Inflamación relacionada con angiopatía amiloide: características clínicas y respuesta al tratamiento en una serie de casos / Cerebral amyloid angiopathy–related inflammation: clinical features and treatment response in a case series

Introducción: La inflamación relacionada con la angiopatía amiloide es una entidad caracterizada por una respuesta inflamatoria alrededor de los depósitos de beta amiloide de la microcirculación cerebral. Métodos: Revisión retrospectiva de una serie de pacientes con inflamación relacionada con angiopatía amiloide, que cumplieran criterios clínico-radiológicos o con diagnóstico histopatológico confirmado. Resultados: Se incluyeron siete pacientes, cinco varones, con edad media de 79 años. El inicio fue agudo o subagudo en seis de los casos. La clínica más frecuente fue deterioro cognitivo (n = 6), alteraciones de conducta (n = 5), crisis epilépticas (n = 5), focalidad neurológica (n = 4) y cefalea (n = 2). El líquido cefalorraquídeo fue anormal en tres de cinco casos (pleocitosis linfocitaria e hiperproteinorraquia). Las imágenes de resonancia magnética cerebral más frecuentes consistieron en microhemorragias (n = 7), hiperintensidades subcorticales en secuencia T2-FLAIR (n = 7) y realce leptomeníngeo (n = 6). La afectación fue bilateral en tres de los casos, con predominio en regiones parieto-occipitales (n = 5). Se realizó una tomografía por emisión de positrones (PET) de amiloide en dos pacientes, resultando positiva en uno. Se obtuvo la confirmación histopatológica mediante una biopsia en dos de los casos. Todos los sujetos recibieron tratamiento inmunosupresor, objetivándose una respuesta clínica y radiológica inicial favorable, con recaída radiológica en dos de los casos tras la retirada del tratamiento, y mejorando tras la reinstauración. Conclusiones: El diagnóstico resulta imprescindible de cara a iniciar un tratamiento precoz, ya que ha demostrado mejorar el pronóstico y disminuir las recurrencias. Si bien el diagnóstico definitivo es histopatológico, los criterios clínico-radiológicos permiten el diagnóstico de esta entidad sin necesidad de biopsia.(AU)

Introduction: Cerebral amyloid angiopathy–related inflammation (CAA-ri) is an entity characterised by an inflammatory response to β-amyloid deposition in the walls of cerebral microvessels. Methods: We conducted a retrospective review of a series of patients with a diagnosis of CAA-ri according to histopathological study findings or clinical-radiological diagnostic criteria. Results: The study included 7 patients (5 men) with a mean age of 79 years. Disease onset was acute or subacute in 6 patients. The most frequent symptoms were cognitive impairment (n = 6), behavioural alterations (n = 5), epileptic seizures (n = 5), focal neurological signs (n = 4), and headache (n = 2). Cerebrospinal fluid was abnormal in 3 patients (lymphocytic pleocytosis and high protein levels). The most frequent MRI findings were microbleeds (n = 7), subcortical white matter hyperintensities on T2-FLAIR sequences (n = 7), and leptomeningeal enhancement (n = 6). Lesions were bilateral in 3 patients and most frequently involved the parieto-occipital region (n = 5). Amyloid PET studies were performed in 2 patients, one of whom showed pathological findings. Two patients underwent brain biopsy, which confirmed diagnosis. All patients received immunosuppressive therapy. An initially favourable clinical-radiological response was observed in all cases, with 2 patients presenting radiological recurrence after treatment withdrawal, with a subsequent improvement after treatment was resumed. Conclusions: Early diagnosis of CAA-ri is essential: early treatment has been shown to improve prognosis and reduce the risk of recurrence. Although a histopathological study is needed to confirm diagnosis, clinical-radiological criteria enable diagnosis without biopsy.(AU)

Cerebral amyloid angiopathy-related inflammation: clinical features and treatment response in a case series.

INTRODUCTION: Cerebral amyloid angiopathy-related inflammation (CAA-ri) is an entity characterised by an inflammatory response to ß-amyloid deposition in the walls of cerebral microvessels. METHODS: We conducted a retrospective review of a series of patients with a diagnosis of CAA-ri according to histopathological study findings or clinical-radiological diagnostic criteria. RESULTS: The study included 7 patients (5 men) with a mean age of 79 years. Disease onset was acute or subacute in 6 patients. The most frequent symptoms were cognitive impairment (n = 6), behavioural alterations (n = 5), epileptic seizures (n = 5), focal neurological signs (n = 4), and headache (n = 2). Cerebrospinal fluid was abnormal in 3 patients (lymphocytic pleocytosis and high protein levels). The most frequent MRI findings were microbleeds (n = 7), subcortical white matter hyperintensities on T2-FLAIR sequences (n = 7), and leptomeningeal enhancement (n = 6). Lesions were bilateral in 3 patients and most frequently involved the parieto-occipital region (n = 5). Amyloid PET studies were performed in 2 patients, one of whom showed pathological findings. Two patients underwent brain biopsy, which confirmed diagnosis. All patients received immunosuppressive therapy. An initially favourable clinical-radiological response was observed in all cases, with 2 patients presenting radiological recurrence after treatment withdrawal, with a subsequent improvement after treatment was resumed. CONCLUSIONS: Early diagnosis of CAA-ri is essential: early treatment has been shown to improve prognosis and reduce the risk of recurrence. Although a histopathological study is needed to confirm diagnosis, clinical-radiological criteria enable diagnosis without biopsy.

Cerebral amyloid angiopathy-related inflammation: clinical features and treatment response in a case series. / Inflamación relacionada con angiopatía amiloide: características clínicas y respuesta al tratamiento en una serie de casos.

INTRODUCTION: Cerebral amyloid angiopathy-related inflammation (CAA-ri) is an entity characterised by an inflammatory response to ß-amyloid deposition in the walls of cerebral microvessels. METHODS: We conducted a retrospective review of a series of patients with a diagnosis of CAA-ri according to histopathological study findings or clinical-radiological diagnostic criteria. RESULTS: The study included 7 patients (5 men) with a mean age of 79 years. Disease onset was acute or subacute in 6 patients. The most frequent symptoms were cognitive impairment (n = 6), behavioural alterations (n = 5), epileptic seizures (n = 5), focal neurological signs (n = 4), and headache (n = 2). Cerebrospinal fluid was abnormal in 3 patients (lymphocytic pleocytosis and high protein levels). The most frequent MRI findings were microbleeds (n = 7), subcortical white matter hyperintensities on T2-FLAIR sequences (n = 7), and leptomeningeal enhancement (n = 6). Lesions were bilateral in 3 patients and most frequently involved the parieto-occipital region (n = 5). Amyloid PET studies were performed in 2 patients, one of whom showed pathological findings. Two patients underwent brain biopsy, which confirmed diagnosis. All patients received immunosuppressive therapy. An initially favourable clinical-radiological response was observed in all cases, with 2 patients presenting radiological recurrence after treatment withdrawal, with a subsequent improvement after treatment was resumed. CONCLUSIONS: Early diagnosis of CAA-ri is essential: early treatment has been shown to improve prognosis and reduce the risk of recurrence. Although a histopathological study is needed to confirm diagnosis, clinical-radiological criteria enable diagnosis without biopsy.

Short communication: Volatile profile of matured Tronchón cheese affected by oxytetracycline in raw goat milk.

The presence of antibiotics in milk destined for cheese production may affect the biological processes responsible for the formation of volatile compounds, leading to alterations in the characteristic cheese flavor expected by consumers. The aim of this study was to evaluate the effect of the presence of oxytetracycline in goat milk on the volatile profile of ripened cheeses. Traditional mature Tronchón cheeses were manufactured from raw goat milk spiked with different concentrations of oxytetracycline (50, 100, and 200 µg/kg). Cheese made from antibiotic-free goat milk was used as control. We analyzed the residual amounts of the antibiotic and the volatile profile of the experimental cheeses on a fortnightly basis during maturation using liquid chromatography tandem mass spectrometry and then solid-phase microextraction followed by gas chromatography-mass spectrometry. Our results suggested that oxytetracycline was widely transferred from milk to cheese: drug concentrations in the cheeses were 3.5 to 4.3 times higher than those in raw milk. Although the residual amounts of oxytetracycline significantly decreased during maturation (88.8 to 96.5%), variable amounts of residues remained in cheese matured for 60 d (<10 to 79 µg/kg). In general, the presence of oxytetracycline in goat milk did not affect the volatile profile of Tronchón cheeses; volatile profile was significantly modified by ripening time. Still, the presence of oxytetracycline residues in cheeses ripened for 60 d could be of great concern for public health.

Characteristics of ripened Tronchón cheese from raw goat milk containing legally admissible amounts of antibiotics.

The aim of this study was to evaluate the transfer of the most widely used antibiotics in dairy goats from milk to cheese as well as their effect on the cheese-making process and cheese characteristics during ripening. Antibiotic-free milk was spiked individually with 7 veterinary drugs (amoxicillin, benzylpenicillin, cloxacillin, erythromycin, ciprofloxacin, enrofloxacin, and oxytetracycline) at an equivalent concentration of the European Union maximum residue limit. Spiked goat milk was used to make mature Tronchón cheeses, which were analyzed at 0, 30, and 60 d of maturation to determine pH, chemical composition, proteolytic and lipolytic activities, and color and textural properties. A sensory evaluation of 60-d ripened cheeses was carried out. Cheeses from raw antibiotic-free goat milk were made simultaneously to be used as reference. The cheese-making process was unaffected by the presence of most antibiotics evaluated. Only erythromycin and oxytetracycline significantly increased the time required for cheese production (122 ± 29 and 108 ± 25 min, respectively). However, variable amounts of antibiotics, ranging from 7.4 to 68%, were transferred from milk to cheese, with oxytetracycline and quinolones showing the highest retention rates. In general, antibiotic residues present in the cheeses at the beginning of maturation decrease significantly along time. Thus, ß-lactams and erythromycin residues were not detectable after 30 d of ripening. However, relatively high concentrations of enrofloxacin (148 ± 12 µg/kg) and ciprofloxacin (253 ± 24 µg/kg) residues were found in the cheeses after 60 d of maturation. The quality characteristics of the Tronchón cheeses were only slightly affected by such substances, with few significant differences in the free fatty acid concentration and color and textural properties of the cheeses. Results herein indicate that the use of goat milk containing antibiotics, such as quinolones, at the European Union maximum residue limit for cheese production could adversely affect the safety of the final products because relatively high concentrations of these substances could be retained in soft and semi-mature cheeses, making it necessary to assess the risk for consumer health. Studies on the partition of the antibiotic substances during cheese-making, using specific technologies, would be convenient to guarantee the safety of cheese and related products.

Short communication: Drug residues in goat milk after prophylactic use of antibiotics in intravaginal sponges for estrus synchronization.

The aim of this study was to determine whether the prophylactic use of antibiotics in intravaginal sponges used for estrus synchronization in goats may result in the presence of inhibitors in milk and, therefore, of positive results by microbial screening tests. Ninety-eight Murciano-Granadina goats were used, divided into 7 groups of 14 animals. Intravaginal sponges were placed in 6 groups using 2 concentrations of 3 different antibiotics: doxycycline, oxytetracycline, and sulfathiazole-framycetin. The sponges of the control group were placed without antibiotics. Milk samples were collected daily until 7 d posttreatment and analyzed using 3 microbial tests. Positive samples were retested by specific receptor-binding assays to confirm the positive results. Vaginal status was evaluated by visual assessment of the external aspect of the sponges after removal. The microbial test response was not affected by either day posttreatment or dose of antibiotic used, except for oxytetracycline at the higher concentration. Moreover, no positive results were obtained using receptor-binding assays, suggesting that residues, if present in milk, did not exceed the regulatory (safety) levels established for these drugs. The occurrence of soiled sponges was higher in the control group. With respect to the dose of antibiotics used, no significant differences were found for the lower dose administered. However, a significant increase in the percentage of clean sponges was observed for the higher dose of doxycycline. We conclude that the prophylactic use of low doses of doxycycline, oxytetracycline, or sulfathiazole in intravaginal sponges used for synchronization of estrus helps to reduce clinical vaginitis in dairy goats and does not seem to be the cause of positive results in microbial inhibitor tests used to detect antibiotics in goat milk.

Effect In Vitro of Antiparasitic Drugs on Microbial Inhibitor Test Responses for Screening Antibiotic Residues in Goat's Milk.

Microbial inhibitor tests are widely used to screen antibiotic residues in milk; however, these tests are nonspecific and may be affected by various substances capable of inhibiting the growth of the test microorganism. The objective of this study was to determine the effect of antiparasitic drugs in goat's milk on the microbial inhibitor test response. Raw antibiotic-free milk from Murciano-Granadina goats was supplemented with eight concentrations of seven antiparasitic substances (albendazole, 10 to 170 mg/kg; closantel, 1 to 140 mg/kg; diclazuril, 8 to 45 mg/kg; febendazole, 10 to 140 mg/kg; levamisole, 40 to 440 mg/kg; diazinon, 8 to 45 mg/kg; and ivermectin, 40 to 200 mg/kg). Twelve replicates for each concentration were analyzed with three microbial inhibitor tests: BRT MRL, Delvotest SP-NT MSC, and Eclipse 100. The results were interpreted visually (negative or positive). Using a logistic regression model, the concentrations of the antiparasitic drugs producing 5% (IC5), 10% (IC10), and 50% (IC50) positive results were determined. In general, the Eclipse 100 test was less sensitive to the effect of antiparasitic substances; the inhibitory concentrations of almost all the drugs assayed were higher than those for other tests. Conversely, the BRT MRL test was most affected, with high levels of interference at lower antiparasitic drug concentrations. Closantel and diazinon interfered with all microbial tests at lower concentrations than did other drugs (IC5 = 1 to 26 and 12 to 20 mg/kg, respectively), and higher concentrations of levamisole and diclazuril (IC5 = 30 to 240 and 50 to 117 mg/kg, respectively) were required to produce 5% positive results. These findings indicate that microbial inhibitor tests can be affected by elevated concentrations of antiparasitic drugs in goat's milk.

Validation of receptor-binding assays to detect antibiotics in goat's milk.

The suitability of different receptor-binding assays to detect antibiotics in raw goat's milk was investigated. Detection capability of most ß-lactams and tetracyclines assessed applying the Betastar Combo, the SNAP Betalactam, the SNAP Tetracycline, and the Twinsensor tests was at or below maximum residue limits established by European legislation. Regarding test specificity, cross-reactions with antibiotics other than ß-lactams and tetracyclines were not found, and no false-positive results were obtained for the Betastar Combo and the SNAP tests when bulk samples of goat's milk were analyzed. For the Twinsensor test, the false-positive rate was 1%. The performance of the Betastar Combo and the SNAP tests was practically unaffected by the milk quality parameters using individual samples of goat's milk collected at points throughout the entire lactation period (false-positive rate, ≤5%). However, a larger number of positive results were obtained by the Twinsensor test in this type of milk sample (>10%), especially in the last weeks of lactation. Interferences related to the use of the preservative azidiol were not observed in any case. Neither were any significant differences found in relation to the interpretation method (visual versus instrumental) applied. In general, the response of the Betastar Combo, SNAP, and Twinsensor tests was optimal for the analysis of bulk caprine milk; thus, they may be used to monitor milk for the presence of ß-lactam and tetracycline residues in quality control programs.

Quantification of milk yield and composition changes as affected by subclinical mastitis during the current lactation in sheep.

The aim of this work was to quantify, on a half-udder basis, the changes in ewe milk yield and composition caused by unilateral subclinical mastitis within the current lactation. Fluctuations due to production level, infection severity, time from the onset of infection, and lactation curves were also studied. Yield and composition of milk from half-udders of unilateral infected ewes were compared between them and with a set of healthy halves using a mixed model. The experiment was completed with a whole-udder approach on the same animals. To test the effect of intramammary infection (IMI) in the 7 wk following the onset of infection, 20 ewes that acquired unilateral subclinical mastitis during lactation and 40 healthy ewes were used. Another group of 20 unilaterally infected ewes from wk 1 of lactation and other 40 healthy ewes were studied to test the effect of IMI on lactational milk yield and composition. The individual milk loss in ewes infected during lactation was 15% for the 7 wk following the onset of infection, and 6.6% more milk was produced by the uninfected half to compensate milk lost by the infected half. Lactational milk yield loss in ewes infected from wk 1 postpartum was 17%. The changes in milk yield were noticed from the week of infection diagnosis. The production level of animals influenced the milk yield changes caused by IMI in such a way that the more productive ewes lost more milk, although these losses were proportional to their production level. On the other hand, infection severity affected milk loss between glands, being more pronounced as somatic cell count increased. A clear decrease of lactose content and casein:protein ratio due to subclinical IMI was observed and it remained throughout the postinfection period. Improving udder health status is necessary to maintain milk production and quality in dairy ewes during lactation.

Short communication: Goat colostrum quality: litter size and lactation number effects.

The quality of colostrum of Murciano-Granadina goats was studied to establish the transition period and the time when milk can be marketed. Forty-three dairy goats were used: 19 primiparous (15 single births; 4 multiple births) and 24 multiparous (10 single births; 14 multiple births). Samples were collected every 12h during the first week postpartum. Physicochemical parameters and somatic cell count were determined. Analysis of variance with repeated measures was used to study the effect of different factors: postpartum time, litter size, lactation number, their interactions, and production level on colostrum. Postpartum time had a significant effect on all parameters studied, which decreased along the first week of lactation, whereas lactose, pH, and conductivity increased. Based on these results, colostrum secretion takes place until 36 h postpartum (hpp). In relation to other factors of variation studied, the lactation number influenced most colostrum components, whereas the litter size only affected the pH value, protein and lactose content. The production level influenced only the protein and dry matter contents, with an inverse relationship. Milk produced during the period between 36 and 96 hpp is considered transition milk, which should not be commercialized. Milk collected after 4d postpartum (96 hpp) could be marketed, ensuring that its composition does not present a risk in the dairy industry.

Evaluation of the Charm maximum residue limit ß-lactam and tetracycline test for the detection of antibiotics in ewe and goat milk.

The Charm maximum residue limit ß-lactam and tetracycline test (Charm MRL BLTET; Charm Sciences Inc., Lawrence, MA) is an immunoreceptor assay utilizing Rapid One-Step Assay lateral flow technology that detects ß-lactam or tetracycline drugs in raw commingled cow milk at or below European Union maximum residue levels (EU-MRL). The Charm MRL BLTET test procedure was recently modified (dilution in buffer and longer incubation) by the manufacturers to be used with raw ewe and goat milk. To assess the Charm MRL BLTET test for the detection of ß-lactams and tetracyclines in milk of small ruminants, an evaluation study was performed at Instituto de Ciencia y Tecnologia Animal of Universitat Politècnica de València (Spain). The test specificity and detection capability (CCß) were studied following Commission Decision 2002/657/EC. Specificity results obtained in this study were optimal for individual milk free of antimicrobials from ewes (99.2% for ß-lactams and 100% for tetracyclines) and goats (97.9% for ß-lactams and 100% for tetracyclines) along the entire lactation period regardless of whether the results were visually or instrumentally interpreted. Moreover, no positive results were obtained when a relatively high concentration of different substances belonging to antimicrobial families other than ß-lactams and tetracyclines were present in ewe and goat milk. For both types of milk, the CCß calculated was lower or equal to EU-MRL for amoxicillin (4 µg/kg), ampicillin (4 µg/kg), benzylpenicillin (≤ 2 µg/kg), dicloxacillin (30 µg/kg), oxacillin (30 µg/kg), cefacetrile (≤ 63 µg/kg), cefalonium (≤ 10 µg/kg), cefapirin (≤ 30 µg/kg), desacetylcefapirin (≤ 30 µg/kg), cefazolin (≤ 25 µg/kg), cefoperazone (≤ 25 µg/kg), cefquinome (20 µg/kg), ceftiofur (≤ 50 µg/kg), desfuroylceftiofur (≤ 50µg/kg), and cephalexin (≤ 50 µg/kg). However, this test could neither detect cloxacillin nor nafcillin at or below EU-MRL (CCß >30 µg/kg). The CCß for tetracyclines was also lower than EU-MRL for chlortetracycline (ewe milk: ≤ 50 µg/kg; goat milk: 75 µg/kg), oxytetracycline (≤ 50 µg/kg), and tetracycline (≤ 50 µg/kg). Regarding the 4-epimers of these tetracyclines only 4-epioxytetracycline was detected by the Charm MRL BLTET test below EU-MRL (ewe milk: 75 µg/kg; goat milk: ≤ 50 µg/kg). Acidiol had no effect on the performance of the test. The Charm MRL BLTET test could be used routinely with adapted test procedure for the fast screening of ewe and goat milk.

Thermodynamic analysis of the thermal stability of sulphonamides in milk using liquid chromatography tandem mass spectrometry detection.

The present study investigates the kinetics of the degradation of eight sulphonamides in skimmed milk when heated at 60, 70, 80, 90 and 100°C using an LC-MS/MS methodology. To determine the thermal stability of these compounds, the first-order kinetic model was applied and the activation energies, half-lives and degradation percentages were calculated. Application of kinetic equations to the different heat treatments used in dairy processing indicates that sulphonamides are very stable during pasteurisation (63°C; 30 min and 72°C; 15s) as well as UHT sterilisation (140°C; 4s). In contrast, the calculations performed with the kinetic model estimated losses in concentrations between 6.5% (sulfadimethoxine) and 85.1% (sulfamethazine) for the sterilisation at 120°C for 20 min. The existence of thermodynamic compensation was also tested for sulphonamide degradation. Results show that enthalpy and entropy values displayed a good linear relationship, and thermodynamically we can establish that the thermal degradation of sulphonamides in skimmed milk exhibits enthalpy-entropy compensation.

Use chemometric techniques in the optimization of a specific bioassay for betalactams in milk.

AIMS: A microbiological bioassay using Geoacillus stearothermophilus was optimized to detect betalactams at concentrations near to the Maximum Residue Limits (MRLs), with low cross-specificity for tetracycline. METHODS AND RESULTS: A factorial design (3 × 4) was used to evaluate the effects of concentration of spores (2·0 × 10(6), 4·0 × 10(6) and 8·0 × 10(6) spores ml(-1) ) and incubation time (3·0, 3·5, 4·0 and 4·5 h) on the response of the bioassay. Then, desirability function to raise the detection capabilities (CC(ß) ) of tetracyclines and increase sensitivity to betalactams was implemented. Significant effects of Log[S] and incubation time [It] on the CC(ß) of betalactams and tetracyclines were observed. Finally, high value of global desirability (D = 0·853), adequate betalactams CC(ß) (3·8 µg l(-1) of penicillin 'G', 27 µg l(-1) of oxacillin, 8·1 µg l(-1) of ampicillin, 48 µg l(-1) of cloxacillin) and high tetracyclines CC(ß) (5260 µg l(-1) chlortetracycline, 1550 µg l(-1) of oxytetracycline, 1070 µg l(-1) of tetracycline) were calculated. CONCLUSIONS: The application of chemometric tools allows the optimization of a bioassay that detects betalactam residues in milk. The more robust conditions have been achieved in Log[S] = 6·30 and [It] = 4·20 h. SIGNIFICANCE AND IMPACT OF THE STUDY: The logistic regression model and the desirability function are adequate chemometric techniques to improve the properties of the methods, because it is possible to increase sensitivity and decrease cross-specificity simultaneously.

Short communication: Effect of subclinical mastitis on proteolysis in ovine milk.

The aim of this work was to evaluate the effect of intramammary infection (IMI) on the endogenous proteolysis of milk. Four control checks were carried out in the half-udder milk of 10 ewes that acquired unilateral subclinical mastitis. Two of these checks were conducted before the infection was established and 2 after. Ten healthy ewes were tested as a control group. The presence of a subclinical IMI involved an increase of the products of casein hydrolysis, the proteose-peptone (p-p) fraction and minor (m) caseins, and a decrease of ß-casein. As a result, a significant increase in the proteolysis index (PI), calculated as the ratio of m-casein to the sum of caseins (α + ß + κ), took place. α-Casein and κ-casein were not significantly affected by IMI. Correlations confirmed the scenario: log(10) of somatic cell count (SCC) was positively correlated with p-p content and negatively with ß-casein, whereas log(10) SCC was not correlated with α-casein or κ-casein. On the other hand, p-p content was positively correlated with m-casein and PI and negatively with ß-casein, but no correlation was detected between p-p content and α- or κ-casein. Furthermore, between casein fractions, m-casein was only significantly correlated with ß-casein. These results suggest that use of indices of proteolysis of caseins such as p-p, m-casein, and PI, could be applied together with SCC to evaluate the cheese-making quality of milk.

Short communication: Occurrence of aflatoxin M1 in the Manchego cheese supply chain.

The importance of ewe milk lies in the production of high quality cheeses, such as Manchego cheese with a Protected Designation of Origin, whose safety must be guaranteed. In a 2-yr study, 407 bulk tank milk samples from farms and 82 silo milk and curd samples from cheese factories were collected from southeast Spain and tested for aflatoxin M1 (AFM1) using 2 commercial ELISA tests. Of these, 99.3% of the bulk tank samples had AFM1 levels below the European Union (EU) legal limit for milk (50 ng/kg), and well below the limit adopted by the Codex Alimentarius (500 ng/kg). Moreover, 98.8% of the silo milk and curd samples from cheese factories had AFM1 levels below the EU limit for milk. When considering median AFM1 concentrations, an average 4-fold increase was found in the final curd in relation to the corresponding silo milk. Control of AFM1 in Manchega ewe milk would enhance dairy product safety by the possible detection of faults in the manufacture of Manchego cheese.

Heat treatment effects on the antimicrobial activity of macrolide and lincosamide antibiotics in milk.

Antibiotic residues in milk can cause serious problems for consumers and the dairy industry. Heat treatment of milk may diminish the antimicrobial activity of these antibiotic residues. This study analyzed the effect of milk processing (60 °C for 30 min, 120 °C for 20 min, and 140 °C for 10 s) on the antimicrobial activity of milk samples fortified with three concentrations of three macrolides (erythromycin: 20, 40 and 80 µg/liter; spiramycin: 100, 200, and 400 µg/liter; and tylosin: 500, 1,000, and 2,000 µg/liter) and one lincosamide (lincomycin: 1,000, 2,000, and 4,000 µg/liter). To measure the loss of antimicrobial activity, a bioassay based on the growth inhibition of Micrococcus luteus was done. The data were analyzed using a multiple linear regression model. The results indicate that treatment at 120 °C for 20 min produces inactivation percentages of 93% (erythromycin), 64% (spiramycin), 51% (tylosin), and 5% (lincomycin), while treatment at 140 °C for 10 s results in generally lower percentages (30% erythromycin, 35% spiramycin, 12% tylosin, and 5% lincomycin). The lowest loss or lowest reduction of antimicrobial activity (21% erythromycin and 13% spiramycin) was obtained by treatment at 60 °C for 30 min.

Effect of heat treatments on stability of ß-lactams in milk.

The presence of residues of antimicrobial substances in milk may have serious toxicological and technical consequences. To date, few studies have been done to evaluate the effect of heat treatments on ß-lactam residues in milk. However, the few studies that have been conducted estimate losses of antimicrobial activity under different combinations of temperature and time using microbiological methods. The aims of this study were to calculate the kinetic parameters for the degradation of ß-lactam antibiotics in milk and to develop prediction models to estimate the concentration losses of these compounds in conventional dairy heat treatments. To do so, we employed a quantitative HPLC method to calculate losses in concentrations of 10 ß-lactam antibiotics in milk with different combinations of temperature and time. Increasing the temperature from 60°C to 100°C decreased the half-life of amoxicillin (372 to 50 min), ampicillin (741 to 26 min), cloxacillin (367 to 46 min), and penicillin G (382 to 43 min). These increases in temperature caused further degradation in cephalosporins, which was accompanied by a decrease in half-life times to reach very low values; for instance, 4, 5, and 6 min for cefoperazone, cephurexime, and cephapirin, respectively. Kinetic equations were applied to different heat treatments used in dairy processing. Heat treatments at high temperatures and long times (e.g., 120°C for 20 min) led to a further degradation of ß-lactam antibiotics with percentages close to 100% for cefoperazone and cefuroxime. In contrast, when milk was subjected to heat treatments at lower temperatures and times (e.g., 72°C for 15s), the degradation of ß-lactam in milk did not exceed 1% for the 10 antibiotics tested.

Optimization of bioassay for tetracycline detection in milk by means of chemometric techniques.

AIMS: In this study, a microbiological method of dichotomous response using Bacillus cereus was designed and optimized to detect tetracyclines (TCs) at concentrations near to the maximum residue limits (MRLs). METHODS AND RESULTS: In a first stage, the response time of bioassay was reduced to 5 h when the logarithm of spore concentration (log S) was increased. Later, a Plackett Burman design (2(6-3)) was analysed using logistic regression model. This design indicates significant effects of log S and chloramphenicol (CAP) on the detection limit (DL) of TC. Then, the response surfaces (RS) of the TCs DTs as a function of log S and CAP were plotted using a Dohlert design and the logistic regression model. These RS show a linear decrease with the raise of CAP and a quadratic effect of log S. Finally, the DTs of TC (109 µg l(-1)) and oxytetracycline (100 µg l(-1)) were adjusted to their MRLs through the desirability function. CONCLUSIONS: By successive application of experimental design techniques could be optimized a bioassay for the detection of TC residues in milk. The best conditions have been achieved when the assay was made with log S = 5·12 and CAP = 470 µg l(-1). SIGNIFICANCE AND IMPACT OF THE STUDY: Experimental design techniques together with the logistic regression model and the desirability function represent an adequate tool for the optimization of a bioassay with binary response.