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Bull. Eur. Ass. Fish Pathol.

, 27(1) 2007, 2

Comparative histopathology of Streptococcus


iniae and Streptococcus agalactiae-infected
tilapia
C.-Y. Chen1, C.-B. Chao2 and P.R. Bowser1*
1
Aquatic Animal Health Program, Department of Microbiology and Immunology, College of
Veterinary Medicine, Cornell University, Ithaca, New York 14853 USA; 2 Institute for Animal Disease
Prevention and Control, Kaohsiung, Taiwan.

Abstract
In this study, the microscopic pathology resulting from infection of tilapia with Streptococcus
iniae or S. agalactiae was compared. Pathological changes associated with infection of tilapia by
either pathogen include: pericarditis, epicarditis, myocarditis, endocarditis, and meningitis. Large
numbers of cocci were present in tissues and in the circulation of S. agalactiae-infected, but not in
S. iniae-infected fish. Lesions similar to those caused by S. agalactiae, including the presence of
large numbers of intralesional bacteria, can be reproduced by intraperitoneal injection of a
relatively high dose in of S. iniae. This suggests that the pathogenesis of different streptococcal
infections in tilapia may be similar. However, tilapia may control natural S. iniae infections more
effectively, resulting in a more chronic form of disease compared to that caused by S. agalactiae.

Introduction 2002). All of these fish-pathogenic streptococci


The genus Streptococcus includes many are closely related and may share similar
important human and animal pathogens and pathogenic determinants with each other and
was first reported in fish in 1957 (Hoshina et the human pathogens S. pyogenes a n d
al., 1958). Streptococcus iniae, the most S. agalactiae.
commonly reported streptococcal pathogen of
fish, having been documented in tilapia in The pathology of S. iniae infection generally
Japan, Israel, the US, and Taiwan (Bowser et includes meningitis, epi/myocarditis and
al., 1998; Eldar et al., 1994; Kitao et al., 1981; septicemia, with enlargement or necrosis in
Perera et al., 1994; Plumb, 1999; Tung et al., spleen and kidney. Interestingly, infections
1985) and in rainbow trout and tilapia in Israel caused by other streptococci have similar
(Eldar et al., 1994; 1995). Phylogenetically, pathology, despite the different species of host
S. iniae is closely related to S. parauberis, both or pathogen involved. In this study we
being clustered with S. agalactiae and examined the pathology caused by natural
S. dysgalactiae, and the whole group clustering S. iniae or S. agalactiae infections in tilapia and
with S. pyogenes according to sequence compared those observations to the pathology
similarity in their 16S rRNA gene (Facklam, associated with experimental infection of
tilapia with S. iniae.

* Corresponding authors E-mail: prb4@cornell.edu


Bull. Eur. Ass. Fish Pathol., 27(1) 2007, 3

Materials and methods 222, Sigma, St. Louis, Missouri, USA), and
Sample collection processed for histological evaluation as
Naturally infected tilapia were collected from previously described. For intraperitoneal and
different outbreaks in the United States and intravascular infection trials, fish were placed
southern Taiwan. The tilapia in the United under light anesthesia with MS-222 and a
States were pure strain Oreochromis niloticus. 100 l suspension containing 107 CFU/mL S.
Tilapia collected in Taiwan are referred to as iniae strain 97057 cells was injected into the
hybrid tilapia because of extensive cross peritoneum or the heart of each test fish. The
breeding in that commercial environment. bacteria used to produce the experimental
Several fish with apparent signs of disease, infection were cultured in either 100 mL
including lethargy, corneal opacity, and dark (immersion infection) or 3 mL (intraperitoneal
coloration were submitted for disease and intravascular infections) Brain Heart
diagnosis. Standard bacterial isolation was Infusion Broth (BHI broth, Becton Dickinson
performed from kidney or spleen on Brain and Company, Sparks, Maryland, USA) for
Heart Infusion (BHI) agar or Blood Agar 48 h, centrifuged, and washed twice in tilapia
(Trypticase Soy Agar plus 5 % sheep blood, phosphate buffer saline (TPBS, 0.01 M pH 7.3
Becton Dickinson and Company, Sparks, phosphate buffer supplemented with 150 mM
Maryland, USA). Internal organs were fixed NaCl), and then re-suspended in TPBS for use.
in 10 % neutral buffered formalin, embedded
Identification of pathogens
in paraffin, sectioned and stained with
Bacterial isolation from diagnostic cases was
hematoxylin and eosin stains for histological
attempted from spleen or trunk kidney of
evaluation (Luna, 1968).
moribund fish. All bacterial isolates meeting
Artificial infections the following requirements were included in
Experimental infection trials were conducted this study. Bacterial growth consisted of
with S. iniae to compare the resulting white, pinpoint raised colonies on BHI agar
pathology with that observed in natural or Blood Agar after 24 to 48 h of incubation.
S. iniae or S. agalactiae outbreaks. Streptococcus Further, microscopic examination revealed
iniae strain 97057 (Cornell University) isolated gram-positive chained cocci after Grams stain
from infected tilapia (Bowser et al., 1998) was procedure. Twenty isolates that met these
used as the model pathogen. In immersion requirements were then subjected to two
infections, pure strain O. niloticus, approxi- polymerase chain reaction (PCR) iden-
mately 200 g, were immersed in S. iniae strain tifications.
97057 suspension at the concentration of 107
DNA was extracted and purified following
CFU/mL in tank water for 30 min, and then
procedures previously described (Chao et al.,
the fish were transferred to a 100 L
2002). A set of primers was used to amplify
experimental tank for observation. Fish
the bacterial 16S ribosomal RNA gene
showing dark coloration and lethargy within
sequence according to Weinsburg et al. (1991).
1 week were collected, euthanized with an
PCR products were recovered from the gel,
overdose of tricaine methanesulfonate (MS-
Bull. Eur. Ass. Fish Pathol., 27(1) 2007, 4

sequences used include S. agalactiae JCM5671


(Genbank accession number AB023574),
S. bovis ATCC43143 (AF104114), S. dysgalactiae
(AB002513), S. equi ATCC 43079 (AB002516),
S. iniae (X58316), S. parauberis (X89967),
S . pneumoniae (AF003930), and S. suis
(AF009509). Staphylococcus aureus (AF015929)
was also included as an outgroup. ClustalX
software (Thompson et al., 1997) version 1.81
was used for multiple lignment of the
sequences and generation of the bootstrap
neighborhood-joint tree. The phylogenetic
tree was displayed using TreeView version
1.6.5, available from R. D. M. Page (Institute
of Biomedical and Life Sciences, University
of Glasgow, Glasgow, UK), at http://
taxonomy.zoology.gla.ac.uk/rod/rod.html.

Results
Identification of pathogens
Figure 1. Phylogenetic tree of selected members of Sequence analysis of the 16S rRNA gene was
Streptococcus indicating relationship between tilapia performed on one typical isolate from each
isolates and other streptococci. Tilapia-pathogenic
S. iniae and S. agalactiae were sequenced and outbreak. The obtained sequences were
compared to other sequences available in Genbank compared to the sequences available in
using a 1356-bp sequence of 16S rRNA gene
Genbank (Figure 1). One group of our isolates
sequence. The Staphylococcus aureus (Sta. aureus) was
used as an outgroup. had very high similarity to S. agalactiae, and
another group clustered with S. iniae. The
cloned into pGEM-T, and transfected into similarities were approximately 99 %. All
Escherichia coli strain DH5a using Transform isolates in the first group were Lancefield
Aid Bacterial Transformation System (MBI group B; while none of the isolates in the
Fermentas Inc., Hanover, New Hampshire, second group were typable. Therefore, the
USA). DNA sequences were then determined first group was identified as S. iniae and the
by an ABI Prism 377-96 DNA Sequencer second group S. agalactiae. Interestingly, all the
(Perkin-Elmer, Wellesley, Massachusetts, important fish pathogenic streptococci
USA) with an ABI Prism Big Dye Terminator (S. dysgalactiae, S. difficile, S. parauberis, S. iniae)
Cycle Sequencing Ready Reaction Kit. The form a separated cluster from other
obtained sequences were compared with streptococci. This suggests that these species
available sequences from other streptococci are evolutionarily closely related and may
in Genbank, and selected sequences were apply a similar strategy in pathogenesis.
used to construct a phylogenetic tree. The
Bull. Eur. Ass. Fish Pathol., 27(1) 2007, 5

Pathology of natural infections


Gross pathological signs of S. iniae and
S. agalactiae infection in tilapia were similar
and could not be used for differential disease
diagnosis. Fish were lethargic with dark
coloration in both infections and some fish
would show sudden erratic swimming
movements. Microscopically, proliferation of
lymphoid tissue was observed in the kidneys
of fish infected by either bacterium. Kidneys
of S. iniae-infected tilapia showed accu-
mulations of eosinophilic material in the
cytoplasm of the tubular cells, with the nuclei
displaced to the side. In S. agalactiae-infected
tilapia, dissolution of some tubules could be
found. Cocci were seen surrounding renal
tubules, within interstitial cells, and within
glomeruli. Fibrin precipitation and lympho-
cyte infiltration were noted in necrotic foci.
In addition, in fish infected with S. agalactiae,
bacterial cells were distributed throughout the
spleen. The head kidney of fish infected with
S. agalactiae had similar necrotic changes with
presence of bacteria as that found in the
spleen. Melanomacrophage centres were
more evident in head kidneys in both S. iniae
and S. agalactiae-infected fish than in
uninfected control fish.
Figure 2. A. Histopathology of S. iniae infection in
In contrast, bacterial cells were rarely tilapia liver demonstrating vacuolation of
hepatocytes and numerous pycnotic nuclei in
observed in the liver of S. iniae infected fish pancreatic tissue. B. Histopathology of S. agalactiae
(Figure 2). Necrotic changes were observed infection in tilapia liver demonstrating vacuolation
of hepatocytes, with colonies of cocci (arrows).
along the hepatic arteries and near the Vacuolation is likely due to excess energy in the
capsule. Fish infected by either bacterium had ration of these cultured fish and not associated with
variable vacuolation of the hepatocytes, which the bacterial infections. Bars = 10 m.

is not uncommon in commercially raised


Visceral pericarditis was a common finding
tilapia and should not be considered
in both infections. In S. iniae infection, the
pathological changes due to infections but
epicardium was congested and thickened,
rather to feeding a diet with excessive caloric
with an associated infiltration of lymphocytes
content.
and macrophages. Numerous cocci and
Bull. Eur. Ass. Fish Pathol., 27(1) 2007, 6

necrotic debris were being seen in epicardium, Streptococcus agalactiae infection also resulted
but were rarely distributed between cardiac in necrotic foci between the mucosa and
muscle fibers. In S. agalactiae-infected tilapia, submucosa. Pathological changes in the
bacterial cells were not limited to the intestine due to the infection by either
epicardium but were also widely distributed bacterium were generally mild, with scattered
among or attached to cardiac muscle fibers individual cell necrosis.
or found in necrotic foci diffusely or as
Although bacterial cells of S. iniae were rarely
colonies. High numbers of bacterial cells were
found in internal organs, they were present
observed in the circulation, in fish infected
in very high numbers in the peritoneal cavity.
with S. agalactiae but not for fish infected with
Peritonitis, with a thick layer of fibrin, necrotic
S. iniae. Inflammatory cell infiltration with
debris, and bacteria, was seen in both
some associated necrosis of the epicardium
infections. This finding correlates well with
and wall of the bulbus arteriosus was common
the fact that necrosis was always more severe
in S. agalactiae-infected tilapia. Valvular
in the superficial regions of liver or spleen.
endocarditis with large intralesional colonies
of cocci were seen in both infections. We observed a moderate infiltration of
inflammatory cells in ovary of S. iniae-infected
Both infections lead to meningitis in tilapia,
tilapia. In the specimens examined, testis
which correlates to clinical findings that fish
appeared to be less affected.
showed erratic behaviour and lethargy.
Histologically, S. iniae infection caused a No significant pathological changes were
thickening of the meninges with an associated found in the gills in S. iniae-infected tilapia,
infiltration of lymphocytes and macrophages. with the exception of external parasites (e.g.
Areas with ischemia-like lesions were seen in Trichodina), or lesions similar to epitheliocystis
the brain; a typical lesion associated with on the gill lamella. Bacterial colonies were
streptococcal meningitis in other animals. observed in the gill of S. agalactiae-infected
Streptococcus agalactiae induced similar, but tilapia, especially near the tip of gill lamella.
more severe lesions. Within the optic lobe, Lesions in the skeletal muscle were rare in the
small colonies of S. agalactiae were sometimes S. iniae infection. In S. agalactiae infected fish,
observed in the capillaries. bacterial cells were observed in the
musculature and were associated with a
Gastroenteritis was seen in both infections.
moderate necrosis.
Streptococcus iniae invaded the submucosa and
serosa, causing local extensive inflammation Pathology in experimental infections
with fibrin, edema, and distention of the Experimental infection trials resulted in
submucosa. In the mucosa, bacterial cells variable results with respect to the severity
aggregated on the luminal surface and of disease signs observed in the tilapia
resulted in necrosis. Streptococcus agalactiae challenged with S. iniae. Although some fish
cells were usually observed between died within 24 h and some developed
glandular tissue, sometimes causing necrosis darkened body coloration within days, over
and dissolution of individual glands.
Bull. Eur. Ass. Fish Pathol., 27(1) 2007, 7

half of the fish challenged did not develop any Perera et al. (1998) investigated S. iniae
sign of disease in either immersion or IP- infection in hybrid tilapia and reported
injected groups. Even among those fish that meningitis, granulomas in liver, epicarditis,
displayed dark coloration few showed and myocarditis. Those descriptions are
histological changes. Pathological changes similar to our findings. Chang & Plumb (1996)
were only significant in moribund fish, with infected tilapia with two non-hemolytic and
such changes as meningitis, infiltration of one -hemolytic streptococci. Findings similar
lymphocytes, and macrophages in internal to ours, including pericarditis, infiltration of
organs. However, histopathology in acute macrophages and lymphocytes into internal
S. iniae infections, either through intraperi- organs, hyaline deposition in tubular cells in
toneal or intravascular injection of the kidney, and meningitis were documented.
pathogen, was m o r e s i m i l a r t o t h e They did report meningitis, and macrophages
pathology in natural S. agalactiae than to with engulfed bacteria in the spleen, heart,
S. iniae infections, and characterized by the and ovary, which we did not observe.
presence of numerous cocci in most organs. Pericarditis, meningitis, and some
macrophages with engulfed bacteria were
Discussion found in cage-cultured tilapia, O. mossambicus
In this study, we found that S. iniae and infected with a -hemolytic streptococcal
S. agalactiae lead to different microscopic infection in Taiwan (Tung et al., 1985). These
lesions in infected fish, even though they have findings also agree with those of our S. iniae
similar gross clinical signs and basic infection studies. Streptococcus iniae infection
bacteriological characteristics. The histo- in juvenile yellowtail leads to infiltration of
pathology of streptococci infection in tilapia cocci-laden macrophages, and granulomas in
has been described in the literature (Miyazaki the third ventricle, meninges, cerebellum,
et al., 1984; Tung et al., 1985; Eldar et al., 1995; hepatic capsule, and peritoneum (Kaige et al.,
Chang & Plumb 1996; Bowser et al., 1998; 1984). Eldar & Ghittino (1999) compared
Perera et al., 1998). Unfortunately, many of the histopathology of rainbow trout infected with
early studies did not provide identification Lactococcus garvieae and S. iniae, which
to species of the pathogens under study, and resulted in similar histology in brain, liver,
usually the pathogens were only roughly kidney, and heart. Their findings were also
characterized by hemolysis reaction and similar to findings in our study for tilapia
Lancefield serotyping. Since variation in infected with S. iniae.
hemolytic ability was reported within even a
In S. agalactiae-infected tilapia, the high
single streptococcal strain (Bowser et al.,
number of bacteria observed in the circulation
1998), the ability to make definitive
and internal organs suggest that either the
judgements from information found in early
pathogen can interfere with the immune
literature may be open to question due to lack
system, or grow faster than the immune
of pathogen identification.
system can eliminate them. Recently Mn-de-
pendent superoxide dismutase in S. agalactiae
Bull. Eur. Ass. Fish Pathol., 27(1) 2007, 8

has been shown to enable this bacterium to intracellular cocci. Although there seems to
evade hydrogen superoxide secreted by be a contradiction in generalization of
macrophages (Poyart et al., 2001). This could pathological changes across different fish
explain why tilapia could not exert an species, it does, in fact, reveal that the outcome
effective immune defense mechanism to of infection depends not only on the pathogen,
eliminate S. agalactiae. This suggests that, even but also on how well the host can react to that
though useful in the diagnosis of natural pathogen. It is probable that, if the host has a
infection, the different pathological changes more effective immune function or is
we observed in natural infections were not generally in a better state of health, it will
specific to each disease. We speculate that better control the infection and have
tilapia innate immunity may control S. iniae granuloma formation. If the growth of the
more effectively than S. agalactiae. However, pathogen cannot be controlled, ubiquitous
S. agalactiae may have additional mechanisms presence of bacterial cells in various tissues
to invade tilapia and successfully multiply in will be found. Further immunology-oriented
tilapia tissues. study is required to clarify this point before
we can understand the nature of streptococcus
Even though we have observed distinct
infection in fish.
histological changes in tilapia for both S. iniae
and S. agalactiae infections, the same may not
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