Infected Quarters Research Articles

Clinical and molecular characterization of both methicillin-resistant andsensitive staphylococcus aureus mastitis

This study targeted bovine mastitis as a possible source of livestock-associated methicillin-resistant Staphylococcus aureus (MRSA), to identify clinical signs associated with MRSA- and non-MRSA-associated mastitis. Thirty-eight mastitis cases (68 infected quarters) were investigated. Gram-positive cocci-shaped isolates were selected based on Baird Parker agar growth as well as Gram-stained bacterial smears. Molecular screening for Staphylococcus aureus (S. aureus) yielded 17 isolates, of which five (29.41%) were methicillin resistant. The five isolates were mecA positive, but mecC negative. Multilocus sequence typing (MLST) indicated that sequence type 1 (ST1) was the identified type of all isolates of MRSA. S. aureus-associated cases showed different clinical forms of mastitis, including subclinical, acute, chronic, and gangrenous. Additionally, subclinical mastitis was the only detected condition associated with MRSA, which may represent a potential hidden risk for humans. Phenotypically, isolates of MRSA showed resistance to all of the tested β-lactam antimicrobials, with marked resistance to tetracycline and gentamycin. Based on our knowledge, this is the first report to identify MRSA ST1 in Egypt. Bovine mastitis could be a source for the dissemination of MRSA to humans and other animals. Additionally, while methicillin-resistance may have no effect on the clinical outcome of mastitis, it does affect therapeutic success, particularly when β-lactam antimicrobials are used.

The occurrence of mastitis and its effect on the milk malondialdehyde concentrations and blood enzymatic antioxidants in dairy cows

Early identification of mastitis is a serious challenge for dairy farmers and veterinarians in ensuring the health of an animal and the hygienic quality of the produced milk. The purpose of this study was to detect the occurrence and aetiology of mastitis in a dairy herd of 15 milked cows localised in a farm in west Slovakia. During the complex investigation, 606 quarter milk samples were examined (6 quarters were discarded) and classified based on the clinical status, the presence of abnormal udder secretions, the result of the California mastitis test (CMT), the somatic cell count (SCC) and the bacteriological identification of the pathogens causing the intramammary infection (IMI). The study was augmented by the detection of malondialdehyde (MDA) in the milk and the measurements of the blood enzymatic activities of glutathione peroxidase (GPx) and superoxide dismutase (SOD) as potential biomarkers for the udder health screening. A positive CMT score was recorded in 19.5% (118) of the examined quarters and 12.5% (76) of the quarters were infected with bacterial pathogens causing latent mastitis (LM; 1.3%), subclinical mastitis (SM; 8.3%), and clinical mastitis (CM; 2.9%). The most commonly isolated bacteria from the infected quarters were coagulase-negative staphylococci (55.2%), Staphylococcus aureus (11.8%) and streptococci (10.5%). The concentration of MDA and SCC were significantly higher from both the SM and CM cases than in the milk samples from the healthy cows, while the blood activities of SOD and GPx were lower in the cows with CM compared to the healthy cows. The higher MDA concentrations in the SM and CM milk observed in this study showed the presence of an oxidative stress in the infected milk, accompanied by a decrease in the antioxidative enzymatic activity in the blood of the cows. Therefore, the measurement of the milk MDA concentration and the activity of the blood SOD and GPx may prove insightful for the better screening of the udder health in the early diagnosis of mastitis.

Effects of chronic Staphylococcus aureus infection on immunological parameters and functionality of macrophages isolated from bovine mammary secretions

The aim of this study was to characterize the effects of chronic S. aureus intramammary infection (IMI) on local innate and adaptive immune response during active involution. Cows in late lactation that were either uninfected or with chronic naturally acquired S. aureus IMI were included in this study. The levels of interleukin (IL)-1β, IL-6 and IL-4 were significantly higher in mammary secretions of S. aureus-infected quarters compared with uninfected at d 7, 14 and 21 of involution. Lactoferrin (Lf), total IgG and S. aureus specific IgG1 levels were significantly lower in mammary secretions of infected quarters compared with uninfected during the first three weeks of involution. The amount of intracellular reactive oxygen species (ROS) produced per macrophage, was significantly higher in mammary secretions of infected quarters compared with uninfected at d 14 post drying off. Nitrite production was significantly higher in phagocytes from infected mammary secretions compared with uninfected at d 7 and 14 post drying off. Chronic S. aureus IMI altered normal secretion composition during bovine mammary gland involution. The high IL-1β and IL-6 levels and increased functionality of macrophages in mammary secretions of infected quarters could be a result of the chronic inflammatory environment triggered by the presence of viable bacteria in mammary tissue. The lower levels of total and S. aureus specific antibodies and other immune factors in mammary secretion during this period may reduce the natural defense potential of the gland contributing to S. aureus persistence.

Staphylococcus aureus and bovine mastitis: molecular typing of methicillinresistance and clinical description of infected quarters

This study targeted Staphylococcus aureus (S. aureus)-mastitis of bovine as a possible source of livestock-associated methicillin-resistant Staphylococcus aureus (MRSA), to describe clinical signs of mastitis associated with MRSA and methicillin-sensitive Staphylococcus aureus (MSSA). The study area was the Gharbia and Kafrelsheikh governorates, in the central and northern regions of the Egyptian Delta. Clinical examination of animals was performed to detect clinical mastitis (CM) and clinically normal cases were tested by the California mastitis test (CMT) to identify subclinical mastitis (SCM). Accordingly, 38 mastitis cases (68 infected quarters) were detected. Milk samples were collected aseptically and were cultured on Baird Parker agar. Fifty nine Gram-positive cocci-shaped isolates were selected and preserved. In addition, 33 Staphylococcus spp. isolates originated from bovine mastitis at the same study area were obtained from Animal Health Research Institute (AHRI). The cocci-shaped Gram-positive bacteria and AHRI Staphylococcus spp. isolates were used for molecular identification of S. aureus and MRSA. Molecular screening had yielded 17 S. aureus isolates, from which five isolates (29.41%) were MRSA and 12 isolates (70.59%) were MSSA. The five MRSA isolates were mecA positive, but mecC negative. Multilocus sequence typing (MLST) of the five MRSA isolates indicated that all were sequence type 1 (ST1). S. aureus-associated cases showed different clinical forms of mastitis, including subclinical, acute, chronic, and gangrenous. However, subclinical mastitis was the only detected form associated with MRSA, which may represent a potential hidden risk for humans. Phenotypic antimicrobial resistance pattern of MRSA isolates showed resistance to all of the tested β-lactam antimicrobials, with marked orgaresistance to tetracycline and gentamycin. Based on our knowledge, this is the first report to identify MRSA ST1 in Egypt. Bovine mastitis could be a source for the dissemination of MRSA to humans and other animals. Additionally,while methicillin-resistance may have no effect on the clinical severity of mastitis, it does affect therapeutic success, particularly when β-lactam antimicrobials are used.

Correlation between Tail Length and Rate of Infected Quarters in Montbéliarde and Ameliorated Dairy Cattle, Algeria

Correlation between Tail Length and Rate of Infected Quarters in Montbéliarde and Ameliorated Dairy Cattle, Algeria

The bovine acute phase protein α1-acid glycoprotein (AGP) can disrupt Staphylococcus aureus biofilm

Staphylococcus aureus biofilm-related infections are of clinical concern due to the capability of bacterial colonies to adapt to a hostile environment. The present study investigated the capability of the acute phase protein alpha 1-acid glycoprotein (AGP) to a) disrupt already established S. aureus biofilm and b) interfere with the biofilm de novo production by using Microtiter Plate assay (MtP) on field strains isolated from infected quarters by assessing. The present study also investigated whether AGP could interfere with the expression of bacterial genes related to biofilm formation (icaA, icaD, icaB, and icaC) and adhesive virulence determinants (fnbA, fnbB, clfA, clfB, fib, ebps, eno) by quantitative real-time PCR (qPCR). The results provided the evidence that AGP could disrupt the biofilm structure only when it was already developed, but could not prevent the de novo biofilm formation. Moreover, AGP could interfere with the expression levels of genes involved in biofilm formation in a dose- and strain-dependent way, by upregulating, or downregulating, icaABC genes and fnbB, respectively. The results presented in this study provide new insights about the direct antibacterial activity of AGP in bovine milk. It remains to be demonstrated the molecular bases of AGP mechanism of action, in particular for what concerns the scarce capability to interact with the de novo formation of biofilm.

Short communication: Intra- and inter-individual milk microbiota variability in healthy and infected water buffalo udder quarters

The concept that ruminant mammary gland quarters are anatomically and physiologically unrelated has been recently challenged by immunological evidence. How this interdependence reflects on individual quarter milk microbiota is unknown. The aim of the present study was to cover this gap by investigating the interdependence of quarters among the same mammary gland at the milk microbiota level using next-generation sequencing of the V4-16S rRNA gene. A total of 52 samples were included in this study and classified as healthy or affected by subclinical mastitis. Extraction of DNA, amplification of the V4-16S rRNA gene, and sequencing using Ion Torrent Personal Genome Machine (Thermo Fisher Scientific, Waltham, MA) were carried out. We found that the intra-individual variability was lower than the inter-individual one. The present findings further support at milk microbiota level the hypothesis of the interdependence of quarters, as previously demonstrated following immunological studies, suggesting that individual factors (e.g., immunity, genetics) may have a role in modulating milk microbiota.

Performance of a milk leukocyte differential test for decision-making in a selective dry cow therapy program

The objectives of this study were 1) to determine the operating characteristics of a commercial milk leukocyte differential (MLD) test to detect intramammary infections in quarters of late-lactation dairy cows as compared to bacteriological culture and 2) to evaluate the milk production and udder health parameters between cows treated following blanket vs selective dry cow therapy (DCT) using the MLD test results. In a first experiment, the MLD test was compared to the bacteriological culture results (gold standard) of 363 quarters from 94 cows. The sensitivity, specificity, and predictive values for the identification of infection using the MLD test were determined. Sensitivity ranged from 44% to 77%, and specificity from 54% to 92%. In the second experiment blanket DCT was compared to selective DCT based on the results of MLD test, and treating only positive quarters; a total of 328 cows were randomly assigned to 1 of the 2 treatment groups. The proportion of quarters positive to bacteriological culture, and the incidence rate of moderate and severe cases of clinical mastitis events, did not differ between treatment groups. Results of these experiments provide information to support decision-making in a selective DCT program in low-SCC herds using the MLD test.

Diagnostic value of composite milk sample vs single quarter milk sample for the detection of Staphylococcus aureus intra-mammary infections in cattle

Staphylococcus aureus (S. aureus) is one of the most important causes of mastitis in dairy cattle. Control and eradication programs of S. aureus intra-mammary infections (IMI) are based on different factors included the correct detection and management of the infected cows. The present study aimed at evaluating the efficacy of composite milk samples (CMS) analysis, compared to quarter milk samples (QMS) analysis, for the bacteriological detection of S. aureus intra-mammary infections. During 2016, 661 CMS (hygienically collected) and 2644 QMS (aseptically collected) were obtained from 661 cows in 5 herds. All the samples were submitted to S. aureus bacteriological culture and somatic cell count (SCC) analysis.QMS bacteriological analysis on blood agar plates was able to detect 236 cows excreting S. aureus, while the bacteriological analysis of CMS, using selective agar, identified 229 positive cows. The concordance was 95% with an excellent Cohen's κ (0.89). Relative sensitivity and specificity of CMS vs QMS, considered as the reference test, were 91.5% ± 2.1 and 96.9% ± 1.3 (CI 95%), respectively. In addition, the relative sensitivity of CMS improved as the number of infected quarters per cow and the number of colony forming units (cfu) per sample increased.The predictive value of CMS results was better when paired with SCC data, in particular CMS showed better negative predictive value when SCC was <200,000 cells/mL and better positive predictive value when SCC was>200,000 cells/mL. The probability for a cow to be S. aureus positive was 56.4% in case of SCC > 200,000 cells/mL, while it was 18.6% in case of SCC < 200,000 cells/mL. The average SCC in CMS was significantly higher in positive cows and the value rose as the number of infected quarters per cow increased.Given the intermittent excretion of S. aureus in milk from dairy cows, it could be more advantageous to carry out several serial CMS, rather than few QMS, being CMS an easier to collect and less expensive milk sampling method. Thus, bacteriological examination of CMS, combined with SCC data of the same sample, could be extremely useful for the success of S. aureus IMI control plans, because repeated CMS are easier to be performed and could be more easily proposed to the farmers.

Expert evaluation of different infection types in dairy cow quarters naturally infected with Staphylococcus aureus or Streptococcus agalactiae

The purpose of this study was to improve the diagnostic recommendations for Staphylococcus aureus and Streptococcus agalactiae control using bacterial culture (BC), polymerase chain reaction (PCR) and somatic cell count (SCC) as diagnostic methods. The study was carried out in three steps: firstly, diagnostic test patterns for naturally infected quarters with Staph. aureus (24 quarters) and Strep. agalactiae (16 quarters) were created by sampling the quarters each day for 21 days and analysing the daily quarter milk samples using BC, PCR and SCC. Secondly, 30 mastitis experts were asked to group and describe the diagnostic test patterns and to establish a diagnosis for each group. The experts’ statements regarding the groups they established were subsequently examined using qualitative content analysis to assign “infection types” to the statements. Lastly, the test performance was estimated for BC, PCR and SCC using generalised logistic regression models with the interpreted statements as a reference for infection.The experts mainly identified the Staph. aureus quarter-patterns as persistent infections, while some had more dynamic patterns. Strep. agalactiae quarter-patterns mainly involved persistent infection, yet some appeared hard to diagnose and were assigned to almost all different infection types, while experts did not agree on the interpretation.Estimates of Se for detection of Staph. aureus infection were 95.9% [93.7; 97.3] for BC, 99.5% [98.3; 99.8] for PCR, and 96.1% [94.0; 97.5] for SCC. The corresponding Sp estimates were 74.5% [65.7; 81.7], 66% [57.2; 73.8] and 43.7% [36.2; 51.5] for BC, PCR and SCC, respectively.The Se estimates of BC and PCR for Strep. agalactiae infection were 100% [83.5; 100] and 99.9% [99.6; 100], respectively, whereas the Se of SCC detecting Strep. agalactiae infection was only 34.3% [26.4; 43.3]. This indicated that Strep. agalactiae-positive BC and PCR test results were more important than SCC results to the experts when diagnosing a quarter as infected. The Sp estimates of BC, PCR and SCC for Strep. agalactiae infection were 99% [72.8; 100], 97.7% [62.1; 99.9], and 65.7% [56.7; 73.7], respectively.We conclude that PCR and BC are highly sensitive in the detection of persistent and new infections as defined by the experts, although the Se was not always 100%. An accepted lower Sp suggests that experts place less emphasis on false-positive results. We recommend that efforts are made to develop consistent terminology to characterise intramammary infections over time so that the course of infection can be taken into account at diagnosis.

Prophylactic Potential of Tri-sodium Citrate on Subclinical Mastitis in Cow in Different Housing System

The present research work was carried out to evaluate prophylactic potential of Tri-sodium citrate powder in subclinical mastitis in milking cows. The study involved twelve HF cross dairy cows in which each diagnosed with at least one subclinical mastitis quarter. The six (n=6) cows from conventional and six (n=6) cows from loose housing system with raised SCC were administered Tri-sodium citrate powder @ 30 mg/kg B.W. orally BID for five days. The SCC values of infected quarters were reduced significantly (P < 0.05) on 28th day after Tri-sodium citrate therapy as compared to value on day ‘O’ before therapy both in loose housing and conventional housing system. The percent efficacy in improvement of infected quarter after administration of Tri-sodium citrate powder in conventional and loose housing system was 63.63 % and 58.33 % respectively. Thus results indicated that tri-sodium citrate having prophylactic potential in controlling mastitis in cows in both conventional and loose housing system.

Incidence and Associated Risk Actors of Heifer Mastitis in Dairy Farms at Mekelle Town, Northern Ethiopia

A longitudinal study was conducted from November 2013 to May 2014 in Mekelle town, Northern Ethiopia, to assess the incidence of heifer mastitis, to isolate and identify the causative pathogens and their susceptibility to different antimicrobials and also identify risk factors associated with occurrence of heifer mastitis. Twenty - five heifers were included from 4 d airy farms and 11 cooperative small holder’s dairy farms during the study period. Total of 100 quarters were examined to detect clinical and subclinical mastitis by physical examinations of udder and milk and California mastitis Test (CMT), respectively. T he incidence of mastitis per gland at risk was 64 % and the spontaneous cure rate of infected quarters was 33 %. Incidence of heifers’ intra - mammary infections (IMI) in the study area was highest at calving (28%). Infection per quarter revealed that 49% we re mastitis positive of which 8.2% of their quarters were clinical and 45/49 (91.8%) quarters were sub clinical type of mastitis. The incidence rate per quarters were statistically significant (p &lt;0.05) and higher in rear quarters than front quarters. The univariate logistic regression showed that among potential risk factors considered from the farm attributes, breed, age, practice of milking mastitic cow last, housing nature, hand wash before and in between milking and udder hygiene had significant effect on the prevalence of subclinical mastitis. Coagulase negative Staphylococci (CNS) (54.5%) and Staphylococcus aureus (21.8%) , were the predominant bacteria. Relatively most of the isolates are susceptible to Chloramphenicol, Gentamycin, Ciprofloxacin and V ancomycin but resistant to Penicillin. The study also revealed that S.aureus, E.coli and S.agalactiae were highly incident at calving. It is concluded that prevalence of CNS, lack of hygienic practice and infestation of ticks should give serious attention.Intramammary treatment of prepartum heifer with appropriate antimicrobials may help to reduce the incidence of mastitis at calving and subsequent lactation period.

Methicillin-resistant Staphylococcus aureus CC22-MRSA-IV as an agent of dairy cow intramammary infections

Methicillin-resistant S. aureus (MRSA) lineages have become major responsible of healthcare- and community-associated infections in human population. Bovine MRSA are sporadically detected in the dairy herd, but its presence enhances the risk of zoonosis. Some lineages are able to lose the specific host tropism, being easily transmitted from animals to humans and vice-versa. The present study aims at clarifying the epidemiology of MRSA intramammary infections in a closed dairy herd, which was running a mastitis control program since years. Quarter milk samples were collected from all lactating cows once a week for 9 weeks and bacteriologically tested. At the end of the follow-up period, also a self-taken nasal swab of the milker was analysed. Three cows (12.5%) were MRSA positive, a fourth showed a transient infection and MRSA was isolated also from the milker’s nose. Somatic cell counts of infected quarters fluctuated from 1000 to 1,800,000 cells/mL. The isolates were genotyped using DNA microarrays and identified as the epidemic UK-EMRSA-15 grouping in CC22. All strains carried the genes for β-lactam and macrolide resistance. The milker isolate differed from cow isolates mainly for the absence of the untruncated β-haemolysin and the presence of the immune evasion cluster. The milker had been volunteering in a nursing home since months, thus playing the role of MRSA vector into the herd. Our results showed the adaptive capacity of such MRSA to the bovine host. Therefore, we suggest that CC22-MRSA should be regarded as a potential cause of reverse zoonosis in dairy cattle herds.

Ability of milk pH to predict subclinical mastitis and intramammary infection in quarters from lactating dairy cattle

Milk pH is increased in lactating dairy cattle with subclinical mastitis (SCM) and intramammary infection (IMI). We hypothesized that milk pH testing provides an accurate, low-cost, and practical on-farm method for diagnosing SCM and IMI. The main objective was to evaluate the clinical utility of measuring milk pH using 3 tests of increasing pH resolution: Multistix 10 SG Reagent Strips for Urinalysis (Multistix strips, Bayer HealthCare Inc., Elkhart, IN), pH Hydrion paper (Microessential Laboratory, Brooklyn, NY), and Piccolo plus pH meter (Hanna Instruments, Woonsocket, RI), for diagnosing SCM and IMI in dairy cattle. Quarter foremilk samples were collected from 115 dairy cows at dry off and 92 fresh cows within 4 to 7 d postcalving. Quarter somatic cell count (SCC) was measured using a DeLaval cell counter (DeLaval, Tumba, Sweden), with SCM defined as SCC >200,000 cells/mL and IMI defined as SCC >100,000 cells/mL and the presence of microorganisms at ≥10 cfu/mL of milk. Milk pH was measured at 37°C using the 3 test methods. The Hydrion pH paper performed poorly in diagnosing SCM and IMI. Receiver operating curve analysis provided optimal pH cutpoints for diagnosing SCM for the pH meter (dry off, ≥6.67; freshening, ≥6.52) and Multistix strips (dry off and freshening, ≥7.0). Test performance of the pH meter and Multistix strips was poor to fair based on area under the receiver operating curve, sensitivity, specificity, positive likelihood ratio, and kappa coefficient. The pH meter and Multistix strips performed poorly in diagnosing IMI at dry off and freshening. We concluded that milk pH does not provide a clinically useful method for diagnosing SCM or IMI in dairy cattle.

Method for short-term prediction of milk yield at the quarter level to improve udder health monitoring

Udder health problems are often associated with milk losses. These losses are different between quarters, as infected quarters are affected both by systemic and pathogen-specific local effects, whereas noninfected quarters are only subject to systemic effects. To gain insight in these losses and the milk yield dynamics during disease, it is essential to have a reliable reference for quarter-level milk yield in an unperturbed state, mimicking its potential yield. We developed a novel methodology to predict this quarter milk yield per milking session, using an historical data set of 504 lactations collected on a test farm by an automated milking system from DeLaval (Tumba, Sweden). Using a linear mixed model framework in which covariates associated with the linearized Wood model and the milking interval are included, we were able to describe quarter-level yield per milking session with a proportional error below 10%. Applying this model enables us to predict the milk yield of individual quarters 1 to 50 d ahead with a mean prediction error ranging between 8 and 20%, depending on the amount of historical data available to estimate the random effect covariates for the predicted lactation. The developed methodology was illustrated using 2 examples for which quarter-level milk losses are calculated during clinical mastitis. These showed that the quarter-level mixed model allows us to gain insight in quarter lactation dynamics and enables to calculate milk losses in different situations.

Subclinical intramammary infection does not affect bovine milk ethanol stability

The present study hypothesized that intramammary infection (IMI) might reduce milk ethanol stability (MES), mainly when IMI is caused by major pathogens. Thus, this study evaluated the effect of IMI on bovine MES using a natural exposure experimental design. Ninety-four lactating cows from five dairy herds were selected once they were determined to have an IMI, based on milk bacteriological culturing with positive isolation and somatic cell count (SCC) &gt; 200×103 cells/mL in two out of three composite milk samples collected during three consecutive weeks. After selection, cows were sampled a second time (within two weeks) for evaluation at mammary quarter level (n = 326): milk yield (kg/quarter/day), MES, composition (fat, protein, lactose, casein, total solids and solids-non-fat), and bacteriologic culture. The effect of subclinical mastitis on MES was tested by two models: 1) comparison of healthy vs. infected quarters; and 2) comparison of contralateral mammary quarter within cow. The only milk composition variable associated with MES was lactose (r = 0.18; P &lt; 0.01). Subclinical IMI did not affect MES when the comparison was performed using both models (1 and 2). Likewise, MES did not change when infected quarters were sorted into two groups of pathogens (major, minor and infrequent; and contagious, environmental, minor and infrequent) and compared with healthy mammary quarters. Considering the results of both models, subclinical IMI did not affect MES of dairy cows.

Elimination of selected mastitis pathogens during the dry period

We aimed to evaluate the elimination of 4 different mastitis pathogens, Streptococcus agalactiae, Mycoplasma bovis, Staphylococcus aureus, and Streptococcus uberis, from infected udder quarters during the dry period using quantitative PCR. The second purpose of this study was to evaluate the association between milk haptoglobin (Hp) concentration and the presence of udder pathogens (Strep. agalactiae, Staph. aureus, M. bovis, and Strep. uberis) in udder quarter milk samples before and after dry period. Aseptic udder quarter milk samples (n = 1,001) were collected from 133 dairy cows at dry off and at the first milking after calving from 1 large dairy herd. Bacterial DNA of Strep. agalactiae, Staph. aureus, Strep. uberis, and M. bovis in the udder quarter milk samples was identified with commercial quantitative PCR analysis Mastitis 4B (DNA Diagnostic A/S, Risskov, Denmark). Milk Hp concentration (mg/L) was measured from udder quarter milk samples. The elimination rates during the dry period for M. bovis, Staph. aureus, Strep. agalactiae, and Strep. uberis were 86.7, 93.6, 96.2, and 100.0%, respectively. The new IMI rate was 3.0% for M. bovis, 2.9% for Staph. aureus, 2.4% for Strep. agalactiae, and 3.1% for Strep. uberis. The milk Hp concentration was significantly higher in udder quarter milk samples with blood and in samples positive for Strep. agalactiae at dry off and for Staph. aureus postcalving. Elevated milk Hp concentration was not associated with the presence of M. bovis in the udder quarter milk samples. In conclusion, elimination of Staph. aureus, Strep. agalactiae, and Strep. uberis during the dry period was high; the elimination of M. bovis from infected udder quarters was lower, but probably spontaneous. Additionally, milk Hp concentration may be used as a marker for udder inflammation when combined with the bacteriological results at dry off and postpartum.

Assessment of acoustic pulse therapy (APT), a non-antibiotic treatment for dairy cows with clinical and subclinical mastitis.

Clinical and subclinical mastitis affects 30% of cows and is regarded as the most significant economic burden on the dairy farm reducing milk yield and quality and increasing culling rate. A proprietary Acoustic Pulse Therapy (APT) device was developed specifically for treating dairy cows. The APT device was designed to produce deep penetrating acoustic pulses that are distributed over a large treated area at a therapeutic level. This paper presents findings from a clinical assessment of this technology for the treatment of dairy cows with subclinical and clinical mastitis. In subclinical mastitis, a group of 116 cows from 3 herds were identified with subclinical intramammary infection and enrolled in the study; 78 cows were assigned to the treatment group and 38 cows to the control group. Significant differences (P<0.001) were found where 70.5% of the cows in the treatment group returned to normal milk production, compared with only 18.4% of the control group. Daily milk yields of the treated cows increased significantly (P<0.05) and the percentage of cows with log somatic cell count under 5.6 cells/mL was significantly higher (P<0.001). Milk of the infected quarters appeared normal with lactose greater than 4.8%, but this difference was not significant. Of the treated cows with identified bacteria, 52.6% of the quarters were cured, while in the control group only 25.0% (P<0.001). Specifically, all cows identified with Escherichia coli in the treatment group were cured, with 66.6% cured with no intervention in the control. Spontaneous cure of glands infected with coagulase negative staphylococci (CNS) and Streptococci was low while treatment successfully increased the cure of CNS from 13.3% to 53.8% and that of Streptococci from 18.2% to 36.4%. Of the 4 cows identified with Staphylococcus aureus, 3 were cured. The clinical mastitis study group included 29 infected cows that were submitted either to a gold standard antibiotic treatment subgroup of 16 cows (n = 16) or to an APT treatment subgroup of 13 cows (n = 13). A cure of 18.7% was shown for the antibiotic treatment, of which logSCC returned to <5.6 cell/mL and 56.2% were culled. A cure of 76.9% was shown for the APT treatment with only one cow culled (7.7%).

Detection of tetracycline and other antimicrobial residues in milk from cows with clinical mastitis treated by combination therapy.

The purpose of this study was to determine tetracycline and other antimicrobial residues in milk from dairy cows treated for clinical mastitis. Data on cow health and drugs used were obtained from the farm veterinarians. A milk sample from each affected udder quarter of each cow was taken for bacterial identification before treatment. All 35 cows in the study were treated with an intramammary product containing tetracycline, neomycin, bacitracin, and prednisolone (tetracycline injector) and also simultaneously with other drugs via the intramammary or parenteral route. The withhold period of the 'tetracycline product' was eight milkings but, in all cases, longer withhold periods were prescribed due to off-label application of additional combinations of drugs. Milk samples from treated udder quarters, taken two milkings before and two after the prescribed withhold period, were analysed for antimicrobial residues. Additionally, milk samples were taken from untreated healthy udder quarters to check if any crossover of drugs had occurred. Three screening tests were used for antimicrobial detection. In 15 (42·9%) cows the milk samples from the treated infected quarters contained tetracycline residues above the maximum residue limit (MRL) after the prescribed withhold period and, in two cases (5·7%) the same was true for neomycin and cefquinome residues. Cephalexin above the MRL was detected in only one case (2·9%). Beta-lactams did not exceed the MRL after the prescribed withhold period. Antimicrobial residues were not detected in milk samples from untreated quarters. Cow condition including parity, decreased milk production and severity of mastitis significantly influenced the excretion of antimicrobials in milk (P ≤ 0·05). No significant differences were found between the tetracycline positive and tetracycline negative cows regarding the bacteria involved, combination antimicrobial therapy, or treatment interval and duration.

Characterization of quorum sensing system in Clostridium chauvoei

Clostridium chauvoei causes fatal black quarter infection in cattle and buffaloes. The quorum sensing (QS) system, a bacterial cell to cell communication process, of the pathogen was characterized in the current study. The results indicated that C. chauvoei lacked luxS (autoinducer-2) based quorum sensing as detected by the sensor strain Vibrio harveyi BB170. This was supported by absence of luxS gene in C. chauvoei genome. However, the genomic analysis indicated the presence of agrBD system in all three genomes of C. chauvoei available at the NCBI database. The AgrD, which synthesizes QS messenger auto-inducing peptide, was a 44 amino acid protein which shared 59% identity and 75% similarity with AgrD of C. perfringens strain 13 and 56% identity (20% coverage) with Staphylococcus aureus N315. The functional cysteine amino acid was conserved in all the strains. The genomic organisation further suggests the presence of diguanylate cyclase, a gene responsible for synthesis of secondary messenger cyclic di-GMP, at 3’ immediate downstream of agrD gene. The real time expression analysis for agrD gene indicated that expression was better at 37 °C (1.9–3.7 fold increase) compared to a higher temperature of 40 °C. However, stable expression was observed at different growth stages (log and early stationary phase) with 0.8–1.4 fold changes in expression pattern. The results indicate the presence of a constitutively expressed agrBD quorum sensing system in C. chauvoei.