Impact of transport conditions and underlying disease on post-stocking survival of juvenile Lates calcarifer.

Diseases caused by pathogens commonly occurring in the aquatic environment or those that are non-host specific are prevalent and threaten the rapid growth of tropical aquaculture. This study investigates causes of mortality in 12 batches of newly stocked juvenile Lates calcarifer from three different hatcheries. Cytology based on Diff-Quik™-stained tissue and blood smears provides rapid diagnosis of possible causes of mortality, while histopathology and haematology provide a better understanding of how prolonged transport and fish with existing chronic disease are more likely to experience elevated mortality post-stocking. Our findings showed that accumulation of ammonia during prolonged transport causes extensive damage to epithelial barriers in gastrointestinal tracts and depressed immunity due to marked hypoglycaemia, predisposing fish to acute Streptococcosis. Lates calcarifer with chronic bacterial enteritis developed severe hypoglycaemia, had low circulating total plasma protein, and suffered high mortality within 24 hours post-stocking. Hypoglycaemia and low circulating blood proteins disrupt osmoregulation and exacerbate dehydration, which is fatal in fish in sea water. Dying L. calcarifer tested PCR positive for scale drop disease virus (SDDV) at 28 days post-stocking showed a 10-fold elevation of white blood cell counts, severe vasculitis, and obstruction of blood supply to major organs. Destruction of important immune organs such as spleen is a hallmark of SDDV infection that explains high incidences of opportunistic Vibrio harveyi infections in 61% of fish with SDDV. Overall, this study reiterates the importance of stocking disease-free fish and reducing transport stress.

The haematology of clinically healthy, farmed juvenile Asian seabass (Lates calcarifer Bloch)-reference intervals, and indicators of subclinical disease.

This study establishes the blood reference intervals (RIs) for clinically healthy and farmed juvenile Asian seabass (Lates calcarifer), within 4-6 weeks after stocking into flow-through, marine aquaculture systems. The 90% percentile RIs (n = 156, mean bodyweight 41.8 g) are as follows: glucose (GLU) 2.4-11.3 mmol/L, haematocrit (Hct) 18.9%-39.2%, haemoglobin concentration (Hb) 56.0-85.0 g/L, total plasma protein (TPP) 56.0-77.0 g/L, total red blood cell (RBC) count 4.1-11.2 × 1012 /L, total white blood cell (WBC) count 5.3-69.9 × 109 /L, total lymphocytes 4.7-51.4 × 109 /L, monocytes 0.3-16.2 × 109 /L and heterophils count 0.6-8.4 × 109 /L. Pearson's method analysis showed weak but significantly positive correlations between fish bodyweight and Hct, Hb, TPP and total RBC count (p < 0.05). Histopathology of 42 of the 156 clinically healthy fish used to derive the RIs, with blood values within the 90% percentile range, did not exhibit any abnormal pathology. In contrast, histopathology from a different group of clinically healthy L. calcarifer (n = 72, mean bodyweight 31.3 g) with blood values falling outside of these established 90% percentile RIs showed that 25% of these fish had severe, chronic granulomatous enteritis, and 13% had severely depleted lipid stores in their liver. Point biserial correlation analysis of blood values from this second group of 72 fish showed that elevated total WBC, monocyte and heterophil counts and reduced Hct levels are significantly associated (p < 0.05) with the occurrence of severe, chronic granulomatous enteritis and depleted lipid stores in their liver. Reduced blood GLU and TPP levels in the second group of fish were significantly associated with fish that had depleted lipid stores in liver (p < 0.05), corroborating a period of malnutrition. This study is among the first to establish blood RIs for clinically healthy, farmed juvenile L. calcarifer and detection of subclinical diseases in fish to support early intervention.

PCR, in-situ hybridization, and phylogenetic analysis suggest that 'big belly' disease in barramundi, Lates calcarifer (Bloch), is caused by a novel Vibrio species.

'Big belly' disease is a chronic, granulomatous bacterial enteritis and peritonitis, first reported in 3- to 4-week-old Asian seabass or barramundi, Lates calcarifer Bloch fry. Affected fry are emaciated and have a swollen abdomen, and the condition is referred to as 'skinny pot-belly' or 'big belly' disease. In this study, histopathological examinations of diseased fish from a batch of 2-month-old, 6- to 8-cm L. calcarifer fingerlings, kept in seawater recirculating aquaculture systems, showed pathology resembling 'big belly' disease. Ethanol-fixed tissues were tested positive using specific PCR primers based on 16S rRNA genes. In situ hybridization using dioxygenin-labelled positive PCR products on formalin-fixed paraffin-embedded tissues showed positive reactions with intralesional, clusters of the large, 'big belly' coccobacilli. A phylogenetic tree constructed based on analyses of these 16S rRNA gene PCR products from five positive fish suggests that the 'big belly' bacterium is most likely a novel Vibrio species.

Scale Drop Disease Virus (SDDV) and Lates calcarifer Herpes Virus (LCHV) Coinfection Downregulate Immune-Relevant Pathways and Cause Splenic and Kidney Necrosis in Barramundi Under Commercial Farming Conditions.

Marine farming of barramundi (Lates calcarifer) in Southeast Asia is currently severely affected by viral diseases. To better understand the biological implications and gene expression response of barramundi in commercial farming conditions during a disease outbreak, the presence of pathogens, comparative RNAseq, and histopathology targeting multiple organs of clinically "sick" and "healthy" juveniles were investigated. Coinfection of scale drop disease virus (SDDV) and L. calcarifer herpes virus (LCHV) were detected in all sampled fish, with higher SDDV viral loads in sick than in healthy fish. Histopathology showed that livers in sick fish often had moderate to severe abnormal fat accumulation (hepatic lipidosis), whereas the predominant pathology in the kidneys shows moderate to severe inflammation and glomerular necrosis. The spleen was the most severely affected organ, with sick fish presenting severe multifocal and coalescing necrosis. Principal component analysis (PC1 and PC2) explained 70.3% of the observed variance and strongly associated the above histopathological findings with SDDV loads and with the sick phenotypes, supporting a primary diagnosis of the fish being impacted by scale drop disease (SDD). Extracted RNA from kidney and spleen of the sick fish were also severely degraded likely due to severe inflammation and tissue necrosis, indicating failure of these organs in advanced stages of SDD. RNAseq of sick vs. healthy barramundi identified 2,810 and 556 differentially expressed genes (DEGs) in the liver and muscle, respectively. Eleven significantly enriched pathways (e.g., phagosome, cytokine-cytokine-receptor interaction, ECM-receptor interaction, neuroactive ligand-receptor interaction, calcium signaling, MAPK, CAMs, etc.) and gene families (e.g., tool-like receptor, TNF, lectin, complement, interleukin, chemokine, MHC, B and T cells, CD molecules, etc.) relevant to homeostasis and innate and adaptive immunity were mostly downregulated in sick fish. These DEGs and pathways, also previously identified in L. calcarifer as general immune responses to other pathogens and environmental stressors, suggest a failure of the clinically sick fish to cope and overcome the systemic inflammatory responses and tissue degeneration caused by SDD.

Haematozoa of wild catfishes in northern Australia.

Very little is known about the diversity, prevalence, or pathogenicity of haematozoa in Australian freshwater fishes. Blood smears from 189 native catfishes, of six different species, from northern Australia were examined for haematozoa. Haematozoan infections were observed only in fishes from Queensland, at an overall prevalence of 0.191 (95% CI = 0.134-0.265). Intraerythrocytic haemogregarines were present in Neoarius graeffei from the Brisbane River at a prevalence of 0.35 (0.181-0.567). Trypanosomes were present in Tandanus species from four rivers, at prevalences ranging from 0.111 (0.020-0.330) to 1 (0.635-1), and in N. graeffei from one river in Queensland, at a prevalence of 0.063 (0.003-0.305). The haematozoans observed appeared to have little impact on their hosts. Tandanus spp. were significantly more likely to be infected with trypanosomes, suggesting a high parasite-host specificity. This is the first widespread survey of wild Australian freshwater catfishes for haematozoa, resulting in the first report of haemogregarines from Australian freshwater fish, and the first report of trypanosomes from Neoarius graeffei and Tandanus tropicanus.

Cryptosporidium in fish: alternative sequencing approaches and analyses at multiple loci to resolve mixed infections.

Currently, the systematics, biology and epidemiology of piscine Cryptosporidium species are poorly understood. Here, we compared Sanger ‒ and next-generation ‒ sequencing (NGS), of piscine Cryptosporidium, at the 18S rRNA and actin genes. The hosts comprised 11 ornamental fish species, spanning four orders and eight families. The objectives were: to (i) confirm the rich genetic diversity of the parasite and the high frequency of mixed infections; and (ii) explore the potential of NGS in the presence of complex genetic mixtures. By Sanger sequencing, four main genotypes were obtained at the actin locus, while for the 18S locus, seven genotypes were identified. At both loci, NGS revealed frequent mixed infections, consisting of one highly dominant variant plus substantially rarer genotypes. Both sequencing methods detected novel Cryptosporidium genotypes at both loci, including a novel and highly abundant actin genotype that was identified by both Sanger sequencing and NGS. Importantly, this genotype accounted for 68·9% of all NGS reads from all samples (249 585/362 372). The present study confirms that aquarium fish can harbour a large and unexplored Cryptosporidium genetic diversity. Although commonly used in molecular parasitology studies, nested PCR prevents quantitative comparisons and thwarts the advantages of NGS, when this latter approach is used to investigate multiple infections.

Genetic diversity of Cryptosporidium in fish at the 18S and actin loci and high levels of mixed infections.

Cryptosporidium is an enteric parasite that infects humans and a wide range of animals. Relatively little is known about the epidemiology and taxonomy of Cryptosporidium in fish. In the present study, a total of 775 fish, belonging to 46 species and comprising ornamental fish, marine fish and freshwater fish were screened for the prevalence of Cryptosporidium by PCR. The overall prevalence of Cryptosporidium in fish was 5.3% (41/775), with prevalences ranging from 1.5 to 100% within individual host species. Phylogenetic analysis of these Cryptosporidium isolates as well as 14 isolates from previous studies indicated extensive genetic diversity as well as evidence for mixed infections. At the 18S locus the following species were identified; Cryptosporidium molnari-like genotype (n=14), Cryptosporidium huwi (n=8), piscine genotype 2 (n=4), piscine genotype 3-like (n=1), piscine genotype 4 (n=2), piscine genotype 5 (n=13), piscine genotype 5-like (n=1) and five novel genotypes (n=5). At the actin locus, species identification agreed with the 18S locus for only 52.3% of isolates sequenced, indicating high levels of mixed infections. Future studies will need to employ both morphological characterization and deep sequencing amplicon-based technologies to better understand the epidemiological and phylogenetic relationships of piscine-derived Cryptosporidium species and genotypes, particularly when mixed infections are detected.

Cryptosporidium huwi n. sp. (Apicomplexa: Eimeriidae) from the guppy (Poecilia reticulata).

The morphological, biological, and molecular characteristics of Cryptosporidium piscine genotype 1 from the guppy (Poecilia reticulata) are described, and the species name Cryptosporidium huwi n. sp. is proposed to reflect its genetic and biological differences from gastric and intestinal Cryptosporidium species. Oocysts of C.huwi n. sp. over-lap in size with Cryptosporidium molnari, measuring approximately 4.4-4.9 µm (mean 4.6) by 4.0-4.8 µm (mean 4.4 µm) with a length to width ratio of 1.04 (0.92-1.35) (n = 50). Similar to C.molnari, C.huwi n. sp. was identified in the stomach only and clusters of oogonial and sporogonial stages were identified deep within the epithelium. However, phylogenetic analysis of 18S rRNA sequences indicated that C. huwi n. sp. exhibited 8.5-9.2% and 3.5% genetic distance from C.molnari isolates and piscine genotype 7 respectively. At the actin locus, the genetic distance between C.huwi n. sp. and C.molnari was 16.6%. The genetic distance between C.huwi n. sp. and other Cryptosporidium species at the 18S locus was 13.2%-17% and at the actin locus was 18.9%-26.3%. Therefore C. huwi n. sp. is genetically distinct from previously described Cryptosporidium species.