Molecular and Cellular Response to Experimental Anisakis pegreffii (Nematoda, Anisakidae) Third-Stage Larval Infection in Rats

Background: Anisakiasis is a zoonotic disease caused by accidental ingestion of live Anisakis spp. third-stage larvae present in raw or undercooked seafood. Symptoms of this emerging infectious disease include mild-to-severe abdominal pain, nausea, and diarrhea. Some patients experience significant allergic reactions.Aims: In order to better understand the onset of anisakiasis, we aimed to: (i) histopathologically describe severe inflammatory/hemorrhagic infection site lesions in Sprague-Dawley rats experimentally infected with Anisakis pegreffii larvae; and (ii) qualitatively and quantitatively characterize the transcriptomes of affected tissues using RNA-Seq.Methodology: The experiment was performed on 35 male rats, sacrificed at 5 time points (6, 10, 18, 24, and 32 h post-infection). Gastric intubation was performed with 10 A. pegreffii larvae (N = 5 infected rats per time point) or 1.5 ml of saline (external control N = 2 rats). 16 pools, seven for muscle tissues and nine for stomach tissues, were created to obtain robust samples for estimation of gene expression changes depicting common signatures of affected versus unaffected tissues. Illumina NextSeq 500 was used for paired-end sequencing, while edgeR was used for count data and differential expression analyses.Results: In total, there were 1372 (855 up and 517 down) differentially expressed (DE) genes in the Anisakis-infected rat stomach tissues, and 1633 (1230 up and 403 down) DE genes in the muscle tissues. Elicited strong local proinflammatory reaction seems to favor the activation of the interleukin 17 signaling pathway and the development of the T helper 17-type response. The number of DE ribosomal genes in the Anisakis-infected stomach tissue suggests that A. pegreffii larvae might induce ribosomal stress in the early infection stage. However, the downstream pathways and post-infection responses require further study. Histopathology revealed severe inflammatory/hemorrhagic lesions caused by Anisakis infection in the rat stomach and muscle tissues in the first 32 h. The lesion sites showed infiltration by polymorphonuclear leukocytes (predominantly neutrophils and occasional eosinophils), and to a lesser extent, macrophages.Conclusion: Understanding the cellular and molecular mechanisms underlying host responses to Anisakis infection is important to elucidate many aspects of the onset of anisakiasis, a disease of growing public health concern

Community Parameters and Genome-Wide RAD-Seq Loci of Ceratothoa oestroides Imply Its Transfer between Farmed European Sea Bass and Wild Farm-Aggregating Fish

Wild fish assemblages that aggregate within commercial marine aquaculture sites for feeding and shelter have been considered as a primary source of pathogenic parasites vectored to farmed fish maintained in net pens at an elevated density. In order to evaluate whether Ceratothoa oestroides (Isopoda, Cymothoidae), a generalist and pestilent isopod that is frequently found in Adriatic and Greek stocks of farmed European sea bass (Dicentrarchus labrax), transfers between wild and farmed fish, a RAD-Seq (restriction-site-associated DNA sequencing)-mediated genetic screening approach was employed. The double-digest RAD-Seq of 310 C. oestroides specimens collected from farmed European sea bass (138) and different wild farm-aggregating fish (172) identified 313 robust SNPs that evidenced a close genetic relatedness between the “wild” and “farmed” genotypes. ddRAD-Seq proved to be an effective method for detecting the discrete genetic structuring of C. oestroides and genotype intermixing between two populations. The parasite prevalence in the farmed sea bass was 1.02%, with a mean intensity of 2.0 and mean abundance of 0.02, while in the wild fish, the prevalence was 8.1%; the mean intensity, 1.81; and the mean abundance, 0.15. Such differences are likely a consequence of human interventions during the farmed fish’s rearing cycle that, nevertheless, did not affect the transfer of C. oestroides

Characteristics of immune response of dolphin as a definitive and rat as an accidental host to Anisakis spp. infection

Pripadnici roda Anisakis Dujardin, 1845 tvore skupinu nametničkih oblića složenog životnog ciklusa koji uključuje morske sisavce kao krajnje domaćine, planktonske rakove kao intermedijarne domaćine te ribe i glavonošce kao paratenične domaćine. Konzumacijom sirovih ili termički nedovoljno obrađenih morskih proizvoda kontaminiranima živim ličinkama trećeg stadija čovjek može postati slučajni domaćin, pri čemu može doći do razvoja bolesti poznate kao anisakijaza, koja je prepoznata kao rastući javnozdravstveni problem. U prvom dijelu istraživanja je provedena histopatološka i imunofluorescencijska analiza lezija probavila krajnjeg domaćina. Lezije uzrokovane prisutnošću nametnika su uključivale izraženi ulcerativni gastritis s mješovitim upalnim infiltratom i čestom prisutnošću bakterija, blagi do umjereni granulomatozni gastritis te jaku piogranulomatoznu upalu. Imunofluorescencijsko bojanje nije proizvelo pozitivne signale za primijenjena protutijela. U drugom dijelu istraživanja, na štakorskom modelu anisakijaze je karakteriziran rani upalni odgovor slučajnog domaćina korištenjem histopatološke analize, imunofluorescencije (CD3, CD4, CD68, iNOS, S100A8/A9) i transmisijske elektronske mikroskopije te mjerenjem ekspresije upalnih biljega (Il1b, Il6, Il18, Ccl2, Icam1, Mmp9), mikroRNA i količine metilirane DNA. Migracija Anisakis ličinki je uzrokovala umjerena do opsežna krvarenja u vezivnom tkivu podsluznice probavila i epimizija/perimizija mišića. U želucu i mišiću je bio prisutan umjeren do obilan upalni infiltrat, dominiran neutrofilima i makrofagima, dok je u crijevu zabilježen samo blagi upalni infiltrat. Lezije su bile karakterizirane prisutnošću CD3+, CD4+, CD68+, iNOS+ i S100A8/A9+ stanica. Il6, Il1b i Ccl3 su pokazali izrazito jaku ekspresiju u želucu u visceralnom masnom tkivu. Ukupno su tri miRNA bile diferencijalno eksprimirane; dvije u želucu (miRNA-451-5p i miRNA-223-3p) te dvije u crijevu (miRNA-451-5p i miRNA-672-5p). Nije zabilježena statistički značajna razlika u količini metilirane DNA između zaraženog i nezaraženog tkiva. Navedeno istraživanje ukazuje da Anisakis spp. u slučajnom domaćinu potiče izraženi upalni odgovor sa značajnom ekspresijom određenih proupalnih citokina i miRNA.Members of the genus Anisakis comprise a group of parasitic nematodes with indirect life cycle, that utilize marine mammals as definitive hosts, planktonic crustaceans as intermediate hosts and fish and cephalopods as paratenic hosts. Following ingestion of raw or undercooked seafood contaminated with live third stage larvae, humans can become accidental hosts, thus contracting a disease termed anisakiasis, being recognized as an emerging public health concern. In the first part of the research histopathological and immunofluorescence analysis of gastrointestinal tract lesions of the final host was performed. Anisakis spp.-induced lesions included severe ulcerative gastritis with mixed inflammatory infiltrate often associated with colonies of bacteria, mild to moderate granulomatous gastritis and severe pyogranulomatous inflammation. Immunofluorescent staining did not yield positive signals for the applied antibodies. In the second part of the research we characterized early inflammatory response in rat model of anisakiasis by means of histopathological analysis, immunofluorescence (CD3, CD4, CD68, iNOS, S100A8/A9) and transmission electron microscopy, in addition to measuring inflammatory markers (Il1b, Il6, Il18, Ccl2, Icam1, Mmp9) and microRNA expression, as well as global DNA methylation status. Anisakis larval migration caused moderate to extensive haemorrhages in submucosal and epimysial/perimysial connective tissue of the muscles. In stomach and muscle, moderate to abundant mixed inflammatory infiltrate was present, dominated by neutrophils and macrophages, while only mild infiltration was seen in intestine. Lesions were characterized by the presence of CD3+, CD4+, CD68+, iNOS+ i S100A8/A9+ cells. Il6, Il1b and Ccl3 showed particularly strong expression in stomach and visceral adipose tissue. In total, three miRNAs were differentially expressed; two in stomach (miRNA-451-5p and miRNA-223-3p) and two in intestine (miRNA-451-5p and miRNA-672-5p). No changes in global DNA methylation were observed between infected and uninfected tissues. This study shows, that Anisakis infection induces strong inflammatory response in accidental host with marked induction of specific proinflammatory cytokines and miRNA expression

Characteristics of immune response of dolphin as a definitive and rat as an accidental host to Anisakis spp. infection

Pripadnici roda Anisakis Dujardin, 1845 tvore skupinu nametničkih oblića složenog životnog ciklusa koji uključuje morske sisavce kao krajnje domaćine, planktonske rakove kao intermedijarne domaćine te ribe i glavonošce kao paratenične domaćine. Konzumacijom sirovih ili termički nedovoljno obrađenih morskih proizvoda kontaminiranima živim ličinkama trećeg stadija čovjek može postati slučajni domaćin, pri čemu može doći do razvoja bolesti poznate kao anisakijaza, koja je prepoznata kao rastući javnozdravstveni problem. U prvom dijelu istraživanja je provedena histopatološka i imunofluorescencijska analiza lezija probavila krajnjeg domaćina. Lezije uzrokovane prisutnošću nametnika su uključivale izraženi ulcerativni gastritis s mješovitim upalnim infiltratom i čestom prisutnošću bakterija, blagi do umjereni granulomatozni gastritis te jaku piogranulomatoznu upalu. Imunofluorescencijsko bojanje nije proizvelo pozitivne signale za primijenjena protutijela. U drugom dijelu istraživanja, na štakorskom modelu anisakijaze je karakteriziran rani upalni odgovor slučajnog domaćina korištenjem histopatološke analize, imunofluorescencije (CD3, CD4, CD68, iNOS, S100A8/A9) i transmisijske elektronske mikroskopije te mjerenjem ekspresije upalnih biljega (Il1b, Il6, Il18, Ccl2, Icam1, Mmp9), mikroRNA i količine metilirane DNA. Migracija Anisakis ličinki je uzrokovala umjerena do opsežna krvarenja u vezivnom tkivu podsluznice probavila i epimizija/perimizija mišića. U želucu i mišiću je bio prisutan umjeren do obilan upalni infiltrat, dominiran neutrofilima i makrofagima, dok je u crijevu zabilježen samo blagi upalni infiltrat. Lezije su bile karakterizirane prisutnošću CD3+, CD4+, CD68+, iNOS+ i S100A8/A9+ stanica. Il6, Il1b i Ccl3 su pokazali izrazito jaku ekspresiju u želucu u visceralnom masnom tkivu. Ukupno su tri miRNA bile diferencijalno eksprimirane; dvije u želucu (miRNA-451-5p i miRNA-223-3p) te dvije u crijevu (miRNA-451-5p i miRNA-672-5p). Nije zabilježena statistički značajna razlika u količini metilirane DNA između zaraženog i nezaraženog tkiva. Navedeno istraživanje ukazuje da Anisakis spp. u slučajnom domaćinu potiče izraženi upalni odgovor sa značajnom ekspresijom određenih proupalnih citokina i miRNA.Members of the genus Anisakis comprise a group of parasitic nematodes with indirect life cycle, that utilize marine mammals as definitive hosts, planktonic crustaceans as intermediate hosts and fish and cephalopods as paratenic hosts. Following ingestion of raw or undercooked seafood contaminated with live third stage larvae, humans can become accidental hosts, thus contracting a disease termed anisakiasis, being recognized as an emerging public health concern. In the first part of the research histopathological and immunofluorescence analysis of gastrointestinal tract lesions of the final host was performed. Anisakis spp.-induced lesions included severe ulcerative gastritis with mixed inflammatory infiltrate often associated with colonies of bacteria, mild to moderate granulomatous gastritis and severe pyogranulomatous inflammation. Immunofluorescent staining did not yield positive signals for the applied antibodies. In the second part of the research we characterized early inflammatory response in rat model of anisakiasis by means of histopathological analysis, immunofluorescence (CD3, CD4, CD68, iNOS, S100A8/A9) and transmission electron microscopy, in addition to measuring inflammatory markers (Il1b, Il6, Il18, Ccl2, Icam1, Mmp9) and microRNA expression, as well as global DNA methylation status. Anisakis larval migration caused moderate to extensive haemorrhages in submucosal and epimysial/perimysial connective tissue of the muscles. In stomach and muscle, moderate to abundant mixed inflammatory infiltrate was present, dominated by neutrophils and macrophages, while only mild infiltration was seen in intestine. Lesions were characterized by the presence of CD3+, CD4+, CD68+, iNOS+ i S100A8/A9+ cells. Il6, Il1b and Ccl3 showed particularly strong expression in stomach and visceral adipose tissue. In total, three miRNAs were differentially expressed; two in stomach (miRNA-451-5p and miRNA-223-3p) and two in intestine (miRNA-451-5p and miRNA-672-5p). No changes in global DNA methylation were observed between infected and uninfected tissues. This study shows, that Anisakis infection induces strong inflammatory response in accidental host with marked induction of specific proinflammatory cytokines and miRNA expression

Characteristics of immune response of dolphin as a definitive and rat as an accidental host to Anisakis spp. infection

Pripadnici roda Anisakis Dujardin, 1845 tvore skupinu nametničkih oblića složenog životnog ciklusa koji uključuje morske sisavce kao krajnje domaćine, planktonske rakove kao intermedijarne domaćine te ribe i glavonošce kao paratenične domaćine. Konzumacijom sirovih ili termički nedovoljno obrađenih morskih proizvoda kontaminiranima živim ličinkama trećeg stadija čovjek može postati slučajni domaćin, pri čemu može doći do razvoja bolesti poznate kao anisakijaza, koja je prepoznata kao rastući javnozdravstveni problem. U prvom dijelu istraživanja je provedena histopatološka i imunofluorescencijska analiza lezija probavila krajnjeg domaćina. Lezije uzrokovane prisutnošću nametnika su uključivale izraženi ulcerativni gastritis s mješovitim upalnim infiltratom i čestom prisutnošću bakterija, blagi do umjereni granulomatozni gastritis te jaku piogranulomatoznu upalu. Imunofluorescencijsko bojanje nije proizvelo pozitivne signale za primijenjena protutijela. U drugom dijelu istraživanja, na štakorskom modelu anisakijaze je karakteriziran rani upalni odgovor slučajnog domaćina korištenjem histopatološke analize, imunofluorescencije (CD3, CD4, CD68, iNOS, S100A8/A9) i transmisijske elektronske mikroskopije te mjerenjem ekspresije upalnih biljega (Il1b, Il6, Il18, Ccl2, Icam1, Mmp9), mikroRNA i količine metilirane DNA. Migracija Anisakis ličinki je uzrokovala umjerena do opsežna krvarenja u vezivnom tkivu podsluznice probavila i epimizija/perimizija mišića. U želucu i mišiću je bio prisutan umjeren do obilan upalni infiltrat, dominiran neutrofilima i makrofagima, dok je u crijevu zabilježen samo blagi upalni infiltrat. Lezije su bile karakterizirane prisutnošću CD3+, CD4+, CD68+, iNOS+ i S100A8/A9+ stanica. Il6, Il1b i Ccl3 su pokazali izrazito jaku ekspresiju u želucu u visceralnom masnom tkivu. Ukupno su tri miRNA bile diferencijalno eksprimirane; dvije u želucu (miRNA-451-5p i miRNA-223-3p) te dvije u crijevu (miRNA-451-5p i miRNA-672-5p). Nije zabilježena statistički značajna razlika u količini metilirane DNA između zaraženog i nezaraženog tkiva. Navedeno istraživanje ukazuje da Anisakis spp. u slučajnom domaćinu potiče izraženi upalni odgovor sa značajnom ekspresijom određenih proupalnih citokina i miRNA.Members of the genus Anisakis comprise a group of parasitic nematodes with indirect life cycle, that utilize marine mammals as definitive hosts, planktonic crustaceans as intermediate hosts and fish and cephalopods as paratenic hosts. Following ingestion of raw or undercooked seafood contaminated with live third stage larvae, humans can become accidental hosts, thus contracting a disease termed anisakiasis, being recognized as an emerging public health concern. In the first part of the research histopathological and immunofluorescence analysis of gastrointestinal tract lesions of the final host was performed. Anisakis spp.-induced lesions included severe ulcerative gastritis with mixed inflammatory infiltrate often associated with colonies of bacteria, mild to moderate granulomatous gastritis and severe pyogranulomatous inflammation. Immunofluorescent staining did not yield positive signals for the applied antibodies. In the second part of the research we characterized early inflammatory response in rat model of anisakiasis by means of histopathological analysis, immunofluorescence (CD3, CD4, CD68, iNOS, S100A8/A9) and transmission electron microscopy, in addition to measuring inflammatory markers (Il1b, Il6, Il18, Ccl2, Icam1, Mmp9) and microRNA expression, as well as global DNA methylation status. Anisakis larval migration caused moderate to extensive haemorrhages in submucosal and epimysial/perimysial connective tissue of the muscles. In stomach and muscle, moderate to abundant mixed inflammatory infiltrate was present, dominated by neutrophils and macrophages, while only mild infiltration was seen in intestine. Lesions were characterized by the presence of CD3+, CD4+, CD68+, iNOS+ i S100A8/A9+ cells. Il6, Il1b and Ccl3 showed particularly strong expression in stomach and visceral adipose tissue. In total, three miRNAs were differentially expressed; two in stomach (miRNA-451-5p and miRNA-223-3p) and two in intestine (miRNA-451-5p and miRNA-672-5p). No changes in global DNA methylation were observed between infected and uninfected tissues. This study shows, that Anisakis infection induces strong inflammatory response in accidental host with marked induction of specific proinflammatory cytokines and miRNA expression

In vitro culture of the zoonotic nematode Anisakis pegreffii (Nematoda, Anisakidae)

Abstract Background Anisakiasis is a foodborne disease caused by the third-stage larvae (L3) of two species belonging to the genus Anisakis: Anisakis pegreffii and Anisakis simplex sensu stricto. Both species have been the subject of different -omics studies undertaken in the past decade, but a reliable in vitro culture protocol that would enable a more versatile approach to functional studies has never been devised. In nature, A. pegreffii shows a polyxenous life-cycle. It reproduces in toothed whales (final host) and disseminates embryonated eggs via cetacean faeces in the water column. In the environment, a first- (L1) and second-stage larva (L2) develops inside the egg, and subsequently hatched L2 is ingested by a planktonic crustacean or small fish (intermediate host). In the crustacean pseudocoelom, the larva moults to the third stage (L3) and grows until the host is eaten by a fish or cephalopod (paratenic host). Infective L3 migrates into the visceral cavity of its paratenic host and remains in the state of paratenesis until a final host preys on the former. Once in the final host’s gastric chambers, L3 attaches to mucosa, moults in the fourth stage (L4) and closes its life-cycle by becoming reproductively mature. Methods Testing two commercially available media (RPMI 1640, Schneider’s Drosophila) in combination with each of the six different heat-inactivated sera, namely foetal bovine, rabbit, chicken, donkey, porcine and human serum, we have obtained the first reliable, fast and simple in vitro cultivation protocol for A. pegreffii. Results Schneider’s Drosophila insect media supplemented with 10% chicken serum allowed high reproducibility and survival of adult A. pegreffii. The maturity was reached already at the beginning of the third week in culture. From collected eggs, hatched L2 were maintained in culture for 2 weeks. The protocol also enabled the description of undocumented morphological and ultrastructural features of the parasite developmental stages. Conclusions Closing of the A. pegreffii life-cycle from L3 to reproducing adults is an important step from many research perspectives (e.g., vaccine and drug–target research, transgenesis, pathogenesis), but further effort is necessary to optimise the efficient moulting of L2 to infective L3. Graphical Abstrac

Interplay between proinflammatory cytokines, miRNA, and tissue lesions in Anisakis-infected Sprague-Dawley rats.

BackgroundAnisakiasis is an emerging public health problem, caused by Anisakis spp. nematode larvae. Anisakiasis presents as variable and unspecific gastrointestinal and/or allergic clinical symptoms, which accounts for the high rate of misdiagnosed cases.Methodology/principal findingsThe aim of this study was to characterize the early cellular (6-72 h p.i.) and molecular (6 h p.i.) immune response and general underlying regulatory mechanism in Anisakis infected rats. Each Sprague-Dawley rat was infected with 10 Anisakis spp. larvae by gastric intubation. Tissues with visible lesions were processed for: i) classic histopathology (HE), immunofluorescence (CD3, iNOS, S100A8/A9), and transmission electron microscopy (TEM); ii) target genes (Il1b, Il6, Il18, Ccl3, Icam1, Mmp9) and microRNA (Rat Immunopathology MIRN-104ZF plate, Quiagen) expression analysis; and iii) global DNA methylation. Histopathology revealed that Anisakis larval migration caused moderate to extensive hemorrhages in submucosal and epimysial/perimysial connective tissue. In stomach and muscle, moderate to abundant mixed inflammatory infiltrate was present, dominated by neutrophils and macrophages, while only mild infiltration was seen in intestine. Lesions were characterized by the presence of CD3+, iNOS+, and S100A8/A9+ cells. The greatest number of iNOS+ and S100A8/A9+ cells was seen in muscle. Il6, Il1b, and Ccl3 showed particularly strong expression in stomach and visceral adipose tissues, but the order of expression differed between tissues. In total, three miRNAs were differentially expressed, two in stomach (miRNA-451 and miRNA-223) and two in intestine (miRNA-451 and miRNA-672). No changes in global DNA methylation were observed in infected tissues relative to controls.Conclusions/significanceAnisakis infection induces strong immune responses in infected rats with marked induction of specific proinflammatory cytokines and miRNA expression. Deciphering the functional role of these cytokines and miRNAs will help in understanding the anisakiasis pathology and controversies surrounding Anisakis infection in humans

Rat and fish peripheral blood ltes rrespond distinctively to amatoda, anisakidae extract

Infective third-stage larvae (L3) of the marine nematode Anisakis pegreffii cause inflammation and clinical symptoms in humans, their accidental host, that subside and self-resolve in couple of weeks after L3 die. To characterise the differences in an early immune response of a marine vs terrestrial host, we stimulated peripheral blood leukocytes (PBLs) of fish (paratenic host) and rat (accidental, human-model host) with A. pegreffii crude extract and analysed PBLs transcriptomes 1 and 12 h post-stimulation. Fish and rat PBLs differentially expressed 712 and 493 transcripts respectively, between 1 and 12 h post-stimulation (False Discovery Rate, FDR<0.001, logFC>2). While there was difference in the highest upregulated transcripts between two time-points, the same Gene Ontologies, Biological processes (intracellular signal transduction, DNA-dependent transcription and DNA-regulated regulation of transcription) and Molecular functions (ATP and metal ion binding) were enriched in the two hosts, showing incrementing dynamic between 1 and 12 h. This suggests that the two distinct hosts employ qualitatively different transcripts cascade only to achieve the same effect, at least during an early innate immunity response. Activation of later immunity elements and/or a combination of other host's intrinsic conditions may contribute to the death of L3 in the terrestrial host