Thiamin dynamics during the adult life cycle of Atlantic salmon (Salmo salar).

Thiamin is an essential water-soluble B vitamin known for its wide range of metabolic functions and antioxidant properties. Over the past decades, reproductive failures induced by thiamin deficiency have been observed in several salmonid species worldwide, but it is unclear why this micronutrient deficiency arises. Few studies have compared thiamin concentrations in systems of salmonid populations with or without documented thiamin deficiency. Moreover, it is not well known whether and how thiamin concentration changes during the marine feeding phase and the spawning migration. Therefore, samples of Atlantic salmon (Salmo salar) were collected when actively feeding in the open Baltic Sea, after the sea migration to natal rivers, after river migration, and during the spawning period. To compare populations of Baltic salmon with systems without documented thiamin deficiency, a population of landlocked salmon located in Lake Vänern (Sweden) was sampled as well as salmon from Norwegian rivers draining into the North Atlantic Ocean. Results showed the highest mean thiamin concentrations in Lake Vänern salmon, followed by North Atlantic, and the lowest in Baltic populations. Therefore, salmon in the Baltic Sea seem to be consistently more constrained by thiamin than those in other systems. Condition factor and body length had little to no effect on thiamin concentrations in all systems, suggesting that there is no relation between the body condition of salmon and thiamin deficiency. In our large spatiotemporal comparison of salmon populations, thiamin concentrations declined toward spawning in all studied systems, suggesting that the reduction in thiamin concentration arises as a natural consequence of starvation rather than to be related to thiamin deficiency in the system. These results suggest that factors affecting accumulation during the marine feeding phase are key for understanding the thiamin deficiency in salmonids.

Characterization of a Novel Infectious Pancreatic Necrosis Virus (IPNV) from Genogroup 6 Identified in Sea Trout (Salmo trutta) from Lake Vänern, Sweden.

In November 2016, infectious pancreatic necrosis virus (IPNV) was isolated from a broodstock female of landlocked sea trout (Salmo trutta) in Lake Vänern in Sweden. VP2 gene sequencing placed the IPNV isolate in genogroup 6, for which pathogenicity is largely unknown. Lake Vänern hosts landlocked sea trout and salmon populations that are endangered, and thus the introduction of new pathogens poses a major threat. In this study we characterized the novel isolate by conducting an infection trial on three salmonid species present in Lake Vänern, whole genome sequencing of the isolate, and prevalence studies in the wild sea trout and salmon in Lake Vänern. During the infection trial, the pathogenicity of the Swedish isolate was compared to that of a pathogenic genogroup 5 isolate. Dead or moribund fish were collected, pooled, and analyzed by cell culture to identify infected individuals. In the trial, the Swedish isolate was detected in fewer sample pools in all three species compared to the genogroup 5 isolate. In addition, the prevalence studies showed a low prevalence (0.2-0.5%) of the virus in the feral salmonids in Lake Vänern. Together the data suggest that the novel Swedish IPNV genogroup 6 isolate is only mildly pathogenic to salmonids.

Non-lethal sampling for the detection of Renibacterium salmoninarum by qPCR for diagnosis of bacterial kidney disease.

Bacterial kidney disease (BKD), caused by Renibacterium salmoninarum (Rs), can be transmitted both horizontally and vertically and there is no available cure or prophylaxis. The control of BKD requires continuous surveillance, which is challenging in aquaculture as well as in programs for conservation and restoration of salmonid fish strains. BKD is a notifiable disease in Sweden and is monitored through the mandatory health control program using a polyclonal ELISA for detection of the Rs p57 protein in kidney. Fish must be killed for sampling, an obvious disadvantage especially regarding valuable broodfish. The present study shows that gill-/cloacal swabs collected in vivo for real-time PCR (qPCRgc ), allow a sensitive and specific detection of Rs. The sensitivity of qPCRgc was estimated to 97.8% (credible interval (ci) 93.8%-100%) compared to 98.3% (ci 92.7%-100%) and 48.8% (ci 38.8%-58.8%) of kidney samples for qPCR (qPCRk ) and ELISA (ELISAk ) respectively, by use of the Bayesian Latent Class Analysis (BLCA). Since the goal of the program is eradication of BKD the most sensitive test is preferrable. Using qPCRgc instead of ELISAk will result in a lower false negative rate and can be useful for surveillance in aquaculture and in breeding programs with valuable fish. However, a higher false positive rate warrants confirmatory lethal testing before a previously Rs negative farm is subject to restrictions.

Assessing the presence and spread of Renibacterium salmoninarum between farmed and wild fish in Sweden.

Bacterial kidney disease (BKD) can be a devastating bacterial infection in salmonids, and it is present in aquaculture throughout the world. BKD is caused by the Gram-positive facultative intracellular bacterium Renibacterium salmoninarum (R. salmoninarum) that is spread both horizontally and vertically. Disease signs include external ulcerations and blisters and internal signs such as organ swelling, granulomas, petechiae and ascites. In Sweden, BKD accounts for a significant income loss in aquacultures due to expensive decontamination of the facility and increased disease susceptibility for the immunocompromised fish leading to higher mortality rates. In addition, uncontrolled spread in aquaculture may threaten the survival of wild fish populations. The aim of our study was to investigate the prevalence of R. salmoninarum in wild salmonids caught in Swedish waters where net pen farms with a recent history of BKD are present. Four rivers with at least one BKD-positive or recently BKD-positive farm were selected. In addition, we evaluated the use of environmental DNA (eDNA) for surveillance and monitoring of ongoing infections at these locations. In total, 1058 fish were sampled from four different river systems, and of them 52 (4.9%) were positive for R. salmoninarum by antigen ELISA. Surprisingly, these fish were not evenly distributed between the four river systems, but 50 were caught in the same river (Ljungan). This accounts for an alarmingly high rate of 17% R. salmoninarum-positive samples in wild salmonids in this area. This number is far above what was expected and clearly shows the risk with an open farming system as well as the importance of effective health monitoring programmes to avoid an uncontrolled spread of the disease. The use of eDNA for monitoring BKD is somewhat difficult to evaluate. Few of the water samples analysed were PCR positive for R. salmoninarum (2 of 38) and those were collected where no ELISA positive fish were identified. In addition to water, sediment samples were collected under a net pen farm that had recently slaughtered all fish due to ongoing R. salmoninarum infections. Sediment samples are more promising than water as 4 of 5 samples at one farming facility where positive for R. salmoninarum. Thus, sediment samples may be valuable for monitoring potential ongoing BKD in farms, without the need to sacrifice valuable fish.

A multi-biomarker study on Atlantic salmon (Salmo salar L.) affected by the emerging Red Skin Disease in the Baltic Sea.

For half a decade, the Atlantic salmon in the Baltic Sea has been facing severe health issues. Clinical signs like haemorrhage, erosions and ulcerative/necrotic skin conditions in returning adults have been reported from different Swedish rivers. These primary disease signs precede a secondary, terminal fungal infection. As initial investigations of the disease did not provide conclusive answers regarding the pathogenesis, this study was initiated to gain insight into a possible link between this so-called Red Skin Disease and anthropogenic influences. Therefore, returning salmon were caught in rivers along the Swedish coast and different tissues were sampled. The focus was put on the measurements of a battery of biomarkers as well as biochemical and haematological parameters, which were analysed using multivariate statistics. The main findings were a severe osmotic haemodilution, an immune response and an alteration of the carbohydrate metabolism in diseased fish. Furthermore, oxidative stress does not seem to be a likely factor in the pathogenesis. Concluding, certain changes in physiological parameters were shown to be indicative for the disease patterns, while others were ruled out as significant factors. Thus, this study contributes to the understanding of the Red Skin Disease and may act as a hypothesis generator for future studies.

Presence and genetic variability of Piscine orthoreovirus genotype 1 (PRV-1) in wild salmonids in Northern Europe and North Atlantic Ocean.

Piscine orthoreovirus genotype 1 (PRV-1) is widespread in farmed Atlantic salmon (Salmo salar L.) populations in northern Europe, Canada and Chile. PRV-1 occurs in wild fish in Norway and Canada; however, little information of its geographical distribution in wild populations is currently available, and the effect of PRV-1 infection in wild populations is currently unknown. In this study, we present the findings of a survey conducted on 1,130 wild salmonids sampled in Denmark, Sweden, Ireland, Faroe Islands, France, Belgium and Greenland between 2008 and 2017. PRV-1 is reported for the first time in wild salmonids in Denmark, Sweden, Faroe Island and Ireland. The annual PRV-1 prevalence ranged from 0% in France, Belgium and Greenland to 43% in Faroe Islands. In total, 66 samples tested positive for PRV-1, including Atlantic salmon broodfish returning to spawn and Atlantic salmon collected at the feeding ground north of Faroe Islands. The phylogenetic analysis of S1 sequences of the PRV-1 isolates obtained in this survey did not show systematic geographical distribution. This study sheds light on the spread and genetic diversity of the virus identified in populations of free-living fish and provides rationale for screening wild broodfish used in restocking programmes.

High applicability of a novel method for gp60-based subtyping of Cryptosporidium meleagridis.

Cryptosporidium meleagridis is a common cause of cryptosporidiosis in avian hosts and the third most common species involved in human cryptosporidiosis. Sequencing of the highly polymorphic 60-kDa glycoprotein (gp60) gene is a frequently used tool for investigation of the genetic diversity and transmission dynamics of Cryptosporidium. However, few studies have included gp60 sequencing of C. meleagridis. One explanation may be that the gp60 primers currently in use are based on Cryptosporidium hominis and Cryptosporidium parvum sequence data, potentially limiting successful amplification of the C. meleagridis gp60 gene. We therefore aimed to design primers for better gp60 subtyping of C. meleagridis. Initially, ∼1,440 bp of the gp60 locus of seven C. meleagridis isolates were amplified using primers flanking the open reading frame. The obtained sequence data (∼1,250 bp) were used to design primers for a nested PCR targeting C. meleagridis. Twenty isolates (16 from human and 4 from poultry) previously identified as C. meleagridis by analysis of small subunit (SSU) rRNA genes were investigated. Amplicons of the expected size (∼900 bp) were obtained from all 20 isolates. The subsequent sequence analysis identified 3 subtype families and 10 different subtypes. The most common subtype family, IIIb, was identified in 12 isolates, represented by 6 subtypes, 4 new and 2 previously reported. Subtype family IIIe was found in 3 isolates represented by 3 novel, distinct subtypes. Finally, IIIgA31G3R1 was found in 1 human isolate and 4 poultry isolates, all originating from a previously reported C. meleagridis outbreak at a Swedish organic farm.

Cryptosporidium parvum IId family: clonal population and dispersal from Western Asia to other geographical regions.

In this study, 111 Cryptosporidium parvum IId isolates from several species of animals in China, Sweden, and Egypt were subtyped by multilocus sequence typing (MLST). One to eleven subtypes were detected at each of the 12 microsatellite, minisatellite, and single nucleotide polymorphism (SNP) loci, forming 25 MLST subtypes. Host-adaptation and significant geographical segregation were both observed in the MLST subtypes. A clonal population structure was seen in C. parvum IId isolates from China and Sweden. Three ancestral lineages and the same RPGR sequence were shared by these isolates examined. Therefore, the present genetic observations including the higher nucleotide diversity of C. parvum IId GP60 sequences in Western Asia, as well as the unique distribution of IId subtypes (almost exclusively found in Asia, Europe, and Egypt) and in combination with the domestication history of cattle, sheep, and goats, indicated that C. parvum IId subtypes were probably dispersed from Western Asia to other geographical regions. More population genetic structure studies involving various C. parvum subtype families using high-resolution tools are needed to better elucidate the origin and dissemination of C. parvum in the world.

Unusual cryptosporidiosis cases in Swedish patients: extended molecular characterization of Cryptosporidium viatorum and Cryptosporidium chipmunk genotype I.

Most human cases of cryptosporidiosis are caused by Cryptosporidium parvum or Cryptosporidium hominis, but the use of molecular diagnostic methods has revealed that several other less common species or genotypes can also be involved. Here, we describe two unusual causes of cryptosporidiosis, one being the recently described species Cryptosporidium viatorum and the other Cryptosporidium chipmunk genotype I. Two Swedish patients who were infected with C. viatorum had travelled to Kenya and Guatemala, respectively, and two others had been infected with Cryptosporidium chipmunk genotype I in Sweden. None of these four patients were immunocompromised, and all four showed classical symptoms of cryptosporidiosis. We performed extensive molecular characterization, including analysis of four loci. The two C. viatorum isolates were found to differ slightly at the 70-kDa heat shock protein locus, which may indicate a local geographical variation in this species that has previously been described exclusively on the Indian subcontinent.