New insight into avian malaria vectors in New Zealand.

BACKGROUND: Mosquitoes (Culicidae) are vectors for most malaria parasites of the Plasmodium species and are required for Plasmodium spp. to complete their life cycle. Despite having 16 species of mosquitoes and the detection of many Plasmodium species in birds, little is known about the role of different mosquito species in the avian malaria life cycle in New Zealand. METHODS: In this study, we used nested polymerase chain reaction (PCR) and real-time PCR to determine Plasmodium spp. prevalence and diversity of mitochondrial cytochrome b gene sequences in wild-caught mosquitoes sampled across ten sites on the North Island of New Zealand during 2012-2014. The mosquitoes were pooled by species and location collected, and the thorax and abdomens were examined separately for Plasmodium spp. DNA. Akaike information criterion (AIC) modeling was used to test whether location, year of sampling, and mosquito species were significant predictors of minimum infection rates (MIR). RESULTS: We collected 788 unengorged mosquitoes of six species, both native and introduced. The most frequently caught mosquito species were the introduced Aedes notoscriptus and the native Culex pervigilans. Plasmodium sp DNA was detected in 37% of matched thorax and abdomen pools. When considered separately, 33% of abdomen and 23% of thorax pools tested positive by nested PCR. The MIR of the positive thorax pools from introduced mosquito species was 1.79% for Ae. notoscriptus and 0% for Cx. quinquefasciatus, while the MIR for the positive thorax pools of native mosquito species was 4.9% for Cx. pervigilans and 0% for Opifex fuscus. For the overall MIR, site and mosquito species were significant predictors of Plasmodium overall MIR. Aedes notoscriptus and Cx. pervigilans were positive for malaria DNA in the thorax samples, indicating that they may play a role as avian malaria vectors. Four different Plasmodium lineages (SYAT05, LINN1, GRW6, and a new lineage of P (Haemamoeba) sp. AENOT11) were identified in the pooled samples. CONCLUSIONS: This is the first detection of avian Plasmodium DNA extracted from thoraxes of native Culex and introduced Aedes mosquito species in New Zealand and therefore the first study providing an indication of potential vectors in this country.

Presence and diversity of mixed avian Plasmodium spp. infections in introduced birds whose distribution overlapped with threatened New Zealand endemic birds.

Aims: To determine the presence of infection and co-infection of Plasmodium lineages in introduced birds at translocation sites for the North Island saddleback (Philesturnus rufusater), to investigate their role as Plasmodium spp. reservoirs.Methods: Blood samples were collected from introduced bird species, with a special focus on blackbirds (Turdus merula) and song thrushes (Turdus philomelos), at six locations in the North Island of New Zealand that were the origin, or translocation sites, for North Island saddleback. Where available, blood smears were examined, and blood samples were tested using nested PCR with subsequent sequence analysis, for the presence of Plasmodium spp.Results: Of the 55 samples tested using PCR analysis, 39 (71%) were positive for Plasmodium spp., and 28/40 (62%) blood smears were positive for Plasmodium spp. Overall, 31 blood samples were from blackbirds with 28/31 (90%) samples positive for Plasmodium spp. Six distinct avian Plasmodium lineages were identified, including three cosmopolitan lineages; Plasmodium vaughani SYAT05 was detected in 16 samples, Plasmodium matutinum Linn1 in 10 samples and Plasmodium elongatum GRW6 in eight samples. Mixed infections with more than one lineage were detected in 12 samples. Samples from two Australian magpies (Gymnorhina tibicen) were positive for Plasmodium. sp. lineage MYNA02, previously not identified in New Zealand.Conclusions and clinical relevance: This is the first report from New Zealand in which specific Plasmodium spp. mixed infections have been found in introduced birds. Co-infections with several cosmopolitan Plasmodium lineages were identified, as well as the first report in New Zealand of an exotic avian Plasmodium sp. lineage, in Australian magpies. Whilst the role of introduced birds in maintaining and spreading pathogenic avian malaria in New Zealand is unclear, there is a potential infection risk to native birds, especially where distributions overlap.

Seroprevalence of antibodies to Rickettsia typhi in the Waikato region of New Zealand.

The first reported New Zealand-acquired case of murine typhus occurred near Auckland in 1989. Since then, 72 locally acquired cases have been recorded from northern New Zealand. By 2008, on the basis of the timing and distribution of cases, it appeared that murine typhus was escalating and spreading southwards. To explore the presence of Rickettsia typhi in the Waikato region, we conducted a seroprevalence study, using indirect immunofluorescence, Western blot, and cross-adsorption assays of blood donor samples. Of 950 human sera from Waikato, 12 (1·3%) had R. typhi antibodies. The seroprevalence for R. typhi was slightly higher in northern Waikato (1·4%) compared to the south (1·2%; no significant difference, χ 2 P = 0·768 at P < 0·05). Our results extend the reported southern range of R. typhi by 140 km and indicate it is endemic in Waikato. Evidence of past Rickettsia felis infections was also detected in six sera. Globally, R. felis is an emerging disease of concern and this pathogen should also be considered when locally acquired rickettsiosis is suspected. If public health interventions are to be implemented to reduce the risk of rickettsioses as a significant public health problem, improvements in rickettsial diagnostics and surveillance will be necessary.

Exudative cloacitis in the kakapo (Strigops habroptilus) potentially linked to Escherichia coli infection.

AIM: To investigate the initiating causes of cloacitis (inflammation of the cloaca) in kakapo (Strigops habroptilus). METHODS: Metagenomics using unbiased RNA or DNA sequencing was applied to faecal material from an 11-year-old female kakapo with exudative cloacitis, and a pool of eight birds (male and female aged 1-20 years) with no current signs or history of the disease. Faecal material from the diseased bird was collected pre- and post-treatment. For RNA sequencing, extracted RNA/DNA was subject to DNase, and the remaining RNA reverse transcribed to cDNA and subject to multiple displacement amplification prior to sequencing. RESULTS: No significant alignment to any known avian virus sequence was obtained from any faecal samples. However significant BLAST alignments to five bacteriophages known to infect enterobacteria were obtained. Strong evidence was obtained for the presence of the bacteriophage Escherichia phage TL-2011b, a bacteriophage known to occur in Escherichia coli causing outbreaks of foodborne disease in humans, in the sample from the diseased bird, but not the non-diseased pool. Differences in E. coli community structure between the diseased bird and the non-diseased pool were also apparent. CONCLUSIONS: Escherichia coli infection of human origin is suggested as a possible cause of exudative cloacitis, although confirmatory work is required to test this hypothesis.

The role of multiple wildlife hosts in the persistence and spread of bovine tuberculosis in New Zealand.

AIM: To explore how the inclusion of multi-host dynamics affects the predicted prevalence of bovine tuberculosis (TB) in possums and other host species following the current best practice for control of TB in large difficult and remote areas, to identify which host species are responsible for changes in predicted prevalence, and whether TB can persist in possum-free host communities. METHODS: Multi-host TB models were constructed, comprising three host species with density-dependent population growth, density-dependent disease transmission and susceptible and infected classes. Models were parameterised for two case studies of current concern in New Zealand, namely chronic TB persistence in a possum-deer-pig complex in extensive forest, and in a possum-pig-ferret complex in unforested semi-arid shrub and grasslands. Persistence of TB in the face of best practice possum control was evaluated from model simulations, and the contribution of different hosts to persistence of TB was assessed by removing each host species in turn from the simulations. A sensitivity test explored how different parameter values affected modelled persistence of TB. RESULTS: The forest multi-host model-predicted amplification of TB prevalence due to the presence of pigs. The presence of pigs and/or deer did not jeopardise the success of best practice possum control in eradicating TB from the system, as pigs and deer are effectively end-hosts for TB. Sensitivity analyses indicated these interpretations were robust to uncertainty in model parameter values. The grassland system model predicted that the multi-host species complex could potentially lead to failure of eradication of TB under possum-only control, due to TB persisting in ferret and pig populations in the absence of possum hosts through reciprocal scavenging, resulting in spillback transmission to possums once their populations had started to recover from control. CONCLUSIONS: With respect to management of TB, for modelled forest habitats, 15 years of effective possum control was predicted to eradicate TB from the possum-deer-pig host community, indicating the current focus on possum-only control is appropriate for such areas. For grassland model systems, TB was predicted to persist in the ferret-pig host complex in the absence of possums, potentially jeopardising the effectiveness of possum-only control programmes. However this outcome depended on the occurrence and rate of pigs acquiring TB from ferrets, which is unknown. Thus some estimation of this transmission parameter is required to enable managers to assess if multi-host disease dynamics are important for their TB control programmes.

Local effects of a global problem: modelling the risk of parasite-induced mortality in an intertidal trematode-amphipod system.

The interactive effects of climate change and parasitism are of concern because of potentially important consequences for host populations, communities and entire ecosystems. In marine environments, the absence of historic baseline data on parasitism and disease limits our ability to make realistic predictions about these consequences. Here, we adapt a simulation model developed for a Northern Hemisphere intertidal host-parasite system to a comparable system in the Southern Hemisphere. The entire life cycle of the intertidal trematode parasite Maritrema novaezealandensis was modelled in order to investigate the interactive effects of parasitic infections and increasing temperatures on the population dynamics of the amphipod host Paracalliope novizealandiae. Despite uncertainties associated with the model and its parameterisation, most temperature increases that were predicted to cause the collapse of the modelled amphipod population in the long term lay within the range of predicted warming for the study area. The high vulnerability of the amphipods in the modelled system illustrates a potentially important ecological mechanism by which consequences of a global problem might manifest on the local scale.

Percutaneous interdigital injection of Mycobacterium bovis as a model for tuberculous lesion development in wild brushtail possums (Trichosurus vulpecula).

Brushtail possums (Trichosurus vulpecula) are the major wildlife reservoir of Mycobacterium bovis, the causative agent of bovine tuberculosis (BTB), in New Zealand. Primary diagnosis of BTB in wild possums is by palpation to detect peripheral lymphadenomegaly followed by necropsy examination, which frequently identifies gross tuberculous lesions in the peripheral lymph nodes and lungs. Experimental infection studies were conducted with wild possums in an attempt to emulate field BTB, focussing on percutaneous administration of virulent M. bovis in the paws. In a preliminary study, viable M. bovis bacilli were recovered from lymph nodes draining fore- or hindlimbs 12 days after percutaneous injection. Subsequently, 21 wild possums were injected interdigitally with 500 colony forming units (cfu) of M. bovis, radio-collared and released; 17/18 possums recaptured 8 weeks later had an established M. bovis lymphatic infection, with 16 having culture-positive gross lesions in the superficial and/or deep axillary lymph nodes. A dual-site infection model was established, involving simultaneous interdigital injection of 100 cfu of M. bovis into front and rear paws of 19 wild possums; this identified that the average degree of lymphadenitis involved 30-fold enlargement of the draining lymph node by 7-8 weeks post injection (wpi). A time-course study demonstrated establishment of M. bovis infection in peripheral lymph nodes of 9/11 possums at 3-5 wpi of doses ranging from 60 to 190 cfu, but with no development of gross lesions; by 7 weeks, 8/8 animals injected similarly had both an established infection and gross lesions of peripheral lymph nodes. The incidence and progression of peripheral lesion development, together with indications of sequential infection of the lungs, liver and mesenteric lymph nodes(MLNs), indicates that a low-dose percutaneous M. bovis infection model is likely to emulate natural disease in possums.

Do different parasite species interact in their effects on host fitness? A case study on parasites of the amphipod Paracalliope fluviatilis.

There is a gap in our understanding of the relative and interactive effects of different parasite species on the same host population. Here we examine the effects of the acanthocephalan Acanthocephalus galaxii, an unidentified cyclophyllidean cestode, and the trematodes Coitocaecum parvum and Microphallus sp. on several fitness components of the amphipod Paracalliope fluviatilis, using a combination of infection surveys and both survival and behavioural trials. In addition to significant relationships between specific parasites and measures of amphipod survival, maturity, mating success and behaviour, interactions between parasite species with respect to amphipod photophilia were also significant. While infection by either A. galaxii or C. parvum was associated with increased photophilia, such increases were negated by co-infection with Microphallus sp. We hypothesize that this is due to the more subtle manipulative effect of A. galaxii and C. parvum being impaired by Microphallus sp. We conclude that the low frequency at which such double infections occur in our sampled population means that such interactions are unlikely to be important beyond the scale of the host individual. Whether or not this is generally true, implying that parasitological models and theory based on single parasite species studies do generally hold, requires cross-species meta-analytical studies.

Parasite spillback: a neglected concept in invasion ecology?

While there is good evidence linking animal introductions to impacts on native communities via disease emergence, our understanding of how such impacts occur is incomplete. Invasion ecologists have focused on the disease risks to native communities through "spillover" of infectious agents introduced with nonindigenous hosts, while overlooking a potentially more common mechanism of impact, that of "parasite spillback." We hypothesize that parasite spillback could occur when a nonindigenous species is a competent host for a native parasite, with the presence of the additional host increasing disease impacts in native species. Despite its lack of formalization in all recent reviews of the role of parasites in species introductions, aspects of the invasion process actually favor parasite spillback over spillover. We specifically review the animal-parasite literature and show that native species (arthropods, parasitoids, protozoa, and helminths) account for 67% of the parasite fauna of nonindigenous animals from a range of taxonomic groups. We show that nonindigenous species can be highly competent hosts for such parasites and provide evidence that infection by native parasites does spillback from nonindigenous species to native host species, with effects at both the host individual and population scale. We conclude by calling for greater recognition of parasite spillback as a potential threat to native species, discuss possible reasons for its neglect by invasion ecologists, and identify future research directions.

Oral vaccination reduces the incidence of tuberculosis in free-living brushtail possums.

Bovine tuberculosis (Tb) caused by Mycobacterium bovis has proved refractory to eradication from domestic livestock in countries with wildlife disease reservoirs. Vaccination of wild hosts offers a way of controlling Tb in livestock without wildlife culling. This study was conducted in a Tb-endemic region of New Zealand, where the introduced Australian brushtail possum (Trichosurus vulpecula) is the main wildlife reservoir of Tb. Possums were trapped and vaccinated using a prototype oral-delivery system to deliver the Tb vaccine bacille Calmette-Guerin. Vaccinated and control possums were matched according to age, sex and location, re-trapped bimonthly and assessed for Tb status by palpation and lesion aspiration; the site was depopulated after 2 years and post-mortem examinations were conducted to further identify clinical Tb cases and subclinical infection. Significantly fewer culture-confirmed Tb cases were recorded in vaccinated possums (1/51) compared with control animals (12/71); the transition probability from susceptible to infected was significantly reduced in both males and females by vaccination. Vaccine efficacy was estimated at 95 per cent (87-100%) for females and 96 per cent (82-99%) for males. Hence, this trial demonstrates that orally delivered live bacterial vaccines can significantly protect wildlife against natural disease exposure, indicating that wildlife vaccination, along with existing control methods, could be used to eradicate Tb from domestic animals.

Avian malaria (Plasmodium spp) in yellow-eyed penguins: investigating the cause of high seroprevalence but low observed infection.

AIM: To investigate the cause of a high seroprevalence of antibodies to Plasmodium spp known to cause avian malaria, but extremely low levels of observed infection, in yellow-eyed penguins, Megadyptes antipodes. METHODS: A polymerase chain reaction (PCR) test specific for malarial parasites was applied to DNA extracted from blood samples collected from 143 yellow-eyed penguins from an area where seroprevalence for malarial antibodies was known to be high but no parasites were observed in blood smears. RESULTS: None of the samples tested positive for malarial parasite DNA using the PCR test. Assuming a sensitivity of 90% for this test, this means that prevalence of infection was 95% likely to be <2.3% in this population during this sampling period. CONCLUSIONS: Serological studies of a population of adult yellow-eyed penguins indicated a high level of exposure to avian malaria parasites, but a correspondingly high level of infection was not observed and no evidence of malarial parasite DNA was found in the current study. Discrepancies between these findings and historical records of Plasmodium spp found in blood smears and post mortem may be explained either by inaccuracy of the serological test used, or by infection occurring in juveniles which is subsequently cleared in surviving adults.

Parasite-induced surfacing in the cockle Austrovenus stuchburyi: adaptation or not?

Parasite manipulation of host behaviour is a compelling example of the extended phenotype. However, in many cases, such manipulation may be incorrectly assumed. Previous work has demonstrated that Austrovenus stuchburyi cockles stranded on mud-flat surfaces due to an inability to re-burrow both contain significantly more metacercariae of the trematode Curtuteria australis and are predated by the definitive host of this parasite at a faster rate than burrowed cockles. These results have been interpreted as strong evidence for a manipulation of cockle behaviour by the trematode to facilitate transmission to the definitive host. The model presented here, however, indicates that the selective advantage to the parasite of the altered host behaviour is currently of a negligible level at our study site that is highly unlikely to have been realized as an adaptation over evolutionary time. Hence, there are no grounds on which the more parsimonious explanation, that the altered host behaviour observed is simply an incidental side-effect of infection, can be rejected. We thus maintain that for any change in the behaviour of infected hosts to be confirmed as potentially a parasite trait that has evolved in response to selection, the adaptive benefit taking into account the entire parasite life cycle may need to be considered.

Stochasticity accelerates nematode egg development.

Day-degrees models of nematode development assume that temperature stochasticity has no effect on the development rate of infective stages as long as the mean temperature is held constant. This assumption was tested in this study. Unembryonated Heterakis gallinarum eggs were subjected to nocturnal and diurnal daily temperature cycles at 12 and 17 C. respectively, and embryonation was compared with eggs subjected to similar stochastic daily cycles, in which random normal variations in the temperature were added to the 2 temperatures. The prediction that there is no effect of stochasticity was refuted. Embryonation of eggs subjected to variable daily cycles occurred significantly earlier than that of eggs subjected to deterministic daily cycles, suggesting that stochastic variation in temperature accelerated embryonation even though mean temperatures were the same. These findings show that the development time of H. gallnarum eggs is decreased by stochastic variation in temperature, which may have important implications for the effects of climate change on parasite availability.

Patterns of parasite aggregation in the wild European rabbit (Oryctolagus cuniculus).

Understanding the factors controlling the distribution of parasites within their host population is fundamental to the wider understanding of parasite epidemiology and ecology. To explore changes in parasite aggregation, Taylor's power law was used to examine the distributions of five gut helminths of the wild rabbit. Aggregation was found to be a dynamic process that varied with year, season, host sex, age class, and myxomatosis. Yearly and seasonal changes are thought, in the main, to be the result of variations in weather conditions acting upon infectious stages (or intermediate hosts). Evidence in support of this was the comparatively low degree of fluctuation in the aggregation of the pinworm, Passalurus ambiguus, as the infectious stage of this parasite is likely to be less susceptible to environmental variation. Host age had a marked effect on the level of aggregation of all parasites, but this effect varied between parasite species. P. ambiguus, Trichostrongylus retortaeformis and Cittotaenia denticulata aggregation were lower in adult than juvenile rabbits whilst Graphidium strigosum and Mosgovoyia pectinata aggregation tended to increase with age. Host immunity is thought to be responsible for these differences. Differences in aggregation for different parasites were also seen when the rabbit population was split into males and females. Myxomatosis had a marked effect on helminth distribution with substantially less aggregation in rabbits showing clinical signs of the disease.

Differential impact of a shared nematode parasite on two gamebird hosts: implications for apparent competition.

If the deleterious effects of non-specific parasites are greater on vulnerable host species than on reservoir host species then exclusion of the vulnerable host through apparent competition is more likely. Evidence suggests that such a mechanism occurs in interactions between the ring-necked pheasant (Phasianus colchicus), the grey partridge (Perdix perdix), and their shared caecal nematode Heterakis gallinarum. Modelling of the system predicts that the reduced parasite impact on the pheasant compared to the partridge results in the force of infection transmitted from pheasants to partridges being sufficient to cause partridge exclusion. Since the parasite impacts are currently estimated from correlational work, controlled infections were conducted to experimentally compare the impact of H. gallinarum on the two hosts and verify cause and effect. While challenged partridges showed reduced mass gain, decreased food consumption, and impaired caecal activity, in comparison to controls, the only detectable effect of parasite challenge on the pheasant was impaired caecal activity. The impact of H. gallinarum on challenged partridges conforms with previous correlational data, supporting the prediction that parasite-mediated apparent competition with the ring-necked pheasant may result in grey partridge exclusion. However, the observed decrease in the caecal activity of challenged pheasants could imply that H. gallinarum may also have an impact on the fecundity and survival of pheasants in the wild, particularly if food is limiting. If this is the case, the associated decrease in the force of infection to which the partridge is exposed may be sufficient to change the model prediction from partridge exclusion to pheasant and partridge coexistence.

Strategies for nematode transmission: selective migration of Trichostrongylus tenuis infective larvae.

Successful transmission of macroparasites is dependent on exposure of susceptible hosts to free-living infective stages. When these hosts are herbivores that feed mostly on a single food plant then natural selection should favour those infective larvae that selectively ascend this main food plant. Red grouse feed predominantly on heather, Calluna vulgaris, so we predict that the infective larvae (L3) of the caecal nematode Trichostrongylus tenuis selectively locate and ascend heather plants. To determine whether the presence of heather influences the horizontal dispersal of T. tenuis L3 across soil, the movement of L3 across trays of soil with and without heather was investigated in the laboratory. More T. tenuis L3 were recovered from soil when heather was present, implying that larval migration may be influenced by chemical cues produced by heather plants. This was investigated in a second experiment, in which the horizontal dispersal of T. tenuis larvae was examined in the presence of heather and grass vegetation. This trial was repeated with larvae of a second species, Haemonchus contortus, a nematode whose hosts feed on a wide range of grass and shrub species. Significantly more larvae of both nematode species were recovered in the region of the heather than the grass or controls. This implies that T. tenuis and H. contortus L3 exhibit selective migration towards heather, perhaps reflecting a general response to plant cues which may be stronger for heather than for grass.

The role of oxygen availability in the embryonation of Heterakis gallinarum eggs.

The importance of oxygen availability in the embryonation of the infective egg stages of the gastrointestinal nematode parasite Heterakis gallinarum was studied in the laboratory. Unembryonated H. gallinarum eggs were kept under either aerobic conditions by gassing with oxygen, or anaerobic conditions by gassing with the inert gas nitrogen, under a range of constant temperatures. Oxygenated eggs embryonated at a rate influenced by temperature. Conversely, eggs treated with nitrogen showed no embryonation although when these eggs were transferred from nitrogen to oxygen gas after 60 days of treatment, embryonation occurred. This demonstrated that oxygen is an essential requirement for H. gallinarum egg development, although undeveloped eggs remain viable, even after 60 days in low oxygen conditions. The effects of climate on the biology of free-living stages studied under constant laboratory conditions cannot be applied directly to the field where climatic factors exhibit daily cycles. The effect of fluctuating temperature on development was investigated by including an additional temperature group in which H. gallinarum eggs were kept under daily temperature cycles between 12 and 22 degrees C. Cycles caused eggs to develop significantly earlier than those in the constant mean cycle temperature, 17 degrees C, but significantly slower than those in constant 22 degrees C suggesting that daily temperature cycles had an accelerating effect on H. gallinarum egg embryonation but did not accelerate to the higher temperature. These results suggest that daily fluctuations in temperature influence development of the free-living stages and so development cannot be accurately predicted on the basis of constant temperature culture.

The role of shared parasites in the exclusion of wildlife hosts: Heterakis gallinarum in the ring-necked pheasant and the grey partridge.

1. A two-host shared-macroparasite model was parameterized from the results of infection and transmission experiments, to investigate whether apparent competition between the ring-necked pheasant (Phasianus colchicus) and the grey partridge (Perdix perdix), mediated via the shared nematode Heterakis gallinarum, could theoretically cause partridge exclusion. 2. Both the model created and the experiments conducted show that the bulk of H. gallinarum infection to partridges, when they occur in the same locations as pheasants, will be from the pheasants and not from the partridges themselves. This is due to R0 for the parasite being 1·23 when infecting pheasants, but only 0·0057 when infecting partridges. Thus, when the pheasant is present in the model the partridge population is impacted by the shared parasite but, when the pheasant is absent, the parasite is lost from the system. 3. Based on best available parameter estimates, the observed impact of H. gallinarum on the grey partridge may be sufficient to cause exclusion when the pheasant is present in the model. This supports the hypothesis that the UK grey partridge decline observed over the past 50 years may be partly due to apparent competition with pheasants. 4. Habitat separation between the two host species, where it decreases the rate of H. gallinarum transmission from the pheasant to the partridge, may allow them to co-exist in the field in the presence of the parasite. We predict, however, that grey partridge exclusion would still occur if separation was less than 43%.