Reptile & Amphibian Forums

I bought two green tree monitors (V.prasinus) and my friend who is a retired vet told me of two zoos that had green tree monitors die from septicaemia caused by bacteria in feeder mice. The first zoo fed frozen mice and after the deaths of their monitors they had an investigation and wrote up a report detaling it. The second zoo fed frozen rat pups and their CB juv female died of septicaemia caused by the same bacteria that was in the mice at the first zoo. My friend suggested that I tell the supplier that I got my green tree monitors from and send him the report. I did and he said that I should post it here on kingsnake so everyone can see it. I do not know how to put it up other than copying the text and pasting it. There are pics that go with the report but they will not copy. If you want the full report with pics, send me your email and I will email the report as an attachment to you.

Veterinary Microbiology 95 (2003) 283–293
Septicaemia in emerald monitors (Varanus prasinus
Schlegel 1839) caused by Streptococcus
agalactiae acquired from mice
U. Hetzel a,∗, A. König b, A.Ö. Yildirim c,
Ch. Lämmlerd, A. Kipar a,1
a Institut für Veterinär-Pathologie, Justus-Liebig-Universität Gießen, Frankfurter Straße 96,
35392 Gießen, Germany
b Institut für Hygiene und Infektionskrankheiten der Tiere, Justus-Liebig-Universität Gießen,
Frankfurter Straße 89, 35392 Gießen, Germany
c Medizinische Klinik II, Justus-Liebig-Universität Gießen, Aulweg 123, 35392 Gießen, Germany
d Institut für Pharmakologie und Toxikologie, Justus-Liebig-Universität Gießen,
Frankfurter Straße 107, 35392 Gießen, Germany
Received 24 January 2003; received in revised form 2 May 2003; accepted 20 May 2003
Abstract
The present study was performed to investigate both the identity and the source of the bacteria
responsible for a fatal septicaemia observed in a group of three subadult emerald monitors (Varanus
prasinus Schlegel 1839). The emerald monitors were necropsied and examined by light microscopy,
including immunohistology, and by electron microscopy. Tissue samples were additionally submitted
for bacteriological, virological and parasitological examinations. The virological and parasitological
results were noncontributory, whereas the bacteriological investigation resulted in the isolation of
gram-positive cocci which were characterized biochemically and serologically and by molecular
analysis. The death of the emerald monitorswas caused by a partially leukocyte-associated septicaemic
infection with streptococci of serological group B of serotype V. Phenotypically and genotypically
identical group B streptococci were isolated from the intestine of subadult mice, obtained from the
feed used for the monitors. The genotypical characterization included an identical DNA fingerprint
of strains of both origins, indicating the epidemiological relation between the feeding mice and the
infections of the monitors.
© 2003 Elsevier B.V. All rights reserved.
Keywords: Emerald monitor; Varanus prasinus; Reptile; Septicaemia; Group B streptococci; Feeding mice
∗ Corresponding author. Tel.: 49-641-9938-226; fax: 49-641-9938-209.
E-mail address: udo.hetzel@vetmed.uni-giessen.de (U. Hetzel).
1 Present address: Department of Veterinary Pathology, Faculty of Veterinary Science, University of Liverpool,
Crown Street, Liverpool L69 7ZJ, UK.
0378-1135/$ – see front matter © 2003 Elsevier B.V. All rights reserved.
doi:10.1016/S0378-1135(03)00184-6
284 U. Hetzel et al. / Veterinary Microbiology 95 (2003) 283–293
1. Introduction
Emerald or green tree monitors (Varanus prasinus Schlegel 1839) are insectivorous
and carnivorous monitor species. Their natural habitats are the arboreal regions of subtropical
and tropical rainforests of New Guinea. Adults, reaching a body size of about
100 cm body length, prey upon insects and small mammals and lay their eggs in arboreal
termite nests (Bennett, 1995; Eidenmüller, 1997). Because of their intense colour
and small body size, emerald monitors are popular as pets and are often bred in
captivity.
The present report describes the findings, associated with a septicaemia, induced by group
B streptococci of serotype V, in three subadult, captive bred emerald monitors which died
suddenly.
2. Material and methods
2.1. Animals and tissue processing
Three emerald monitors were necropsied 12–24 h after death. Tissue samples obtained
from brain, spinal cord, lungs, heart, liver, spleen, kidneys, small and large intestines, skeletal
muscles and skin were routinely prepared for light microscopical examinations. Representative
tissue samples were additionally investigated by transmission electron microscopy.
About 165 frozen subadult dead mice of the remaining stock of feeding animals, used as
nourishment of the monitors prior to their death, were dissected and their gastrointestinal
tract collected for bacteriological examination.
2.2. Bacteriological, virological and parasitological examination
Routine bacteriological examination was performed on lung, liver, kidney and intestinal
specimens of the three emerald monitors and on the gastrointestinal tract of the 165 mice.
Virological examination (cell culture) was performed with tissue specimens from liver and
intestines of the emerald monitors. Parasitological examination was performed on the large
intestinal content of the three animals.
2.3. Light microscopy
Sections (5
m) of paraffin wax-embedded tissue specimens were subjected to haematoxylin
and eosin and Giemsa stains and to immunohistochemical examinations.
Immunohistochemical examination was performed to detect streptococci (Streptococcus
agalactiae) in tissues of the emerald monitors, applying a mouse anti-S. agalactiae IgG1
monoclonal antibody (QED Biosciences Inc., San Diego, CA). Deparaffinized sections were
incubated with the primary antibody (diluted 1:10 000 in tris-buffered saline, pH 7.6) for
12–16 h. Detection was achieved by the peroxidase–anti-peroxidase method (Sternberger
et al., 1970); 3,3-diaminobenzidine served as chromogene.
U. Hetzel et al. / Veterinary Microbiology 95 (2003) 283–293 285
2.4. Transmission electron microscopy
Semithin sections (0.75
m) were prepared from glutardialdehyde postfixed, epoxy resinembedded
tissue specimens and stained with toluidine blue. Ultrathin sections (75 nm) were
routinely stained with uranyl acetate/lead citrate and examined with an EM10 C electron
microscope (Carl Zeiss GmbH, Oberkochen, Germany).
2.5. Phenotypic and genotypic characterization of the isolated streptococci
The streptococci isolated from both emerald monitors and mice were investigated for
biochemical properties; for CAMP reactivity on sheep blood agar in the presence of a
-toxin-producing Staphylococcus aureus strain (Lämmler and Hahn, 1994); by Lancefield
serogrouping with a latex agglutination test system (Streptococci Identification Test,
DR0585A, Oxoid,Wesel, Germany); for colony morphology in soft agar media; for pigmentation
in GBS Islam agar (Oxoid) supplemented with 50 ml/l sterile horse serum (Wibawan
and Lämmler, 1991; Wibawan et al., 1991), for haemagglutinating properties (Wibawan
et al., 1993a) and for hyaluronidase enzyme activity (Yildirim et al., 2002a,c). The bacteria
were serotyped by determination of specific polysaccharide and protein antigens
(Wibawan and Lämmler, 1990) and investigated by polymerase chain reaction for the
genes rib and bag, encoding the protein antigens Rib and c (Yildirim et al., 2002a,b).
Molecular analysis included the amplification of a S. agalactiae-specific part of the gene
encoding the 16S rRNA, the 16S–23S rDNA intergenic spacer region, the CAMP factor
gene cfb, the hyaluronidase-encoding gene hylB, the insertion element IS1548 and the
laminin and C5a peptidase-encoding genes lmb and scpB, respectively (Yildirim et al.,
2002a,b).
Epidemiological relations were determined by macrorestriction analysis of the chromosomal
DNA of the isolates, using the rare cutting enzyme SmaI, and subsequent separation
of the fragments by pulsed-field gel electrophoresis (Soedarmanto et al., 1996).
3. Results
3.1. Case details and gross pathology
Two male and one female 9-month-old, subadult captive bred emerald monitors with a
body length of 60–75 cm and a body weight of 35–40 g, developed lethargy and inappetence
and died approximately 24–48 h after ingesting subadult dead mice and crickets (Gryllus
bimacularius and Achaeta domestica).
At necropsy, all three emerald monitors were in good body condition. The gastrointestinal
tracts were moderately full of digesta. Moderate to severe, petechial to ecchymotic haemorrhage
was observed in the heart, lungs, liver, kidneys, small intestine, ventral subcutis,
skeletal muscles and abdominal fat bodies. A moderate haemopericardium (0.5–0.8 ml) and
a moderate alveolar oedemawas present. One monitor exhibited a multifocal fibrinonecrotic
inflammation of the small intestine.
286 U. Hetzel et al. / Veterinary Microbiology 95 (2003) 283–293
3.2. Microbiology
Bacteriological investigation of lung, kidney, liver and intestine resulted in the isolation
of streptococci of serological group B (GBS) in moderate to high numbers from all tissues
in all three monitors. Citrobacter freundii, Proteus sp. and Pseudomonas aeruginosa were
each isolated from all organs of one or two animals in variable amounts.
Virological examination yielded negative results in all three emerald monitors. Parasitological
examination identified adult Oxyuridae in low numbers in two, and a moderate
number of Isospora sp. oocysts in one monitor.
From the gastrointestinal tract of 9 of the 165 subadult mice streptococci with weak
-haemolysis were isolated either in low numbers (n = 8) or sporadically (n = 1).
3.3. Phenotypical and genotypical characteristics of GBS
The streptococci isolated from the three emerald monitors and the subadult feeding mice
(n = 9), respectively, were comparatively investigated. They were weakly -haemolytic and
were biochemically and serologically identified as S. agalactiae belonging to Lancefield
serogroup B. The isolated GBS grew in fluid media, producing an uniform turbidity of
the growth media, and in soft agar, forming diffuse colonies. In addition, the GBS were
CAMP-positive, displayed an orange–red pigmentation and did not haemagglutinate rabbit
erythrocytes. The species identification was confirmed by amplification of species-specific
parts of the 16S rRNA gene, the 16S–23S rDNA intergenic spacer region and the CAMP
factor gene cfb. All 12 group B streptococcal isolates were classified as serotype V without
a corresponding protein antigen. The lack of protein antigens was confirmed by a negative
PCR result for the protein antigens Rib- and c-encoding genes rib and bag. In addition, all
isolated GBS displayed a positive reaction in the hyaluronidase plate test, yielded a hylB
gene amplicon of 3.3 kb and were negative for the insertion element IS1548 and the genes
lmb and scpB (Table 1).
Table 1
Cultural, biochemical, serological and genotypic properties of group B streptococci (S. agalactiae), isolated from
three emerald monitors and nine feeding mice
Properties Results
Weak -haemolysis Lactose, inositol, mannitol, sorbitol − Glucose, maltose, sodium hippurate, saccharose, salicin, fructose Group B streptococcal polysaccharide antigen Growth in fluid medium Uniform turbidity
Growth in soft agar Diffuse colonies
CAMP factor Orange–red pigmentation Haemagglutination of rabbit erythrocytes − S. agalactiae 16S rRNA gene, 16S–23S rDNA intergenic spacer region, cfb gene PCR for IS1548, lmb and scpB − Hyaluronidase plate test and PCR for hylB gene (3.3 kb)
U. Hetzel et al. / Veterinary Microbiology 95 (2003) 283–293 287
Fig. 1. Identical pulsed-field electrophoretic restriction pattern of group B streptococci, isolated from emerald
monitors (1–3) and mice (4–8) after macrorestriction analysis of the chromosomal DNA with the restriction
enzyme SmaI. M lambda DNA/HindIII fragments at 0.1–200 kb and lambda DNA concatemeres at 50–1000 kb
(Sigma, Deisenhofen, Germany) as standards.
A macrorestriction analysis of the bacterial chromosomal DNA resulted in a uniform
SmaI restriction pattern of all 12 isolates, indicating their clonal identity (Fig. 1).
3.4. Light microscopy, immunohistochemistry and transmission electron microscopy
In all three emerald monitors, coccoid bacteria were found as clumps within the cytoplasm
of numerous intravascular monocytes and singularly within few heterophils in
arteries and veins of variable diameters in heart, lung, liver (including sinusoids), kidneys
(including glomerular tufts), spleen, stomach and intestines, ovaries or testes, adrenal
glands, skeletal muscle and abdominal fat bodies (Figs. 2 and 3). The coccoid bacteria
were additionally found as cell-free clumps and chains within cardiac atrial and ventricular
lumina, within blood vessel lumina, often accumulating within hyaline thrombi and
attaching to the endothelium (Fig. 3). Multifocal to coalescing haemorrhage was seen in
subcutis and subcutaneous, thoracic and abdominal wall skeletal muscle layers, accompanied
by focal degeneration of muscle fibres. Lungs exhibited focal alveolar haemorrhage
and accumulations of bacteria-laden macrophages, livers showed moderate hepatocellular
lipid accumulation.
Both cell-free and intracellular coccoid bacteria displayed a strong positive immunohistochemical
reaction with the monoclonal antibody against S. agalactiae (Fig. 3).
Ultrastructurally, the bacteria were spherical to ovoid (coccoid), ranged between 1 and
2
min diameter and exhibited a distinct cell wall with microfilaments and capsule (Fig. 4).
Within leukocytes, bacteria were located in cytoplasmatic vacuoles (phagosomes); they
formed pairs as well as short chains and displayed signs of replication (Fig. 4).
288 U. Hetzel et al. / Veterinary Microbiology 95 (2003) 283–293
Fig. 2. Emerald monitor. Heart: myriads of coccoid bacteria are found within circulating leukocytes (arrowheads)
and as cell-free clumps (arrow) in the cardiac chamber. Haematoxylin-eosin stain. Bar = 80
m.
Fig. 3. Emerald monitors. (a) Lung: numerous bacteria-laden monocytes are found within pulmonary capillaries.
(b) Kidney: bacteria-laden monocytes (arrow) are found within glomerular tuft as well as in small vessels.
Immunohistochemical demonstration of S. agalactiae. Papanicolaou’s haematoxylin counterstain. Bar = 40
m.
U. Hetzel et al. / Veterinary Microbiology 95 (2003) 283–293 289
Fig. 4. Emerald monitor. Cardiac chamber: ultrastructural demonstration of bacteria, located within a cytoplasmic
vacuole (phagolysosome) in a monocyte and cell-free. Bar = 3.8
m. Inset: bacteria exhibit a distinct cell wall
with microfilaments (arrow) and capsule (arrowhead) and form pairs and short chains. Bar = 2.4
m.
4. Discussion
In the present study, streptococci of serological group B (S. agalactiae, group B streptococci,
GBS) of serotype V were isolated from three subadult emerald monitors which died
as a result of septicaemia. Identical bacteria were isolated from mice, originating from the
feed for the monitors.
Bacteriologically, the GBS were identified by conventional bacteriological methods, such
as their biochemical properties, a positiveCAMPreaction, an orange–red pigmentation, and
by serogrouping. They did not haemagglutinate rabbit erythrocytes, which is, together with a
negative lactose reaction and the orange–red pigmentation, a typical feature of human rather
than bovine isolates of this bacterial species (Wibawan et al., 1993a; Lämmler and Hahn,
1994). The species identification of the GBS was also confirmed by PCR amplification of
species-specific parts of the 16S rRNA gene, the 16S–23S rDNA intergenic spacer region
and the CAMP factor gene cfb. This corresponded with previous studies which did not
indicate any sequence variation in these genes or gene segments (Lämmler et al., 1998;
Abdulmawjood and Lämmler, 1999; Hassan et al., 2000; Yildirim et al., 2002a–c). The
isolates grew in fluid media with a uniform turbidity of the growth medium, formed diffuse
colonies in soft agar and could be classified as serotype V. According to previous studies
(Wibawan and Lämmler, 1991;Wibawan et al., 1993b), these growth properties are typical
for a more hydrophilic surface of GBS which carry polysaccharide antigens.
290 U. Hetzel et al. / Veterinary Microbiology 95 (2003) 283–293
The identical phenotype of the isolated GBS, also including biochemical, pigmentary and
growth properties and their classification as serotype V, indicated that all 12 GBS isolates
from emerald monitors and from feeding mice had a clonal relation. This was additionally
confirmed by a positive hyaluronidase reaction and by the genotypic properties, such as
a hylB gene amplicon of 3.3 kb and the negative reaction for IS1548 as well as for the
genes lmb and scpB, encoding the laminin-binding protein and C5a peptidase, respectively,
and by DNA fingerprinting. After macrorestriction analysis of the chromosomal DNA by
pulsed-field gel electrophoresis, all isolates yielded identical SmaI restriction patterns, indicating
the epidemiological relation between the GBS isolated from the emerald monitors
and the feeding mice. The hylB gene amplicon size of 3.3 kb appears to be the typical gene
size for hyaluronidase-positive GBS (Granlund et al., 1998; Rolland et al., 1999; Yildirim
et al., 2002c). However, the lack of the genes lmb and scpB is characteristic for bovine
isolates, whereas GBS of human origin are usually positive for both genes (Franken et al.,
2001). Like previously characterized GBS isolated from pigs and nutrias (Wibawan et al.,
1993b), the GBS investigated in the present study exhibited features of both bovine and
human isolates.
In humans, GBS are primarily commensal organisms colonising the gastrointestinal and
genital tract, but can also cause systemic infections in newborns, infants, pregnant women
and nonpregnant adults (Schuchat, 1998; Spellerberg, 2000; Tettelin et al., 2002). In susceptible
hosts, the vaginal, rectal and respiratory mucosa is the portal of GBS entry (Shen
et al., 2000). In nonpregnant adults, GBS represent nosocomial infectious agents, inducing,
e.g. bacteriaemia, soft tissue infection, peri- and endocarditis, pneumonia and urosepsis in
debilitated patients (Elliott et al., 1998; Schuchat, 1998; Farley, 2001).
The cases of GBS septicaemia in emerald monitors presented in this study bear some similarities
to group B streptococcal disease in neonates (early onset disease) and nonpregnant
human adults, in so far as it presents as a primary, partially leukocyte-associated bacteraemia
(Spellerberg, 2000; Farley, 2001). Although other pathogenic or facultative pathogenic bacteria,
such as C. freundii, Proteus sp. and P. aeruginosa, were additionally isolated from the
emerald monitors, the generally high number of GBS observed within circulating leukocytes
as well as free within the blood suggest that GBS were the major causative agent.
However, the GBS septicaemia in emerald monitors might have developed in the context
of polymicrobial bacteraemia, a feature which is apparent in approximately 1/3 of GBS
septicaemia cases in nonpregnant human adults (Schuchat, 1998). In emerald monitors,
bacteria were phenotypically identified as streptococci by light and transmission electron
microscopy, the latter based on the size and morphological features like microfilaments and
capsule (Cheville, 1994). Furthermore, the bacteria showed a strong immunohistochemical
reaction with a monoclonal antibody against S. agalactiae. According to the manufacturer,
this antibody cross-reacts with S. dysgalactiae; however, the latter was not identified by
bacteriology.
GBS are able to survive intracellularly in different eukaryotic cells, such as macrophages,
endothelial cells and respiratory epithelial cells (Rubens et al., 1991, 1992;Valentin-Weigand
et al., 1997; Nizet et al., 1997), a feature which seems to correlate with their invasiveness
(Spellerberg, 2000). Additionally, GBS can augment spreading by impairing their phagocytosis
via an antiphagocytic capsule (Rubens et al., 1987) and by inducing apoptosis in
macrophages (Fettucciari et al., 2000). In the emerald monitors examined in this study,
U. Hetzel et al. / Veterinary Microbiology 95 (2003) 283–293 291
intracellular GBS were located in phagosomes, indicating their uptake by phagocytosis and
showed intracellular replication.
It is likely that the portal of infection for the emerald monitors was the intestine, where
bacteria from the gastrointestinal tract of feeding mice would be exposed to mucosal surfaces
and could be taken up by phagocytes. As the morphological studies on the emerald
monitors suggested, subsequent bacteraemiawas likely mediated by macrophages and polymorphonuclear
cells. Previous experimental studies on mice revealed that these are the first
immune cells interacting with GBS (Cornacchione et al., 1998).
GBS have long been known as causative agents of bovine and caprine mastitis (Lämmler
and Hahn, 1994; Keefe, 1997) and are taxonomically classified as S. agalactiae. GBS have
also been isolated from other animal species, such as camels, monkeys, pigs, nutrias, horses,
cats, dogs and fish, occasionally in association with pathological processes (Wilkinson et al.,
1973; Finch and Martin, 1984; Hommez et al., 1991;Wibawan et al., 1993b; Lämmler et al.,
1998; Bekele and Molla, 2001; Yildirim et al., 2002a,b).
GBS are subdivided into nine different serotypes (Ia, Ib, II–VIII), based on variations of
capsular polysaccharide antigens (Wibawan and Lämmler, 1990; Schuchat, 1999). During
the last decade, GBS of serotype V have been identified as a major cause of group B streptococcal
disease both in newborns and nonpregnant adults (Blumberg et al., 1996; Schuchat,
1998; Elliott et al., 1998; Tyrrell et al., 2000). This study, however, shows that infection
with GBS of serotype V can also cause fatal septicaemia in reptiles. Mice which usually
serve for experimental studies on GBS (Cornacchione et al., 1998; Fettucciari et al., 2000),
can be naturally infected. As GBS have to be considered as zoonotic agents, reptiles and
mice-like humans, a variety of mammal species and fish (Euzéby, 2003)—represent potential
reservoirs. Furthermore, reptile breeders and owners should be aware of the potential
source of infection feeding mice might represent.
Acknowledgements
The authors wish to thank Dr. R. Marschang and Dr. C. Bauer for performing the virological,
respectively, parasitological examinations, and Dr. S. Estuningsih and Mrs. A. Artelt
for technical assistance with immunohistochemical tests. We are grateful to Mr. S. Hännig
for providing the monitors and mice.
References
Abdulmawjood, A., Lämmler, C., 1999. Amplification of 16S ribosomalRNAgene sequences for the identification
of streptococci of Lancefield group B. Res. Vet. Sci. 67, 159–162.
Bekele, T., Molla, B., 2001. Mastitis in lactating camels (Camelus dromedarius) in Afar Region, north-eastern
Ethiopia. Berl. Münch. Tierärztl. Wochenschr. 114, 169–172.
Bennett, D., 1995. Warane der Welt Welt der Warane. Edition Chimaira, Frankfurt, pp. 112–114.
Blumberg, H.M., Stephens, D.S., Modansky, M., Erwin, M., Elliot, J., Facklam, R.R., Schuchat, A., Baughman,
W., Farley, M.M., 1996. Invasive group B streptococcal disease: the emergence of serotype V. J. Infect. Dis.
173, 365–373.
Cheville, N., 1994. Ultrastructural Pathology. Iowa State University Press, Ames, pp. 617–710.
292 U. Hetzel et al. / Veterinary Microbiology 95 (2003) 283–293
Cornacchione, P., Scaringi, L., Fettuciari, K., Rosati, E., Sabatini, R., Orefici, G., Von Hunolstein, C., Modesti,
A., Minelli, F., Marconi, P., 1998. Group B streptococci persist inside macrophages. Immunology 93, 86–95.
Eidenmüller, B., 1997. Warane; Lebensweise, Pflege, Zucht. Herpetan, Offenbach, pp. 79–81.
Elliott, J.A., Farmer, K.D., Facklam, R.R., 1998. Sudden increase in isolation of group B streptococci, serotype
V, is not due to emergence of a new pulsed-field gel electrophoresis type. J. Clin. Microbiol. 36, 2115–2116.
Euzéby, J.P., 2003. DBV, Dictionnaire de Bactériologie Vétérinaire. http://www.bacterio.cict.fr/bacdico/
garde.html.
Farley, M.M., 2001. Group B streptococcal disease in nonpregnant adults. Clin. Infect. Dis. 33, 556–561.
Fettucciari, K., Rosati, E., Scaringi, L., Cornacchione, P., Migliorati, G., Sabatini, R., Fetriconi, I., Rossi, R.,
Marconi, P., 2000. Group B Streptococcus induces apoptosis in macrophages. J. Immunol. 165, 3923–3933.
Finch, L.A., Martin, D.L., 1984. Human and bovine group B streptococci: two distinct populations. J. Appl.
Bacteriol. 57, 273–278.
Franken, C., Haase, G., Brandt, C.,Weber-Heynemann, J., Martin, S., Lämmler, C., Podbielski, A., Lütticken, R.,
Spellerberg, B., 2001. Horizontal gene transfer and host specificity of beta-haemolytic streptococci: the role
of a putative composite transposon containing scpB and lmb. Mol. Microbiol. 41, 925–935.
Granlund, M., Oberg, L., Sellin, M., Norgren, M., 1998. Identification of a novel insertion element, IS1548, in
group B streptococci, predominantly in strains causing endocarditis. J. Infect. Dis. 177, 967–976.
Hassan, A.A., Abdulmawjood, A., Yildirim, A.O., Fink, K., Lämmler, C., Schlenstedt, R., 2000. Identification of
streptococci isolated from various sources by determination of cfb gene and other CAMP-factor genes. Can.
J. Microbiol. 46, 946–951.
Hommez, J., Devrise, L.A., Castryck, F., Miry, C., 1991. Beta-hemolytic streptococci from pigs: bacteriological
diagnosis. Zentralbl. Veterinärmed. 38, 441–444.
Keefe, G.P., 1997. Streptococcus agalactiae mastitis: a review. Can. Vet. J. 38, 429–437.
Lämmler, C., Hahn, G., 1994. Streptokokken. In: Blobel, H., Schließer, T. (Eds.), Handbuch der bakteriellen
Infektionen bei Tieren. Band II/2: Streptokokken-Infektionen und Rotlauf. 2. Auflage. Gustav Fischer Verlag,
Jena/Stuttgart, 1994, pp. 7–141.
Lämmler, C., Abdulmawjood, A., Weiss, R., 1998. Properties of serological group B streptococci of dog, cat and
monkey origin. Zentralbl. Veterinärmed. 45, 561–566.
Nizet, V., Kim, K.S., Stins, M., Chi, E.Y., Nguyen, D., Rubens, C.E., 1997. Invasion of brain microvascular
endothelial cells by group B streptococci. Infect. Immun. 65, 5047–5081.
Rolland, K., Marois, C., Siquier, V., Cattier, B., Quentin, R., 1999. Genetic features of Streptococcus agalactiae
strains causing severe neonatal infections, as revealed by pulsed-field gel electrophoresis and hylB gene
analysis. J. Clin. Microbiol. 37, 1892–1898.
Rubens, C.E., Wessels, M.R., Heggen, L.M., Kasper, D.L., 1987. Transposon mutagenesis of type III group B
streptococcus: correlation of capsule expression with virulence. Proc. Natl. Acad. Sci. USA 84, 7208–7212.
Rubens, C.E., Raff, H.V., Jackson, J.C., Chi, E.Y., Bielitzki, J.T., Hillier, S.L., 1991. Pathophysiology and
histopathology of group B streptococcal sepsis in Macaca nemestrina primates induced after intraamniotic
inoculation: evidence for bacterial cellular invasion. J. Infect. Dis. 164, 320–330.
Rubens, C.E., Smith, S., Hulse, M., Chi, E.Y., van Belle, G., 1992. Respiratory epithelial cell invasion by group
B streptococci. Infect. Immun. 60, 5157–5163.
Schuchat, A., 1998. Epidemiology of group B streptococcal disease in the United States: shifting paradigms. Clin.
Microbiol. Rev. 11, 497–513.
Schuchat, A., 1999. Group B streptococcus. Lancet 353, 51–56.
Shen, X., Lagergard, T., Yang, Y., Lindblad, M., Fredriksson, M., Holmgren, J., 2000. Systemic and mucosal
immune responses in mice after mucosal immunization with group B streptococcus type III capsular
polysaccharide-cholera toxin B subunit conjugate vaccine. Infect. Immun. 68, 5749–5755.
Soedarmanto, I., Pasaribu, F.H.,Wibawan, I.W.T., Lämmler, C., 1996. Identification and molecular characterization
of serological group C streptococci isolated from diseased pigs and monkeys in Indonesia. J. Clin. Microbiol.
34, 2201–2204.
Spellerberg, B., 2000. Pathogenesis of neonatal Streptococcus agalactiae infections. Microb. Infect. 2, 1733–1742.
Sternberger, L.A., Hardy Jr., P.H., Cuculis, J.J., Meyer, G.H., 1970. The unlabeled antibody enzyme method
of immunohistochemistry: preparation and properties of soluble antigen–antibody complexes (horseradish
peroxidase–antihorseradish peroxidase) and its use in identification of spirochetes. J. Histochem. Cytochem.
18, 315–333.
U. Hetzel et al. / Veterinary Microbiology 95 (2003) 283–293 293
Tettelin, H., Masignani, V., Cieslewicz, M.J., Eisen, J.A., Peterson, S., 2002. Complete genome sequence and
comparative genomic analysis of an emerging human pathogen, serotype V Streptococcus agalactiae. Proc.
Natl. Acad. Sci. USA 99, 12391–12396.
Tyrrell, G.J., Senzilet, L.D., Spika, J.S., Kertesz, D.A., Alagaratnam, M., Lovgren, M., Talbot, J.A., 2000. Invasive
disease due to group B streptococcal infection in adults: results from a Canadian population-based active
laboratory surveillance study—1996. Sentinel health unit surveillance system site coordinators. J. Infect. Dis.
182, 168–173.
Valentin-Weigand, P., Jungnitz, H., Zock, A., Rohde, M., Chhatwal, G.S., 1997. Characterization of group B
streptococcal invasion in HEp-2 epithelial cells. FEMS Microbiol. Lett. 147, 69–74.
Wibawan, I.W.T., Lämmler, C., 1990. Distribution of B streptococcal type antigens among streptococci of
serological groups B, G and L. Zentralbl. Bakteriol. 273, 471–477.
Wibawan, I.W.T., Lämmler, C., 1991. Influence of capsular neuraminic acid on properties of streptococci of
serological group B. J. Gen. Microbiol. 137, 2721–2725.
Wibawan, I.W.T., Lautrou, Y., Lämmler, C., 1991. Antibiotic resistance patterns and pigment production of
streptococci of serological group B isolated from bovines and humans. J. Vet. Med. B 38, 731–736.
Wibawan, I.W.T., Lämmler, C., Seleim, R.S., Pasaribu, F.H., 1993a. A haemagglutinating adhesin of group B
streptococci isolated from cases of bovine mastitis mediates adherence to HeLa cells. J. Gen. Microbiol. 139,
2173–2178.
Wibawan, I.W.T., Lämmler, C., Smola, J., 1993b. Properties and type antigen patterns of group B streptococcal
isolates from pigs and nutrias. J. Clin. Microbiol. 31, 762–764.
Wilkinson, H.W., Thacker, L.G., Facklam, R.R., 1973. Nonhemolytic group B streptococci of human, bovine, and
ichthyic origin. Infect. Immun. 7, 496–498.
Yildirim, A.O., Lämmler, C., Weiss, R., 2002a. Identification and characterization of Streptococcus agalactiae
isolated from horses. Vet. Microbiol. 85, 31–35.
Yildirim, A.O., Lämmler, C., Weiss, R., Kopp, P., 2002b. Pheno- and genotypic properties of streptococci of
serological group B of canine and feline origin. FEMS Microbiol. Lett. 212, 187–192.
Yildirim, A.O., Fink, K., Lämmler, C., 2002c. Distribution of the hyaluronate lyase encoding gene hylB and the
insertion element IS1548 in streptococci of serological group B isolated from animals and humans. Res. Vet.
Sci. 73, 131–135.