A survey of oysters (Crassostrea gigas) in New Zealand for Vibrio parahaemolyticus and Vibrio vulnificus

A microbiological survey was conducted to determine the levels of total and pathogenic Vibrio parahaemolyticus (Vp) and Vibrio vulnificus (Vv) in Pacific oysters (Crassostrea gigas) collected from commercial growing areas in the North Island, New Zealand.The survey was intended to be geographically representative of commercial growing areas of Pacific oysters in New Zealand, while selecting the time frame most likely to coincide with the increased abundance of pathogenic vibrio species. Vp was detected in 94.8% of oyster samples examined (n = 58) with a geometric mean concentration of 99.3 MPN/g, while Vv was detected in 17.2% of oyster samples examined with a geometric mean concentration of 7.4 MPN/g. The frequency of Vp positive samples was 1.7 fold greater than reported in a study conducted three decades ago in New Zealand. Potentially virulent (tdh positive) Vp was detected in two samples (3.4%, n = 58) while no trh (another virulence marker) positive samples were detected. 16 S rRNA genotype could be assigned only to 58.8% of Vv isolates (8:1:1 A:B:AB ratio, n = 10). There was a good agreement [98.2% of Vp (n = 280) and 94.4% of Vv (n = 18) isolates] between molecular tests and cultivation based techniques used to identify Vibrio isolates and there was a significant (R² = 0.95, P < 0.001, n = 18) linear relationship between the MPN estimates by real-time PCR and cultivation. There was no significant correlation between any of the environmental parameters tested and Vp or Vv concentrations.     

Effects of resynchronization programs on pregnancy per artificial insemination, progesterone, and pregnancy-associated glycoproteins in plasma of lactating dairy cows

Objectives were to develop a timed artificial insemination (TAI) resynchronization program to improve pregnancy per AI and to evaluate responses of circulating progesterone and pregnancy-associated glycoproteins in lactating cows. Cows (n = 1,578) were presynchronized with 2 injections of PGF_2α, given 14 d apart starting on d 45 ± 3 postpartum, followed by Ovsynch [2 injections of GnRH 7 d before and 56 h after injection of PGF_2α, TAI 16 h after second injection (d 0)]. The Resynch-treated cows received an intravaginal progesterone insert from d 18 to 25, GnRH on d 25, and pregnancy diagnosis on d 32, and nonpregnant cows received PGF_2α., GnRH 56 h later, and TAI 16 h later (d 35). The control cows were diagnosed for pregnancy on d 32 and nonpregnant cows received GnRH, PGF_2α 39 d after TAI, GnRH 56 h later, and TAI 16 h later (d 42). Pregnancy was reconfirmed on d 60 after AI. Ovarian structures were examined in a subset of cows at the time of GnRH and PGF_2α injections. Blood samples for analyses of progesterone and pregnancy-associated glycoproteins were collected every 2 d from d 18 to 30 in 100 cows, and collection continued weekly to d 60 for pregnant cows (n = 43). Preenrollment pregnancies per AI on d 32 did not differ for cows subsequently treated as Resynch (45.8%, n = 814) and control (45.9%, n = 764), and pregnancy losses on d 60 were 6.7 and 4.0%, respectively. Resynchronized service pregnancy per AI (36%, n = 441; 39.5%, n = 412) and pregnancy losses (6.3 and 6.7%) did not differ for Resynch and control treatments, respectively. Days open for pregnant cows after 2 TAI were less for the Resynch treatment than for the control treatment (96.2 ± 0.82 vs. 99.5 ± 0.83 d). Cows in the Resynch treatment had more large follicles at the time of GnRH. The number of corpora lutea did not differ between treatments at the time of PGF_2α. Plasma progesterone for pregnant cows was greater for Resynch cows than for control cows (18-60 d; 6.6 vs. 5.3 ng/mL), and plasma concentrations of progesterone on d 18 were greater for pregnant cows than for nonpregnant cows (5.3 vs. 4.3 ng/mL). Plasma pregnancy-associated glycoproteins during pregnancy were lower for cows in the Resynch treatment compared with control cows on d 39 (2.8 vs. 4.1 ng/mL) and 46 (1.3 vs. 3.0 ng/mL). Cows pregnant on d 32 that lost pregnancy by d 60 (n = 7) had lower plasma concentrations of pregnancy-associated glycoproteins on d 30 than cows that maintained pregnancy (n = 36; 2.9 vs. 5.0 ng/mL). Pregnancy-associated glycoproteins on d 30 (>0.33 ng/mL) were predictive of a positive d 32 pregnancy diagnosis (sensitivity = 100%; specificity = 90.6%). In conclusion, Resynch and control protocols had comparable pregnancy per AI for first and second TAI services, but pregnancy occurred 3.2 d earlier in the Resynch group because inseminations in the Resynch treatment began 7 d before those in the control treatment. Administration of an intravaginal progesterone insert, or GnRH, or both increased progesterone during pregnancy. Dynamics of pregnancy-associated glycoproteins were indicative of pregnancy status and pregnancy loss.     

Risk factors associated with short-term post-treatment outcomes of clinical mastitis

The objectives of this study were to characterize 60-d outcomes after treatment of mild (abnormal milk) and moderate (abnormal milk and abnormal udder) cases of clinical mastitis (CM) occurring in a single quarter of cows on Wisconsin farms (n = 4) and to determine risk factors associated with those outcomes. Duplicate milk samples were collected from the affected quarter of each cow for microbiological analysis at the onset of CM (PRE) and 21 d later (POST). Cows were treated only in the affected quarter using an intramammary product containing 125 mg of ceftiofur. Bacteriological cure was defined as absence of pathogens in the POST sample obtained from the enrolled quarter. Recurrence was defined for the cow when CM occurred after the milk-withholding period for the enrolled case of CM. Retention in the herd was defined when a cow was retained within the herd for the 60-d follow-up period. Somatic cell count reduction (SCCR) was defined at the cow level as somatic cell count (SCC) below 200,000 cells/mL at the Dairy Herd Improvement Association test day occurring between 21 to 55 d post-treatment. The effects of farm, days in milk, parity, severity, microbiological diagnosis at PRE, previous milk yield, previous SCC, previous occurrence of CM and treatment duration on selected post-treatment outcomes were assessed using Chi-squared analysis and logistic regression. Microbiological results at PRE were distributed as: Escherichia coli (n = 14), Klebsiella spp. (n = 11), Enterobacter spp. (n = 8), Serratia spp. (n = 7), other gram-negative species (n = 3), Streptococcus spp. (n = 25), coagulase-negative staphylococci (n = 4); Staphylococcus aureus (n = 1); Streptococcus agalactiae (n = 1), other gram-positive species (n = 9), and culture negative (n = 60). Treated quarters were more likely to experience bacteriological cure when the cow experienced CM for the first time in the lactation and when no pathogen was recovered from PRE milk samples obtained from the enrolled quarter. Parity and bacteriological cure were associated with the probability of recurrence. Greater milk yield at previous Dairy Herd Improvement test was the most important predictor for retention within the herd. When SCC before CM was >200,000 cells/mL the probability of having SCCR after treatment was decreased. When the case experienced bacteriological cure, the cow was less likely to experience recurrent cases and was more likely to have SCCR below 200,000 cells/mL. Post-treatment outcomes, such as recurrence and SCCR, are strongly associated with bacteriological cure and, when monitored, can be used to help determine if a treatment has been successful. Information about the etiology of CM, history of clinical and subclinical mastitis, and parity are useful to review when making strategic treatment decisions.     

Concentrations of 17beta-estradiol in Holstein whole milk

Some individuals have expressed concern about estrogens in food because of their potential to promote growth of estrogen-sensitive human cancer cells. Researchers have reported concentrations of estrogen in milk but few whole milk samples have been analyzed. Because estrogen associates with the fat phase of milk, the analysis of whole milk is an important consideration. The objectives of this study, therefore, were to quantify 17β-estradiol (E₂) in whole milk from dairy cows and to determine whether E₂ concentrations in milk from cows in the second half of pregnancy were greater than that in milk from cows in the first half of pregnancy or in nonpregnant cows. Milk samples and weights were collected during a single morning milking from 206 Holstein cows. Triplicate samples were collected and 2 samples were analyzed for fat, protein, lactose, and somatic cell counts (SCC); 1 sample was homogenized and analyzed for E₂. The homogenized whole milk (3 mL) was extracted twice with ethyl acetate and once with methanol. The extract was reconstituted in benzene:methanol (9:1, vol/vol) and run over a Sephadex LH-20 column to separate E₂ from cholesterol and estrone before quantification using radioimmunoassay. Cows were classified as not pregnant (NP, n = 138), early pregnant (EP, 1 to 140 d pregnant, n = 47), or midpregnant (MP, 141 to 210 d pregnant, n = 21) at the time of milk sampling based on herd health records. Mean E₂ concentration in whole milk was 1.4 ± 0.2 pg/mL and ranged from nondetectable to 22.9 pg/mL. Milk E₂ concentrations averaged 1.3, 0.9, and 3.0 pg/mL for NP, EP, and MP cows, respectively. Milk E₂ concentrations for MP cows were greater and differed from those of NP and EP cows. Milk composition was normal for a Holstein herd in that log SCC values and percentages of fat, protein, and lactose averaged 4.9, 3.5, 3.1, and 4.8, respectively. Estradiol concentration was significantly correlated (r = 0.20) with percentage fat in milk. Mean milk yield was 18.9 ± 0.6 kg for the morning milking. The mean E₂ mass accumulated in the morning milk was 23.2 ± 3.4 ng/cow. Likewise, using the overall mean concentration for E₂ in milk, the mean E₂ mass in 237 mL (8 fluid ounces) of raw whole milk was 330 pg. The quantity of E₂ in whole milk, therefore, is low and is unlikely to pose a health risk for humans.     

Genomic typing of enterococci isolated from bovine mammary glands and environmental sources

Enterococcal isolates (n = 102) from various sources of bovine origin on 1 farm were characterized using pulsed field gel electrophoresis analysis of SmaI restriction patterns. Isolates originated from feed samples (n = 6), bedding samples (n = 15), and bovine quarter-milk samples (n = 81). Isolates collected from milk samples included those from high-somatic cell count cows (n = 42), postpartum milk samples (n = 16), and clinical mastitis samples (n = 23). Species evaluated included Enterococcus faecium (n = 68), Enterococcus casseliflavus (n = 29), and Enterococcus faecalis (n = 5). A total of 20 clusters representing 44 isolates were detected when a similarity cut-off level of 75% was applied to interpret the pulsed field gel electrophoresis results. Fifteen of the clusters contained only isolates from milk samples. Four clusters contained isolates from bedding and milk samples. One cluster contained only isolates from feed samples. Clusters comprised of a single species represented 17 of the 20 total clusters. These results suggest enterococci from bovine origin were genetically diverse, whereas a limited number of isolates from various sources appeared to cluster together.     

Supplementation of progesterone via controlled internal drug release inserts during ovulation synchronization protocols in lactating dairy cows

Our objective was to determine the effect of exogenous progesterone (P4) during a timed artificial insemination (TAI) protocol on pregnancies per AI (P/AI) in dairy cows not previously detected in estrus. Lactating cows (n = 3,248) from 7 commercial dairy herds were submitted to a presynchronization protocol (2 injections of PGF_2α 14 d apart; Presynch), and cows in estrus after the second PGF_2α received AI (EDAI; n = 1,583). Cows not inseminated by 12 to 14 d after the second PGF_2α injection were submitted to a TAI protocol (GnRH on d 0, PGF_2α on d 7, and GnRH + TAI 72 h after PGF_2α). At onset of the TAI protocol, cows were balanced by parity and days in milk and assigned randomly to receive no exogenous P4 (control, n = 803) or a controlled internal drug release (CIDR) insert containing 1.38 g of P4 from d 0 to 7 (CIDR, n = 862). Blood samples were collected at the second PGF_2α injection of the Presynch and on the day of the first GnRH injection of the TAI protocol for P4 determination. When P4 in both samples was <1 ng/mL, cows were classified as anovular, whereas cows having at least 1 sample ≥1 ng/mL were classified as cyclic. Concentration of P4 at 11 to 14 d after AI was determined in a subgroup of cows (n = 453) from 2 herds. Pregnancy was diagnosed at 40 ± 5 and 65 ± 5 d after AI. Proportion of cows inseminated on estrus after the second PGF_2α injection of the Presynch protocol differed among herds (range = 26.7 to 59.8%). Overall P/AI for EDAI cows at 40 ± 5 and 65 ± 5 d were 36.2 and 33.7%, respectively, and pregnancy loss was 8.8%. Proportion of cyclic cows at the onset of the TAI protocol differed among herds (range from 66.5 to 86.3%), but did not differ between treatments (control = 72.4%, CIDR = 74.1%). Treatment affected P/AI at 40 ± 5 (control = 33.3%, CIDR = 38.1%) and 65 ± 5 (control = 30.0%, CIDR = 35.1%) d after AI but did not affect pregnancy loss (8.6%). Cyclic cows had greater P/AI at 40 ± 5 (38.2 vs. 29.3%) and 65 ± 5 d (35.1 vs. 26.1%) after AI, but cyclic status had no effect on pregnancy loss. Treatment affected P4 concentration after AI, with more CIDR cows having P4 ≥1 ng/mL (94.4 vs. 86.9%) and P4 ≥3.2 ng/mL (81.8 vs. 68.0%) at 11 to 14 d after AI compared with control cows. Treatment of cows not previously detected in estrus with a CIDR insert during a TAI protocol increased proportion of cows with functional CL after AI and P/AI.     

Involvement of Clostridium gasigenes and C. algidicarnis in 'blown pack' spoilage of Brazilian vacuum-packed beef

The objectives of this study were to isolate psychrotrophic clostridia from Brazilian vacuum-packed beef cuts (spoiled or not) and to identify the isolates by using 16S rRNA gene sequencing. Anaerobic psychrotrophic microorganisms were also enumerated and samples were collected to verify the incidence of psychrotrophic clostridia in the abattoir environment. Vacuum-packed beef cuts (n = 8 grossly distended and n = 5 non-spoiled) and environmental samples were obtained from a beef packing plant located in the state of São Paulo, Brazil. Each sample was divided in three subsamples (exudate, beef surface and beef core) that were analyzed for vegetative forms, total spore-forming, and sulfide reducing spore-forming, both activated by alcohol and heat. Biochemical profiles of the isolates were obtained using API20A, with further identification using 16S rRNA gene sequencing. The growth temperature and the pH range were also assessed. Populations of psychrotrophic anaerobic vegetative microorganisms of up to 10¹⁰ CFU/(g, mL or 100 cm²) were found in ‘blown pack’ samples, while in non-spoiled samples populations of 10⁵ CFU/(g, CFU/mL or CFU/100cm²) was found. Overall, a higher population of total spores and sulfide reducing spores activated by heat in spoiled samples was found. Clostridium gasigenes (n = 10) and C. algidicarnis (n = 2) were identified using 16S rRNA gene sequencing. Among the ten C. gasigenes isolates, six were from spoiled samples (C1, C2 and C9), two were isolated from non-spoiled samples (C4 and C5) and two were isolated from the hide and the abattoir corridor/beef cut conveyor belt. C. algidicarnis was recovered from spoiled beef packs (C2). Although some samples (C3, C7, C10 and C14) presented signs of ‘blown pack’ spoilage, Clostridium was not recovered. C. algidicarnis (n = 1) and C. gasigenes (n = 9) isolates have shown a psychrotrophic behavior, grew in the range 6.2-8.2. This is the first report on the isolation of psychrotrophic Clostridium (C. gasigenes and C. algidicarnis) in Brazil. This study shows that psychrotrophic Clostridium may pose a risk for the stability of vacuum-packed beef produced in tropical countries during shelf-life and highlights the need of adopting control measures to reduce their incidence in abattoir and the occurrence of ‘blown pack’ spoilage.     

Analysis of furan in heat-processed foods consumed in Korea using solid phase microextraction–gas chromatography/mass spectrometry (SPME–GC/MS)

Furan (C₄H₄O) is a volatile compound formed during the Maillard reaction and was recently classified as a possible human carcinogen (group 2B) by the International Agency for Research on Cancer. It has been reported to occur in various canned and jarred foods that undergo heat treatment. The aim of the present study was to optimise the sample preparation for furan analysis using solid phase microextraction–gas chromatography/mass spectrometry (SPME–GC/MS), according to the food matrix. We also performed the monitoring and risk assessment of furan in various food products. The optimised fibre exposure temperatures, time and amount of sample of liquid, semi solid and paste state foods were 5 g (ml), 50 °C, and 20 min, respectively. The level of furan in canned meat (32.16 ng/g) was the highest among the samples studied. The furan levels in canned fish, canned vegetable, nutritional/diet drinks, canned soups and jarred sauces were 29.40, 22.86, 7.28, 18.54 and 21.52 ng/g, respectively. Furan concentrations in baby food products were between 3.43 and 97.21 ng/g. Exposure estimates (14.59 ng/kg bw/day) of baby foods was the highest among all the tested food samples. However, the exposure estimate of baby foods was lower than that prescribed by the US FDA.     

Diagnosing intramammary infections: comparison of multiple versus single quarter milk samples for the identification of intramammary infections in lactating dairy cows

The objective was to examine the potential benefits of using different combinations of multiple quarter milk samples compared with a single sample for diagnosing intramammary infections (IMI) in dairy cattle. Data used in the analyses were derived from 7,076 samples from 667 quarters in 176 cows in 8 herds in 4 locations (Minnesota/Wisconsin, n = 4; Prince Edward Island, n = 2; Ontario, n = 1; New York, n = 1). Duplicate quarter milk samples were collected at morning milking for 5 consecutive days. Cows were evenly distributed between early postparturient and mid- to late-lactation cows. All samples were frozen for shipping and storage, thawed once, and cultured in university laboratories using standardized procedures consistent with National Mastitis Council guidelines. The presence of specific pathogens was confirmed and identified using the API identification system (bioMerieux, Marcy l’Etoile, France) in each laboratory. A previously developed gold standard was applied to the first sample from d 1, 3, and 5 to classify infected quarters. The data were analyzed separately for coagulase-negative staphylococci (CNS) and Streptococcus spp. Various combinations of test results from d 2 and 4 were used in the test evaluation. These consisted of single samples (n = 4), 2 sets of duplicate samples (2 samples collected on the same day), 2 sets of consecutive samples (2 samples collected 2 d apart), and 2 sets of triplicate samples (2 samples on the same day and a third sample 2 d apart). Series interpretation of duplicate or consecutive samples (i.e., positive = same pathogen isolated from both samples) resulted in the highest specificity (Sp; CNS Sp = 92.1-98.1%; Streptococcus spp. Sp = 98.7-99.6%), but lowest sensitivity (Se; CNS Se = 41.9-53.3%; Streptococcus spp. Se = 7.7-22.2%). Parallel interpretation of duplicate or consecutive samples (i.e., positive = pathogen isolated from either) resulted in the highest Se (CNS Se = 70.8-80.6%; Streptococcus spp. Se = 31.6-48.1%), but lowest Sp (CNS Sp = 72.0-77.3%; Streptococcus spp. Sp = 89.5-93.3%). The difference in estimates between single and duplicate samples was larger than between single and consecutive samples. Overall, triplicate samples provided the best combination of Se and Sp, but compared with a single sample, provided only a modest gain in Sp and little or no gain in Se.     

Analysis of residual oxytetracycline in fresh milk using polymer reversed-phase column

A simple and rapid reversed phase high performance liquid chromatograph (HPLC) method for analysis of oxytetracycline (OTC) was developed and applied in the determination of the antibiotic in fresh milk sample. Isocratic elution was performed with acetic acid:water (pH 4.5):acetonitrile (4:68:28), using a polymer reversed-phase (PLRP) column and UV detection at 354 nm wavelength. The average recoveries of OTC spiked milk at 0.1, 0.2, 0.5 and 100 ng/mL were in excess of 92% with intraday and interday precision between 0.8% and 6.6% respectively. A good linearity was established between the range 100–1000 ng/mL with r² = 0.9995. The limit of detection and quantitation were 30 and 100 ng/mL respectively. The method demonstrated successful application for analysis of 100 milk samples. Two samples out of 70 from livestock keepers tested OTC positive while none of the 30 samples from milk centers tested positive.     

Supplemental progesterone and timing of resynchronization on pregnancy outcomes in lactating dairy cows

The objective was to determine the effect of exogenous progesterone (P₄) in a timed artificial insemination (TAI) protocol initiated at 2 different times post-AI on pregnancies per AI (P/AI) in lactating dairy cows. Cows (n = 1,982) in 5 dairy herds were assigned randomly at a nonpregnancy diagnosis 32 ± 3 d post-AI to 1 of 4 resynchronization (RES) treatments arranged in a 2 × 2 factorial design using the Ovsynch-56 (GnRH, 7 d later PGF_2α, 56 h later GnRH, 16 h later TAI) protocol. Treatments were as follows: cows initiating RES 32 ± 3 d after AI with no supplemental P₄ (d 32 RES-CON; n = 516); same as d 32 RES-CON plus a controlled internal drug release (CIDR) insert containing P₄ at the onset of Ovsynch-56 (d 32 RES-CIDR; n = 503); cows initiating RES 39 ± 3 d after AI (d 39 RES-CON; n = 494); and same as d 39 RES-CON plus a CIDR (d 39 RES-CIDR; n = 491). Cows were inseminated if observed in estrus before TAI. The P/AI was determined 32 and 60 d after TAI. In a subgroup of cows (n = 1,152), blood samples were collected and ovarian structures examined by ultrasonography on the days of the first GnRH (G1) and PGF_2α of Ovsynch-56. Percentage of cows with a corpus luteum (CL) at G1 was unaffected by timing of treatments, but percentage of cows with a CL at PGF_2α was greater for d 32 than for d 39 cows (87.9 vs. 79.4%). In addition, percentage of cows with P₄ ≥1 ng/mL at G1 was unaffected by timing of treatments, but was increased for d 32 compared with d 39 RES cows on the day of the PGF_2α of the RES protocols (86.5 vs. 74.3%). Treatment did not affect ovulation to G1 or P/AI 32 d after RES TAI (d 32 RES-CON = 30.1%, d 32 RES-CIDR = 28.8%, d 39 RES-CON = 27.5%, d 39 RES-CIDR = 30.5%). A greater percentage of d 39 RES cows underwent premature luteolysis during the RES protocol compared with d 32 RES cows. An interaction was detected between day of RES initiation and CIDR treatment, in which the CIDR increased P/AI 60 d after TAI for d 39 (CON = 23.7% vs. CIDR = 28.0%), but not for d 32 (CON = 26.9% and CIDR = 24.2%) cows. Pregnancy loss was unaffected by treatment. In addition, cows had improved P/AI 60 d after TAI when they received a CIDR and did not have a CL (CON-CL = 28.2%, CON-No CL = 19.2%, CIDR-CL = 27.0%, and CIDR-No CL = 26.5%) or had P₄ <1 ng/mL (CON-High P₄ = 27.8%, CON-Low P₄ = 15.0%, CIDR-High P₄ = 25.0%, and CIDR-Low P₄ = 29.4%) at G1, but not if a CL was present or P₄ was ≥1 ng/mL at G1. In conclusion, addition of a CIDR insert to supplement P₄ during the RES protocol increased P/AI for cows initiating RES 39 ± 3 d after AI but not 32 ± 3 d after AI.     

Verocytotoxigenic Escherichia coli O157 in beef and sheep abattoirs in Ireland and characterisation of isolates by Pulsed-Field Gel Electrophoresis and Multi-Locus Variable Number of Tandem Repeat Analysis

This study aimed to investigate verocytotoxigenic Escherichia coli O157 in the largest beef and sheep slaughter plants in Ireland over a one-year period. Samples consisted of pooled rectal swabs (n = 407) and pooled carcass swabs (n = 407) from 5 animals belonging to the same herd or flock and minced meat (n = 91) from the same sampling date. E. coli O157 isolates were characterised using PCR for a range of genes, i.e. 16S, rfbE, fliC, vtx1, vtx2, eaeA and confirmed VTEC O157 isolates were tested for antimicrobial susceptibility and typed using Pulsed-Field Gel Electrophoresis (PFGE) and Multi-Locus Variable Number of Tandem Repeat Analysis (MLVA). VTEC O157 was isolated from 7.6% and 3.9% of bovine rectal and carcass swab samples and from 5.8% and 2.9% of ovine rectal and carcass swab samples respectively. None of the bovine minced meat samples (n = 77) and only one of the 14 ovine minced meat samples was positive for VTEC O157. Following PFGE and MLVA, cross contamination from faeces to carcasses was identified. While PFGE and MLVA identified the same clusters for highly related strains, MLVA discriminated better than PFGE in addition to being more rapid and less labour intensive. Results showed that cattle and sheep presented for slaughter in Ireland harbour VTEC O157, and although the levels entering the food chain are low, this should not be overlooked as possible sources of zoonotic infection; molecular typing was able to demonstrate relationships among strains and could be used to elucidate the sources of human infection.     

Real-time polymerase chain reaction for the quantitative detection of tetA and tetB bacterial tetracycline resistance genes in food

A new, rapid, sensitive and specific method was developed to directly detect and quantify tetA and tetB in food. Both tet genes are two of the most frequently present tetracycline resistance genes in Gram-negative bacteria. A set of primers and Taqman probes was designed for each gene. The standard curves were performed using Escherichia coli BM13 (C600 RifR)/RP4 and E. coli NCTC 50365, which carry tetA and tetB, respectively. Meat and fish samples inoculated with these reference strains were used as a matrix to construct the standard curves for the analysis of 20 samples of chicken meat and 10 samples of hake (Merlucius merlucius). The limits of detection in pure culture were 5 cfu/mL (0.7 log cfu/mL) in the case of tetA, 50 cfu/mL (1.7 log cfu/mL) for tetB and 5 × 10² cfu/g (2.7 log cfu/g) for both genes in food samples. The results obtained by real-time quantitative polymerase chain reaction (qPCR) were compared to counts of tetracycline-resistant bacteria obtained by plating extracts of poultry and hake samples in culture media supplemented with 16 mg/L of tetracycline. Counts of tetracycline-resistant bacteria obtained by qPCR showed a positive correlation, especially interesting when compared with microbiological counts of tetracycline-resistant Enterobacteriaceae in poultry meat (r = 0.5509) and with tetracycline-resistant mesophilic aerobic bacteria in hake samples (r = 0.7146). The obtained results demonstrate that this method could be a useful tool for the direct quantification of the amount of bacterial strains that carry tetA and/or tetB genes in food samples.     

Development and validation of analytical methods for ethyl carbamate in various fermented foods

The aim of this work was to develop and validate analytical methods for ethyl carbamate (EC) in various food matrices. Column chromatography was used for the analysis of EC in kimchi, a fermented soybean paste (doenjang), a fermented fish product (jeotgal), yoghurt, bread, and cheese. To remove the fat in the bread and cheese, a Florisil cartridge was selected. The volume of dichloromethane in the chromatography column was optimised to 60 mL for the kimchi, cheese, and fermented soybean paste. For the bread, jeotgal, and yoghurt, the best recovery rate was found by using 100 mL of dichloromethane. For the accurate analysis of EC in the vinegar, 150 mL of dichloromethane and a neutralisation process (pH = 8.0) were required. In the standard curve of EC, satisfactory linearity (R² = 0.998) was shown. The limit of quantification (LOQ) was 10 ng/mL and the recovery rates ranged from 76.9% to 118.1%. Intra- and inter-assay precision ranged from 3.5% to 34.2% and 3.8% to 41.9%, respectively.     

Clenbuterol residues in pig muscle after repeat administration in a growth-promoting dose

The aim of this study was to determine the level of clenbuterol residues in muscle tissue of pigs after repeat administration in a growth-promoting dose. An anabolic dose of clenbuterol (20 μg/kg body mass per day) was administered orally to experimental group (n = 12) for 28 days, whereas control animals (n = 3) were left untreated. Clenbuterol treated pigs were randomly sacrificed (n = 3) on days 0, 3, 7 and 14 of treatment discontinuation and clenbuterol residues determined in muscle tissue. Determination of residual clenbuterol was by enzyme-linked immunosorbent assay (ELISA) as a screening method and liquid chromatography tandem mass spectrometry (LC-MS/MS) as a confirmation method. The highest clenbuterol content in the muscle of treated animals was recorded on day 0 of treatment cessation (4.40 ± 0.37 ng/g) and significantly (p < 0.05) exceeded the maximum residue limit (MRL) of 0.1 ng/g. On day 3 of withdrawal, it was 0.49 ± 0.22 ng/g and on day 7 0.10 ± 0.02 ng/g (at MRL); on day 14 of treatment discontinuation, clenbuterol content was below the limit of detection (< 0.1 ng/g) in all samples. Administration of clenbuterol as a growth promoter in pig production could lead to residues in meat for human consumption up to 7 days after treatment discontinuation.     

Comparison of two estrus-synchronization protocols and timed artificial insemination in dairy cattle

The objective of this study was to evaluate the effect of the Ovsynch protocol with and without exogenous progesterone on pregnancy rate (PR) in cows in which estrous cycles were previously synchronized with 2 doses of PGF_2α and that were not detected in estrus during the presynchronization period. The study was conducted in Chihuahua, Mexico (8,650 Holstein milking cows; 305-d mature equivalent milk yield = 13,790 kg). On d 47 postpartum, estrous cycles in cows were synchronized by using 2 doses of PGF_2α 14 d apart. Any cow detected in estrus during this presynchronization period was inseminated. Cows not detected in estrus were selected at random and assigned to receive progesterone supplementation or to serve as controls. Controls (n = 594) were subjected to the Ovsynch protocol and cows in the progesterone supplemented treatment (n = 594) were subjected to the Ovsynch protocol plus an intravaginal insert containing 1.9 g of progesterone inserted at the time of the first GnRH injection and removed 7 d later. Progesterone-supplemented cows had a greater PR (31.2%) compared with controls (22.7%). Plasma progesterone concentrations at artificial insemination (AI) were <1 ng/mL and did not differ between treatments. At 14 d post-AI, however, more cows that received progesterone supplementation had concentrations of progesterone >1 ng/mL compared with controls. It was concluded that after a presynchronization period, cows subjected to the Ovsynch program and supplemented with exogenous progesterone had a greater PR and greater concentrations of progesterone after AI than those subjected to the Ovsynch protocol and not supplemented with progesterone.     

Short communication: Aflatoxin M1 in dairy products sold in Şanlıurfa,Turkey

The aim of this study was to detect the presence of aflatoxin M₁ (AFM1) in samples of raw milk (n = 38), UHT milk (n = 12), white pickled cheese (n = 50), and yogurt (n = 50) collected from the ?anl?urfa city markets and locally produced dairy products by ELISA. The mean contamination rates were 56.74 ± 40.32, 43.1 ± 23.19, 103.2 ± 29.13, and 55.28 ± 12.68 ng/kg, respectively, for raw milk, UHT milk, white pickled cheese, and yogurt. According to the data, 21 (55%) raw milk, 3 (25%) UHT milk, 10 (20%) white pickled cheese, and 10 (20%) yogurt samples were contaminated with AFM1 over the acceptable levels (≥50 ng/kg), ranging from 0.82 to 130.89 ng/kg. None of the white pickled cheese samples contained AFM1 levels above the Turkish legal limit (250 ng/kg). Consequently, the AFM1 contamination levels determined in this study in white pickled cheese were not considered to pose a serious public health hazard. However, the AFM1 levels in raw and UHT milk and yogurt samples indicate an increased human health risk in Turkey related to high aflatoxin levels. Therefore, milk and dairy products have to be monitored by the Turkish public health authorities continuously to detect AFM1 contamination.     

Heat stress abatement during the dry period influences metabolic gene expression and improves immune status in the transition period of dairy cows

Heat stress (HT) and photoperiod affect milk production and immune status of dairy cows. The objective was to evaluate the effects of HT abatement prepartum under controlled photoperiod on hepatic metabolic gene expression and cellular immune function of periparturient Holstein cows (n = 21). Cows were dried off 46 d before expected calving date and assigned to treatments by mature equivalent milk production. The treatments were 1) HT and 2) cooling (CL), both imposed during a photoperiod of 14L:10D. Rectal temperature was measured twice daily, whereas respiration rate was measured 3 times/wk at 1500 h during the entire dry period. After calving, cows were housed in a freestall barn with cooling, and milk yield was recorded daily up to 140 d in milk. Liver samples were taken at dry off, −20, 2, and 20 d relative to calving by biopsy. Under a similar schedule, neutrophil function was determined in blood of cows on HT (n = 12) and CL (n = 9). Blood samples were taken on −46, −32, −18, 0, 14, 28, and 42 d relative to calving for measurement of metabolites and were collected twice daily from −7 to 2 d relative to calving for prolactin (PRL) analysis. The HT cows had greater concentrations of PRL at 0 d relative to calving (150 vs. 93; SEM = 11 ng/mL) and had higher afternoon rectal temperatures (39.4 vs. 39.0; SEM = 0.04°C) and elevated respiration rates (78 vs. 56; SEM = 2 breaths/min) during the prepartum period compared with CL cows. Relative to HT cows, CL cows had greater hepatic expression of PRL-R, SOCS-3, and CAV-1 mRNA. Neutrophil oxidative burst was greater in CL cows relative to HT cows at 2 d (61 vs. 42; SEM = 6%) and at 20 d (62 vs. 49; SEM = 5%) relative to calving, and phagocytosis was greater in CL cows at 20 d (47 vs. 33; SEM = 4%) relative to calving compared with HT cows. Humoral response, as measured by IgG secretion against ovalbumin challenge, was greater for CL cows at −32 d (0.44 vs. 0.33; SEM = 0.05 OD) and −21 d (0.60 vs. 0.50 ± 0.04 OD) relative to calving compared with HT cows. These results suggest that HT abatement during the dry period improved innate and acquired immune status as measured by neutrophil function and immunoglobulin secretion against ovalbumin challenge, and altered hepatic gene expression related to PRL signaling in the periparturient period or subsequent lactation.     

Distribution of coagulase-negative Staphylococcus species from milk and environment of dairy cows differs between herds

In many parts of the world, coagulase-negative staphylococci (CNS) are the predominant pathogens causing intramammary infections (IMI) in dairy cows. The cows’ environment is thought to be a possible source for CNS mastitis and this was investigated in the present paper. A longitudinal field study was carried out in 6 well-managed dairy herds to determine the distribution and epidemiology of various CNS species isolated from milk, causing IMI and living freely in the cows’ environment, respectively. In each herd, quarter milk samples from a cohort of 10 lactating cows and environmental samples from stall air, slatted floor, sawdust from cubicles, and sawdust stock were collected monthly (n = 13). Isolates from quarter milk samples (n = 134) and the environment (n = 637) were identified to species level using amplified fragment length polymorphism (AFLP) genotyping. Staphylococcus chromogenes, S. haemolyticus, S. epidermidis, and S. simulans accounted for 81.3% of all CNS milk isolates. Quarters were considered infected with CNS (positive IMI status) only when 2 out of 3 consecutive milk samples yielded the same CNS AFLP type. The species causing IMI were S. chromogenes (n = 35 samples with positive IMI status), S. haemolyticus (n = 29), S. simulans (n = 14), and S. epidermidis (n = 6). The observed persistent IMI cases (n = 17) had a mean duration of 149.4 d (range 63.0 to 329.8 d). The CNS species predominating in the environment were S. equorum, S. sciuri, S. haemolyticus, and S. fleurettii. Herd-to-herd differences in distribution of CNS species were observed in both milk and the environment, suggesting that herd-level factors are involved in the establishment of particular species in a dairy herd. Primary reservoirs of the species causing IMI varied. Staphylococcus chromogenes and S. epidermidis were rarely found in the environment, indicating that other reservoirs were more important in their epidemiology. For S. haemolyticus and S. simulans, the environment was found as a reservoir, suggesting that IMI with these species were possibly environmental in origin.     

Artificial lighting during winter increases milk yield in dairy ewes

In Australia, the supply of sheep milk is reduced during the winter. Housing dairy animals under lights during winter is a simple technique to increase milk yield; however, it is difficult to predict the magnitude of this increase in dairy ewes, because there are few corroborating data. We studied 220 East Friesian crossbred ewes (50 primiparous and 170 multiparous ewes, respectively) that lambed in April to May 2007 (late autumn, southern hemisphere) and were weaned from their lambs within 24 h of parturition and milked exclusively by machine. These ewes were ranked according to their milk production, and ewes producing ≥1,000 mL/d of milk were allocated to 1 of 2 groups. One group of ewes was kept indoors under a long-day photoperiod (16 h of light), whereas the other group was kept indoors under a naturally declining day length. Ewes were maintained under these conditions for 8 wk. Milk yield was measured twice weekly, and ewe weight and condition were measured at weekly intervals. From a subset of ewes (n = 20 per group), milk samples were collected twice weekly at the morning milking to measure milk lipid, protein, and lactose, and blood samples were collected once a week to measure plasma prolactin concentrations. Mean daily milk yield was analyzed as a percentage of preexperimental milk yield because the milk yield of ewes housed under the long photoperiod was lower than that of ewes under a declining day length when the treatments began. Thus, the ewes under a long photoperiod yielded 91.7% of their starting yield by wk 8 of treatment, whereas ewes under a declining day length yielded 76.25% of their initial value (LSD = 5.1), and this divergence in milk yield was apparent by wk 2 of treatment. Mean plasma prolactin levels were greater in ewes housed under the long-day photoperiod (n = 20) compared with control ewes (n = 20) at wk 6 (168 ± 27 vs. 72 ± 19 ng/mL, respectively), wk 7 (125 ± 28 vs. 37 ± 7 ng/mL, respectively), and wk 8 of the experiment (132 ± 35 vs. 31 ± 7 ng/mL, respectively). The composition of the milk was similar between the groups at each time point, and milk from these ewes (n = 20 per group) contained, on average, 6.1 ± 0.05% lipid, 4.8 ± 0.02% protein, and 5.4 ± 0.01% lactose (n = 309 samples). We concluded that ewes increase milk production in response to being housed under a long-day photoperiod during winter.