J Vet Diagn Invest 17:601–605 (2005)
Occurrence of Piscirickettsiosis-like syndrome in tilapia
in the continental United States
Michael J. Mauel, Debra L. Miller, Eloise Styer, Deborah B. Pouder, Roy P. E. Yanong,
Andrew E. Goodwin, Thomas E. Schwedler
Abstract. From 2001 to 2003, tilapia (Oreochromis sp.) farms in Florida, California, and South Carolina
experienced epizootics of a systemic disease causing mortality. The fish exhibited lethargy, occasional exophthalmia, and skin petechia. The gills were often necrotic, with a patchy white and red appearance. Grossly, the
spleen and kidneys were granular with whitish irregular nodules throughout. Granulomatous infiltrates were
observed in kidney, spleen, testes, and ovary tissues, but not in the liver. The granulomas contained pleomorphic
coccoid bacteria, measuring 0.57 ⫾ 0.1 ⫻ 0.8 ⫾ 0.2 ␮m, that were Giemsa-positive, acid-fast-negative, and
Gram-negative. The bacteria had a double cell wall, variable electron-dense and -lucent areas, and were present
in the cytoplasm and within phagolysosomes. The syndrome was associated with cold stress and poor water
conditions. These findings are consistent with an infectious process caused by a Piscirickettsia-like bacterium
described previously in tilapia in Taiwan and Hawaii. This report involves the first identified cases of a piscirickettsiosis-like syndrome affecting tilapia in the continental United States.
Key words: Piscirickettsiosis; Piscirickettsia-like organisms; piscirickettsiosis-like syndrome; Tilapia.
Since the identification of Piscirickettsia salmonis as the
first rickettsia-like organism recognized as a fish pathogen,9
a number of Piscirickettsia-like organisms (PLOs) have been
observed microscopically or isolated in cell culture from a
variety of marine and fresh-water piscine species from locations worldwide.5,11,12,16 Piscirickettsiosis or piscirickettsiosis-like syndromes can be severe, resulting in high mortality and significant economic losses.
The number of reports of piscirickettsiosis, with significant associated economic losses, have been increasing for
the last 5–10 years in British Columbia,2,8 Ireland,16 and
Scotland.10 PLOs have also been observed in and isolated
from seabass in Europe1,7 and California3 during epizootics
characterized by high mortality and severe economic losses.
Tilapia in Taiwan4,6 and Hawaii15 have also been severely
affected by PLO epizootics. Here we report on the first identified cases of PLO epizootics in tilapia in the continental
United States.
Between 2001 and 2003, 3 facilities in the continental
United States (California, South Carolina, and Florida) reported epizootics with signs consistent with a piscirickettsiosis-like syndrome. All 3 facilities had increased mortalities during times of fish stress due to lowered temperature
or water quality. The fish appeared lethargic, with darkened
color, and would crowd toward the middle of the pond or
tank. Occasionally, petechia were observed on the sides of
From the Veterinary Diagnostic and Investigational Laboratory,
The University of Georgia, Tifton, GA 31793 (Mauel, Miller, Styer);
Tropical Aquaculture Laboratory, Department of Fisheries and
Aquatic Sciences, Institute of Food and Agricultural Sciences, University of Florida, Ruskin, FL 33570 (Pouder, Yanong); Aquaculture/
Fisheries Center, University of Arkansas at Pine Bluff, Pine Bluff,
AR 71601 (Goodwin); Clemson University, Clemson, SC 29631
(Schwedler). Current address (Mauel): Thad Cochran National
Warmwater Aquaculture Center, College of Veterinary Medicine,
Mississippi State University, Stoneville, MS 38776.
Corresponding Author: Michael J. Mauel, Thad Cochran National
Warmwater Aquaculture Center, College of Veterinary Medicine,
Mississippi State University, PO Box 197, Stoneville, MS 38776.
the fish. Fins had light fraying, some exophthalmia was observed, and gills were necrotic, with a patchy white and red
appearance. Internally, the spleen and kidneys were extremely granular with whitish irregular nodules throughout. The
liver was a pale reddish tan with patchy lighter areas.
In hematoxylin and eosin (HE) sections, the spleens had
extensive necrosis and multifocal to confluent granulomatous inflammatory infiltrates (Fig. 1). Occasional melanomacrophages were associated with the infiltrates. Splenic parenchyma was replaced by multiple and coalescing granulomas. The granulomas were characterized by central areas
of necrosis and/or vacuolated macrophages mixed with very
few neutrophils surrounded by large (but less vacuolated)
macrophages and fibrosis. Occasionally, a fine, fibrous capsule encompassed the granulomas. The gills had mild blunting and the tips were minimally hypercellular, with increased
secretory mucus cells. Granulomas at the base of the gills
and the gill filaments were multifocally and mildly expanded
by a mixed population of inflammatory cells. The cytoplasmic vacuoles of vacuolated macrophages contained numerous small pleomorphic coccoid bacteria (Fig. 2). The bacteria were Gram-negative, stained Giemsa positive, and acidfast negative. This inflammatory process nearly replaced the
entire normal splenic architecture.
In the winter of 2001, tilapia (Oreochromis mossambicus
⫻ Oreochromis hornum) from an intensive recirculating
aquaculture facility in southern California were observed
swimming slowly and crowding in the middle of the ponds.
The farmer recorded increased mortality of 5–10 times
above normal for 2 weeks. The water quality was as follows:
temperature 22⬚C, ammonia ⬎ 5 mg/liter, pH ⬎ 8.2, salinity
5 ppt, alkalinity 450 ppm, hardness 270 ppm. Pond water
was filtered by passing through four sedimentation earth
ponds, and 50% of the water per day was replaced after
filtering. Fish were fed 3 times daily and the ponds were
harvested by size selection every 3 weeks. To reduce external and gill parasites (Ambiphrya, Epistylis, Cleidodiscus),
the water was treated with potassium permanganate every 2
weeks.
601
602
Brief Communications
Figure 1. Photomicrograph of Haemotoxylin eosin stained spleen from the Southern California case. Note the numerous granulomas
and the replacement of normal splenic tissue with granulomatous infiltrates. Scale bar ⫽ 50 ␮m.
The increased mortality observed by the farmer was a
continuing problem, with the facility having heavier losses
in winter (5–10-fold greater in winter than summer) but losses were also observed in summer, when the fish were
stressed by poor water quality. The mortalities increased
when the temperature was below 25⬚C. The facility reported
that the majority of the mortalities were observed in the juvenile tilapia ranging in weight from 20 to 90 g.
Facility personnel observed white nodules in the viscera
of the tilapia. Spleens submitted in neutral buffered 10%
formalin were embedded in paraffin and sectioned. The sections were stained with H & E, Giemsa, and acid-fast stain.
The splenic lesions were consistent with Piscirickettsia-like
bacterial infections observed in tilapia in Hawaii.15 Piscirickettsia salmonis and the Hawaiian PLO infections have
previously been treated with tetracycline and oxytetracy-
cline. When tilapia were fed oxytetracycline-medicated feed,
mortalities were reduced to near 0 by the fifth day.
In the winter of 2002–2003, tilapia mortalities at a research facility in South Carolina were observed with nodular
lesions in the gills and viscera (Fig. 3). Formalin-fixed
spleen tissue and dead fish were submitted for examination.
Water-quality data were not submitted. Fish were submitted
in 2 groups, based on pen location. In both groups, white
nodules were observed grossly and necrogranulomatous inflammation observed microscopically in the gills, spleen,
and kidney. The liver and remaining organs were unremarkable in group 1 (3 fish); however, in group 2 (3 fish), there
was severe necrogranulomatous inflammation throughout the
liver, multifocally throughout the skeletal muscle, bone,
mildly in the subcutis, epicardium (1 fish), surrounding the
spinal cord (1 fish), and infiltrating the choroid gland.
Figure 2. Photomicrograph of Giemsa-stained granulomatous inflammation in the spleen of a Florida tilapia demonstrating Piscirickettsia-like organism (PLO) bacteria (arrow). Scale bar ⫽ 10 ␮m.
Brief Communications
603
Figure 3. South Carolina tilapia with the gross signs of the severe, chronic stage of the PLO disease. Granulomas in the gills A, and
in the spleen B.
In December 2002, tilapia (O. niloticus ⫻ O. aureus) from
a grow-out facility in Florida were submitted for diagnosis.
Ten fish were collected for examination but only 3 survived
for the 1-hour transport time to the laboratory. The fish were
4–6 months of age, 5–20 cm long, and 70,000 fish had been
stocked in a 260,000-liter tank 3.5 months earlier. Water was
supplied from a well and the water, tested from several locations within the system, had the following conditions: pH
7.5–8.0, salinity 11 ppt, nitrite 0.03–0.11 ppm, total ammonia nitrogen 5.0–6.0 ppm, total alkalinity 136.8 ppm, total
hardness ⬎ 855 ppm.
Grossly, 2 of the 10 fish had petechia on one side and
some fish in the system were observed to be bloated. Some
fish with frayed fins and some with exophthalmia were observed. The gills were necrotic, with a patchy white and red
appearance. Skin had excess mucus and microscopic examination revealed Trichodina, Ambiphrya, and gyrodactylids
on the skin, fins, and gills. There was little food present in
the stomach or intestines and a clear fluid was observed in
the abdomen of 2 out of the 10 fish. Cultures of brain and
kidney tissues on TSA⫹blood were negative. A Lowenstein–
Jensen culture with spleen tissue was negative for mycobacterium.
Tissue samples from both the South Carolina and Florida
cases were routinely processed for transmission electron microscopy and prokaryotic organisms typical of PLOs were
observed within large vacuoles that compressed the host cell
cytoplasm and displaced the host nucleus to one side. The
pleomorphic coccoid bacteria measured 0.57 ⫾ 0.1 ⫻ 0.8 ⫾
0.2 ␮m. The bacteria had a double cell wall, variable electron-dense and -lucent areas, and occurred free in the cytoplasm and within phagolysosomes. PLOs condensed to various degrees were also present in small and large phagolysosomes that did not appear to be located in the same vacuoles as the other PLOs (Fig. 4).
Splenic tissues from both the South Carolina and Florida
cases tested negative for the presence of P. salmonis by P.
salmonis-specific polymerase chain reaction (PCR)14 and P.
salmonis-specific fluorescent antibody assays.13 The tilapia
disease agent (0.57 ⫻ 0.8 ␮m) is smaller than the reported
604
Brief Communications
Figure 4. Transmission electron micrograph of Piscirickettsia-like organisms (PLOs) (arrows) in tilapia spleen macrophage phagolyzosomes. Scale bar ⫽ 3 ␮l.
size of P. salmonis (0.5 ⫻ 1.5 ␮m).9 The combination of
characteristics—different host species, smaller cell size, no
response to P. salmonis-specific PCR and P. salmonis-specific fluorescent antibody assays—indicates that the bacterium causing epizootics in tilapia in these cases is not P.
salmonis.
All of the above cases presented disease signs consistent
with piscirickettsiosis-like syndrome. From the cases described here, we conclude that a Piscirickettsia-like organism is now present in the continental United States. This has
implications for the transport of tilapia and would suggest
the need for gross and microscopic observation of tilapia
splenic tissue prior to transfer, as well as close attention by
aquaculturists to production water-quality conditions, as this
syndrome is expressed during periods of fish stress.
5.
6.
7.
8.
9.
References
10.
1. Athanassopoulou F, Groman D, Prapas, Sabatakou O, T: 2004,
Pathological and epidemiology observations on rickettsiosis in
cultured seabass (Dicentrarchus labrax L.) from Greece. J Appl
Ichthyol 20:525–529.
2. Brocklebank JR, Speare DJ, Armstrong RD, Evelyn T: 1992,
Septicemia suspected to be caused by a rickettsia-like agent in
farmed Atlantic salmon. Can Vet J 33:407–408.
3. Chen MF, Yun S, Marty GD, et al.: 2000. A Piscirickettsia salmonis-like bacterium associated with mortality of white seabass
Atractoscion nobilis. Dis Aquat Org 43:117–126.
4. Chen SC, Tung MC, Chen SP, et al.: 1994, Systemic granulomas
11.
12.
13.
caused by a rickettsia-like organism in Nile tilapia, Oreochromis
niloticus (L.), from southern Taiwan. J Fish Dis 17:591–599.
Chen SC, Wang PC, Tung MC, et al.: 2000, A Piscirickettsia
salmonis-like organism in grouper, Epinephelus melanostigma,
in Taiwan. J Fish Dis 23:415–418.
Chern RS, Chao CB: 1994, Outbreaks of a disease caused by a
rickettsia-like organism in cultured tilapias in Taiwan. Fish
Pathol 29:61–71.
Comps M, Raymond JC, Plassiart GN: 1996, Rickettsia-like organism infecting juvenile sea-bass Dicentrarchus labrax. Bull
Eur Assoc Fish Pathol 16:30–33.
Evelyn TPT: 1992, Salmonid rickettsial septicemia. In: Diseases
of seawater netpen-reared salmonid fishes in the Pacific Northwest, ed. Kent ML. Can Spec Pub Fish Aquat Sci 116. Dept
Fisheries and Oceans, Nanaimo, BC, Canada.
Fryer JL, Lannan CN, Giovannoni SJ, Wood ND: 1992, Piscirickettsia salmonis gen. nov., sp. nov., the causative agent of an
epizootic disease in salmonid fishes. Int J Syst Bacteriol 42:
120–126.
Grant AN, Brown AG, Cox DI, et al.: 1996, Rickettsia-like organism in farmed salmon. Vet Rec 138:423.
Jones SRM, Markham RJF, Groman DB, Cusack RR: 1998, Virulence and antigenic characteristics of a cultured Rickettsialeslike organism isolated from farmed Atlantic salmon Salmo salar
in eastern Canada. Dis Aquat Org 33:25–31.
Khoo L, Dennis PM, Lewbart GA: 1995, Rickettsia-like organisms in the blue-eyed plecostomus, Panaque suttoni (Eigenmann and Eigenmann). J Fish Dis 18:157–164.
Lannan CN, Ewing SA, Fryer JL: 1991, A fluorescent antibody
test for detection of the rickettsia causing disease in Chilean
salmonids. J Aquat Ann Health 3:229–234.
Brief Communications
14. Mauel MJ, Giovannoni SJ, Fryer JL: 1996, Development of
polymerase chain reaction assays for detection, identification,
and differentiation of Piscirickettsia salmonis. Dis Aquat Org
26:189–195.
15. Mauel MJ, Miller DL, Frazier K, et al.: 2003, Characterization
605
of a piscirickettsiosis-like disease in Hawaiian tilapia. Dis Aquat
Org 53:249–55.
16. Rodger HD, Drinan EM: 1993, Observation of a rickettsia-like
organism in Atlantic salmon, Salmo salar L., in Ireland. J Fish
Dis 16:361–369.
J Vet Diagn Invest 17:605–609 (2005)
Seasonal meningoencephalitis in Holstein cattle caused by Naegleria fowleri
Barbara M. Daft, Govinda S. Visvesvara, Deryck H. Read, Hailu Kinde,
Francisco A. Uzal, Michael D. Manzer
Abstract. Primary amoebic meningoencephalitis is a fulminant infection of the human central nervous
system caused by Naegleria fowleri, a free-living amoeba that thrives in artificially or naturally heated water.
The infection usually is acquired while bathing or swimming in such waters. The portal of entry is the olfactory
neuroepithelium. This report describes fatal meningoencephalitis caused by N. fowleri in Holstein cattle that
consumed untreated surface water in an area of California where summer temperatures at times exceed 42⬚C.
In the summers of 1998 and 1999, severe multifocal necrosuppurative hemorrhagic meningoencephalitis was
observed in brain samples from nine 10–20-month-old heifers with clinical histories of acute central nervous
system disease. Olfactory lobes and cerebella were most severely affected. Lesions were also evident in periventricular and submeningeal neuropil as well as olfactory nerves. Naegleria fowleri was demonstrated by
immunohistochemistry in brain and olfactory nerve lesions and was isolated from one brain. Even though
cultures of drinking water did not yield N. fowleri, drinking water was the likely source of the amoeba. The
disease in cattle closely resembles primary amoebic meningoencephalitis in humans. Naegleria meningoencephalitis should be included among differential diagnoses of central nervous system disease in cattle during
the summer season in areas with high ambient temperatures.
Key words: Bovine; encephalitis; Naegleria fowleri; primary amoebic meningoencephalitis.
Infection of the human central nervous system (CNS) with
free-living amoebae was first described in 1966 and is now
recognized worldwide.14 There are 4 genera of amoebae that
cause CNS disease in mammals, namely Acanthamoeba
(several species), Naegleria fowleri, Balamuthia mandrillaris, and the recently described Sappinia diploidea.12 Acanthamoeba and Naegleria are ubiquitous in soil and fresh water, including lakes, streams, and hot springs. These amoebae
have also been isolated from various artificial water sources,
such as swimming pools, tap water, heating and ventilation
units, air conditioners, cooling water, sewage, contaminated
cell cultures, and contact lens-storing fluid.5,15,18 Their cysts
have even been demonstrated in dust during dust storms.13
The habitat of Balamuthia is not known but is believed to
be similar to that of Naegleria and Acanthamoeba.8
Primary amoebic meningoencephalitis (PAM) is the term
From the University of California, Davis, School of Veterinary
Medicine, California Animal Health and Food Safety Laboratory
System, San Bernardino Branch, San Bernardino, CA, 92408 (Daft,
Read, Kinde, Uzal); Davis Central Reference Laboratory, Davis,
CA, 95616 (Manzer); and Centers for Disease Control and Prevention, Division of Parasitic Diseases, Chamblee, GA, 30341 (Visvesvara).
Corresponding Author: Barbara M. Daft, California Animal
Health and Food Safety Laboratory, San Bernardino Branch, 105 W.
Central Avenue, San Bernardino, CA 92408.
for the human disease caused by N. fowleri. It is an acute,
usually fatal, necrotizing, and hemorrhagic meningoencephalitis.2 As of 2001, 200 cases of PAM have been reported in
humans. About half of these were reported in the United
States (G. S. Visvesvara, personal communication). Although CNS infections due to Acanthamoeba and Balamuthia have been recorded in animals, such as dogs, sheep,
cattle, primates, and horses,1,4,9–11,15,16 there has been only 1
report of naturally acquired PAM in animals, namely in a
South American tapir at a zoo in Arizona.6 Primary amoebic
meningoencephalitis has been experimentally induced in
mice, sheep, and monkeys.12,17,18
Naegleria fowleri is thermophilic and tolerates temperatures of up to 45⬚C; hence, the frequent association of PAM
with a history of contact with naturally warm or artificially
heated waters. Most human cases occur in healthy young
individuals after swimming or bathing in pools, ponds, hot
springs, canals, or lakes, although some cases have been
associated with tap water or inhalation of cysts during dust
storms.12 The portal of entry is the olfactory mucosa. The
parasite migrates to the brain via olfactory nerves. Incubation period is short, with onset of clinical signs several days
following exposure. The disease rapidly progresses and usually culminates in death within 5–7 days.7
The life cycle of N. fowleri includes a trophozoite stage
(amoebic form), a temporary flagellate stage, and, in unfa-