Characterization of Lactococcus garvieae and Streptococcus agalactiae in cultured red tilapia Oreochromis sp. in Thailand.

OBJECTIVE: Acute mortality with clinical symptoms of streptococcal-like infections was observed in red tilapia Oreochromis sp. cultured in floating cages in Prachin Buri Province, Thailand, during May 2023. Herein, we identified an emerging pathogen, Lactococcus garvieae, as the etiological agent. METHODS: After bacterial isolation from the brain and kidney of diseased fish, identification was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and the VITEK 2 system. Sequencing of the 16S ribosomal RNA (rRNA) gene and phylogenetic analysis were applied to confirm bacterial species. Antimicrobial susceptibility testing was conducted. Histopathological findings in the brain, kidney, spleen, liver, and heart were evaluated. RESULT: From 20 fish samples, L. garvieae (n = 18 isolates) and Streptococcus agalactiae (n = 2 isolates) were identified. A phylogenetic tree of the 16S rRNA gene revealed that Thai isolates of either L. garvieae or S. agalactiae clustered with reference piscine isolates from intercontinental locations. Our isolates showed resistance against quinolones while being susceptible to other antimicrobials. Histopathological changes demonstrated severe septicemic conditions, with more invasive lesions-especially in the heart and liver-being apparent in L. garvieae-infected fish compared to S. agalactiae-infected fish. CONCLUSION: This study represents the first reported outbreak of L. garvieae with a concurrent S. agalactiae infection in farmed red tilapia in Thailand.

Inflammatory responses to Opisthorchis viverrini infection in animal models: A comparison between susceptible and nonsusceptible hosts in different anatomical locations.

Background: Inflammation caused by Opisthorchis viverrini infection increases the risk of cholangitis, cholecystitis, and leads to bile duct cancer (cholangiocarcinoma or CCA). However, only certain infected individuals are susceptible to CCA, suggesting the involvement of host factors in cancer development. In addition, there are reports indicating differences in the locations of CCA. Aim: This study aims to investigate cellular inflammatory responses in the common bile duct (CB), intrahepatic bile duct (IHB), and gallbladder (GB) in susceptible and non-susceptible hosts following O. viverrini infection. Methods: Thirty Syrian golden hamsters (a susceptible host) and 30 BALB/c mice (a non-susceptible host) infected with O. viverrini were studied at six time points (five animals per group). Histopathological evaluations were conducted on samples from the IHB, CB, and GB. Inflammatory cell infiltration was quantitatively assessed and compared between groups and time points. Statistical analysis was performed using one-way ANOVA, with a significance level of p < 0.05. Results: Inflammation was significantly more pronounced in the IHB compared to the other two biliary locations. In comparison between susceptible and non-susceptible hosts, the intensity of inflammation was higher in the OV+H group than in the OV+M group (p < 0.05). Conclusion: This study highlights the association between host response to inflammation, tissue location, and host susceptibility, with the IHB showing particular susceptibility to inflammation and pathological changes. These findings contribute to our understanding of the increased risk of CCA in susceptible hosts.

Impact of high-pressure processing on hemolymph, color, lipid globular structure and oxidation of the edible portion of blood clams.

Impact of high-pressure processing (HP-P) on hemolymph and lipid globular structures of the edible portion (EP) of blood clams (BC) was investigated. HP-P above 400 MPa decreased heme iron content, while upsurged non-heme iron content. Increasing pressure induced gaps and abnormal hemocyte cell arrangements. However, HP-P at 300 MPa improved and maintained total hemocyte counts, the heme iron content, and a*-value in BC-EP. For lipid globular structures, the mean diameter drastically decreased when an HP-P pressure of 600 MPa was employed. HP-P at higher pressure induced lipid oxidation, along with decreases in monounsaturated and polyunsaturated fatty acids as well as increases in thiobarbituric acid reactive substances and peroxide value. FTIR spectra displayed a reduction in phosphate groups and cis double bonds in lipids from HP-P treated BC, compared to controls. Therefore, HP-P at 300 MPa is recommended for preparing ready-to-cook BC with less tissue damage and lipid oxidation.

Mixed Eimeria and Cryptosporidium infection and its effects on pathology and clinical outcomes in juvenile Asian seabass (Lates calcarifer) cultured in Thailand.

Coccidiosis is an important disease in juvenile fish because of severe intestinal injury during infection. We first reported the mixed infection of intestinal coccidia and its association with health status and pathological findings in juvenile Asian seabass (Lates calcarifer) cultured in Thailand. Two groups of Asian seabass, 60-day fish and 90-day fish, were sampled to investigate prevalence and coccidian infection intensity using morphological characterization and PCR. Phylogenetic analysis of 18S rRNA gene amplified from the intestines revealed Eimeria sp. and Cryptosporidium sp. infection. The prevalence of Eimeria sp. and Cryptosporidium sp. in sampled fish was 100%. Clinical outcomes assessed, using health assessment index (HAI) scoring and semi-quantitative grading of intestinal lesions and inflammation, demonstrated that all fish developed variety of pathology and clinical illness; however, infection intensity in 60-day fish was significantly higher (p < .05) than 90-day fish. The HAI score of 60-day fish was poorer than 90-day fish, which correlated to a high infection intensity (r = .397), analysed by Pearson correlation coefficient. Overproduction of intestinal oxidants contributing to mucosal injury was examined by nitrotyrosine expression. The high production of reactive nitrogen species indicated severe inflammatory response, and intestinal injuries occurred mainly in the 60-day fish.

Unraveling the relationship among inflammatory responses, oxidative damage, and host susceptibility to Opisthorchis viverrini infection: A comparative analysis in animal models.

Background and Aim: Opisthorchis viverrini infection-induced inflammation contributes to cholangiocarcinoma (CCA) development in humans and animals. Inflammation generates free radicals, such as reactive oxygen species and reactive nitrogen species (RNS), which damage the host's DNA. However, only 5% of O. viverrini-infected individuals develop malignancy, suggesting that variations in the inflammatory response of individuals to the parasite may influence susceptibility. Due to limitations in studying human susceptibility, we used an animal model to investigate the profiles of inflammatory reactions, oxidative burst, and irreversible DNA damage. This study aimed to explore the potential role of inflammation and RNS in causing DNA damage that may predispose susceptible hosts and non-susceptible animal models to cancer development in O. viverrini infection. Materials and Methods: This experimental study was conducted on 30 Syrian golden hamsters (OV-H) and 30 BALB/c mice (OV-M) infected with O. viverrini, representing susceptible and non-susceptible models, respectively. Five animals per group were examined at six predetermined time points during the experiment. Biliary tract samples were systematically investigated using histopathological evaluation for inflammatory cell infiltration and immunohistochemical staining for RNS production and markers of DNA damage, including nitrotyrosine and 8-hydroxy-2'-deoxyguanosine. These features were quantified and compared among the experimental groups. Mann-Whitney U-test was used for statistical analysis, with p < 0.05 considered statistically significant. Results: The comparison revealed that the OV-M group exhibited significantly earlier and higher rates of inflammatory cell infiltration during the acute phase, whereas the OV-H group exhibited chronic and more severe inflammation (p < 0.020). Intracellular RNS production and DNA damage were closely associated with the inflammatory response. Conclusion: This study demonstrates differential responses in susceptible and non-susceptible models of O. viverrini infection regarding disease onset and duration, as well as intracellular RNS production and DNA damage caused by inflammation. Persistent inflammation generated oxidatively damaged DNA, which is a distinct pathological characteristic of susceptible hosts and may be critical for CCA development.

Inflammatory cell responses in biliary mucosa during Opisthorchis viverrini infection: Insights into susceptibility differences among hosts.

Background: Individual host susceptibility is believed to be a risk factor in the interaction between the host and the parasite. Since studying time series in humans is limited, animal models are replaced. Aim: This study aims to explore and compare the pattern of inflammatory cell types along the biliary tract and their association with proliferative lesions in the early development of cholangiocarcinoma from susceptible and nonsusceptible animal models. Methods: Thirty male Syrian golden hamsters and 30 BALB/c mice, serving as the susceptible and nonsusceptible animal models, were used in this comparative study. The animals were infected with 50 Opisthorchis viverrini metacercariae via gastric intubation. At days 1, 2, 7, 14, 28, and 56 postinfection (p.i.), five animals were randomly selected from each group and humanely sacrificed. The hepatobiliary tissues were collected and processed for histopathological study. Histochemical and immunohistochemical staining were applied to differentiate the inflammatory cell types. Kruskal-Wallis and Mann-Whitney tests were applied to assess all semi-quantitative and quantitative variables. The correlation between each variable was also analyzed using Spearman rank at a p-value < 0.05. Results: The results demonstrated that mice had different patterns of infiltrating cell types when compared to hamsters. This suggested that the cellular response to the infection in mice occurred earlier than that in hamsters. The response in mice reached its peak at D7 to D14 and then rapidly declined at D28. In contrast, although the inflammatory response in hamsters started slowly, the response reached the peak at D28 and maintained a high level until D56. Significant differences in the number of inflammatory cells between mice and hamsters were seen at D1 (p = 0.047), D7 (p = 0.049), D28 (p = 0.040), and D56 (p < 0.040). Conclusion: The inflammatory responses to O. viverrini infection in the nonsusceptible animal model occurred and declined earlier while the response in the susceptible animal model occurred later in a gradual manner. Both rodents are suitable animal models for the studies of opisthorchiasis susceptibility.

Distinct antibody response in susceptible and non-susceptible hosts of the carcinogenic liver fluke Opisthorchis viverrini infection.

Opisthorchis viverrini is a carcinogenic parasite that can cause bile duct cancer called cholangiocarcinoma. A study of the immune response of this parasite in susceptible and non-susceptible hosts may provide a clue to develop vaccines and immunodiagnostic markers, which are currently not available. Here, we compared the antibody response in susceptible Golden Syrian hamsters and non-susceptible BALB/c mice infected by the liver fluke. In mice, the antibody was detected between 1 and 2 weeks post-infection, whereas it was positive between 2 and 4 weeks post-infection in hamsters. Immunolocalization revealed that the antibody from mice reacts strongly with the tegumental surface and gut epithelium of the worm, while hamster antibody showed a weak signal in the tegument and a comparable signal in the gut of the worm. Immunoblot of the tegumental proteins demonstrated that while hamster antibody showed a broad specificity, mice strongly reacted with a single protein band. Mass spectrometry revealed these immunogenic targets. Recombinant proteins of the reactive targets were produced in the bacterial expression system. The immunoblot of these recombinant proteins confirm the reactivity of their native form. In summary, there is a different antibody response against O. viverrini infection in susceptible and non-susceptible hosts. The non-susceptible host reacts quicker and stronger than the susceptible host.

Impact of Prior Pulsed Electric Field and Chitooligosaccharide Treatment on Trypsin Activity and Quality Changes in Whole and Beheaded Harpiosquillid Mantis Shrimp during Storage in Iced Water.

Harpiosquillid mantis shrimp (Harpiosquilla raphidea) (HMS) without and with beheading pretreated with pulsed electric field (PEF) (15 kV/cm, 800 pulses, 5 min) were soaked in chitooligosaccharide (COS) solution at varying concentrations (0, 1 and 2%, w/v) for 20 min and stored for 3 days in iced water. Changes in the trypsin activity, color, texture, protein pattern, TCA soluble peptide content, histological images, protein secondary structure and microbial load were monitored during the storage. The beheaded HMS pretreated with PEF and soaked in 2% COS solution showed the maximum efficacy in inhibiting trypsin activity and proteolysis, thus retaining muscle proteins, especially myosin heavy chain, actin and troponin T as well as shear force up to day 3. Pronounced muscle destruction in the whole HMS was displayed by a decreased mean grey index and fiber gapping. Such changes were lowered by the beheading and PEF/2% COS treatment (2% COS-BH). Nevertheless, no marked change in the secondary structure including α-helix, ß-sheets, ß-turns and random coil were observed among any of the samples. The microbiological analysis revealed that the total viable count (TVC) was below 6 log CFU/g till day 2 in all samples. Nonetheless, the 2% COS-BH sample had the lowest psychrophilic bacterial count and Enterobacteriaceae count at day 3, compared to the others. Thus, the combination of the prior PEF and 2% COS treatment of beheaded HMS could effectively inhibit proteases, retard the microbial growth and maintain the quality of HMS stored in iced water.

Distinctive location of piscine intestinal coccidiosis in Asian seabass fingerlings.

Background and Aim: Coccidian infection (coccidiosis) is one of the most important causes of illness and death in the fish population, including Asian sea bass. The fingerling developmental stage is sensitive to various infectious agents. Economic losses are sustained by the sea bass aquaculture industry due to coccidiosis annually. However, the related pathological changes in the Asian sea bass fingerlings' three-part intestine remain unknown. This study aimed to investigate the Asian sea bass fingerlings' infection rate, infection location and site, and specific pathological lesions in the small intestinal tissues in a marine cage farming operation. Materials and Methods: A cross-sectional study was conducted on 44 fingerling fishes. Major coccidia proportions were identified morphologically at both the macroscopic and microscopic levels. The infection number was determined based on coccidia presence at various intestinal locations and sites. All areas were assessed for pathological lesions using semi-quantitative grading. Analysis of variance was used to perform all data analyses using the SPSS software. Data were expressed as means ± standard deviation. p < 0.05 was considered statistically significant. Results: All Asian sea bass fingerlings studied were infected with coccidia. Enteritis and mucosal necrosis were distinct lesions found in the anterior intestine, which had the highest infection rate (49.94%), followed by the mid intestine (35.63%), and the posterior intestine (22.43%). The most common coccidian infection site was extracellular (subepithelial), followed by intracytoplasmic, and epicellular sites. Histopathological lesion determination revealed that intestinal tissue inflammation and epithelial injuries were predominantly seen in the anterior gut (p < 0.05). Conclusion: There was a high coccidian infection rate in Asian sea bass fingerlings from marine cage farming operations. Infection and intestinal damage at the anterior intestine, a major site, led to fingerling death. Disease prevention in the nursery should be intensive from the fingerling period to decrease the fatality rate caused by coccidia.

Prevalence of major nematodes and human factors that affect infection in the zebra dove in a closed cage system.

Background and Aim: Roundworms cause infections in the avian population that lead to illness and poor production. The singing zebra dove is an economically important animal in the Indo-Malay region. The prevalence of these parasitic groups in zebra doves is unknown. This study estimated the prevalence and associated human risk factors of gastrointestinal nematode infections in zebra dove farming. Materials and Methods: A cross-sectional survey was conducted from January to April 2021. The study was conducted on 184 doves in three zebra dove farms. Fecal samples were collected from pooled zebra dove droppings. Major proportions and infection intensity of gastrointestinal nematodes were morphologically identified and morphometrically investigated. Associated human factors were assessed through the interview surveys among farmers. Results: Results showed that 36.96% of the zebra doves were infected. The primary nematodes were Ascaridia galli (34.78%), Heterakis gallinarum (6.52%), Trichostrongylus tenuis (2.17%), Syngamus spp. (4.35%), and Amidostomum spp. (2.17%). The primary human factors that contribute to parasitic infection were poor hygiene, food contamination with parasites, and inappropriate deworming. Conclusion: There was a high prevalence of gastrointestinal nematodes in the zebra dove in the close cage system. Human factors played key roles as risk factors, and improves farming management will help reduce parasitic infections. However, these nematodes may contribute to poor health status and poor productivity of zebra doves. Further extensive studies on clinical signs and pathological changes should be conducted.

Biliary Migration, Colonization, and Pathogenesis of O. viverrini Co-Infected with CagA+ Helicobacter pylori.

Co-infection with the cagA strain of Helicobacter pylori exacerbates the pathology of human liver fluke Opisthorchis viverrini (OV) infection leading to cholangiocarcinoma. However, underlying mechanisms remain unclear. We report a significant increase in cagA-positive and cagA-negative H. pylori in the stomach, blood, bile, and in the OV worms of co-infected Syrian golden hamsters at one hour, three hours, and one month, post-infection, compared to hamsters infected with either OV or H. pylori alone. Except in the worms, H. pylori numbers declined at three months post-infection, particularly in the bile fluid of co-infected animals. Both strains of H. pylori were immunohistochemically detected in the tegument of the worm, as well as in the bile duct epithelium when co-infected with O. viverrine, but not in H. pylori infection alone. Interestingly, only the cagA-positive strain was detected in the gut of the worm. Co-infection between cagA-positive H. pylori and O. viverrini resulted in a more severe biliary pathology and decreased E-cadherin expression in vivo and in vitro than those of the cagA-negative strain. These data suggest that O. viverrini acts as a carrier of cagA-positive H. pylori and co-migrates to the bile ducts, whereas O. viverrini facilitates H. pylori colonization and enhances the biliary pathogenesis and carcinogenesis.

Synergistic effects of cagA+ Helicobacter pylori co-infected with Opisthorchis viverrini on hepatobiliary pathology in hamsters.

Human liver fluke infection caused by Opisthorchis viverrini is associated with several biliary diseases including cholangiocarcinoma (CCA). Recently, it was discovered that the liver fluke is a reservoir of Helicobacter pylori, particularly the cagA-positive strain (cytotoxin-associated gene A) in its gut. Given that two carcinogenic pathogens are associated with CCA development, however, the role of cagA-positive H. pylori in opisthorchiasis has not been clarified. The present study was therefore aimed to investigate histopathological changes of the biliary system in hamsters co-infected with O. viverrini and cagA-positive H. pylori or O. viverrini and cagA-negative H. pylori, with controls of O. viverrini, cagA-positive H. pylori, or cagA-negative H. pylori alone, over time. Major histopathological changes were systematically investigated. All pathological features were quantified/semi-quantified and compared among the experimental groups. The results showed that O. viverrini infection groups (O. viverrini, cagA-positive H. pylori and cagA-negative H. pylori) showed a high degree of eosinophil and mononuclear cell infiltration, lymphoid aggregation and granuloma. Specifically, O. viverrini co-infected with cagA-positive H. pylori presented significantly higher inflammatory scores than O. viverrini and O. viverrini with cagA-positive H. pylori. Proliferation and adaptive lesions such as hyperplasia, goblet cell metaplasia and dysplasia were detected only in O. viverrini infection groups. Dysplasia, the precancerous lesion of CCA, was observed in the first-order bile ducts, especially where the inflammation existed and was found earlier and more severely in O. viverrini with cagA-positive H. pylori than other groups. Similarly, the BrdU (bromodeoxyuridine) proliferation index was significantly higher in O. viverrini co-infected with cagA-positive H. pylori than O. viverrini and O. viverrini with cagA-negative H. pylori groups. Periductal fibrosis was a prominent histopathologic feature in chronic infection in O. viverrini infection groups. Multiple logistic regression showed that O. viverrini co-infected with cagA-positive H. pylori and the duration of infection were the most important factors associated with periductal fibrosis (OR 3.02, 95% CI 1.02-9.29, p = 0.04 and OR 3.82, 95% CI 2.61-5.97, p<0.001). This study demonstrates that the liver fluke co-infected with cagA-positive H. pylori induces severe biliary pathology that may predispose to cholangiocarcinogenesis.

RNA Interference as an Approach to Functional Genomics Genetic Manipulation of Opisthorchis viverrini.

The availability of genome and transcriptome data of the liver fluke Opisthorchis viverrini provides the foundation for exploration of gene function and its effect on host-parasite interactions and pathogenesis of O. viverrini-associated bile duct cancer. Functional genomics approaches address the function of DNA at levels of the gene, RNA transcript and protein product using informative manipulations of the genome, epigenome, transcriptome, proteome, microbiome and metabolome. Advances in functional genomics for O. viverrini have thus far focused on RNA interference. The flukes have been transfected with double-stranded RNAs aiming to silence target gene expression. In general, this approach for functional genomics investigation of this pathogen has been found to be tractable and efficient: suppression of messenger RNA expression in O. viverrini results in reduction of protein activity and phenotypic changes. Future perspectives for functional genomics of this liver fluke and close phylogenetic relatives are also discussed.

Opisthorchis viverrini Proteome and Host-Parasite Interactions.

The omics technologies have improved our understanding of the molecular events that underpin host-parasite interactions and the pathogenesis of parasitic diseases. In the last decade, proteomics and genomics in particular have been used to characterize the surface and secreted products of the carcinogenic liver fluke Opisthorchis viverrini and revealed important roles for proteins at the host-parasite interface to ensure that the flukes can migrate, feed and reproduce in a hostile environment. This review summarizes the advances made in this area, primarily focusing on discoveries enabled by the publication of the fluke secreted proteomes over the last decade. Protein families that will be covered include proteases, antioxidants, oncogenic proteins and the secretion of exosome-like extracellular vesicles. Roles of these proteins in host-parasite interactions and pathogenesis of fluke-induced hepatobiliary diseases, including cholangiocarcinogenesis, are discussed. Future directions for the application of this knowledge to control infection and disease will also be discussed.