Epetraborole, a leucyl-tRNA synthetase inhibitor, demonstrates murine efficacy, enhancing the in vivo activity of ceftazidime against Burkholderia pseudomallei, the causative agent of melioidosis.

Burkholderia pseudomallei is the causative agent of melioidosis, which is increasingly being reported worldwide. Mortality rates as high as 40% have been reported based on clinical patient outcomes in the endemic areas of Australia and Thailand. Novel therapies are needed to reduce treatment duration and adverse effects and improve treatment outcomes. Epetraborole, a novel antibiotic, targets leucyl-tRNA synthetase (LeuRS), an essential enzyme that catalyzes the attachment of leucine to transfer RNA. Epetraborole was evaluated for in vitro activity and efficacy in a murine model to assess clinical relevance against Burkholderia pseudomallei infections for possible treatment of melioidosis. Epetraborole was tested against 13 clinically derived and three reference B. pseudomallei strains that have a broad spectrum of susceptibilities to the standard-of-care (SoC) drugs for melioidosis, which showed that epetraborole exhibited minimal inhibitory concentrations of 0.25-4 µg/mL. Ex vivo studies using THP-1 macrophages confirmed the potency of epetraborole and demonstrated synergy between epetraborole and ceftazidime. In the acute pulmonary murine infection model of melioidosis, epetraborole demonstrated equivalent efficacy when delivered orally or subcutaneously, which compared well with the standard-of-care drug ceftazidime. In addition, adding epetraborole to ceftazidime significantly improved antimicrobial activity in this animal model. This work warrants further exploration of epetraborole as a candidate for treating melioidosis and substantiates LeuRS as a clinically relevant drug target in B. pseudomallei.

A lower motor neuron disease in takahe (Porphyrio hochstetteri) is an endoplasmic reticulum storage disease.

AIMS: To investigate the pathogenesis of a disease in takahe (Porphyrio hochstetteri) with intracytoplasmic inclusion bodies in lower motor neurons. METHODS: Four birds aged between 5 and 12 years, from three different wildlife sanctuaries in New Zealand were examined. Of these, only one had signs of spinal dysfunction in the form of paresis. Stained paraffin sections of tissues were examined by light microscopy and immunostained sections of the ventral horn of the spinal cord by confocal microscopy. Epoxy resin sections of the spinal cord from the bird with spinal dysfunction were examined by electron microscopy. RESULTS: Two types of inclusion bodies were noted, but only in motor neurons of the ventral spinal cord and brain stem. These were large globoid eosinophilic bodies up to 5 µm in diameter, and yellow/brown granular inclusions mostly at the pole of the cell. The globoid bodies stained with Luxol fast blue but not with periodic acid Schiff (PAS), or Sudan black. The granular inclusions stained with Luxol fast blue, PAS and Sudan black. Both bodies were slightly autofluorescent. On electron microscopy the globoid bodies had an even electron-dense texture and were bound by a membrane. Beneath the membrane were large numbers of small intraluminal vesicles. The smaller granular bodies were more heterogeneous, irregularly rounded and membrane-bound accumulations of granular electron-dense material, often with electron-lucent vacuoles. Others were more vesicular but contained varying amounts of electron-dense material. The large globoid bodies did not immunostain for lysosomal markers lysosomal associated protein 1 (LAMP1) or cathepsin D, so were not lysosomal. The small granular bodies stained for cathepsin D by a chromogenic method.A kindred matrix analysis showed two cases to be as closely related as first cousins, and another case was almost as closely related to one of them, but the fourth bird was unrelated to any other. CONCLUSIONS: It was concluded that this was an endoplasmic reticulum storage disease due to a specific protein misfolding within endoplasmic reticulum. It was rationalised that the two types of inclusions reflected the same aetiology, but that misfolded protein in the smaller granular bodies had entered the lysosomal system via endoplasmic reticulum autophagy. Although the cause was unclear, it most likely had a genetic aetiology or predisposition and, as such, has clinical relevance.

Efficacy and Improved Resistance Potential of a Cofactor-Independent InhA Inhibitor of Mycobacterium tuberculosis in the C3HeB/FeJ Mouse Model.

AN12855 is a direct, cofactor-independent inhibitor of InhA in Mycobacterium tuberculosis In the C3HeB/FeJ mouse model with caseous necrotic lung lesions, AN12855 proved efficacious with a significantly lower resistance frequency than isoniazid. AN12855 drug levels were better retained in necrotic lesions and caseum where the majority of hard to treat, extracellular bacilli reside. Owing to these combined attributes, AN12855 represents a promising alternative to the frontline antituberculosis agent isoniazid.

Wildlife diseases in New Zealand: recent findings and future challenges.

Our knowledge of diseases in New Zealand wildlife has expanded rapidly in the last two decades. Much of this is due to a greater awareness of disease as a cause of mortality in some of our highly threatened species or as a limiting factor to the successful captive rearing of intensely managed species such as hihi (Notiomystis cincta), kiwi (Apteryx spp.) and kakapo (Strigops habroptilus). An important factor contributing to the increase of our knowledge has been the development of new diagnostic techniques in the fields of molecular biology and immunohistochemistry, particularly for the diagnosis and epidemiology of viral and protozoan diseases. Although New Zealand remains free of serious exotic viruses there has been much work on understanding the taxonomy and epidemiology of local strains of avipox virus and circoviruses. Bacterial diseases such as salmonellosis, erysipelas and tuberculosis have also been closely investigated in wildlife and opportunist mycotic infections such as aspergillosis remain a major problem in many species. Nutritional diseases such as hyperplastic goitre due to iodine deficiency and metabolic bone disease due to Ca:P imbalance have made significant impacts on some captive reared birds, while lead poisoning is a problem in some localities. The increasing use of wildlife translocations to avoid the extinction of threatened species has highlighted the need for improved methods to assess the disease risks inherent in these operations and other intensive conservation management strategies such as creching young animals. We have also become more aware of the likelihood of inbreeding suppression as populations of many species decrease or pass through a genetic bottleneck. Climate change and habitat loss, however, remain the greatest threats to biodiversity and wildlife health worldwide. Temperature changes will affect our wildlife habitats, alter the distribution of disease vectors and wildlife predators, or directly harm threatened species in vulnerable localities.

Clinical and veterinary trypanocidal benzoxaboroles target CPSF3.

African trypanosomes cause lethal and neglected tropical diseases, known as sleeping sickness in humans and nagana in animals. Current therapies are limited, but fortunately, promising therapies are in advanced clinical and veterinary development, including acoziborole (AN5568 or SCYX-7158) and AN11736, respectively. These benzoxaboroles will likely be key to the World Health Organization's target of disease control by 2030. Their mode of action was previously unknown. We have developed a high-coverage overexpression library and use it here to explore drug mode of action in Trypanosoma brucei Initially, an inhibitor with a known target was used to select for drug resistance and to test massive parallel library screening and genome-wide mapping; this effectively identified the known target and validated the approach. Subsequently, the overexpression screening approach was used to identify the target of the benzoxaboroles, Cleavage and Polyadenylation Specificity Factor 3 (CPSF3, Tb927.4.1340). We validated the CPSF3 endonuclease as the target, using independent overexpression strains. Knockdown provided genetic validation of CPSF3 as essential, and GFP tagging confirmed the expected nuclear localization. Molecular docking and CRISPR-Cas9-based editing demonstrated how acoziborole can specifically block the active site and mRNA processing by parasite, but not host CPSF3. Thus, our findings provide both genetic and chemical validation for CPSF3 as an important drug target in trypanosomes and reveal inhibition of mRNA maturation as the mode of action of the trypanocidal benzoxaboroles. Understanding the mechanism of action of benzoxaborole-based therapies can assist development of improved therapies, as well as the prediction and monitoring of resistance, if or when it arises.

The 7-phenyl benzoxaborole series is active against Mycobacterium tuberculosis.

We identified a series of novel 7-phenyl benzoxaborole compounds with activity against Mycobacterium tuberculosis. Compounds had a range of activity with inhibitory concentrations (IC90) as low as 5.1 µM and no cytotoxicity against eukaryotic cells (IC50 > 50 µM). Compounds were active against intracellular mycobacteria cultured in THP-1 macrophages. We isolated and characterized resistant mutants with mutations in NADH dehydrogenase (Ndh) or the regulatory protein Mce3R. Mutations suggest that Ndh may be the target of this series.

Discovery of a Potent and Specific M. tuberculosis Leucyl-tRNA Synthetase Inhibitor: (S)-3-(Aminomethyl)-4-chloro-7-(2-hydroxyethoxy)benzo[c][1,2]oxaborol-1(3H)-ol (GSK656).

There is an urgent need to develop new and safer antitubercular agents that possess a novel mode of action. We synthesized and evaluated a novel series of 3-aminomethyl 4-halogen benzoxaboroles as Mycobacterium tuberculosis (Mtb) leucyl-tRNA synthetase (LeuRS) inhibitors. A number of Mtb LeuRS inhibitors were identified that demonstrated good antitubercular activity with high selectivity over human mitochondrial and cytoplasmic LeuRS. Further evaluation of these Mtb LeuRS inhibitors by in vivo pharmacokinetics (PK) and murine tuberculosis (TB) efficacy models led to the discovery of GSK3036656 (abbreviated as GSK656). This molecule shows potent inhibition of Mtb LeuRS (IC50 = 0.20 µM) and in vitro antitubercular activity (Mtb H37Rv MIC = 0.08 µM). Additionally, it is highly selective for the Mtb LeuRS enzyme with IC50 of >300 µM and 132 µM for human mitochondrial LeuRS and human cytoplasmic LeuRS, respectively. In addition, it exhibits remarkable PK profiles and efficacy against Mtb in mouse TB infection models with superior tolerability over initial leads. This compound has been progressed to clinical development for the treatment of tuberculosis.

A Novel 6-Benzyl Ether Benzoxaborole Is Active against Mycobacterium tuberculosis In Vitro.

We identified a novel 6-benzyl ether benzoxaborole with potent activity against Mycobacterium tuberculosis The compound had an MIC of 2 µM in liquid medium. The compound was also able to prevent growth on solid medium at 0.8 µM and was active against intracellular bacteria (50% inhibitory concentration [IC50] = 3.6 µM) without cytotoxicity against eukaryotic cells (IC50 > 100 µM). We isolated resistant mutants (MIC ≥ 100 µM), which had mutations in Rv1683, Rv3068c, and Rv0047c.

Description of four new species of coccidia (Apicomplexa: Eimeriidae) from brown kiwi, Apteryx mantelli, in New Zealand.

This study used morphological techniques to describe and name four new species of coccidia from the brown kiwi (Apteryx mantelli). Four distinct eimerian oocyst species were recovered that we describe as new species. The largest of these, Eimeria paraurii n. sp. measured 32.2 × 19.8 µm and is morphologically similar to gametocytes previously described histologically in colorectal polyps (Morgan et al. in Parasitol Res 111(4):1689-1699, 2012). Eimeria apteryxii n. sp. measured 23.9 × 14.9 µm and is similar to renal oocysts described histologically in brown, rowi (A. rowii) and Haast tokoeka kiwi (A. australis "Haast") (Morgan et al. in Avian Pathol 42(2):137-146, 2013). Eimeria kiwii n. sp. measured 14.8 × 13.9 µm and resembled gametocytes described previously in kiwi intestinal epithelial cells in brown kiwi (Morgan et al. in Parasitol Res 111(4):1689-1699, 2012). Eimeria mantellii n. sp. measured 17.8 × 10.7 µm and did not appear similar to any coccidia previously described in histological studies in kiwi. These are the first species of Eimeria to be described and named from brown kiwi. Because the morphological descriptions in the present study were determined from a limited number of kiwi droppings from two geographical locations, it is likely that these represent only a portion of Eimeria species present in other populations of both brown kiwi and other Apteryx species from around New Zealand.

A potent antimalarial benzoxaborole targets a Plasmodium falciparum cleavage and polyadenylation specificity factor homologue.

Benzoxaboroles are effective against bacterial, fungal and protozoan pathogens. We report potent activity of the benzoxaborole AN3661 against Plasmodium falciparum laboratory-adapted strains (mean IC50 32 nM), Ugandan field isolates (mean ex vivo IC50 64 nM), and murine P. berghei and P. falciparum infections (day 4 ED90 0.34 and 0.57 mg kg-1, respectively). Multiple P. falciparum lines selected in vitro for resistance to AN3661 harboured point mutations in pfcpsf3, which encodes a homologue of mammalian cleavage and polyadenylation specificity factor subunit 3 (CPSF-73 or CPSF3). CRISPR-Cas9-mediated introduction of pfcpsf3 mutations into parental lines recapitulated AN3661 resistance. PfCPSF3 homology models placed these mutations in the active site, where AN3661 is predicted to bind. Transcripts for three trophozoite-expressed genes were lost in AN3661-treated trophozoites, which was not observed in parasites selected or engineered for AN3661 resistance. Our results identify the pre-mRNA processing factor PfCPSF3 as a promising antimalarial drug target.

Adenomatous hyperplasia of the mucous glands in captive Archey's frogs (Leiopelma archeyi).

AIMS: To describe the gross and light microscopic characteristics of skin lesions observed on the ventral skin of captive Archey's frogs (Leiopelma archeyi) between 2000 and 2012, and to investigate their occurrence, possible aetiology and association with survival. METHODS: Postmortem skin samples were obtained for histological evaluation from 37 frogs, with and without skin lesions, that died while in captivity at Auckland Zoo between 2000 and 2012. Four frogs with skin lesions were biopsied under general anaesthesia and samples used for both light and transmission electron microscopy. The records of 94 frogs held at the University of Otago and Auckland Zoo between 2000-2012 were reviewed, which included some frogs recently collected from the wild. Information about the occurrence of skin lesions, and mortality associated with skin lesions was collated. RESULTS: Grossly the skin lesions varied in appearance; most were circular, pale grey papules, which measured from <0.5-1.5 mm in diameter with no umbilication. The overlying epidermis was not fragile and there was no associated inflammation. Contents often appeared clear or semi-transparent. Lesions were located predominantly on ventral surfaces including trunk, thighs, lower legs and forearms, and gular region, but not on digits. The number ranged from single to multiple, often confluent lesions covering the entire ventral surface of the frog. Histologically the lesions consisted of enlarged proliferating mucous glands that expanded the dermis and elevated the epidermis. They were semi-organised, solid or occasionally cavitated acinar structures with central lumina which sometimes contained mucus. Nuclei showed moderate anisokaryosis and mitotic figures were uncommon. Transmission electron microscopy did not show any infectious agents. Between 2000 and 2012, skin lesions were recorded in 35/94 (37%) frogs. The size and location of skin lesions varied over time, with some resolving and sometimes reappearing. Skin lesions were not associated with an increased risk of death. CONCLUSIONS: The skin lesions had the gross and microscopic characteristics of adenomatous hyperplasia of the dermal mucous glands. CLINICAL RELEVANCE: The aetiology of this adenomatous hyperplasia is unknown, but factors associated with the captive environment are the most likely cause. This is the first description of adenomatous hyperplasia of the cutaneous mucous glands in amphibians.

Discovery of Novel Oral Protein Synthesis Inhibitors of Mycobacterium tuberculosis That Target Leucyl-tRNA Synthetase.

The recent development and spread of extensively drug-resistant and totally drug-resistant resistant (TDR) strains of Mycobacterium tuberculosis highlight the need for new antitubercular drugs. Protein synthesis inhibitors have played an important role in the treatment of tuberculosis (TB) starting with the inclusion of streptomycin in the first combination therapies. Although parenteral aminoglycosides are a key component of therapy for multidrug-resistant TB, the oxazolidinone linezolid is the only orally available protein synthesis inhibitor that is effective against TB. Here, we show that small-molecule inhibitors of aminoacyl-tRNA synthetases (AARSs), which are known to be excellent antibacterial protein synthesis targets, are orally bioavailable and effective against M. tuberculosis in TB mouse infection models. We applied the oxaborole tRNA-trapping (OBORT) mechanism, which was first developed to target fungal cytoplasmic leucyl-tRNA synthetase (LeuRS), to M. tuberculosis LeuRS. X-ray crystallography was used to guide the design of LeuRS inhibitors that have good biochemical potency and excellent whole-cell activity against M. tuberculosis Importantly, their good oral bioavailability translates into in vivo efficacy in both the acute and chronic mouse models of TB with potency comparable to that of the frontline drug isoniazid.

Cryptosporidium and Toxoplasma Parasites Are Inhibited by a Benzoxaborole Targeting Leucyl-tRNA Synthetase.

The apicomplexan parasites Cryptosporidium and Toxoplasma are serious threats to human health. Cryptosporidiosis is a severe diarrheal disease in malnourished children and immunocompromised individuals, with the only FDA-approved drug treatment currently being nitazoxanide. The existing therapies for toxoplasmosis, an important pathology in immunocompromised individuals and pregnant women, also have serious limitations. With the aim of developing alternative therapeutic options to address these health problems, we tested a number of benzoxaboroles, boron-containing compounds shown to be active against various infectious agents, for inhibition of the growth of Cryptosporidium parasites in mammalian cells. A 3-aminomethyl benzoxaborole, AN6426, with activity in the micromolar range and with activity comparable to that of nitazoxanide, was identified and further characterized using biophysical measurements of affinity and crystal structures of complexes with the editing domain of Cryptosporidium leucyl-tRNA synthetase (LeuRS). The same compound was shown to be active against Toxoplasma parasites, with the activity being enhanced in the presence of norvaline, an amino acid that can be mischarged by LeuRS. Our observations are consistent with AN6426 inhibiting protein synthesis in both Cryptosporidium and Toxoplasma by forming a covalent adduct with tRNA(Leu) in the LeuRS editing active site and suggest that further exploitation of the benzoxaborole scaffold is a valid strategy to develop novel, much needed antiparasitic agents.

Antimalarial Benzoxaboroles Target Plasmodium falciparum Leucyl-tRNA Synthetase.

There is a need for new antimalarials, ideally with novel mechanisms of action. Benzoxaboroles have been shown to be active against bacteria, fungi, and trypanosomes. Therefore, we investigated the antimalarial activity and mechanism of action of 3-aminomethyl benzoxaboroles against Plasmodium falciparum Two 3-aminomethyl compounds, AN6426 and AN8432, demonstrated good potency against cultured multidrug-resistant (W2 strain) P. falciparum (50% inhibitory concentration [IC50] of 310 nM and 490 nM, respectively) and efficacy against murine Plasmodium berghei infection when administered orally once daily for 4 days (90% effective dose [ED90], 7.4 and 16.2 mg/kg of body weight, respectively). To characterize mechanisms of action, we selected parasites with decreased drug sensitivity by culturing with stepwise increases in concentration of AN6426. Resistant clones were characterized by whole-genome sequencing. Three generations of resistant parasites had polymorphisms in the predicted editing domain of the gene encoding a P. falciparum leucyl-tRNA synthetase (LeuRS; PF3D7_0622800) and in another gene (PF3D7_1218100), which encodes a protein of unknown function. Solution of the structure of the P. falciparum LeuRS editing domain suggested key roles for mutated residues in LeuRS editing. Short incubations with AN6426 and AN8432, unlike artemisinin, caused dose-dependent inhibition of [(14)C]leucine incorporation by cultured wild-type, but not resistant, parasites. The growth of resistant, but not wild-type, parasites was impaired in the presence of the unnatural amino acid norvaline, consistent with a loss of LeuRS editing activity in resistant parasites. In summary, the benzoxaboroles AN6426 and AN8432 offer effective antimalarial activity and act, at least in part, against a novel target, the editing domain of P. falciparum LeuRS.

Efficacy of anthelmintic treatment in captive-reared black stilts (Himantopus novaezelandiae) released to the wild.

AIMS: To assess the efficacy of praziquantel (PZQ) administered to control helminths in captive-reared critically endangered black stilts (Himantopus novaezelandiae) before their release into the wild and determine the effect on their survival shortly after release. METHODS: Of 77 captive black stilts, 37 were treated with PZQ prior to release into the wild in South Canterbury, New Zealand, in August and September 2007. Faecal helminth egg counts (FEC) were measured before and after anthelmintic treatment, and before and after release to the wild using modified faecal flotation and sedimentation methods. In addition, total helminth counts were determined in 11 of the birds that died following release, as well as four captive and 11 other wild stilts. RESULTS: The efficacy of PZQ against trematodes was 92% and against Capillaria spp. was 34%. No trematode or Capillaria spp. eggs were detected in treated birds 1 day after treatment, but FEC increased 3-5 days after treatment. There were no differences in the total helminth counts for trematodes, cestodes or Capillaria spp. in control or treated birds (p>0.2). Survival did not differ between treatment groups in the August or September releases (p>0.4). Of control and treated birds, 11/17 (65%) and 8/14 (57%) survived to 31 days, respectively, in the August release, and 16/20 (80%) and 10/15 (67%) survived to 84 days, respectively, in the September release. CONCLUSION: Overall, the results suggest that PZQ treatment may be an unnecessary cost and the risks of producing anthelmintic resistance, injuring the birds during processing or producing an unnecessary stress response at the time of release could exceed any likely benefits. CLINICAL RELEVANCE: It is recommended that pre-release anthelmintic treatment of black stilts should be used only if indicated by health screening. Any treatment should incorporate annual efficacy testing to monitor the emergence of anthelmintic resistance.

Tavaborole topical solution, 5% for the treatment of toenail onychomycosis.

Tavaborole topical solution, 5% (tavaborole) is a novel, boron-based, antifungal pharmaceutical agent indicated for treatment of toenail onychomycosis due to the dermatophytes Trichophyton rubrum or Trichophyton mentagrophytes. In preclinical studies, tavaborole effectively penetrated through full-thickness, non-diseased cadaver fingernails, including those with up to four layers of nail polish. Limited systemic absorption was observed following topical application of tavaborole. In phase III clinical trials involving patients with distal subungual onychomycosis affecting 20-60% of a target great toenail, significantly more patients treated with tavaborole versus vehicle achieved completely clear nail with negative mycology following daily application for 48 weeks. Treatment-emergent adverse events reported by at least 1% of patients treated with tavaborole and at a greater frequency versus vehicle included ingrown toenail, exfoliation, erythema and dermatitis. Treatment discontinuations were uncommon. Results from preclinical studies and phase III clinical trials establish tavaborole as a safe and efficacious treatment for toenail onychomycosis.

Investigation of mortalities associated with Salmonella spp. infection in wildlife on Tiritiri Matangi Island in the Hauraki Gulf of New Zealand.

CASE HISTORY: Salmonellosis was suspected as the cause of death in eight wild animals on Tiritiri Matangi Island, in the Hauraki Gulf of New Zealand, between November and September 2011, including three hihi (Notiomystis cincta), a tuatara (Sphenodon punctatus), a masked lapwing (Vanellus miles novaehollandiae), and a saddleback (Philesturnus carunculatus). An outbreak investigation to identify the source and distribution of infection was undertaken over the summer of 2011-2012. CLINICAL AND LABORATORY FINDINGS: Surveillance of five species of forest bird (n=165) in December 2011 returned a single positive result for Salmonella spp. Environmental sampling of 35 key water sources and hihi supplementary feeding stations conducted in December 2011 and March 2012 returned isolates of S. enterica subspecies houtenae and S. enterica serovar Saintpaul from a stream, a dam and a supplementary feeding station. The same serotypes were identified in tissue samples collected from post mortem specimens of the affected birds, and their similarity was confirmed by pulsed-field gel electrophoresis. DIAGNOSIS: Mortality in wildlife associated with infection with S. enterica subspecies houtenae and S. enterica serovar Saintpaul. CLINICAL RELEVANCE: This is the first detection of these Salmonella spp. from wild birds in New Zealand. Our study highlights how active surveillance in response to observed disease emergence (here mortalities) can provide important insight for risk assessment and management within populations of endangered species and inform risk assessment in translocation planning.

Prevalence of and risk factors for coccidiosis in kiwi between 1977 and 2011.

AIMS: To identify risk factors associated with coccidiosis in kiwi (Apteryx spp.) using a retrospective analysis of historical data from kiwi examined post-mortem, and to determine the prevalence of coccidial oocysts in the droppings of wild and captive kiwi. METHODS: Necropsy reports were examined from kiwi submitted to the National Wildlife Mortality Database of New Zealand (Huia) between February 1977 and May 2011. All cases that reported histological examination of one or more hepatic, intestinal or renal tissues were included in the study (n=372). Data collated for analysis included the presence or absence of coccidiosis in one or more tissues, age, host species, habitat, and season of submission. Fisher's exact test was used to determine the association between each independent variable and the prevalence of coccidiosis. Droppings opportunistically collected from wild and captive kiwi between January 2008 and June 2010 were also examined for the presence of coccidial oocysts. RESULTS: Coccidiosis was evident in 47/372 (12.6%) kiwi examined post-mortem and was considered the primary cause of death in 12/47 (26%) infected cases. Examination of 412 droppings from wild and captive kiwi collected over a 29-month period revealed that 98 (23.8%) samples contained coccidia at the time of sampling. Enteric (n=27) coccidiosis was the most common form diagnosed using histology, followed by renal (n=19) and hepatic (n=11), with splenic (n=2) and pulmonary (n=1) infections infrequently seen. Many kiwi demonstrated infections in multiple tissues. The prevalence of coccidiosis was greater in juvenile kiwi (36/148, 24.3%) than adults (8/133, 6.0%) or chicks (2/83, 2.4%) (p<0.001), although there was no difference in mortality between age groups. Season of year was also associated with overall prevalence (p=0.05), with most cases being diagnosed in the autumn and winter. Coccidiosis was histologically evident in four of five species of kiwi examined, and in all host species upon analysis of droppings. Host species or habitat (captive vs. wild) did not influence the prevalence of disease detected histologically. CONCLUSIONS: Age and season were the only factors that influenced the prevalence of coccidiosis in kiwi in this study. Coccidiosis was present in all species of kiwi, and this is the first report of coccidiosis in rowi (Apteryx rowi).

Investigation of an outbreak of craniofacial deformity in yellow-eyed penguin (Megadyptes antipodes) chicks.

AIM: To investigate an outbreak of severe craniofacial deformity in yellow-eyed penguin (Megadyptes antipodes, hoiho) chicks at a single breeding site on the Otago Peninsula in the South Island of New Zealand. METHODS: Morbidity and mortality of yellow-eyed penguins breeding on the coastal regions of Otago was monitored from November 2008 to March 2009. Dead chicks and unhatched eggs were recovered and examined. Between October and December 2008 32 eggs were recorded at 17 nests in the Okia Reserve. Eleven chicks survived to about 90 days of age, of which eight were found to have moderate to severe craniofacial deformity. The six most severe chicks were subject to euthanasia and examined in detail at necropsy, and the remaining two affected chicks were released to the wild after a period of care in a rehabilitation centre. Post-mortem samples were analysed for inorganic and organic toxins. RESULTS: The six deformed chicks all had severe shortening of the mandible and maxilla by 20-50 mm. The rostral and caudal regions of the skull were approximately 40 and 80% of normal length, respectively. Other, more variable lesions included cross bill deformity, malformed bill keratin, microphthalmia with misshapen scleral ossicles and oral soft tissue excess thought to be secondary to bony malformations. During the same year, mild sporadic bill deformities were also reported in 10 unrelated chicks from >167 chicks at other breeding sites on the southern Otago coast. Concentrations of organic toxins and heavy metals in body tissues from affected chicks were apparently similar to those in unaffected chicks on other beaches. CONCLUSIONS: No cause of this outbreak of craniofacial deformity could be established although the high prevalence at a single site suggests that it was due to an unidentified local teratogen.

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.