California Mastitis Test Research Articles

Antimicrobial Resistance Profile and Molecular Characterization of Staphylococcus aureus Isolated from Subclinical Mastitis of Dairy Cows in Mathura Region

Background: Subclinical mastitis is the most underrated yet economically important disease of livestock. It goes undetected by clinical examinations, making routine surveillance and monitoring necessary for its detection. Among causative agents, Staphylococcus aureus is the most crucial one. The present study determines the prevalence, antimicrobial resistance profile and molecular characterization of S. aureus from subclinical mastitis of cattle in the Mathura region. Methods: The present research was conducted during 2019-2021 and different gaushalas and dairies in and around the Mathura region were screened for mastitis by California mastitis test and somatic cell count. The samples positive were further tested by bacterial, biochemical and molecular tests along with an antimicrobial resistance profile. Result: Our research found a significant amount of S. aureus in subclinical samples with the presence of mecA gene suggesting MRSA. The public health importance of S. aureus and emerging resistance against antibiotics demands regular monitoring and effective use of antimicrobial agents against the MRSA isolates.

The first study on antimicrobial resistance of Staphylococcus aureus isolated from raw goat milk associated with subclinical mastitis in Siliragung Subdistrict, East Java, Indonesia.

Raw goat milk is a highly nutritious dairy product and a suitable medium for the growth of antimicrobial-resistant Staphylococcus aureus, the leading cause of subclinical mastitis. This study aimed to investigate the resistance status of S. aureus which isolated from goat milk associated with subclinical mastitis cases in Siliragung Subdistrict, Banyuwangi District, East Java, Indonesia. The S. aureus isolates were recovered from 258 raw goat milk samples from seven different dairy goat farms. Preliminary screening of subclinical mastitis using the California mastitis test, then samples with score +3 and +4 were taken for further isolation and identification, followed by a biochemical test to determine the S. aureus. Moreover, the bacteria susceptibility test against several antimicrobials was done using the disk diffusion method. Based on our findings, a total of 66 (25.58%) raw goat milk samples were tested positive for S. aureus, of which 36.36% were identified as multidrug-resistant. Moreover, S. aureus were also identified as resistant to penicillin (81.82%), ampicillin (65.15%), erythromycin (50.52%), and gentamicin (36.09%). The prevalence of S. aureus isolated from raw goat milk associated with subclinical mastitis in Siliragung Subdistrict, Banyuwangi District, Indonesia, was recorded at 25.58%. Moreover, 36.36% of S. aureus isolates were categorized as resistant to three or more classes of antibiotics. The biosafety and biosecurity procedures during the milking process should be strengthened in dairy goat farms to prevent the transmission of antimicrobial resistance among animals, humans, and environments.

Udder and teat morphometry and its relationship with occurrence of intramammary infections in dairy cattle

Bovine mastitis, an inflammatory disease of the mammary gland, is often caused by bacterial infection and is a major health problem on dairy farms. A cross-sectional study of four herds was conducted to assess udder and teat-related risk factors for the development of intramammary infections in 243 lactating cows. Udder and teat morphometric parameters were determined through visual appraisal method. Intramammary infection was assessed using California mastitis test (CMT), Somatic cell count (SCC) and bacteriology of milk samples. The frequencies of udder conformations were 65.8%, 18.9%, 7.4% and 7.8% for desirable, pendulous, round and goaty-shapes, respectively. Teat conformation revealed that 76.0%, 5.3%, 6.6%, 4.5% and 7.5% for desirable, short, funnel, bottle and conical-shapes, respectively. There was a significant (p<0.01) effect of udder shape and teat-end shape on SCC level and intramammary infections. The mean SCC and mastitis infection levels for undesirable udder was significantly (p<0.05) higher as compared to the desirable shaped udder. Similarly, significant (p<0.05) higher level SCC was found in undesired teats compared to desirable teats. The overall proportion of quarter mastitis infection in all four farms revealed 1.23% for clinical and 36.52% for subclinical mastitis. Herd-wise subclinical mastitis prevalence showed 42.8%, 35.07%, 33.09% and 2.94% for farm A, C, B and C, respectively. The most frequently isolated bacterial species were Staphylococcus aureus (56.34%) followed by Escherichia coli (22.55%), Streptococcus agalactiae (9.86%) and Bacillus spp. (8.45%). In conclusion, undesirable udder and teats conformations were more susceptible to intramammary infection and these traits must be considered accordingly while selecting dairy animals for future milk production. Observed high occurrence of subclinical Staphylococcal mastitis needs improvements in milking hygiene, mammary gland health, regular screening of animals, and use of biosecurity as an intervention tactic to limit the spread of pathogens at dairy farms.

Resistance Profile of Bovine Mastitis Isolates, Presence of the mecA Gene and Identification of ESBL Producing Strains from Small Rural Dairy Properties

Bovine mastitis is an inflammation of the mammary gland in response to invasion by opportunistic agents. Due to the high economic importance of dairy production and the complexity related to animal health, the objective of this work was to identify and evaluate the antibacterial resistance profile of samples of mastitis milk, milking hand and milking equipment from small rural dairy farms belonging to the northwest region of the state of Paraná, Brazil. Five small, non-technical dairy farms in the municipalities of Boa Esperança, Juranda and Tapejara, all belonging to the northwest region of the state of Paraná, Brazil, were selected. The properties had Holstein and/or crossbred herds, carried out a bucket-by-foot milking system and all had the presence of animals with subclinical mastitis confirmed by the California Mastitis Test. Samples of sterile swabs from the milking insufflators, the milking hand and milk samples were collected-and later, isolation tests and phenotypic characterization of the samples, sensitivity tests to antimicrobials and phenotypic tests for the detection of beta-producing strains were performed with extended-spectrum beta-lactamase (ESBL), molecular identification of Staphylococcus aureus isolates and mecA gene research. Of the 199 samples collected from the 15 selected properties in the municipalities of Boa Esperança, Tapejara and Juranda, 72 (36.20%) were classified as multiresistant. Isolated from milkers' hands and milking machines, which phenotypically produce extended-spectrum beta-lactamase (ESBL), the presence of the mecA gene was also observed in 11 isolates of Staphylococcus spp. of milk samples, machines and milking hands. Mastitis can be spread to the herd through the milking process by the milkers' instruments and hands, and adequate management measures can prevent its transmission and the conscious use of antibiotics decreases the prevalence of multidrug-resistant pathogens. In this work, different pathogenic bacteria were detected in mastitic milk, milking equipment and milking hand with a high percentage (36.20%) of isolates classified as multidrug resistant. In addition, the presence phenotypically (ESBL) and molecularly (mecA gene) of isolates carrying resistance genes was also verified. These results directly reflect on the health of the animals, the health of the workers and the health of the respective environment, which can enable the continuity of the propagation of the etiological agents involved in the mastitis infection. The awareness of producers and workers on these properties about the disease, transmission, sanitary aspects and adequate management and treatment are essential for improving milk production and production efficiency.

CALIFORNIA MASTITIS TEST AND IDENTIFICATION OF ANTIBIOTIC-RESISTANT BACTERIA ON DAIRY CATTLE IN “KUD” SEMEN GANDUSARI SUBDISTRICT, BLITAR

Mastitis is an inflammation of the udder that often affects dairy cows in the production phase. The impact of mastitis losses is quite large, including a drastic decrease in milk quantity and quality. Mastitis can be caused by antibiotic-resistant bacteria in dairy cows, including Escherichia coli, Staphylococcus spp or Streptococcus spp. The results of a recent study stated that the prevalence of mastitis in Indonesia reached 83%. The Coliform Mastitis Test (CMT), used worldwide for early detection of mastitis, has a specificity of 80.6% and a sensitivity of 82.4%. Semen Village Unit Cooperative (KUD) is a cooperative that is the center of milk production in Blitar District, which accommodates the milk of dairy cows from smallholder farms in Blitar District. The high incidence of mastitis caused by the presence of bacteria resistant to antibiotics encourages counseling on mastitis and identifying bacteria that cause antibiotic resistance. Extension activities are carried out by presenting material about mastitis caused by the presence of bacteria resistant to antibiotics with lecture techniques (PowerPoint slides), videos, questions, and answers, and demonstrating the California Mastitis Test on milk samples as an early detection test, as well as identifying bacteria that cause antibiotic resistance. This series of counseling activities is an effort to reduce the incidence of mastitis through increased knowledge and understanding of the community.

The prevalence and risk factors of subclinical mastitis in water buffalo (Bubalis bubalis) in Bangladesh

Subclinical mastitis (SCM) in water buffalo is responsible for reduced milk yield and quality. This cross-sectional study was carried out to a) estimate the prevalence of SCM, b) identify risk factors associated with SCM, and c) identify farm-level risk factors associated with bulk milk somatic cell count (BMSCC).The buffalo farms included in this study represented five rearing systems: free-range, semi-free-range, household, semi-intensive, and intensive, providing a total of 3491 functional quarters of 880 lactating buffalo on 248 farms. The California mastitis test score was used to identify SCM. Bulk milk samples (n = 242) were used for farm-level BMSCC. Quarter and buffalo-level risk factors for SCM were measured using questionnaires and observations.The overall SCM prevalence was high at 27.9% at the quarter-level (25th and 75th percentiles: 8.3% and 41.7%) and 51.5% at buffalo-level (25th and 75th percentiles: 33.3% and 66.7%). The geometric mean BMSCC was 217,000 cells/mL of milk (ranging from 36,000-1,213,000 cells/mL), which is low on average, but some farms could improve substantially. The buffalo rearing system, udder location (left versus right), teat shape, udder asymmetry, number of milkers, and having a quarantine facility were associated with buffalo udder health. Our findings suggest that mainly using free-range rearing systems may help decrease the prevalence of SCM primarily by employing buffalo breeding and better farm biosecurity, and udder health control strategies can be designed based on our findings.

STUDY OF PREVALENCE AND CAUSES OF SUB-CLINICAL MASTITIS IN GOATS OF GANDAKI PROVINCE, NEPAL

A study was conducted to determine the prevalence and causes of subclinical mastitis in goats at Goat Research Station (GRS), Bandipur, and National Livestock Breeding Office (NLBO) farm Lampatan, Pokhara. Altogether 61 samples including 43 from GRS and 18 from NLBO were screened for subclinical mastitis using the California Mastitis Test (CMT) reagent, of which positive samples were then aseptically collected in a sterile 10 ml plastic tube and cultured in nutrient agar. Culturepositive samples were Gram-stained to determine their type (gram-positive or gram-negative). In the case of mixed colonies, the culture was sub-cultured. The data were entered and tabular and graphical representation was done in MS-Excel 2010. Results revealed that 26.23% of samples were positive for CMT and of 122 active teats tested, 19 (15.57%) were positive for CMT. Similarly, 23.3% of samples from goats of GRS, and Bandipur were CMT positive whereas 33.3% of samples from goats of NLBO, and Pokhara were positive. On the other hand, the prevalence of subclinical mastitis was 27.27%, 31.03%, and 19.04% for Saanen, Boer, and cross-bred goats, respectively. Considering the samples collected from GRS, 75% were culture-positive of total cultured samples while those from NLBO showed 57.14 % culture positive. In the meantime, overall samples showed 68.42% of total cultured samples to be culture positive. Out of 12 culturepositive samples, 92.30 %( 11/12) were gram-positive and only 7.69 %( 1/12) were gram-negative.

MOLECULAR CHARACTERIZATION SOME BACTERIAL PATHOGENS CAUSING BOVINE MASTITIS WITH SPECIAL REFERENCE TO Mycoplasma bovis

In dairy industry, bovine mastitis is the most prevalent disease, which reduces milk production and causes economic losses. This study was conducted to estimate the prevalence of Mycoplasma bovis and some bacteria causing mastitis in dairy farms and partial sequencing of 16SrRNA target genes and Quinolones Resistance Determining Regions (QRDRs) (gyrA and parC) in M. bovis isolates. 370 milk samples were obtained from farms located in villages in Fayoum governorate, Egypt. The examined milk samples (8,91%) were positive for the California mastitis test (CMT). Multiplex RT-PCR was used for the recognition of microorganisms causing mastitis (Staphylococcus (S.) aureus, Streptococcus species (spp.), Escherichia (E.) coli, and Mycoplasma (M.) bovis) from mastitic milk. The results revealed that E. coli was the most predominant (84.8%) followed by S. aureus (81.8%) while M. bovis was the lowest one (51.5%). Mixed infection with two or more mastitic bacterial agents was also identified. All 33 examined mastitic milk samples were diagnosed with mixed infection with E. coli, S. aureus, Streptococcus spp. and M. bovis (36.36%), E. coli and S. aureus (21.21%), and rephrase E. coli, M. bovis, and Streptococcus spp. (6.06%). The sequence analysis of M. bovis 16SrRNA genes illustrated a high similarity of examined isolates to strains previously deposited in the GenBank recovered from the same locality. The gyrA amino acids showed no substitution but showed 100% similarity with M. bovis isolates worldwide. However, the amino acid sequence of parC, showed substitution at positions 2 (Gln to Arg) (CAG >>CGT), 75 (Ile to Ser) (ATT>>AGC), and 79 (Asn to Asp) (AAC>>GAT). Sequence results can lead to the creation of appropriate treatment and control measures for M. bovis, while multiplex RT-PCR, can be exploited as a standard diagnostic method for major mastitis pathogens.

PREVALENCE OF MASTITIS IN NILI RAVI BUFFALO HERD AT LIVESTOCK EXPERIMENT STATION BHUNIKEY, PATTOKI

Mastitis is one of the major and keen parameter for quality and quantity of milkproduction. The recent study was conducted to estimate the occurrence rate of mastitis in four differentseasons of the year and its therapeutic cure in Llivestock Experiment Station Nili Ravi Bhunikey. Forthis purpose, the total of 1188 milk samples were screened for mastitis through California MastitisTest. Out of total 1188 milk samples, the prevalence rate of mastitis was 9.7% (27/279) in winter, 14%(40/286) in spring , 7.2% (23/320) in summer and 6% (18/303) in autumn respectively. An overallprevalence rate of mastitis was 9 %( 108/1188).The screened positive samples were subjected toculture on culture media. Seven antibiotics were selected for the fortitude of antibiotic sensitivityagainst the cultured organisms. Antibiotics used in current research were Gentamicin, Enrofloxacin,Amoxycillin, Norfloxacin, Tylosin, Oxytetracycline and Chloramphenicol. The efficacy of thedifferent antibiotics was checked by the configuration of the zone of inhibition. The outcome showedthat the incidence of mastitis was greater in spring and lower in autumn. From selected antibiotics,Enrofloxacin and Gentamycine have resulted in more effective against the cultured organism.

Analysis of the efficiency of a rapid test for the early diagnosis of mastitis in cows

This article describes the results of a study of the content of somatic cells in milk using various methods. Milk samples were tested using a standardized apparatus method using a Somatos-V viscometer, from which 3 groups of 20 samples were selected. The formation of groups was based on the content of somatic cells in the samples (up to 200; 200-400 and over 400 thousand/cm3). Further research was aimed at studying the diagnostic properties of the new test, the action of which is based on the suppression of foaming by mastitis milk. All samples were tested using the foam test and the California mastitis test. The coincidence of the results of the study of the studied test systems with the reference method was revealed in relation to samples containing a small (up to 200 thousand/cm3) and an increased (over 400 thousand/cm3) number of somatic cells. In samples with a cell concentration from 200 to 400 thousand/cm3, there are different results in the study by express testing methods. The foam test showed 90% and the California test showed 75% agreement with the results of viscometry. Given the difficulty in diagnosing latent mastitis, the foam test studied in this work may have promising prospects for use on farms for the early detection of sick cows. This method allows more accurately, qualitatively, to determine the increase in the number of somatic cells in milk, which is important in the diagnosis of latent subclinical mastitis and their timely treatment.

Antimicrobial efficacy of Thymus vulgaris extract against some Staphylococcus species isolated from subclinical mastitis in cattle in Basrah province, Iraq.

Thymus vulgaris extracts can play a significant role as alternatives for antimicrobial agents against bovine staphylococcus mastitis. This research's goal was to evaluate the antibacterial properties of an extract from T. vulgaris as an alternative to antibiotics for bovine Staphylococcus mastitis. In addition, it is important to know the effect of the extraction methods (hot alcoholic, cold alcoholic, and hot water extract) on their effectiveness. Two hundred ten cow milk samples from different areas of Basrah province had been suffering subclinical mastitis reported by using the California mastitis test (CMT). Staphylococcus species were identified by conventional microbiological technique, GP24 Kit, and nuc gene. Antimicrobial activity of various concentrations of T. vulgaris extracted (75, 50, 25) mg/ml with different methods of extraction (hot alcoholic, cold alcoholic, and hot water extract). Out of 210 samples, 99 (47.1%) were positive for the CMT, and the identification rate of Staphylococci spp. by conventional microbiological technique and GP24 kit was 78 (78.8%). Out of 78 isolates of Staphylococcus spp. 48 (61.5%) were identified as Staphylococcus aureus, by using both molecular techniques using PCR and miniaturized Kit GP24 and employing the miniature GP24, the remaining 30 (38.5%) were determined to be different species of Staphylococcus. Antibacterial activity of various concentrations of T. vulgaris extracted (75, 50, 25) mg/ml with different methods of extraction revealed that hot alcoholic extract (100%) was more effective than cold alcoholic extract (66.7%), whereas there is no effect on the bacteria species with the hot water extract. Thymus vulgaris extracts can play a significant role as alternatives for antimicrobial agents against bovine staphylococcus mastitis.

Isolation and Determination of Antibacterial Sensitivity Characteristics of Staphylococcus aureus from Lactating Cows in West Shewa Zone, Ethiopia.

Staphylococcus (S.) aureus is one of the etiologies of bovine mastitis, hindering milk production and productivity in dairy farms. This study was aimed at assessing the distribution of bovine mastitis and the isolation rate of S. aureus in milked cows of West Shewa Zone. The clinical mastitis was diagnosed by physical methods including observation and palpation, whereas the subclinical mastitis was tested by the California mastitis test (CMT). All of the cows tested for mastitis were aseptically sampled (teat-milk) for bacteriology. The bacterium was primarily identified based on colony characterization, catalase, coagulase tests, and Gram stain reaction. Finally, MALDI-TOF Biotyper confirmed the species. The antibacterial sensitivity characteristics of the isolates to different antibacterial drugs were tested by the disk diffusion method. The drugs were selected based on the frequent usage in veterinary medicine in the study area. By using particular primers, the presence of the resistance (mecA and blaZ), and thermonuclease (nuc) genes were determined by polymerase chain reaction (PCR). The data were analyzed by R statistical software. The associations between the dependent variables (prevalence of mastitis and S. aureus) and the explanatory variables were analysed by chi-square (χ 2) and logistic regression tests at a 95% confidence interval (CI). Accordingly, 258 lactating cows were examined, of which 97 (37.6%) were mastitis positive. Of these mastitis positive cows, 59 (60.8%) were subclinical and 38 (39.2%) were clinical. Among the 258 milk samples, 43 (16.7%) were positive for S. aureus. According to the results of the current investigation, subclinical mastitis was significantly more prevalent than clinical mastitis (p < 0.05). The disease was found varied with the lactation stage of the animal, milking with washed hand, udder washing before milking, and tick infestation of the teat. In comparison to animals from farms with lower number of lactating cows, the prevalence of the bacteria was significantly higher in animals managed in farms with large (OR = 12.58, 95% CI = 2.33-68.54, and p < 0.05) and medium (OR = 12.58, 95% CI = 2.33-68.54, and p < 0.05) population of lactating cows per herd. The isoation rate of the bacterium was also found significantly higher in tick-infested cows (OR = 27.69, 95% CI = 9.71-93.01, and p < 0.05) than tick free cows. The antibiogram tests revealed that the isolates resisted penicillin G and tetracycline group drugs (oxytetracycline and tetracycline). Moreover, the nuc gene was confirmed to be present in all of the examined isolates. However, they were not found harboring blaZ and mecA genes. We concluded that S. aureus is sustaining as a main causative agent of bovine mastitis, and they were resistant to the frequently used antibiotics in public and veterinary medicines in the study areas. Therefore, research-based interventions need to be taken in action to combat the pathogen.

Clinical and Subclinical Mastitis in Ruminants: A Review of Etiological agents, Diagnosis, Clinical Management and Risk factors

Ruminant mastitis is a welfare and economic problem of livestock and the dairy industry. It is a major animal health problem in countries where livestock are raised for the purpose of milk production. This Review focuses on ruminant mastitis, etiologic agents, diagnostic test and associated risk factors. In this narrative review, a comprehensive literature search of all relevant articles was performed to identify articles published in Science Direct, Web of Science, PubMed, Scopus, MEDLINE, Google Scholar, LISTA and AJOL. English articles published from 2010 to 2023 were reviewed and used. The findings revealed that even though Staphylococcus aureus, Non-aureus Staphylococci species, and Streptococci are the major mastitis pathogens. Others like Escherichia coil, Klebsiella, Citrobacter, Enterobacter, Pseudomonas, Proteus, Serratia, Bacillus, and Corynebacterium etc were isolated from milk samples. Diagnosis was observed to be based on observation of milk and cardinal signs of inflammation of the mammary glands, the use of diagnostic test, like bacterial culture, Strip cup test, White side test (WST), Surf field mastitis test (SFMT), and California Mastitis test (CMT). Molecular typing techniques including PCR, Sequencing, MALDI-TOF MS, MLST, PFGE, spa typing etc have also been observed important role in characterizing agents of mastitis. Major risk factor observed is age, parity and stage of lactations. Clinical management of this condition in ruminants is based on the understanding of the etiology, and treatment options.

Virulence genes and antibiotic resistance profiles of Staphylococcus aureus isolated from bovine mastitis milk samples.

The increasing importance of antibiotic resistance shows the need for determining indices of the epidemiology of infection. This study aimed to determine the virulence genes and antibiotic resistance profiles of Staphylococcus aureus isolated from bovine mastitis cases. A total of 200 cattle were selected based on California Mastitis Test (CMT) results, and the samples were cultured in the laboratory. Grown colonies were examined by conventional phenotypic methods and confirmed using PCR amplification of 16S rRNA gene. The prevalence of the virulence genes was also defined. The results of phenotypic and molecular tests were compared using SPSS software by McNemar test. Then, the confirmed isolates were tested for antibiotic susceptibility using the disc diffusion method. Of the 200 positive CMT cattle, 24 animals were positive for S. aureus and confirmed using 16S rRNA gene amplification. Statistical analysis showed that the phenotypic and genotypic tests of hemolysin genes were not significantly different (P>0.01). PCR analysis revealed the presence of coa and clfa genes in more than half of the cases. Overall, nine genetic profiles of virulence factors were found among S. aureus isolates. The highest and lowest resistance rates were against penicillin and gentamicin, respectively. Our findings showed a high rate of antibiotic resistance. So, accurate and fast diagnosis and antimicrobial susceptibility tests should be considered before prescribing the drugs.

Assessment of udder skin surface temperature changes in relation to mastitis in crossbred dairy cows in different farms in Kerala

The aim of the present study was to assess the infrared thermographic profile in relation to mastitis in lactating dairy cows. In this study, 20 lactating crossbred dairy cows each were monitored for a period of 21 days in four dairy farms. The udder skin surface temperature (USST) was recorded using infrared thermography (IRT) during the study period at diurnal time intervals. Milk samples were collected at the time of afternoon milking from each quarter and tested for California Mastitis Test (CMT). It was observed that higher USST was noticed in udders with clinical mastitis followed by non-mastitis and subclinical mastitis, and the skin surface temperature of non-mastitis and clinical mastitis udders were significantly higher than subclinical mastitis affected udders. The results of this study indicate the limitations in the use of IRT of udders for early detection of mastitis across all regions.

Molecular characterization of Streptococcus agalactiae and Streptococcus dysgalactiae causing bovine mastitis in the southern region of Bangladesh.

This study was conducted to validate polymerase chain reaction (PCR) as a confirmatory diagnostic tool to find out the presence and frequency of Streptococcus agalactiae (S. agalactiae) and Streptococcus dysgalactiae (S. dysgalactiae) in mastitic milk samples obtained from dairy cows in the southern region of Bangladesh. A total of 196 samples of bovine milk were collected from various dairy farms in the Chattogram metropolitan area of the southern part of Bangladesh. DNA extracted from isolates obtained by culturing California mastitis test (CMT)-positive mastitic milk samples (n = 146) on 5% sheep blood agar was used as a template for PCR. Two sets of specific primers based on the 16S rRNA gene were used to discriminate between S. agalactiae and S. dysgalactiae. Four PCR products were subjected to sequencing, followed by phylogenetic analysis. The PCR analyses revealed that out of the 146 CMT-positive milk samples tested, 29 samples were positive for S. agalactiae (19.86%), while 26 samples were positive for S. dysgalactiae (17.81%). Further sequence analysis of the corresponding PCR products and bioinformatics analysis verified the results. The study proves the efficiency of PCR as a useful diagnostic approach to determine the presence and prevalence of S. agalactiae and S. dysgalactiae in mastitic milk samples obtained from dairy cows.

Development of a prototype milk screening reagent for the detection of bovine subclinical mastitis using common detergents and Hibiscus sabdariffa petals

Mastitis is an inflammatory condition of the mammary gland of female animals which can either be clinical or subclinical. Subclinical mastitis is that which cannot be diagnosed by physical assessment of the udder, however, it continues to reduce the quality and quantity of the milk leading to economic losses. Diagnosis and treatment of subclinical mastitis is therefore of paramount importance to the farmer, however, the commercially available California Mastitis Test (CMT) reagent commonly used to detect subclinical mastitis is expensive and not readily accessible especially in the rural agrarian communities. This work was conducted to develop a test reagent from locally and readily available materials that can be used by rural farmers in the detection of subclinical mastitis. Different concentrations (1.5%, 3% and 6%) of different household detergents dissolved in water extract of Hibiscus sabdariffa (zobo) have been explored for their suitability to detect somatic cells in cow milk. The reagents were mixed with mastitic and nonmstatitic milk while the test conducted with CMT served as control to which the results from formulated reagents were compared for sensitivity. It was found that a 3% detergent concentration in zobo can be used to obtain gel formation and a colour change from red to dark green corresponding to somatic cell counts and pH respectively, much like the CMT reagent. The test conducted revealed 53.57%, 46.42%, 42.85% and 42.85% of cows’ fore-quarter milk samples were positive to CMT, detergents A, B and C respectively. The combination of household detergent and water extract of Zobo can therefore be an alternative to CMT reagent in the detection of subclinical mastitis, especially in the rural areas. This indicates the potential of using these test reagents for subclinical mastitis screening. Further studies are suggested to standardize and patent this reagent as an alternative for subclinical mastitis diagnostic test.

Determination of antibiotic resistance and biofilm formation in Klebsiella strains isolated from bovine mastitis cases

Mastitis is diseases of dairy cows with a high economic impact. Bovine mastitis is caused by a wide range of bacterial pathogens. As one of the major environmental pathogens Klebsiella ssp. was investigated in this study by some phenotypic characteristics like antibiotic resistance patterns and biofilm formation properties. A number of 483 cows by dairy farms around the Konya were examined by California Mastitis Test (CMT) producing 36 positive in terms of subclinical mastitis. A further 19 samples from clinical mastitic udders were also collected. Samples were inoculated onto Trypticase Soy Agar medium enriched with sheep blood and incubated aerobicaly for 24-48 hours at 37 °C. By morphological, biochemical and cultural charcateristics 14 isolates out of 37 coliform were identified as Klebsiella ssp. The double disc synergy method and Congo Red Agar test were used to perform antibiotic susceptibility and in vitro slime forming properties, respectively. Resistances to the antibiotics Ampicillin, Carbenicillin, Cephotaxime, Chloramphenicol, Erythromycin, Gentamicin, Neomycin, Oxytetracycline, Sulphamethoxazole/Trimpethoprim, Amoxicillin-Clavulanate and Imipenem were 78.5%, 78.5%, 35.7%, 42.8%, 100%, 7%, 7%, 50%, 14%, 21% and 7%, respectively. Three of the total isolets produced biofilm only. This appears to the first report on ESBL producing Klebsiella ssp from subclinical cases of bovine mastitis in Konya, Türkiye. Presently, two numbers of antimicrobial combinations to treat bovine cases are recommended by this work. In conclusion, because of costly challange nature of Klebsiella caused bovine mastitis implemantation of an effective mastitis control program should be used in local farms from Konya.

Immunopathological Responses to the Bovine Mastitis Associated with Staphylococcus Species Infection

Bovine mastitis is a disease that concerns animals' welfare and increases the economic production losses. Bacterial agents such as Staphylococcus species are the main causative agent of bovine mastitis. This bacterial agent expresses some inflammatory cytokines that might enhance the cell-mediated, which may promote the pathogenesis of mastitis. The objective of the current study was to investigate the bovine innate immune response circulating levels of pro-inflammatory and anti-inflammatory cytokines. A total of 10 mL of milk specimens were collected randomly from 100 clinically mastitic cows, and another 20 clinically healthy cows were considered as a control group for the California Mastitis test. The microbiological cultures of milk specimens were performed. The interleukins (ILs)that involved IL-4, IL-6, and IL-10 were detected using the ELISA test for the evaluation of the pro-inflammatory bovine mastitis pathophysiology. The results of this study showed that Staphylococcus aureus detection was in 31.2% of mastitic milk and 8.7% of non-mastitic milk specimens; and the coagulase-negative Staphylococcus was detected in 14.8% and 18.7% in the mastitic and non-mastitic milk specimens, respectively. The IL-6 level was shown significantly higher (P&lt;0.05)in the specimens of mastitic milk (194±12.8 pg/mL) compared to the non-mastitic milk (31±2.9 pg/mL). In conclusion, the elevated level of expression of IL-6 cytokine in the milk of cows with mastitis suggested that IL-6 might be used as a potentially suitable biomarker for early bovine mastitis diagnosis

Cytological and microbiological examination of bovine milk in Prototheca-infected dairy herd

In healthy milk the somatic cell count (SCC) is less than 100.000 cells per mL, although in the presence of infectious agents, especially pathogenic bacteria, SCC can be extremely high. Cytological appearance of inflammation in mammary gland depends on the origin and the time-course of the inflammatory response. According to predominant cell type there can be suppurative, pyogranulomatous, granulomatous and lymphocytic inflammation. Prototheca spp. as facultative pathogen creates chronic inflammation in mammary gland with high SCC and reduced milk production. The aim of our study was to elaborate a practical and accurate laboratory procedure to enhance the precision of milk somatic cell differentiation, and to provide quick and easy-to-perform detection for the presence of infectious algae in mastitis cases. In 95 quarter samples SCC was determined by California Mastitis Test, cellular density estimation and somatic cell differentiation were carried out by microscopically evaluation, while presence of Prothoteca spp. was investigated with the help of specific culture medium (PIM agar). According to CMT results, SCC values showed 67.3% prevalence of mastitis, from which 6.3% was clinical and 93.7% subclinical. Comparing CMT results and estimated cellularity values indicated tight correlation. In CMT negative quarters 60% of somatic cells were neutrophil granulocytes, while in CMT positive but Prototheca spp. negative cases these cells were present in 74%. Interestingly in Prototheca spp. positive samples only 60.25% of cells were neutrophil granulocytes, while macrophages and lymphocytes were present in 17% and 18.3%, respectively. Moreover, remarkable cytological pattern was notified in some samples that were positive for Prototheca spp., and statistical analysis showed tight correlation between infectious algae and the ratio of macrophages (p=0.005). In conclusion laboratory techniques applied in our study seems to be helpful supplementary methods to reveal pathological background of mastitis as well as to monitor the progression of inflammatory reactions.