Short communication: Characterization of an atypical maltose-negative Staphylococcus aureus through the use of phenotypic and molecular techniques.

The most clinically relevant staphylococci in veterinary medicine are those that are coagulase-positive, namely Staphylococcus aureus. During microbiological udder health monitoring (2009-2018), a new S. aureus strain (coagulase-positive and maltose-negative) was discovered as an emerging udder pathogen during routine examinations of South African dairy herds. This study challenged the conventional microbiological diagnosis of staphylococci by comparing its results to those of the MALDI-TOF mass spectrometry and 16S rRNA sequencing. Both of these tests confirmed that the maltose-negative staphylococcus (MNS), identified as Staphylococcus pseudintermedius by conventional microbiology, was S. aureus ST2992. Multi locus sequence typing was performed on 3 of the MNS isolates and indicated that these isolates were of single origin. These strains tested positive for both MALA and MALR genes (control: S. aureus ATCC 25923). Although the α-glucosidase gene was present, it was not expressed phenotypically. The latter may be attributed to the abnormal stop codon identified in the MALA gene sequence of S. aureus ST2992 (GenBank accession number, MN531305). The newly identified MNS has a field behavior different to that of maltose-positive S. aureus, and more similar to the low virulence of non-aureus staphylococci.

Sensitivity and specificity of a hand-held milk electrical conductivity meter compared to the California mastitis test for mastitis in dairy cattle.

Screening tests for mastitis can play an important role in proactive mastitis control programs. The primary objective of this study was to compare the sensitivity and specificity of milk electrical conductivity (EC) to the California mastitis test (CMT) in commercial dairy cattle in South Africa using Bayesian methods without a perfect reference test. A total of 1848 quarter milk specimens were collected from 173 cows sampled during six sequential farm visits. Of these samples, 25.8% yielded pathogenic bacterial isolates. The most frequently isolated species were coagulase negative Staphylococci (n=346), Streptococcus agalactiae (n=54), and Staphylococcus aureus (n=42). The overall cow-level prevalence of mastitis was 54% based on the Bayesian latent class (BLC) analysis. The CMT was more accurate than EC for classification of cows having somatic cell counts >200,000/mL and for isolation of a bacterial pathogen. BLC analysis also suggested an overall benefit of CMT over EC but the statistical evidence was not strong (P=0.257). The Bayesian model estimated the sensitivity and specificity of EC (measured via resistance) at a cut-point of >25 mΩ/cm to be 89.9% and 86.8%, respectively. The CMT had a sensitivity and specificity of 94.5% and 77.7%, respectively, when evaluated at the weak positive cut-point. EC was useful for identifying milk specimens harbouring pathogens but was not able to differentiate among evaluated bacterial isolates. Screening tests can be used to improve udder health as part of a proactive management plan.

Trends in udder health and emerging mastitogenic pathogens in South African dairy herds.

The aim of this study was to retrospectively analyse the results of milk samples obtained from South African dairy herds during the period 1996 to April 2007 in order to identify possible trends in isolates of microorganisms and their pathogenicity under field conditions. Milk samples were obtained from 7 of the 9 provinces in South Africa where there are low numbers of dairy cows. Although there is scientific limitation to a country wide survey, such as the variation in herd size, management skills, parity, milk yield, milking frequency and other parameters, the size of this database helps to give a fair indication of general udder health in South Africa. Cytology and routine bacteriology were performed on 379,000 milk samples of lactating cows and bacteriology on 11,946 samples from non-lactating cows. According to the results obtained, mastitis did not decrease in South Africa over the test period. The prevalence of mastitis and teat canal infection was lowest in 2002. Mastitis and teat canal infection increased from 2002 to 2006 from 8.1% and 24.1% to 15.4 and 30.0% respectively. The percentage of mastitogenic pathogens isolated from cows over these years also varied. Previously unknown or almost eradicated mastitogenic pathogens such as alphabeta haemolytic Staphylococcus aureus which is thought to be of human origin, Streptococcus agalactiae and Enterococcus canis were responsible for numerous mastitis outbreaks seen in the test samples. Coagulase-negative staphylococci were the most frequently isolated bacteria in milk samples from both lactating and dry cows, followed by Staphylococcus aureus and Streptococcus agalactiae. Although Staphylococcus aureus remained the principal mastitogenic pathogen in South Africa, owing to its chronic nature and resultant economic losses, most cases of mastitis were caused by coagulase-negative staphylococci. This finding increases the importance of coagulase-negative staphylococci (formerly described as a minor pathogen) significantly. Isolations of Streptococcus agalactiae peaked between 2000 and 2005 and decreased again by 2007. Coagulase-negative staphylococcal isolates increased from 2002 and were still on the increase in 2007. Streptococcus agalactiae, Streptococcus uberis and Enterococcus canis were isolated more frequently from milk samples of lactating cows compared with dry cows, while Enterococcus faecalis was isolated more frequently from dry cow samples.

Intramammary antibiotic withdrawal periods for dairy goats compared to those for dairy cattle.

This study investigated the withdrawal periods (WP) of two intramammary antibiotics Cloxamast LC (Intervet SA) and Spectrazol Milking Cow (Schering-Plough Animal Health) in dairy goats and compared them to those recommended for use in cattle. The WP for Cloxamast LC, measured by the Thermo Resistant Inhibitory Substances (TRIS) test, was 60 h in composite samples, 56 h in udder half samples, and the dye was visible for up to 56 h. The WP was significantly shorter than the 72 h recommended WP for use in cattle. It was however significantly longer when the 24 h safety margin (48 h) was subtracted from the recommended WP for cattle. For Spectrazol Milking Cow the antibiotics could be detected by the TRIS test for 61 h in composite samples and 59 h in udder half samples. This did not differ significantly from the recommended 60 h WP for cattle. However, it was significantly longer than that recommended for use in cattle without the 24 h safety margin. There was no significant difference in WP between infected and non-infected udder halves, while there was a weak positive correlation between WP and stage of lactation (R2 = 0.253). There was a moderate positive correlation (R2 = 0.583) between the TRIS test and the presence of dye in milk in udder half samples and between WP in both udder half and composite milk samples (R2 = 0.456). Weak to moderate positive correlations were present between milk yield and the WP in both udder half (R2 = 0.414) and composite (R2 = 0.262) milk samples. Significant differences (P < 0.001) were also observed between the milk yield of udder halves with and without palpable udder damage and between samples that tested TRIS positive and negative on both composite (P = 0.008) and udder half samples (P < 0.001). There was no significant difference between the milk yield of samples with or without dye. There was a significant difference in milk yield between infected and non-infected udder halves (P = 0.054) and a weak negative correlation between milk yield and stage of lactation (R2 = -0.379).

Value of tests for evaluating udder health in dairy goats: somatic cell counts, California Milk Cell Test and electrical conductivity.

The value of electric conductivity (EC), California Milk Cell Test (CMCT) and somatic cell count (SCC) as diagnostic tools was investigated in dairy goats. Conductivity colour reading correlated with SCC. Milk samples with conductivity colour red had significantly higher SCC than those with conductivity colours green and orange (P < 0.001). There were moderate positive correlations between CMCT (R2 = 0.470), and conductivity score and CMCT and conductivity colour readings (R2 = 0.597). Conductivity scores were significantly (P< 0.001) higher during and after intra-mammary treatment with Cloxamast LC and conductivity colours were significantly different between treatment and control groups (P< 0.001). There was a weak positive correlation between conductivity colour and stage of lactation (R2 = 0.317) and a moderately positive correlation between conductivity score and stage of lactation (R2 = 0.523). A moderately negative correlation was shown between milk yield and conductivity score (R2 = -0.426) and between milk yield and conductivity colour (R2 = -0.433). Moderate positive correlations were present between CMCT and SCC (R2 = 0.689) and between CMCT and stage of lactation (R2 = 0.459). CMCT ratings were significantly different (P < 0.001) for the intra-mammary treatment groups. CMCT ratings for infected and non-infected udder halves (P = 0.008) were significantly different; as were those for infected and non-infected udder halves and for left and right udder halves separately (P= 0.010). CMCT ratings for milk samples with SCC above and below 750 x 10(3) cells per ml were significantly different (P < 0.001) as well as for milk from treated and control udder halves with SCC below or above 750 x 10(3) cells per ml (P < 0.001). CMCT was found to be more accurate for indicating the absence of mastitis than for diagnosing it. There were significant differences in log SCC between treatment and control groups, during and after treatment. Infected udder halves had significantly higher log SCC than non-infected udder halves before and after treatment, but not during treatment. There was a moderate positive correlation between stage of lactation and SCC (R2 = 0.438).

Intramammary antibiotics in dairy goats: withdrawal periods of three intramammary antibiotics compared to recommended withdrawal periods for cows.

Intramammary antibiotics are registered and tested for use in dairy cattle. This study investigated withdrawal periods of three intramammary antibiotics (Curaclox LC [Norbrook Pharmacia AH]), Spectrazol Milking Cow (Schering-Plough Animal Health) and Rilexine 200 LC (Logos Agvet [Virbac]) in dairy goats and compared them to withdrawal periods recommended for use in cattle. Three trials were carried out in two different herds. The withdrawal periods for Curaclox LC in eight relatively low producing Saanen goats were 74.3 h (+/- 19.21) measured by Thermo-Resistant Inhibitory Substances (TRIS), 90.0 h (+/- 16.97) measured by colour dye, 99.4 h (+/- 9.07) for cloxacillin measured by Parallux Beta Lactam Assay test (IDEXX distributors), and 92.6 h (+/- 11.41) for ampicillin measured by Parallux. The withdrawal period for Curaclox LC recommended for use in cattle (72 h) was significantly shorter than the withdrawal periods as measured by colour dye (P < 0.001), Parallux testing for cloxacillin (P < 0.001) and ampicillin (P = 0.003). There was a significant difference in withdrawal periods as measured by TRIS (P = 0.009) and colour dye (P = 0.036). The mean withdrawal periods measured on 12 relatively high producing Saanen and Saanen-Toggenburg crossbreed dairy goats measured by TRIS, colour dye and Parallux for ampicillin and cloxacillin were, however, shorter at 42.0 h (+/- 7.077), 64.5 h (+/- 60.26), 77.3 h (+/- 13.56) and 70.7 h (+/- 12.65), respectively. These withdrawal periods for Curaclox LC were significantly longer than the withdrawal periods recommended for cattle as measured by TRIS (P < 0.001) and colour dye (P < 0.001). The combined withdrawal periods for Curaclox LC in the two trials as measured by TRIS, colour dye, Parallux testing for Cloxacillin and Ampicillin were 58.64h (+/- 24.31), 75.8 h (+/- 17.70), 87.0 h (+/- 16.10) and 80.3 h (+/- 16.23), respectively. The withdrawal period, when data of the two trials were combined, measured by TRIS (P < 0.001) was significantly longer than the withdrawal period recommended for use in cattle. The mean withdrawal period as measured by TRIS for Spectrazol Milking Cow for seven relatively low producing Saanen dairy goats was 95.3 h (+/- 17.23). This was significantly (P < 0.001) longer than the withdrawal period recommended for use in cattle (60 h) for Spectrazol Milking Cow. The mean withdrawal period as measured by TRIS for Rilexine 200 LC for 20 relatively high producing Saanen and Saanen-Toggenburg crossbreed dairy goats was 36.9 h (+/- 9.943) and was significantly (P < 0.001) shorter than the withdrawal period as recommended for use in cattle (96 h). There were also significant differences in all the one-sample t-tests between withdrawal periods for goats and recommended withdrawal periods for cattle without the 24 h safety margin.

Intramammary antibiotics in dairy goats: effect of stage of lactation, parity and milk volume on withdrawal periods, and the effect of treatment on milk compositional quality.

The length of the antibiotic withdrawal period after intramammary treatment was influenced by the milk yield of dairy goats during this trial. Shorter withdrawal periods were seen in relatively high yielding dairy goats (production above 1.5 l per day) compared to low producers (less than 1.3 l per day). High yielding goats treated with Curaclox LC (Norbrook [Pharmacia AH]) had a withdrawal period of 42 h, while low yielding goats, treated with the same product, had a withdrawal period of 74 h. The recommended withdrawal period for Curaclox LC for use in cattle is 72 h. Relatively high yielding goats treated with Rilexine 200 LC (Logos Agvet [Virbac]) had a significantly shorter withdrawal period (37 h) than that recommended for use in cattle (96 h). Low yielding goats treated with Spectrazol Milking Cow (Schering-Plough Animal Health) had a significantly longer (95 h) withdrawal period than that recommended for use in cattle (60 h). Withdrawal periods were also influenced by stage of lactation and parity. There was a moderate positive correlation between lactation number and withdrawal period, as measured by TRIS (R2 = 0.621), and a moderate negative correlation between stage of lactation and withdrawal period (R2 = -0.669). In Trials 1, 2 and 3 combined there was a moderate negative correlation between withdrawal period and volume (R2 = -0.511) and a strong positive correlation between withdrawal period and lactation number (R2 = 0.720). The differences in percentage milk fat, protein and lactose before, during and after treatment were not statistically significant except in Trial 3 (Curaclox LC and Rilexine 200 LC) where protein and lactose differed significantly. In Trial 2 (Spectrazol Milking Cow) milk fat percentages differed significantly between treatment and control groups as did protein percentages in Trial 3. These differences are however, not biologically meaningful.

The influence of intramammary antibiotic treatment, presence of bacteria, stage of lactation and parity in dairy goats as measured by the California Milk Cell Test and somatic cell counts.

The California Milk Cell Test (CMCT) and somatic cell counts (SCC) on their own were not reliable methods in the identification of subclinical mastitis in the dairy goats studied and should be accompanied by microbiological tests. However, CMCT and SCC were indicators of irritation of the udder parenchyma. In healthy goats Spectrazol Milking Cow (Schering-Plough AH) caused the least and Curaclox LC (Norbrook (ARK AH)) the most irritation of parenchyma after intramammary treatment. The effects of Rilexine 200 LC (Logos Agvet (Virbac)) were intermediate. There was a highly significant difference (P < 0.001) in the mean log SCC between treated and control groups for goats treated with Curaclox LC and Rilexine 200 LC but no significant difference was present in the mean log SCC of treatment and control groups for goats treated with Spectrazol Milking Cow at the 07:00 and at 19:00 samplings. The CMCT was an indicator of the level of SCC in goat milk. The CMCT was more useful in confirming the absence of infection, rather than in diagnosing mastitis.

Withdrawal periods and tissue tolerance after intramammary antibiotic treatment of dairy goats with clinical mastitis.

The aim of this study was to determine withdrawal periods (WP) and tissue irritation after administration of three intramammary antibiotics [Curaclox LC (Norbrook (ARK AH)], Spectrazol Milking Cow (Schering-Plough AH) and Rilexine 200 LC [Logos Agvet (Virbac)] in goats with clinical mastitis. Withdrawal periods in goats with clinical mastitis treated with Curaclox LC, were not significantly different from those recommended for use in cows (72 h) with (67 h) or without (48 h) the 24 h mandatory safety margin while Spectrazol caused a significantly longer withdrawal period (122 h) than that recommended for use in cattle with (60 h) and without (36 h) the 24h safety margin. The withdrawal period of clinical mastitis cases treated with Rilexine 200 LC was 48 h compared to the 96 h recommended for use in cows. A linear model of regression with factors influencing the WP in goats with clinical mastitis was as follows: WP = 30.21 + 4.692 (sampling time) + 22.11 (udder pathology) - 13.6 (floccules) - 0.00649 (milk yield). Somatic Cell Counts (SCC) of milk from udder halves with clinical mastitis ranged from 7,053 x 10(3) to 7,948 x 10(3) cells per ml without isolations of bacteria and between 6,476 x 10(3) and 8,479 x 10(3) cells per ml with isolations of bacteria. Most of the variation in SCC could not be explained and the California Milk Cell Test (CMCT) and SCC on their own were not reliable methods for mastitis diagnosis. However, CMCT and SCC were indicators of udder irritation. In goats without clinical mastitis, Spectrazol Milking Cow caused the least tissue irritation followed by Rilexine 200 LC and Curaclox LC. For goats with clinical mastitis, Rilexine 200 LC caused the least irritation, followed by Curaclox LC while Spectrazol Milking Cow caused the most irritation.