Systemic Histomoniasis in a Leucistic Indian Peafowl (Pavo cristatus) from

Southern Brazil
Author(s): Mariana de Mello Zanim Michelazzo, João Pedro Sasse, Marielen de Souza, Victor Hugo
Brunaldi Marutani, Ana Angelita Sampaio Baptista, João Luis Garcia, Amauri Alcindo Alfieri, and
Selwyn Arlington Headley
Source: Avian Diseases, 61(3):325-329.
Published By: American Association of Avian Pathologists
https://doi.org/10.1637/11583-010617-RegR
URL: http://www.bioone.org/doi/full/10.1637/11583-010617-RegR

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AVIAN DISEASES 61:325–329, 2017

Systemic Histomoniasis in a Leucistic Indian Peafowl (Pavo cristatus) from

Southern Brazil
Mariana de Mello Zanim Michelazzo,A João Pedro Sasse,B Marielen de Souza,C Victor Hugo Brunaldi Marutani,A
Ana Angelita Sampaio Baptista,C João Luis Garcia,B Amauri Alcindo Alfieri,D and Selwyn Arlington HeadleyAE
A
Laboratory of Animal Pathology, Department of Veterinary Preventive Medicine, Universidade Estadual de Londrina, Paraná, Brazil
B
Laboratory of Parasitology, Department of Veterinary Preventive Medicine, Universidade Estadual de Londrina, Paraná, Brazil
C
Laboratory of Avian Medicine, Department of Veterinary Preventive Medicine, Universidade Estadual de Londrina, Paraná, Brazil
D
Multi-User Animal Health Laboratory, Molecular Biology Unit, Department of Veterinary Preventive Medicine, Universidade Estadual de
Londrina, Paraná, Brazil
Received 10 January 2017; Accepted 11 April 2017; Published ahead of print 20 June 2017

SUMMARY. The pathological and molecular findings associated with Histomonas meleagridis are described in a leucistic Indian
peafowl (Pavo cristatus) from Southern Brazil. The most significant gross findings were multifocal necrotizing hepatitis and
diphtheric typhlitis. Histopathologic evaluation of the liver, ceca, kidney, spleen, and small intestine revealed systemic
histomoniasis (SH) associated with intralesional and intravascular accumulations of histomonad organisms consistent with H.
meleagridis. PCR was used to amplify the DNA of H. meleagridis from the liver, ceca, small intestine, spleen, lungs, and kidneys.
Direct sequencing and phylogenetic analyses confirmed that the isolate of the flagellated trichomonad identified from this
investigation is more phylogenetically related to H. meleagridis than Tetratrichomonas gallinarum, Tritrichomonas foetus, and
Dientamoeba fragilis. These results confirmed the occurrence of SH in this peafowl and add to the diagnosis of this disease in birds
from Brazil. This report might represent the first complete identification of spontaneous histomoniasis in a peafowl due to
pathological and molecular characteristics and one of the few documented cases of SH in non-commercial birds.

RESUMEN. Histomoniasis Sistémica en un pavo real blanco (Pavo cristatus) en el Sur de Brasil.
Los hallazgos patológicos y moleculares asociados con Histomonas meleagridis se describen en un pavo real blanco (Pavo cristatus)
en el sur de Brasil. Los hallazgos macroscópicos más significativos fueron hepatitis necrotizante multifocal y tiflitis diftérica. La
evaluación histopatológica del hı́gado, ciegos, riñón, bazo e intestino delgado reveló histomoniasis sistémica (SH) asociada con
acumulaciones intralesionales e intravasculares de organismos compatibles con H. meleagridis. Se utilizó PCR para amplificar el
ADN de H. meleagridis del hı́gado, sacos ciegos, intestino delgado, bazo, pulmones y riñones. La secuenciación directa y los análisis
filogenéticos confirmaron que el aislamiento de la tricomonas flageladas que fueron identificadas en esta investigación estaban
filogenéticamente más relacionadas con H. meleagridis y no con Tetratrichomonas gallinarum, Tritrichomonas foetus y Dientamoeba
fragilis. Estos resultados confirmaron la presencia de histomoniasis sistémica en este pavo real y se suman al diagnóstico de esta
enfermedad en aves de Brasil. Este informe podrı́a representar la primera identificación completa de la histomoniasis espontánea en
un pavo real debido a caracterı́sticas patológicas y moleculares y uno de los pocos casos documentados de histomoniasis sistémica en
aves no comerciales.
Key words: Histomonas meleagridis, trichomonad, diagnostic pathology, molecular diagnostics
Abbreviations: BLAST ¼ Basic Local Alignment Search Tool; ITS ¼ Internal Transcribed Spacer; rRNA ¼ small-subunit
ribosomal RNA; SH ¼ systemic histomoniasis

Histomoniasis is a fatal disease caused by the flagellate protozoan
parasite Histomonas meleagridis, which produces lesions predomi-
nantly in the ceca and liver (14), but occasionally in the bursa of
Fabricius, kidney, and spleen (24,25) in a wide range of gallinaceous
birds. Histomoniasis is also referred to as histomonosis, blackhead,
enterohepatitis, and infectious typhlohepatitis (14,24,25), occurs in
all continents (24), and is described predominantly in chickens and
turkeys (14,16,24). However, few reports of blackhead were located
in the peafowl; these include experimental (21) and spontaneous
(13) infections. There is also a retrospective study that used cases
from two U.S. diagnostic laboratories (4). Although blackhead has a
worldwide distribution, few published descriptions of this disease in
birds from Brazil were located when major databases were searched;
these reports include histomoniasis in free-range chickens (1),
E
Corresponding author. E-mail: selwyn.headley@uel.br
Humane Care of Animals. The owner authorized the use of this bird for
scientific purposes.
turkeys (2,28), and a conference proceeding that described the
pathological alterations in a peafowl, Pavo cristatus (26).
By taxonomy, H meleagridis is a flagellated trichomonad that is
similar to Dientamoeba fragilis and Tritrichomonas foetus (17).
Moreover, phylogenetic studies of the small subunit ribosomal
RNA (rRNA) (9) and 5.8S rRNA genes with the flanked ITS regions
(8,9,20) have demonstrated that these organisms are closely related. In
addition, with the proposed new classification system based on
molecular phylogenetics and ultrastructural characteristics (3),
Histomonas is now classified within the family Dientamoebidae, order
Tritrichomonadida, class Tritrichomonadea. Additional examples of
trichomonad flagellate organisms within the Dientamoebidae family
are members of the Dientamoeba and Protrichomonas genera (3).
Since the first description of blackhead in 1895 (22,25), there
have been numerous publications on all aspects of this disease. It is
now known that the cecal worm, Heterakis gallinarum, plays an
important role in the dissemination and maintenance of the
biological cycle of H. meleagridis (11,23,25,29), and control of this
intestinal nematode was fundamental for reduced infectious rates of

325
326 M. M. Z. Michelazzo
blackhead worldwide (23). However, histomoniasis is being
considered as a re-emerging disease due to the increased use of the
free-range production system (16), the prohibition of dimetridazole
as the drug of choice for the treatment of blackhead, and the ban of
Nifursol as a growth promoter in the poultry industry of Europe and
the United States (12,24). Additionally, most of these new cases of
histomoniasis have been described in birds reared under the free-
range system (12,16), because they are more likely to ingest H.
gallinarum. Intriguingly, parasitism by the cecal nematode in free-
range chickens from different geographical regions of Brazil varies
between 22 to 86% (30), whereas comparatively few cases of
histomoniasis are documented in birds within this continental
country. This paper describes the pathological and molecular
findings associated with a spontaneous systemic infection of H.
meleagridis in a leucistic Indian peafowl (Pavo cristatus) from
Southern Brazil, adds to the identification of this parasitic disease of
birds in this country, and complements a previous report (26).

MATERIALS AND METHODS

Case description. A 5-month-old Indian peafowl (Pavo cristatus) was
submitted for routine necropsy evaluation at the Laboratory of Animal
Pathology, Universidade Estadual de Londrina, Southern Brazil, after it
was found dead. The owner relayed that the peafowl was reared under a
semi-intensive system, shared yard space with other peacocks (n ¼ 3) and
domestic chickens (Gallus domesticus, n ¼ 10). He further indicated that
the bird demonstrated apathy, drooped wings, yellow feces, anorexia,
and depression one day prior to death. The owner also relayed that the
peafowl arrived at the present location 20 days before the onset of
clinical manifestations and was previously housed at a nursery
maintained by a rural shop. Further, the other birds that cohabited
the same yard space were reportedly asymptomatic.
Necropsy and histological staining. The bird was submitted for
routine necropsy soon after death. Tissues fragments (i.e., brain, liver,
spleen, kidney, lung, cecum, and small intestine) were collected and
fixed by immersion in 10% buffered formalin solution and routinely Fig. 1. Gross findings associated with histomoniasis in a peafowl.
processed for histopathologic evaluation with hematoxylin and eosin (A) There is multifocal to coalescing, necrotizing hepatitis. (B) Observe
(H&E) stain. Selected sections from all tissues previously mentioned, the severely thickened walls of the opened ceca with accumulation of
diphtheritic exudate. Scale in cm.
with exception of the brain, were subjected to the periodic acid–Schiff
(PAS) histochemical staining technique. In addition, fragments of all
tissues mentioned above were collected freshly at necropsy and stored at sequences were then generated by the CAP3 program (http://asparagin.
-80 C until used in molecular diagnostics. Fecal samples were collected cenargen.embrapa.br/cgi-bin/phph/cap3.pl), after which the partial
for parasitological evaluation. nucleotide sequences were compared by using the Basic Local Alignment
Molecular identification of H. meleagridis. Genomic DNA was Search Tool (BLAST) program (http://www.ncbi.nlm.nih.gov/BLAST)
extracted (PureLink Genomic DNA MiniKit, Invitrogen, Carlsbad, CA) with similar sequences deposited in GenBank.
from fresh fragments of the liver, ceca, small intestine, spleen, lungs, and Phylogenetic tree, nucleotide sequence identity table, and sequence
kidneys and used in a PCR assay designed to amplify a partial fragment alignments based on the 5.8S rRNA gene and the flanking ITS1 and
of the 5.8S rRNA gene and the internal transcribed spacer regions (ITS1 ITS2 regions of selected trichomonad flagellate organisms were then
and ITS2) of trichomonad flagellate as described (8). A positive control created using MEGA 7 (19), constructed by the maximum likelihood
consisted of protozoan DNA from an unpublished report; nuclease-free method based on the Jukes–Cantor mode, by using 1000 bootstrapped
water (Invitrogen Corporation) was used as the negative control in the
data sets. Toxoplasma gondii was used as the outgroup.
PCR assay. All PCR products were separated by electrophoresis in 2%
agarose gels, stained with Sybr Safe (Invitrogen,), visualized under UV
light, and photographed using a gel documentation system (Safe Imager,
RESULTS
Invitrogen).
The amplified PCR products were then purified and submitted for Pathological and parasitological findings. The significant gross
direct sequencing by using the DYEnamic ET Dye Terminator Cycle lesions were restricted to the liver and cecum, all other tissues were
Sequencing Kit (GE Healthcare, Little Chalfont, UK) with the forward
not severely affected. There were multifocal to coalescing, caseous
and reverse primers in the 3500 Genetic Analyzer (Applied Biosystems,
Carlsbad, CA). The obtained sequences were examined for quality nodules, that varied from 0.5 cm to 1.2 cm in diameter, and
analysis of chromatogram readings by using the PHRED software randomly distributed throughout the capsular and sectioned surfaces
(http://asparagin.cenargen.embrapa.br/phph); sequences were only ac- of the liver (Fig. 1A). Several similar caseous nodules were observed
cepted if base quality was equal to or greater than 20. Consensus at the serosal surface of the ceca. On opening the ceca, it was
 Systemic histomoniasis in a peafowl 327

Fig. 2. Histopathologic findings associated with H. meleagridis in a peafowl. Observe the ‘‘starry-sky’’ appearance due to intralesional
accumulations of histomonad organisms in the (A) liver, (B) cecum, and (D) spleen. (C) There are numerous intralesional organisms consistent with
H. meleagridis at the mucosa of the small intestine; these organisms are highlighted in the insert. A and B, periodic acid–Schiff stain; C and D;
hematoxylin and eosin stain. Bar, A, B, and C, 50 lm; D, 20 lm; insert, 10 lm.

observed that the walls were several thickened with accumulations of
caseosa material adhered to the serosal surfaces of the organ resulting
in diphtheric typhlitis (Fig. 1B).
Significant histopathologic alterations were observed in the liver,
ceca, small intestine, kidney, and spleen. Histopathologic evaluation
of the liver revealed severe, multifocal to coalescing necrotizing
hepatitis. These necrotic areas (lytic or caseous) contained
accumulations of normal and degenerated histiocytes and hetero-
phils admixed with tissue debris associated with intersessional
intracytoplasmic trophozoites consistent with H. meleagridis. These
trophozoites were non-flagellated, amoeboid-like, and irregularly
shaped, with an unstained halo around a centrally located nucleus,
and varied between 2.3 lm and 8.2 lm in diameter. In some areas,
trophozoites contained eccentric fusiform nuclei with intracytoplas-
mic basophilic debris. In addition, trophozoites were also located
distant from the necrotic areas within normally looking hepatic
parenchyma and imparted a ‘‘starry-sky’’ appearance to the organ
(Fig. 2A). Histopathology of the ceca revealed severe, acute,
transmural necrotizing typhlitis associated with intralesional and
intravascular trophozoites with similar morphology as observed in
the liver. The ‘‘starry-sky’’ appearance was also observed within the
cecal mucosa (Fig. 2B). In addition, there were severe transmural
necrotizing enteritis (Fig. 2C), discrete multifocal necrotizing
nephritis, and splenitis associated with similar intralesional histo-
monad organisms. The ‘‘starry-sky’’ feature was also seen in the
spleen (Fig. 2D). Flagellated organisms consistent with H.
meleagridis were also observed admixed with inflammatory exudate
within submucosal vessels of the small intestine.
Internal parasites were not observed during necropsy, and
evidence of concomitant parasitism was not identified with routine
parasitological evaluations.
Molecular characterization of H. meleagridis. PCR was used to
amplify the 350bp fragment of the 5.8S rRNA gene and the adjacent
ITS1 and ITS2 regions from all tissue fragments evaluated.
However, good quality sequences were only obtained from the
ceca. The nucleotide sequence of the isolate (H. meleagridis UEL-1)
obtained from this study is deposited in GenBank (Accession #
KY426802). BLAST analyses revealed that the isolate identified
during this investigation had an identity of 99% with similar strains
of H. meleagridis (KJ863549.1, and KJ863552.1) deposited in
GenBank.
The phylogenetic analysis revealed two distinct branches when the
selected trichomonad organisms were compared: one consisting of
isolates of H. meleagridis, T. gallinarum, and Trichomonas spp., and
the other formed by isolates of T. foetus and D. fragilis. However,
both branches were distinct from T. gondii, used as the outgroup.
Nevertheless, within the branch that contained strains of H.
meleagridis and T. gallinarum, the isolate derived from this study
328 M. M. Z. Michelazzo
Fig. 3. Molecular phylogenetic analysis by the maximum likelihood method based on the 5.8S rRNA gene and the ITS regions of selected
flagellated organisms; evolutionary analyses were conducted in MEGA7. The GenBank Accession numbers of the organisms used are given; the strain
identified during this investigation is highlighted (black dot). Toxoplasma gondii was used as the outgroup to provide stability to the tree.
clustered with other strains of H. meleagridis and were distinct from
those of T. gallinarum and Trichomonas spp. (Fig. 3).
DISCUSSION
The pathological findings observed in this peafowl are consistent
with previous descriptions of H. meleagridis in domestic chickens
and turkeys (14,16,25,29). However, a conformation of histomo-
niasis in multiple tissues of this bird was achieved with the PCR
assay that targeted the 5.8S rRNA gene and flanking ITS regions of
flagellated trichomonad; similar findings have been described
(8,9,20). Consequently, these results confirmed the participation
of H. meleagridis in the development of the lesions observed in
multiple organs of this peafowl and reinforce the findings of a
previous report that was based only on pathological findings (26). As
far as the authors are aware, this report might represent one of the
few complete identifications of spontaneous histomoniasis in a
peafowl due to pathological and molecular characteristics. Three
previous descriptions were located: one experimental induced
infectious study that confirmed the disease based on pathological
findings (21), a five-year epidemiological report that identified H.
meleagridis DNA in two peacocks (13), and a retrospective study
that identified H. meleagridis DNA in five peacocks (4). Addition-
ally, one of the peacocks from the retrospective study contained
DNA of H. meleagridis and T. gallinarum resulting in a co-infection
(4). Nevertheless, the results herein described represent the few
published data (1,2,28) of this important disease of birds from
Brazil. Collectively, these findings might suggest that the occurrence
of histomoniasis in the peacock is reduced. Alternatively, more
peacocks must be investigated to understand the dynamics of
histomoniasis in this species.
The finding of histomonads in the liver, ceca, kidney, spleen, and
small intestine of this peafowl resulting in systemic histomoniasis
(SH) is significant, because in most cases the disease is restricted to
the liver and ceca of affected birds (7,16,24,25). Nevertheless, SH
was described in outbreaks of turkeys with typical histopathologic
lesions occurring in the liver, ceca, spleen, lungs, kidneys, and the
pancreas (18,27). In addition, there are several descriptions of the
concurrent dissemination of H. meleagridis to the bursa of Fabricius
in chickens (6,18,27). Moreover, similar findings of SH were
described in experimentally infected turkeys and chickens with
additional lesions occurring in the small intestine (10,15), spleen
(7,15), and bursa of Fabricius (15). However, we did not locate
descriptions of SH in birds that are non-chicken and non-turkeys;
therefore, this report might represent one of the few documented
cases of SH in non-commercial birds.
Although the exact pathogenesis associated with SH is not fully
elucidated (27), vascular dissemination has been incriminated (5) as
the most likely form of spread to other tissues, because the lymphatic
system of birds is not well developed (27). Vascular dissemination in
SH seems to be related to the unique integrated portal system of
birds that consists of the coccygeo-mesenteric, common mesenteric,
pancreatico-duodenal, and splenic veins, all of which meet in the
hepatic parenchyma (24). These anatomical features of birds will
then support the theory that the distribution of H. meleagridis in SH
probably occurs by infected macrophages via the vascular system to
multiple organs (27) and would explain the occurrence of flagellated
histomonad in the submucosal vessels of the small intestine and ceca
of this peafowl and in other organs.
The polygenetic analyses clearly demonstrated that the strain of
H. meleagridis identified in this peafowl was more phylogenetically
related to other isolates of this flagellated trichomonad and were
different from those of T. gallinarum, T. foetus, and D. fragilis.
 Systemic histomoniasis in a peafowl
These findings are in accordance with previous studies (8,9,20),
confirming that this molecular approach is efficient to differentiate
between species of flagellate trichomonad organisms.
In conclusion, intralesional and intravascular organisms consistent
with H. meleagridis were observed in multiple organs of a peafowl
with gross demonstration of histomoniasis in the liver and ceca.
Molecular diagnosis confirmed the pathological findings, adds to the
identification of this flagellated histomonad in this bird species, and
provides additional evidence of the occurrence of histomoniasis in
commercial and non-commercial birds from Brazil.
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ACKNOWLEDGMENT
A. A. Alfieri, J. L. Garcia and S. A. Headley are recipients of the
National Council for Scientific and Technological Development
(CNPq; Brazil) fellowships and grants.