Short communication

Transmission and tissue distribution of Pseudoloma

neurophilia (Microsporidia) of zebrafish, Danio rerio

(Hamilton)

M L Kent and J K Bishop-Stewart

Center for Fish Disease Research, Department of Microbiology, Oregon State University, Corvallis, OR, USA

Keywords: microsporidia, Pseudoloma neurophilia,tissue distribution, transmission, zebrafish.

There has recently been a dramatic increase in theuse of zebrafish, Danio rerio (Hamilton), as alaboratory model to study basic mechanisms ofvertebrate developmental genetics (Postlethwait &Talbot 1997). Based on diagnostic examinationsconducted through the Zebrafish InternationalResource Center on 27 zebrafish research facilities,the most common disease in this fish is neuralmicrosporidiosis (http://www.zfin.org/zf_info/stckctr/health.html). This infection was first reported in1980 in France (de Kinkelin 1980), and we haverecently assigned it to a new genus and species,Pseudoloma neurophilia (Matthews, Brown, Larison,Bishop-Stewart, Rogers & Kent 2001). The infec-tion is most common in the spinal chord and hindbrain. It also occurs in ventral root ganglia andsomatic muscle, where it is associated withinflammation. These fish are usually severelyemaciated and may exhibit scoliotic changes.

We systematically evaluated several fish to betterelucidate the distribution of the infection andassociation with lesions. Fish from previous diag-nostic cases were selected as follows: five infectedfemales, five infected males and five fish with diffuse,chronic myositis. These categories were assignedbased on haematoxylin and eosin (H & E)-stainedsections. Fish were killed with an overdose of

MS-222, the abdomen was dissected open, and thefish were preserved whole in Dietrich’s solution. Fishwere processed for histology and sagittal sectionswere obtained to best demonstrate the centralnervous system and associated structures. Fish weresplit in half and each side was processed. Fouradjacent sections from each fish were examined; twostained with H & E and two with Fungi-Fluor stain(Polysciences, Warrington, PA http://www.polys-ciences.com), a fluorescent stain that specificallybinds to chitin (Berlin, Conteas, Ash, Sorvillo, Jacob,Yatabe & Peter 2000). Sections were deparaffinized,and stained with Fungi-Fluor staining solution A for1 min. Slides were then counterstained with solutionB (blocking agent) for 1 min, and then slides wererinsed with distilled water for 1 min. Slides wereobserved as water mounts under coverslips sealedwith paraffin using a fluorescent microscope witha 4¢,6¢-diamidino-2-phenylindole hydrochloride(DAPI filter) (250–400 nm).

Several special stains have been described formicrosporidian spores, including fluorescent stains(Weber, Schwartz & Deplazes 1999). For fishmicrosporidia, Guzman, Enriquez & Schottelius(2001) used Calcofluor White (Sigma, St Louis,MO, USA) for detecting Nucleospora salmonis intissue smears. Fungi-Fluor stain was very effectivefor visualization of spores (Fig. 1), and spores weredetected in several organs by the method whilst theywere not seen in adjacent sections stained with H &E (Table 1). Free spores were common in thekidney and ovaries, and many more spores wereassociated with the somatic muscle lesions thanwere seen in H & E-stained sections. Intact

Journal of Fish Diseases 2003, 26, 423–426

Correspondence Prof M L Kent, Center for Fish Disease

Research, Department of Microbiology, 220 Nash Hall, Oregon

State University, Corvallis, Oregon, 97331-3804, USA

(e-mail: michael.kent@orst.edu)

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Figure 1 Pseudoloma neurophilia in zebrafish tissues stained with Fungi-Fluor. S ¼ spores. Bar ¼ 10 lm, unless otherwise indicated.

(A) Xenomas in brain (bar ¼ 25 lm), (B) free spores in kidney, (C) xenoma in muscle (bar ¼ 25 lm), (D) spores in muscle associated

with myositis, (E) spores in ovarian tissue, 0 ¼ oocytes, (F) spores in retina of eye and (G) high magnification of spores.

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Journal of Fish Diseases 2003, 26, 423–426 M L Kent and J K Bishop-Stewart Pseudoloma in zebrafish

xenomas were occasionally found in the muscle,indicating that non-neural tissues may be a site ofdevelopment. While the ovaries of females wereoften infected, no spores were found within eggs.However, we found spores in an egg of onezebrafish from our diagnostic cases that was notincluded in Table 1 (Fig. 2). The parasite was notdetected in one fish with severe, diffuse myositis.This suggests that this lesion might also be causedby other agents, although it is possible that sporeswere present but undetected.

Fish microsporidia in general are thought to betransmitted directly by the ingestion of spores (seereview by Shaw & Kent 1999). Transovarialtransmission is another route of infection employedby microsporidia (Dunn, Terry & Smith 2001) and

this has been suggested for a few fish microsporidia(Summerfelt & Warner 1970; Docker, Devlin,Richard, Khattra & Kent 1997).

We examined the ability of Pseudoloma to betransmitted by direct exposure to infectious materi-al. Fish were held in aquaria with flowingdechlorinated city water at 28 �C. Fifty adultzebrafish from a wholesale distributor were usedin this experiment. No fish obtained from thisdistributor had previously been found to be infec-ted. A total of 25 fish were exposed to the infectedspinal chords collected from 35 infected fish, while25 fish were not exposed and held as controls. Toprepare the inoculum, infected spinal chords wereremoved, mixed with 0.85% sterile saline, andpassed through a 100 lm nylon screen. Fish were

Figure 2 Zebrafish egg replete with spores

of Pseudoloma neurophilia (H & E,

bar ¼ 10 lm).

Table 1 Organ distribution of Pseudoloma neurophilia from diagnostic cases. Five females (F), five males (M) and five additional fish

with myositis (My) examined in histological section and stained with either haematoxylin and eosin or Fungi-Fluor

Fish CNS Muscle Nerves Gill Gut Liver Kidney Spleen Heart Gonads Skin

F1 +/+* 0/0 ++/0 0/0 0/0 0/0 0/+ 0/0 0/0 0/0 0/++

F2 ++/+++ 0/+ +/++ 0/0 0/0 0/+ 0/++ 0/0 0/0 0/++ 0/0

F3 +/+ 0/0 0/0 0/0 0/0 0/0 0/0 N N 0/++ 0/0

F4 ++/++ 0/0 0/+ 0/+ 0/+ 0/0 0/+ 0/0 0/0 0/++ 0/0

F5 +++/+++ 0/0 0/++ 0/0 0/+ 0/0 0/0 0/0 0/0 0/0 0/0

M1 +++/+++ 0/0 0/+ 0/0 0/0 0/0 ++/++ 0/0 0/0 0/0 0/0

M2 ++/++ 0/+ 0/++ 0/0 0/0 0/0 0/+++ 0/0 0/0 0/0 0/+

M3 ++/++ 0/0 0/++ 0/0 0/0 0/0 0/++ 0/0 0/0 0/0 0/0

M4 ++/++ 0/0 0/++ 0/0 0/0 0/0 0/++ 0/0 0/0 0/0 0/0

M5 ++/++ 0/0 0/++ 0/0 0/0 0/0 0/++ 0/0 0/0 0/0 0/0

My1 0/0 0/0 0/++ 0/0 0/0 0/0 0/++ 0/0 0/0 0/0 0/0

My2 0/++ 0/+ 0/++ 0/+ 0/0 0/0 0/++ 0/0 0/+ 0/0 0/0

My3 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0

My4 ++/++ +/++ 0/++ 0/0 0/0 0/0 0/++ 0/0 0/0 0/0 0/0

My5 ++/++ +/+ 0/++ 0/0 0/0 0/0 0/++ 0/0 0/0 0/0 0/0

* Haematoxylin and eosin/Fungi-Fluor. +, Few spores (1–10 seen in tissue); ++, several spores observed (spores seen in most fields of view); +++,

heavy infection (numerous free spores or xenomas); N, not present in sections; CNS, central nervous system.

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Journal of Fish Diseases 2003, 26, 423–426 M L Kent and J K Bishop-Stewart Pseudoloma in zebrafish

exposed in a 16 L aquarium. Water flow was turnedoff, the inoculum added and then water flowresumed after 24 h. Exposed and control fish(10 fish per group) were examined by histology(sections stained with H & E) at 8 and 20 weeks,and the 10 control fish at 20 weeks post-exposurewere also examined with the Fungi-Fluor stain tofurther assure the absence of spores.

We observed the infection in three fish at8 weeks post-exposure. After 20 weeks post-expo-sure, 100% (10/10) of the fish were infected, whilecontrol fish remained negative throughout thestudy. Fish were moderately or heavily infected,but did not exhibit myositis or clinical disease.

Our ability to transmit the disease by water borneexposure (presumably per os) is consistent withother fish microsporidia that have been shown to betransmissible directly. Zebrafish readily devour deadtank mates, which would provide a direct source ofthe infection.

Observation of the infection in an egg andovaries indicates that transovarial (vertical) trans-mission may occur. Transmission of microsporidiafrom one generation to the next with eggs or sexualproducts has been suggested for Loma salmonae(cf. Docker et al. 1997) and Pleistophora ovariae(cf. Summerfelt & Warner 1970). However, truevertical transmission (i.e. transmission of theinfection to embryos within eggs) for theseinfections has not been confirmed. Perhaps Pseudo-loma is spread to progeny with sexual products,rather than directly within eggs as is suspected withL. salmonae and infectious haematopoietic necrosisvirus of salmonids (Groff & LaPatra 2000). Thismay account for the wide distribution of theinfection because research institutions often sharespecific lines or mutants by shipping eggs.

Chlorine treatment of eggs at 25 ppm for 10 minis a routine practice in zebrafish laboratories(Westerfield 2000), but microsporidian spores arerelatively resistant to chlorine (Khalifa, El Temsahy& Abou El Naga 2001). We have worked exten-sively with one large facility that maintains a strictquarantine procedure in which only chlorine disin-fected eggs enter the facility and we recently detectedheavy infections by P. neurophilia in their sentinelfish. Even if chlorine eliminates viable spores thatoccur outside the eggs following spawning, sporeswithin eggs would probably survive and could thusbe a source of infection for tank mates. Studies areplanned to evaluate the concentrations of chlorineneeded to kill spores of Pseudoloma.

Acknowledgement

Supported in part by the Zebrafish InternationalResource Center (NIH grant P40 RR12546).

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Received: 15 January 2003Accepted: 31 March 2003

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Journal of Fish Diseases 2003, 26, 423–426 M L Kent and J K Bishop-Stewart Pseudoloma in zebrafish