L. Howe

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.

A live attenuated equine infectious anemia virus proviral vaccine with a modified S2 gene provides protection from detectable infection by intravenous virulent virus challenge of experimentally inoculated horses.

ABSTRACT 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, EIAVUKΔS2, 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 EIAVUKΔS2 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, EIAVPV. This level of protection was observed in each of the six experimental horses challenged with the reference virulent EIAVPV by using a low-dose multiple-exposure protocol (three administrations of 10 median horse infectious doses [HID50], intravenous) designed to mimic field exposures and in all three experimentally immunized ponies challenged intravenously with a single inoculation of 3,000 HID50. 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.

Concurrent avian malaria and avipox virus infection in translocated South Island saddlebacks (Philesturnus carunculatus carunculatus).

Abstract CASE HISTORY: Outbreaks of mortality in South Island saddlebacks (Philesturnus carunculatus carunculatus) that had been translocated to two offshore islands in the Marlborough Sounds of New Zealand were investigated during the summer of 2002 and 2007. Both outbreaks were associated with a severe decrease in numbers of saddlebacks of up to 60% of approximately 200 birds. CLINICAL AND PATHOLOGICAL FINDINGS: Many of the surviving birds were in poor condition, and had skin lesions on the legs and head. Necropsy showed pale liver and lungs, and a swollen spleen. Histopathology revealed schizonts resembling Plasmodium spp. within the cytoplasm of many hepatocytes and splenic histiocytes. The skin lesions consisted of epithelial proliferations containing numerous Bollinger bodies typical of avipox virus (APV) infection. Two different APV were isolated, using PCR, from two different birds exhibiting skin lesions. Each isolate had 100% sequence homology with APV members from either Clade A or Clade B. In addition, PCR analysis revealed that the Plasmodium elongatum present in infected birdsbelonged to a strain that was endemic in the population of North Island saddlebacks (Philesturnus carunculatus rufusater). DIAGNOSIS: Concurrent infections with Plasmodium spp. haemoparasites and APV were identified as the likely cause of death in the birds examined. CONCLUSIONS AND CLINICAL RELEVANCE: Although the Plasmodium spp. identified is thought to be endemic to saddlebacks in New Zealand, the affected birds were likely to be immunocompromised by concurrent APV infection or through lack of genetic diversity. Both the introduced mosquito Culex quinquefasicatus and the native mosquito Culex pervigilans are likely vectors for both these diseases, and the provision of water supplies less favourable to mosquito-breeding is recommended.

Transient immune suppression of inapparent carriers infected with a principal neutralizing domain-deficient equine infectious anaemia virus induces neutralizing antibodies and lowers steady-state virus replication

The genetic variation of equine infectious anaemia virus (EIAV) clearly affects the antigenic properties of the viral envelope; however, effects on immunogenicity remain undefined, although widely assumed. Here, the immunogenicity is reported of a novel, neutralization-resistant, pony-isolate envelope EIAV(PV564DeltaPND) that contains a 14-residue deletion in the designated principal neutralizing domain (PND) of the gp90 protein. Two ponies inoculated with a chimeric virus, EIAV(DeltaPND), containing the EIAV(PV564DeltaPND) envelope in a reference provirus strain, remained asymptomatic through 14 months post-inoculation, producing high steady-state levels of envelope-specific antibodies but no detectable serum-neutralizing antibodies. Consequent dexamethasone-induced immune suppression produced characteristic EIA that resolved concomitantly with the development of high-titre, strain-specific, neutralizing antibodies and a 100-fold reduction in steady-state virus loads. These results demonstrate: natural variations in the EIAV envelope have profound effects on both antigenic and immunogenic properties; the PND is not required for neutralizing antibody responses; and transient immune suppression can enhance established host immunity to achieve more effective control of steady-state lentivirus replication.

Discerning an Effective Balance between Equine Infectious Anemia Virus Attenuation and Vaccine Efficacy

ABSTRACT Among the diverse experimental vaccines evaluated in various animal lentivirus models, live attenuated vaccines have proven to be the most effective, thus providing an important model for examining critical immune correlates of protective vaccine immunity. We previously reported that an experimental live attenuated vaccine for equine infectious anemia virus (EIAV), based on mutation of the viral S2 accessory gene, elicited protection from detectable infection by virulent virus challenge (F. Li et al., J. Virol. 77:7244-7253, 2003). To better understand the critical components of EIAV vaccine efficacy, we examine here the relationship between the extent of virus attenuation, the maturation of host immune responses, and vaccine efficacy in a comparative study of three related attenuated EIAV proviral vaccine strains: the previously described EIAVUKΔS2 derived from a virulent proviral clone, EIAVUKΔS2/DU containing a second gene mutation in the virulent proviral clone, and EIAVPRΔS2 derived from a reference avirulent proviral clone. Inoculations of parallel groups of eight horses resulted in relatively low levels of viral replication (average of 102 to 103 RNA copies/ml) and a similar maturation of EIAV envelope-specific antibody responses as determined in quantitative and qualitative serological assays. However, experimental challenge of the experimentally immunized horses by our standard virulent EIAVPV strain by using a low-dose multiple exposure protocol (three inoculations with 10 median horse infective doses, administered intravenously) revealed a marked difference in the protective efficacy of the various attenuated proviral vaccine strains that was evidently associated with the extent of vaccine virus attenuation, time of viral challenge, and the apparent maturation of virus-specific immunity.

An outbreak of avian malaria in captive yellowheads/mohua (Mohoua ochrocephala)

Abstract CASE HISTORY: Eight mohua, or yellowheads (Mohoua ochrocephala), were held in a large open aviary over the summer months of 2003–2004, following their capture for captivebreeding purposes. Two birds died of transportation trauma shortly after arrival, one became ill and died a month later, and another four died within a 2-week period in February 2004. The eighth bird also became ill at this time but survived for a year following treatment with chloroquine and doxycycline. CLINICAL AND PATHOLOGICAL FINDINGS: The affected birds were depressed, lethargic and dyspnoeic. Necropsy of three birds showed a slightly pale and swollen liver and spleen. Impression smears of the liver of one bird revealed schizonts resembling Plasmodium spp. within the cytoplasm of many hepatocytes, which was confirmed histopathologically. Similar protozoal organisms were seen within splenic histiocytes and pulmonary endothelial cells of 5/6 birds. Electron microscopy identified these as protozoal schizonts containing merozoites of similar size and structure to those of Plasmodium spp. DIAGNOSIS: The birds were infected with a protozoal haemoparasite resembling Plasmodium spp.; asexual stages within hepatocytes and endothelial cells of the lung and spleen were typical of this organism. CONCLUSIONS AND CLINICAL RELEVANCE: The mohua captured from west Otago were highly susceptible to avian malaria as they came from an isolated population that was likely to be naïve and have had no previous contact with this organism. The birds were probably infected by bites from mosquitoes feeding off local populations of blackbirds subsequently found to be infected with Plasmodium spp.

Detection of papillomaviral DNA sequences in a feline oral squamous cell carcinoma

Oral squamous cell carcinomas (OSCCs) are common and often fatal feline neoplasms. Factors that predispose to neoplasm development in cats are poorly defined. Around 25% of human OSCCs are caused by papillomaviruses (PVs). To determine if PVs are associated with OSCCs in cats, three sets of consensus primers were used to evaluate 20 feline OSCCs and 20 non-neoplastic feline oral lesions for the presence of PV DNA. Papillomaviral sequences were detected within one OSCC, but no non-neoplastic lesion. Sequencing of the amplified DNA revealed a previously unreported PV that was most similar to human PV type 76. This is the first time PV DNA has been amplified from the oral cavity of a cat. However, while these results suggest that feline gingival epithelial cells can be infected by PVs, they do not support a causal association between viral infection and the development of feline OSCCs.

An atypical genotype of Toxoplasma gondii as a cause of mortality in Hector's dolphins (Cephalorhynchus hectori)

Hectors dolphins (Cephalorhynchus hectori) are a small endangered coastal species that are endemic to New Zealand. Anthropogenic factors, particularly accidental capture in fishing nets, are believed to be the biggest threat to survival of this species. The role of infectious disease as a cause of mortality has not previously been well investigated. This study investigates Toxoplasma gondii infection in Hectors dolphins, finding that 7 of 28 (25%) dolphins examined died due to disseminated toxoplasmosis, including 2 of 3 Mauis dolphins, a critically endangered sub-species. A further 10 dolphins had one or more tissues that were positive for the presence of T. gondii DNA using PCR. Genotyping revealed that 7 of 8 successfully amplified isolates were an atypical Type II genotype. Fatal cases had necrotising and haemorrhagic lesions in the lung (n=7), lymph nodes (n=6), liver (n=4) and adrenals (n=3). Tachyzoites and tissue cysts were present in other organs including the brain (n=5), heart (n=1), stomach (n=1) and uterus (n=1) with minimal associated inflammatory response. One dolphin had a marked suppurative metritis in the presence of numerous intra-epithelial tachyzoites. No dolphins had underlying morbillivirus infection. This study provides the first evidence that infectious agents could be important in the population decline of this species, and highlights the need for further research into the route of entry of T. gondii organisms into the marine environment worldwide.

PRESENCE AND SEASONAL PREVALENCE OF PLASMODIUM SPP. IN A RARE ENDEMIC NEW ZEALAND PASSERINE (TIEKE OR SADDLEBACK, PHILESTURNUS CARUNCULATUS)

The conservation and management of Saddlebacks (Philesturnus carunculatus) and other New Zealand birds, currently relies on the translocation of individuals to predator-free sites. Avian malaria has been identified as one of the diseases to be tested for prior to translocations in New Zealand, with the aim of translocating disease-free individuals. We describe avian malaria lineages and their seasonal prevalence in 2007–2008 in Saddlebacks from Mokoia Island, a source of birds for translocations, and investigate their pathogenicity. Three lineages of avian malaria were found at low prevalence (≥10.6%) and parasitemia (all but one infection were below 1/10,000 erythrocytes), typical of chronic infections. Two lineages clustered with previously identified lineages of Plasmodium relictum and one with a lineage of Plasmodium (Huffia) elongatum. Prevalence of malaria infection was higher in the spring with no significant difference in prevalence between juvenile and adult birds. We found no effect of stress on infections or any indication of pathogenicity.

Avian malaria in New Zealand

Avian malaria parasites of the genus Plasmodium have the ability to cause morbidity and mortality in naïve hosts, and their impact on the native biodiversity is potentially serious. Over the last decade, avian malaria has aroused increasing interest as an emerging disease in New Zealand with some endemic avian species, such as the endangered mohua (Mohua ochrocephala), thought to be particularly susceptible. To date, avian malaria parasites have been found in 35 different bird species in New Zealand and have been diagnosed as causing death in threatened species such as dotterel (Charadrius obscurus), South Island saddleback (Philesturnus carunculatus carunculatus), mohua, hihi (Notiomystis cincta) and two species of kiwi (Apteryx spp.). Introduced blackbirds (Turdus merula) have been found to be carriers of at least three strains of Plasmodium spp. and because they are very commonly infected, they are likely sources of infection for many of New Zealands endemic birds. The spread and abundance of introduced and endemic mosquitoes as the result of climate change is also likely to be an important factor in the high prevalence of infection in some regions and at certain times of the year. Although still limited, there is a growing understanding of the ecology and epidemiology of Plasmodium spp. in New Zealand. Molecular biology has played an important part in this process and has markedly improved our understanding of the taxonomy of the genus Plasmodium. This review presents our current state of knowledge, discusses the possible infection and disease outcomes, the implications for host behaviour and reproduction, methods of diagnosis of infection, and the possible vectors for transmission of the disease in New Zealand.