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.

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.

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.

Avian polyomavirus identified in a nestling Gouldian finch (Erythrura gouldiae) in New Zealand.

Abstract CASE HISTORY: Four 4-5-month-old nestlings and one adult in a commercial aviary of 53 Gouldian finches (Erythrura gouldiae) died over a 2-week period in July 2000. PATHOLOGICAL FINDINGS: One nestling was necropsied and showed bronze-tinged skeletal muscles, a swollen liver with haemorrhagic margins and numerous haemorrhages on serosal surfaces. The histological lesions included multifocal hepatic necrosis and haemorrhage associated with the presence of large clear or basophilic intranuclear inclusions in hepatocytes and Kupffer cells suggestive of avian polyomavirus infection. Similar inclusion bodies were present in splenic histiocytes. MOLECULAR BIOLOGY: DNA was subsequently extracted from archived portions of liver, spleen, gizzard, heart, lung and kidney. A broad spectrum nested PCR was used to detect polyomavirus which sequence analysis confirmed as finch polyomavirus. DIAGNOSIS: Avian polyomavirus. CLINICAL RELEVANCE: Avian virus infections such as polyomavirus should be suspected in cases of sudden death in nestlings, particularly in susceptible species such as psittacine and passerine birds. The archiving of tissues from unconfirmed disease outbreaks provides a valuable resource for retrospective investigations.

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).

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.

Enteric coccidiosis in the brown kiwi (Apteryx mantelli).

Enteric coccidiosis may cause significant morbidity and mortality in juvenile brown kiwi (Apteryx mantelli). Morphology of sporulated oocysts indicates that at least two Eimeria species are able to infect the brown kiwi. A histological study of the endogenous stages of coccidia was undertaken in the intestinal tracts of ten naturally infected young kiwi. Sequential sectioning of the entire intestinal tract allowed identification and recording of the distribution of the various coccidial life stages. Macromeronts measuring 268 × 162 µm when mature were found mainly within the lamina propria of the proximal one third of the small intestine. A smaller form of lamina propria meront was also identified (8.7 × 6.4 µm) with a similar distribution to the macromeronts. Small meronts (4.4 × 3.8 µm) were also identified in mucosal epithelial cells, with the overall peak in distribution within the intestinal tract being distal to the lamina propria meronts. Three morphologically distinctive gametocytes were identified. Type A gametocytes contained within epithelial cells shared the same distribution as the epithelial meronts. Polyps containing large numbers of type B gametocytes within the distal intestinal tract were found in two cases, and type C gametocytes were identified throughout the entire intestinal tract in one case only. The observational nature of this study precludes complete knowledge of the parasite life cycles using histology alone. However, it is likely that each of the three morphologically distinct gametocytes represents a separate species of enteric coccidia.

Salt gland adenitis associated with bacteria in Blue Penguins (Eudyptula minor) from Hauraki Gulf (auckland, New Zealand).

Three Blue Penguins (Eudyptula minor) were rescued between July 2006 and January 2007 off the east coast of Auckland, New Zealand. They were taken to a rehabilitation center, where they subsequently died in May 2007 and were submitted for necropsy. There was unilateral enlargement of the salt glands with disseminated small, pale, and firm foci in all birds. Histologic examination of the affected glands demonstrated the presence of multifocal granulomas and areas of severe squamous metaplasia of the collecting ducts. The remaining gland had areas of hyperplasia, dysplasia, and necrosis with a severe granulomatous inflammatory reaction. Intralesional gram-negative bacteria were detected, but, unfortunately, bacterial culture was unrewarding. No further cases were observed in penguins in the subsequent year, and the primary cause of the salt gland adenitis remains uncertain.

Crystal structures of the human and fungal cytosolic Leucyl-tRNA synthetase editing domains: A structural basis for the rational design of antifungal benzoxaboroles.

Leucyl-tRNA synthetase (LeuRS) specifically links leucine to the 3' end of tRNA(leu) isoacceptors. The overall accuracy of the two-step aminoacylation reaction is enhanced by an editing domain that hydrolyzes mischarged tRNAs, notably ile-tRNA(leu). We present crystal structures of the editing domain from two eukaryotic cytosolic LeuRS: human and fungal pathogen Candida albicans. In comparison with previous structures of the editing domain from bacterial and archeal kingdoms, these structures show that the LeuRS editing domain has a conserved structural core containing the active site for hydrolysis, with distinct bacterial, archeal, or eukaryotic specific peripheral insertions. It was recently shown that the benzoxaborole antifungal compound AN2690 (5-fluoro-1,3-dihydro-1-hydroxy-1,2-benzoxaborole) inhibits LeuRS by forming a covalent adduct with the 3' adenosine of tRNA(leu) at the editing site, thus locking the enzyme in an inactive conformation. To provide a structural basis for enhancing the specificity of these benzoxaborole antifungals, we determined the structure at 2.2 A resolution of the C. albicans editing domain in complex with a related compound, AN3018 (6-(ethylamino)-5-fluorobenzo[c][1,2]oxaborol-1(3H)-ol), using AMP as a surrogate for the 3' adenosine of tRNA(leu). The interactions between the AN3018-AMP adduct and C. albicans LeuRS are similar to those previously observed for bacterial LeuRS with the AN2690 adduct, with an additional hydrogen bond to the extra ethylamine group. However, compared to bacteria, eukaryotic cytosolic LeuRS editing domains contain an extra helix that closes over the active site, largely burying the adduct and providing additional direct and water-mediated contacts. Small differences between the human domain and the fungal domain could be exploited to enhance fungal specificity.

Hyperplastic goitre and mortality in captive-reared black stilts ( Himantopus novaezelandiae ).

AIM: To study the gross, histopathological and clinico-pathological findings in cases of hyperplastic goitre in sub-adult captive- reared black stilts following their release on riverbeds in the south Canterbury region of New Zealand. METHODS: Necropsies were undertaken on the recovered carcasses of 48 black stilts over a 3-year period (1997-1999). The cause of death was determined, and thyroid glands were examined histopathologically and compared with those of free-living pied stilts. Concentrations of triiodothyronine (T3) and thyroxine (T4) in the serum of sub-adult and adult stilts were measured before and after iodine supplementation. RESULTS: The main causes of death of captive-reared black stilts following release were trauma, predation and starvation. An increase in size of the thyroid gland due to follicular hyperplasia and dilation was seen in all birds with intact thyroid glands (n=27). Dysplastic follicular changes such as epithelial desquamation, lipid deposition and haemorrhage were common in a large proportion of individuals with goitre. Dietary supplementation with iodine greatly improved survival rates in sub-adults following release, and significantly increased concentrations of T3 and T4 in serum. CONCLUSIONS: Subclinical goitre due to thyroid hyperplasia and dysplasia was the cause of hypothyroidism and this contributed to the poor survival of released sub-adult black stilts raised in captivity. Iodine supplementation of the diet of captive adults and sub-adults resulted in increased concentrations of T3 and T4 in serum and improved survivability.

Non-systemic erosive stomatitis of unknown aetiology in a dairy cow herd in New Zealand.

CASE HISTORY: Veterinarians from the Investigation and Diagnostic Centre (IDC), Wallaceville, New Zealand, investigated a novel vesicular disease in a 397-cow dairy herd, characterised by erosive stomatitis. CLINICAL AND PATHOLOGICAL FINDINGS: The investigation commenced with a report of erosive stomatitis in four dairy cows. The herd was examined that day and 30/397 (8%) adult cows were found to be affected. Two weeks later, the oral cavity of 180 cows from one management group were re-examined, and it was estimated that 80% of this group had healing erosive lesions. During the course of the investigation, intact vesicles were observed on the muzzle of two affected animals. None of the affected animals was systemically ill and there was no decrease in milk production. DIAGNOSIS: No infectious aetiological agent was detected using virus isolation, polymerase chain reaction (PCR), electron microscopy (EM) and serological tests, for any exotic infectious vesicular disease or any endemic cause of vesicular disease. CLINICAL RELEVANCE: Lesions of erosive stomatitis occurring in cattle must be differentiated from vesicular disease during exotic disease investigations.

An antifungal agent inhibits an aminoacyl-tRNA synthetase by trapping tRNA in the editing site.

Aminoacyl-transfer RNA (tRNA) synthetases, which catalyze the attachment of the correct amino acid to its corresponding tRNA during translation of the genetic code, are proven antimicrobial drug targets. We show that the broad-spectrum antifungal 5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole (AN2690), in development for the treatment of onychomycosis, inhibits yeast cytoplasmic leucyl-tRNA synthetase by formation of a stable tRNA(Leu)-AN2690 adduct in the editing site of the enzyme. Adduct formation is mediated through the boron atom of AN2690 and the 2'- and 3'-oxygen atoms of tRNA's3'-terminal adenosine. The trapping of enzyme-bound tRNA(Leu) in the editing site prevents catalytic turnover, thus inhibiting synthesis of leucyl-tRNA(Leu) and consequentially blocking protein synthesis. This result establishes the editing site as a bona fide target for aminoacyl-tRNA synthetase inhibitors.

Aspergillosis in a North Island robin (Petroica longipes).

CASE HISTORY: A 3-year-old female North Island robin (Petroica longipes) was found dead on Tiritiri Matangi Island during the breeding season. CLINICAL FINDINGS: The bird was in poor condition, and there was a 13 x 8 mm granulomatous mass in the thoracic cavity causing displacement of the heart and left lung. Histologically, the mass was a large granuloma infiltrated with fungal hyphae, and the liver contained multifocal aggregates of inflammatory cells. DIAGNOSIS: Thoracic aspergillosis and multifocal hepatitis. CLINICAL RELEVANCE: Determining the causes of death in populations of wild birds is often hampered by a lack of recovery of carcasses, autolysis and poor clinical history. In this case, the life history of the bird was known and recovery of the body was relatively swift. This is the first published description of aspergillosis in a free-living North Island robin.

Degradation of a Caulobacter soluble cytoplasmic chemoreceptor is ClpX dependent.

In order to determine whether ClpXP-mediated proteolysis is a common mechanism used to regulate the chemotaxis machinery during the cell cycle of Caulobacter crescentus, we have characterized a soluble cytoplasmic chemoreceptor, McpB. The mcpB gene lies adjacent to the major chemotaxis operon, which encodes 12 chemotaxis proteins, including the membrane chemoreceptor McpA. Like McpA, McpB possesses a C-terminal CheBR docking motif and three potential methylation sites, which we suggest are methylated. The McpB protein is degraded via a ClpX-dependent pathway during the swarmer-to-stalked cell transition, and a motif, which is 3 amino acids N-terminal to the McpB CheBR docking site, is required for proteolysis. Analysis of the degradation signal in McpB and McpA reveals a common motif present in the other four chemoreceptors that possess CheBR docking sites. A green fluorescent protein (GFP) fusion bearing 58 amino acids from the C terminus of McpA, which contains this motif, is degraded, suggesting that the C-terminal sequence is sufficient to confer ClpXP protease susceptibility.

Squamous cell carcinoma in a tuatara (Sphenodon punctatus).

CASE HISTORY: A mature male tuatara was presented with a history of recurrent cloacal prolapse. CLINICAL AND PATHOLOGICAL FINDINGS: The prolapsed tissue included a 12 mm diameter mass, which histologically showed cords and nodules of neoplastic epithelial cells. DIAGNOSIS: The mass was diagnosed as a squamous cell carcinoma with ulceration of the overlying stratified squamous epithelium and diffuse inflammation of the surrounding dermis. CLINICAL RELEVANCE: This case presented a rare opportunity to investigate squamous cell carcinoma in a long-lived lower vertebrate species. Future recurrence or metastasis of the mass may add useful information to the current base of knowledge of the behaviour of malignant neoplasms in reptiles.

Proteolysis of the Caulobacter McpA chemoreceptor is cell cycle regulated by a ClpX-dependent pathway.

Proteolysis is involved in cell differentiation and the progression through the cell cycle in Caulobacter crescentus. We have constitutively expressed the transmembrane chemoreceptor McpA from a multicopy plasmid to demonstrate that McpA degradation is modulated during the cell cycle. The level of McpA protein starts to decrease only when the swarmer cells differentiate into stalked cells. The reduction in McpA protein levels is maintained until the stalked cells develop into predivisional cells, at which point the level returns to that observed in swarmer cells. The cell-cycle-regulated degradation of McpA does not require the last 12 C-terminal amino acids, but it does require three amino acids (AAL) located 15 residues away from the C terminus. The ClpXP protease is essential in C. crescentus for viability, and thus, we tested McpA degradation in xylose conditional mutants. The effect on McpA degradation occurred within two generations from the start of ClpX depletion. The conditional mutants' growth rate was only slightly affected, suggesting that ClpX is directly involved in McpA proteolysis.

Multiple endocrine neoplasia type 1 in a crossbred dog.

Multiple endocrine neoplasia type 1 was diagnosed in a 12-year-old male crossbred dog. Relevant history included polyuria and polydipsia of four months' duration. Physical examination revealed abdominal enlargement, seborrhoea and polypnoea. Diagnostic tests indicated hypercalcaemia, elevated serum alkaline phosphatase and alanine aminotransferase, an exaggerated response to adrenocorticotropic stimulation of the adrenal gland, lack of cortisol suppression with a low dose dexamethasone suppression test and suppression of cortisol secretion with a high dose dexamethasone test. An enlarged right parathyroid gland was removed surgically and confirmed histopathologically to be a parathyroid adenoma. The pituitary-dependent hyperadrenocorticism was treated successfully with mitotane for 14 months before the patient was euthanased for an unrelated problem.

Pulmonary Mycobacterium bovis infection in a dog.

AIM: To describe the clinical course of a dog infected with Mycobacterium bovis causing a granulomatous pneumonia. CLINICAL FINDINGS: The dog initially presented with a persistent cough, inappetence and weight loss. Clinical findings included a fever, dyspnoea and tachypnoea, with haematological evidence of a mild neutrophilia and hypoalbuminaemia. Radiographs of the chest demonstrated a concomitant pneumothorax, pleural effusion, and a consolidated area within the left caudal lung lobe. An exploratory thoracotomy revealed this to be a ruptured granulomatous lesion. Subsequent histopathological, microbiological and genetic studies identified M. bovis as the causal agent. CLINICAL SIGNIFICANCE: Mycobacterium bovis infections should be included in the differential diagnosis of pulmonary disease and pleural effusions in dogs living in regions of New Zealand known to have a high incidence of mycobacterial infection in wildlife and farm animals.

Progressive myelopathy and neuropathy in New Zealand Huntaway dogs.

AIM: To investigate the nature and cause of a progressive ataxia in three 20-month-old Huntaway dogs that were litter mates. METHODS: Affected dogs were examined before they were humanely killed and submitted to necropsy. Selected formalin-fixed tissues were examined by light and electron microscopy. RESULTS: The lesions were those of axon and myelin degeneration within sensory, proprioceptive and motor tracts of the spinal cord and to a lesser degree some peripheral nerves. CONCLUSION: A progressive myelopathy and neuropathy, tentatively described as a central-peripheral distal axonopathy, was present in all 3 dogs. The cause was not determined but was likely to be either genetic or nutritional. CLINICAL RELEVANCE: In the early stages of this disease, careful examination may be necessary to distinguish the signs of ataxia from orthopaedic disease such as hip dysplasia. Affected animals are unlikely to be of use as working dogs.