Feline Bowenoid in situ carcinoma (BISC) is a rare disease that presents as multiple discrete plaques of epidermal hyperplasia and dysplasia. Two studies using immunohistochemistry revealed papillomaviral antigens in 11% and 47% of BISCs. Additionally, a recent study detected papillomaviral DNA in 24% of BISC lesions. To further investigate the association between papillomaviruses and BISC, polymerase chain reaction using consensus primers was used to detect papillomaviral DNA in 18 formalin-fixed samples of BISC. Papillomaviral DNA was amplified from 11 of the samples but from none of the controls. Six amplicons were sequenced; one was homologous with a papillomavirus from a human patient with multiple cutaneous squamous cell carcinomas and the other five showed weak homology to human papillomavirus type 17. These five sequences were > 96% homologous over a 235 bp sequence, indicating the presence in all five BISCs of one papillomavirus type distinct from any previously sequenced and more closely related to human than animal papillomaviruses. The results confirm an association between BISC and papillomaviruses, and as all six papillomavirus sequences identified are closely related to human papillomaviruses, it is possible that the virus is transmitted from humans to cats or vice versa.

Squamous cell carcinomas (SCCs) are common skin tumours of cats. Previous studies have suggested that papillomaviral (PV) DNA is detectible within some feline SCCs. A PV DNA sequence has been previously amplified from five feline bowenoid in situ carcinomas (BISCs). Primers specific for this sequence were used in a nested polymerase chain reaction to compare PV detection rates in SCCs to rates within non-SCC skin lesions. Papillomaviral DNA was amplified from 20 of 20 BISC, 17 of 20 invasive SCC and 3 of 17 non-SCC controls. The rate of PV amplification from feline cutaneous SCCs was significantly higher than from non-SCC lesions. These results confirm that feline cutaneous SCCs are associated with PV infection. In humans, there is evidence that PVs promote SCC development within sun-exposed skin. The demonstrated association between PVs and feline cutaneous SCCs suggests, but does not prove, that PVs may also promote feline SCC development. If PVs are oncogenic in cats, prevention of PV infection may reduce feline cutaneous SCC development. To the authors' knowledge, this is the first time that PV DNA has been amplified from a non-SCC sample of feline skin.

Avian malaria is caused by intracellular mosquito-transmitted protist parasites in the order Haemosporida, genus Plasmodium. Although Plasmodium species have been diagnosed as causing death in several threatened species in New Zealand, little is known about their ecology and epidemiology. In this study, we examined the presence, microscopic characterization and sequence homology of Plasmodium spp. isolates collected from a small number of New Zealand introduced, native and endemic bird species. We identified 14 Plasmodium spp. isolates from 90 blood or tissue samples. The host range included four species of passerines (two endemic, one native, one introduced), one species of endemic pigeon and two species of endemic kiwi. The isolates were associated into at least four distinct clusters including Plasmodium (Huffia) elongatum, a subgroup of Plasmodium elongatum, Plasmodium relictum and Plasmodium (Noyvella) spp. The infected birds presented a low level of peripheral parasitemia consistent with chronic infection (11/15 blood smears examined). In addition, we report death due to overwhelming parasitemia in a blackbird, a great spotted kiwi and a hihi. These deaths were attributed to infections with either Plasmodium spp. lineage LINN1 or P. relictum lineage GRW4. To the authors’ knowledge, this is the first published report of Plasmodium spp. infection in great spotted and brown kiwi, kereru and kokako. Currently, we are only able to speculate on the origin of these 14 isolates but consideration must be made as to the impact they may have on threatened endemic species, particularly due to the examples of mortality.

Equine infectious anemia virus (EIAV) infection of horses is characterized by well-defined waves of viremiaassociated with the sequential evolution of distinct viral populations displaying extensive envelope gp90 variation; however, a correlation of in vivo envelope evolution with in vitro serum neutralization phenotype remains undefined. Therefore, the goal of the present study was to utilize a previously defined panel of natural variant EIAV envelope isolates from sequential febrile episodes to characterize the effects of envelope variation during persistent infection on viral neutralization phenotypes and to define the determinants of EIAV envelope neutralization specificity. To assess the neutralization phenotypes of the sequential EIAV envelope variants, we determined the sensitivity of five variant envelopes to neutralization by a longitudinal panel of immune serum from the source infected pony. The results indicated that the evolution of the EIAV envelope sequences observed during sequential febrile episodes produced an increasingly neutralization-resistant phenotype. To further define the envelope determinants of EIAV neutralization specificity, we examined the neutralization properties of a panel of chimeric envelope constructs derived from reciprocal envelope domain exchanges between selected neutralization-sensitive and neutralization-resistant envelope variants. These results indicated that the EIAV gp90 V3 and V4 domains individually conferred serum neutralization resistance while other envelope segments in addition to V3 and V4 were evidently required for conferring total serum neutralization sensitivity. These data clearly demonstrate for the first time the influence of sequential gp90 variation during persistent infection in increasing envelope neutralization resistance, identify the gp90 V3 and V4 domains as the principal determinants of antibody neutralization resistance, and indicate distinct complex cooperative envelope domain interactions in defining sensitivity to serum antibody neutralization.

Previous evaluations of inactivated whole-virus and envelope subunit vaccines to equine infectious anemia virus (EIAV) have revealed a broad spectrum of efficacy ranging from highly type-specific protection to severe enhancement of viral replication and disease in experimentally immunized equids. Among experimental animal lentivirus vaccines, immunizations with live attenuated viral strains have proven most effective, but the vaccine efficacy has been shown to be highly dependent on the nature and severity of the vaccine virus attenuation. We describe here for the first time the characterization of an experimental attenuated proviral vaccine, EIAV UK ⌬S2, based on inactivation of the S2 accessory gene to down regulate in vivo replication without affecting in vitro growth properties. The results of these studies demonstrated that immunization with EIAV UK ⌬S2 elicited mature virus-specific immune responses by 6 months and that this vaccine immunity provided protection from disease and detectable infection by intravenous challenge with a reference virulent biological clone, EIAV PV . This level of protection was observed in each of the six experimental horses challenged with the reference virulent EIAV PV by using a low-dose multiple-exposure protocol (three administrations of 10 median horse infectious doses [HID 50 ], intravenous) designed to mimic field exposures and in all three experimentally immunized ponies challenged intravenously with a single inoculation of 3,000 HID 50 . In contrast, naïve equids subjected to the low-or high-dose challenge develop a detectable infection of challenge virus and acute disease within several weeks. Thus, these data demonstrate that the EIAV S2 gene provides an optimal site for modification to achieve the necessary balance between attenuation to suppress virulence and replication potential to sufficiently drive host immune responses to produce vaccine immunity to viral exposure.

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Yellow-eyed penguins on Stewart Island were identified with a Leucocytozoon spp. of a novel lineage in association with a high regional incidence of chick mortality (n=32, 100% mortality) during the November 2006 to January 2007 breeding season. Fourteen chicks from Stewart Island were examined post-mortem and histologically for Leucocytozoon infection. In addition, a survey of blood to detect Leucocytozoon spp. infections using PCR was performed on 107 yellow-eyed penguins from 4 distinct nesting areas on the South Island (Oamaru, Otago Peninsula, and Catlins) (n=95), and Stewart Island (n=12). The results of the study revealed that 2 of the 14 (14%) chicks necropsied showed severe, disseminated megaloschizont formation in the liver, spleen, lung, kidney and other tissues characteristic of leucocytozoonosis. Eighty-three percent (83%) of blood samples collected from Stewart Island penguins contained Leucocytozoon DNA, whereas samples from the 3 other nesting areas were negative for the blood parasite. Leucocytozoon spp. DNA sequences isolated from blood and tissues of adults (n=10) and chicks (n=7) were similar and grouped with other published Leucocytozoon spp. sequences but in a distinct cluster together with closely related isolates from a Western march harrier (Circus aerginosus) and common loon (Gavia immer). These findings suggest that yellow-eyed penguins on Stewart Island are infected with a regionally isolated, host-specific Leucocytozoon spp. which may contribute to the high chick mortality observed during this period.

1 Avian malaria and related haemosporidian parasites (genera Haemoproteus, Plasmodium, and 2 Leucocytozoon) affect bird demography, species range limits, and community structure, yet they 3 remain unsurveyed in most bird communities and populations. We conducted a community-level 4 survey of these vector-transmitted parasites in New Mexico, USA, to describe their diversity, 5 abundance, and host associations. We focused on the breeding-bird community in the transition 6 zone between piñon-juniper woodland and ponderosa pine forests (elevational range: 2150-2460 7 meters). We screened 186 birds representing 49 species using both standard PCR and microscopy 8 techniques to detect infections of all three avian haemosporidian genera. We detected infections 9 in 68 out of 186 birds (36.6%), the highest proportion of which were infected with Haemoproteus 10 (20.9%), followed by Leucocytozoon (13.4%), then Plasmodium (8.0%). We sequenced mtDNA 11 for 77 infections representing 43 haplotypes (25 Haemoproteus, 12 Leucocytozoon, 6 12 Plasmodium). When compared to all previously known haplotypes in the MalAvi and GenBank 13 databases, 63% (27) of the haplotypes we recovered were novel. We found evidence for host 14 specificity at the avian clade and species level, but this specificity was variable among parasite 15 genera, in that Haemoproteus and Leucocytozoon were each restricted to three avian groups (out 16 of six), while Plasmodium occurred in all groups except non-passerines. We found striking 17 variation in infection rate among host species, with nearly universal infection among vireos and 18 no infection among nuthatches. Using rarefaction and extrapolation, we estimated the total avian 19 haemosporidian diversity to be 70 haplotypes (95% CI: 43-98); thus, we may have already 20 sampled ~60% of the diversity of avian haemosporidians in New Mexico pine forests. It is 21 possible that future studies will find higher diversity in microhabitats or host species that are 22 under-sampled or unsampled in the present study. Fortunately, this study is fully extendable via 23 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2997v2 | CC BY 4.0 Open Access | rec