A Novel Gonadotropic Microsporidian Parasite (Microsporidium clinchi n. sp.) Infecting a Declining Population of Pheasantshell Mussels (Actinonaias pectorosa) (Unioinidae) from the Clinch River, USA

Freshwater mussels of the order Unionida are among the most endangered animal groups globally, but the causes of their population decline are often enigmatic, with little known about the role of disease. In 2018, we collected wild adult pheasantshell (Actinonaias pectorosa) and mucket (Actinonaias ligamentina) during an epidemiologic survey investigating an ongoing mussel mass mortality event in the Clinch River, Virginia and Tennessee, USA. Histopathology and transmission electron microscopy showed a novel microsporidian parasite primarily infecting the ovary of pheasantshell. Sequencing of the small subunit rRNA gene produced a 1333 bp sequence with the greatest similarity to Pseudonosema cristatellae (AF484694.1; 86.36%; e-value = 0), a microsporidium infecting the freshwater bryozoan (Cristatella mucedo). Microsporidia were observed in 65% (17/26) of the examined female pheasantshell (A. pectorosa) and in no (0/2) female muckets (A. ligamentina) and occurred at mortality and non-mortality sites. Our findings indicate that a novel parasite, Microsporidium clinchi n. sp., is present in pheasantshell in the Clinch River, and while likely not a cause of mass mortality, could reduce fecundity and recruitment in this declining population and threaten the success of reintroductions. Surveillance of M. clinchi n. sp. and evaluation of broodstock and their progeny for microsporidia would therefore be prudent.

First Report of Enterocytozoon hepatopenaei Infection in Pacific Whiteleg Shrimp (Litopenaeus vannamei) Cultured in Korea

The consumption of cultured crustaceans has been steadily increasing, and Pacific whiteleg shrimp (Litopenaeus vannamei) are major cultivated invertebrates worldwide. However, shrimp productivity faces a variety of challenges, mainly related to outbreaks of lethal or growth retardation-related diseases. In particular, hepatopancreatic microsporidiosis caused by the microsporidian parasite Enterocytozoon hepatopenaei (EHP) is an important disease associated with growth retardation in shrimp. Here, we report the detection of EHP through histopathological, molecular and electron microscopy methods in the hepatopancreas of Pacific whiteleg shrimp with growth disorder in a South Korean farm. Phylogenetic analysis showed a clade distinct from the previously reported EHP strains isolated in Thailand, India, China and Vietnam. An EHP infection was not associated with inflammatory responses such as hemocyte infiltration. Although EHP infection has been reported worldwide, this is the first report in the shrimp aquaculture in Korea. Therefore, an EHP infection should be managed and monitored regularly for effective disease control and prevention.

A review of ocular Encephalitozoon cuniculi infection in the rabbit

Encephalitozoon cuniculi is a microsporidian parasite affecting rabbits and other mammals, including immunocompromised humans. One clinical manifestation of E. cuniculi is the ocular form, which typically presents with phacoclastic uveitis, a yellow-white granuloma and a cataract. This literature review focuses on the aetiopathogenesis, presentation, diagnosis and treatment options for ocular E. cuniculi with emphasis on phacoemulsification, the gold standard treatment.

Development of a Quantitative PCR Method for Specific and Quantitative Detection of Enterocytospora artemiae, a Microsporidian Parasite of Chinese Grass Shrimp (Palaemonetes sinensis)

Enterocytospora artemiae (EAM) mainly parasitizes the hepatopancreas of Palaemonetes sinensis. Serious infection leads to hepatopancreatic lesions, which greatly reduce the vitality of P. sinensis. Currently, EAM is detected via conventional PCR methods. However, conventional PCR has low sensitivity and cannot be used for accurate quantitative detection of EAM or its parasitic activity in host tissues. In this study, we designed a pair of specific primers based on the sequence of the ribosomal protein S9 gene (RPS9; GenBank accession number: MZ420734) to establish and optimize a SYBR Green I real-time fluorescent quantitative PCR detection method for EAM. Only EAM appeared as a bright and single target band, whereas other microorganisms did not, indicating that the primer for RPS9 had high specificity. This method displayed optimum amplification effects at an annealing temperature of 55°C, and the melting curve of the product produced a single peak. The established method showed a good linear relationship from 2.2 × 108 to 2.2 × 101 copies/μL. The relationship between the number of cycle thresholds (Ct) and the logarithm of the initial template amount (x) conformed to Ct = −3.281 log x + 36.543 (R2 = 0.998). Amplification efficiency was 101.737%, and the lower limit of detection sensitivity was 2.2 × 101 copies/μL. Good intra- and inter-group repeatability was observed within the linear range. The sensitivity of this method was more than 200 times higher than that of nested PCR. Thus, detection data obtained using this method may be useful as a technical reference for rapid and accurate identification of EAM infection and for the prevention and control of EAM during P. sinensis breeding.

Genome and Evolutionary Analysis of Nosema ceranae: A Microsporidian Parasite of Honey Bees

Microsporidia comprise a phylum of single cell, intracellular parasites and represent the earliest diverging branch in the fungal kingdom. The microsporidian parasite Nosema ceranae primarily infects honey bee gut epithelial cells, leading to impaired memory, suppressed host immune responses and colony collapse under certain circumstances. As the genome of N. ceranae is challenging to assembly due to very high genetic diversity and repetitive region, the genome was re-sequenced using long reads. We present a robust 8.8 Mbp genome assembly of 2,280 protein coding genes, including a high number of genes involved in transporting nutrients and energy, as well as drug resistance when compared with sister species Nosema apis. We also describe the loss of the critical protein Dicer in approximately half of the microsporidian species, giving new insights into the availability of RNA interference pathway in this group. Our results provided new insights into the pathogenesis of N. ceranae and a blueprint for treatment strategies that target this parasite without harming honey bees. The unique infectious apparatus polar filament and transportation pathway members can help to identify treatments to control this parasite.

Clinical Investigation and Molecular Diagnosis of Microsporidian Enterocytozoon hepatopenaei (EHP) in Shrimps from Selangor, Malaysia

In recent years, shrimp aquaculture production in Southeast Asia countries was highly infected by, microsporidian parasite, EHP. Recently, shrimps from farms located in Selangor were encountering growth retardation and it highly concerned us to carry out a clinical investigation in both farms (Location A and B). EHP infected P. monodon samples were collected from both locations. A total of 43 shrimp samples were collected and diagnosed via PCR using the 18S rRNA gene. Environmental parameters were found relatively higher than the advised values. It indicates that the farms are highly infected and toxic which had led to growth retardation and mortality. However, no significant differences were observed (except water temperatures) between the locations. The phylogenetic analysis expressed EHP detected from Malaysia formed in one clade and all the reference sequences clustered based on geographical continents (Asian-Latin America). Minimum-spanning network analysis shows that Malaysia samples Location A are genetically related with other Asian EHP samples even though the total positive rate of EHP infection was lower. However, samples from Location B were not clustered within the EHP clade and shows the highest genetic variance within Malaysia and among Asian samples. This preliminary investigation results confirmed the incidence of EHP outbreak in Malaysia.

A potential prokaryotic and microsporidian pathobiome that may cause shrimp white feces syndrome (WFS)

White feces syndrome (WFS) in shrimp cultivation ponds is characterized by the occurrence of shrimp with abnormal, white intestines (midguts) combined with large floating mats of white, shrimp fecal strings. The etiology for WFS is complex, similar to diarrhea in humans. EHP-WFS is a type of WFS characterized by massive quantities of spores from the microsporidian parasite Enterocytozoon hepatopenaei (EHP) together with mixed, unidentified bacteria in the shrimp hepatopancreas, midgut and fecal strings. However, WFS does not always develop in shrimp with severe EHP infections in controlled laboratory challenges. Further, in EHP-WFS outbreak ponds, some shrimp show white midguts (WG) while others in the same pond show grossly normal midguts (NG). We hypothesized that comparison of the microbial flora between WG and NG from the same EHP-WFS pond would reveal probable combinations of microbes significantly associated with EHP-WFS. To test this hypothesis, we selected a pond exhibiting a severe EHP-WFS outbreak in cultivated Penaeus vannamei and used a combination of microscopic and microbial profiling analyses to compare WG and NG samples. By histology, EHP plasmodia and spores were confirmed in the hepatopancreas (HP) and midgut of WG and NG shrimp, but pathological severity and spore quantity was higher in the WG shrimp. In addition, intestinal microbiomes in WG shrimp were less diverse and had higher abundance of bacteria from the genera Vibrio and Propionigenium. Propionigenium quantity in the HP of WG shrimp was significantly higher (P = 1.08e-5) than in NG shrimp (4,506 vs. 3 copies /100 ng DNA, respectively). These findings supported our hypothesis by revealing two candidate bacterial genera that should be tested in combination with EHP as a potential eukaryote-prokaryote pathobiome that causes EHP-WFS in P. vannamei.

A New Genus Of Microsporidian Parasite (Hepatosporidae; Microsporidia) Found In The Oocytes Of Ribbon Worms From The North Pacific Genus Maculaura (Heteronemertea; Nemertea)

An intracellular microsporidian parasite was first observed within oocytes of Maculaura alaskensis, a small pilidiophoran nemertean, commonly found on sandflats along the Pacific coast of North America. Infected oocytes have large vesicles containing dozens to hundreds of diplokaryotic, ellipsoid spores measuring 1.3 by 2.3 μm. A partial small subunit nuclear ribosomal 18S gene sequence isolated from the microsporidian does not match any known microsporidian sequences in the public databases. Phylogenetic analysis groups it with Hepatospora eriocheir in a sister clade to the Enterocytozoonidae. All the known life stages of this parasite are contained within a membranous envelope. This microsporidian was identified in M. alaskensis, Maculaura aquilonia, Maculaura oregonensis, and Maculaura cerebrosa in Coos Bay, Oregon, in M. alaskensis from Newport, Oregon, and in M. aquilonia collected in Juneau, Alaska. This is, to our knowledge, the first species of microsporidia found to directly infect nemertean host cells.

Effects of Prebiotics and Probiotics on Honey Bees (Apis mellifera) Infected with the Microsporidian Parasite Nosema ceranae

Nosema ceranae is a microsporidian fungus that parasitizes the midgut epithelial cells of honey bees, Apis mellifera. Due to the role that midgut microorganisms play in bee health and immunity, food supplementation with prebiotics and probiotics may assist in the control of N. ceranae. The dietary fiber prebiotics acacia gum, inulin, and fructooligosaccharides, as well as the commercial probiotics Vetafarm Probotic, Protexin Concentrate single-strain (Enterococcus faecium), and Protexin Concentrate multi-strain (Lactobacillus acidophilus, L. plantarum, L. rhamnosus, L. delbrueckii, Bifidobacterium bifidum, Streptococcus salivarius, and E. faecium) were tested for their effect on N. ceranae spore loads and honey bee survivorship. Bees kept in cages were inoculated with N. ceranae spores and single-dose treatments were administered in sugar syrup. Acacia gum caused the greatest reduction in N. ceranae spore numbers (67%) but also significantly increased bee mortality (62.2%). However, Protexin Concentrate single-strain gave similarly reduced spore numbers (59%) without affecting the mortality. In a second experiment, multiple doses of the probiotics revealed significantly reduced spore numbers with 2.50 mg/mL Vetafarm Probotic, and 0.25, 1.25, and 2.50 mg/mL Protexin Concentrate single-strain. Mortality was also significantly reduced with 1.25 mg/mL Protexin Concentrate single-strain. N. ceranae-inoculated bees fed 3.75 mg/mL Vetafarm Probotic had higher survival than N. ceranae-inoculated bees, which was similar to that of non-inoculated bees, while N. ceranae-inoculated bees fed 2.50 mg/mL Protexin Concentrate single-strain, had significantly higher survival than both N. ceranae-inoculated and non-inoculated bees. Protexin Concentrate single-strain is promising as it can reduce N. ceranae proliferation and increase bee survivorship of infected bees, even compared to healthy, non-infected bees.