Presence and diversity of mixed avian Plasmodium spp. infections in introduced birds whose distribution overlapped with threatened New Zealand endemic birds.

Aims: To determine the presence of infection and co-infection of Plasmodium lineages in introduced birds at translocation sites for the North Island saddleback (Philesturnus rufusater), to investigate their role as Plasmodium spp. reservoirs.Methods: Blood samples were collected from introduced bird species, with a special focus on blackbirds (Turdus merula) and song thrushes (Turdus philomelos), at six locations in the North Island of New Zealand that were the origin, or translocation sites, for North Island saddleback. Where available, blood smears were examined, and blood samples were tested using nested PCR with subsequent sequence analysis, for the presence of Plasmodium spp.Results: Of the 55 samples tested using PCR analysis, 39 (71%) were positive for Plasmodium spp., and 28/40 (62%) blood smears were positive for Plasmodium spp. Overall, 31 blood samples were from blackbirds with 28/31 (90%) samples positive for Plasmodium spp. Six distinct avian Plasmodium lineages were identified, including three cosmopolitan lineages; Plasmodium vaughani SYAT05 was detected in 16 samples, Plasmodium matutinum Linn1 in 10 samples and Plasmodium elongatum GRW6 in eight samples. Mixed infections with more than one lineage were detected in 12 samples. Samples from two Australian magpies (Gymnorhina tibicen) were positive for Plasmodium. sp. lineage MYNA02, previously not identified in New Zealand.Conclusions and clinical relevance: This is the first report from New Zealand in which specific Plasmodium spp. mixed infections have been found in introduced birds. Co-infections with several cosmopolitan Plasmodium lineages were identified, as well as the first report in New Zealand of an exotic avian Plasmodium sp. lineage, in Australian magpies. Whilst the role of introduced birds in maintaining and spreading pathogenic avian malaria in New Zealand is unclear, there is a potential infection risk to native birds, especially where distributions overlap.

Development of a rapid HRM qPCR for the diagnosis of the four most prevalent Plasmodium lineages in New Zealand.

Although wildlife rehabilitation and translocations are important tools in wildlife conservation in New Zealand, disease screening of birds has not been standardized. Additionally, the results of the screening programmes are often difficult to interpret due to missing disease data in resident or translocating avian populations. Molecular methods have become the most widespread method for diagnosing avian malaria (Plasmodium spp.) infections. However, these methods can be time-consuming, expensive and are less specific in diagnosing mixed infections. Thus, this study developed a new real-time PCR (qPCR) method that was able to detect and specifically identify infections of the three most common lineages of avian malaria in New Zealand (Plasmodium (Novyella) sp. SYAT05, Plasmodium elongatum GRW6 and Plasmodium spp. LINN1) as well as a less common, pathogenic Plasmodium relictum GRW4 lineage. The assay was also able to discern combinations of these parasites in the same sample and had a detection limit of five parasites per microlitre. Due to concerns relating to the presence of the potentially highly pathogenic P. relictum GRW4 lineage in avian populations, an additional confirmatory high resolution (HRM) qPCR was developed to distinguish between commonly identified P. elongatum GRW6 from P. relictum GRW4. The new qPCR assays were tested using tissue samples containing Plasmodium schizonts from three naturally infected dead birds resulting in the identified infection of P. elongatum GRW6. Thus, these rapid qPCR assays have shown to be cost-effective and rapid screening tools for the detection of Plasmodium infection in New Zealand native birds.

New insight into avian malaria vectors in New Zealand.

BACKGROUND: Mosquitoes (Culicidae) are vectors for most malaria parasites of the Plasmodium species and are required for Plasmodium spp. to complete their life cycle. Despite having 16 species of mosquitoes and the detection of many Plasmodium species in birds, little is known about the role of different mosquito species in the avian malaria life cycle in New Zealand. METHODS: In this study, we used nested polymerase chain reaction (PCR) and real-time PCR to determine Plasmodium spp. prevalence and diversity of mitochondrial cytochrome b gene sequences in wild-caught mosquitoes sampled across ten sites on the North Island of New Zealand during 2012-2014. The mosquitoes were pooled by species and location collected, and the thorax and abdomens were examined separately for Plasmodium spp. DNA. Akaike information criterion (AIC) modeling was used to test whether location, year of sampling, and mosquito species were significant predictors of minimum infection rates (MIR). RESULTS: We collected 788 unengorged mosquitoes of six species, both native and introduced. The most frequently caught mosquito species were the introduced Aedes notoscriptus and the native Culex pervigilans. Plasmodium sp DNA was detected in 37% of matched thorax and abdomen pools. When considered separately, 33% of abdomen and 23% of thorax pools tested positive by nested PCR. The MIR of the positive thorax pools from introduced mosquito species was 1.79% for Ae. notoscriptus and 0% for Cx. quinquefasciatus, while the MIR for the positive thorax pools of native mosquito species was 4.9% for Cx. pervigilans and 0% for Opifex fuscus. For the overall MIR, site and mosquito species were significant predictors of Plasmodium overall MIR. Aedes notoscriptus and Cx. pervigilans were positive for malaria DNA in the thorax samples, indicating that they may play a role as avian malaria vectors. Four different Plasmodium lineages (SYAT05, LINN1, GRW6, and a new lineage of P (Haemamoeba) sp. AENOT11) were identified in the pooled samples. CONCLUSIONS: This is the first detection of avian Plasmodium DNA extracted from thoraxes of native Culex and introduced Aedes mosquito species in New Zealand and therefore the first study providing an indication of potential vectors in this country.

Coccidia species in endemic and native New Zealand passerines.

New Zealand native passerines are hosts to a large variety of gastrointestinal parasites, including coccidia. Coccidian parasites are generally host-specific, obligate intracellular protozoan parasites. In passerine birds, members of the genus Isospora are most common. Under natural conditions, these parasites seldom pose a threat, but stressors such as quarantine for translocation, overcrowding, or habitat changes may cause an infection outbreak that can severely affect wild populations. Although coccidia are important pathogens and have caused mortalities in kiwi (Apteryx spp.) and hihi (Notiomystis cincta), their prevalence, epidemiology, life cycles, and taxonomic relationships are still widely unknown in native New Zealand songbirds. Over a period of 3 years (2007-2009), we examined 330 fecal samples of six native passerine species: tui (Prosthemadera novaeseelandiae), North Island saddleback (Philesturnus carunculatus rufusater), North Island robin (Petroica longipes), silvereye (Zosterops lateralis), and fantail (Rhipidura fuliginosa). The overall prevalence by flotation of coccidian infection in the New Zealand bird species examined was 21-38 %, 21 % in North Island robin, 38 % in tui, and 25 % in saddleback. Similar to prior studies in other countries, preliminary sequencing results suggest that coccidia in passerines in New Zealand are members of the family Eimeriidae, unlike the phenotypically similar genus Cystisospora of mammals. Using molecular methods, we identified at least five new genetically distinct Isospora species in the examined birds (three in tui and one each in saddlebacks and North Island robins).

Avian malaria in New Zealand.

Avian malaria parasites of the genus Plasmodium have the ability to cause morbidity and mortality in naïve hosts, and their impact on the native biodiversity is potentially serious. Over the last decade, avian malaria has aroused increasing interest as an emerging disease in New Zealand with some endemic avian species, such as the endangered mohua (Mohua ochrocephala), thought to be particularly susceptible. To date, avian malaria parasites have been found in 35 different bird species in New Zealand and have been diagnosed as causing death in threatened species such as dotterel (Charadrius obscurus), South Island saddleback (Philesturnus carunculatus carunculatus), mohua, hihi (Notiomystis cincta) and two species of kiwi (Apteryx spp.). Introduced blackbirds (Turdus merula) have been found to be carriers of at least three strains of Plasmodium spp. and because they are very commonly infected, they are likely sources of infection for many of New Zealand's endemic birds. The spread and abundance of introduced and endemic mosquitoes as the result of climate change is also likely to be an important factor in the high prevalence of infection in some regions and at certain times of the year. Although still limited, there is a growing understanding of the ecology and epidemiology of Plasmodium spp. in New Zealand. Molecular biology has played an important part in this process and has markedly improved our understanding of the taxonomy of the genus Plasmodium. This review presents our current state of knowledge, discusses the possible infection and disease outcomes, the implications for host behaviour and reproduction, methods of diagnosis of infection, and the possible vectors for transmission of the disease in New Zealand.

Coccidiosis in hihi/stitchbirds (Notiomystis cincta) due to coccidia of the Eimeriidae.

AIM: To describe the pathology of coccidiosis in hihi and to provide preliminary data on the taxonomy of the coccidia involved using molecular methods. METHODS: In an initial study from 1994 to 1997, gross and histopathological examinations were performed on 12 dead juvenile hihi from the National Wildlife Centre (NWC) at Mt. Bruce. In a second study during 2008-2010 DNA from sporulated oocysts and liver tissue was used for PCR analysis and sequencing. Faecal samples were also obtained from infected hihi from the NWC and examined for coccidial oocysts, which were then sporulated in the laboratory in 1994-1997 and 2007-2009. In addition, a post mortem was performed on a dead adult hihi from the NWC in 2008, and 18 archived hihi tissues from 11 individual birds stored at the Institute of Veterinary, Animal and Biomedical Sciences (IVABS) were used for DNA extraction. RESULTS: Severe gross and histopathological changes in the intestine and occasionally in the liver were found in the 12 dead birds examined. The morphological characteristics of the sporulated oocysts suggested that two types of coccidia were present. PCR analysis and sequencing of extracted DNA supported the existence of at least two different coccidia species in hihi. These were genetically more closely related to the genus Eimeria than to the morphologically similar genus Cystisospora (formerly Isospora) of mammals. In addition, one liver tissue sample that was examined post mortem was positive for at least two different coccidia species of the family Eimeriidae according to sequencing results, and the presence of extraintestinal coccidian stages was confirmed. CONCLUSIONS: Preliminary morphological and sequencing results suggest that two types of eimeriid coccidia are present and at least one of these commonly has extraintestinal stages. CLINICAL RELEVANCE: Coccidiosis in hihi is a serious disease capable of causing mortalities in juvenile and adult birds in captive situations. Treatment and control of the disease will be difficult as the extraintestinal stages of the organism are likely to be refractile to oral treatment.