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.

What Are Sheep Doing? Tri-Axial Accelerometer Sensor Data Identify the Diel Activity Pattern of Ewe Lambs on Pasture.

Monitoring activity patterns of animals offers the opportunity to assess individual health and welfare in support of precision livestock farming. The purpose of this study was to use a triaxial accelerometer sensor to determine the diel activity of sheep on pasture. Six Perendale ewe lambs, each fitted with a neck collar mounting a triaxial accelerometer, were filmed during targeted periods of sheep activities: grazing, lying, walking, and standing. The corresponding acceleration data were fitted using a Random Forest algorithm to classify activity (=classifier). This classifier was then applied to accelerometer data from an additional 10 ewe lambs to determine their activity budgets. Each of these was fitted with a neck collar mounting an accelerometer as well as two additional accelerometers placed on a head halter and a body harness over the shoulders of the animal. These were monitored continuously for three days. A classification accuracy of 89.6% was achieved for the grazing, walking and resting activities (i.e., a new class combining lying and standing activity). Triaxial accelerometer data showed that sheep spent 64% (95% CI 55% to 74%) of daylight time grazing, with grazing at night reduced to 14% (95% CI 8% to 20%). Similar activity budgets were achieved from the halter mounted sensors, but not those on a body harness. These results are consistent with previous studies directly observing daily activity of pasture-based sheep and can be applied in a variety of contexts to investigate animal health and welfare metrics e.g., to better understand the impact that young sheep can suffer when carrying even modest burdens of parasitic nematodes.

Aspects of the development of Ixodes anatis under different environmental conditions in the laboratory and in the field.

BACKGROUND: Numerous laboratory and fewer field-based studies have found that ixodid ticks develop more quickly and survive better at temperatures between 18 °C and 26 °C and relative humidity (RH) between 75 and 94%. Ixodes anatis Chilton, 1904, is an endophilic, nidicolous species endemic to North Island brown kiwi (Apteryx mantelli) (NIBK) and the tokoeka (Apteryx australis), and little is known about the environmental conditions required for its development. The aims of this study were to determine and compare the conditions of temperature and RH that ensure the best survival of the kiwi tick and the shortest interstadial periods, in laboratory conditions and outdoors inside artificial kiwi burrows. METHODS: Free-walking engorged ticks were collected off wild kiwi hosts and placed in the laboratory under various fixed temperature and humidity regimes. In addition, sets of the collected ticks at different developmental stages were placed in artificial kiwi burrows. In both settings, we recorded the times taken for the ticks to moult to the next stage. RESULTS: Larvae and nymphs both showed optimum development at between 10 °C and 20 °C, which is lower than the optimum temperature for development in many other species of ixodid ticks. However, larvae moulted quicker and survived better when saturation deficits were < 1-2 mmHg (RH > 94%); in comparison, the optimum saturation deficits for nymph development were 1-10 mmHg. CONCLUSIONS: Our results suggest that the kiwi tick has adapted to the stable, but relatively cool and humid conditions in kiwi burrows, reflecting the evolutionary consequences of its association with the kiwi.

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.

Gastrointestinal nematode infection affects overall activity in young sheep monitored with tri-axial accelerometers.

Animals suffering from parasitism typically display altered grazing behaviour and a voluntary reduction in feed intake. These changes are potentially important as indicators of disease. Recent advances in sensor technologies provide the opportunity to objectively measure animal activity while on pasture. Tri-axial accelerometers measure body movement in terms of acceleration, which can then be used to estimate physical activity over time. This study investigated if tri-axial measures of overall activity can be used to assess the impact of gastrointestinal nematode (GIN) infection in young sheep. To address this, the overall activity, faecal nematode egg count (FEC) and body weight of two treatment groups of Romney X Suffolk ram lambs were compared. Animals were monitored for four days using tri-axial accelerometer sensors mounted on a ram mating harness after 42-days grazing on contaminated pasture. On Day 0, all lambs were given anthelmintics. Subsequently, a Suppressive Treatment Group (n = 12) was treated with anthelmintics every two weeks. An Untreated Group (n = 12) did not receive further anthelmintics. Overall activity levels were monitored from Day 42 - 46. Activity level was calculated as vectorial dynamic body acceleration (VeDBA). Anthelmintic treatment had a significant effect on FEC but there was no evidence found for a treatment effect on body weight growth over the 42-day period. An effect of treatment and lamb starting weight on overall activity was found (beta = -0.74, 95 % CI -1.17 to -0.30, p =  0.002), identifying a negative impact of parasitism on activity in heavier animals. These results highlight the usefulness of this approach in assessing the effect of GIN parasitism on sheep monitored remotely. If a threshold value of activity could be determined, it could provide a useful tool for farmers and managers that serves as an early indicator of parasitism in sheep.

The Pacific Biosciences de novo assembled genome dataset from a parthenogenetic New Zealand wild population of the longhorned tick, Haemaphysalis longicornis Neumann, 1901.

The longhorned tick, Haemaphysalis longicornis, feeds upon a wide range of bird and mammalian hosts. Mammalian hosts include cattle, deer, sheep, goats, humans, and horses. This tick is known to transmit a number of pathogens causing tick-borne diseases, and was the vector of a recent serious outbreak of oriental theileriosis in New Zealand. A New Zealand-USA consortium was established to sequence, assemble, and annotate the genome of this tick, using ticks obtained from New Zealand's North Island. In New Zealand, the tick is considered exclusively parthenogenetic and this trait was deemed useful for genome assembly. Very high molecular weight genomic DNA was sequenced on the Illumina HiSeq4000 and the long-read Pac Bio Sequel platforms. Twenty-eight SMRT cells produced a total of 21.3 million reads which were assembled with Canu on a reserved supercomputer node with access to 12 TB of RAM, running continuously for over 24 days. The final assembly dataset consisted of 34,211 contigs with an average contig length of 215,205 bp. The quality of the annotated genome was assessed by BUSCO analysis, an approach that provides quantitative measures for the quality of an assembled genome. Over 95% of the BUSCO gene set was found in the assembled genome. Only 48 of the 1066 BUSCO genes were missing and only 9 were present in a fragmented condition. The raw sequencing reads and the assembled contigs/scaffolds are archived at the National Center for Biotechnology Information.

Factors affecting abundance of different stages of the endophilic tick Ixodes anatis in brown kiwi (Apteryx mantelli) shelters.

Ixodes anatis is a species of endophilic (nidicolous) tick species parasitizing brown kiwi (Apteryx mantelli). Even though they are endemic to New Zealand like their host, very little is known about these ticks or their population dynamics and relationships with their hosts. We conducted a study from May 2013 to June 2014 to evaluate the effect of shelter location (one of three gullies), habitat (forest, scrub and pasture) and type (tree, soil and surface) on the abundance of the different life stages of I. anatis. In total, 12,172 ticks were collected from 63 shelters, which were sampled monthly for 11 months over the 14 month period. Un-engorged larvae predominated over other stages accounting for 87.2% of the samples collected. We found that location, habitat in which the shelters were located, and the type of shelter were significant predictors of I. anatis abundance. Tree shelters in forests had significantly higher tick abundance than those in scrub and pasture. Tree and soil shelters in general had significantly more ticks than surface shelters. Shelters located in Kauri Bush a drier site, had higher abundances than those in wetter sites. While some of these changes can be explained with the movement of the host, we believe more research needs to be done on the effect of shelters' microclimate on I. anatis' life cycle.

Establishment of Cooperia oncophora in calves.

The establishment rate of Cooperia oncophora related to host age and previous infection was investigated in young calves. Calves of similar age were kept on a feed pad and allocated into multiple groups, based on their age and weight. Two groups (each n = 16) received trickle infections with an ivermectin-susceptible C. oncophora isolate of 2000 or 10,000 infective stage larvae per week while another group (n = 16) was kept as an uninfected control. At intervals over a period of 11 months, two animals from each group were challenged with 15,000 infective stage larvae of an ivermectin-resistant isolate, 25 days later orally treated with ivermectin and 5 days after that slaughtered for worm counts. On three occasions additional calves (n = 2), subjected to the high trickle infection rate, received an ivermectin treatment to remove the existing worm burden, prior to challenge as above. Further calves (n = 4) of similar age were introduced at the beginning and the end of the experiment to determine the effect of larval age on establishment rate. The establishment in the two trickle infection groups declined to <10% within the first three months, which was significantly different from the control group. In the animals receiving the high trickle infection, but an anthelmintic treatment before challenge the establishment rate was not significantly different from the controls. Over the duration of the experiment establishment in the control group declined from 53% to <20%, which was similar to the decrease recorded at the beginning and the end of the experiment in the animals to determine the effect of larval age. The findings indicate that an existing C. oncophora burden had a strong effect on the establishment of incoming larvae in the trickle infected groups, but this was not observed if the existing burden was removed before the final challenge. The decline in establishment rate in the control group was attributed to the age of the larvae and not the age of the calves per se.

Control options for Neospora caninum--is there anything new or are we going backwards?

Recent work has highlighted and enumerated the economic annual losses due to Neospora caninum abortions worldwide, which should provide strong motivation for the control of bovine neosporosis. However, with the recent withdrawal from sale of the only commercially available vaccine, control options for N. caninum have become more restricted. While researchers continue to work on developing alternative efficacious vaccines, what are the control options presently available for the cattle industries? At the practical level, recommendations for 'Test-and-cull', or 'not breeding from seropositive dams' stand diametrically opposed to alternative options put forward that suggest a primary producer is better advised to keep those cows in the herd that are already seropositive, i.e., assumed to be chronically infected, and indeed those that have already aborted once. Treatment with a coccidiostat has been recommended as the only economically viable option, yet no such treatment has gained official, regulatory approval. Dogs are central to the life cycle of N. caninum and have repeatedly been associated with infection and abortions in cattle by epidemiological studies. Knowledge and understanding of that pivotal role should be able to be put to use in control programmes. The present review canvasses the relevant literature for evidence for control options for N. caninum (some of them proven, many not) and assesses them in the light of the authors' knowledge and experience with control of N. caninum.

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.

Potential contribution of P-glycoproteins to macrocyclic lactone resistance in the cattle parasitic nematode Cooperia oncophora.

Resistance against macrocyclic lactones such as ivermectin is widespread among parasitic gastrointestinal nematodes of small ruminants and is rapidly increasing in cattle parasites. ABC transporters of the subfamily B, the so-called P-glycoproteins (Pgps) have been frequently implicated in ivermectin resistance and are a major cause of multi-drug resistance in protozoa and helminths. The Pgp inhibitor verapamil (VPL) dramatically enhanced susceptibility of the cattle parasitic nematode Cooperia oncophora to ivermectin in vitro as measured in a larval developmental assay and a larval migration inhibition assay using third stage larvae. Moreover, VPL completely restored susceptibility to ivermectin in a resistant isolate resulting in virtually identical dose-response curves of susceptible and resistant isolates in the presence of VPL. Further characterisation of the molecular mechanisms resulting in Pgp-mediated ivermectin resistance is still hampered by the lack of molecular and biochemical information for Pgps of parasitic nematodes. Using PCR with degenerate primers, fragments of four different C. oncophora Pgps could be amplified and the Conpgp-2, previously implicated in macrocyclic lactone resistance in Haemonchus contortus, and Conpgp-3 full-length cDNAs were obtained by RACE PCR. The pgp sequences presented here contribute important data required to systematically screen resistant C. oncophora isolates for up- or down-regulation of Pgps and for the detection of single nucleotide polymorphisms in Pgps to detect selection of specific Pgp alleles by anthelmintics as early as possible.

Comparison of the FLOTAC technique with the McMaster method and the Baermann technique to determine counts of Dictyocaulus eckerti L1 and strongylid eggs in faeces of red deer (Cervus elaphus).

The FLOTAC flotation technique has been introduced as a new diagnostic tool to detect parasitic elements from faeces. Samples from naturally infected young deer were used for counting Dictyocaulus larvae and strongylid eggs. The FLOTAC technique, using 11 different flotation solutions with specific gravities (sg) between 1.20 and 1.45, was compared with the Baermann technique and the saturated sodium chloride (sg 1.20)-based McMaster method. In addition, a comparison was made between the FLOTAC technique with magnesium sulphate (sg 1.28) and the Baermann technique for larval recovery from faeces that were examined on the day of collection or after 7 days storage at 4 degrees C. On the whole egg counts between the FLOTAC using different flotation solutions and the McMaster were unremarkable. In contrast, variations of larval counts were detected between different flotation solutions as well as with the Baermann technique. Most flotation solutions with a specific gravity of 1.20 floated significantly fewer lungworm larvae (p < 0.05) compared to flotation solutions with a higher specific gravity. Magnesium sulphate (sg 1.28) consistently produced the highest mean larval counts in all conducted experiments. Larval counts using magnesium sulphate (sg 1.28) were higher than with the Baermann technique both on the day of collection and after 7 days. Overall, the use of magnesium sulphate (sg 1.28) with FLOTAC for larval counts resulted in higher counts than the Baermann recovery technique and was the better choice of those flotation solutions examined. Furthermore, magnesium sulphate (sg 1.28) was also reliable for strongylid egg detection with the FLOTAC apparatus.