574 Brief Communications

portant in human and animal health. In: Microbial survival in
the environment, pp. 45–47. Springer-Verlag, Berlin, Germany.
10. Munro R, Ross H, Cornwell C, Gilmour J: 1992, Disease con-
ditions affecting common seals (Phoca vitulina) around the
Scottish mainland, September–November 1988. Sci Total En-
viron 115:67–82.
11. Porter JF, Wardlaw AC: 1993, Long-term survival of Bordetella
bronchiseptica in lakewater and in buffered saline without add-
ed nutrients. FEMS Microbiol Lett 110:33–36.
12. Register KB, Ackermann MR: 1997, A highly adherent phe-
notype associated with virulent Bvg1-phase swine isolates of
Bordetella bronchiseptica grown under modulating conditions.
Infect Immun 65:5295–5300.
13. Register KB, Boisvert AM, Ackermann MR: 1997, Use of ri-
botyping to distinguish isolates of Bordetella bronchiseptica. Int
J Syst Bacteriol 47:678–683.
14. Register KB, Magyar T: 1999, Optimized ribotyping protocol
applied to Hungarian Bordetella bronchiseptica isolates; iden-
tification of two novel ribotypes. Vet Microbiol 69:277–285.
15. Register KB, Sacco RE, Foster G: 2000, Ribotyping and restric-
tion endonuclease analysis reveal a novel clone of Bordetella
bronchiseptica in seals. J Vet Diagn Invest 12:535–540.

J Vet Diagn Invest 15:574–576 (2003)

A case of yellow fever in a brown howler (Alouatta fusca) in Southern Brazil

Eliza Simone Viégas Sallis, Vera Lúcia Reis Souza de Barros, Shana Letı́cia Garmatz,
Rafael Almeida Fighera, Dominguita Lühers Graça1

Abstract. Many brown howlers (Alouatta fusca) have died in a 3-month period in a subtropical forest in
Southern Brazil. One was examined after a systemic illness. According to clinical signs, and necropsy and
histopathology findings, yellow fever virus (YFV) infection was suspected. Tissue sections from liver, kidney,
and lymphoid organs were screened by immunohistochemistry for YFV antigens. Cells within those tissues
stained positively with a polyclonal antibody against YFV antigens (1:1,600 dilution), and yellow fever was
diagnosed for the first time in the brown howler in the area.

Yellow fever virus (YFV) infection is an acute ar-
thropod-borne Flavivirus (family Togaviridae) infec-
tion that causes fever, jaundice, albuminuria, and hem-
orrhage. It occurs in 2 forms: urban and sylvan. There
are 2 types of endemic areas: humid forests and emerg-
ing zones where urban and sylvan forms intermingle.3
The vectors are Aedes aegypti in urban areas,9 A. al-
bopictus in suburban areas,10 and tree-hole–breeding
mosquitoes (Haemagogus spp.) in the forests.9 The vi-
rus circulates in the forests in mosquito vectors caus-
ing scattered epizootics in nonimmune monkeys. Al-
ternatively, transmission may be vertical from the fe-
male mosquito to her offspring, allowing virus surviv-
al from one rainy season to the next in A. aegypti
eggs.3 Yellow fever remains endemic in many regions
of Africa and South America, despite the existence of
an effective vaccine.2,9 Currently, YFV infection may
be confused with other similar hemorrhagic condi-
tions.2,4,9
In humans, severe necrotic lesions are seen at au-
topsy2 in many organs, particularly the liver. Micro-
scopically, YFV infection is characterized by midzonal
necrosis of the liver with microvesicular fatty change
From the Departamento de Patologia, Universidade Federal de
Santa Maria, 97105-900 Santa Maria, Brazil (Sallis, Garmatz, Figh-
era, Graça), and the Instituto Evandro Chagas, Rod. BR-316, Km 7,
67030-000 Ananindeua, PA, Brazil (de Barros). 1 Corresponding au-
thor.
of hepatocytes, the presence of apoptotic bodies
(Councilman bodies), renal tubular degeneration, and
splenic lymphoid necrosis.5,6,8
Up to 80 brown howlers (Alouatta fusca) are sus-
pected to have died from March 2001 to May 2001 at
the border between Brazil and Argentina. People of
the subtropical forest of the area reported that many
dead monkeys had fallen from the trees. They ob-
served that the monkeys had yellow mucosae and skin.
A yellow fever outbreak was suspected. One of the
dead monkeys was necropsied in May 2001 at the De-
partment of Pathology, Universidade Federal de Santa
Maria, Southern Brazil.
The necropsied brown howler was a thin, young
adult female monkey in poor body condition. All mu-
cosae and large vessel intimae and the liver were jaun-
diced. The right kidney had an irregular area at the
surface, and the spleen had uneven borders. The stom-
ach was full of nondigested green leaves, fibers, and
seeds. The intestines contained feces coated in dry,
yellow mucus. The urinary bladder contained yellow
urine with white floccular strands. Formalin-fixed, par-
affin-embedded tissues were sectioned at 5 mm and
stained with hematoxylin and eosin (HE). By light mi-
croscopy, massive coagulation necrosis of most hepa-
tocytes and fatty degeneration of the remaining cells
was observed in the liver (Fig. 1). Scattered apoptotic
hepatocytes were present. Renal tubular cells had de-
 Brief Communications
Figure 1. Liver; brown howler (Alouatta fusca). Massive coag-
ulative necrosis of the hepatocytes. HE. 1603.
Figure 2. Spleen, brown howler (Alouatta fusca). Necrosis of
lymphoblasts in the follicles (.). HE.1603.
generative changes, and hyaline casts were noted with-
in the lumen of many tubules. An extensive area of
subacute interstitial nephritis in the right kidney cor-
responded to the irregular area seen grossly. The lym-
phoid follicles of the splenic white pulp had variable
central necrosis (Fig. 2). No lesions were found in oth-
er organs.
The history, gross findings, and histological changes
were highly suggestive of yellow fever. Paraffin-em-
bedded sections of liver, kidney, and spleen were sub-
mitted for immunohistochemistry to the Evandro Cha-
gas Institute at Belém, Pará, Northern Brazil. A mouse
polyclonal antibody (dilution 1:1,600) and a commer-
cially available Streptavidin–alkaline phosphatase
complexa that reacted with the biotin linked with the
secondary antibody were used for YFV antigen detec-
tion. The specific substrate for the alkaline phospha-
tase was Histomark Red.b The slides were counter-
stained with Mayer hematoxylin and mounted in En-
tellan.c Positive and negative controls were run with
575
Figure 3. Liver; brown howler (Alouatta fusca). Yellow fever
virus antigen (red reaction) was detected in hepatocytes. Immuno-
histochemistry, alkaline phosphatase, red substrate. Mayer hematox-
ylin counterstain. 4003.
every test. Renal tubular epithelial cells and wide-
spread hepatocytes stained positive for YFV (Fig. 3).
Cases of yellow fever and dengue, both Flavivirus
infections, overlap geographically in some areas of
South America and, clinical differentiation of these in-
fections is usually difficult.4 The severe hepatic lesions
of yellow fever were once considered pathognomonic.1
Historically, in dengue, the low production of viral
progeny by infected hepatocytes usually resulted in
limited foci of infection, whereas foci tend to be mas-
sive in yellow fever infection. However, in recent
years, dengue has been shown to be capable of more
extensive cytopathic effect.7 An immunohistochemical
method is required for confirmation of either diagno-
sis.2
In the monkey studied in this report, jaundice was
marked, but other typical gross lesions, e.g., hemor-
rhages and serous effusions9 were absent. Histopath-
ologic changes in the liver were severe and consistent
with the extensive hepatic destruction reported for yel-
low fever.9
In Brazil, urban yellow fever has been curtailed for
more than 50 years by controlling the vector, Aedes
sp.10 The sylvatic cycle still occurs in outbreaks that
reflect vector movement and changing climatic con-
ditions.6 After diagnosis in this brown howler, sanitary
measures were immediately taken to halt the vector
spread to the surrounding urban areas, and the human
population around the forest was vaccinated because
of the potential zoonotic hazard.
Acknowledgement. We thank Dr. L. A. V. Pereira
for photographic processing.
Sources and manufacturers
a. GIBCO-BRL Life Tech. Inc., Gaithersburg, MD.
b. Kirkegaard and Perry Labs Inc., Gaithersburg, MD.
576 Brief Communications

c. Merck, Darmstadt, Germany.
References
1. Brés PLJ: 1986, A century of progress in combating yellow
fever. Bull WHO 64:775–786.
2. De Brito T, Siqueira SAC, Santos RTM, et al.: 1992, Immuno-
histochemical detection of viral antigens in the liver, kidney and
heart. Pathol Res Pract 188:177–181.
3. Fontenille D: 1999, Transmission et conservation du virus amar-
il dans la nature. Bull Soc Pathol Exot 92:435.
4. Hall WC, Crowell TP, Watts DM, et al.: 1991, Demonstration
of yellow fever and dengue antigens in formalin-fixed paraffin–
embedded human liver by immunohistochemical analysis. Am
J Trop Med Hyg 45:408–417.
5. Ishak KG, Walker DH, Coetzer JAW, et al.: 1982, Viral hem-
orrhagic fevers with hepatic involvement: pathologic aspects
with clinical correlations. Prog Liv Dis 7:495–499.
6. Lovejoy TE: 1993, Global change and epidemiology: nasty syn-
ergies. In: Emerging viruses, ed. Morse SS, pp. 261–268. Ox-
ford University Press, New York, NY.
7. Marianneau P, Steffan A-M, Royer C, et al.: 1998, Differing
infection patterns of dengue and yellow fever viruses in a human
hepatoma cell line. Infect Dis 178:1270–1278.
8. Monath TP: 1985, Flaviviruses. In: Virology, ed. Fields BN,
Knipe DM, Melnick JL, Chanock RM, Roizman B, Shape RE,
eds, pp. 955–1004. Raven Press, New York, NY.
9. Monath TP: 1998, Yellow fever. In: Zoonoses, ed. Palmer SR,
Soulsby L, Simpson DIH, eds., pp. 487–498. University Press,
Oxford Great Britain.
10. Mondet B, da Rosa APAT, Vasconcelos, PFC: 1996, Les risques
d’épidémisation urbaine de la fièvre jaune ao Brésil par les vec-
teurs de la dengue. Bull Soc Pathol Exot 89:107–114.

J Vet Diagn Invest 15:576–579 (2003)

In vitro evaluation of the susceptibility of Edwardsiella ictaluri, etiological agent of

enteric septicemia in channel catfish, Ictalurus punctatus (Rafinesque), to florfenicol

Anissa McGinnis1, P. Gaunt, T. Santucci, R. Simmons, R. Endris

Abstract. In vitro studies were conducted to assess the sensitivity of Edwardsiella ictaluri, the etiological
agent of enteric septicemia of catfish (ESC), to the antibacterial drug florfenicol (FFC). Twelve different E.
ictaluri isolates from cases submitted between 1994 and 1997 to the Thad Cochran National Warmwater Aqua-
culture Center fish diagnostic laboratory (Stoneville, MS) were used for testing. These isolates originated from
channel catfish (Ictalurus punctatus) infected with E. ictaluri through natural outbreaks of ESC in the com-
mercial catfish ponds in Mississippi. Seven hundred sixty-seven additional cultures of E. ictaluri were obtained
from channel catfish infected experimentally with E. ictaluri. In some of these experimental infections, FFC
was used for treatment. These cultures of E. ictaluri were identified by morphological and biochemical tests.
Kirby–Bauer zones of inhibition (in mm) for FFC against E. ictaluri were determined using standard methods.
The minimum inhibitory concentration (MIC) of FFC was determined for the natural outbreak E. ictaluri isolates
and arbitrarily selected experimental cultures. The zones of inhibition for FFC tested with E. ictaluri ranged
from 31 to 51 mm. The MIC for FFC tested with E. ictaluri was consistently 0.25 mg/ml. Edwardsiella ictaluri
tested in these studies were highly sensitive to FFC in vitro.

Edwardsiella ictaluri, first isolated in 1976,2 is the
etiological agent of enteric septicemia of catfish
(ESC). The disease is capable of causing high mortal-
ities in channel catfish (Ictalurus punctatus) and is
considered the most significant factor affecting com-
mercial catfish aquaculture. Outbreaks of ESC peak
when water temperatures are 22–28 C.13,14 For suc-
cessful treatment of fish exhibiting signs of ESC, im-
mediate diagnosis and treatment with oral antibiotics
are recommended while the majority of the fish are
From the Department of Pathobiology and Population Medicine,
Mississippi State University College of Veterinary Medicine, Delta
Research and Extension Center, PO Box 197, Stoneville, MS 38776
(McGinnis, Gaunt, Santucci), and the Schering Plough Animal
Health, 1095 Morris Avenue, Union, NJ 07083 (Simmons, Endris).
1 Corresponding author.
still feeding.12 Currently, the only antibiotics approved
for use with food fish in the United States are oxytet-
racycline (Terramycint)a and a sulphadimethoxine and
ormetoprim combination (Romet-30t). b However,
there are reports of bacterial resistance to these anti-
biotics.7,11,12 In addition, palatability problems have
been reported with sulphadimethoxine and ormetoprim
combinations. 8 An alternative treatment for ESC
would be beneficial to the industry.
Previous in vitro research with fish pathogens in-
dicates that most fish pathogenic bacteria are sensitive
to the antibiotic florfenicol (FFC). Studies involving
the bacterial species Photobacterium damsela (subsp.
Piscicida), Vibrio anguillarum, Aeromonas hydrophi-
la, Aeromonas salmonicida, and Edwardsiella tarda
determined that the minimum inhibitory concentra-
tions (MICs) were less than or equal to 1.6 mg/ml for