Total infectome investigation of diphtheritic stomatitis in yellow-eyed penguins (Megadyptes antipodes) reveals a novel and abundant megrivirus.

First identified in 2002, diphtheritic stomatitis (DS) is a devastating disease affecting yellow-eyed penguins (Megadyptes antipodes, or hoiho in te reo Maori). The disease is associated with oral lesions in chicks and has caused significant morbidity and mortality. DS is widespread among yellow-eyed penguin chicks on mainland New Zealand yet appears to be absent from the subantarctic population. Corynebacterium spp. have previously been suspected as causative agents yet, due to inconsistent cultures and inconclusive pathogenicity, their role in DS is unclear. Herein, we used a metatranscriptomic approach to identify potential causative agents of DS by revealing the presence and abundance of all viruses, bacteria, fungi and protozoa - together, the infectome. Oral and cloacal swab samples were collected from presymptomatic, symptomatic and recovered chicks along with a control group of healthy adults. Two novel viruses from the Picornaviridae were identified, one of which - yellow-eyed penguin megrivirus - was highly abundant in chicks irrespective of health status but not detected in healthy adults. Tissue from biopsied oral lesions also tested positive for the novel megrivirus upon PCR. We found no overall clustering among bacteria, protozoa and fungi communities at the genus level across samples, although Paraclostridium bifermentans was significantly more abundant in oral microbiota of symptomatic chicks compared to other groups. The detection of a novel and highly abundant megrivirus has sparked a new line of inquiry to investigate its potential association with DS.

Ultrasonographic features of the regressing yolk sac of normal captive Brown kiwi chicks (Apteryx mantelli) vary with age.

Brown kiwi (Apteryx mantelli) are a culturally significant ratite species endemic to New Zealand. Chicks can develop diseases of the yolk sac which can be fatal if not promptly recognized and treated. Ultrasound examination of the yolk sac is an important component in the evaluation of chicks with suspected yolk sac disease. However, there are currently no reports of the normal ultrasonographic appearance of the regressing yolk sac in this species. The objective of this prospective reference interval study was to describe the normal ultrasonographic appearance of the yolk sac in brown kiwi chicks. Focused coelomic sonographic examinations were performed on 29 healthy chicks from >1 day of age until the yolk sac was completely absorbed or was <1 mL in volume. 41.6% (57/137) of yolks were round/ovoid in shape and 58.6% (80/137) had lobulated margins with invaginations. All yolks (137/137) were hyperechoic relative to adjacent coelomic structures. The heterogeneity of the echogenicity of the yolk was significantly associated with age (increased heterogeneity in younger birds) and was homogeneous, mildly heterogeneous, and moderately heterogeneous in 33% (46/138), 46% (63/138), and 21% (29/138) of all examinations respectively. Intrasac anechoic pockets were seen in 14% (20/139) of yolk sacs. The rate of yolk sac resorption slowed with age. In all examinations identifying a completely absorbed yolk sac or a yolk sac <1 mL in volume, chicks were less than 21 days old. The results of this study provide a reference of normal for the sonographic appearance of the yolk sac in captive brown kiwi chicks.

Molecular characterisation and additional morphological descriptions of Eimeria spp. (Apicomplexa: Eimeriidae) from brown kiwi (Apteryx mantelli Bartlett).

Brown kiwi (Apteryx mantelli Bartlett), a ratite endemic to New Zealand, is currently listed as "Vulnerable" under the IUCN classification system due to predation by introduced mammals. Operation Nest Egg (ONE) raises chicks and juveniles in predator-proof enclosures until they are large enough to defend themselves. These facilities experience an environmental accumulation of coccidial oocysts, which leads to severe morbidity and mortality of these kiwi. Four species of coccidia have been morphologically described from sporulated oocysts with additional opportunistic descriptions of endogenous stages. This research continues the morphological descriptions of these species of Eimeria with an additional novel morphotype also morphologically described. It also provides the first genetic characterisation targeting the mitochondrial cytochrome c oxidase I (COI) gene. Based on these findings, it was determined there are at least five morphotypes of Eimeria that infect brown kiwi and co-infections are common at the ONE facilities surveyed. The COI amplicon targeted for this study was sufficient to provide differentiation from other members of this genus. Sanger sequencing yielded ambiguous bases, indicating the need for more in-depth sequencing.

A novel gyrovirus is abundant in yellow-eyed penguin (Megadyptes antipodes) chicks with a fatal respiratory disease.

Yellow-eyed penguins (Megadyptes antipodes), or hoiho in te reo Maori, are predicted to become extinct on mainland Aotearoa New Zealand in the next few decades, with infectious disease a significant contributor to their decline. A recent disease phenomenon termed respiratory distress syndrome (RDS) causing lung pathology has been identified in very young chicks. To date, no causative pathogens for RDS have been identified. In 2020 and 2021, the number of chick deaths from suspected RDS increased four- and five-fold, respectively, causing mass mortality with an estimated mortality rate of >90%. We aimed to identify possible pathogens responsible for RDS disease impacting these critically endangered yellow-eyed penguins. Total RNA was extracted from tissue samples collected during post-mortem of 43 dead chicks and subject to metatranscriptomic sequencing and histological examination. From these data we identified a novel and highly abundant gyrovirus (Anelloviridae) in 80% of tissue samples. This virus was most closely related to Gyrovirus 8 discovered in a diseased seabird, while other members of the genus Gyrovirus include Chicken anaemia virus, which causes severe disease in juvenile chickens. No other exogenous viral transcripts were identified in these tissues. Due to the high relative abundance of viral reads and its high prevalence in diseased animals, it is likely that this novel gyrovirus is associated with RDS in yellow-eyed penguin chicks.

Toxoplasma gondii Exposure Prevalence in Little Spotted Kiwi (Apteryx owenii).

Toxoplasma gondii has been reported as a cause of morbidity and mortality in New Zealand's native avifauna, including the ground-dwelling Kiwi (Apteryx spp.). To better understand the extent of T. gondii infection in Little Spotted Kiwi (Apteryx owenii), a prevalence survey of kiwi living inside a 200-ha predator-proof mainland ecosanctuary (Zealandia Te Mara a Tane, Wellington, New Zealand) was undertaken. Antibodies to T. gondii were detected by a latex agglutination test (LAT) with a cutoff positive titer of ≥1:64, and T. gondii DNA was detected by PCR. In total, 16/19 (84.2%) birds tested were positive for T. gondii by LAT (10/11), PCR (10/19), or both (4/11). Antibody titers ranged from 1:32 to ≥1:2,048. These results suggest widespread exposure of T. gondii in this population of Little Spotted Kiwi and, in conjunction with earlier reports of toxoplasmosis causing mortality in kiwi, raise important questions as to the effect this parasite may be having on this rare endemic species. Further information on the epidemiology of T. gondii infections within free-living and managed kiwi populations is urgently needed.

Comparing the Mini-FLOTAC and centrifugal faecal flotation for the detection of coccidia (Eimeria spp.) in kiwi (Apteryx mantelli).

Coccidia (Eimeria spp.) in brown kiwi (Apteryx mantelli) cause significant morbidity and mortality in captive rearing facilities. Monitoring the abundance of this parasite in individual birds is crucial for successful management of kiwi. This research compares the abilities of centrifugal faecal flotations (CFF) and a modified Mini-FLOTAC protocol to detect oocysts. We hypothesised that the Mini-FLOTAC would detect higher oocyst counts. Kiwi dropping samples (n = 10) were homogenized in MgSO4 (SG 1.28) and oocyst counts made with CFFs and Mini-FLOTAC counting chambers, with three replicates for each method. For CFF, 0.5 g of droppings were examined using standard methods. Mini-FLOTAC counts were made using a modified sample preparation compared with the manufacturer's protocol but still used a 1:20 dilution of droppings. Oocysts were quantified using light microscopy at ×100-300 magnification. A linear mixed-effects model by REML showed that oocyst per gram estimates via the Mini-FLOTAC method were 3.2 times higher (95% CI 2.4-4.5, p < 0.01) than the CFF results. This increased detection likely represents a more accurate estimation of parasite shedding and should be considered for use in research or applications requiring more accuracy, cost-effectiveness, or accessibility than the CFF provides.

Post-release breeding success of oil-rehabilitated and non-rehabilitated little blue penguins, Eudyptula minor, following the M/V Rena oil spill, New Zealand.

During the 2011 M/V Rena oil spill in Tauranga, New Zealand, 383 little blue penguins (LBP, Eudyptula minor) were oiled, rescued and rehabilitated, with 90.6% surviving to be released back to the wild. We monitored the post-release breeding success of rehabilitated LBPs to assess the effectiveness of the rehabilitation process. Breeding success was monitored during the first breeding season post-release (2012-13) to assess productivity for both rehabilitated and control penguins (breeding pairs of rehabilitated or non-oiled (control) birds). Egg laying, hatching and fledging success rates for pairs of rehabilitated and control penguins were both within ranges reported for other LBP colonies in Australia and New Zealand. The only significant difference between rehabilitated and control pairs was that hatching success was reduced in the first season after release for rehabilitated pairs. Overall, rehabilitation appeared to reverse most potential negative effects of oiling on the post-release breeding success of rehabilitated LBP.

Survival rates of oil-rehabilitated and non-rehabilitated little penguins after the C/V Rena oil spill, New Zealand.

We monitored the post-release survival of little penguins (Eudyptula minor) rehabilitated after the 2011 C/V Rena oil spill in New Zealand to assess the effectiveness of the rehabilitation process. Surveys were conducted over a 23-month period after the spill to assess whether survival differed between rehabilitated and control penguins. Survival probabilities from mark-recapture analyses were lower for both oiled and control penguins in the first six months of the study (monthly probability 0.92) but increased and remained high thereafter (monthly probabilities 0.97-1.0). Importantly, survival did not differ significantly between oiled and control birds throughout the study. Post-release survival of rehabilitated birds was not influenced by the degree of oiling, body mass (at admission or release), blood parameters (admission packed cell volume, total protein or blood glucose) or the duration of captivity. Rehabilitation therefore appeared to successfully reverse the negative effects of oiling on the post-release survival of treated penguins.

Efficacy of seawater for washing oiled birds during an oil spill response.

Aquatic pollution events can be detrimental to the survival of wildlife, particularly birds. To decontaminate affected birds, large quantities of fresh water are required. A recent study using seabird feathers, demonstrated that seawater wash/rinse can effectively remove oil from feathers. However to determine whether seawater was effective for live birds, we used 36 mallard ducks to replicate the oiled feather wash/rinse study. We investigated the time and volume of water used, bird water-proofing scores after daily swims and a barbule amalgamation index (BAI), for feathers collected at stages throughout the process. Results indicate that for oiled mallard ducks, the use of seawater for decontamination wash/rinse was effective. Seawater wash however, took longer and used a greater quantity of water. Time to birds being waterproof, was not significantly different between groups. The use of seawater has worldwide application for oiled wildlife response activities particularly in areas where freshwater supplies are limited.

DIPHTHERITIC STOMATITIS IN YELLOW-EYED PENGUINS (MEGADYPTES ANTIPODES) IN NEW ZEALAND.

Diphtheritic stomatitis is a seasonal disease that has been recognized as a syndrome in Yellow-eyed Penguin ( Megadyptes antipodes ) chicks in New Zealand for >10 yr. It was present in about 50% of 234 chicks examined since 2002 and is characterized by a thick serocellular exudate in the oral cavity of 1-4-wk-old chicks. The syndrome includes inanition, weight loss, and death in many affected birds. Microscopically, the lesions varied in severity. Most affected chicks had severe, locally extensive, ulcerative stomatitis with large amounts of exudate containing numerous bacteria; a smaller number had mild focal lesions with smaller amounts of exudate and bacteria. Although Corynebacterium amycolatum has been consistently isolated from the oral lesions, it was also present in the oral cavity of 34% of normal adult penguins and their chicks and is not known to possess diphtheritic toxins. A primary viral pathogen was therefore suspected, and intracytoplasmic inclusion bodies were occasionally seen in oral mucosal epithelial cells. No herpesvirus DNA was detected with PCR. Avipoxvirus DNA and an unidentified virus-like agent were detected in some early oral lesions, but could not be confirmed in subsequent testing. Electron microscopy on early affected epithelium with intracytoplasmic inclusion bodies was unrewarding. Our findings raise the possibility that the disease is caused by an unknown primary virus infection followed by secondary Corynebacterium invasion, but this requires confirmation. The means of transmission has not been established but insect vectors are suspected.

Extra-intestinal coccidiosis in the kiwi (Apteryx spp.).

Despite significant conservation intervention, the kiwi (Apteryx spp.) is in serious population decline. To increase survival in the wild, conservation management includes rearing of young birds in captivity, safe from introduced mammalian predators. However, an increase in density of immunologically naïve kiwi increases the risk of exposure to disease, including coccidia. Intestinal coccidiosis has recently been described in the kiwi, and although extra-intestinal coccidiosis was first recognized in kiwi in 1978, very little is known about this disease entity. This study used archived histological tissues and reports from routine necropsies to describe the pathology of naturally occurring extra-intestinal coccidiosis. At least 4.5% of all kiwi necropsied during 1991 to 2011 (n=558) were affected by extra-intestinal coccidiosis, and it is estimated that it caused death in 0.9 to 1.2% of kiwi in the study group. Four forms were recognized: renal, hepatic, and, less commonly, splenic and pulmonary. At necropsy, renal coccidiosis was associated with miliary white streaks and foci through the kidneys, renomegaly, and renal pallor or congestion. Renal meronts and gametocytes were confined to the distal convoluted tubules and collecting ducts, and were associated with renal tubular necrosis and tubular obstruction. Hepatic miliary pinpoint foci were present throughout the hepatic parenchyma associated microscopically with macromeronts measuring 304×227 µm. In two cases, clusters of splenic meronts were identified, and a similar lesion was identified in the pulmonary interstitium of another case. Juvenile, captive kiwi were most often affected with extra-intestinal coccidiosis, illustrating an increased expression of disease with population manipulation for conservation purposes.

The role of lead in a syndrome of clenched claw paralysis and leg paresis in Swamp Harriers (Circus approximans).

We investigated the hypothesis that lead poisoning was the cause of the clinical syndrome of clenched feet paralysis and leg paresis in wild raptors. Swamp Harriers (Circus approximans) are one of three extant native raptor species in New Zealand. Harriers with the syndrome were found to have statistically significantly higher blood lead concentrations than those without clenched feet (t-test; t=-4.06, df=5, P=0.01). However, elevated blood lead concentrations were also present in 60% of wild harriers without the clinical syndrome of clenched feet paralysis and leg paresis. There were features of the response to chelation treatment, electroneurodiagnostics, and pathology that were inconsistent with lead poisoning as reported in other birds of prey. We conclude that lead may be a factor in the expression of this clinical syndrome of clenched claw paralysis but that other factors not identified in our study play a role in the expression of the disease.