Quantitative genetics of disease resistance in vaccinated and unvaccinated Atlantic salmon (Salmo salar L.).

Furunculosis (Aeromonoas salmonicida) is an important disease in Atlantic salmon (Salmo salar) farming. Vaccination and selective breeding for increased resistance to the disease on the basis of challenge tests of unvaccinated fish are used as complementary prophylactic methods. An important issue is whether genetic predisposition to infection is consistent across vaccinated and unvaccinated fish. Hence, the main objective of this study was to determine the magnitude of the genetic associations (correlations) between resistance to furunculosis in vaccinated and unvaccinated fish, and to estimate the magnitude of the correlation of resistance to furunculosis with resistance to the viral diseases infectious pancreatic necrosis (IPN) and infectious salmon anaemia (ISA). Sub-samples of unvaccinated and vaccinated salmon from 150 full-sib families were subjected to separate cohabitation challenge tests. Substantial genetic variation was found in resistance to furunculosis in both the unvaccinated (heritabilities of 0.51 ± 0.05) and vaccinated (0.39 ± 0.06) fish. However, the genetic correlation between resistance to furunculosis in the two groups was low (0.32 ± 0.13), indicating a weak genetic association between resistance in the two groups. Hence, the current selection strategy on the basis of challenge tests of unvaccinated fish is likely to produce low genetic improvement in resistance to furunculosis under field conditions, where fish are vaccinated with an effective vaccine. Evidence was found of significantly favourable genetic associations of resistance to furunculosis in unvaccinated (but less so for vaccinated) fish with resistance to both IPN and ISA (unvaccinated fish), indicating that vaccination 'mask' genetic associations between resistance to different diseases.

A sequential threshold cure model for genetic analysis of time-to-event data.

In analysis of time-to-event data, classical survival models ignore the presence of potential nonsusceptible (cured) individuals, which, if present, will invalidate the inference procedures. Existence of nonsusceptible individuals is particularly relevant under challenge testing with specific pathogens, which is a common procedure in aquaculture breeding schemes. A cure model is a survival model accounting for a fraction of nonsusceptible individuals in the population. This study proposes a mixed cure model for time-to-event data, measured as sequential binary records. In a simulation study survival data were generated through 2 underlying traits: susceptibility and endurance (risk of dying per time-unit), associated with 2 sets of underlying liabilities. Despite considerable phenotypic confounding, the proposed model was largely able to distinguish the 2 traits. Furthermore, if selection is for improved susceptibility rather than endurance, the error of applying a classical survival model was nonnegligible. The difference was most pronounced for scenarios with substantial underlying genetic variation in endurance and when the 2 underlying traits were lowly genetically correlated. In the presence of nonsusceptible individuals, the method provides a novel and more accurate tool for utilization of time-to-event data, and has also been proven successful when applied to zero-inflated longitudinal binary data.

The zoonotic potential of Giardia and Cryptosporidium in Norwegian sheep: a longitudinal investigation of 6 flocks of lambs.

Faecal samples collected from lambs on 6 Norwegian farms on 2 separate occasions during spring/summer 2008 (approximately 550 samples collected at each occasion) were examined for Cryptosporidium oocysts and Giardia cysts by immunofluorescent antibody test. Overall prevalence at the first sampling was approximately 23% for Giardia and 15% for Cryptosporidium, and at the second sampling approximately 31% for Giardia and 24% for Cryptosporidium, with substantial between-farm variation on each sampling occasion. To assess the potential public health significance of these infections, molecular analyses were conducted on 42 Giardia isolates and 42 Cryptosporidium isolates, with PCR targeted at one or both of two genes (Giardia: glutamate dehydrogenase and beta-giardin genes; Cryptosporidium: SSU rRNA and actin genes) for each parasite. Of the Giardia isolates, 41 were Assemblage E (non-zoonotic) and 1 was Assemblage B (zoonotic). Of the Cryptosporidium isolates, 35 were cervine genotype (potentially zoonotic) and 7 C. xiaoi (non-zoonotic). These results suggest that sheep in Norway are unlikely to be an important reservoir of zoonotic Giardia in Norway, but might have some public health significance with respect to Cryptosporidium.

Molecular and epidemiological investigations of cryptosporidiosis in Cuban children.

Molecular and epidemiological studies of Cryptosporidium infections amongst 28 Cuban children (aged 2-8 years) with diarrhoea are described. As few of the younger infected children but most of the older infected children had been breastfed, short-term protection from maternal antibodies passed to infants during breastfeeding may result in a lack of cryptosporidial infection in infancy. This protection of breastfeeding children may, however, result in such children developing less anti-Cryptosporidium immunity of their own (than their bottle-fed counterparts), so that, by school age, the children who had been breastfed are those most likely to be found infected. In the present study, in contrast with the observations made during a previous study of cryptosporidiosis in Cuban children, vomiting was rare (7%) whereas abdominal pain was common (57%). These differences in expression of symptoms between studies may be age-related. As seen in other studies from similar countries, including those of the Caribbean and Latin America, C. hominis was found to predominate, the results of the successful molecular analyses revealing 10 C. hominis infections but no C. parvum. Subgenotyping (at the gp60 locus) indicated that the C. hominis infections included a wide range of subtypes, with isolates from three subtype families (Ia, Ib and Id) being detected.

Giardia infections in Cuban children: the genotypes circulating in a rural population.

Stool samples containing Giardia duodenalis cysts were collected from 95 primary-school children in central Cuba, and preserved by storing at -20 degrees C in 70% ethanol. Clinical data were collected for each child. Although 57% of the children were asymptomatic, the remaining 43% each reported between one and three symptoms. Following cyst quantification and isolation, molecular analyses were attempted on all cyst isolates, with the focus on the parasite's beta-giardin and glutamate-dehydrogenase (gdh) genes. Unfortunately, the cyst-preservation procedure appeared to have a deleterious effect on the cysts, since genotyping data could only be obtained for 20 of the 95 isolates. These data indicated, however, an approximately equal distribution between assemblage A (nine isolates) and assemblage B (11 isolates). Children found to be excreting relatively large numbers of cysts were more likely to be symptomatic than children who were excreting fewer cysts, and children with Giardia isolates from assemblage B were more likely to have symptomatic infections than children with isolates from assemblage A. Although considerable sequence variability was seen in the assemblage-B isolates, the assemblage-A isolates were relatively genetically homogeneous. This is the first publication from the Caribbean in which the Giardia genotypes circulating within the population have been identified, the first from the Americas providing information on associations between clinical presentation and the assemblage of the infecting Giardia, and the first to indicate that levels of cyst excretion may have clinical significance.

Development and use of a pepsin digestion method for analysis of shellfish for Cryptosporidium oocysts and Giardia cysts.

Investigation of shellfish for Cryptosporidium oocysts and Giardia cysts is of public health interest because shellfish may concentrate these pathogens in their bodies, and because shellfish are frequently eaten raw or lightly cooked. To date, the methods used for the analysis of shellfish for these parasites are based on those originally designed for water concentrates or fecal samples; the reported recovery efficiencies are frequently relatively low and the amount of sample examined is small. Here, we describe the development and use of a pepsin digestion method for analyzing shellfish samples for these parasites. The conditions of the isolation method did not affect subsequent parasite detection by immunofluorescent antibody test, and allowed examination of 3-g samples of shellfish homogenate, with recovery efficiencies from blue mussel homogenates of between 70 and 80%, and similar recoveries from horse mussel and oyster homogenates. Although exposure of the parasites to the conditions used in the technique affected their viability, as assessed by vital dyes, the maximum reduction in viability after 1-h incubation in digestion solution was 20%. In a preliminary survey of shellfish collected from the Norwegian coast, Cryptosporidium oocysts were detected in blue mussel homogenates in 6 (43%) of 14 batches and Giardia cysts in 7 (50%) of these batches. However, this relatively high occurrence, compared with other surveys, may be due to the higher recovery efficiency of the new method, and the relatively large sample size analyzed. A more comprehensive study of the occurrence of these parasites in shellfish would be of pertinence to the Norwegian shellfish industry.

Family association between immune parameters and resistance to Aeromonas hydrophila infection in the Indian major carp, Labeo rohita.

Seven innate immune parameters were investigated in 64 full-sib families (the offspring of 64 sires and 45 dams) from two year-classes of farmed rohu carp (Labeo rohita). Survival rates were also available from Aeromonas hydrophila infection (aeromoniasis) recorded in controlled challenge tests on a different sample of individuals from the same families. Due to strong confounding between the animal additive genetic effect and the family effects (common environmental+non-additive genetic), reliable additive (co)variance components and hence heritabilities and genetic correlations could not be obtained for the investigated parameters. Therefore, estimates of the association of challenge test survival with the studied immune parameters were obtained as product moment correlations between family least square means. These correlations revealed statistically significant (p<0.05) negative correlations of survival with bacterial agglutination titre (-0.48), serum haemolysin titre (-0.29) and haemagglutination titre (-0.34); and significant positive correlation with ceruloplasmin level (0.51). The correlations of survival to aeromoniasis with myeloperoxidase activity, superoxide production and lysozyme activity were found to be not significantly different from zero (p>0.05). Assuming that the negatively correlated candidate traits are not favourable as indirect selection criteria, the results suggest that ceruloplasmin level could potentially be a marker for resistance to aeromoniasis in rohu. The use of this immune parameter as an indirect selection criterion for increased resistance to aeromoniasis in rohu will, however, require that the parameter shows significant additive genetic variation and a significant genetic correlation with survival. Further studies are therefore needed to obtain a reliable heritability estimate for ceruloplasmin and its genetic correlation with survival from aeromoniasis.

Giardia cysts in sewage influent in Bergen, Norway 15-23 months after an extensive waterborne outbreak of giardiasis.

AIMS: In autumn/winter 2004, a large outbreak of waterborne giardiasis occurred in Bergen, Norway. Over 1 year later, the concentrations and genotypes of Giardia cysts occurring in sewage influent were studied to investigate the impact of the outbreak event on Giardia infections in the community. METHODS AND RESULTS: Sewage influent samples from four sewage treatment works (STW) serving Bergen were analysed for Giardia cysts on four occasions between 15 and 23 months after the outbreak. Cysts genotypes were investigated at one to three genes. Data from influent analysis from one of the STW before the outbreak, and from patient faecal samples analysed during the outbreak, provided baseline comparative data. Relatively high concentrations of Giardia cysts of diverse genotypes, both from Assemblages A and B, were detected at all STW. CONCLUSIONS: Comparison of data suggests that although Giardia cyst concentrations in sewage influent returned to pre-outbreak levels within 18 months after the outbreak peak, the genetic composition of the isolates remained significantly influenced by the Assemblage B isolate associated with the outbreak. SIGNIFICANCE AND IMPACT OF THE STUDY: Genotypes associated with an extensive outbreak of giardiasis continued to occur in Giardia infections in Bergen's population many months after the outbreak was considered to be over.

Giardia duodenalis cysts isolated from wild moose and reindeer in Norway: genetic characterization by PCR-rflp and sequence analysis at two genes.

There are few genotyping studies of Giardia duodenalis isolates from cervid hosts, although a previous study suggested that cervids may be a source of infection for humans and cattle. Giardia duodenalis isolates collected from wild moose (Alces alces) and reindeer (Rangifer tarandus) in Norway during 2002 and 2003 were characterized by polymerase chain reaction-restriction fraction length polymorphism (PCR-RFLP) at the beta-giardin gene, and sequence analysis at both the beta-giardin and glutamate dehydrogenase (gdh) genes. All results suggested that these isolates (n=25) belonged to assemblage A. Three different restriction patterns were obtained with PCR-RFLP, one of which has previously been associated with assemblage A. At the beta-giardin gene, sequences from six reindeer isolates and one moose isolate were identical to a previously published assemblage A sequence from G. duodenalis cysts isolated from dairy calves. The other 10 moose isolates could be divided into five groups, with between two and 14 single nucleotide polymorphisms (SNPs) from the published genotype A2. At the gdh gene, three different sequences were obtained, differing from each other by between one and 15 SNPs and which have all been previously published as genotype A1, but with different specific hosts. Grouping of the isolates based on the sequences from both genes gave complex results; whereas all the G. duodenalis isolates from reindeer grouped together, two moose isolates, which had identical sequences at the beta-giardin gene, had sequences that differed from each other by 15 SNPs at the gdh gene. The results of these studies, together with the large Norwegian populations of these cervids and the amount of fecal matter they produce, indicate that moose and reindeer may be significant reservoirs of G. duodenalis infection in Norway, which may be of importance to veterinary and public health.

First record of natural Tritrichomonas foetus infection of the feline uterus.

Tritrichomonas foetus was found in the uterus of a cat with pyometra and in the faeces of three other cats in the same household, one of which had chronic diarrhoea. This is the first report of a feline uterine infection with T. foetus and also the first time T. foetus has ever been diagnosed in animals in Norway. The diagnosis was made by microscopic examination and sequencing studies.

Molecular characterisation of Giardia isolates from clinical infections following a waterborne outbreak.

OBJECTIVES: During autumn/winter 2004/2005 an outbreak of waterborne giardiasis occurred in Bergen, Norway. Genetic characterisation at 2 genes of Giardia duodenalis isolates from samples from the outbreak peak showed significant variations between isolates. Characterisation of further isolates from patients diagnosed in the subsequent months was conducted to determine whether isolates with particular sequences might predominate, or whether the sequence variation would continue. METHODS: Genetic characterisation was conducted on 63 isolates from patients diagnosed in the 12 months subsequent to the outbreak peak. RESULTS: At the beta-giardin gene and glutamate dehydrogenase gene, particular isolate sequences within Assemblage B, gradually predominated over time. These sequences had not been the most frequently identified amongst 21 isolates from the outbreak peak. Nor were they apparently associated with a particular sequence at the triose phosphate isomerase gene. CONCLUSIONS: The predominance of particular sequences at the beta-giardin gene and glutamate dehydrogenase gene over time suggests that these sequences may be associated with enhanced transmission characteristics such as higher virulence, greater cyst environmental resistance, increased proliferation, or a combination of these factors. Alternatively greater association with clinical disease may have led to increased submission of samples with these sequences. Whether these sequences may be associated with particular symptom characteristics such as overt clinical disease, infection persistence or unresponsiveness to treatment warrants further study.

Fate of Cryptosporidium oocysts and Giardia cysts in the Norwegian aquatic environment over winter.

We investigated the survival of Cryptosporidium oocysts and Giardia cysts during winter in an aquatic environment (approximate temperature measurements between 1 and 7 degrees C) in Norway, using morphology and uptake of dyes as indicators of viability. Previous research has shown that in the terrestrial environment, shear forces caused by freeze and thaw cycles probably cause the parasites to be inactivated. Such forces occurred infrequently in the aquatic environment, as freezing of the water around the parasites was not observed during the study period (although freezing of the water surface did occur). The rate of decline in viability (log(10) N (t)/N (0)) was similar in control and experimental environments for both parasites; no Cryptosporidium oocysts with viable morphology were detected after approximately 20 weeks and no Giardia cysts with apparently viable morphology could be detected after 1 month. These results suggest that infection with these parasites in Norway is not usually from transmission stages that have over-wintered in the Norwegian environment.

Occurrence of Cryptosporidium oocysts and Giardia cysts in sewage in Norway.

Samples of sewage influent from 40 sewage treatment works (STW) throughout Norway were examined for Cryptosporidium oocysts and Giardia duodenalis cysts. Both parasites were detected frequently (80% of STW were Cryptosporidium positive; 93% of STW were Giardia positive) and at maximum concentrations of > 20,000 parasites/liter. The data suggest giardiasis is more widespread, and/or occurs with greater infection intensity, than cryptosporidiosis in Norway. STW serving higher person equivalents were more likely to be positive and had higher parasite concentrations. Parasite concentrations were used to estimate the proportion of contributing populations that could be clinically infected. For Cryptosporidium, the highest estimates were up to 5 per 100,000 individuals for two populations in eastern Norway. For Giardia, the highest estimate was 40 infected per 100,000 persons (approximately five times the usual national annual average) contributing to an STW in western Norway. As this population experienced a large waterborne giardiasis outbreak 6 months after sampling, it can be speculated that regular challenge with Giardia may occur here. Most Giardia isolates in sewage influent were assemblage A, although some assemblage B isolates were detected. There was substantial heterogeneity, but most samples contained isolates similar to genotype A3. Removal efficiencies at two STW with secondary treatment processes were estimated to be approximately 50% for Cryptosporidium and > 80% for Giardia. An STW with minimal treatment had negligible removal of both parasites. Many STW in Norway have minimal treatment and discharge effluent into rivers and lakes, thus, risk of contamination of water courses by Cryptosporidium and Giardia is considerable.

Cryptosporidium parvum infections in Bergen, Norway, during an extensive outbreak of waterborne giardiasis in autumn and winter 2004.

During a large waterborne giardiasis outbreak in Norway, many diarrheic patients were found to have Cryptosporidium infections. Gene sequencing identified these infections as Cryptosporidium parvum infections, although they were not identical. Whether these infections were due to a simultaneous outbreak of waterborne cryptosporidiosis or reflected background levels not normally detected is discussed.

Application of genotyping during an extensive outbreak of waterborne giardiasis in Bergen, Norway, during autumn and winter 2004.

During the autumn and winter of 2004 and 2005, an extensive outbreak of waterborne giardiasis occurred in Bergen, Norway. Over 1,500 patients were diagnosed with giardiasis. Analysis of water from the implicated source revealed low numbers of Giardia cysts, but the initial contamination event probably occurred up to 10 weeks previously. While sewage leakage from a residential area is now considered to be the probable source of contamination, during the episode waste from one particular septic tank was thought to be a possible source. Genotyping of cysts from the septic tank demonstrated that they were assemblage A cysts, although the sequences were not identical to any previously published sequences. For the beta-giardin gene, the closest published subgenotype was subgenotype A3; for the gdh gene, the closest published subgenotype was subgenotype A2. Genotyping of cysts from 21 patient samples revealed that they were assemblage B cysts; thus, the septic tank was unlikely to be the contamination source. Sequencing of the beta-giardin and gdh genes from patient samples and a comparison of the sequences gave complex results. For the beta-giardin gene, three isolates had sequences identical to subgenotype B3 sequences. However, other isolates had between one and four single-nucleotide polymorphisms (SNPs). For the gdh gene, none of the sequences were identical to the sequence published for subgenotype B3, and the sequences had between one and three SNPs. One isolate, which was identical to subgenotype B3 at the beta-giardin gene, was more similar to subgenotype B2 at the gdh gene. Grouping the isolates on the basis of SNPs resulted in different groups for the two genes. The results are discussed in relation to giardiasis in Norway and to other Giardia genotyping studies.

The potential for acquiring cryptosporidiosis or giardiosis from consumption of mung bean sprouts in Norway: a preliminary step-wise risk assessment.

The current work evolved from a microbial survey of fruits and vegetables conducted in Norway between 1999 and 2001. This survey found that mung bean sprouts were more likely to be contaminated with Cryptosporidium and Giardia than the other produce included in the survey. To support this observation and to demonstrate to public health officials that this might be a risk warranting further attention, a simple risk assessment was initiated. Assuming that 60,000 people in Norway consume a single serving of bean sprouts per week, and contamination levels are similar to those found in the survey, it was calculated that there could be in the order of 20 cases of Giardia or Cryptosporidium infection per 100,000 population attributable to consumption of mung bean sprouts. A number of assumptions were made for the calculations, including parasite factors (e.g. viability, genotype), product factors (e.g. extent of product contamination) and host factors (e.g. composition and extent of consumer group). These assumptions and areas of uncertainty, where further data would improve the risk assessment, are highlighted throughout. Not only does the risk assessment identify new areas of research, but it also demonstrates how risk assessment can be used as a tool to try to influence public health surveillance.

Full scale validation of helminth ova (Ascaris suum) inactivation by different sludge treatment processes.

The Norwegian sewage sludge regulation requires disinfection (hygienisation) of all sludges for land application, and one of the criteria is that disinfected sludge should not contain viable helminth ova. All disinfection processes have to be designed and operated in order to comply with this criterion, and four processes employed in Norway (thermophilic aerobic pre-treatment, pre-pasteurisation, thermal vacuum drying in membrane filter presses and lime treatment) have been tested in full scale by inserting semipermeable bags of Ascaris suum eggs into the processes for certain times. For lime treatment supplementary laboratory tests have been conducted. The paper presents the results of the experiments, and it could be concluded that all processes, except lime treatment, could be operated at less stringent time-temperature regimes than commonly experienced at Norwegian plants today.

Effects of the Norwegian winter environment on Giardia cysts and Cryptosporidium oocysts.

The structural integrity of Cryptosporidium oocysts and Giardia cysts in the Norwegian winter environment was investigated. During winter 2001/2002, Cryptosporidium oocysts and Giardia cysts were placed in the upper layers of soil in different matrices contained in chambers and exposed to the Norwegian climate. Morphological characteristics and inclusion/exclusion of vital dyes were monitored and compared to refrigerated controls. Reduction in parasite numbers was recorded for all parasites, geographical locations, and matrices. Shear forces generated during freeze-thaw cycles are postulated to have disintegrated the parasites exposed to the Norwegian winter and retrospective laboratory studies support this theory. Increased dye inclusion, possibly indicative of viability loss, was also noted. The refrigerated control parasites exhibited no decline in numbers, and alteration in dye inclusion characteristics for refrigerated parasites was slower. Cryptosporidium oocysts were apparently more robust than Giardia cysts; differences between isolates were also noted. These results suggest Cryptosporidium oocysts and Giardia cysts do not persist in the Norwegian terrestrial environment over winter, and when detected, will have been excreted since the previous winter. Differences in the morphological characteristics, matrix effects, and the possible relationship of the dye data to parasite survival are discussed in relation to further studies.

Occurrence of parasites on fruits and vegetables in Norway.

Between August 1999 and January 2001, samples of various fruits and vegetables obtained within Norway were analyzed by published methods for parasite contamination. Neither Cyclospora oocysts nor Ascaris (or other helminth) eggs were detected on any of the samples examined for these parasites. However, of the 475 samples examined for Cryptosporidium oocysts and Giardia cysts, 29 (6%) were found to be positive. No samples were positive for both parasites. Of the 19 Cryptosporidium-positive samples. 5 (26%) were in lettuce, and 14 (74%) in mung bean sprouts. Of the 10 Giardia-positive samples, 2 (20%) were in dill, 2 (20%) in lettuce, 3 (30%) in mung bean sprouts, 1 (10%) in radish sprouts, and 2 (20%) in strawberries. Mung bean sprouts were significantly more likely to be contaminated with Cryptosporidium oocysts or Giardia cysts than the other fruits and vegetables. Concentrations of Cryptosporidium and Giardia detected were generally low (mean of approximately 3 [oo]cysts per 100 g produce). Although some of the contaminated produce was imported (the majority, if sprouted seeds are excluded), there was no association between imported produce and detection of parasites. Crvptosporidium oocysts and Giardia cysts were also detected in water samples concerned with field irrigation and production of bean sprouts within Norway. This is the first time that parasites have been detected on vegetables and fruit obtained in a highly developed. wealthy country, without there being an outbreak situation. These findings may have important implications for global food safety.

Factors affecting recovery efficiency in isolation of Cryptosporidium oocysts and Giardia cysts from vegetables for standard method development.

While recently published techniques for recovering parasites from fruits and vegetables demonstrate a marked increase in efficiency and utility, there is still scope for further improvement in developing a standard method, particularly with difficult, but important, sample matrices such as bean sprouts. Herein, a number of parameters used in published techniques are investigated more closely. While sample size reduction may improve recovery efficiency because of a range of factors, it is important to keep the sample large enough for detection of low-level contamination. Age of sample is also important, and samples should be as fresh as possible. Elution procedures may contribute to losses of Giardia and should be more thoroughly investigated. Improved immunomagnetic separation techniques currently coming onto the market also have the potential to increase recovery efficiency substantially, even with difficult samples such as aged bean sprouts. However, merely increasing magnetic strength of the capturing magnet does not affect recovery efficiency, which must be reliant on a superior bead system, buffering system, or both.