Clinical Mastitis Milk Samples Research Articles

Trueperella Pyogenes—Strain Diversity and Occurrence in Dairy Herds

Trueperella (T.) pyogenes is a mastitis-causing pathogen formerly known to cause severe clinical mastitis (CM), especially during the summer, leading to milk losses and low recovery rates. Unfortunately, its transmission behavior within herds is unclear. The diversity and occurrence of T. pyogenes were monitored to gain an initial insight into the infection transmission behavior of T. pyogenes in dairy herds and to lay a foundation for following targeted investigations. CM milk samples were collected from German herds, and one Swedish farm was sampled for isolates from subclinical mastitis. All in all, 151 T. pyogenes isolates from 16 herds were isolated, identified by MALDI TOF analysis and typed with RAPD PCR. Of these, 17 isolates originated from subclinical mastitis cases. We found that T. pyogenes mastitis occurred year-round, and clinical mastitis cases were caused by multiple strains (31 affected animals/28 strains). Instances of multiple cows being infected with the same T. pyogenes strain were rare and typically only involved a small number of animals at a time. However, if several quarters of a cow were affected, it was likely the same strain. Unlike clinical infections, subclinical T. pyogenes infections, in one investigated farm, harbored a dominant strain. Additionally, we found that T. pyogenes infections tended to persist and stay within a herd for a minimum of 7 months in the same or different cows.

Antioxidative Sirt1 and the Keap1-Nrf2 Signaling Pathway Impair Inflammation and Positively Regulate Autophagy in Murine Mammary Epithelial Cells or Mammary Glands Infected with Streptococcus uberis.

Streptococcus uberis mastitis in cattle infects mammary epithelial cells. Although oxidative responses often remove intracellular microbes, S. uberis survives, but the mechanisms are not well understood. Herein, we aimed to elucidate antioxidative mechanisms during pathogenesis of S. uberis after isolation from clinical bovine mastitis milk samples. S. uberis's in vitro pathomorphology, oxidative stress biological activities, transcription of antioxidative factors, inflammatory response cytokines, autophagosome and autophagy functions were evaluated, and in vivo S. uberis was injected into the fourth mammary gland nipple of each mouse to assess the infectiousness of S. uberis potential molecular mechanisms. The results showed that infection with S. uberis induced early oxidative stress and increased reactive oxygen species (ROS). However, over time, ROS concentrations decreased due to increased antioxidative activity, including total superoxide dismutase (T-SOD) and malondialdehyde (MDA) enzymes, plus transcription of antioxidative factors (Sirt1, Keap1, Nrf2, HO-1). Treatment with a ROS scavenger (N-acetyl cysteine, NAC) before infection with S. uberis reduced antioxidative responses and the inflammatory response, including the cytokines IL-6 and TNF-α, and the formation of the Atg5-LC3II/LC3I autophagosome. Synthesis of antioxidants determined autophagy functions, with Sirt1/Nrf2 activating autophagy in the presence of S. uberis. This study demonstrated the evasive mechanisms of S. uberis in mastitis, including suppressing inflammatory and ROS defenses by stimulating antioxidative pathways.

Accuracy of an AI-based automated plate reading mobile application for the identification of clinical mastitis-causing pathogens in chromogenic culture media

Using on-farm microbiological culture (OFC), based on chromogenic culture media, enables the identification of mastitis causing pathogens in about 24 h, allows rapid decision making on selective treatment and control management measures of cows with clinical mastitis (CM). However, accurate interpretation of OFC results requires trained and experienced operators, which could be a limitation for the use of OFC in dairy farms. Our hypothesis was that AI-based automated plate reading mobile application can analyze images of microorganisms’ colonies in chromogenic culture media with similar diagnostic performance as a trained specialist evaluator. Therefore, the aim of the present study was to evaluate the diagnostic accuracy of an AI-based application (Rumi; OnFarm, Piracicaba, São Paulo, Brazil) for interpreting images of mastitis causing microorganism colonies grown in chromogenic culture media. For this study two trials were organized to compare the results obtained using an AI-based application Rumi with the interpretation of: (1) a trained specialist, using MALDI-TOF MS as the gold standard; (2) farm personnel users (FPU). In trial 1, a total of 476 CM milk samples, from 11 farms located in São Paulo (n = 7) and Minas Gerais (n = 4), southeast Brazil, were inoculated in chromogenic culture media plates (Smartcolor 2, OnFarm, Piracicaba, São Paulo, Brazil) by specialists under lab conditions, and digital images were recorded 24 h after incubation at 37 °C. After that, all the 476 digital images were analyzed by the Rumi and by another specialist (who only had access to the digital images) and the diagnostic accuracy indicators sensitivity (Se) and specificity (Sp) were calculated using MALDI-TOF MS microbiological identification of the isolates as the reference. In Trial 2, a total of 208 CM milk samples, from 150 farms from Brazil, were inoculated in chromogenic culture media plates by FPU, and the results of microbiological growth were visually interpreted by FPU under on-farm conditions. After visual interpretation, results were recorded using an OnFarmApp application (herd manage application for mastitis by OnFarm, Piracicaba, São Paulo, Brazil), and the images of the chromogenic culture plates were captured by the OnFarmApp to be evaluated by Rumi and Bayesian Latent Class Models were performed to compare Rumi and the FPU. In Trial 1, Rumi presented high and intermediate accuracy results, with the only exception of the low Enterococcus spp.’s Se. In comparison with the specialist, Rumi performed similarly in Se and Sp for most groups of pathogens, with the only exception of non-aureus staphylococci where Se results were lower. Both Rumi and the specialist achieved Sp results > 0.96. In Trial 2, Rumi had similar results as the FPU in the Bayesian Latent Class Model analysis. In conclusion, the use of the AI-based automated plate reading mobile application can be an alternative for visual interpretation of OFC results, simplifying the procedures for selective treatment decisions for CM based on OFC.

Prevalence and antibiotic susceptibility profile of Staphylococcus aureus in clinical and subclinical mastitis milk samples

Background: Staphylococcus aureus is the most commonly isolated bacterial pathogen in clinical and subclinical mastitis among cows. This study aimed to investigate the prevalence of Staphylococcus aureus, including MRSA (Methicillin-resistant Staphylococcus aureus), in cows with clinical and subclinical mastitis in Cumilla and Chattogram regions. Additionally, antimicrobial resistance patterns were identified. Methods: A total of 429 milk samples were collected from different teats of 125 cows with clinical and subclinical mastitis across 15 farms. Standard bacteriological methods were applied to isolate and identify Staphylococcus aureus. Confirmation of Staphylococcus aureus was achieved through PCR for the presence of the nuc gene. Antimicrobial susceptibility testing was conducted for 11 antimicrobials. Isolates showing resistance to cefoxitin and oxacillin underwent mecA gene screening to identify MRSA. Multivariable logistic regression models were used to identify risk factors associated with the presence of Staphylococcus aureus Results: The study revealed that 13.54% (95% CI, 5.00–38.78%) of mastitis-infected cows tested positive for Staphylococcus aureus. Staphylococcus aureus isolates showed the highest resistance to penicillin, cloxacillin, and streptomycin (61.54%, 53.85%, and 53.85%, respectively), while manifesting the least resistance to ciprofloxacin, ceftriaxone, and sulfamethoxazole + trimethoprim (92%, 77%, and 77%, respectively), and gentamycin (69.23%). The prevalence of MRSA was 2.08% (95% CI, 1.55–38.34%). Conclusion: This study represents the first report of MRSA in clinical and subclinical mastitis milk samples in Bangladesh. The findings emphasize the importance of ongoing monitoring of Staphylococcus aureus and MRSA in mastitis cases, considering their prevalence and patterns of antimicrobial resistance.

Phenotypic and genotypic investigation of antimicrobial resistance and extended-spectrum beta-lactamase production among Escherichia coli isolated from bovine mastitis

Escherichia coli has been one of the most highlighted pathogens isolated from clinical cases of mastitis. Unfortunately, the deficiency of appropriate antimicrobial stewardship and overuse of antimicrobial agents have increased antimicrobial resistance in animal isolates. The current study aimed to demonstrate the prevalence of extended-spectrum beta-lactamase (ESBL) production, resistance genes, and multi drug resistant (MDR) among E. coli isolates from cases of clinical mastitis. For this purpose, 40 E. coli isolates were collected from clinical mastitis milk samples from five farms in Mashhad, Iran. The antimicrobial susceptibility testing (AST) was applied to 15 antimicrobial agents of veterinary and human medicine interest. Also, ESBL production was evaluated using a doubledisc synergy test (DDST). The distribution of 20 resistance genes was sought among E. coli isolates by six multiplexPCR and three uniplex-PCR assays. The highest sensitivity was identified against imipenem and amikacin (100%). On the other hand, the highest resistance was observed for tetracycline and trimethoprim-sulfamethoxazole (70% - 72.5%), respectively. According to the AST and DDST tests, one isolate was confirmed as ESBL-producing and MDR. In addition, the most frequent resistance genes were blaTEM and AmpC (100% each). The qnrA encoding resistance to quinolones was similarly prevalent and detected in 50% of the isolates. In conclusion, at least three resistant genes were detected in 28 isolates (70%), but the majority of isolates were sensitive against most of the tested antibiotics. This fact might relate to the low expression of these genes within the isolates. The horizontal gene transfer of the present genes may confer resistance to other related bacterial species in humans or domestic animals.

The Efficiency of Centrifugation in the Detection of Mollicutes in Bovine Milk

Mastitis is one of the main diseases that affects dairy cattle. It is characterized by a multifactorial disease with multiple etiologies including microorganisms such as bacteria, viruses, fungi, yeasts, and algae. Mastitis caused by Mycoplasma spp. results in a contagious nature of infection and has been studied much in Brazil. The objectives of this research were (1) to investigate in clinical mastitis milk samples, by conventional PCR, the presence of the following mycoplasma species: Mycoplasma (M.) bovis, M. bovirhinis, M. bovigenitalium, M. californicum, and M. alkalescens and (2) compare the PCR Mollicutes detection rates using previous concentration and homogenization steps of the milk samples. Of the 993 clinical mastitis milk samples analyzed, 13.7% (136/993) and 8.0% (80/993) were positive for the Mollicutes class after concentration and homogenization, respectively, and all of them were PCR-negative for the five species investigated. Of the 70 expansion milk tanks analyzed, all of them were PCR-negative for the Mollicutes class. The importance of the previous concentration of milk in the routine of molecular analysis was evidenced when compared to the results obtained only with homogenization.

Comparative Genomic Analyses of Lactococcus garvieae Isolated from Bovine Mastitis in China.

Lactococcus garvieae is an emerging zoonotic pathogen, but there are few reports regarding bovine mastitis. The prevalence of L. garvieae represents an increasing disease threat and global public health risk. Thirty-nine L. garvieae isolates were obtained from 2,899 bovine clinical mastitis milk samples in 6 provinces of China from 2017 to 2021. Five clonal complexes were determined from 32 multilocus sequence types (MLSTs) of L. garvieae: sequence type 46 (ST46) was the predominant sequence type, and 13 novel MLSTs were identified. All isolates were resistant to chloramphenicol and clindamycin, but susceptible to penicillin, ampicillin, amoxicillin-clavulanic acid, imipenem, ceftiofur, enrofloxacin, and marbofloxacin. Based on genomic analyses, L. garvieae had 6,310 genes, including 1,015 core, 3,641 accessory, and 1,654 unique genes. All isolates had virulence genes coding for collagenase, fibronectin-binding protein, glyceraldehyde-3-phosphate dehydrogenase, superoxide dismutase, and NADH oxidase. Most isolates had lsaD and mdtA antimicrobial resistance (AMR) genes. Based on COG (Clusters of Orthologous Genes database) results, the functions of defense, transcription and replication, and recombination and repair were enhanced in unique genes, whereas functions of translation, ribosomal structure, and biogenesis were enhanced in core genes. The KEGG functional categories enriched in unique genes included human disease and membrane transport, whereas COG functional categories enriched in core genes included energy metabolism, nucleotide metabolism, and translation. No gene was significantly associated with host specificity. In addition, analysis of core genome single nucleotide polymorphisms (SNPs) implied potential host adaptation of some isolates in several sequence types. In conclusion, this study characterized L. garvieae isolated from mastitis and detected potential adaptations of L. garvieae to various hosts. IMPORTANCE This study provides important genomic insights into a bovine mastitis pathogen, Lactococcus garvieae. Comprehensive genomic analyses of L. garvieae from dairy farms have not been reported. This study is a detailed and comprehensive report of novel features of isolates of L. garvieae, an important but poorly characterized bacterium, recovered in the past 5 years in 6 Chinese provinces. We documented diverse genetic features, including predominant sequence type ST46 and 13 novel MLSTs. Lactococcus garvieae had 6,310 genes, including 1,015 core, 3,641 accessory, and 1,654 unique genes. All isolates had virulence genes coding for collagenase, fibronectin-binding protein, glyceraldehyde-3-phosphate dehydrogenase, superoxide dismutase, and NADH oxidase and resistance to chloramphenicol and clindamycin. Most isolates had lsaD and mdtA antimicrobial resistance genes. However, no gene was significantly associated with host specificity. This is the first report that characterized L. garvieae isolates from bovine mastitis and revealed potential host adaptations of L. garvieae to various hosts.

The Prevalence of Escherichia coli Derived from Bovine Clinical Mastitis and Distribution of Resistance to Antimicrobials in Part of Jiangsu Province, China

Bovine mastitis is often taken as one of the most common diseases in dairy farms, which its pathophysiology leads to a reduction of milk production and its quality. The penetration of pathogenic bacteria into the mammary gland, through either a contagious or environmental approach, has been determined the way of infection. The mastitis derived bacteria have become a challenge in practice, since the increasing exposure of antimicrobial. In order to identify characteristics of the epidemiological regulation and drug resistance of the pathogenic bacteria of bovine mastitis in northern Jiangsu, 156 clinical mastitis milk samples were collected from 3 large-scale farms for the epidemiological investigation and analysis of the drug resistance of E. coli. The bacteria were positively isolated in a total of 143 milk samples. The results showed that 78 strains of E. coli were detected, with a prevalence rate of 26.99%, followed by 67 strains of K. pneumoniae, with a prevalence of 23.19%, and 38 strains of Staphylococcus, with a prevalence of spp. 13.15%. In addition, 78 strains of E. coli isolated from bovine mastitis were tested for susceptibility to 8 kinds of antibiotics. It was shown that gentamicin and tetracycline were the most effective against E. coli, with the susceptibility rate of 83.3%, followed by streptomycin and ciprofloxacin, with 73.1% and 71.8% respectively. However, β-lactams including penicillin, cefothiophene, and amoxicillin showed serious resistance to E. coli isolates. There were 12 drug resistance genes detected by PCR, including β-lactam (blaTEM, blaCTX-M, and blaSHV), aminoglycoside (armA and armB), tetracycline (tetA, tetB, and tetC), and quinolone (qnrS, qepA, oqxA, and oqxB) related genes. Notably, all E. coli isolates carried blaTEM gene (100%). The detection rate of blaCTX-M was 53.8%, followed by the detection of blaSHV (20.5%), armA (9.0%), tetA (26.9%), tetB (2.6%), tetC (20.5%), qnrS (29.5%), oqxA (37.2%) and oqxB (1.3%). The present study provides crucial information on the distribution of bovine mastitis derived bacterial pathogens in Jiangsu province, as well as highlighting the antimicrobial resistance which might help to improve the efficiency of antibiotics treatment on bovine mastitis.

Novel aadA5 and dfrA17 variants of class 1 integron in multidrug-resistant Escherichia coli causing bovine mastitis.

Mobile genetic elements (MGEs) are associated with the emergence of multidrug resistance in extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae. This study explores the role of class 1 integrons and IS26 elements in breaching taxonomic barriers. A total of 110 E. coli bacteria were isolated from 300 clinical mastitis milk samples. The 98% E. coli isolates were extended-spectrum beta-lactamase- producers. About 83% of these isolates carried co-resistance for fluoroquinolones. The co-existence of (extended-spectrum beta-lactamase + quinolone resistance determining region mutations) and (extended-spectrum beta-lactamase + plasmid-mediated quinolone resistance genes) was found in 76% and 44% of isolates, respectively. The MGEs were detected in 88% of isolates with IS26 in 82% and class 1 integrase in 40% of isolates. The types of class 1 integron gene cassettes detected includes dfrA7, (dfrA17 + aadA5), and (dfrA1 + aadA1). We discovered 2 and 4 novel variants of the dfrA17 and aadA5 genes, respectively. We report a variant of aadA5 with mutation E235G in the Indian subcontinent earlier reported only in a human clinical isolate from Belgium. About 19 isolates carried IS26 linked to integrase gene intI1 with an internal deletion of 265bp in the 5`CS of integrase gene intI1, earlier reported only in E. coli ST131 isolates from human clinical, wastewater samples. This study suggests intercontinental dissemination of antibiotic resistant genes (ARGs) across different microbiomes via mobile genetic elements. KEY POINTS: • The role of mobile genetic elements in the emergence of multidrug-resistant E. coli in bovine mastitis. • Novel variants of the aadA5 (aminoglycoside adenyl transferase) and dfrA17 (dihydrofolate reductase) genes were identified in pathogenic E. coli isolated from bovine mastitis in class 1 integron gene cassette. • Sequence analysis of mobile genetic components revealed the physical connection between IS26 and intI1 genes with an internal deletion in 5'CS of class 1 integrase.

The protective role of caffeic acid on bovine mammary epithelial cells and the inhibition of growth and biofilm formation of Gram-negative bacteria isolated from clinical mastitis milk.

As a first-line barrier against bacterial infection of mammary tissues, bovine mammary epithelial cells (bMECs) are generally believed to be involved in the immune response due to exogenous stress. Due to the escalating crisis of antibiotic resistance, there is an urgent need for new strategies to combat pathogenic bacteria-infected bovine mastitis. In this study, isolated bMECs and Institute of Cancer Research (ICR) mice were used for Escherichia coli infection and caffeic acid (CA) pretreatment experiments in vitro and in vivo. The inhibitory effect of CA on bacterial growth and biofilm formation was also demonstrated with bacteria strains isolated from mastitis-infected milk. It was demonstrated that CA supplementation prohibits the growth of the predominant strains of bacteria isolated from clinical bovine mastitis milk samples. CA was found to disrupt the biofilm formation of E. coli B1 in a sub-minimum inhibitory concentration (sub-MIC) and inhibited the adherence property of E. coli on bMECs by decreasing the staining of bacteria on cell surfaces in vitro. In addition, CA was found to attenuate proinflammatory and oxidative responses in cells infected with E. coli. The pretreatment of bMECs with CA also restored altered lipid homeostasis caused by E. coli stimulation. The protective role of CA was further confirmed via the administration of CA in mice followed by representative Gram-negative bacterial infection. Collectively, these findings highlight the potential of CA to mediate Gram-negative infections and indicate that it has the potential to be developed as a novel antibacterial drug.

Detection of virulence genes and determination of the antimicrobial susceptibility of Escherichia coli isolates with mastitis in Mashhad, Iran – a short communication

The purpose of this study was to determine the virulence genes and antimicrobial resistance patterns of Escherichia coli isolated from milk samples of cows with bovine mastitis. Forty-seven E. coli isolates from clinical mastitis milk samples, from five dairy farms in Northeast of Iran, were subjected to multiplex PCR to determine virulence genes stx1, stx2, eaeA, hlyA, sta, F4, F17, fliC, and rfbE. In addition, antimicrobial susceptibility was assessed by applying disk diffusion methods. The eaeA and stx1 genes were most frequently detected in 42 (89.3%) and 34 (72.3%) isolates, respectively. However, the least frequent gene was F41 as it was found in only one isolate (2.1%). Furthermore, 9 out of 47 isolates were hlyA positive, and four isolates harbored the sta gene. The antimicrobial susceptibility demonstrated the highest resistance against lincomycin (100%) and neomycin (91.4%). Since these bacteria represent a high-risk pathogen on farms, the emergence of multiple antibiotic-resistant and pathogenic E. coli strains should be of great concern for public health.

Development of an on-site lateral flow immune assay based on mango leaf derived colloidal silver nanoparticles for rapid detection of Staphylococcus aureus in milk.

In order to ensure food safety, screening food samples for the presence of pathogens has been categorised as a legal testing item throughout the globe. One of the most prevalent zoonotic bacteria transmitted through dairy milk is Staphylococcus aureus. Given the limitations of the conventional detection methods, in the current study we desigined a competitive lateral flow immune assay (LFIA) using colloidal silver nanoparticles derived from mango leaves for the detection of Staphylococcus aureus in cow milk. SpA, a recombinant protein of Staphylococcus aureus, was used to raised hyperimmune sera used for developing the assay followed by conjugation with the synthesized nanoparticles. To increase the specificity of the assay, the milk samples were prenriched with selective agar exclusively require for Staphyloccocus aureus. The assay was found to be completed within 7-8h by observing test and control lines in LFIA strips. The developed assay was found to specifically detect the bacteria as low as 1000cfu/ml of milk samples. With a total 230 number of raw and clinical mastitis milk samples, the assay was validated and achieved relative accuracy, specificity, and sensitivity values of 97.39, 98.03, and 96.1%, respectively. The developed LFIA, which uses economically feasible and stable silver nanoparticles derived from mango leaves, has the potential for routine screening of milk samples for the presence of Staphylococcus aureus, especially in low-resource settings, allowing for early diagnosis, which facilitates effective treatment for the dairy animals and prevents the transmission of the disease in consumers.

Molecular Epidemiology of Klebsiella pneumoniae from Clinical Bovine Mastitis in Northern Area of China, 2018–2019

Klebsiella pneumoniae ( K. pneumonia , KpI) is a predominate inducement of bovine mastitis, which is associated with high mortality and milk yield reduction. However, data is lacking on the molecular characteristics of bovine K. pneumoniae , limiting the risk assessment of its transmission through the food chain. Herein, we investigated the prevalence of K. pneumoniae in 6301 clinical mastitis (CM) milk samples from dairy cattle in northern area of China. In total, 183 K. pneumoniae isolates were recovered, with detection rates of 3.0% and 2.8% in 2018 and 2019, respectively. Like human clinical K. pneumoniae , all CM K. pneumoniae isolates belonged to one of three phylogroups: KpI ( n = 143), Klebsiella. quasipneumoniae subsp. similipneumoniae (KpII-B) ( n = 37), and Klebsiella variicola (KpIII) ( n = 3). We detected the extended-spectrum β-lactamase-encoding genes bla SHV-2a , bla CTX-M-14 , and bla CTX-M-15 , as well as clpC , lpfA , lacI , lacZ , lacY , and the fecABDEIR operon in the KpI isolates, which may contribute to their pathogenicity and host adaptability in cows. The high prevalence of KpI in dairy farms may be problematic, as it showed relatively higher rates of antibiotic resistance and virulence gene carriage than the KpII-B and KpIII isolates. Furthermore, we observed distinct differences in population structure between CM- and human infection-associated KpI isolates, with the genes associated with invasive infection in humans rarely being observed in bovine isolates, indicating that few CM-associated K. pneumoniae isolates pose a threat to human health. Nevertheless, bovine KpII-B isolates shared a high level of nucleotide sequence identity with isolates from human infections and frequently carried the nitrogen-fixation gene nif , suggesting an association between KpII-B isolates from cattle and humans, and plant-derived bacteria.

A Comparative Study on Changes in Total and Differential Milk Cell Counts, Activity, and Expression of Milk Phagocytes of Healthy and Mastitic Indigenous Sahiwal Cows.

Milk somatic cell counts (SCCs) have been used as a gold standard to monitor mammary health as well as an indicator of raw milk quality. The present work was undertaken to compare the changes in the milk SCC, milk differential leukocyte counts (DLCs), phagocytic activity (PA) of milk neutrophils and macrophages (by nitroblue tetrazolium assay), extracellular trap formation (PicoGreen assay) and mRNA expression of various genes in milk neutrophils and macrophages (reverse transcription–polymerase chain reaction), and milk plasma cortisol concentration (enzyme-linked immunosorbent assay) in healthy, subclinical mastitis (SCM), and clinical mastitis (CM) cows. Milk was collected from healthy, SCM, and CM cows grouped based on their SCCs and California mastitis test with eight cows in each group. Milk SCC was estimated by SCC counter, and DLC was done after staining the milk slide under a microscope at 100×. Total SCCs in healthy, SCM, and CM cows were on an average of 128.30, 300.3, and 694.40 × 103 cells/mL, respectively. Milk DLCs indicated a lower percentage of macrophage and lymphocytes and a higher (p < 0.05) percentage of neutrophils in SCM and CM compared to healthy milk. The percentage of mature segmented neutrophils was lower, whereas immature band neutrophils were higher (p < 0.05) in the SCM and CM groups as compared to healthy cows. The viability, in vitro PA, and extracellular trap formation of neutrophils were lower (p < 0.05) in SCM and CM milk samples as compared to healthy samples. However, the PA of macrophage remained unchanged in all the studied groups. The relative mRNA expression of Toll-like receptors (TLR2, TLR4), myeloperoxidase, and interleukin 2α (IL-2α) receptor (CD25) were minimum in healthy samples and increased (p < 0.05) with the progress of mammary inflammation. However, CD44 decreased (p < 0.05), and CD62L remained unchanged in mastitis as compared to healthy cows. Plasma cortisol concentrations were higher (p < 0.05) in mastitis as compared to healthy cows and were negatively correlated with the number of milk macrophages and the functions of milk phagocytes. Estimation of total SCC, milk DLC, and activity of milk phagocytes is essential for effective control and prevention of incidence of mastitis in dairy cows.

In-vitro potential of crude extracts of selected garden herbs for mastitis management in Zambia

Introduction: The advent of bovine mastitis associated antimicrobial resistance has necessitated development of alternative herbal remedies. In this study, we tested, in-vitro, the antimicrobial efficacy of three crude extracts: (1) aqueous, (2) ethanol and (3) ethyl acetate extracts of Zambian cultivated Aloe barbadensis Miller (Aloe vera) and Curcuma longa (Turmeric) and also made comparisons to synthetic anti-mastitis formulations. Methods: The active compounds of these herbs were obtained as crude extracts using water, ethanol and ethyl acetate as solvents. The test microbes comprised of ten standard controls including Escherichia coli and Pseudomonas aeruginosa and 84 field isolates (all gram positive bacteria) from clinical and subclinical mastitis milk samples from selected districts of the Southern Province of Zambia belonging to the genera Staphylococcus, Streptococcus, Bacillus and Lactobacillus. The agar dilution method was used to determine the antimicrobial activity by observing the sensitivity which was graded as sensitive, intermediate or resistant. Commercial synthetic anti-mastitis preparations served as positive controls and their sensitivity was compared to that of the herbal preparations. The Minimum Inhibitory Concentrations (MIC) of the herbal formulation was also determined using two-fold serial dilutions. Results: The agar dilution test results of the field isolates indicated 18.8% resistance, 25.3% intermediate and 56% sensitivity for the herbal extracts, while the synthetic anti-mastitis preparation averaged 16.8% resistance, 4.75% intermediate and 78.5% sensitivity. The gram negative controls tested were generally resistant. On average, the agar dilution trials revealed; 41.8% resistance, 13% intermediate and 45.3% sensitivity for the herbal formulation while the synthetic antimicrobials produced 23.5% resistance, 5.2% intermediate and 71.3% sensitivity. Mean MIC was 11.5mg/ml at the 5% serial dilution for the herbal extract compared to the Cephalexin (semi-synthetic antimicrobial) MIC for S. aureus at 0.5mg/L or 0.0005mg/ml. Conclusion: The herbs Aloe vera and Curcuma longa from Zambia showed antimicrobial efficacy on bovine mastitis causing microbes and may be used as a cheaper alternative to synthetic anti-mastitis preparations and as an aid to fight antimicrobial resistance to the benefit of the most affected and resource limited small-scale dairy farmers.

Molecular characterization of antimicrobial resistance in Klebsiella pneumoniae isolated from Brazilian dairy herds

In this observational study, phenotypic and genotypic patterns of antimicrobial resistance (AMR) in Klebsiella pneumoniae isolated from intramammary infections, clinical mastitis, fresh feces, rectal swabs, animal hindlimbs, and bulk tank milk samples from Brazilian dairy herds were investigated. In addition, we identified specific genetic variants present among extended-spectrum β-lactamase (ESBL) producers. We obtained 169 isolates of K. pneumoniae from 2009 to 2011 on 24 Brazilian dairy farms located in 4 Brazilian states. The AMR profile of all isolates was determined using disk-diffusion assays. The antimicrobial panel included drugs commonly used as mastitis treatment in Brazilian dairy herds (gentamicin, cephalosporins, sulfamethoxazole-trimethoprim, tetracycline) as well as antimicrobials of critical importance for human health (meropenem, ceftazidime, fluoroquinolones). The K. pneumoniae isolates resistant to tetracycline, fluoroquinolones, sulfamethoxazole-trimethoprim, or chloramphenicol were screened for presence of drug-specific AMR genes [tet, qnr, aac(6')-Ib, floR, catA2, cm1A, dfr, sul] using PCR. In addition, we identified ESBL genes present among ESBL-producers by using whole genome sequencing. Genomes were assembled and annotated, and patterns of AMR genes were investigated. Resistance was commonly detected against tetracycline (22.5% of all isolates), streptomycin (20.7%), and sulfamethoxazole-trimethoprim (9.5%). Antimicrobial resistance rates were higher in K. pneumoniae isolated from intramammary infections in comparison with isolates from feces (19.2 and 0% of multidrug resistance in intramammary and fecal isolates, respectively). In contrast, no difference in AMR rates was observed when contrasting hind limbs and isolates from intramammary infections. The genes tetA, sul2, and floR were the most frequently observed AMR genes in K. pneumoniae resistant to tetracycline, sulfamethoxazole-trimethoprim, and chloramphenicol, respectively. The tetA gene was present exclusively in isolates from milk. The genes blaCTX-M8 and blaSHV-108 were present in 3 ESBL-producing K. pneumoniae, including an isolate from bulk tank milk. The 3 isolates were of sequence type 281 and had similar mobile genetic elements and virulence genes. Our study reinforced the epidemiological importance and dissemination of blaCTX-M-8 pST114 plasmid in food-producing animals in Brazil.

Development and evaluation of 4 loop-mediated isothermal amplification assays to detect mastitis-causing bacteria in bovine milk samples

Farmers prefer fast, sensitive, and on-site tests for treatment decisions on mastitis. Due to the time to results of the currently available diagnostic tools, these are rarely used for that purpose. Genotypic tests that do not require a growth step may be suitable for on-site testing, for example loop-mediated isothermal amplification (LAMP), which has been described as a sensitive test that can be used on-site. Therefore, this study aimed to develop and evaluate LAMP assays for the detection of a subset of mastitis-causing pathogens, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, and Streptococcus spp., in milk from cows with clinical mastitis. Furthermore, a generic nucleic acid lateral flow immunoassay (NALFIA) was evaluated as a potential on-site readout of the LAMP assays. For each assay of LAMP and NALFIA, the limit of detection and analytical specificity were determined using isolates, and the diagnostic specificity was determined using selected samples with known etiology. In addition, the diagnostic specificity of LAMP was determined using field samples with unknown etiology at testing. Bacteriological culture with identification by mass spectrometry was used as a reference method. The 4 assays had a kappa ≥0.73 with the reference method when testing the selected samples, but ≥0.47 when testing field samples. After correcting for prevalence, kappa was ≥0.80 for the E. coli, K. pneumoniae, and Staph. aureus assays. The Streptococcus spp. assay had a kappa of 0.47 (0.48 after correction) with the reference method, probably caused by the assay broadly targeting a genus instead of a particular species. The NALFIA readout was found to have kappa ≥0.81 for the E. coli, Staph. aureus, and Streptococcus spp. assays at a generic runtime, but for the K. pneumoniae assay a shorter runtime could be used. In conclusion, LAMP is a promising method for fast on-site tests for mastitis-causing pathogens if the current elaborate method for sample preparation is replaced by a simplified protocol. The NALFIA is an easy and reliable readout for on-site use, with the observation that for the current assay designs a generic runtime is not yet possible for the chosen set of pathogens. If associated with a simple and fast sample preparation protocol, the combination of LAMP and NALFIA has the potential to enable fast and reliable on-site testing of clinical mastitis milk samples.

Prevalence, etiology, and economic impact of clinical mastitis on large dairy farms in China

This study investigated the continuous monthly prevalence of bovine clinical mastitis (CM) and the distribution of causative pathogens among 36,619 CM milk samples from large dairy farms across seven Chinese provinces from 2015 to 2017 using data from routine CM recording systems. Based on treatment period and cost per cow, withdrawal period, daily milk production, and milk value data from each farm in 2017, we calculated the economic impact of CM at the farm level with 2578–9044 lactating cows per farm. Results showed a wide variation in monthly prevalence of CM (0.6 %–18.2 %) among the seven farms over the study period, indicating regional and temporal differences in the occurrence of CM in China. Enterobacteriaceae were the predominant pathogens across all farms from six provinces except Shandong, in which the Streptococcus spp. was the most prevalent. However, the distribution of various Enterobacteriaceae species differed among farms, and Streptococcus species distribution was strongly associated (Pearson’s coefficient, 68.4 %) with location. Monthly economic losses associated with CM showed clear variation, ranging from 12,000–76,000 USD/farm/month. Sensitivity analysis showed that economic loss at the farm level was most sensitive to variation in the prevalence of CM, followed by antibiotic treatment period and daily milk production per cow. To our knowledge, this is the longest running study of CM and the first estimation of its economic impacts in China. Our findings highlight the considerable costs associated with mastitis, and indicate that preventive measures and regional and timely treatment of CM are needed.

Antimicrobial Study of Clinical Mastitic Milk Samples of Cows in and Around Bikaner

Mastitis, an inflammatory condition of the mammary gland, is one of the most economically important diseases of dairy herds. This disease is more prevalent in high yielding dairy cattle. It is caused due to colonization of pathogenic bacteria causing series of events that lead to major alteration in the mammary tissues and composition of milk secreted from the tissue cells (Batavani et al., 2007; Goel et al., 2008). Clinical mastitis is recognized by abnormal milk, varying degree of mammary gland inflammation (redness, heat, swelling and pain) with or without illness of the cow. Mastitis in dairy animals leads to heavy economic losses due to reduction in milk production, altered composition, and cost of treatment of affected animals. The clinical mastitis in different cases can be identified by external symptoms such as udder swelling, touch, pain, redness and hardness of the udder. The objective of the present study was to assess the sensitivity of bacterial isolates from mastitic milk samples of cows to some selected antibiotics.

Amount of Milk Neutrophil Percentage and Associated CD Molecular Changes on the Compositional and Technological Properties of Milk

Background:Changes occurring in the activity and expression of neutrophils and their transmigration through the blood mammary barrier owing to a mammary infection affecting milk quality and outcome of mastitis.Objective:To understand the role played by various neutrophil molecules on mastitis and milk quality.Methods:18 Karan Fries cows of similar parity, milk yield and lactation stage were selected and screened for mastitis based upon milk Somatic Cell Counts (SCC) as well as California mastitis test and divided into 3 groups of 6 eachi.e., healthy, sub-clinical (SCM), Clinical Mastitis (CM). Milk samples were analyzed for milk composition and technological properties. Milk neutrophils were isolated and their percentage, Phagocytic Activity (PA), viability were estimated. Activities of neutrophil enzymesi.e. Elastase 2, Collagenase and Cathepsin G were analyzed using ELISA. Relative mRNA expression of cell surface molecules like selectin (CD-62L), integrin (CD-11b), chemokine receptors (CXCR1 and CXCR2), CD-44 and chemotactic factor (IL-8) in milk neutrophil were also studied.Results:In the present study, neutrophil percentage (%) was significantly (p˂0.05) higher in SCM and CM milk samples compared to healthy milk samples, whereas neutrophil PA, viability were significantly (p˂0.05) lower in both samples contrast to healthy samples. Activities of Elastase 2 and Collagenase were significantly (p˂0.05) more in SCM milk. There was a significant (p˂0.05) difference in protein, pH, and lactose between healthy, SCM and SCM and CM milk. Significantly (p˂0.05) high Electrical Conductivity (EC) was observed in CM milk than SCM and CM milk. No significant changes in milk fat, Solid Not Fat (SNF) and density were found among any of the groups. The relative mRNA expression of CXCR1, CXCR2 and IL-8 were significantly (p˂0.05) high in milk neutrophils with the progression of SCM and CM, whereas significantly higher expression of CD11b CD-11b was found only in CM cows but there was no change in the expression of CD62L CD-62L in any of the groups. Expression of CD-44 molecule increased significantly in SCM cows, whereas it decreased significantly in clinically infected mastitis cows.Conclusion:This study highlights the changes occurring in the activity of milk neutrophils in healthy, subclinical and clinical mastitis crossbred cows.