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.

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.

Comparison of PCR assays to detect Toxoplasma gondii oocysts in green-lipped mussels (Perna canaliculus).

Toxoplasma gondii is recognised as an important pathogen in the marine environment, with oocysts carried to coastal waters in overland runoff. Currently, there are no standardised methods to detect T. gondii directly in seawater to assess the extent of marine ecosystem contamination, but filter-feeding shellfish may serve as biosentinels. A variety of PCR-based methods have been used to confirm presence of T. gondii DNA in marine shellfish; however, systematic investigations comparing molecular methods are scarce. The primary objective of this study was to evaluate analytical sensitivity and specificity of two nested-PCR (nPCR) assays targeting dhps and B1 genes and two real-time (qPCR) assays targeting the B1 gene and a 529-bp repetitive element (rep529), for detection of T. gondii. These assays were subsequently validated for T. gondii detection in green-lipped mussel (Perna canaliculus) haemolymph using oocyst spiking experiments. All assays could reliably detect 50 oocysts spiked into mussel haemolymph. The lowest limit of detection was 5 oocysts using qPCR assays, with the rep529 primers performing best, with good correlation between oocyst concentrations and Cq values, and acceptable efficiency. Assay specificity was evaluated by testing DNA from closely related protozoans, Hammondia hammondi, Neospora caninum, and Sarcocystis spp. Both nPCR assays were specific to T. gondii. Both qPCR assays cross-reacted with Sarcocystis spp. DNA, and the rep529 primers also cross-reacted with N. caninum DNA. These studies suggest that the rep529 qPCR assay may be preferable for future mussel studies, but direct sequencing is required for definitive confirmation of T. gondii DNA detection.

Investigation of Toxoplasma gondii and association with early pregnancy and abortion rates in New Zealand farmed red deer (Cervus elaphus).

This study tested for association between Toxoplasma gondii and pregnancy and abortion to investigate sub-optimal reproduction in farmed red deer (Cervus elaphus). Sera from a sub-sample (n = 2304) of pregnant and non-pregnant hinds in early gestation at first pregnancy scan (scan 1) and approximately at the end of second trimester at second pregnancy scan (scan 2) were tested for T. gondii antibodies using a validated ELISA. Foetuses and/or uteri from pregnant, non-pregnant, and aborting hinds at scan 1, scan 2, or weaning were tested for T. gondii DNA by nested PCR. At scan 1, 31.1% of 861 rising two-year-old (R2) and 28.3% of 357 mixed-aged (MA, ≥ 2 years) hinds were sero-positive. There was no association between scan 1 serology and non-pregnancy at animal (R2, p = 0.05 and MA, p = 0.43) or herd level (R2, p = 0.37). Toxoplasma gondii DNA was detected in 3/18 placenta and 4/18 foetal brains from aborting R2 hinds and 15/157 R2 and 3/21 MA uteri from non-pregnant hinds at scan 1. At scan 2, sero-prevalence was higher (odds ratio = 1.6, 95% CI = 1.04-2.48) in aborted (34.3% of 268) than in non-aborted (23.5% of 446) R2 hinds (p = 0.03) and 7.9% of abortions between scans were attributable to T. gondii exposure. Within-herd sero-prevalence at scan 2 was positively associated with daily abortion rate in R2 herds with aborted hinds (p < 0.001) but not in MA herds (p = 0.07). Toxoplasma gondii DNA was detected in 27/169 uteri, 2/20 cotyledons, and 1/5 foetal brains from aborted hinds at scan 2 and in uteri from 5/33 hinds not rearing a calf to weaning. Toxoplasma gondii RFLP genotyping of five loci revealed a unique type I/III genotype pattern, TgRDNZ1, in a foetal brain sample, not been previously reported in deer. These findings provide serological and molecular evidence that T. gondii infection is associated with abortion in red deer, possibly in all three trimesters.

First report of Toxoplasma gondii sporulated oocysts and Giardia duodenalis in commercial green-lipped mussels (Perna canaliculus) in New Zealand.

Pollution of marine ecosystems with the protozoan parasites Toxoplasma gondii, Cryptosporidium spp. and Giardia duodenalis can be studied using bivalve shellfish as biosentinels. Although evidence suggests that these parasites are present in New Zealand coastal waters, the extent of protozoal pollution has not been investigated. This study used optimised molecular methods to detect the presence of Cryptosporidium spp., G. duodenalis and T. gondii in commercially sourced green-lipped mussel (Perna canaliculus), an endemic species found throughout coastal New Zealand. A nested polymerase chain reaction was validated for detection of T. gondii DNA and applied to 104 commercially sourced mussels. Thirteen mussels were positive for T. gondii DNA with an estimated true prevalence of 16.4% using Bayesian statistics, and the presence of T. gondii in mussels was significantly associated with collection during the summer compared with that in the winter (P = 0.003). Consumption of contaminated shellfish may also pose a health risk for humans and marine wildlife. As only sporulated T. gondii oocysts can be infectious, a reverse transcriptase-polymerase chain reaction was used to confirm presence of a sporozoite-specific marker (SporoSAG), detected in four mussels. G. duodenalis assemblage B, known to be pathogenic in humans, was also discovered in 1% mussels, tested by polymerase chain reaction (n = 90). Cryptosporidium spp. was not detected in the sampled mussel haemolymph. Results suggest that New Zealand may have high levels of coastal contamination with T. gondii, particularly in summer months, and that naturally exposed mussels can ingest and retain sporulated oocysts, further establishing shellfish consumption as a health concern.

Investigation of a mortality cluster in wild adult yellow-eyed penguins (Megadyptes antipodes) at Otago Peninsula, New Zealand.

We investigated an epidemic mortality cluster of yellow-eyed penguins (Megadyptes antipodes) that involved 67 moribund or dead birds found on various beaches of the Otago Peninsula, New Zealand, between 21 January and 20 March 2013. Twenty-four carcases were examined post-mortem. Histological lesions of pulmonary, hepatic and splenic erythrophagocytosis and haemosiderosis were found in 23 of 24 birds. Fifteen birds also had haemoglobin-like protein droplets within renal tubular epithelial cells. Despite consistent histological lesions, a cause of death could not be established. Virology, bacteriology and molecular tests for avian influenza, avian paramyxovirus-1, avipoxvirus, Chlamydia psittaci, Plasmodium spp., Babesia spp., Leucocytozoon spp. and Toxoplasma gondii were negative. Tissue concentrations of a range of heavy metals (n = 4 birds) were consistent with low level exposure, while examination of proventricular contents and mucus failed to detect any marine biotoxins or Clostridium botulinum toxin. Hepatic concentrations of total polycyclic aromatic hydrocarbons (PAHs) (n = 5 birds) were similar to background concentrations of polycyclic aromatic hydrocarbons previously found in yellow-eyed penguins from the South Island of New Zealand, but there were significantly higher concentrations of 1-methylnapthelene and 2-methylnapthelene in the birds found dead in this mortality cluster. The biological significance of this finding is unclear. A temporal investigation of the epidemic did not indicate either a common source or propagative epidemic pattern. Although our investigation did not definitively implicate a toxic or infectious agent, we could not rule out causes such as toxic marine organisms or mycoplasmosis. Further investigations should therefore by carried out in the event of future mortality clusters.

Brucellosis in Endangered Hector's Dolphins (Cephalorhynchus hectori).

Brucella spp infections of marine mammals are often asymptomatic but have been associated with reproductive losses and deaths. Zoonotic infections originating from marine isolates have also been described. Hector's dolphins (Cephalorhynchus hectori) are an endangered species with a declining population, and the role of infectious disease in population dynamics is not fully understood. In this study, 27 Hector's dolphins found dead around the New Zealand coastline between November 2006 and October 2010 were evaluated for lesions previously associated with cetacean brucellosis. Tissues were examined using histological, immunohistochemical, and molecular (polymerase chain reaction [PCR]) techniques. Seven of 27 dolphins (26%) had at least 1 tissue that was positive on PCR for Brucella spp. Lesions consistent with brucellosis were present in 10 of 27 (37%) dolphins, but in 8 of these dolphins Brucella infection could not be demonstrated in lesional tissues. Two dolphins (7%) were diagnosed with active brucellosis: 1 female with placentitis and metritis, and 1 stillborn male fetus. Brucella identified in these 2 dolphins had genetic similarity (99%) to Brucella pinnipedialis. The omp2a gene amplicon from the uterus of the female had 100% homology with ST27 genotype isolates from a human in New Zealand and a bottlenose dolphin of Pacific origin. The remaining 5 PCR-positive dolphins were assessed as having asymptomatic or latent infection. While most Brucella infections identified in this study appeared to be subclinical, the finding of 2 dolphins with reproductive disease due to Brucella infection suggests that this disease has the potential to affect reproductive success in this species.

Using a common commensal bacterium in endangered Takahe as a model to explore pathogen dynamics in isolated wildlife populations.

Predicting and preventing outbreaks of infectious disease in endangered wildlife is problematic without an understanding of the biotic and abiotic factors that influence pathogen transmission and the genetic variation of microorganisms within and between these highly modified host communities. We used a common commensal bacterium, Campylobacter spp., in endangered Takahe (Porphyrio hochstetteri) populations to develop a model with which to study pathogen dynamics in isolated wildlife populations connected through ongoing translocations. Takahe are endemic to New Zealand, where their total population is approximately 230 individuals. Takahe were translocated from a single remnant wild population to multiple offshore and mainland reserves. Several fragmented subpopulations are maintained and connected through regular translocations. We tested 118 Takahe from 8 locations for fecal Campylobacter spp. via culture and DNA extraction and used PCR for species assignment. Factors relating to population connectivity and host life history were explored using multivariate analytical methods to determine associations between host variables and bacterial prevalence. The apparent prevalence of Campylobacter spp. in Takahe was 99%, one of the highest reported in avian populations. Variation in prevalence was evident among Campylobacter species identified. C. sp. nova 1 (90%) colonized the majority of Takahe tested. Prevalence of C. jejuni (38%) and C. coli (24%) was different between Takahe subpopulations, and this difference was associated with factors related to population management, captivity, rearing environment, and the presence of agricultural practices in the location in which birds were sampled. Modeling results of Campylobacter spp. in Takahe metapopulations suggest that anthropogenic management of endangered species within altered environments may have unforeseen effects on microbial exposure, carriage, and disease risk. Translocation of wildlife between locations could have unpredictable consequences including the spread of novel microbes between isolated populations.

Four cases of fatal toxoplasmosis in three species of endemic New Zealand birds.

Four cases of fatal toxoplasmosis in three endemic New Zealand avian species are reported. Between 2009 and 2012, two kereru (Hemiphaga novaeseelandiae), one North Island brown kiwi (Apteryx mantelli), and one North Island kaka (Nestor meridionalis) were submitted for necropsy examination. On gross postmortem, the kiwi had marked hepatosplenomegaly while the kaka and two kereru had swollen, slightly firm, deep-red lungs. Histologically there was extensive hepatocellular necrosis in the liver of the kiwi while the kaka and kereru showed severe fibrinous bronchointerstitial pneumonia. In the kiwi, protozoal organisms were present within both hepatocytes and Kupffer cells of the liver and within the epithelial cells and macrophages of the interstitium of the lungs in the kaka and two kereru. The diagnosis of toxoplasmosis was confirmed with immunohistochemistry and PCR of paraffin-embedded formalin-fixed tissue of the liver, lungs, or both. Genotyping of up to seven markers revealed that an atypical Type II isolate of Toxoplasma gondii was present in at least three of the cases. This study provides evidence that T. gondii can cause mortality in these endemic species and suggests further research is needed to determine the full extent of morbidity and mortality caused by this parasite in New Zealand's unique avifauna.

Disease screening of three breeding populations of adult exhibition budgerigars (Melopsittacus undulatus) in New Zealand reveals a high prevalence of a novel polyomavirus and avian malaria infection.

Disease surveillance is vital to the management of New Zealand's endemic and threatened avian species. Three infectious agents that are potential threats to New Zealand's endemic birds include avian polyomavirus (APV), beak and feather disease virus (BFDV), and avian malaria. All three agents have been reported in New Zealand; however, possible reservoir populations have not been identified. In this communication, we report the first study of APV, BFDV, and avian malaria in introduced adult exhibition budgerigars (Melopsittacus undulatus) in New Zealand. Blood samples were collected from 90 living adult budgerigars from three breeding locations in the North Island of New Zealand. An overall APV prevalence of 22% was determined using a broad-spectrum nested PCR that amplified the major capsid protein VP1 gene of polyomavirus. Phylogenetic analysis of the VP1 gene revealed a unique isolate of APV, which had a sequence divergence of 32% to previously reported budgerigar fledgling disease strains and 33% to the recently reported New Zealand finch isolate. All of the budgerigars sampled were found to be PCR negative for BFDV, and an overall prevalence of 30% was detected by PCR for avian malaria. Sequencing revealed the presence of ubiquitous malarial strains and also the potentially destructive Plasmodium relictum strain. The results of this study suggest that both APV and avian malaria are present in New Zealand adult budgerigars, and our study highlights the need for further studies to determine whether these pathogens in captive bird populations may be a threat or spill over into New Zealand's endemic and threatened avifauna and whether prevention and control methods need to be implemented.

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.

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.

Malaria parasites (Plasmodium spp.) infecting introduced, native and endemic New Zealand birds.

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.

The Pathology of Fatal Avian Malaria Due to Plasmodium elongatum (GRW6) and Plasmodium matutinum (LINN1) Infection in New Zealand Kiwi (Apteryx spp.).

Avian malaria caused by Plasmodium species is a known cause of mortality in avifauna worldwide, however reports within New Zealand kiwi (Apteryx spp.) are scant. Postmortem reports from kiwi were obtained from the Massey University/Te Kunenga ki Purehuroa School of Veterinary Science Pathology Register from August 2010-August 2020. Gross lesions were described from postmortem reports, and archived H.E.-stained slides used for histological assessment. Nested PCR testing was performed on formalin-fixed paraffin-embedded tissue samples to assess the presence of Plasmodium spp. and Toxoplasma gondii DNA and cases with a PCR-positive result were sequenced to determine the lineage involved. Of 1005 postmortem reports, 23 cases of confirmed or suspected avian malaria were included in this study. The most consistent gross lesions included splenomegaly, hepatomegaly, and interstitial pneumonia with oedema. Histological lesions were characterised by severe interstitial pneumonia, pulmonary oedema, interstitial myocarditis, hepatic sinusoidal congestion and hypercellularity, and splenic macrophage hyperplasia and hyperaemia/congestion with numerous haemosiderophages. Cytoplasmic meronts were consistently found within endothelial cells of a variety of tissues, and within tissue macrophages of the liver, lung and spleen. A diagnosis of avian malaria was confirmed via PCR testing in 13 cases, with sequencing revealing P. matutinum (LINN1) and P. elongatum (GRW6) as the species involved. This is the largest case series describing the pathology of avian malaria as a cause of mortality in endemic New Zealand avifauna.

PREVALENCE AND PATHOGEN LOAD OF EIMERIA IN WILD YELLOW-EYED PENGUINS (MEGADYPTES ANTIPODES) AND THE MORPHOLOGIC CHARACTERIZATION OF A NOVEL EIMERIA SPECIES.

Coccidia infections in wild birds rarely cause clinical signs; however, disease and mortality can occur with predisposing environmental and host conditions. The Yellow-eyed Penguin (Megadyptes antipodes) is an endangered species endemic to New Zealand that has seen significant ongoing population decline. The aim of this study was to examine the host-pathogen dynamics of coccidian parasites in two wild populations of Yellow-eyed Penguin: the mainland (South Island) population and the sub-Antarctic (Enderby Island) population. There was weak evidence for a difference in the prevalence of the Eimeria sp. in birds from Enderby Island (76.6%; 36/47; 95% confidence interval [CI] 62.78-86.4%) and the South Island of New Zealand (58.54%; 24/41; 95% CI 43.37-72.24%). The mean pathogen load in penguins on Enderby Island was 9,723 oocysts/g of feces (SE=5831 oocysts/g) and from the South Island of New Zealand was 1,050 oocysts/g (SE=398 oocysts/g). No evidence of an association was found between pathogen load and body weight in either study population. The morphology of the sporulated coccidial oocysts was consistent with a novel species of Eimeria. There was statistically significant variation between the oocysts collected from the two sites in all measurements apart from the oocyst wall thickness. However, the standard technique of assessing linear regressions of the length and width of oocysts from both sampling sites was 0.80, and therefore above the standard R2>0.5 used to indicate variation within a single population of oocysts, suggesting that only a single species of Eimeria was present at both sampling locations. The prevalence and pathogen load of Eimeria sp. was substantially higher than previous reports of coccidial oocysts in Yellow-eyed Penguins and free-living Sphenisciformes globally. This host-parasite relationship deserves further investigation, as the impact of this novel organism on the population remains unclear.

The same papillomavirus is present in feline sarcoids from North America and New Zealand but not in any non-sarcoid feline samples.

Feline sarcoids are uncommon dermal neoplasms that are associated with papillomavirus (PV) infection. A single PV type, designated feline sarcoid-associated PV (FeSarPV), was detected in 9 feline sarcoids from North America. As FeSarPV has only been detected within feline sarcoids, the epidemiology of the infection remains unknown. The present study used polymerase chain reaction (PCR) to investigate whether this PV is also present within sarcoids from New Zealand cats. Additionally, as PVs are often host-specific, it was hypothesized that FeSarPV may often asymptomatically infect cats but rarely cause disease. To test this hypothesis, specific PCR primers were designed to investigate the presence of FeSarPV DNA within 120 samples from the skin and mouth of cats without sarcoids. Feline sarcoids from both New Zealand and North America contained FeSarPV DNA sequences. However, FeSarPV DNA was not detected within any non-sarcoid feline sample. To the authors' knowledge, this is the first time that FeSarPV has been reported in a country outside North America. As FeSarPV does not asymptomatically infect cats, feline sarcoids are likely due to cross-species infection. Although the reservoir host of FeSarPV is unknown, the host is present and has contact with cats, in both New Zealand and North America.

An EIAV field isolate reveals much higher levels of subtype variability than currently reported for the equine lentivirus family.

BACKGROUND: Equine infectious anemia virus (EIAV), a lentivirus that infects horses, has been utilized as an animal model for the study of HIV. Furthermore, the disease associated with the equine lentivirus poses a significant challenge to veterinary medicine around the world. As with all lentiviruses, EIAV has been shown to have a high propensity for genomic sequence and antigenic variation, especially in its envelope (Env) proteins. Recent studies have demonstrated Env variation to be a major determinant of vaccine efficacy, emphasizing the importance of defining natural variation among field isolates of EIAV. To date, however, published EIAV sequences have been reported only for cell-adapted strains of virus, predominantly derived from a single primary virus isolate, EIAVWyoming (EIAVWY). RESULTS: We present here the first characterization of the Env protein of a natural primary isolate from Pennsylvania (EIAVPA) since the widely utilized and referenced EIAVWY strain. The data demonstrated that the level of EIAVPA Env amino acid sequence variation, approximately 40% as compared to EIAVWY, is much greater than current perceptions or published reports of natural EIAV variation between field isolates. This variation did not appear to give rise to changes in the predicted secondary structure of the proteins. While the EIAVPA Env was serologically cross reactive with the Env proteins of the cell-adapted reference strain, EIAVPV (derivative of EIAVWY), the two variant Envs were shown to lack any cross neutralization by immune serum from horses infected with the respective virus strains. CONCLUSION: Taking into account the significance of serum neutralization to universal vaccine efficacy, these findings are crucial considerations towards successful EIAV vaccine development and the potential inclusion of field isolate Envs in vaccine candidates.

Felis catus papillomavirus type 2 virus-like particle vaccine is safe and immunogenic but does not reduce FcaPV-2 viral loads in adult cats.

Felis catus papillomavirus type 2 (FcaPV-2) commonly infects the skin of domestic cats and has been associated with the development of skin cancer. In the present study, a FcaPV-2 virus-like particle (VLP) vaccine was produced and assessed for vaccine safety, immunogenicity, and impact on FcaPV-2 viral load. This is the first report of the use of a papillomavirus VLP vaccine in domestic cats. The FcaPV-2 VLP vaccine was given to ten adult cats that were naturally infected with FcaPV-2, and a further ten naturally infected cats were sham vaccinated as a control group. The rationale for vaccinating cats already infected with the virus was to induce neutralizing antibody titers that could prevent reinfection of new areas of skin and reduce the overall viral load, as has been demonstrated in other species. Reducing the overall FcaPV-2 viral load could reduce the risk for subsequent PV-associated cancer. The vaccine in this study was well-tolerated, as none of the cats developed any signs of local reaction or systemic illness. In the treatment group, the geometric mean anti-papillomavirus endpoint antibody titers increased significantly following vaccination from 606 (95% CI 192-1913) to 4223 (2023-8814), a 7.0-fold increase, although the individual antibody response varied depending on the level of pre-existing antibodies. Despite the immunogenicity of the vaccine, there was no significant change in FcaPV-2 viral load in the treatment group compared to the control group, over the 24 week follow-up period. A possible reason is that FcaPV-2 was already widespread in the basal skin layer of these adult cats and so preventing further cells from becoming infected had no impact on the overall viral load. Therefore, these results do not support the use of a FcaPV-2 VLP vaccine to reduce the risk for PV-associated cancer in cats in which FcaPV-2 infection is already well established. However, these results justify future studies in which the vaccine is administered to younger cats prior to FcaPV-2 infection becoming fully established.

Amplification of papillomaviral DNA sequences from a high proportion of feline cutaneous in situ and invasive squamous cell carcinomas using a nested polymerase chain reaction.

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.

Pregnancy and mid-term abortion rates in farmed red deer in New Zealand.

This paper describes pregnancy and mid-term abortion rate data from an investigation of sub-optimal reproductive performance in New Zealand farmed red deer. For 2 years, 87 yearling (YL) and 71 mixed-age adult (MA) herds from 85 farms were observed with 15 YL and seven MA herds observed both years. Hinds were pregnancy diagnosed using ultrasonography in the late autumn-early winter (PD1) and again late winter-early spring (PD2) and mid-term daily abortion rates (DAR) were calculated. Overall, 85.8% of 22,130YL (range, 7.0%-100% between herds) and 93.3% of 36,223 MA hinds (range, 39.8%-100% between herds) were pregnant at PD1. The mean interval between ultrasonic assessments was 90 and 87 days in YL and MA herds, respectively. Mid-term abortions occurred in 305 (2.8%) hinds from 60 (73%) YL herds and 92 (1.2%) hinds from 36 (61%) MA herds. The mean mid-term DAR, was greater (P = 0.009) in YL (mean 0.043%, range 0.005%-0.213%, 95% CI = 0.034-0.053) than MA herds (mean 0.025%, range 0.007%-0.101%, 95% CI = 0.018-0.032). In herds with hinds that aborted, the mean DAR was greater in small than large YL herds (0.055% compared with 0.033%, P = 0.023), but not MA herds. At PD1, 46 YL and 12 MA hinds had ultrasonographic evidence of abortions from 22 herds. This, combined with the 1.2% to 7.1% foetal loss in three herds pregnancy diagnosed earlier than the usual PD1 date as a pilot to test for earlier abortion, supports that abortion can occur prior to normal pregnancy assessment dates, contributing to sub-optimal pregnancy rates. Abortion rates were not consistent within herd between years for either YL or MA herds. Thus, early and mid-term abortion is prevalent in New Zealand farmed deer constituting a significant production cost, warranting further investigation into causation.