Serum metabolome differences associated with subclinical intramammary infection caused by Streptococcus agalactiae and Prototheca spp. in multiparous dairy cows.

Mastitis is one of the most significant diseases in dairy cows and causes several economic losses. Somatic cell count (SCC) is often used as an indirect diagnostic tool for mastitis, especially for subclinical mastitis (SCM) where no symptoms or signs can be detected. Streptococcus agalactiae is one of the main causes of contagious mastitis, and Prototheca spp. is an alga-inducing environmental mastitis that is not always correlated with increased milk SCC. The aim of this study was to evaluate the changes in the metabolomic profile of blood in relation to subclinical intramammary infection (IMI) in dairy cows. In addition, differences resulting from the etiologic agent causing mastitis were also considered. Forty Holstein-Friesian dairy cows in mid and late lactation were enrolled in this cross-sectional design study. Based on the bacteriological examination of milk, the animals were divided into 3 groups: group CTR (control group; n = 16), group A (affected by SCM with IMI caused by Strep. agalactiae; n = 17), and group P (affected by SCM with IMI caused by Prototheca spp.; n = 7). Blood samples from the jugular vein were collected in tubes containing clot activator; the serum aliquot was stored until metabolomic analysis by 1H-nuclear magnetic resonance spectroscopy. Statistical analysis was conducted by fitting a linear model with the group as the fixed effect and SCC as the covariate. Forty-two metabolites were identified, and among them 10 were significantly different among groups. Groups A and P showed greater levels of His and lactose and lower levels of acetate, Asn, and dimethylamine compared with group CTR. Group A showed high levels of Val, and group P showed high levels of Cit and methylguanidine, as well as lower levels of 3-hydroxybutyrate, acetone, allantoin, carnitine, citrate, and ethanol. These metabolites were related to ruminal fermentations, energy metabolism, urea synthesis and metabolism, immune and inflammatory response, and mammary gland permeability. These results suggest systemic involvement with subclinical IMI and that the metabolic profile of animals with SCM undergoes changes related to the etiological agent of mastitis.

Quarter-level analyses of the associations among subclinical intramammary infection and milk quality, udder health, and cheesemaking traits in Holstein cows.

In this study, we investigated associations among subclinical intra-mammary infection (IMI) and quarter-level milk composition, udder health indicators, and cheesemaking traits. The dataset included records from 450 Holstein cows belonging to three dairy herds. After an initial screening (T0) to identify animals infected by Streptococcus agalactiae, Streptococcus uberis, Staphylococcus aureus, and Prototheca spp., 613 quarter milk samples for 2 different sampling times (T1 and T2, 1 mo after T1) were used for analysis. Milk traits were analyzed using a hierarchical linear mixed model including the effects of days in milk, parity and herd, and bacteriological and inflammatory category [culture negative with somatic cell count (SCC) <200,000 cells/mL; culture negative with SCC ≥200,000 cells/mL; or culture positive]. All udder health indicators were associated with increased SCC and IMI at both sampling times. The largest effects were detected at T2 for milk lactose (-7% and -5%) and milk conductivity (+9% and +8%). In contrast, the increase in differential SCC (DSCC) in samples with elevated SCC was larger at T1 (+17%). Culture-negative samples with SCC ≥200,000 cells/mL had the highest SCC and greatest numbers of polymorphonuclear-neutrophils-lymphocytes and macrophages at both T1 and T2. Regarding milk cheesemaking ability, samples with elevated SCC showed the worst pattern of curd firmness at T1 and T2. At T2, increased SCC and IMI induced large decreases in recoveries of nutrients into the curd, in particular recovered protein (-14% and -16%) and recovered fat (-12% and -14%). Different behaviors were observed between Strep. agalactiae and Prototheca spp., especially at T2. In particular, samples that were positive for Strep. agalactiae had higher proportions of DSCC (+19%) compared with negative samples with low SCC, whereas samples that were positive for Prototheca spp. had lower DSCC (-11%). Intramammary infection with Prototheca spp. increased milk pH compared with culture-negative samples (+3%) and negative samples that had increased SCC (+2%). The greatest impairment in curd firmness at 30 min from rennet addition was observed for samples that were positive for Prototheca spp. (-99% compared with negative samples, and -98% compared with negative samples with high SCC). These results suggest that IMI caused by Prototheca spp. have detrimental effects on milk technological traits that deserve further investigation of the mechanisms underlying animals' responses to infection.

Different distribution of antimicrobial resistance genes and virulence profiles of Staphylococcus aureus strains isolated from clinical mastitis in six countries.

Staphylococcus aureus is recognized worldwide as one of the main contagious mastitis agents in cattle and can express a set of antimicrobial resistance genes and virulence-associated genes that explain the wide range of outcomes of intramammary infections. Staphylococcus aureus strains are heterogeneous: their different resistance and virulence patterns, associated with host-level factors and treatment factors, are related to the severity of infection. The aim of this study was to determine phenotypic antibiotic susceptibility, occurrence of selected antimicrobial resistance genes and other virulence genes in 93 S. aureus strains isolated from clinical mastitis in 6 countries: Argentina, Brazil, Germany, Italy, the United States (New York State), and South Africa. These isolates were tested against a total of 16 drugs (amoxicillin-clavulanate, ampicillin, cefazolin, cefoperazone, cefquinome, enrofloxacin, erythromycin, gentamicin, kanamycin, lincomycin, oxacillin, penicillin, rifampin, spiramycin, sulfamethoxazole/trimethoprim, tylosin) by minimum inhibitory concentration (MIC) assay, and examined for the presence of 6 antibiotic-resistance genes (blaZ, mecA, mecC, ermA, ermB, ermC) and 6 virulence-associated genes (scn, chp, sak, hla, hlb, sea) via PCR analysis. The phenotypic results of this study revealed the presence of 19.4% penicillin-resistant strains, whereas 22.6% of the strains were classified as having resistance (5.4%) or intermediate resistance (17.2%) to erythromycin. Most (96.8%) of the isolates were inhibited by cephalosporins, and all were susceptible to amoxicillin-clavulanate. Two strains (1 from Germany, 1 from Italy) were resistant to oxacillin and were positive for mecA. Among the other antimicrobial resistance genes, the most frequently detected was blaZ (46.2%), and 32.3% of the isolates were positive for erm genes: ermC (21.5%) and ermB (10.8%). The most prevalent virulence gene was hla (100%), followed by hlb (84.9%) and sea (65.6%). These results show a low prevalence of antibiotic multidrug resistance in S. aureus isolates, even if the detection of selected antimicrobial resistance genes did not always correspond with the occurrence of phenotypic antibiotic resistance; the immune evasion cluster gene prevalence was quite low in the samples analyzed.

Transfer of passive immunity in dairy calves: the effectiveness of providing a supplementary colostrum meal in addition to nursing from the dam.

Failed transfer of passive immunity (FTPI) in dairy calves - which is often due to the low amount of colostrum provided within a few hours after birth - remains a crucial issue. Enabling dairy calves to nurse colostrum from their dams could be useful in increasing intake and thus avoiding FTPI, but further potential effects on the health and welfare of both calves and dams should also be considered. In this study, 107 calf-dam pairs from two Italian dairy farms were alternately assigned to one of the following colostrum provision methods (CPMs): 'hand-fed method' (HFM) - the calf was separated from the dam immediately after birth and colostrum was provided by nipple-bottle (n = 50); 'nursing method' (NM) - the calf nursed colostrum from the dam for the first 12 h of life without farmer assistance (n = 30); and 'mixed method' (MM) - the nursing calf received a supplementary colostrum meal by nipple-bottle (n = 27). Serum of calves (1 to 5 days of age) and samples of their first colostrum meal were analysed by electrophoresis to assess immunoglobulin (Ig) concentration. Additionally, behavioural indicators of separation distress (calf and dam vocalisations; calf refusal of the first meal after separation; undesirable dam behaviour at milking) in the following 24 h were recorded as binary variables (Yes/No), and the health status of calves (disease occurrence and mortality) and dams (postpartum disorders and mastitis occurrence) were monitored for the first 3 months of life and 7 days after parturition, respectively. The lowest FTPI occurrence (calf serum Ig concentration <10.0 g/l) was found in the MM (11.1%) and the HFM (22.0%) compared with the NM (60.0%) (P<0.05), and the highest percentage of calves with optimal transfer of passive immunity (serum Ig concentration ≥16.0 g/l) was observed in the MM (55.6%). The lowest calf-dam separation distress was observed in the HFM (P<0.05). The highest calf disease occurrence was recorded in the HFM (64.0%) and the lowest in the NM (33.3%), with an intermediate value for the MM (44.4%) (P<0.05). No effect of the CPM was observed on dam health or calf mortality (P>0.05). The results of this study indicated that providing calves with a supplementary colostrum meal in addition to nursing from the dam (MM) is truly effective in maximizing passive immunity transfer. Anyway, specific strategies should be studied to minimise calf-dam separation distress.

Factors associated with passive immunity transfer in dairy calves: combined effect of delivery time, amount and quality of the first colostrum meal.

Despite the well-known importance of an adequate colostral immunoglobulin (Ig) transfer to calf health and survival, failed transfer of passive immunity (FTPI) remains a widespread problem in dairy farming. The aim of this study was to investigate the management factors associated with FTPI in newborn calves, evaluating particularly the combined effect of delivery time, amount and quality of the first colostrum meal. The study was conducted from March to August 2014 on 21 Italian dairy farms. Farmers were asked as first to answer a farm-level questionnaire on calf management. Blood sampling was then performed on overall 244 calves (1 to 5 days of age) born from Holstein cows, and a sample of the first colostrum meal of each calf was collected. Individual information on calves and the respective colostrum management were recorded. Serum and colostrum Ig concentrations were assessed by electrophoresis. A mixed effects multivariable logistic regression model was used to investigate the association of the variables obtained from both the management questionnaire and the individual calf data with FTPI (calf serum Ig concentration 87.6 g/l) within 1.0 h from birth. Considerable improvements are still needed about colostrum management for newborn calves in dairy farms. The results of this study will help in developing farm-specific programs for reducing the occurrence of FTPI.

Occurrence of methicillin-resistant Staphylococcus aureus in dairy cattle herds, related swine farms, and humans in contact with herds.

In this study we investigated the circulation of methicillin-resistant Staphylococcus aureus (MRSA) in 2 dairy cattle farms (farm A and B), previously identified as MRSA-positive in bulk tank milk samples, and epidemiologically related to swine farms. Collected specimens included quarter milk samples and nasal swabs from dairy cows, pig nasal swabs collected at both the farm and slaughterhouse level, environmental dust samples, and human nasal swabs from the farms' owners and workers. The prevalence of MRSA was estimated at the herd level by testing quarter milk samples. The prevalence of MRSA was 4.8% (3/63; 95% confidence interval=0-10.2%) and 60% (33/55; 95% confidence interval=47.05-72.95) in farm A and B, respectively. In farm A, MRSA was also isolated from humans, pigs sampled at both farm and slaughterhouse level, and from environmental samples collected at the pig facilities. The dairy cattle facilities of farm A tested negative for MRSA. In farm B, MRSA was isolated from environmental dust samples in both the cattle and pig facilities, whereas nasal swabs collected from cows and from humans tested negative. Sixty-three selected MRSA isolates obtained from different sources in farm A and B were genetically characterized by multilocus sequence typing, spa-typing, ribosomal spacer-PCR, and also tested for the presence of specific virulence genes and for their phenotypical antimicrobial susceptibility by broth microdilution method. Different clonal complex (CC) and spa-types were identified, including CC398, CC97, and CC1, CC already reported in livestock animals in Italy. The MRSA isolates from quarter milk of farm A and B mostly belonged to CC97 and CC398, respectively. Both lineages were also identified in humans in farm A. The CC97 and CC398 quarter milk isolates were also identified as genotype GTBE and GTAF by ribosomal spacer-PCR respectively, belonging to distinct clusters with specific virulence and resistance patterns. The GTBE and GTAF clusters also included swine, environmental, and human isolates from both farms. A high heterogeneity in the genetic and phenotypic profiles was observed in environmental isolates, in particular from farm B. These results demonstrate the possibility of a dynamic sharing and exchange of MRSA lineages or genotypes between different species and farm compartments in mixed-species farms. The risk of transmission between swine and related dairy cattle herds should be considered. Our findings also confirm the zoonotic potential of livestock-associated MRSA and underline the importance of applying biosecurity measures and good hygiene practices to prevent MRSA spread at the farm level and throughout the food production chain.

Randomized noninferiority field trial comparing 2 first-generation cephalosporin products at dry off in quarters receiving an internal teat sealant in dairy cows.

The study objective was to compare 2 commercial dry cow mastitis products at the quarter level, with concurrent internal teat sealant application, evaluating the cure risk difference, odds of a cure, odds of a new intramammary infection (NIMI) during the dry period, and risk for a clinical mastitis (CM) case between calving and 60d in milk (DIM). A total of 590 cows (2,360 quarters) from 8 commercial dairy herds in Italy were enrolled and randomized to 1 of the 2 treatments at dry off: Cefovet A (CF; 250mg of cephazoline; Merial Italia SpA, Milan, Italy), and Cepravin (CP; 250mg of cephalonium dehydrate MSD Animal Health Srl, Segrate, Italy). Quarter milk samples were collected before dry cow therapy treatment at dry off, 2 to 9 DIM, and 10 to 17 DIM. Quarter milk samples from CM cases were collected during the first 60 DIM. Noninferiority analysis was used to evaluate the effect of treatment on the risk difference of a bacteriological cure during the dry period, the primary outcome. The odds of cure, developing a NIMI during the dry period, and the risk of a CM event within 60 DIM were evaluated with multivariable logistic regression and hazard analysis, respectively. The overall crude quarter-level prevalence of NIMI at dry off was 15.3%. The most common pathogen isolated from milk samples at dry-off was coagulase-negative staphylococci. Noninferiority analysis showed no effect of treatment on the risk difference for a cure between dry off and both postpartum samples, difference was 0.013. The least squares means from the multivariable model evaluating the odds of cure was 94% for CF and 95%for CP. We observed no effect of treatment on the odds for the presence of a NIMI at 2 to 9 DIM (least squares means: CF=0.09 and CP=0.07), nor did we note a difference in risk of experiencing a CM event between calving and 60 DIM (hazard ratio=0.8). In conclusion, no difference was observed between the 2 products evaluated when assessing the aforementioned outcomes in quarters also receiving an internal teat sealant.

Short communication: In vitro antimicrobial susceptibility of Mycoplasma bovis isolates identified in milk from dairy cattle in Belgium, Germany, and Italy.

The objective of this study was to assess the in vitro antimicrobial susceptibility of 73 isolates of Mycoplasma bovis isolated from milk of dairy cattle herds of Belgium, Germany, and Italy. Minimal inhibitory concentration (MIC) values were determined by the microbroth dilution method for the following antimicrobials: erythromycin, spiramycin, tilmicosin, tylosin, lincomycin, enrofloxacin, doxycycline, oxytetracycline, florfenicol, and tiamulin. Macrolides, florfenicol, oxytetracycline, and enrofloxacin, were chosen because they represent antimicrobials families commonly used in several countries for treatment of M. bovis, and their MIC values in cattle population are reported in several studies, allowing a comparison with previous data. Doxycycline and tiamulin were selected to assess the susceptibility of M. bovis to new antimicrobials, because they are not registered in the European Union for the treatment of dairy cattle. Among the agents of the different antimicrobial classes, the macrolides showed the highest concentration to inhibit 90% of isolates (MIC90), all above the highest concentration tested: >8µg/mL for erythromycin, >16µg/mL for spiramycin, and >32µg/mL for tilmicosin and tylosin. Also the MIC90 of lincomycin was above the highest concentration tested (>32µg/mL), but the distribution of the MIC values was almost perfectly bimodal: 41 isolates had a MIC ≤0.5µg/mL and 30 isolates >32µg/mL. Oxytetracycline had a 2-fold higher concentration to inhibit 50% of isolates (2 vs. 0.5µg/mL) and 1-fold higher MIC90 (4 vs. 2µg/mL) than doxycycline. Enrofloxacin and florfenicol had both a MIC90 of 2µg/mL, whereas tiamulin had a MIC90 of 0.5µg/mL. Significant differences on the MIC values were found among the 3 countries for several antimicrobials: compared with Germany, Belgium and Italy showed significantly higher MIC for lincomycin, spiramycin, and tylosin, and lower for oxytetracycline and florfenicol. The Belgian isolates showed the lowest MIC for enrofloxacin compared with Germany and Italy. The MIC results obtained in our study suggest the presence of a high level of resistance of M. bovis isolates originating from milk to macrolides in all countries involved in this study. On the contrary, a low level of resistance was found against the antimicrobials that are not used in cattle, such as tiamulin and doxycycline, highlighting a possible link between antimicrobial treatments and development of resistance in the studied M. bovis population.

Short communication: Methicillin-resistant Staphylococcus aureus in bulk tank milk of dairy cows and effect of swine population density.

The methicillin-resistant Staphylococcus aureus (MRSA) has recently frequently been reported in dairy cattle, usually with low prevalence. The livestock-associated MRSA (LA-MRSA) ST398 is especially involved in cases of subclinical and clinical mastitis. Swine carry LA-MRSA without clinical symptoms and are considered its reservoir and shedder. People exposed to swine are particularly at risk of LA-MRSA colonization. Environments with relevant livestock density are a demonstrated risk factor for humans to be carriers of a LA-MRSA. This work investigated dairy farms located in an area with a high livestock density, mainly represented by swine. Bulk tank milk samples from 224 dairy farms were collected, and their status was defined as MRSA-positive or MRSA-negative based on culture on chromogenic medium. The number of fattening swine and of fattening swine herds was calculated in an area of 3 km around each dairy farm through georeferencing. The probability of a Staphylococcus aureus-positive dairy farm to be MRSA positive based on the extent of potential infective pressure due to swine density was calculated. Both the number of swine herds and the number of swine were associated with the MRSA status of dairy herds. The 9 MRSA isolated were typed by multi-locus sequence typing and spa-typing, and characterized for their virulence factors and antimicrobial resistance profiles. The ST and spa-types detected are consistent with those present in the Italian swine population. Virulence and resistance profiles are mostly consistent with the types detected. This work provides the first evidence of the epidemiological challenge exerted by the density of the swine population on MRSA in dairy cows.

Antimicrobial susceptibilities and random amplified polymorphic DNA-PCR fingerprint characterization of Lactococcus lactis ssp. lactis and Lactococcus garvieae isolated from bovine intramammary infections.

In total, 181 streptococci-like bacteria isolated from intramammary infections (IMI) were submitted by a veterinary clinic to Quality Milk Production Services (QMPS, Cornell University, Ithaca, NY). The isolates were characterized by sequence analysis, and 46 Lactococcus lactis ssp. lactis and 47 Lactococcus garvieae were tested for susceptibility to 17 antibiotics. No resistant strains were found for ß-lactam antibiotics widely used in clinical practice (penicillin, ampicillin, and amoxicillin), and all minimum inhibitory concentrations (MIC) were far from the resistance breakpoints. Eight strains had MIC intermediate to cefazolin. The random amplification of polymorphic DNA (RAPD)-PCR fingerprint patterns showed a slightly higher heterogeneity for Lc. lactis ssp. lactis isolates than for Lc. garvieae isolates.

Effect of feeding fine maize particles on the reticular pH, milk yield and composition of dairy cows.

The particle size of cereal grains has been found to modulate the rate of passage from the rumen and the digestibility of starch and neutral detergent fibre (NDF), but few studies have examined its impact on reticular pH. The study aimed to evaluate the effect of feeding finely ground maize on the risk of ruminal acidosis, milk yield and composition. Twelve Holstein-Friesian cows were assigned to one of two experimental groups and fed according to a cross-over design. Diets were isoenergetic and isonitrogenous and were characterised by the same NDF and ADF, differing only in maize particle size. In the control diet (Ct), the maize meal was ground to 1.0 mm, whereas in the experimental diet, it was finely ground (Fg) to 0.5 mm. The pH and temperature of the reticulum were continuously measured in eight cows throughout the trial using indwelling sensors. Dry matter intake was higher in cows offered Fg diet than in Ct (19.0 vs. 20.3 kg/day; p = 0.067). However, milk yield (p = 0.855) and the 3.5% fat-corrected milk (FCM) (p = 0.724) did not show any differences between the diets. Casein (2.48 vs. 2.57%; p = 0.035) and crude protein (CP) (3.18 vs. 3.31%; p = 0.021) resulted higher in Fg. Similarly, starch digestibility increased in animals offered Fg diet versus Ct (0.94 vs. 0.98; p = 0.078). Among the reticular parameters, the Fg-fed cows spent a significantly higher time below the 5.5 pH threshold (15 vs. 61 min/day; p = 0.047) and had an average daily variation in reticular pH characterised by a lower nadir pH (5.95 vs. 5.72; p < 0.001) and a higher pH range (0.79 vs. 0.94; p = 0.003). In this study, grain particle size affected the risk of the onset of ruminal acidosis. Therefore, it should be carefully considered when formulating rations.

Effect on quarter milk somatic cell count and antimicrobial susceptibility of Staphylococcus rostri causing intramammary infection in dairy water buffaloes.

In many parts of the world, coagulase-negative staphylococci (CNS) are the predominant cause of intramammary infections (IMI) in dairy cows and in water buffaloes, as well. A longitudinal field study was carried out on one well-managed dairy water buffalo herd to determine the prevalence and distribution of CNS and a recently described CNS-species, Staphylococcus rostri, in milk samples to explore its relevance for buffaloes' udder health throughout lactation, and to gain insight into the susceptibility of the latter species toward commonly used antimicrobials. Twice weekly quarter milk samples from a cohort of 11 lactating water buffaloes were collected over an 8-mo period. The CNS (n=109; 76.2% of all culture-positive samples) were the predominant pathogens causing IMI, followed by Corynebacterium bovis (n=11; 7.6%) and Streptococcus spp. (n=9; 6.2%) other than Stretococcus uberis (n=2; 1.4%). Thirty-seven hemolytic staphylococci suspected to be Staphylococcus aureus were further differentiated using transfer DNA-intergenic spacer-PCR and rpoB-gene sequencing because they were coagulase-negative. Thirty-three of those isolates were identified as Staph. rostri, whereas 2 others were identified as Staphylococcus epidermidis. None of the Staph. rostri isolates displayed resistance to the antimicrobial agents tested. Mean quarter milk somatic cell count (qSCC) of all samples collected throughout lactation was 20,970 cells/mL. The qSCC at sampling of quarters infected with Staph. rostri (34,466 cells/mL) and CNS other than Staph. rostri (34,813 cells/mL) were significantly higher than the qSCC of noninfected quarters (20,287 cells/mL), yet not significantly different from each other. These findings provide novel insight into the prevalence and distribution, antimicrobial susceptibility, and relevance of Staph. rostri compared with other CNS species causing IMI in water buffaloes. Further studies are needed to pinpoint the relevance, niches, and transmission routes of Staph. rostri, as well as other CNS in water buffaloes.

Old and new diagnostic approaches for Q fever diagnosis: correlation among serological (CFT, ELISA) and molecular analyses.

The objective of this study was to evaluate the performance of the complement fixation test (CFT) with respect to ELISA for the serological diagnosis of Q fever and to assess the role of serology as a tool for the identification of the shedder status. During 2009-2010, sera from 9635 bovines and 3872 small ruminants (3057 goats and 815 sheep) were collected and analyzed with CFT and ELISA. In addition, 2256 bovine, 139 caprine and 72 ovine samples (individual and bulk tank milk samples, fetuses, vaginal swabs and placentae) were analyzed with a real-time PCR kit. The relative sensitivity (Se) and specificity (Sp) of CFT with respect to ELISA were Se 26.56% and Sp 99.71% for cattle and Se 9.96% and Sp 99.94% for small ruminants. To evaluate the correlation between serum-positive status and shedder status, the ELISA, CFT and real-time PCR results were compared. Due to the sampling method and the data storage system, the analysis of individual associations between the serological and molecular tests was possible only for some of the bovine samples. From a statistical point of view, no agreement was observed between the serological and molecular results obtained for fetus and vaginal swab samples. Slightly better agreement was observed between the serological and molecular results obtained for the individual milk samples and between the serological (at least one positive in the examined group) and molecular results for the bulk tank milk (BTM) samples. The CFT results exhibited a better correlation with the shedder status than did the ELISA results.

Standardization of bulb and root sample sizes for the Allium cepa test.

Although the Allium cepa test has been widely used to identify potentially cytotoxic and genotoxic pollutants in aquatic environments, variable non-standardized choices have been made regarding the number of plant bulbs and roots analyzed. We propose numbers for bulbs and roots per bulb when comparing the frequencies of micronuclei, mitotic anomalies and mitotic index with this test. Roots that had been treated with aqueous solutions, such as water samples collected in August 2007 from the Paraíba do Sul River at the Brazilian cities of Tremembé and Aparecida; negative and positive controls were used for bioassays. The presence of pollutants in the river water had been presumed based on our previous cytological data and an official report by the São Paulo State Environmental Agency (Brazil) on presence of fecal contaminants (Tremembé and Aparecida) and elevated dissolved aluminium (Aparecida) in the water under study. The sampling of ten bulbs and five roots per bulb was found adequate for comparative studies to evaluate with the A. cepa test the potential damage inflicted by pollutants in aquatic environments. Furthermore, even one bulb and one root per bulb was sufficient in discerning this damage, thereby shortening the time required to attain a statistically confident comparative evaluation. However, to allow for the use of statistical programs based on the evaluation of average values, and to avoid criticism based on genetic variability, we propose that three bulbs and three roots per bulb be considered as standard sample sizes for the A. cepa test.

Evaluation of the cytotoxic and genotoxic potential of water from the River Paraíba do Sul, in Brazil, with the Allium cepa L. test.

This work investigated the cytotoxic and genotoxic potential of water from the River Paraíba do Sul (Brazil) using Allium cepa roots. An anatomo-morphological parameter (root length), mitotic indices, and frequency of micronuclei were analysed. Eight bulbs were chosen at random for treatment for 24 to 120 hours with the River water collected in the years of 2005 and 2006 from sites in the cities of Tremembé and Aparecida (São Paulo state, Brazil). Daily measurements of the length of the roots grown from each bulb were carried out throughout the experiment. Mitotic index (MI) and frequency of micronuclei (MN) were determined for 2000 cells per root, using 3-5 root tips from other bulbs (7-10). Only in the roots treated with samples of the River water collected in 2005 in Tremembé city was there a decrease in the root length growth compared to the respective control. However, a reduction in MI values was verified for both sites analysed for that year. Considering the data involving root length growth and especially MI values, a cytotoxic potential is suggested for the water of the River Paraíba do Sul at Tremembé and Aparecida, in the year of 2005. On the other hand, since in this year the MN frequency was not affected with the river water treatments, genotoxicity is not assumed for the river water sampled at the aforementioned places.

Evaluation of the cytotoxic and genotoxic potential of water from the River Paraíba do Sul, in Brazil, with the Allium cepa L. test / Avaliação do potencial citotóxico e genotóxico da água do Rio Paraíba do Sul através do teste Allium cepa

This work investigated the cytotoxic and genotoxic potential of water from the River Paraíba do Sul (Brazil) using Allium cepa roots. An anatomo-morphological parameter (root length), mitotic indices, and frequency of micronuclei were analysed. Eight bulbs were chosen at random for treatment for 24 to 120 hours with the River water collected in the years of 2005 and 2006 from sites in the cities of Tremembé and Aparecida (São Paulo state, Brazil). Daily measurements of the length of the roots grown from each bulb were carried out throughout the experiment. Mitotic index (MI) and frequency of micronuclei (MN) were determined for 2000 cells per root, using 3-5 root tips from other bulbs (7-10). Only in the roots treated with samples of the River water collected in 2005 in Tremembé city was there a decrease in the root length growth compared to the respective control. However, a reduction in MI values was verified for both sites analysed for that year. Considering the data involving root length growth and especially MI values, a cytotoxic potential is suggested for the water of the River Paraíba do Sul at Tremembé and Aparecida, in the year of 2005. On the other hand, since in this year the MN frequency was not affected with the river water treatments, genotoxicity is not assumed for the river water sampled at the aforementioned places.

Este trabalho é parte de uma investigação sobre o potencial citotóxico e genotóxico da água do Rio Paraíba do Sul (Brasil) utilizando raízes de Allium cepa. Foi analisado um parâmetro anátomo-morfológico (crescimento das raízes), bem como o índice mitótico e a frequência de micronúcleos. Aleatoriamente oito bulbos foram submetidos aos tratamentos de 24 a 120 horas com água do Rio proveniente dos pontos de coleta das cidades de Tremembé e Aparecida nos anos de 2005 e 2006. O comprimento das raízes de cada bulbo foi acompanhado diariamente ao longo do experimento. O índice mitótico (IM) e a frequência de micronúcleos (MN) foram determinados pela análise de 2.000 células por raiz, sendo utilizadas 3-5 raízes de outros bulbos (7-10). Somente as raízes tratadas com amostras de água coletadas em 2005 na cidade de Tremembé, apresentaram decréscimo no comprimento das raízes quando comparadas com o controle. Entretanto, foi observada redução do IM nas raízes tratadas com água de ambos os pontos de coleta no mesmo ano. Considerando os dados de crescimento de raiz e especialmente IM, um potencial citotóxico é sugerido para a água do Rio Paraíba do Sul em Tremembé e Aparecida, no ano de 2005. Por outro lado, para este mesmo ano, a frequência de micronúcleos não foi alterada; assim, a genotoxicidade não foi assumida para a água do Rio nos pontos mencionados.

Idiopathic chronic fatigue and chronic fatigue syndrome: a comparison of two case-definitions.

The aim of the study was to compare the signs and symptoms of individuals meeting two different definitions of chronic fatigue syndrome (CFS). Ninety-four patients fitting the eligibility criteria for idiopathic fatigue were enrolled into the study. Of the 94 patients, 48 met the 1988 definition of CFS, 20 the 1994 (but not the 1988) definition of CFS, and 26 met neither definition. The 1994 defined cases were more likely than 1988 defined cases, and non-syndromal individuals to be male, married, and high school educated. The 1994 cases were less likely than 1988 cases to present acute onset, self reported sore throat, mild fever lymphadenopathy, pharyngitis. In conclusion, the 1994 criteria increased the number of patients classified as CFS; however, those who fit only the 1994 criteria were less likely to have an acute symptomatic onset and signs and symptoms suggestive of an infectious process.

[Auditory brain stem evoked potentials in the evaluation of chronic fatigue syndrome]. / I potenziali evocati uditivi troncoencefalici nella valutazione della sindrome da fatica cronica.

The Chronic Fatigue Syndrome (CFS) was formally defined to describe disabling fatigue of multifactorial ethology with depression and immunologic dysfunctions linked to some currently recognized infectious agents. In most cases neurophysiological tests reveal abnormalities. In this paper the Authors use low (11 pps) and high (51-71 pps) frequency ABR to evaluate the electrophysiological function of auditory brainstem responses. Eighteen patients with suspected CFS, between the ages of 17 and 63, were examined. Eleven subjects had clinically diagnosed "true" CFS (CDC criteria modified by Fukuda). The 11 pps frequency test did not reveal a high number of abnormalities in the patients in question. However, the high frequency stimulation test (with 51 and 71 pps) which was statistically significant (P = 0.009) revealed numerous aberrations in 7 patients; absence of the first wave in 1 case, in 5 numerous wave gap delays and in 1 patient absence of the first wave and numerous wave gap delays. The high frequency test did not show many abnormalities for the 4 remaining patients. For the 7 "non CFS" subjects, the clinical-audiological comparison showed no statistical significance (P = 0.920). The Authors hypothesize that the absence of the first wave in the CFS Subject may well indicate a cyto-neural junction disease in the organ of Corti. The combined analysis of clinical and audiological data showed that the described tests are more reliable when employed in dealing with patients with clinically assessed "true" CFS.