Note
Molecular identification of Cryptosporidium isolates from pet birds in Japan Niichiro ABE 1, Ikuko MAKINO 2, Atsushi KOJIMA 3 1
Department of Microbiology, Osaka City Institute of Public Health and Environmental Sciences 2 3
Fujisawa Avian Clinic, Fujisawa, Kanagawa
Little Animal and Bird Clinic Little Bird, Setagaya-ku, Tokyo
ABSTRACT Cryptosporidium spp. are important pathogens for humans and animals. Cases of infection by C. parvum, C. hominis, C. meleagridis, C. andersoni, and C. muris with zoonotic potential have also been reported in domestic and wild birds. Recent studies have revealed the presence of at least 13 host-adapted genotypes in birds. It is generally difficult to discern their oocysts accurately by morphology among Cryptosporidium species and genotypes. In Japan, 2 species(C. baileyi and C. meleagridis)and 2 genotypes(avian genotypes Ⅲ and Ⅴ)have been identified molecularly in pet(cockatiels and peach-faced lovebirds)and domestic(chickens)birds, but the presence of other species and/or genotypes in birds remains unclear. In this study, we attempted to identify 7 isolates from 3 cockatiels, 1 budgerigar, 1 masked lovebird, 1 Pacific parrotlet, and 1 Java sparrow, which were raised by individual owners, using sequence analysis of Cryptosporidium actin locus. Analysis identified avian genotype V in cockatiels and a budgerigar, avian genotype Ⅲ in a masked lovebird, C. galli in a Pacific parrotlet, and C. baileyi in a Java sparrow. This report is the first of a study identifying the presence of avian genotype Ⅴ and C. baileyi in budgerigar and Java sparrow in Japan, respectively. This study also demonstrated Pacific parrotlet(Forpus coelestis)as a new host record of C. galli. Key words:Cryptosporidium, avian genotype Ⅴ, avian genotype Ⅲ, C. baileyi, C. galli.
Seven species of Cryptosporidium, an important
renal or cloacal illness[7, 13, 17].It is generally difficult
pathogen in humans and animals, have been reported
to discriminate accurately among Cryptosporidium
in domestic birds: C. meleagridis, C. baileyi, C. galli,
species and genotypes using light microscopy because
C. parvum, C. hominis, C. muris, and C. andersoni
of the morphological similarity of their oocysts. The
[17].The five species aside from C. baileyi and C. galli
host specificity of Cryptosporidium genotypes found
have also been recognized as zoonotic Cryptosporidium
in birds remains unclear. Moreover, it is important
[21]. In addition to these valid species, the following
to identify the avian isolates accurately, particularly
13 genotypes are known: avian genotypes Ⅰ-Ⅵ
pet birds that are in close contact with humans in
(identified in many avian species), goose genotypes
everyday life and which could be a source of human
(Branta canadensis)Ⅰ-Ⅴ, black duck(Anas rubripes)
infection. Cryptosporidium parasite was first isolated
genotype, and Eurasian woodcock(Scolopax rusticola)
from chickens in domestic birds[10].Later, this isolate
genotype[6, 17]. Among these species and genotypes,
was identified as C. baileyi using multilocus sequence
C. meleagridis, C. baileyi, C. galli, avian genotype
analysis[11]. Cryptosporidium parasites were also
Ⅲ , and possibly avian genotype V are recognized as
found histopathologically in Japanese quail[14] , but
important pathogens associated with mortality, weight
they have not been identified. Recently, several isolates
loss, diarrhea, respiratory illness, chronic vomiting, and
from pet birds in Japan were identified molecularly.
― 19 ― Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Molecular identification of Cryptosporidium isolates from pet birds in Japan
Infection of C. baileyi, C. meleagridis, avian genotype
using a QIAamp DNA Stool Mini Kit and DNeasy
Ⅲ or Ⅴ in cockatiels and peach-faced lovebirds has
Blood and Tissue Kit(Qiagen GmbH, Hilden, Germany) ,
been reported[1-3, 13]. This study identified seven
respectively, according to the manufacturer’s instructions.
isolates from five species of pet birds using sequence
The Cryptosporidium actin gene fragment(approximately
analysis of Cryptosporidium actin locus.
1100 bp) was amplified following the nested PCR
Fecal samples from seven pet birds(three cockatiels,
protocol[23]. PCR amplification was performed in a
one budgerigar, one masked lovebird, one Pacific
volume of 50μl containing 1× PCR buffer, 2 mM MgCl2,
parrotlet, and one Java sparrow)were collected during
250μM of each dNTP, 0.5μM of each primer, 1.25
2012–2014 at Little Animal and Bird Clinic Little Bird
units of TaKaRa Ex Taq Hot Start Version(Takara
and Fujisawa Avian Clinic(Table 1). They were found
Shuzo Co. Ltd., Otsu, Shiga, Japan), and 5μl of DNA
to be positive for Cryptosporidium infection by light
sample. Reactions were performed using a GeneAmp
microscopy using sucrose centrifugal flotation. All
PCR System 9700 thermocycler(Applied Biosystems,
birds had been kept at separate households. The two
CA, U.S.A.).The PCR products were purified using the
cockatiels(original hosts of isolate codes 0306-01 and
QIAquick Gel Extraction or QIAquick PCR Purification
120420-1)died during treatment. One cockatiel(0306-
Kit(QIAGEN GmbH, Hilden, Mettmann, Germany),
01)was provided for autopsy. Small pieces of each
and were sequenced in both directions on an automated
internal organ(crop, proventriculus, small intestine,
sequencer(ABI 3130; Applied Biosystems, Foster City,
cloaca, trachea, kidney, liver)collected using disposable
Carlsbad, California, U.S.A.) . Sequence chromatograms
tweezers were put into individual 1.5-ml tubes
from each strand were inspected using the SEQUENCHER
containing 70% ethanol. These samples were provided
Version 4.1(Gene Codes Corp., Ann Arbor, MI, U.S.A.).
for identification of the location of Cryptosporidium
Nucleotide similarity searching of the obtained partial
found in a fecal sample of this cockatiel by PCR sequence
gene sequences was performed using the FASTA
analysis, as reported previously [3]. DNAs were
program(EMBL; http://www.ebi.ac.uk/ Tools/fasta33/
extracted and purified from fecal and tissue samples
nucleotide.html) .
Table 1. Cryptosporidium isolates from pet birds identified in the present study Isolate code 0306-01
0016-01
120420-1
3503-58
733-3
0018-03
0224-01 a
Host detailsa Nymphicus hollandicus; cockatiel; 2 months; emaciation, diarrhea and death Nymphicus hollandicus; cockatiel; 3 months; dispepsia Nymphicus hollandicus; cockatiel; 2 months; emaciation, diarrhea and death Melopsittacus undulatus; budgerigar; 2 months; diarrhea and vomiting Agapornis personata; masked lovebird; 6 years; no symptom Forpus coelestis; Pacific parrotlet; >2 years; diabetes and proventriculus distension Padda oryzivora; Java sparrow; 2 months; diarrehea
Identification at actin locus avian genotype Ⅴ
avian genotype Ⅴ
avian genotype Ⅴ
avian genotype Ⅴ
avian genotype Ⅲ
100% identity(978/978 bp)to those from avian genotype Ⅴ(AB471660, AB471661, JQ320301) 100% identity(978/978 bp)to those from avian genotype Ⅴ(AB471660, AB471661, JQ320301) 100% identity(978/978 bp)to those from avian genotype Ⅴ(AB471660, AB471661, JQ320301) 100% identity(978/978 bp)to those from avian genotype Ⅴ(AB471660, AB471661, JQ320301) 100% identity(1000/1000 bp)to those from avian genotype Ⅲ(AB471655-AB471659) 100%(987/987 bp, 921/921 bp)or 99.9%(961/962
C. galli
bp, 927/928 bp)identity to those from C. galli (AY163901, EU53265-EU543267, EU543265)
C. baileyi
Descibed in order of scientific name, common name, age of the bird, and clinical symptoms.
― 20 ― Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Most highest nucleotide sequence identity
100% identity(990/990 bp)to those from C. baileyi (AF382346, EU741840-EU741852)
Niichiro ABE, Ikuko MAKINO, Atsushi KOJIMA
Partial actin gene nucleotide sequences(978–1000
resulted from an infection of avian genotype V because
bp) were obtained from all isolates examined in
we were unable to examine other pathogens such as
this study. Sequences from four isolates from three
Candida spp., Escherichia coli, Salmonella spp., and
cockatiels and one budgerigar were identical among
various viruses that are recognized as concurrent
those isolates and were also identical to those of
pathogens of intestinal cryptosporidiosis [5, 12].
Cryptosporidium avian genotype V(Table 1).The PCR
The site of infection in avian hosts of this genotype
was positive in six samples(crop, proventriculus, small
has been found by scanning electron microscopical
intestine, cloaca, trachea, kidney)except for the liver.
or histopathological examination to be the ileum,
The sequences from those amplicons were mutually
cecum, ureter, and cloaca[7, 9]. In the present study,
identical and also identical to that of avian genotype
Cryptosporidium DNA was detected in the trachea,
V(AB471660) . The sequence of the isolate from a
crop, proventriculus, small intestine, cloaca, and kidney
masked lovebird was identical to that of avian genotype
collected from a dead cockatiel(0306-01). Recently, we
Ⅲ. The sequences of the two isolates from a Pacific
also identified the trachea and cloaca as the possible
parrotlet and a Java sparrow were also identical to
location of this genotype in avian hosts by PCR for
those of C. galli and C. baileyi, respectively(Table 1).
actin and 18S ribosomal DNA loci using the tissue
Results demonstrated that intragenotype or intraspecies
DNA samples from a severely emaciated dead cockatiel
variations are low or absent at the actin locus in
[3]. Considering both the present and the previous[3]
Cryptosporidium[23]. Therefore, we identified the
PCR data, we strongly presume the possible location
present isolates as Cryptosporidium avian genotypes Ⅲ,
of this genotype as the trachea, which is true also for
V, C. galli, and C. baileyi.
C. baileyi. Further histological examination of trachea
Cryptosporidium avian genotype V, recently proposed
in birds infected with avian genotype V must be
as a new Cryptosporidium species(C. avium) [9], was
conducted to examine this possibility.
first found in fecal samples from two cockatiels caged
In Japan, C. baileyi has been identified only in
at the same pet shop in Japan[2]. Subsequently, it has
domestic chickens and pet cockatiels [1, 11]. In
been identified in cockatiels and a budgerigar in China
addition, the avian genotype V has been identified only
[19, 24]and in a blue-fronted parrot in Brazil[16], in
in pet cockatiels[2, 3]. Therefore this report is the
a pet Major Mitchell’s cockatoo in U.S.A.[7]. Recently,
first of a study identifying the presence of C. baileyi
we also identified this genotype in a cockatiel[3]. In
and avian genotype V in Java sparrow and budgerigar,
the present study, the infection with this genotype was
respectively in Japan. Reports of the literature show
confirmed in the three cockatiels. Although we were
that C. galli has been identified in at least 32 avian
unable to perform a follow-up survey of the origin, avian
species[4, 6, 8, 15, 16, 18-20, 22], but it has not been
genotype V might be widely distributed throughout
found in the Pacific parrotlet (Forpus coelestis).
the cockatiel population of Japan. The pathogenicity
Therefore, the present report also identified a new host
of this genotype in avian hosts has remained unclear
of C. galli.
because of a lack of clinical reports associated with
About 5,000 psittaciform birds have been introduced
this genotype infection[2, 9, 16, 19, 24]. All birds
annually into Japan during the most recent five years
infected with this genotype examined in the present
(2011–2015). These birds are mainly imported from
study showed mainly digestive symptoms(Table 1). A
Belgium, the United States of America, the Philippines,
recent report has also described a fatal case with renal
and Singapore(This information is available at the
and cloacal cryptosporidiosis caused by this genotype
following URLS, but all descriptions at these sites are in
in a Major Mitchell’s cockatoo(Cacatua leadbeteri) [7],
Japanese: http://www.mhlw.go.jp/stf/ seisakunitsuite/
suggesting possible pathogenicity of avian genotype
bunya/0000069864.html). In addition, a few birds are
V. However, it was not possible to ascertain whether
bred in households or breeder farms in Japan. Because
the digestive symptoms found in the present study
no survey of Cryptosporidium infection in birds has
― 21 ― Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Molecular identification of Cryptosporidium isolates from pet birds in Japan
been performed in exporting countries and/or in Japan,
Mitchell’s cockatoo(Lophochroa leadbeateri). J.
the origins of Cryptosporidium- parasite-infected pet
Zoo. Wildl. Med. 46:934-937.
birds in Japan remain unclear. Global epizootiological
8. da Silva, D. C., Homem, C. G., Nakamura, A. A.,
studies of Cryptosporidium infection in pet birds must
Teixeira, W. F. P., Perri, S. H. V. and Meireles, M.
be undertaken to control Cryptosporidium infection
V. 2010. Physical, epidemiological, and molecular
among pet bird populations, and to clarify the infection
evaluation of infection by Cryptosporidium galli in
sources and routes of transmission.
Passeriformes. Parasitol. Res. 107:271-277. 9. Holubová, N., Sak, B., Horčičková, M., Hlásková, L., Kvĕtoňová, D., Menchaca, S., McEvoy, J. and Kváč
REFERENCES
M. 2016. Cryptosporidium avium n. sp.(Apicomplexa:
1. Abe, N. and Iseki, M. 2004. Identification of
Cryptosporidiidae)in birds. Parasitol. Res. 115:
Cryptosporidium isolates from cockatiels by direct sequencing of the PCR-amplified small subunit
2243-2251. 10. Itakura, C., Goryo, M. and Uemura, T. 1984.
ribosomal RNA gene. Parasitol. Res. 92:523-526.
Cryptosporidial infection in chickens. Avian Pathol.
2. Abe, N. and Makino, I. 2010. Multilocus genotypic analysis of Cryptosporidium isolates from cockatiels,
13:487-499. 11. Kimura, A., Suzuki, Y. and Matsui, T. 2004.
Japan. Parasitol. Res. 106:1491-1497.
Identification of the Cryptosporidium isolates from
3. Abe, N., Matsuo, K. and Makino, I. 2015. Ascaridia
chickens in Japan by sequence analyses. J. Vet.
nymphii n. sp.(Nematoda: Ascaridida)from the
Med. Sci. 66:879-881.
alimentary tract of a severely emaciated dead
12. Lindsay, D. S., Blagburn, B. L., Hoerr, F. J. and
cockatiel Nymphicus hollandicus. Parasitol. Res.
Smith, P. C. 1991. Cryptosporidiosis in zoo and pet
114:4281-4288.
birds. J. Protozool. 38:180S-181S.
4. Antunes, R. G., Simoes, D. C., Nakamura, A. A.
13. Makino, I., Abe, N. and Reavill, D. R. 2010.
and Meireles, M. V. 2008. Natural infection with
Cryptosporidium avian genotype Ⅲ as a possible
Cryptosporidium galli in canaries(Serinus canaria) ,
causative agent of chronic vomiting in peach-faced
in a cockatiel(Nymphicus hollandicus) , and in lesser
lovebirds(Agapornis roseicollis). Avian Dis. 54:
seed-finches(Oryzoborus angolensis)from Brazil.
1102-1107.
Avian Dis. 52:702-705.
14. Murakami, S., Miyama, M., Ogawa, A., Shimada, J.
5. Blagburn, B. L., Lindsay, D. S., Hoerr, F. J., Atlas, A.
and Nakane, T. 2002. Occurrence of conjunctivitis,
L. and Toivio-Kinnucan, M. 1990. Cryptosporidium
sinusitis and upper region tracheitis in Japanese
sp. infection in the proventriculus of an Australian
quail(Coturnix coturnix japonica), possibly caused
diamond firetail finch (Staganoplura bella:
by Mycoplasma gallisepticum accompanied by
Passeriformes, Estrilgidae). Avian Dis. 34:1027-
Cryptosporidium sp. infection. Avian Pathol. 31:
1030.
363-370.
6. Chelladurai, J. J., Clark, M. E., Kváč, M., Holubová,
15. Nakamura, A. A., Simões, D. C., Antunes, R. G., da
N., Khan, E., Stenger, B. L., Giddings, C. W. and
Silva, D. C. and Meireles, M. V. 2009. Molecular
McEvoy, J. 2016. Cryptosporidium galli and novel
characterization of Cryptosporidium spp. from fecal
Cryptosporidium avian genotype VI in North
samples of birds kept in captivity in Brazil. Vet.
American red-winged blackbirds (Agelaius
Parasitol. 166:47-51.
phoeniceus) . Parasitol. Res. 115:1901-1906.
16. Nakamura, A. A., Homem, C. G., da Silva, A. M.
7. Curtiss, J. B., Leone, A. M., Wellehan, J. F. X.,
J. and Meireles, M. V. 2014. Diagnosis of gastric
Emerson, J. A., Howerth, E. W. and Farina, L.
cryptosporidiosis in birds using a duplex real-time
L. 2015. Renal and cloacal cryptosporidiosis
PCR assay. Vet. Parasitol. 205:7-13.
(Cryptosporidium avian genotype V)in a major
17. Nakamura, A. A. and Meireles, M. V. 2015.
― 22 ― Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016
Niichiro ABE, Ikuko MAKINO, Atsushi KOJIMA
Cryptosporidium infections in birds - a review.
Guimarães, M. B., Souza, S. O., Allegretti, L.,
Braz. J. Vet. Parasitol. 24:253-267.
Sinhorini, J. A., Duarte, V. V. and Soares, R. M.
18. Ng, J., Pavlasek, I. and Ryan, U. 2006. Identification
2011. Genotyping of Cryptosporidium spp. from
of novel Cryptosporidium genotypes from avian
free-living wild birds from Brazil. Vet. Parasitol.
hosts. Appl. Environ. Microbiol. 72:7548-7553.
175:27-32.
19. Qi, M., Wang, R., Ning, C., Li, X., Zhang, L., Jian,
23. Sulaiman, I. M., Lal, A. A. and Xiao, L. 2002.
F., Sun, Y. and Xiao, L. 2011. Cryptosporidium spp.
Molecular phylogeny and evolutionary relationships
in pet birds: genetic diversity and potential public
of Cryptosporidium parasites at the actin locus. J.
health significance. Exp. Parasitol. 128:336-340.
Parasitol. 88:388-394.
20. Ryan, U. M., Xiao, L., Read, C., Sulaiman, I. M.,
24. Zhang, X. X., Zhang, N. Z., Zhao, G. H., Zhao, Q.
Monis, P., Lal, A. A., Fayer, R. and Pavlasek, I.
and Zhu, X. Q. 2015. Prevalence and genotyping of
2003. A redescription of Cryptosporidium galli
Cryptosporidium infection in pet parrots in North
Pavlasek, 1999(Apicomplexa: Cryptosporidiidae)
China. BioMed. Res. Int. 2015:1-6.
from birds. J. Parasitol. 89:809-813. 21. Ryan, U., Fayer, R. and Xiao, L. 2014. Cryptosporidium species in humans and animals: current understanding and research needs. Parasitology 141:1667-1685. 22. Sevá, A. P., Funada, M. R., Richtzenhain, L.,
Correspondence:Niichiro ABE, Osaka City Institute of Public Health and Environmental Sciences, Tennoji–ku, Osaka 5430026, Japan. E-mail:
[email protected]
遺伝子解析による小鳥由来 Cryptosporidium 株の同定 阿部 仁一郎 1、牧野 幾子 2、小嶋 篤史 3 1 2
大阪市立環境科学研究所微生物保健グループ、
ふじさわアビアンクリニック、3 鳥と小動物の病院リトル・バード
要 約 クリプトスポリジウムはヒトと動物に寄生する原虫で、鳥類では人獣共通寄生性 5 種と少なくとも 13 の遺伝子型 の報告がある。日本では、C. baileyi(オカメインコ、鶏)、C. meleagridis(オカメインコ)、avian genotype Ⅲ(コ ザクラインコ)、Ⅴ(オカメインコ)が確認されていたが他種寄生状況は不明であった。今回、本原虫の actin 遺伝子 領域における塩基配列の相同性検索により鳥類由来 7 株の同定を試みたところ、オカメインコ由来 3 株とセキセイイ ンコ由来 1 株を avian genotype Ⅴ、 キエリクロボタンインコ由来 1 株を avian genotype Ⅲ、 マメルリハ由来 1 株を C. galli、文鳥由来 1 株を C. baileyi と同定した。国内のセキセイインコと文鳥に avian genotype V と C. baileyi が各々 寄生していることを初めて確認し、マメルリハを C. galli の新宿主として報告した。 Key words:Cryptosporidium、avian genotype Ⅴ、avian genotype Ⅲ、C. baileyi、C. galli
― 23 ― Jpn. J. Vet. Parasitol. Vol. 15. No. 1 2016