Molecular identification of Cryptosporidium isolates from pet birds in [PDF]

difficult to discern their oocysts accurately by morphology among Cryptosporidium species and genotypes. In. Japan, 2 sp

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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

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Niichiro ABE, Ikuko MAKINO, Atsushi KOJIMA

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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

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