Advance Publication

The Journal of Veterinary Medical Science

Accepted Date: 23 October 2019 J-STAGE Advance Published Date: 4 November 2019

©2019 The Japanese Society of Veterinary Science Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Wildlife Science

NOTE

Detection of avian haemosporidia from captive musophagid birds at a zoological garden in Japan

Masayoshi KAKOGAWA 1, 2), Ayana ONO 3), Mizue INUMARU 3), Yukita SATO 3) *,

Mitsuhiko ASAKAWA 2)

1) Kobe Animal Kingdom, Kobe, Hyogo 650-0047, Japan

2) Graduate School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu,

Hokkaido 069-8501, Japan

3) Laboratory of Biomedical Science, Department of Veterinary Medicine, College of

Bioresource Sciences, Nihon University, Fujisawa, Kanagawa 252-0880, Japan

*Corresponding author Prof. Dr. Yukita SATO E-mail: [email protected] Laboratory of Biomedical Science, Department of Veterinary Medicine, College of Bioresource Sciences, Nihon University, Fujisawa, Kanagawa 252-0880, Japan Phone: +81-466-84-3378; Fax: +81-466-84-3445

Running head: HAEMOSPORIDIA IN CAPTIVE MUSOPHAGID BIRD

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1 ABSTRACT

2

3 One captive musophagid bird at a zoological garden in Japan showed clinical

4 symptoms and was found to be infected with avian haemosporidia. We subsequently

5 collected blood from all musophagid birds kept in the garden and examined for avian

6 haemosporidia using both microscopic and molecular examination. Only Haemoproteus

7 gametocytes were observed in the blood of two Guinea turaco (Tauraco persa). Three

8 genetic lineages of Haemoproteus were identified from three Guinea turacos and one

9 genetic lineage of Leucocytozoon was identified from a grey plantain-eater (Crinifer

10 piscator). Detected Haemoproteus lineages were all identical and completely different

11 from those previously reported in Japan, suggesting that these birds were infected in

12 their original habitat. This is the first record of Haemoproteus infection in Guinea

13 turacos.

14

15

16

17 Key words: Haemoproteus, Japan, Leucocytozoon, musophagid bird, zoo

18

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19 Avian haemosporidia, which includes the genera Plasmodium, Haemoproteus,

20 and Leucocytozoon, are common avian blood protozoa that are found in numerous bird

21 species worldwide [14]. Among them, avian Plasmodium causes so-called “avian

22 malaria” in host birds; it is a highly pathogenic and lethal infectious disease, especially

23 for several reported naïve bird species [1, 14]. Penguins are typical those naïve species

24 for avian malaria, showing critical symptoms as observed among captive penguins at

25 zoological gardens and aquariums [2, 5, 7, 15]. Moreover, Haemoproteus and

26 Leucocytozoon infection also seriously affects captive birds [4, 6, 8, 12]. Because many

27 endangered species are kept in zoological gardens and aquariums, it is necessary to

28 monitor the prevalence of those protozoans to promote proper care of these birds and

29 improve their conservation efforts. Some infection cases of those avian haemosporidia

30 were reported among wild and captive birds in Japan [10, 11]. For example, one captive

31 white eared-pheasant (Crossoptilon crossoptilon) in a zoological garden of Japan were

32 found to be infected with avian malaria parasite, P. juxtanucleare with clinical

33 observation of lethargy and weakness [11]. However, avian haemosporidia prevalence in

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34 captive wild birds at zoological gardens and aquariums in Japan has not sufficiently

35 been demonstrated.

36 At Kobe Animal Kingdom, a zoological garden located in the west part of Japan,

37 Haemoproteus infection was found in a captive Guinea turaco (Tauraco persa) with

38 clinical symptoms in December 2015. This zoological garden also kept other

39 musophagid birds at that time, but avian haemosporidia prevalence in the zoo was

40 unknown. Therefore, in this study, we examined the blood of the infection-positive bird

41 and other captive musophagid birds to determine their infection status.

42 First, protozoa were detected in the blood of one individual (No. 1, Table 1) that

43 was kept at Kobe Animal Kingdom in Kobe Prefecture, Japan in December 2015. Then,

44 blood samples of captive musophagid birds were obtained from the wing veins of six

45 individuals, which included one male and three female Guinea turaco (Tauraco persa)

46 in May 2016, one female grey plantain-eater (Crinifer piscator), and one female violet

47 turaco (Musophaga violacea) in June 2016. Those blood collections were implemented

48 as usual health check procedure for kept birds in this zoological garden. Collected blood

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49 was used for both microscopic and molecular examination. Blood smears were stained

50 with Diff-Quick solution for morphological observation. DNA was extracted from blood

51 samples and used for nested PCR to amplify haemosporidia mitochondrial DNA, and

52 amplified DNA were sequenced as described previously [9]. Obtained nucleotide

53 sequences were aligned by Clustal W program and compared at 479 bp with sequences

54 in the GenBank database (https://www.ncbi.nlm.nih.gov/genbank/) and the MalAvi

55 database [3] using the Basic Local Alignment Search Tool (BLAST,

56 http://www.ncbi.nlm.nih.gov/BLAST/) to construct molecular phylogenetic trees by

57 MEGA6 software.

58 Gametocytes that were morphologically similar to Haemoproteus were observed

59 from blood smears of two Guinea turacos (Fig. 1) but could not be identified to species

60 (Table 1, Fig. 2). One Guinea turaco that was previously infected with Haemoproteus

61 was still positive for Haemoproteus (No. 1, Table 1). No Plasmodium or Leucocytozoon

62 gametocytes were found in any of the blood smears of the studied birds. Four DNA

63 sequences were obtained from four birds, which included three identical Haemoproteus

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64 lineages from three Guinea turacos and one Leucocytozoon lineage from a western

65 plantain-eater (Table 1). Those Haemoproteus and Leucocytozoon DNA sequences were

66 deposited in GenBank and assigned the accession numbers LC271257 and LC271258,

67 respectively. Plasmodium DNA was not amplified from any of the studied samples

68 (Table 1). Two phylogenetic trees were obtained for Haemoproteus and Leucocytozoon

69 (Figs 2 and 3, respectively).

70 In this study, we found that Haemoproteus gametocytes from two captive Guinea

71 turacos were identical to the Haemoproteus genetic lineages amplified from three

72 Guinea turacos. One Haemoproteus-positive Guinea turaco showed clinical symptoms

73 including anorexia or diarrhea, which indicated possible pathogenicity in this bird

74 species caused by infection. These captive musophagid birds in this zoological garden

75 were kept with other bird species such as Ramphastidae in the semi free-ranging area

76 and visitors were allowed to feed to these captive birds directly. These rearing

77 environment might be possible stress factors to those captive birds occasionally,

78 inducing some symptoms for infected individuals. The detected Haemoproteus lineages

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79 from Guinea turacos were not found in other birds in Japan, and these captive Guinea

80 turacos were introduced to the studied zoological garden in 2006; therefore, we suggest

81 that these protozoa-positive birds were infected in their region of origin. However, some

82 lethal cases were reported when non-adapted bird species were infected with

83 Haemoproteus spp. [4, 8]. Consequently, it is also necessary to identify and estimate the

84 prevalence of avian Haemoproteus in the native habitats of Guinea turacos.

85 Haemoproteus gametocyte detection indicated that the protozoans multiplied in at

86 least two Guinea turacos (Nos. 1 and 2), and those birds may be reservoirs of

87 Haemoproteus for other captive individuals during the blood-sucking season of vectors,

88 such as biting midges or louse flies [14]. Individual No. 1 was likely infected with

89 Haemoproteus from at least December 2015 to May 2016, because it first showed

90 clinical symptoms of Haemoproteus infection in December 2015 with observation of

91 gametocytes of Haemoproteus in the blood film; therefore, continuous examination of

92 blood with special attention to body condition is needed for this and other individuals to

93 improve treatment strategies for infected individuals and conservation of affected

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94 species. Some medical treatments can be applied to those infected birds, i.e.,

95 symptomatic treatments at first, and then usage of anti-protozoal drugs such as

96 chloroquine and primaquine after detection of haemosporidia.

97 Contrary to the observed Haemoproteus infection prevalence, we only detected

98 Leucocytozoon DNA from the western plantain-eater, but we did not detect any

99 gametocytes. The detected lineage was genetically similar to those from free-ranging

100 raptors worldwide, including in Japan (Fig. 2). However, the reservoir birds, areas of

101 infection origin, and pathogenicity to host birds are unknown. Because raptors are also

102 kept at Kobe Animal Kingdom and black flies (Simuliidae), which are vectors of

103 Leucocytozoon, are distributed in Japan [13], it is necessary to determine if those

104 captive raptors can be reservoirs. Because the prevalence of haemosporidia in arthropod

105 vectors and wild bird hosts both inside and outside of the studied zoological garden

106 have not been examined, future research should demonstrate if there is transmission and

107 consider adequate strategies to prevent protozoan infection.

108

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109 ACKNOWLEDGMENTS

110 This study was partially supported by the Japan Society for the Promotion of Science,

111 KAKENHI(26450484), the Strategic Research Base Development Program

112 “International Research on the Management of Zoonosis in Globalization and Training

113 for Young Researchers” from the MEXT of Japan and Nihon University Research

114 Grants.

115

116 REFERENCES

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137 8. Halpern, N., and Bennett, G. F. 1983. Haemoproteus and Leucocytozoon infections

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139 9. Hellgren, O., Waldenström, J., and Bensch, S. 2004. A new PCR assay for

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145 Matsumoto, R., Suda, A., and Ueda, M. 2007. Plasmodium (Bennettinia)

146 juxtanucleare infection in a captive white eared-pheasant (Crossoptilon

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148 12. Ortiz-Catedral, L., Brunton, D., Stidworthy, M., F., Elsheikha, H., M., Pennycott,

149 T., Schulze, C., Braun, M., Wink, M., Gerlach, H., Pendl, H., Gruber, A., D., Ewen,

150 J., Pérez-Tris, J., Valkiūnas, G., and Olias, P. 2019. Haemoproteus minutus is highly

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154 Leucocytozoon lovati from probable vectors, black flies (Simuliudae) collected in

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156 14. Valkiūnas, G. 2005. Avian malaria parasites and other haemosporidia. CRC Press,

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160 Figure legends

161

162 Figure 1. Gametocytes of Haemoproteus sp. found from two captive Guinea turacos

163 (Tauraco persa) as indicated by arrows (Left: No.1, Right No. 2). Bar=10 μm.

164

165 Figure 2. Phylogenetic status of detected lineages from 3 captive Guinea turacos

166 (Tauraco persa). All DNA sequences of detected lineages were identical each other and

167 classified as genus Haemoproteus.

168

169 Figure 3. Phylogenetic status of a detected lineage from captive Western plantain-eater

170 (Crinifer piscator). Detected lineage was classified as genus Leucocytozoon but no

171 parasites were found in the blood film.

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Table 1. Detection and identification of avian haemosporidia from captive musophagid birds.

DNA detection and identification Microscopic Individual birds idetification Plasmodium Haemoproteus Leucocytozoon Guinea turaco (Tauraco persa ) No. 1 - + - Haemoproteus

Guinea turaco (Tauraco persa ) No. 2 - + - Haemoproteus

Guinea turaco (Tauraco persa ) No. 3 - + - -

Guinea turaco (Tauraco persa ) No. 4 - - - -

Grey plantain-eater (Crinifer piscator ) - - + -

Violet turaco (Musophaga violacea ) - - - - Fig. 1 Fig. 2

Lineage from Guinea turaco (Tauraco persa) No.1 on present study 99 Lineage from Guinea turaco (Tauraco persa) No.2 on present study 85 Lineage from Guinea turaco (Tauraco persa) No.3 on present study

Haemoproteus sp. from captive Great Blue Turaco (Corythaeola cristata) in Singapore

Haemoproteus paranucleophilus

Haemoproteus sp. from Siberian blue robin (Luscinia cyane) in Japan

53 56 Haemoproteus balmorali

56 Haemoproteus sp. from Blue-and-White Flycatcher (Cyanoptila cyanomelana) in Japan

59 Haemoproteus sp. from Rufous-collared Sparrow (Zonotrichia capensis) in Peru

Haemoproteus sp. from Carmelite Sunbird (Chalcomitra fuliginosa) in Gabon 66 Haemoproteus micronuclearis

Haemoproteus sp. from Scarlet tanager (Piranga olivacea) in Columbia 73 83 Haemoproteus sp. from Galapagos Penguin (Spheniscus mendiculus) in Equator

Haemoproteus pallidus

Haemoproteus lanii

Haemoproteus syrnii

Leucocytozoon schoutedeni

0.02 Fig. 3

67 Leucocytozoon sp. from Red kite (Milvus milvus) in Spain

77 Leucocytozoon sp. from Steller's sea eagle (Haliaeetus pelagicus) in Japan

80 Lineage from Western plantain-eater (Crinifer piscator) on present study

Leucocytozoon sp. from Great egret (Ardea alba) in Japan

97 Leucocytozoon sp. from Japanese night heron (Gorsachius goisagi) in Japan

Leucocytozoon schoutedeni

99 Leucocytozoon fringillinarum

Leucocytozoon majoris

Leucocytozoon toddi

Heamoproteus columbae

0.02