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

Comparison of intestinal bacterial communities in asymptomatic and diseased Asian seabass (Lates calcarifer) with chronic enteritis and mixed bacterial infections

Asian seabass (Lates calcarifer) is a major aquaculture food fish species in Singapore. Farming of this species is increasingly threatened by frequent outbreaks of infectious diseases, resulting in mortality exceeding 50–70%. In this study, we investigated the comparative gut bacterial microbiota using 16S rRNA metasequencing between asymptomatic and diseased juvenile fish collected during a disease outbreak soon after stocking. Mild to severe chronic granulomatous enteritis was observed histopathologically in both asymptomatic and diseased fish. Kidneys of diseased fish tested PCR positive for the ‘big belly’ novel Vibrio spp., Streptococcus iniae and Vibrio harveyi. These bacteria were also readily detected by PCR in water samples corresponding to tanks fish were sampled from. Potentially beneficial microbes that promote gut health such as Firmicutes, Bacteroidota and Actinobacteriota were the dominant phyla in the intestinal microbiota of asymptomatic fish. Moreover, the bacteria with probiotic potential such as Lactobacillus only presented in asymptomatic fish, and Weissella was unique and prevalent (47.59%) in asymptomatic fish during the recovery phase of the disease outbreak, making them candidate biomarkers for monitoring health status of L. calcarifer. Conversely, diseased fish showed reduced diversity of their gut microbiome, with high abundance of members of the phylum Proteobacteria. Vibrio was the most dominant genus (87.3%) and Streptococcus iniae was only detected in diseased fish. These findings provide a baseline study for understanding changes in intestinal microbiota in newly stocked fish with mixed bacterial infection, biomarker assisted health monitoring, and future host-derived probiotics screening in L. calcarifer

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

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

Diseases of Asian seabass (or barramundi), Lates calcarifer Bloch

Other than the study by Griffiths (2009) on gill diseases, there has been no comprehensive study and report on the major diseases of Asian seabass (or barramundi) Lates calcarifer Bloch. It is a food fish species of growing importance in Asia and Australia. This study investigates some of the major diseases encountered in the various stages of the culture of L. calcarifer, at the histopathological, ultrastructural and molecular levels. Culture practices can have significant impacts on fish health. Disease outbreaks are influenced by factors involving the host, environment and pathogen. Current knowledge on diseases of L. calcarifer, and these factors which may influence disease outbreaks are discussed in Chapter 1. This is the first report of an intestinal Eimeria infection in L. calcarifer. The Eimeria infection was associated with severe pathology and significant mortality in the absence of other pathogens. It was detected in diseased L. calcarifer in all five nurseries in Ca Mau, Vietnam. Although these were small scale nurseries which stocked an average of 3000 to 5000 fish at any one time, a mortality rate of up to 30% was reported and is the cause of significant economic losses for these nurseries. Moderate to heavy Eimeria infestation were observed in greater than 80% of diseased fish examined. This high rate of Eimeria infestation is suspected to be linked to the low daily water exchange rates practised in these nurseries. However, the examination of only diseased fish does not allow the determination of prevalence. A systemic iridovirus infection was concurrently observed in some of the fishes but was not consistently present when compared to the Eimeria infection. Molecular analysis showed that the Eimeria of L. calcarifer from Vietnam formed clades with the Eimeria detected in L. calcarifer cultured in Australia, but clustered separately from other known Eimeria species. Although Cryptosporidium was detected in these L. calcarifer tissues, it could not be demonstrated histologically or ultrastructurally, suggesting a low grade infestation or perhaps an environmental contaminant in fish tissues tested. In situ hybridization using labeled PCR products showed that labeled DNA probes generated from 18S PCR products could not be used to distinguish between closely related genera such as Cryptosporidium and Eimeria. Future investigation to determine the origin, transmission and risk factors associated with this Eimeria infestation in L. calcarifer are needed. ‘Scale drop syndrome’ is a novel disease first reported in L. calcarifer in Penang, Malaysia in 1992. Cases with similar gross and clinical presentations were observed in Singapore in 2002, 2006 and 2009. Affected fish have loose scales, which dropped off easily when handled. The disease was initially observed in 100-300g fish, and later in larger fish up to 5kg bodyweight. Cumulative mortalities of 40 to 50% were reported by farms, posing significant economic losses of larger more valuable fish. This investigation forms the first pathological description of ‘scale drop syndrome’ (SDS) in L. calcarifer. To aid recognition of new cases for study, a case definition was developed for ‘scale drop syndrome’ in L. calcarifer as a systemic vasculitis associated with tissue necrosis in all major organs including the skin, with apparent targeting of cells of epithelial origin. Attempts to isolate or detect the causative agent(s) by cell culture, PCR and immunohistochemistry have proven unsuccessful. Further studies to elucidate the definitive aetiology, isolate the causal agent(s) and reproduce the disease will help better understanding and control of SDS. Although systemic iridoviral disease has been previously reported in many freshwater and marine fish species, this study forms the first report of this disease in L. calcarifer. Systemic iridoviral disease was observed in 5 to 20g L. calcarifer usually 2 to 3 weeks post-transfer into sea cages at two farms. Inclusion bodies suggestive of a systemic iridovirus infection were observed in clinically healthy L. calcarifer from the land-based nursery of one of these two farm; the presence of an iridovirus infection was supported by positive PCR results using Red Sea bream iridovirus (RSIV) primer 1. The presence of inclusions was not accompanied by any tissue necrosis in these clinically healthy fish. This finding suggested that the systemic iridovirus infection occurred before stocking at sea, and did not originate from wild fish or older fish in adjacent sea cages as initially suspected by this farm. Immunohistochemistry on tissues of clinical cases of systemic iridovirus gave positive results using the Red Sea bream iridovirus monoclonal antibody (RSIV M10), although intensity varied between tissues, possibly related to varying exposure of different tissues to fixation chemicals. Inclusion bodies in clinically healthy fish from the same farm did not show positive reaction with RSIV M10. This may be due to a lack of antigenic expression by the viral infected cells at this early stage of infection. Viral nervous necrosis (VNN) is a serious disease of hatchery reared L. calcarifer fry in this study. Mortalities of 50 to 100% were reported in 3wo fry. VNN can be difficult to diagnose in older fry, where it can be associated with few vacuolations or an absence of viral inclusions ‘Pot belly disease’ (PBD) was previously reported in L. calcarifer fry less than 1g, in association with an intracellular coccobacillus infection and mortalities of 80 to 100%. In this study, PBD was observed in 120g L. calcarifer at two sea cage farms, in association with significant granulomatous enteritis. The extent of the granulomatous enteritis is likely to have an effect on affected fish. It was observed concurrently with systemic iridoviral disease at one farm and nocardiosis at another farm. Diagnosis by histopathology and the lack of other confirmatory tests for PBD may result in underdiagnosis of this disease. The epidemiology of PBD needs further study to establish origin and modes of transmission, to facilitate better disease control. Diseases associated with infections by ubiquitous bacteria such as Vibrio, Tenacibaculum were commonly observed in L. calcarifer post-handling. Tenacibaculosis and vibriosis often occurred concurrently with other diseases such as streptococcosis, systemic iridviral disease or PBD. Streptococcosis can affect fish up to 3kg bodyweight, resulting in significant mortalities greater than 40 to 50%. Like SDS, because streptococcosis can affect up to market size fish, they can cause considerable economic losses. Although vaccines against Streptococcosis are available, conflicting views are held on the efficacy of Streptococcus vaccines by various research groups. Overall, the South-east Asian L. calcarifer farms which practiced vaccination against Streptococcus iniae reported a reduction of mortality, especially in fish greater than 1 to 1.5kg bodyweight. Nocardiosis has been reported as an emerging disease in marine food fish species caused by acid fast filamentous branching bacterium. Although nocardiosis was observed histopathologically in L. calcarifer at two sea cage farms, the numbers of samples examined were small and no other tests were attempted due to lack of suitable samples. More intensive and extensive study is needed to determine the significance of nocardiosis in L. calcarifer. Chronic granulomatous enteritis was not uncommon in the cases submitted to the Fish Health Laboratory in Perth. Although the peritonitis was associated with heavy bacteria infection, it is unclear if these are secondary invaders. Schipps, Bosmans & Humphreys (2009) reported that Vibrio harveyi and Photobacterium damsela damsela vaccinations appeared to be not efficacious, suggesting that these bacteria were not the primary cause of the disease. It is well recognized that disease outbreaks in farmed fish are influenced by the interaction between host, the environment and pathogens. While serious diseases are often reported in association with specific aquatic pathogens, not much is known about the risk factors which trigger fish disease outbreaks. Disease outbreaks often occur after stressful events such as net transfers, recent handling or poor water quality. In fact, diseases are often caused by ubiquitous pathogens that are commonly present in the culture environment. Although further research is necessary to gather more information to improve diagnosis and management of specific diseases, general health management strategies can be applied at the various stages in the culture of L. calcarifer to minimize disease outbreaks. This is discussed for L. calcarifer in Chapter 6. Observations of types of disease agents may be influenced by site conditions or the types of tests or materials examined. For example, some parasites may be more prevalent in certain sites where intermediate hosts abound, or loosely attached ectoparasites may be lost unless wet mount microscopic examinations of fresh tissues were carried out. The study of emerging diseases such as scale drop syndrome (SDS) or pot belly disease (PBD) in L. calcarifer has been hampered by lack of confirmatory diagnostic tools and inadequate knowledge on critical epidemiological factors such as mode of transmission or potential reservoirs. While ideally identification and isolation of the causal agent will help fulfil Koch’s postulates, it may be possible to improve the understanding of disease via cohabitation or infectivity trials using tissue homogenates from diseased fish when pure isolates are not available. There is a need to conduct research to not only establish a definitive aetiology, but also to identify risk factors to facilitate successful disease control. The successful management of disease in aquaculture does not lie in any one strategy but an integrated management of all risks encountered during the culture cycle against disease occurrence or incursions

The pathology associated with putative algal toxicosis in red snapper, Lutjanus species (Bloch 1790)

Reports of toxic algae-related fish kills are on the rise. Toxic algae may predispose to secondary infectious disease or poor growth, and have a significant impact on aquaculture (Andersen et al., 2016; Noga, 1998; Place et al., 2012). This case was presented for investigation into persistent low-grade mortality in red snapper, Lutjanus species Bloch, which did not respond to several therapeutic interventions. Histopathological examinations suggest exposure to toxic algae

A novel treatment against the monogenean parasite, Gyrodactylus turnbulii, infecting guppies (Poecilia reticulata), using a plant-based commercial insecticide Timor C

Monogenean infections are a common cause of fish morbidity and mortality in aquaculture. In most cases, treatment relies on the application of toxic or unapproved chemicals. Therefore, alternative treatments against monogenean infections in fish that are safe and environmentally friendly are needed. In the present study, the efficiency of Timor C, a plant-based commercial insecticide, was investigated against Gyrodactylus turnbulii, a monogenean parasite infecting guppies (Poecilia reticulata). Since G. turnbulii primarily infects the fins and skin, with a predilection for the caudal fin, tail clips of heavily parasitized guppies were exposed in vitro to Timor C at various concentrations and parasite detachment and death were recorded over time. There was a positive correlation between time to detachment and Timor C concentration, with complete detachment within 60 min of exposure at a concentration of 50 ppm. Withdrawal of Timor C afterward did not lead to recovery of the exposed parasites. Treatment of infected fish for 24 h at 10 and 20 ppm was effective in lab-based trials, reducing the infection prevalence from 100% at time 0 to 22% post-treatment, and the infection rate to an average of less than one parasite per fish, as compared to an average of about five parasites per fish in the control. Repeated weekly treatment application for three consecutive weeks appeared safe for the fish, and did not cause an adverse effect to the biofiltration system, as determined by examining nitrification activity, compared to non-treated controls. Treatment was successfully tested in three separate applications at a commercial closed recirculating guppy farm, where a significant reduction in infection rates was achieved. Timor C was not toxic to Artemia at concentrations ranging from 10 to 100 ppm over 24 h of exposure. This study demonstrates the effectiveness of Timor C as a treatment against G. turnbulii infection in guppies and potentially against similar parasites in other aquacultural species

A novel treatment against the monogenean parasite, Gyrodactylus turnbulii, infecting guppies (Poecilia reticulata), using a plant-based commercial insecticide Timor C

Monogenean infections are a common cause of fish morbidity and mortality in aquaculture. In most cases, treatment relies on the application of toxic or unapproved chemicals. Therefore, alternative treatments against monogenean infections in fish that are safe and environmentally friendly are needed. In the present study, the efficiency of Timor C, a plant-based commercial insecticide, was investigated against Gyrodactylus turnbulii, a monogenean parasite infecting guppies (Poecilia reticulata). Since G. turnbulii primarily infects the fins and skin, with a predilection for the caudal fin, tail clips of heavily parasitized guppies were exposed in vitro to Timor C at various concentrations and parasite detachment and death were recorded over time. There was a positive correlation between time to detachment and Timor C concentration, with complete detachment within 60 min of exposure at a concentration of 50 ppm. Withdrawal of Timor C afterward did not lead to recovery of the exposed parasites. Treatment of infected fish for 24 h at 10 and 20 ppm was effective in lab-based trials, reducing the infection prevalence from 100% at time 0 to 22% post-treatment, and the infection rate to an average of less than one parasite per fish, as compared to an average of about five parasites per fish in the control. Repeated weekly treatment application for three consecutive weeks appeared safe for the fish, and did not cause an adverse effect to the biofiltration system, as determined by examining nitrification activity, compared to non-treated controls. Treatment was successfully tested in three separate applications at a commercial closed recirculating guppy farm, where a significant reduction in infection rates was achieved. Timor C was not toxic to Artemia at concentrations ranging from 10 to 100 ppm over 24 h of exposure. This study demonstrates the effectiveness of Timor C as a treatment against G. turnbulii infection in guppies and potentially against similar parasites in other aquacultural species

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