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

One captive musophagid bird at a zoological garden in Japan showed clinical symptoms and was found to be infected with avian haemosporidia. We subsequently collected blood from all musophagid birds kept in the garden and examined for avian haemosporidia using both microscopic and molecular examination. Only Haemoproteus gametocytes were observed in the blood of two Guinea turaco (Tauraco persa). Three genetic lineages of Haemoproteus were identified from three Guinea turacos and one genetic lineage of Leucocytozoon was identified from a grey plantain-eater (Crinifer piscator). Detected Haemoproteus lineages were all identical and completely different from those previously reported in Japan, suggesting that these birds were infected in their original habitat. This is the first record of Haemoproteus infection in Guinea turacos.

ABSTRACT. One captive musophagid bird at a zoological garden in Japan showed clinical symptoms and was found to be infected with avian haemosporidia. We subsequently collected blood from all musophagid birds kept in the garden and examined for avian haemosporidia using both microscopic and molecular examination. Only Haemoproteus gametocytes were observed in the blood of two Guinea turaco (Tauraco persa). Three genetic lineages of Haemoproteus were identified from three Guinea turacos and one genetic lineage of Leucocytozoon was identified from a grey plantain-eater (Crinifer piscator). Detected Haemoproteus lineages were all identical and completely different from those previously reported in Japan, suggesting that these birds were infected in their original habitat. This is the first record of Haemoproteus infection in Guinea turacos.

KEY WORDS:
Haemoproteus, Japan, Leucocytozoon, musophagid bird, zoological garden Avian haemosporidia, which includes the genera Plasmodium, Haemoproteus, and Leucocytozoon, are common avian blood protozoa that are found in numerous bird species worldwide [14]. Among them, avian Plasmodium causes so-called "avian malaria" in host birds; it is a highly pathogenic and lethal infectious disease, especially for several reported naïve bird species [1,14]. Penguins are typical those naïve species for avian malaria, showing critical symptoms as observed among captive penguins at zoological gardens and aquariums [2,5,7,15]. Moreover, Haemoproteus and Leucocytozoon infection also seriously affects captive birds [4,6,8,12]. Because many endangered species are kept in zoological gardens and aquariums, it is necessary to monitor the prevalence of those protozoans to promote proper care of these birds and improve their conservation efforts. Some infection cases of those avian haemosporidia were reported among wild and captive birds in Japan [10,11]. For example, one captive white eared-pheasant (Crossoptilon crossoptilon) in a zoological garden of Japan were found to be infected with avian malaria parasite, P. juxtanucleare with clinical observation of lethargy and weakness [11]. However, avian haemosporidia prevalence in captive wild birds at zoological gardens and aquariums in Japan has not sufficiently been demonstrated.
At Kobe Animal Kingdom, a zoological garden located in the west part of Japan, Haemoproteus infection was found in a captive Guinea turaco (Tauraco persa) with clinical symptoms in December 2015. This zoological garden also kept other musophagid birds at that time, but avian haemosporidia prevalence in the zoo was unknown. Therefore, in this study, we examined the blood of the infection-positive bird and other captive musophagid birds to determine their infection status.
First, protozoa were detected in the blood of one individual (No. 1, Table 1) that was kept at Kobe Animal Kingdom in Kobe Prefecture, Japan in December 2015. Then, blood samples of captive musophagid birds were obtained from the wing veins of six individuals, which included one male and three female Guinea turaco (Tauraco persa) in May 2016, one female grey plantain-eater (Crinifer piscator), and one female violet turaco (Musophaga violacea) in June 2016. Those blood collections were implemented as usual health check procedure for kept birds in this zoological garden. Collected blood was used for both microscopic and molecular examination. Blood smears were stained with Diff-Quick solution for morphological observation. DNA was extracted from blood samples and used for nested PCR to amplify haemosporidia mitochondrial DNA, and amplified DNA were sequenced as described previously [9]. Obtained nucleotide sequences were aligned by Clustal W program and compared at 479 bp with sequences in the GenBank database (https://www.ncbi.nlm.nih.gov/genbank/) and the MalAvi database [3] using the Basic Local Alignment Search Tool (BLAST, http://www.ncbi.nlm.nih.gov/BLAST/) to construct molecular phylogenetic trees by MEGA6 software.
Gametocytes that were morphologically similar to Haemoproteus were observed from blood smears of two Guinea turacos ( Fig. 1) but could not be identified to species (Table 1, Fig. 2). One Guinea turaco that was previously infected with Haemoproteus was still positive for Haemoproteus (No. 1, Table 1). No Plasmodium or Leucocytozoon gametocytes were found in any of the blood smears of the studied birds. Four DNA sequences were obtained from four birds, which included three identical Haemoproteus lineages from three Guinea turacos and one Leucocytozoon lineage from a western plantain-eater (Table 1). Those Haemoproteus and Leucocytozoon DNA sequences were deposited in GenBank and assigned the accession numbers LC271257 and LC271258, respectively. Plasmodium DNA was not amplified from any of the studied samples ( Table 1). Two phylogenetic trees were obtained for Haemoproteus and Leucocytozoon (Figs. 2 and 3, respectively).
In this study, we found that Haemoproteus gametocytes from two captive Guinea turacos were identical to the Haemoproteus genetic lineages amplified from three Guinea turacos. One Haemoproteus-positive Guinea turaco showed clinical symptoms including anorexia or diarrhea, which indicated possible pathogenicity in this bird species caused by infection. These captive musophagid birds in this zoological garden were kept with other bird species such as Ramphastidae in the semi free-ranging area and visitors were allowed to feed to these captive birds directly. These rearing environment might be possible stress factors to those captive birds occasionally, inducing some symptoms for infected individuals. The detected Haemoproteus lineages from Guinea turacos were not found in other birds in Japan, and these captive Guinea turacos were introduced to the studied zoological garden in 2006; therefore, we suggest that these protozoa-positive birds were infected in their region of origin. However, some lethal cases were reported when non-adapted bird species were infected with Haemoproteus spp. [4,8]. Consequently, it is also necessary to identify and estimate the prevalence of avian Haemoproteus in the native habitats of Guinea turacos.
Haemoproteus gametocyte detection indicated that the protozoans multiplied in at least two Guinea turacos (Nos. 1 and 2), and those birds may be reservoirs of Haemoproteus for other captive individuals during the blood-sucking season of vectors, such as biting midges or louse flies [14]. Individual No. 1 was likely infected with Haemoproteus from at least December 2015 to May 2016, because it first showed clinical symptoms of Haemoproteus infection in December 2015 with observation of gametocytes of Haemoproteus in the blood film; therefore, continuous examination of blood with special attention to body condition is needed for this and other individuals to improve treatment strategies for infected individuals and conservation of affected species. Some medical treatments can be applied to those infected birds, i.e., symptomatic treatments at first, and then usage of anti-protozoal drugs such as chloroquine and primaquine after detection of haemosporidia.
Contrary to the observed Haemoproteus infection prevalence, we only detected Leucocytozoon DNA from the western plantaineater, but we did not detect any gametocytes. The detected lineage was genetically similar to those from free-ranging raptors worldwide, including in Japan (Fig. 2). However, the reservoir birds, areas of infection origin, and pathogenicity to host birds are unknown. Because raptors are also kept at Kobe Animal Kingdom and black flies (Simuliidae), which are vectors of Leucocytozoon, are distributed in Japan [13], it is necessary to determine if those captive raptors can be reservoirs. Because the prevalence of haemosporidia in arthropod vectors and wild bird hosts both inside and outside of the studied zoological garden have not been examined, future research should demonstrate if there is transmission and consider adequate strategies to prevent protozoan infection.   3. Phylogenetic status of a detected lineage from captive Western plantain-eater (Crinifer piscator). Detected lineage was classified as genus Leucocytozoon but no parasites were found in the blood film.