Pathogenicity associated with an infestation of the marine leech parasite Pterobdella arugamensis in farmed fish.

The marine leech Pterobdella arugamensis is a hematophagous parasite, and the extent of injury to the host largely depends on the number of attached leeches. This study aimed to assess the pathogenicity of marine leeches in Asian seabass (Lates calcarifer) and tiger grouper (Epinephelus fuscoguttatus) fingerlings under laboratory conditions. Five groups of healthy Asian seabass and tiger grouper were exposed to varying numbers of marine leeches (0, 1, 10, 30, or 70 per fish) for 7 d. Infested Asian seabass and tiger grouper both showed pathological changes even with only 1 leech, manifesting as clinical signs like haemorrhages. The cumulative mortality at 7 d post-exposure (dpe) was 11 or 33% for Asian seabass infested with 1 or 10 marine leeches, respectively. Fish with 30 or 70 marine leeches showed higher rates of mortality (56%). A similar trend was seen in tiger grouper, with mortality rates reaching 78% in fish with 30 or 70 marine leeches, and 56 or 33% in fish with 10 leeches or 1 leech, respectively. Factorial analysis of mortality after 7 dpe between both species showed significant differences (2-way ANOVA p = 0.001) when exposed to varying numbers of marine leeches. The haematocrit values differed significantly between Asian seabass or tiger grouper infested with either 0 or 1 marine leech and those infested with 10, 30, or 70 marine leeches (1-way ANOVA, p = 0.0001). This suggests that marine leech infestation has a measurable impact on both species. Consequently, fish farmers should promptly address leech infestation upon discovery in their cages.

Detection of Enterocytozoon hepatopenaei (EHP) (microsporidia) in several species of potential macrofauna-carriers from shrimp (Penaeus vannamei) ponds in Malaysia.

Infection by the microsporidian parasite Enterocytozoon hepatopenaei (EHP) has become a significant problem in the shrimp cultivation industry in Asian countries like Thailand, China, India, Vietnam, Indonesia, and Malaysia. The outbreak of this microsporidian parasite is predominantly related to the existence of macrofauna-carriers of EHP. However, information about potential macrofauna-carriers of EHP in rearing ponds is still limited. In this study, the screening of EHP in potential macrofauna-carriers was conducted in farming ponds of Penaeus vannamei in three states in Malaysia, namely Penang, Kedah, and Johor. A total of 82 macrofauna specimens (phyla: Arthropoda, Mollusca, and Chordata) were amplified through a polymerase chain reaction (PCR) assay targeting genes encoding spore wall proteins (SWP) of EHP. The PCR results showed an average prevalence of EHP (82.93%) from three phyla (Arthropoda, Mollusca and Chordata). The phylogenetic tree generated from the macrofauna sequences was revealed to be identical to the EHP-infected shrimp specimens from Malaysia (MW000458, MW000459, and MW000460), as well as those from India (KY674537), Thailand (MG015710), Vietnam (KY593132), and Indonesia (KY593133). These findings suggest that certain macrofauna species in shrimp ponds of P. vannamei are carriers of EHP spores and could be potential transmission vectors. This study provides preliminary information for the prevention of EHP infections that can be initiated at the pond stage by eradicating macrofauna species identified as potential vectors.

Occurrence of Enterocytozoon hepatopenaei (EHP) infection on Penaeus vannamei in one rearing cycle.

The microsporidian parasite Enterocytozoon hepatopenaei (EHP) is an emerging problem in the marine shrimp industry, primarily in Asian countries such as China, Thailand, India, Malaysia, Indonesia, and Vietnam. A screening was conducted to investigate the prevalence of EHP after a fixed period of culturing for 1 rearing cycle in 3 states of Malaysia. The screening stages covered Penaeus vannamei post larvae (PL) and after 14-30, 31-50, 51-70, and 71-90 d of culture in 1 production cycle. A total of 279 samples were amplified using a PCR assay targeting the gene encoding a spore wall protein (SWP) of EHP. The EHP infection was initially detected in the hatchery and increased to 96.6% after the shrimp were transferred to the pond. The positive EHP sequence showed 91 to 100% similarity to sequences from India, Thailand, Vietnam, Indonesia, and Latin America. EHP infection increased throughout 1 rearing cycle due to factors such as the cannibalistic feeding habits of shrimp and the presence of unknown vectors or carriers of EHP in the culture ponds. Hence, the finding from the current study will be fundamental for other studies concerning EHP.

Expression profile of immune-related genes in Lates calcarifer infected by Cryptocaryon irritans.

Cryptocaryon irritans causes Cyptocaryonosis or white spot disease in a wide range of marine fish including Lates calcarifer (Asian seabass). However, the immune response of this fish to the parasite is still poorly understood. In this study, quantitative polymerase chain reaction (qPCR) was performed to assess the expression profile of immune-related genes in L. calcarifer infected by C. irritans. A total of 21 immune-related genes encoding various functions in the fish immune system were utilized for the qPCR analysis. The experiment was initiated with the infection of juvenile fish by exposure to theronts from 200 C. irritans cysts, and non-infected juvenile fish were used as controls. Spleen, liver, gills and kidney tissues were harvested at three days post-infection from control and infected fish. In addition, organs were also harvested on day-10 post-infection from fish that had been allowed to recover from day-4 up to day-10 post-infection. L. calcarifer exhibited pathological changes on day-3 post-infection with the characteristic presence of white spots on the entire fish body, excessive mucus production and formation of a flap over the fish eye. High quality total RNA was extracted from all tissues and qPCR was performed. The qPCR analysis on the cohort of 21 immune-related genes of the various organs harvested on day-3 post-infection demonstrated that most genes were induced significantly (p < 0.05) in all tissues, particularly liver (11/21 genes) and kidney (11/21). The expression profile demonstrated that induction of the MHC Class IIα gene was the highest compared to the other genes followed by serum amyloid A, CC chemokine and hepcidin-2 precursor genes. In fish that were allowed to recover from the C. irritans infection (10 days post-infection), expression of the immune-related genes was down-regulated to levels similar to the control fish. These results provide insights into the interaction between C. irritans and L. calcarifer and suggest that the innate immune system plays an important role in early defence against parasite infection allowing the fish to eventually recover from the infection.

Cryptocaryon irritans infection induces the acute phase response in Lates calcarifer: a transcriptomic perspective.

Cryptocaryoniasis (also known as marine white spot disease) is mediated by Cryptocaryon irritans. This obligate ectoparasitic protozoan infects virtually all marine teleosts, which includes Lates calcarifer, a highly valuable aquaculture species. Little is known about L. calcarifer-C. irritans interactions. This study was undertaken to gain an informative snapshot of the L. calcarifer transcriptomic response over the course of C. irritans infection. An in-house fabricated cDNA microarray slides containing 3872 probes from L. calcarifer liver and spleen cDNA libraries were used as a tool to investigate the response of L. calcarifer to C. irritans infection. Juvenile fish were infected with parasites for four days, and total RNA was extracted from liver tissue, which was harvested daily. We compared the transcriptomes of C. irritans-infected liver to uninfected liver over an infection period of four days; the comparison was used to identify the genes with altered expression levels in response to C. irritans infection. The greatest number of infection-modulated genes was recorded at 2 and 3 days post-infection. These genes were mainly associated with the immune response and were associated in particular with the acute phase response. Acute phase proteins such as hepcidin, C-type lectin and serum amyloid A are among the highly modulated genes. Our results indicate that an induced acute phase response in L. calcarifer toward C. irritans infection is similar to the responses observed in bacterial infections of teleosts. This response demonstrates the importance of first line defenses in teleost innate immune responses against ectoparasite infection.

Prevalence, mean intensity and site preference of Caligus rotundigenitalis Yü, 1933 (Copepoda: Caligidae) on cage cultured crimson snapper (Lutjanus erythropterus Bloch, 1790) from Bukit Tambun, Penang, Malaysia.

Snapper had been cultured in Malaysia since 1980 due to the fry availability and the high demand. However, details on the caligids infestation were not properly documented. This study was carried out to determine the prevalence, mean intensity and site preference of Caligus rotundigenitalis (Caligidae, Siphonostomatoida) a parasitic copepod on cage cultured crimson snapper, Lutjanus erythropterus from Bukit Tambun, Penang, Malaysia. A total of 70 specimens of cultured snapper were examined based on different infestation sites such as head, body as well as operculum. The specimens were separated into three groups according to the size of the fish. C. rotundigenitalis was found to be the only species infesting L. erythropterus with the prevalence and the mean intensity of 81.4% and 5.6±4.4, respectively. There was a significant difference between the prevalence of site infestation of the body and inner operculum sites. The prevalence of C. rotundigenitalis was highest on inner operculum of the fish followed by the body and head. However, there was no significant difference in the distribution of C. rotundigenitalis over the different infestation sites derived from the three groups. The information obtained from this study can be used for more effective control measures of ectoparasitic copepod infestation in floating cages.

Pathology Associated with White Spot Virus (WSV) Infection in Wild Broodstock of Tiger Prawns (Penaeus monodon).

A total of six wild broodstocks of tiger prawns, Penaeus monodon, were found positive for White Spot Virus (WSV) with an IQ2000 detection kit. Using histopathology, the intranuclear inclusion of haemocyte due to WSV infection was observed in the epithelium cells of the antennal gland, stomach and gills. This result confirmed that the wild broodstocks were positive with WSV without showing any white spot. Additionally, histopathological examination also revealed an accumulation of haemocytes around the hepatopancreatic tubules resulting from bacterial infection. Encapsulation and nodule formation, as well as related necrosis, were also observed around the hepatopancreatic tubules infected with a metazoan parasite. Encysted tylocephalum larval cestodes were observed in the hepatopancreas, with haemocytic aggregation being observed around the infected tubules. These findings showed some bacterial and parasitic infections which, in addition to the viral infection itself, could contribute to the 80% mortality rate in wild broodstocks infected with WSV.

Infestation of gill copepod Lernanthropus latis (Copepoda: Lernanthropidae) and its effect on cage-cultured Asian sea bass Lates calcarifer

Twenty Asian sea bass Lates calcarifer from a floating cage in Bt. Tambun, Penang were examined for the presence of parasitic gill copepod, Lernanthropus latis. The prevalence of L. latis was 100% with the intensity of infection ranging from 1 to 18 parasites per host or 3.75 of mean intensity. Female parasites having oblong cephalothorax and egg-strings were seen mainly on the entire gill of examined Asian sea bass. The infected gill of Asian sea bass was pale and had eccessive mucus production. Under light and scanning electron microscopies (SEM), L. latis was seen grasping or holding tightly to the gill filament using their antenna, maxilla and maxilliped. These structures are characteristically prehensile and uncinate for the parasite to attach onto the host tissue. The damage was clearly seen under SEM as the hooked end of the antenna was embedded into the gill filament. The parasite also has the mandible which is styliform with eight teeth on the inner margin. The pathological effects such as erosion, haemorrhages, hyperplasia and necrosis along the secondary lamellae of gill filaments were seen and more severe at the attachment site. The combined actions of the antenna, maxilla and maxilliped together with the mandible resulted in extensive damage as L. latis attached and fed on the host tissues.

Screening of parasitic and IHHNV infections in wild giant freshwater prawn Macrobrachium rosenbergii from Rejang River at Kuching, Sarawak.

A preliminary survey of parasitic and infectious hypodermal and haematopoietic necrosis virus (IHHNV) infections in giant freshwater prawn from the Damak Sea of Rejang River, Kuching, Sarawak was conducted. Symptoms of black spots/patches on the rostrum, carapace, pleopods or telson were observed in most of the 107 samples collected. Parasitic examination revealed sessiline peritrichs such as (Zoothamnium sp.), nematode larvae, gregarine stage and cocoon of leech with prevalences of 1.2%, 1.2%, 5% and 17% respectively. Under histopathological examination, changes like accumulation of hemocytes around hepatopancreatic tubules due to vibriosis, basophilic intranuclear inclusions in the epithelium and E-cell of hepatopancreatic tubules as a result of HPV were seen through the section. No positive infection of IHHNV was detected in 78 samples. As such, the wild giant freshwater prawns in Damak Sea of Rejang River in Kuching are IHHNV-free though infections of parvo-like virus and bacteria were seen in histopathology.