3.1. Macroscopic examination and presumptive diagnosis
According to the farmer, fish mortality of unknown cause has occurred in the last two years with various symptoms affecting cultured fish at all stages. Clinical signs of this recent unknown disease resembling streptococcosis were noted, such as marked hemorrhage, corneal opacities, spinning near the water surface, erosion of the caudal fin, and exophthalmos (eye protrusion). The obtained specimens were about 5 months old and had a body length of approximately 13 cm. The diseased fish showed external lesions with intraocular haemorrhages and exophthalmos and internal changes such as necrotic and hemorrhagic liver (Figure S1). Presumptive diagnosis by microscopic examination of Gram stain of stamp-smeared tissues of the brain and head kidney revealed numerous Gram-positive cocci bacteria in both smeared tissues (representative results in brain in Figure S2A).
3.2. Recovery of S. suis from naturally diseased fish
The predominant bacterial colonies on 24 hours incubated TSA plates were small whitish colonies (Figure S2B) that were Gram-positive cocci. One representative isolate, namely 3112, was used for further investigation. PCR amplification of 16S rDNA and gdh gene using DNA extracted from bacterium 3112 as template yielded amplicon products of approximately 1.5 kb and 0.7 kb, respectively (Figure S3A). DNA sequencing and BLAST analysis identified bacterium 3112 as Streptococcus suis based on 99.67-99.87% nucleotide identity of the 16S rDNA sequence and 98.18-98.48% nucleotide identity and 100% amino acid sequence identity of the gdh gene to the top 10 BLAST hits of various isolates of S. suis complete genomes available in the GenBank database. The partial 688 bp DNA sequence of the gdh gene of S. suis 3112 is shown in Figure S3B while the sequence of the partial 16S rDNA (1.5 kb) was deposited in the GenBank database under accession number OK356401. Phylogenetic analysis based on 16S rDNA sequences revealed that S. suis 3112 isolated from the diseased snakeskin gourami belonged to the same cluster as S. suis from pigs and cattle (Figure 1).
3.3. S. suis is highly pathogenic to both juvenile and adult snakeskin gourami
The results of experimental challenge showed that S. suis exhibited significant virulence in snakeskin gourami and the mortality rate depended on the size and dose (Figure 2). The high dose of S. suis, which was rapidly lethal without clear clinical signs, caused 67.5% mortality in small fish within 3 days post-infection (dpi) and slower mortality in big fish with the same percentage after 5 dpi. The low dose of S. suis was also highly virulent, but the disease developed quite slowly, with mortality of 42.5 and 60% in small and big fish, respectively, at one week after injection. Of note, mortality in small fish at high and low doses ceased at 8 and 10 dpi, respectively, whereas mortality in big fish persisted up to 15 dpi at both high and low doses. At the end of the experiment, the mean cumulative mortality and standard error of the mean (SEM) of small and big fish at low and high doses were 50% (±5.0), 75% (±0.00), 80% (±0.00) and 92.5% (±2.5), respectively. Statistical analysis of differences between treatments showed that mortality was significantly higher in big fish than in small fish when the same low dose was administered (p<0.05, Table 1).
Starting at 3 dpi, after acute death, fish showed lethargic behavior and erratic swimming, swirling or loss of balance, ‘C’- or ‘S’ - shaped posture (Figure S4). The clinical symptoms that developed after injection with S. suis are shown in Figure 2 and Table 1. The clinical signs in experimentally infected fish in all groups were lethargy, swimming disorders, loss of appetite, corneal opacity/exophthalmos, and skin haemorrhages/erosions, and internal signs were enlarged liver and splenomegaly (Figure 3). Although clinical signs were similar, small fish died acutely, but mortality, macroscopic lesions, and histopathology were less variable than in big fish. S. suis was successfully recovered as pure colonies from the head kidney, liver, and spleen of moribund and dead fish after challenge. The gdh PCR results of DNA extracted from the isolated bacteria and DNA extracted from the internal organs of the experimental fish are also shown in Figure S5 and Table 1. No morbidity or mortality was observed in the control small fish group until 21 dpi, while two out of 40 individuals in the control big fish group (5%) died at 3 dpi without major clinical signs but with some injuries on their bodies. This could be because the fish were aggressive and fighting each other, which led to the death of some fish. S. suis was not recovered from the internal organs of these fish as well as gdh PCR results that were negative and histological findings revealed no abnormal signs (data not shown).
3.4. Histopathology of snakeskin gourami experimentally infected with S. suis
Similar histological changes were observed in multiple organs of both small and big fish. The detailed changes in individual organ are described below.
Liver: Unlike the normal liver, which is made up of densely packed hepatocytes with polyhedral shapes (Figure 4A), the infected liver showed congestion in hepatoportal and sinusoidal blood vessels, severe hepatic lipidosis with large number of lipid vacuoles in both small (Figure 4B) and big fish (Figure 4C). Affected hepatocytes exhibited atrophy, lipid storage loss and chromatin margination (Figure 4B1). Notably, all affected big fish had multiple coalescing granulomas in the liver that occupied a large portion of the tissue parenchyma (Figure 4C), which was explicitly not presented in small fish. Granulomas, which may have necrotic cores, typically progress through characteristic stages with overall progressive fibrosis. The main part of granulomas contained large bacterial colonies in their necrotic centers, tissue death (necrosis), accumulation of pigmented macrophages, and were surrounded by fibroblast-like cells (Figure 4C1).
Kidney: The most notable changes were severely diminished hemopoietic tissue accompanied by interstitial haemorrhage and congestion of the intertubular blood vessels (Figure 4E and F). Obvious necrosis of tubular epithelial cells was observed (Figure 4E1). Glomerular necrosis with dilatation of Bowman's space was observed in most cases (data not shown). Typical granulomas and increased melanoma macrophage centres (MMc) were observed only in the kidneys of the big fish (Figure 4F), but not in the kidneys of the small fish (Figure 4E).
Spleen: The splenic lesions showed a marked reduction in the amount of red pulp and red blood cells (Figure 4H and I). Pigmented macrophage aggregations were also noted in certain areas (Figure 4H). The hematopoietic components were degenerated and necrotized (Figure 4H1). An increasing number of MMc and their multifocal areas of necrosis as well as typical granulomas were observed only in the spleen of the big fish (Figure 4I and I1).
Brain: The examined sections showed that the affected brain had severe meningeal congestion (Figure 5B and D). The brain parenchyma was degenerated and meningitis was characterized by infiltration with mononuclear cells resembling lymphocytes around the area of congestion (Figure 5B1). It is noteworthy that severe blood vessel congestion was observed in the cerebellum compared to other parts of the brain (Figure 5C).
Eye: The eyes of many fish examined showed marked pathology, with varying degrees of severity of blood congestion in the lens cortex (Figure 6B). The retina was ulcerated with thickened stromal layers, including advanced lesions with disruption and separation of retinal fibres and vacuolization in the stromal layers (Figure 6B1).