Detection of Aeromonas hydrophila possessing aerolysin gene using gold nanoparticle probe.

Objective: The aerolysin (aerA) is a virulence indicator used to identify the pathogenicity of the Aeromonas strain. Targeting a pathogen's crucial virulence gene for detection is essential, as it determines the potential threat to the host. This study aimed to develop a gold nanoparticle (AuNP) probe for detecting the gene aerA in Aeromonas hydrophila among field samples. Materials and Methods: Kidney samples among both healthy and sick Nile tilapias in five provinces of Luzon Island were collected for bacterial analysis. Screening using specific primers targeting aerA was conducted in parallel with testing the AuNPs probe on the same sample set. The positive control provided by BFAR-NFLD, confirmed by polymerase chain reaction (PCR) assay, was used as a positive sample containing the target gene. Results: The AuNP probe demonstrated a computed accuracy of 81.32%, sensitivity of 100%, and specificity of 81.26%. Among the 257 reactions, 59 were false positives, while no false negative results were observed. The AuNP probe could detect aerA at levels as low as 30 ng/µl. The low prevalence of the target gene may be attributed to the use of general media instead of specific media like Rimler-Shotts agar. Conclusion: The established colorimetric detection method for A. hydrophila with the aerA gene offers a swift alternative to PCR, negating the requirement for advanced equipment like a thermal cycler.

Probiotic effect of Bacillus spp. isolated from African nightcrawler (Eudrilus eugeniae) on the performance of Nile Tilapia (Oreochromis niloticus L.).

This study evaluated the effects of the probiotic Bacillus spp. isolated from African nightcrawler (Eudrilus eugeniae) on the growth and survival performances of Nile tilapia (Oreochromis niloticus). A total of 400 Nile tilapia fingerlings (1.76 ± 0.07 g) were equally and randomly distributed into 20 glass aquaria and divided into five experimental groups with four replicates. Each experimental group was fed with a commercial diet (control) supplemented (at 108 CFU·g-1) with one of the four Bacillus spp. (ANSCI9, BFAR9, RM3, and RM10) at five percent of their body weight. After 30 days of feeding, the surviving fish were challenged with Aeromonas hydrophila to observe their survival. The treatment groups supplemented with BFAR9 (2.73 ± 0.26 g) and RM10 (3.15 ± 0.30 g) showed significantly higher (P < 0.05) absolute growth than the control (2.20 ± 0.16 g) group. Furthermore, those given with RM10 had better (P < 0.05) specific growth rate (SGR) (1.60 ± 0.10%·day-1) relative growth rate (RGR) (181.39 ± 18.16%) and feed conversion ratio (FCR) (1.99 ± 0.13) compared to the control group (SGR = 1.29 ± 0.07%·day-1; RGR = 129.84 ± 9.77%; FCR = 2.60 ± 0.16). The challenge test revealed that all Bacillus spp.-treated groups showed significantly higher (P < 0.05) survival rates (ANSCI9 = 82.5 ± 8.16%; BFAR9 = 80.0 ± 10.00%; RM3 = 77.5 ± 20.82%; RM10 = 85.0 ± 10.00%) than the control group (55.00 ± 19.15%), with the highest relative level of protection recorded for RM10 (66.67%). These results revealed that the probiotic Bacillus spp. isolated from E. eugeniae improved the growth, feed utilization, waste excretion, and the disease resistance of Nile tilapia.

Anisakis spp. in fishery products from Japanese waters: Updated insights on host prevalence and human infection risk factors.

The nematodes of the genus Anisakis are among the most relevant parasitic hazards in fishery products since they are responsible for human infection and allergy cases. In a food safety and epidemiological perspective, several marine hosts from different locations around Japan were examined to characterize the parasitism of Anisakis larvae. Chum salmon (Oncorhynchus keta) and Alaska pollock (Gadus chalcogrammus) showed the highest overall prevalence (100%), followed by blue mackerel (Scomber australasicus) (97.5%), Pacific cod (Gadus macrocephalus) (80%), chub mackerel (Scomber japonicus) (60.1%), Japanese flying squid (Todarodes pacificus) (17%) and Japanese pilchard (Sardinops sagax melanostictus) (2%). In Pacific krill (Euphausia pacifica), apart from one Hysterothylacium aduncum larva, no Anisakis specimens were detected. Anisakis simplex sensu stricto was molecularly identified (PCR-RFLP) for the first time in Japanese flying squid and Japanese pilchard distributed in the Northwestern Pacific ocean. That was the most frequent parasitic species detected followed by A. pegreffii, mostly in the western areas of Japan, hybrid genotypes between the two sibling species as well as A. typica and A. berlandi. Surprisingly, A. simplex s.s. was the most abundant species in one batch of chub mackerel from the East China Sea and A. pegreffii was the main species found in one batch from the Pacific coast of Aomori, which seems to indicate that the ranges of these two sibling species might be more variable than previously thought.

Selection and screening of bacteria from African nightcrawler, Eudrilus eugeniae (Kinberg, 1867) as potential probiotics in aquaculture.

Earthworms are used as an alternative protein source in aquaculture. These organisms serve as an ideal and favorable site for bacterial growth and activity. Hence, in our present study, we isolated and screened potential probiotic bacteria from African nightcrawler (Eudrilus eugeniae). Among 45 bacterial isolates, four (ANSCI9, BFAR9, RM3, and RM10) were selected based on their hydrophobicity, hydrolytic enzyme production, pH and fish bile tolerance, aggregation, and antimicrobial properties. The selected isolates showed good hydrophobicity (≥ 30%) and enzyme production (≥ 10 mm clearing zones), tolerance to pH and fish bile, and inhibitory properties against pathogenic microorganisms. The isolates were identified as Bacillus sp. RM3 (MH919306), Bacillus sp. RM10 (MH919308), Bacillus sp. ANSCI9 (MH919310) and Bacillus sp. BFAR9 (MH919302). These isolates were individually incorporated in the diets of Nile tilapia (Oreochromis niloticus) fingerlings for 14 days to assess their biosafety. The results showed that the survival rates in all treated groups (98.75 ± 2.5 to 100.00 ± 0.0%) were not significantly different (P < 0.05) from the control group (commercial diet) (96.25 ± 2.5%), suggesting that isolates have no adverse effect on the host. This study revealed the presence of potential probiotic microorganisms in E. eugeniae that are beneficial to the aquaculture industry.

Anisakis Dujardin, 1845 infection (Nematoda: Anisakidae) in Pygmy Sperm Whale Kogia breviceps Blainville, 1838 from west Pacific region off the coast of Philippine archipelago.

Cetaceans are definitive hosts of anisakid nematodes known to cause human anisakidosis. Despite the reported strandings of different cetaceans in the Philippines, studies on anisakids from these definitive hosts are limited. Here, the morphologically and molecularly identified anisakid species, specifically those of the genus Anisakis Dujardin, 1845 in stranded Pygmy Sperm Whale Kogia breviceps Blainville, 1838 in the west Pacific region off Philippine waters are presented. Morphological data using SEM and LM revealed multi-infections with different Anisakis species belonging to Anisakis type I and type II groups. Molecularly, PCR-RFLP on the ITS rDNA and sequence data analyses of both ITS rDNA and mtDNA cox2 regions identified those from Anisakis type I group as A. typica (Diesing, 1860), whereas those from type II group as A. brevispiculata Dollfus, 1968, and A. paggiae Mattiucci et al. (Syst Parasitol 61:157-171, 2005). This is the first record of Anisakis infection from this host stranded in the west Pacific region off the coast of Philippine waters and new geographical record for A. paggiae.

Gonad-infecting philometrid Philometra philippinensis sp. nov. (Nematoda, Philometridae) from the bigeye barracuda Sphyraena forsteri Cuvier (Sphyraenidae) off Mariveles, Bataan Province, Philippine archipelago.

We conducted a parasitological study to identify nematodes infecting the gonads of bigeye barracuda Sphyraena forsteri Cuvier (Sphyraenidae) caught in the West Philippine Sea, off the Mariveles coast, Bataan Province, the Philippine archipelago. Males and very tiny nongravid female philometrid nematodes were collected from the ovaries of the fish. Based on light and scanning electron microscope observations, and supported by molecular analyses of the partial nucleotide sequences of the 18S small subunit ribosomal DNA, the philometrid nematode was determined to be a new species belonging to the genus Philometra Costa, 1845 in the family Philometridae (Nematoda: Dracunculoidea), and was named P. philippinensis sp. nov. This new species was clearly distinguishable morphologically from other congeners that parasitize the gonads of marine fishes. Males have unique sickle-shaped spicules and gubernaculum, and a vulva-like cloacal opening. Following Dentirumai philippinensis Quiazon et Moravec, 2013 and Philometra robusta Moravec, Möller et Heeger, 1992, this is the third philometrid species and the first gonad-infecting philometrid from the Philippine archipelago.

Anisakis species (Nematoda: Anisakidae) of Dwarf Sperm Whale Kogia sima (Owen, 1866) stranded off the Pacific coast of southern Philippine archipelago.

Anisakid nematodes in the Pacific region of the Philippine archipelago still remain unexplored. This study was carried out to identify anisakid species from one of their final hosts, the Kogiid whale (Dwarf Sperm Whale, Kogia sima) stranded off the southern part (Davao Gulf) of the Philippine archipelago. Anisakid worms were initially identified morphologically using light and scanning electron microscopy, whereas identification to species level was carried out molecularly using PCR-RFLP and sequencing of the ITS (ITS1-5.8s rRNA-ITS2) and mtDNA cox2 regions. Parasitological study revealed new geographical records for the presence of two Anisakis species (A. brevispiculata and A. typica) and two unknown Anisakis species that are genetically close, at mtDNA cox2 region, to A. paggiae and A. ziphidarum. Based on the molecular data on both genes, the current findings suggest possible occurrence of local variations or sibling species of A. paggiae and A. ziphidarum in the region. Given that Anisakis species have not been reported in the Philippine archipelago, their presence in the Dwarf Sperm Whale inhabiting this region indicates high possibility of Anisakis infections in the marine fishes, cephalopods and other intermediate hosts within the Philippine waters.

Molecular characterization and comparison of four Anisakis allergens between Anisakis simplex sensu stricto and Anisakis pegreffii from Japan.

It remains unclear whether allergens are the same among the sibling species of Anisakis simplex sensu lato. This study was carried out to compare the amino acid sequences of three major (Ani s 1, 2 and 12) and one minor (Ani s 9) Anisakis allergens between A. simplex s.s. and Anisakis pegreffii. We found 2 (out of 163), 1 (out of 869), and 29 (out of 266) amino acid variable sites for Ani s 1, 2, and 12, respectively. However, as both intra- and inter-species variations were present at the same amino acid positions, no amino acid variations clearly distinguished the two sibling species. IgE-binding epitopes (Ani s 1) and a binding motif of human leukocyte antigen (Ani s 2 and 9) demonstrated by previous studies were conserved. The similarities of the amino acid sequences of the allergens indicate possible similar allergy-associated health risks in humans infected with or accidentally ingesting either Anisakis species.

Distribution of Anisakis species larvae from fishes of the Japanese waters.

Human anisakiasis is caused by the consumption of raw, marinated or undercooked fish and squid infected with nematodes of the genus Anisakis Dujardin, 1845. In view of food safety, this study was carried out to examine the distribution of Anisakis species in marine fishes within Japanese waters. Seven fish species from six localities were collected and examined for Anisakis infection. Morphological and molecular (ITS region and mtDNA cox2 gene) characterization revealed the presence of two, among the three sibling species of Anisakis simplex, viz. A. simplex sensu stricto (s.s.) and A. pegreffii. Distribution data were collated with the results from the previous researches to better understand Anisakis distribution in Japanese waters. Distributions of Anisakis species were found to be locality-specific rather than host-specific, particularly between the two major species, A. simplex s.s. and A. pegreffii. Anisakis simplex s.s. is mainly found in fishes from northern Japan to Pacific sides, whereas A. pegreffii is in fishes from the Sea of Japan to East China Sea sides.

Experimental challenge of Anisakis simplex sensu stricto and Anisakis pegreffii (Nematoda: Anisakidae) in rainbow trout and olive flounder.

The third-stage larvae of Anisakis simplex sensu lato (s.l.) are found in many marine fishes. To ensure food safety, it is important to determine whether these larvae are present in the body muscle of commercial fish species. However, there is little information regarding the tissue specificity of Anisakis and two of its sibling species, A. simplex sensu stricto (s.s.) and Anisakis pegreffii, that are common in marine fish in Japanese waters. We orally challenged rainbow trout (Oncorhynchus mykiss (Walbaum)), and olive flounder (Paralichthys olivaceus (Temminck and Schlegel)) with L3 larvae of these two sibling species and monitored infection for 5weeks. In rainbow trout, A. simplex s.s., but not A. pegreffii larvae, migrated into the body muscle. A small number of freely moving A. pegreffii larvae were recovered within the body cavity. In olive flounder, A. simplex s.s. larvae were found in both the body cavity and body muscle. A. pegreffii larvae were found only in the body cavity and primarily encapsulated in lumps. Our results indicate that there are differences in the sites of infection and host specificity between the two sibling species of A. simplex s.l.

Morphological differences between larvae and in vitro-cultured adults of Anisakis simplex (sensu stricto) and Anisakis pegreffii (Nematoda: Anisakidae).

Proper identification of Anisakis species infecting host fishes is very important to both human health and fish disease diagnosis. The foremost problem in the identification of Anisakis larvae in fishes is that L3 larvae cannot be easily differentiated morphologically, especially between A. simplex (sensu stricto) (s.s.) (Rudolphi, 1809) and A. pegreffii Campana-Rouget et Biocca, 1955. Instead, molecular means such as allozyme, mitochondrial DNA (mtDNA) cox2 region and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analyses had been successfully used. In this study, morphological differences of L3 larvae collected from fishes and in vitro-cultured L4 larvae and adult A. simplex (s.s.) and A. pegreffii were evaluated. Anisakis larvae were collected from 7 different host fishes within Japan. Undamaged A. simplex (s.s.) and A. pegreffii collected from Oncorhynchus keta (Walbaum) and Scomber japonicus Houttuyn, respectively, were used for in vitro-culture in order to obtain L4 and adult stages. Species identification was confirmed by PCR-RFLP analysis of the ITS region (ITS1-5.8S-ITS2) of ribosomal DNA and by mtDNA cox2 gene sequencing. Results revealed that L3, L4 and adult stages of A. simplex (s.s.) and A. pegreffii are morphologically distinguishable based on ventriculus length, wherein the former has longer ventriculus (0.90-1.50 mm) than the latter (0.50-0.78 mm). For oesophagus/ventriculus ratio, these two species are distinguishable only during L4 and adult stages. Also, adult male A. simplex (s.s.) and A. pegreffii were found to be distinguishable by differences in the distribution pattern of the caudal papillae, particularly the 3rd pair of distal papillae.

Taxonomical study into two new species of Philometra (Nematoda: Philometridae) previously identified as Philometra lateolabracis (Yamaguti, 1935).

Males of the nematode Philometra lateolabracis (Yamaguti, 1935), the type species of the genus Philometra Costa, 1845, were discovered for the first time in gonads of its type host, the Japanese seaperch, Lateolabraxjaponicus (Cuvier). Morphological comparisons carried out between the collected male and female P. lateolabracis with the male and female philometrid nematodes previously reported as P. lateolabracis infecting chicken grunt, Parapristipoma trilineatum (Thunberg), and red sea bream, Pagrus major (Temminck et Schlegel), revealed that the latter represent two new species, Philometra isaki sp. n. and Philometra madai sp. n., respectively. Molecular comparison of ITS2 rDNA between P. lateolabracis and P. madai supported the morphological conclusion that the two nematodes obtained from different fish species should be assigned to different species.

Philometra sawara sp. n. and a redescription of Philometra sciaenae Yamaguti, 1941 and Philometra nemipteri Luo, 2001 (Nematoda: Philometridae): a morphological and molecular approach.

Morphological data and molecular analyses are used to describe the taxonomy of philometrid nematodes of the genus Philometra Costa, 1845, found in the gonads of marine fishes in Japan. A new Philomnetra species, P. sawara sp. n., is described based on male and female specimens collected from the gonads of Scomberomorus niphonius (Cuvier) (Japanese Spanish mackerel). Two additional species, Philometra nemipteri Luo, 2001 and Philometra sciaenae Yamaguti, 1941, are confirmed as valid species and are redescribed based on specimens collected from the gonads of Nemipterus virgatus (Houttuyn) (golden threadfin bream) and Pennahia argentata (Houttuyn) (silver croaker), respectively. Male P. nemipteri are first reported and described in this study. Redescriptions of female P. nemipteri and male and female P. sciaenae were also necessary based on our morphological observations. A molecular comparison of the internal transcribed spacer 2 (ITS2) region of the ribosomal DNA between P. sawara, P. nemipteri, P. sciaenae, and previously reported philometrid nematodes from the genus Philometra and Philometroides Yamaguti, 1935 supports the conclusion that the three Philometra species in the current study are independent. An ITS2-derived neighbour-joining tree, consisting of both the current specimens and previously described Philometra and Philometroides species, is also presented.