Detailed analysis of mortality rates in the female progeny of 1,001 Holstein bulls allows the discovery of new dominant genetic defects.

Reducing juvenile mortality in cattle is important for both economic and animal welfare reasons. Previous studies have revealed a large variability in mortality rates between breeds and sire progeny groups, with some extreme cases due to dominant mutations causing various syndromes among the descendants of mosaic bulls. The purpose of this study was to monitor sire-family calf mortality within the French and Walloon Holstein populations, and to use this information to detect genetic defects that might have been overlooked by lack of specific symptoms. In a population of heifers born from 1,001 bulls between 2017 and 2020, the average sire-family mortality rates were of 11.8% from birth to 1 year of age and of 4.2, 2.9, 3.1, and 3.2% for the perinatal, postnatal, preweaning, and postweaning subperiods, respectively. After outlining the 5 worst bulls per category, we paid particular attention to the bulls Mo and Pa, because they were half-brothers. Using a battery of approaches, including necropsies, karyotyping, genetic mapping, and whole-genome sequencing, we described 2 new independent genetic defects in their progeny and their molecular etiology. Mo was found to carry a de novo reciprocal translocation between chromosomes BTA26 and BTA29, leading to increased embryonic and juvenile mortality because of aneuploidy. Clinical examination of 2 calves that were monosomic for a large proportion of BTA29, including an orthologous segment deleted in human Jacobsen syndrome, revealed symptoms shared between species. In contrast, Pa was found to be mosaic for a dominant de novo nonsense mutation of GATA 6 binding protein (GATA6), causing severe cardiac malformations. In conclusion, our results highlight the power of monitoring juvenile mortality to identify dominant genetic defects due to de novo mutation events.

Influence of production, reproduction, morphology, and health traits on true and functional longevity in French Holstein cows.

In the long term, resilient animals are able to maintain their normal biological processes when confronted with environmental perturbations, reducing their risk of being culled. Therefore, longevity can be proposed as an indicator of long-term resilience. Decisions to remove a given dairy cow from the herd are mainly related to low milk production (i.e., voluntary culling) or to reasons other than production (i.e., involuntary culling). The aptitude of animals to delay any culling is defined as true longevity (TL), whereas functional longevity (FL) is the ability to avoid involuntary culling. The aim of the study was to investigate the influence of production, reproduction, morphology, and health traits on TL and FL, to identify risk factors for culling. Data included 278,217 lactations from 122,461 Holstein Friesian cows reared in 640 herds. The length of productive life, calculated as the time between first calving and culling, or censoring, was used as the measure of longevity. Survival analysis was performed using proportional hazards models assuming a piecewise Weibull distribution of the baseline hazard function, with or without adjustment for milk production to evaluate FL and TL. Insemination status, calving ease, mastitis, somatic cell count, displaced abomasum, and udder depth had significant relationships with TL and FL. Differences in estimates of relative risk between TL and FL showed that milk production often influenced culling decisions: farmers are more prone to cull animals with low production even when they had good other characteristics. The culling risk factors identified in the present study can be used to study resilience in dairy cattle and to improve genetic evaluations of functional or total longevity.

Copper sulfate and sodium selenite lipid-microencapsulation modifies ruminal microbial fermentation in a dual-flow continuous-culture system.

Undesirable interactions between trace mineral elements and ruminal contents may occur during digestion when mineral salts are supplemented. Antimicrobial effects of copper sulfate (CuSO4) may affect ruminal digestibility of nutrients when fed as a source of copper (Cu), while sodium selenite (Na2SeO3) may be reduced in the rumen to less available forms of selenium (Se). Our objective was to evaluate if protection of CuSO4 and Na2SeO3 by lipid-microencapsulation would induce changes on ruminal microbial fermentation. We used 8 fermentors in a dual-flow continuous-culture system in a 4 × 4 duplicated Latin square with a 2 × 2 factorial arrangement of treatments. Factors were CuSO4 protection (unprotected and protected by lipid-microencapsulation) and Na2SeO3 protection (unprotected and protected by lipid-microencapsulation). Treatments consisted of supplementation with 15 mg/kg of Cu and 0.3 mg/kg of Se from either unprotected or protected (lipid-microencapsulated) sources, as follows: (1) Control (unprotected CuSO4 + unprotected Na2SeO3); (2) Cu-P (protected CuSO4 + unprotected Na2SeO3); (3) Se-P (unprotected CuSO4 + protected Na2SeO3); (4) (Cu+Se)-P (protected CuSO4 + protected Na2SeO3). All diets had the same nutrient composition and fermentors were fed 106 g of dry matter/d. Each experimental period was 10 d (7 d of adaptation and 3 d for sample collections). Daily pooled samples of effluents were analyzed for pH, NH3-N, nutrient digestibility, and flows (g/d) of total N, NH3-N, nonammonia N (NAN), bacterial N, dietary N, and bacterial efficiency. Kinetics of volatile fatty acids was analyzed in samples collected daily at 0, 1, 2, 4, 6, and 8 h after feeding. Main effects of Cu protection, Se protection, and their interaction were tested for all response variables. Kinetics data were analyzed as repeated measures. Protection of Cu decreased acetate molar proportion, increased butyrate proportion, and tended to decrease acetate:propionate ratio in samples of kinetics, but did not modify nutrient digestibility. Protection of Se tended to decrease NH3-N concentration, NH3-N flow, and CP digestibility; and to increase flows of nonammonia N and dietary N. Our results indicate that protection of CuSO4 may increase butyrate concentration at expenses of acetate, while protection of Na2SeO3 tended to reduce ruminal degradation of N. Further research is needed to determine the effects of lipid-microencapsulation on intestinal absorption, tissue distribution of Cu and Se, and animal performance.

Estimation of genetic parameters for dairy traits and somatic cell score in the first 3 parities using a random regression test-day model in French Alpine goats.

Lactation curve shape can affect an animal's health, feed requirements, and milk production throughout the year. We implemented a random regression model for the genetic evaluation of lactation curve shapes of dairy traits in French Alpine goats for their first 3 parities. Milk, fat, and protein yields, fat and protein contents, somatic cell score, and fat/protein ratio were considered. The data consisted of test-day records from 49,849 first lactation Alpine goats during their first 3 lactations. The reference model used a Legendre polynomial of order 2 for each parity to describe the genetic and permanent environmental effects, and was compared with a model that combined the second and third parities. A rank reduction of the variance-covariance matrix was also performed using an eigenvalue decomposition for each parity from the 2 models. Genetic parameters were consistent between the models tested. With a reduction to rank 2 and combining the second and third parities, the first 2 principal components correctly summarized the genetic variability of milk yield level and persistency, with a near-nil correlation between the 2, and with a much shorter computation time than the reference model. A favorable correlation of +0.43 between milk yield persistency and fat/protein ratio persistency at the beginning of the lactation was found from buck estimated breeding values.

Effect of rumen-protected B vitamins and choline supplementation on health, production, and reproduction in transition dairy cows.

The objectives were to determine the effects of a rumen-protected blend of B vitamins and choline (RPBC) on the incidence of health disorders, milk yield, and reproduction in early lactation and the effects on gene expression and liver fat infiltration. A randomized controlled trial in 3 commercial dairy herds (n = 1,346 cows with group as the experimental unit; experiment 1) and a university research herd (n = 50 cows with cow as the experimental unit; experiment 2) evaluated the use of 100 g/cow per d of commercially available proprietary RPBC supplement (Transition VB, Jefo, St. Hyacinthe, Quebec, Canada), or a placebo, fed 3 wk before to 3 wk after calving. In experiment 2 liver biopsies were taken at 4 and 14 ± 1 d in milk to measure triacylglycerol concentrations and expression of 28 genes selected to represent relevant aspects of liver metabolism. Treatment effects were assessed using multivariable mixed logistic regression models for binary health and reproductive outcomes; linear regression models for milk yield, dry matter intake, and liver outcomes; and survival analysis for time insemination and pregnancy. In experiment 1, treatment did not have an effect on the incidence of hyperketonemia (blood ß-hydroxybutyrate ≥ 1.2 mmol/L; cumulative incidence to 3 wk postpartum of 28 to 30%), clinical health disorders, or udder edema. The prevalence of anovulation at 8 wk postpartum was 11% in the treatment group and 23% in the control but did not differ statistically given group-level randomization. Pregnancy at first insemination (33 and 35%) and median time to pregnancy to 200 d in milk (96 and 97 d) were not different between treatment and control, respectively. No difference was observed between treatment groups in milk yield or components through the first 3 Dairy Herd Improvement Association test days (44 kg/d in both groups, accounting for parity and components). In experiment 2, there were no differences between treatment groups in feed intake. Mean blood ß-hydroxybutyrate was lower at wk 3 in RPBC (0.6 vs. 0.9 ± 0.12 mmol/L) with no difference between treatments for mean blood concentrations of fatty acids (wk -1 or 1) and ß-hydroxybutyrate at wk 1 or 2. The gene for acyl-CoA oxidase 1 (ACOX1) had lower mRNA abundance in RPBC with no difference between treatments for the other genes, but the expression of half of the genes assessed differed with days in milk. Liver triacylglycerol was lower in primiparous cows at 4 d in milk in RPBC (2.0 vs. 4.4 ± 1.2%) but not at 14 d in milk (2.2 vs. 3.2 ± 0.97%) with no treatment effect in multiparous cows (4.6 ± 0.8%). Accounting for parity, days in milk, fat and protein percentages, repeated test days, and a random effect of cow, no significant difference was observed between treatments in milk yield across the first 3 Dairy Herd Improvement Association tests (41.2 ± 1.3 in RPBC vs. 38.0 ± 1.4 kg/d in control). Under the diet and management conditions of the field study including low prevalence of clinical health disorders, in experiment 1 we did not detect a benefit of RPBC, but in experiment 2 liver fat content decreased in primiparous cows.

Genetic analysis of calf health in Charolais beef cattle.

The objective of this study was to investigate the factors that influence calf health and survival in Charolais cattle. Data from 2,740 calves, originating from 16 French farms and observed from birth until 30 d of age, were analyzed using models that took account of direct genetic, maternal genetic, and common environmental effects. Both direct and maternal genetic parameters were estimated for birth weight (BW), calving ease (CE), neonatal vitality (NV), survival at 30 d (Surv), and umbilical infection and diarrhea at different ages (0 to 5 d: Umb1 and Diar1; 6 to 20 d: Umb2 and Diar2; and 21 to 30 d: Umb3 and Diar3). The heritability values for direct and maternal genetic effects were, 0.026 (SE = 0.027) and 0.096 (SE = 0.042) for Surv, 0.280 (SE = 0.063) and 0.063 (SE = 0.038) for BW, 0.129 (SE = 0.041) and 0 for CE, 0.073 (SE = 0.035) and 0 for NV, 0.071 (SE = 0.038) and 0.017 (SE = 0.026) for Umb1, 0 and 0.082 (SE = 0.029) for Umb2, 0 and 0.044 (SE = 0.030) for Diar1, 0.016 (SE = 0.022) and 0.012 (SE = 0.026) for Diar2, and 0.016 (SE = 0.028) and 0 for Diar3, respectively. Significant genetic variability in beef cattle was thus revealed for five calf health traits: NV, Surv, Diar1, Umb1, and Umb2. In addition, for three traits (Surv, Diar1, and Umb2), maternal genetic effects clearly contributed more to health performance than direct genetic effects. Estimates of genetic correlation between traits varied markedly (from 0 to 1 in absolute values) depending on the traits in question, the age for a given trait, and the type (direct or maternal) of the genetic effects considered. These results suggest that not all health traits in Charolais cattle can be improved simultaneously, and breeders will therefore have to prioritize certain traits of interest in their breeding objectives. Overall, our results demonstrate the potential utility of collecting and integrating data on calf diseases, NV and survival in future beef cattle breeding programs. To ensure appropriate biological and genetic evaluations of calf health performance, it is important to accurately describe the phenotypes for diarrhea and umbilical infections (in terms of age ranges) and account for maternal genetic and common environmental effects that explain calf health performance traits. Further investigation and improved data collection are now necessary to maximize the efficiency of breeding schemes designed to simultaneously improve production and health traits.

Rapid Discovery of De Novo Deleterious Mutations in Cattle Enhances the Value of Livestock as Model Species.

In humans, the clinical and molecular characterization of sporadic syndromes is often hindered by the small number of patients and the difficulty in developing animal models for severe dominant conditions. Here we show that the availability of large data sets of whole-genome sequences, high-density SNP chip genotypes and extensive recording of phenotype offers an unprecedented opportunity to quickly dissect the genetic architecture of severe dominant conditions in livestock. We report on the identification of seven dominant de novo mutations in CHD7, COL1A1, COL2A1, COPA, and MITF and exploit the structure of cattle populations to describe their clinical consequences and map modifier loci. Moreover, we demonstrate that the emergence of recessive genetic defects can be monitored by detecting de novo deleterious mutations in the genome of bulls used for artificial insemination. These results demonstrate the attractiveness of cattle as a model species in the post genomic era, particularly to confirm the genetic aetiology of isolated clinical case reports in humans.

Individual responses of dairy cows to a 24-hour milking interval.

Some dairy farmers opt to omit one milking, either incidentally or weekly, without changing other milking times. This practice entails an extended milking interval of 24h (24h-MI), which is associated with a decrease in milk yield. This decrease varies among cows and could be partly due to factors such as stage of lactation and milk yield level. The aim of this study was to describe the average and individual responses in terms of loss and carryover effects of a 24h-MI on milk yield. The influence of factors such as parity, stage of lactation, and milk yield potential were investigated, together with response repeatability. Our trial used 292 Holstein-Friesian cows, and consisted of 3 successive periods: 1 wk of twice-daily milking (TDM) as a control, one 24h-MI, and then 13d of TDM. The number of observations per cow ranged from 1 to 9, with no more than three 24h-MI per lactation. The 24h-MI reduced milk yield by 23% (7.8 kg on average) and milk lactose content by 2.6g/kg on the 24h-MI day. Milk fat and protein content, and somatic cell score increased by 3.0 g/kg, 0.5 g/kg, and 0.4 units, respectively. No significant carryover effect was found of a 24h-MI on milk yield or milk composition 2 wk after resumption of TDM. Milk yield loss and recovery varied widely (coefficient of variation 62%), and the relationship between milk loss and milk recovery showed substantial variation (residual standard deviation 2.1 kg/d). Cows with a greater milk potential level lost more milk yield but recovered more milk, with no influence on recovery:loss ratio. Cows in early lactation recovered the lost milk yield faster. Repeatability of the responses to a 24h-MI was 44% for milk yield loss (kg/d), 57% for relative milk yield loss (%), 33% for milk yield recovery (kg/d), and 0% for milk recovery:loss ratio (%), suggesting a genetically determined ability to limit loss when one milking is omitted. To conclude, a 24h-MI caused higher milk yield losses than reported in previous studies. Stage of lactation, estimated potential milk yield level, and parity explained the cows' response to the 24h-MI, but did not account for all the individual variability.

Inbreeding and crossbreeding parameters for production and fertility traits in Holstein, Montbéliarde, and Normande cows.

Breed differences and nonadditive genetic effects for milk production traits, somatic cell score (SCS), conception rate (CR), and days to first service (DFS) were estimated for Holstein × Montbéliarde and Holstein × Normande crossbreds, using an animal model adapted from the French genetic evaluation and extended to across-breed analysis. Inbreeding and breed differences were estimated from all purebred recorded cows. Only records from 1,137 herds with Holstein × Montbéliarde crossbred cows and from 1,033 herds with Holstein × Normande crossbred cows were used to estimate crossbreeding parameters. In these herds, crossbred cows represented about 13% of the total number of recorded animals compared with <1% when all herds were considered. Compared with the Montbéliarde and Normande breeds, the Holstein breed was genetically superior for production [+951kg and +2,444kg for 305-d mature-equivalent (305ME) milk, +40kg and +102kg for 305ME fat, +17kg and +54kg for 305ME protein, respectively] and inferior for fertility traits (-12 and -9% for CR, respectively). Inbreeding depression caused loss of yield for production traits (from -32 to -41kg of 305ME milk, -1.4 to -1.7kg of 305ME fat, and -1.1 to -1.3kg of 305ME protein per inbreeding percentage), a small increase in SCS (+0.001 to 0.006) and DFS (+0.12d), and a decrease in CR (-0.27 to -0.44%). Favorable heterosis effects were found for all traits (+494 to 524kg of 305ME milk, +21 to 22kg of 305ME fat, +15 to 16kg of 305ME protein, -0.05 to -0.04 SCS, +2 to 3% for CR, and -3 to 6d of DFS), to such a point that F1 crossbreds could compete with Holstein cows for milk production while having a better fertility. However, recombination losses suggested that some F1 heterosis was lost for backcross cows.

Characterization of French dairy farm environments from herd-test-day profiles.

Genotype by environment interactions are ignored in national genetic evaluations of dairy cattle. However, some breeders consider that the genetic merit of top sires is not correctly estimated for use in their own particular environment or with their own herd management. With the objective of later investigating genotype by environment interactions at the national level, we studied the relationship between herd management (e.g., feeding system, herd size, production, workforce) and herd-test-day (HTD) profiles for milk yield and contents of fat and protein using a database of 934 herds. Herd-test-day profiles, estimated using a test-day model, are thought to reflect herd management because they represent the part of production due only to month-to-month variations in environmental conditions of production; that is, those related mainly to feeding and climate conditions. Herd clustering based on results from a factor analysis of descriptors of HTD profiles was performed. Each cluster of herds was then characterized in terms of herd management. Three herd clusters were identified. Cluster 1 was characterized by low HTD profiles for milk yield and protein and fat contents as well as a low level of intensification. Cluster 2 was defined by a high milk yield HTD profile and a high level of intensification. Cluster 3 consisted of herds with HTD profiles showing high protein and fat contents but it was difficult to link this cluster to any specific herd management. The use of clusters based on HTD profiles as descriptors of distinct herd management systems will be used in a genotype by environment interaction study in France.

A general method to validate breeding value prediction software.

The validity of national genetic evaluations depends on the quality of input data, on the model of analysis, and on the correctness of genetic evaluation software. A general strategy was developed to validate national breeding value prediction software: performances from a real data file were replaced with simulated ones, created from simulated fixed and random effects and residuals in such a way that BLUP estimates from the evaluation software must be equal to the simulated effects. This approach was implemented for a multiple-trait model and a random regression test-day model. An example was presented on test-day observations analyzed with a random regression animal model including a lactation curve described as a sum of fixed polynomial regression and fixed spline regression on days in milk, and with genetic and permanent environmental effects modeled by using Legendre polynomials of order 2. Residuals had heterogeneous variances, and phantom parent groups were included. This method can be easily extended to other linear models. The comparison of genetic evaluation results with simulated true effects is used to demonstrate the great efficiency and usefulness of the proposed method.

Environmental effects on lactation curves included in a test-day model genetic evaluation.

A large number of environmental factors affect the daily milk production of a cow. Lactation curves included in the French test-day model are modelled as a function of days in milk with semi-parametric curves (splines). The proper modelling of environmental effects in the test-day analysis was investigated using test-day records collected from the first three lactations of French Montbéliarde cows from 1988 to 2005. Four lactation-curve effects describing calving month, length of dry period, age at calving and gestation defined within parity-class were fitted. The shape of lactation curves did not depend on year of calving, which can be modelled as a constant over the whole lactation. To reduce computational requirements and time, data were pre-adjusted in a first step for fixed effects with no year interaction, and then used for genetic evaluation. Correlations for each lactation between 305-day estimates of genetic and permanent environment effects computed using pre-adjustment factors obtained at a 4-year interval were virtually one. The use of a two-step procedure had a very limited impact on the estimates of genetic and permanent environment effects. The minimum correlations with values estimated with a one-step procedure were 0.9984 and 0.9974, respectively. The knowledge of systematic environmental effects affecting the cow daily yield through lactation curves offers interesting perspectives to predict future daily milk production.

Estimation of genetic correlations among countries in international dairy sire evaluations with structural models.

The increase in the number of participating countries and the lack of genetic ties between some countries has lead to statistical and computational difficulties in estimating the genetic (co)variance matrix needed for international sire evaluation of milk yield and other traits. Structural models have been proposed to reduce the number of parameters to estimate by exploiting patterns in the genetic correlation matrix. Genetic correlations between countries are described as a simple function of unspecified country characteristics that can be mapped in a space of limited dimensions. Two link functions equal to the exponential of minus the Euclidian distance between the coordinates of two countries and the exponential of minus the square of this Euclidian distance were used for the study on international simulated and field data. On simulated data, it was shown that structural models might allow an easier estimation of genetic correlations close to the border of the parameter space. This is not always possible with an unstructured model. On milk yield data, genetic correlations obtained from 22 countries for structural models based on 2 and 7 dimensions, respectively, were analyzed. Only a structural model with a large number of axes gave reasonable estimates of genetic correlations compared with correlations obtained for an unstructured model: 76.7% of correlations deviated by less than 0.030. Such a model reduces the number of parameters from 231 genetic correlations to 126 coordinates. On foot angle data, large deviations were observed between genetic correlations estimated with an unstructured model and correlations estimated with a structural model, regardless of the number of axes taken into account.

Principal components and factorial approaches for estimating genetic correlations in international sire evaluation.

The increasing number of participating countries and the lack of genetic links among some of them lead to statistical and computational difficulties in estimating the genetic (co)variance matrix needed for international sire evaluation of milk yield. Reparameterization using principal components or factorial approaches is proposed to exploit patterns in the genetic correlation matrix in order to reduce the number of parameters to be estimated without much loss of information. A 2-step approach was used. First, the genetic matrix between 8 or 9 "base" countries was used to determine a reduced number of principal components or factors. Then, the contributions of the remaining countries to these principal components or factors were computed. The resulting genetic correlations for the 18 countries were compared with the "reference" genetic correlations obtained with a classical model. The impact of using reparameterized genetic correlation matrices on breeding value prediction was investigated for both approaches. A better agreement between predicted breeding values and stability of their rankings was found when an approximate factor analysis was used, whatever the number of factors considered. The estimation of genetic correlations among 18 countries using an approximate factorial approach with 5 factors taken into account led to a reduction of the number of parameters to estimate from 171 to 80. The average absolute deviation of the correlations estimated with an approximate factorial approach from the "reference" genetic correlations was 0.014, which is considered very satisfactory in light of the computational ease.

[2 cases of paresthetic meralgia of the femoral cutaneous nerve]. / Dos casos de meralgia parestésica del nervio femorocutáneo.

We report two cases of meralgia paresthetica. The first patient was 35-year-old pregnant woman who reported dull pain on the front and side of her thigh presenting at 6 weeks gestation. The symptoms disappeared in 1 week with conservative treatment and no other symptoms developed. The second patient was a 56-year-old woman, obese, who reported intense, burning pain with numbness and occasional bouts of piercing pain. The symptoms were resistant to treatment with non-steroidal antiinflamatory drugs or mild opiates, but were relieved by injection of local anesthetics and corticosteroids. Meralgia paresthetica is mononeuropathy that affects the lateral femoral cutaneous nerve. It can occur during pregnancy or in other contexts. The most common cause is entrapment of the nerve at the anterior superior iliac spine or during its passage through the inguinal ligament. The most common symptom is numbness at the front of the thigh, although burning, formication, tingling, or other forms of paresthesia may also be present. The condition is usually self-limiting, although uncomfortable. Diagnosis is based on careful neurological examination. Conservative treatment is preferred and is based on limiting periods of standing and avoidance of tight clothing. Oral analgesics can be prescribed if necessary and weight loss is required for obese patients. Surgical ablation of the nerve is a last resort.

Microbial agents associated with waterborne diseases.

Many classes of pathogens excreted in feces are able to initiate waterborne infections. There are bacterial pathogens, including enteric and aquatic bacteria, enteric viruses, and enteric protozoa, which are strongly resistant in the water environment and to most disinfectants. The infection dose of viral and protozoan agents is lower than bacteria, in the range of one to ten infectious units or oocysts. Waterborne outbreaks of bacterial origin (particularly typhoid fever) in the developing countries have declined dramatically from 1900s. Therefore, some early bacterial agents such as Shigella sonnei remains prevalent and new pathogens of fecal origin such as zoonotic C. jejuni and E. coli O157:H7 may contaminate pristine waters through wildlife or domestic animal feces. The common feature of these bacteria is the low inoculum (a few hundred cells) that may trigger disease. The emergence in early 1992 of serotype O139 of V. cholerae with epidemic potential in Southeast Asia suggests that other serotypes than V. cholerae O1 could also getting on epidemic. Some new pathogens include environmental bacteria that are capable of surviving and proliferating in water distribution systems. Other than specific hosts at risk, the general population is refractory to infection with ingested P. aeruginosa. The significance of Aeromonas spp. in drinking water to the occurrence of acute gastroenteritis remains a debatable point and has to be evaluated in further epidemiological studies. Legionella and Mycobacterium avium complex (MAC) are environmental pathogens that have found an ecologic niche in drinking and hot water supplies. Numerous studies have reported Legionnaires' disease caused by L. pneumophila occurring in residential and hospital water supplies. M. avium complex frequently causes disseminated infections in AIDS patients and drinking water has been suggested as a source of infection; in some cases the relationship has been proven. More and more numerous reports show that Helicobacter pylori DNA can be amplified from feces samples of infected patients, which strongly suggests fecal-to-oral transmission. Therefore, it is possible that H. pylori infection is waterbome, but these assumptions need to be substantiated. Giardiasis has become the most common cause of human waterborne disease in the U.S. over the last 30 years. However, as a result of the massive outbreak of waterborne cryptosporidiosis in Milwaukee, Wisconsin, affecting an estimated 403,000 persons, there is increasing interest in the epidemiology and prevention of new infection disease caused by Cryptosporidium spp. as well as monitoring water quality. The transmission of Cryptosporidium and Giardia through treated water supplies that meet water quality standards demonstrates that water treatment technologies have become inadequate, and that a negative coliform no longer guarantees that water is free from all pathogens, especially from protozoan agents. Substantial concern persists that low levels of pathogen occurrence may be responsible for the endemic transmission of enteric disease. In addition to Giardia and Cryptosporidium, some species of genera Cyclospora, Isospora, and of family Microsporidia are emerging as opportunistic pathogens and may have waterborne routes of transmission. More than 15 different groups of viruses, encompassing more than 140 distinct types can be found in the human gut. Some cause illness unrelated with the gut epithelium, such as Hepatitis A virus (HAV) and Hepatitis E virus (HEV). Numerous large outbreaks have been documented in the U.S. between 1950 and 1970, and the incidence rate has strongly declined in developing countries since the 1970s. Hepatitis E is mostly confined to tropical and subtropical areas, but recent reports indicate that it can occur at a low level in Europe. A relatively small group of viruses have been incriminated as causes of acute gastroenteritis in humans and fewer have proven to be true etiologic agents, including rotavirus, calicivirus, astrovirus, and some enteric adenovirus. These enteric viruses have infrequently been identified as the etiologic agents of waterborne disease outbreaks, because of inadequate diagnostic technology, but many outbreaks of unknown etiology currently reported are likely due to viral agents. Actually, Norwalk virus and Norwalk-like viruses are recognized as the major causes of waterborne illnesses world-wide. The global burden of infectious waterborne disease is considerable. Reported numbers highly underestimate the real incidence of waterborne diseases. The most striking concern is that enteric viruses such as caliciviruses and some protozoan agents, such as Cryptosporidium, are the best candidates to reach the highest levels of endemic transmission, because they are ubiquitous in water intended for drinking, being highly resistant to relevant environmental factors, including chemical disinfecting procedures. Other concluding concerns are the enhanced risks for the classic group of debilitated subjects (very young, old, pregnant, and immunocompromised individuals) and the basic requirement of to take specific measures aimed at reducing the risk of waterborne infection diseases in this growing, weaker population.

Advances in the bacteriology of the coliform group: their suitability as markers of microbial water safety.

Advances in the elaboration of novel genomic types of beta-galactosidase-positive Enterobacteriaceae and comprehensive studies of their habitats have resulted in an innovative approach to the assessment of the merits and shortcomings of the thermotrophic and fecal species Escherichia coli and all other coliforms as markers of the microbiological safety of water. As one of the consequences, it is recommended to abolish the "technical" designation fecal coliforms because their current method of detection will result in the isolation of thermotrophic organisms that have been demonstrated, beyond a doubt, to be of environmental, rather than uniquely enteric origin. Additional population studies have demonstrated that none of the coliforms can function as reliable markers for all enteric pathogens (index organisms sensu Ingram), nor be of use in validating adequate processing for safety of raw water, which represents the indicator function of markers, as defined by Ingram. Future studies along these lines will have to provide the data required to assess the suitability of additional markers for the reliable monitoring of drinking water for microbiological safety.

Bacteriophages as indicators of enteric viruses and public health risk in groundwaters.

Low concentrations of all types of bacteriophages in groundwater limit their power to predict the presence of enteric viruses. There is little concordance in the literature regarding phage detection methods, thus making comparisons extremely difficult. Different authors have used different hosts, phage concentration methods, and end-point determinations. Also, markedly different volumes of sample have been employed, varying from 1 litre to 400 l. Bacteriophage concentration methods are not reproducible. There has been marked variability among groups in the natural substrates used (for example, beef extract), the type of adsorbing filter used, centrifugation instruments and conditions, and the delivery of the concentrate to the host cells. There is no consensus on the best bacterial host strain. Currently, several are employed with each showing differential sensitivities and specificities. In particular, host stability must be considered. Host stability has two components: the ability of the host to continue to be receptive to the bacteriophage after continued sub-culture, and the lack of lysogenic or temperate bacteriophage in the host cell line which may be randomly and unpredictably activated. There is a lack of consistent recovery of bacteriophages from individual faecal specimens. In particular, only approximately 3% of individual humans carry the FRNA phages. While there is some evidence to indicate that the phages multiply in sewage, it is not clear how they do so since the host pili should not be produced at lower temperatures. These ecological factors need to be understood. Of all the phages thus far studied, Bacteroides fragilis HSP40 has the highest recovery rate from individual people. However, Bacteroides, being an anaerobe, is a difficult host for routine laboratory analysis. Methods for the enumeration of F(+)-specific phages and Bacteroides phages are complex, time-consuming, costly and not reproducible. Conversely, somatic coliphage methods are simpler and results can be available in 4-6 h. The occurrence of phages and viruses in groundwater depends on physicochemical characteristics that control their fate and transport in the groundwater/aquifer environment. There are very little actual data taken from the field that allow an understanding of the ecology and life span of phages in their natural environment. Moreover, the ability of phages to serve as a source of food for other microbes needs to be understood. There has been a lack of association of bacteriophage recovery with gastroenteritis outbreaks due to enteric viruses. There is only a small epidemiological database concerning the occurrence of enteric viruses in groundwater.

Pseudomonas gessardii sp. nov. and Pseudomonas migulae sp. nov., two new species isolated from natural mineral waters.

Twenty-five non-identified fluorescent Pseudomonas strains isolated from natural mineral waters were previously clustered into three phenotypic subclusters, XIIIb, XVa and XVc. These strains were characterized genotypically in the present study. DNA-DNA hybridization results and DNA base composition analysis revealed that these strains were members of two new species, for which the names Pseudomonas gessardii sp. nov. (type strain CIP 105469T) and Pseudomonas migulae sp. nov. (type strain CIP 105470T) are proposed. P. gessardii included 13 strains from phenotypic subclusters XVa and XVc. P. migulae included 10 strains from phenotypic subcluster XIIIb. The levels of DNA-DNA relatedness ranged from 71 to 100% for P. gessardii and from 74 to 100% for P. migulae. The G + C content of the DNA of each type strain was 58 mol%. DNA similarity levels, measured with 67 reference strains of Pseudomonas species, were below 55%, with delta Tm values of 13 degrees C or more. The two new species presented basic morphological characteristics common to all pseudomonads. Various phenotypic features were found to differentiate them: P. gessardii strains utilized L-arabitol, myo-inositol, adonitol, xylitol and meso-erythritol as carbon sources, whereas P. migulae strains assimilated L-arabinose, D-xylose, D-saccharate, meso-tartrate, tricarballylate, D-glucuronate, D-galacturonate, phenylacetate and histamine. The complete 16S rRNA sequences of each type strain were determined and compared with those of the type strains of Pseudomonas species. Finally, a phylogenetic tree was inferred from sequence analysis and demonstrated that the two new species fell into the 'Pseudomonas fluorescens intrageneric cluster'. To date, their clinical significance is unknown.

Taxonomic study of bacteria isolated from Lebanese spring waters: proposal for Pseudomonas cedrella sp. nov. and P. orientalis sp. nov.

Deoxyribonucleic acid relatedness studies (S1 nuclease method) have shown that 15 strains isolated from three Lebanese spring waters, belonging to the genus Pseudomonas, formed two homogeneous DNA groups, with a within-group DNA relatedness ranging from 70 to 100%. These groups are referred to as Pseudomonas cedrella sp. nov. and Pseudomonas orientalis sp.nov. These strains were previously grouped on the basis of a numerical analysis in phenons Ve, Vd, Vg, and VI. DNA relatedness with 65 strains representing 24 species of the genus Pseudomonas sensu stricto was below 50%. The highest DNA binding value (50%) was found with P. marginalis species. A comparison of the complete 16S rRNA gene sequences of the strains representing the two new deoxyribonucleic acid hybridization groups, i.e., strains CFML 96-198T and CFML 96-170T, and the sequence of other strains of the genus Pseudomonas revealed that these strains (CFML 96-198T and CFML 96-170T) fell within the 'Pseudomonas fluorescens intrageneric cluster'. The G+C contents of the DNA of P. cedrella CIP 105541T and P. orientalis CIP 105540T were 59 and 60 mol%, respectively. The two species can be differentiated from each other by the fact that P. cedrella strains hydrolyze erythritol and D-lyxose. P. cedrella grouped together a total of nine strains from phenotypic groups Ve, Vg, and VI. P. orientalis grouped together six strains from both phenotypic groups Vd and Ve.