified as Mycoplasma by the digitonin test and by a specific PCR. At species level, one strain isolated from a bulk tank milk sample was identified as M. bovis. The remaining two strains isolated from bulk tank milk samples were identified as M. bovigenitalium, while the two strains isolated from milk of individual cows were identified as M. alkalescens. These results show that not only M. bovis is present in Chilean dairy herds, but also other pathogenic species not previously described in Chile such as M. bovigenitalium and M. alkalescens, which pose a potential risk for dairy herds in southern Chile.

Technical note: Development of a closed-tube isothermal multiple self-matching-initiated amplification assay for visual detection of Staphylococcus aureus in milk samples

Staphylococcus aureus is one of the most common mastitis-causing bacteria in dairy cows. It is associated with reduced production performance in animals and with huge financial losses for the dairy industry worldwide. An accurate and sensitive method for the early diagnosis and identification of Staph. aureus in milk samples is essential. The present study aimed to establish a closed-tube isothermal multiple self-matching-initiated amplification (IMSA) technique for visual confirmation of the presence of Staph. aureus targeting the nuc conserved sequence gene. The specific primers successfully amplified the target sequence within 45 min at 63°C reaction temperature and using the optimal components of the reaction system. The positive amplicon showed bright green fluorescence under UV light when mixed with the chromogenic substrate SYBR Green I, and the negative samples remained orange in color. We observed fluorescence and a ladder-like pattern in the IMSA amplicon for all Staph. aureus strains, and we observed no significant change for the non-Staph. aureus strains. The IMSA assay had high specificity compared with loop-mediated isothermal amplification (LAMP): it confirmed the presence of all 7 Staph. aureus strains, and we found no false-positive results for the 12 non-Staph. aureus strains. The lower limit of detection for the IMSA assay was 1 × 102 cfu/mL, 10-fold more sensitive than the results obtained using LAMP. We also successfully applied the IMSA assay to confirm the presence of Staph. aureus in milk samples of cows with mastitis, and the results were consistent with those of LAMP and real-time PCR. The present study reports the use of IMSA to confirm the presence of Staph. aureus and provides a potentially useful method for rapid preliminary screening for Staph. aureus.

Discovery and quantification of Irgacure-907 in commercial cow milk by using untargeted liquid chromatography high-resolution mass spectrometry

Food safety control can be realized through contaminant monitoring and regulation in food matrices and food-associated products. Untargeted profiling methods can provide comprehensive results through screening and identification of unexpected compound. In this study, we have successfully employed the untargeted profiling method to screen unexpected compounds, and identified an unexpected contaminant, Irgacure-907, in two of seven commercial cow milk samples. The Irgacure-907 was further confirmed and quantified as 7.9 ± 0.4 ng mL−1 and 21.2 ± 1.9 ng mL−1 through standard addition in two commercial cow milk samples. This is the first study to use untargeted profiling to screen unknown contaminants present in commercial milk. Moreover, the presence of Irgacure-907 in commercial milk has not been reported previously in Taiwan. This untargeted profiling approach can be used to reveal other unknown food contaminants in various food matrices in the future and is beneficial to public health worldwide.

Monitoring of Antimicrobial Resistance in diagnostic Veterinary laboratory in Dar es Salaam, Tanzania

Antimicrobial resistance (AMR) is a global one health issue threatening our ability to treat bacterial infections in humans and animals. Surveillance of AMR is important in order to estimate the size of the problem, to identify targets for measures and to evaluate the effect of implemented measures. The study was conducted to determine how microbiological samples were collected, results interpreted and the number of samples collected for bacterial culture and sensitivity testing; to identify bacteria frequently isolated from milk and avian samples; and to determine the trend of samples submitted for bacterial culture, sensitivity testing and AMR prevalence. A retrospective study was conducted to collect AMR data by extracting information from laboratory logbooks and laboratory information system from 2010-2017. About 90% of samples were submitted by farmers, and then registered into laboratory registration systems at the reception. A total of 4157 samples were collected for bacterial culture, which included 3571 milk samples collected from cows, 555 samples obtained from live or dead chicken, and 31 samples collected from other animal species. Four hundred and thirty (430) samples requested bacterial culture and sensitivity testing, of which 346 (80.5%) were from cow milk samples, 53 (12.3%) from avian samples, and 31 (7.2%) from other animal species. The common bacterial isolates were Micrococcus, E. coli, Salmonella, Staphylococcus, Enterobacter, and Bacillus species. The use of diagnostics and detection of drug susceptibility is important to support rational use of antibiotics and tracking of AMR.

Monitoring of Antimicrobial Resistance in diagnostic Veterinary laboratory in Dar es Salaam, Tanzania

Antimicrobial resistance (AMR) is a global one health issue threatening our ability to treat bacterial infections in humans and animals. Surveillance of AMR is important in order to estimate the size of the problem, to identify targets for measures and to evaluate the effect of implemented measures. The study was conducted to determine how microbiological samples were collected, results interpreted and the number of samples collected for bacterial culture and sensitivity testing; to identify bacteria frequently isolated from milk and avian samples; and to determine the trend of samples submitted for bacterial culture, sensitivity testing and AMR prevalence. A retrospective study was conducted to collect AMR data by extracting information from laboratory logbooks and laboratory information system from 2010-2017. About 90% of samples were submitted by farmers, and then registered into laboratory registration systems at the reception. A total of 4157 samples were collected for bacterial culture, which included 3571 milk samples collected from cows, 555 samples obtained from live or dead chicken, and 31 samples collected from other animal species. Four hundred and thirty (430) samples requested bacterial culture and sensitivity testing, of which 346 (80.5%) were from cow milk samples, 53 (12.3%) from avian samples, and 31 (7.2%) from other animal species. The common bacterial isolates were Micrococcus, E. coli, Salmonella, Staphylococcus, Enterobacter, and Bacillus species. The use of diagnostics and detection of drug susceptibility is important to support rational use of antibiotics and tracking of AMR.

Genotoxic impact of emerging contaminant amoxicillin residue on zebra fish (Danio rerio) embryos

The widest-spectrum, most-consumed β-lactam antibiotic amoxicillin (AMX) is used to treat bovine mastitis that is caused primarily by many bacteria. Excessive use of antibiotics can lead to established residual contamination of the milk even after pasteurization. The amount of antibiotic residue above Maximum Residue Limit (MRL) has a negative impact on both public health and the environment. Therefore, the objective of this study is to determine the concentration of amoxicillin residue (AMXR) in raw and pasteurized milk samples of cow suffered from mastitis, by the standard methods of HPLC compared to pure AMX drug and effect of the said residue on the developmental toxicity and genotoxicity of zebra fish at 72 hpf and 48 hpf embryo, respectively. Results obtained by HPLC showed that AMXR exhibits 574.89 and 250.75 times higher concentration in the raw and pasteurized milk than MRL in compare to pure AMX drug. This current study showed that AMXR decreased the body length and yolk sac region, while the pure AMX drug-treated group showed increased height and length of the yolk sac and shorter body length relative to the other groups. The Comet Assay measured the DNA damage caused by AMXR. The group where AMXR were applied showed the highest percentage of tail DNA and tail moment relative to other groups. So, here AMXR is considered as the genotoxic contaminant that is emerging and affect on public health.

THE DEVELOPMENT OF THE METHOD FOR THE DETERMINATION OF SULFONAMIDES AND TRIMETOPRIM RESIDUAL QUANTITIES IN MILK SAMPLES BY UPLC-MS/MS METHOD AND ITS VERIFICATION THROUGH INTERLABORATORY TESTING

This manuscript presents the results of development and adoption of the confirmatory UPLC- MS/MS method for determination of sulfonamides and trimethoprim residues in whole and powdered cow's milk samples. Sample preparation includes a recovery with water (in the case of milk powder), separation of milk fat by centrifugation at 4 °C, proteins precipitation with acetonitrile using ultrasonic intensification, extraction of analytes with a two-component mixture of organic solvents from weak acidic phosphate buffer solution, concentration of obtained extracts, degreasing and reconstitution with the mobile phase. The use of tandem-mass spectrometric detector in multiple reaction monitoring mode provides high specificity for all ten sulfonamides, which molecules contain the same structural elements, and trimethoprim, as three ion transitions were used for each of the target analytes. The approach of matrix matched calibration on the milk matrix was applied, and it allowed us to disregard taking into consideration the absolute extraction of analytes during sample preparation, as well as the influence of sample matrix components on analytes ionization. The main advantages of the developed technique are simplicity, rapidity and cost effective sample preparation, fast procedure of sample analysis (the time of chromatographic separation is 2.0 min), high selectivity, and sensitivity at the level of ≤ 0.5 μg/kg (as compared with the maximum residues level of sulfonamides in dairy products, viz. 100 μg/kg). According to the European Commission Decision 2002/657/EC, the values of CCα and CCβ for sulfonamides and trimethoprim in milk were calculated. The capability of the method was confirmed on the basis of the established validation parameters, using spiked milk samples and "added – found" approach. The method was tested during the analysis of spiked samples of whole and powdered cow's milk (CV ~ 20%). The developed technique was verified by interlaboratory comparative testing of milk powder samples for sulfonamides content. The UPLC-MS/MS method for the determination of sulfonamides and trimethoprim residues in whole and milk powder samples can be used both for routine laboratory analysis of dairy products and for the examination of suspicious or questionable samples.

Isolation and Characterization of Lactic Acid Bacteria From Milk and Their Effects on the Pathogenic Bacteria

Background: Probiotics are “live microbial cells” that are beneficial for human and animal health. Lactobacilli are such a diverse group of bacteria with similar metabolic and physiological characteristics, and constitute important and beneficial gut microflora. During carbohydrate fermentation, lactobacilli produce lactic acid as an end product in metabolism. Hence, lactobacilli have high significance to be used as probiotics in the food industry, because of their acidifying properties. Also, lactobacilli are considered “safe”, owing to their ubiquitous presence in the food. Many researchers provided evidence for the presence of lactobacilli in milk sources. Thus, the present study aimed to isolate and characterize different lactobacilli strains from milk sources and analyze their “probiotic potential”. Materials and methods: Forty-one lactobacilli isolates were obtained from raw cow milk. Then, the strains were characterized by morphological identification and biochemical tests. Besides, probiotic potentials were evaluated with the bile tolerance test, antibiotic susceptibility test, and determining suitable pH for the optimal growth of lactobacilli. The lactobacilli isolates were also analyzed for their probiotic characteristics and the release of antimicrobial substances. Their antimicrobial activities against pathogenic strains were assessed by determining the minimum inhibitory concentration, with the help of agar diffusion methods. Results: From 50 milk samples, 41 lactobacilli isolates were obtained, out of which five lactobacilli strains were identified as Lactobacillus casei, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus acidophilus, and Lactobacillus lactis. Moreover, 35 isolates showed an inhibitory effect. These strains were able to survive and grow in 0.5% to 2.5% bile salt concentrations. Lactic acid bacteria were susceptible to antibiotics, and 35 isolates obtained from raw milk showed an inhibitory effect against pathogenic bacteria. The observed minimum inhibitory concentration ranged from 50 to 100 µL and varied between the different pathogens. Conclusion: Out of 41 Lactobacillus isolates obtained from cow milk samples, 35 were identified with probiotic characteristics. Hence, this study highlighted the novel probiotic bacteria and validated the antimicrobial properties of the Lactobacillus spp against pathogenic bacteria.

Use of ELISA technique for detection of streptomycin residues in milk

This study involved examination of 45 milk samples were distributed as: 15 imported milk powder, 10 imported liquid milk, 10 local cow's milk, 10 local sheep's milk. These samples were collected from September 2010 to April 2011 at different markets in Mosul city (Iraq) in order to detect level of streptomycin residue using Enzyme Linked ImmunoSorbent Assay (ELISA) technique. The results showed that the percentage of positive samples of streptomycin residue in milk was 53.3%, and as follow 15.6% in the local sheep's milk samples, 13.3% in the local cow's milk samples, 11.1% in the imported liquid milk samples and 13.3% in the imported milk powder samples with mean value of 22.8, 20.1, 12.6 and 19.9 µg/kg milk, respectively. From these results, it is clear that all samples of milk had residual streptomycin level within the permissible limits and it is regarded as safe for human consumption.

Monitoring of Antibiotic Residues in Chicken Meat, Cow and Buffalo Milk Samples in Nepal

In animal products, antibiotic residue is a serious concern for public health in the Himalayan nation, Nepal. A cross-sectional study on antibiotic residues in chicken meat, milk samples of cows, and buffaloes was conducted in selected districts of different provinces of Nepal. Altogether 126 chicken meat samples, 42 each from Kailali (Farwestern Province), Kaski (Gandaki Province), and Nuwakot (Bagmati Province) districts were tested for tetracycline, sulphonamides, penicillin, gentamicin, streptomycin residues by the ELISA method. At Kaski district approximately 57 % (24/42), 50 % (21/42) and 83% (35/42) of meat samples were tested positive for gentamicin (mean-ppb:11.5 μg/Kg/L), tetracycline (mean-ppb:1.44 μg/Kg/L), and fluoroquinolones (mean-ppb: 11.46 μg/ Kg/L) residues respectively. In Kailali and Nuwakot district, 48% (20/42) and 50 % (21/ 42) of meat samples deemed positive for sulphonamides (mean-ppb:15.9 μg/ Kg/L) and penicillin (mean-ppb: 0.39 μg/ Kg/L) residues, respectively. Of the total 168 tests performed in milk samples from Kavre district, 55% (93/168) of the milk samples were tested positive to antibiotic residues for gentamicin, streptomycin, and sulphonamides. The chicken meat samples had antibiotic residues within the national maximum residue limit (MRL); however, milk samples exceeded the national MRL for sulphonamides residues (mean-ppb: 26.44 μg/ Kg/L). The effective surveillance for antibiotic residues in animal products should be implemented strictly in Nepal.&#x0D; Int. J. Appl. Sci. Biotechnol. Vol 8(3): 355-362

Front face synchronous fluorescence as a tool for the quality assurance of Greek milk

This research focuses on implementing the low cost and rapid front face synchronous fluorescence (SyFS) in order to ensure the quality assurance of Greek milk. Specifically, samples originated from the Greek domestic production of goat, sheep, cow, as well as foreign cow milk samples and adulterated cow milk samples. SyFS spectra were acquired in the excitation area of 250–500 nm with (Δλ)= 100 nm. Greek and foreign cow milk samples were differentiated based on intensity variations at wavelengths 350–515 nm, 540–579 nm, and 580–600 nm. The emissions at these wavelength positions correspond to tryptophan, vitamin A, and riboflavin. The supervised model with 94 samples exhibited p-value = 7,98E-11, RMSEE= 0,29171, RMSEcv= 0,29284 and RMSEP= 0,98013, AUROC for Greek samples= 0,61 and AUROC for foreign= 0,85. We differentiated milk samples according to the animal type with PCA and OPLS-DA models of 107 samples exhibiting RMSEE= 0,225842, RMSEcv= 0,228054 and RMSEP= 0,518635, AUROC for sheep samples= 0,99, AUROC for goat samples= 0,98 and AUROC for cow samples= 0,96. In fact, the emission band 350–591 nm characterized sheep milk and corresponds to aminoacids and fatty acids, cow milk was related to the 350–600 nm emission band related to the b-carotene and to the goat milk the emission bands 350–505 nm and 520–600 nm were attributed to tryptophan, NADH and Rivoflabin. Finally, we investigated whether SyFS coupled with chemometrics may provide preliminary evidence on adulterated cow milk samples. All models were validated with permutation testing, p-values and ROC curves.

Milk from healthy or infected cattle as a source of multi-drug resistant, AmpC β-lactamase-producing Escherichia coli

Drinking raw unpasteurized milk or products made from raw milk is a recent trend observed among the people due to their belief in enhancing nutritional properties and better taste. It poses a great risk of microbial transmission along with antibiotic resistance gene pool (extended spectrum beta-lactamase or ampC) among the consumers. The study was conducted to detect the occurrence of AmpC producing E. coli in raw milk samples collected from healthy and mastitic cattle in West Bengal. In total, 450 cow milk samples were collected from all the six agro-climatic zones of West Bengal. The samples were collected from healthy as well as infected cattle suffering with clinical or sub-clinical mastitis, irrespective of age and breed. The collected milk samples were tested with MAST INDEX reagent for the detection of sub-clinical mastitis. E. coli isolates obtained from cattle milk samples (n=450) were subjected to cefoxitin-cloxacillin double disc synergy test and PCR based confirmation of blaAmpC, class I integron and CITM genes. All the ACBL producing E. coli isolates were subjected for their resistance to other antimicrobials. All the levofloxacin and Co-Trimoxazole resistant ACBL-producing isolates were studied for PMQR, sul1, sul2, sul3 genes. E. coli was isolated from 205 collected milk samples (205/450, 45.6%). The phenotypical test detected 231/383 isolates (60.3%) were AMPC producers which were also found to possess blaAmpC. Comparison of ACBL-producing E. coli occurrence in cattle with mastitis, sub-clinical mastitis and healthy cattle revealed significantly higher occurrence rate in infected cattle than the healthy ones (p<0.05). Phenotypical antibiotic resistance pattern of the isolates showed resistance against penicillin-G (100%), cefoxitin (100%), ampicillin (42%), co-trimoxazole (13.5%) and levofloxacin (5.2%). Out of 30 co-trimoxazole resistant isolates identified, 25 (83%) and seven (30.4%) isolates possessed sul1 and sul2 gene, respectively, whereas, four (13.3%) isolates possessed both sul1 and sul2 genes. Three levofloxacin resistant isolates possessed qnrS gene. This is the first systemic study with substantial numbers of milk samples from healthy and infected cattle depicting occurrence of ACBL-producing E. coli and linkage present in the isolates between the genes encoding ACBL and quinolone or sulphonamide resistance.

Antimicrobial and Antibiofilm Capacity of Chitosan Nanoparticles against Wild Type Strain of Pseudomonas sp. Isolated from Milk of Cows Diagnosed with Bovine Mastitis.

Bovine mastitis (BM) is the most prevalent bacterial infection in the livestock sector, affecting the dairy industry greatly. The prevention and treatment of this disease is mainly made via antibiotics, but the increasing antimicrobial resistance of pathogens has affected the efficiency of conventional drugs. Pseudomonas sp. is one of the pathogens involved in this infection. The therapeutic rate of cure for this environmental mastitis-causing pathogen is practically zero, regardless of treatment. Biofilm formation has been one of the main virulence mechanisms of Pseudomonas hence presenting resistance to antibiotic therapy. We have manufactured chitosan nanoparticles (NQo) with tripolyphosphate (TPP) using ionotropic gelation. These NQo were confronted against a Pseudomonas sp. strain isolated from milk samples of cows diagnosed with BM, to evaluate their antimicrobial and antibiofilm capacity. The NQo showed great antibacterial effect in the minimum inhibitory concentrations (MIC), minimum bactericidal concentration (MBC) and disk diffusion assays. Using sub lethal concentrations, NQo were tested for inhibition of biofilm formation. The results show that the nanoparticles exhibited biofilm inhibition and were capable of eradicate pre-existing mature biofilm. These findings indicate that the NQo could act as a potential alternative to antibiotic treatment of BM.

Carbohydrate Fermentation Profile and Physiological Studies of Lactic Acid Bacteria from Native Raw Cow Milk

The aim of this study was to identify and evaluate the physiological studies of lactic acid bacteria from native raw cow milk. Cow milk samples were collected purposively from four different locations in Ibadan. The proximate analysis, pH and acidity of the milk samples were examined using standard procedures. Lactic acid bacteria (LAB) were isolated, characterized, and identified using both morphological, biochemical test, and Analytical profile index (API) system. The identified LAB were subjected to various physiological conditions such as growth at different temperature (15, 30, 45°C), pH (4, 6, 8) and NaCl concerntrations (4, 6, and 8%).The heterotrophic counts ranged between 3.1 ×107to 4.2 ×107CFU/mL and lactic acid bacteria counts ranged from 2.2× 107 to 3.8 × 107 CFU/mL. Thirty-five LAB isolates were randomly picked and identified as Lactobacillus (57.15%), Streptococcus (14.29%), Leuconostoc (8.57%), Pediococcus (8.57%), Lactococcus (5.71%) and Enterococcus (5.71%). The LAB isolates were further identified as Lactobacillus plantarum, Leuconostoc mesenteriodes, Lactococcus lactis, Lactobacillus acidiophilus and Lactobacillus bulgaricus based on API 50 test kit, and were able to grow at different physiological paramaters. This study shows that Lactobacillus strains isolated from raw cow milk had better physiological attributes. These LAB could be recommended for further assessment such as evaluation of probiotic potential properties and genomic analysis.

Sensitive immunoassays based on specific monoclonal IgG for determination of bovine lactoferrin in cow milk samples

Lactoferrin (LF), a bioactive multifunctional protein of the transferrin family, is found mainly in the secretions of all mammals, especially in milk. In the present study, a hybridoma cell (LF8) secreting IgG against bovine LF was screened, and the purified LF8 mAb showed high specificity and affinity to bovine LF. The linear range of ic-ELISA to detect LF was 9.76 ~ 625 ng/mL, with a limit of detection (LOD) of 0.01 ng/mL. The average recovery of intra- and inter-assay were (104.45 ± 4.12)% and (107.13 ± 4.72)%, respectively. The LOD of colloidal gold- and AuNFs-based strip by naked eye were 9.7 and 2.4 ng/mL, respectively, and the detection time was less than 10 min without any samples pretreatment and expensive equipment. The developed ELISA and lateral flow immunosensors based on specific IgG could be used directly for rapid detection of the bovine LF content in cow milk samples.

Raw milk and fecal microbiota of commercial Alpine dairy cows varies with herd, fat content and diet.

The factors that influence the diversity and composition of raw milk and fecal microbiota in healthy commercial dairy herds are not fully understood, partially because the majority of metataxonomic studies involve experimental farms and/or single factors. We analyzed the raw milk and fecal microbiota of 100 healthy cows from 10 commercial alpine farms from the Province of Trento, Italy, using metataxonomics and applied statistical modelling to investigate which extrinsic and intrinsic parameters (e.g. herd, diet and milk characteristics) correlated with microbiota richness and composition in these relatively small traditional farms. We confirmed that Firmicutes, Ruminococcaceae and Lachnospiraceae families dominated the fecal and milk samples of these dairy cows, but in addition, we found an association between the number of observed OTUs and Shannon entropy on each farm that indicates higher microbiota richness is associated with increased microbiota stability. Modelling showed that herd was the most significant factor affecting the variation in both milk and fecal microbiota composition. Furthermore, the most important predictors explaining the variation of microbiota richness were milk characteristics (i.e. percentage fat) and diet for milk and fecal samples, respectively. We discuss how high intra-herd variation could affect the development of treatments based on microbiota manipulation.

Comprehensive Assessment of Essential and Potentially Toxic Trace Elements in Bovine Milk and Their Feeds in Different Agro-climatic Zones of Sri Lanka.

Fresh milk is an important source of essential mineral supplement for humans. However, the levels of trace elements in milk are an important component of its safety and quality. Trace elements also act as a bio-indicator of agricultural pollution. Samples of raw cow milk (n= 68), animal feed [forage (n= 36) and concentrates (n= 14)], and water (n= 35) were collected from different agro-climatic regions of Sri Lanka. The concentrations of 15 trace elements including toxic heavy metals such as Cd, Pb, and As were quantified using inductively coupled plasma mass spectrometry after microwave-assisted digestion. Among the studied trace elements, the mean elemental concentrations of Se, Cd, As, and Cu in cow milk were 18.1, 1.45, 7.35, and 71.7μgL-1, respectively. The mean concentrations of these elements in forage were 0.216, 0.066, 0.046, and 9.21mgkg-1, and in concentrate feed were 0.329, 0.202, 0.229, and 2.28mgkg-1, respectively. The As content of the studied milk was well below the maximum permissible level while 15% of the samples had Pb exceeding the permissible limit of 20μgL-1. However, As, Cd, and Pb levels in drinking water provided to animals were well below the WHO permissible limits. The results of this study revealed that the trace elements in cow milk depend mainly on the feed, and hence, levels of these contaminants in feed materials should be monitored. Although due to the consumption of milk, the populations have a minor exposure to trace elements and also minimum health risks, a detailed study with a large number of samples is highly recommended.