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It has come to our attention that the name of one of the authors in our manuscript was incorrectly spelled ‘Jinyoung Byan’; the correct spelling is ‘Jinyoung Byun’ as in the author list above. In addition, the excel files of the supplementary tables were not included during the online publication of our article. These have now been made available online. We apologize for any inconvenience caused.

Background Bovine fasciolosis is an economically important livestock disease in Europe, and represents a particular challenge for organic farms, where cattle are grazed extensively and the use of anthelmintic is limited. A two-year longitudinal study was conducted on two conventional and two organic Danish dairy farms to examine the current temporal trend of F. hepatica infection on-farm, and to gather data of practical relevance for parasite control. Data were collected both at the herd and individual level using currently available diagnostic methods: a commercial serum antibody ELISA, a commercial copro-antigen ELISA, faecal egg counts, and monthly bulk tank milk (BTM) ELISA. The temporal patterns (animal age, farm-level temporal trends and seasonality) in the animal-level test results were analysed by generalised additive mixed models (GAMM). Results Patterns of infection differed substantially between the farms, due to different grazing management and anthelmintic use. However, animals were first infected at the age of 1.5–2 years (heifers), and most at-risk animals sero-converted in autumn, suggesting that summer infections in snails prevail in Denmark. Our results also suggest that the lifespan of the parasite could be over 2 years, as several cows showed signs of low grade infection even after several years of continuous indoor housing without access to freshly-cut grass. The serum antibody ELISA was able to detect infection first, whereas both copro-antigen ELISA and faecal egg counts tended to increase in the same animals at a later point. Decreasing BTM antibody levels were seen on the two farms that started anthelmintic treatment during the study. Conclusions While important differences between farms and over time were seen due to varying grazing management, anthelmintic treatment and climatic conditions, the young stock was consistently seen as the main high-risk group and at least one farm also had suspected transmission (re-infection) within the lactating herd. Careful interpretation of test results is necessary for older cows as they can show persistent infections several years after exposure has stopped. Rigorous treatment regimens can reduce BTM ELISA values, but further research is needed to develop a non-medicinal approach for sustainable management of bovine fasciolosis. Electronic supplementary material The online version of this article (10.1186/s13071-018-3248-z) contains supplementary material, which is available to authorized users.

Purpose: To investigate the prevalence of Cryptosporidium spp. infection and Toxoplasma gondii antibodies in farmed and feral mink in Denmark. Methods: We examined meat juice from 235 feral mink and 306 farmed mink for T. gondii antibodies, and faecal samples from 113 feral mink and 166 farmed mink for Cryptosporidium oocyst excretion. Meat juice was analysed using a commercial indirect enzyme-linked immunosorbent assay and oocyst excretion was identified by a modified Ziehl–Neelsen method. Results: All farmed mink tested sero-negative, while 53.6% of feral mink were T. gondii sero-positive. The probability of being sero-positive for T. gondii was not associated with recent escapes from farms (p = 0.468), but was significantly higher for male feral mink (64.2%) than female feral mink (42.5%) (p = 0.0008). Only one feral mink and four farmed mink (2.4%) excreted Cryptosporidium oocysts. Conclusion: Farmed mink were all T. gondii sero-negative, whereas approximately half the feral mink were sero-positive. Cryptosporidium prevalence in farmed and feral mink were low. Overall, the public health risk of transmission of these two parasites via mink in Denmark is low.

International audience; Trichuris trichiura and T. suis are whipworms of humans and pigs, respectively, but it has recently been suggested that humans may be infected with multiple genotypes or species of Trichuris and cross-infection with Trichuris of pig origin has also been reported. In addition, the species status of Trichuris in non-human primates is unsettled and it is unknown how many whipworm species we share with other primates. Herein, we inferred the phylogeny of Trichuris collected from human, baboon and pig based on nuclear (18S and beta-tubulin) and mitochondrial (cox1) genes and evaluated the use of three PCR linked restriction fragment length polymorphism (PCR-RFLP) to identify worms. We found that all baboon worms clustered with human worms and that all these primate worms are different from T. suis. In general, there was an agreement between the phylogeny established based on the nuclear and mtDNA genes. However, we found evidence for non-targeted cox1 gene amplification for a subset of the human worms and suggest the presence of mitochondrial pseudogenes (numts) of pig cox1 gene in the human Trichuris genome. In conclusion, phylogenetic characterization of human whipworm based on the cox1 gene alone may be problematic without suitable preceded measures to avoid the numts amplification.

Summary Aqueous environmental DNA (eDNA) is an emerging efficient non-invasive tool for species inventory studies. To maximize performance of downstream quantitative PCR (qPCR) and next-generation sequencing (NGS) applications, quality and quantity of the starting material is crucial, calling for optimized capture, storage and extraction techniques of eDNA. Previous comparative studies for eDNA capture/storage have tested precipitation and ‘open’ filters. However, practical ‘enclosed’ filters which reduce unnecessary handling have not been included. Here, we fill this gap by comparing a filter capsule (Sterivex-GP polyethersulfone, pore size 0·22 μm, hereafter called SX) with commonly used methods. Our experimental set-up, covering altogether 41 treatments combining capture by precipitation or filtration with different preservation techniques and storage times, sampled one single lake (and a fish-free control pond). We selected documented capture methods that have successfully targeted a wide range of fauna. The eDNA was extracted using an optimized protocol modified from the DNeasy® Blood & Tissue kit (Qiagen). We measured total eDNA concentrations and Cq-values (cycles used for DNA quantification by qPCR) to target specific mtDNA cytochrome b (cyt b) sequences in two local keystone fish species. SX yielded higher amounts of total eDNA along with lower Cq-values than polycarbonate track-etched filters (PCTE), glass fibre filters (GF) or ethanol precipitation (EP). SX also generated lower Cq-values than cellulose nitrate filters (CN) for one of the target species. DNA integrity of SX samples did not decrease significantly after 2 weeks of storage in contrast to GF and PCTE. Adding preservative before storage improved SX results. In conclusion, we recommend SX filters (originally designed for filtering micro-organisms) as an efficient capture method for sampling macrobial eDNA. Ethanol or Longmire's buffer preservation of SX immediately after filtration is recommended. Preserved SX capsules may be stored at room temperature for at least 2 weeks without significant degradation. Reduced handling and less exposure to outside stress compared with other filters may contribute to better eDNA results. SX capsules are easily transported and enable eDNA sampling in remote and harsh field conditions as samples can be filtered/preserved on site.

Schistosomiasis is a waterborne, infectious disease with high morbidity and significant economic burdens affecting more than 250 million people globally. Disease control has, with notable success, for decades focused on drug treatment of infected human populations, but a recent paradigm shift now entails moving from control to elimination. To achieve this ambitious goal more sensitive diagnostic tools are needed to monitor progress towards transmission interruption in the environment, especially in low-intensity infection areas. We report on the development of an environmental DNA (eDNA) based tool to efficiently detect DNA traces of the parasite Schistosoma mansoni directly in the aquatic environment, where the non-human part of the parasite life cycle occurs. To our knowledge, this is the first report of the successful detection of S. mansoni in freshwater samples using aquatic eDNA. True eDNA was detected in as few as 10 cercariae/L water in laboratory experiments. The field applicability of the method was tested at known transmission sites in Kenya, where comparison of schistosome detection by conventional snail surveys (snail collection and cercariae shedding) with eDNA (water samples) showed 71% agreement between the methods. The eDNA method furthermore detected schistosome presence at two additional sites where snail shedding failed, demonstrating a higher sensitivity of eDNA sampling. We conclude that eDNA provides a promising new tool to significantly improve the environmental surveillance of S. mansoni. Given the proper method and guideline development, eDNA could become an essential future component of the schistosomiasis control tool box needed to achieve the goal of elimination.SignificanceAccurate detection and delineation of schistosomiasis transmission sites will be vital in on-going efforts to control and ultimately eliminate one of the most neglected tropical parasitic diseases affecting more than 250 million people worldwide. Conventional methods to detect parasites in the environment are cumbersome and have low sensitivity. We therefore developed an environmental DNA (eDNA) based method for schistosome detection in aquatic environments. Aquatic eDNA showed higher sensitivity than conventional snail surveys. We conclude that eDNA is a promising non-invasive and sensitive tool for environmental surveillance of schistosomiasis transmission. As the efforts and aims to control the disease are transitioning towards complete transmission interruption, this could be the robust and cost-effective surveillance tool needed in the “end game” of schistosomiasis.

Schistosomiasis is a water-based, infectious disease with high morbidity and significant economic burdens affecting >250 million people globally. Disease control has, with notable success, for decades focused on drug treatment of infected human populations, but a recent paradigm shift now entails moving from control to elimination. To achieve this ambitious goal, more sensitive diagnostic tools are needed to monitor progress toward transmission interruption in the environment, especially in low-intensity infection areas. We report on the development of an environmental DNA (eDNA)-based tool to efficiently detect DNA traces of the parasite Schistosoma mansoni directly in the aquatic environment, where the nonhuman part of the parasite life cycle occurs. This is a report of the successful detection of S. mansoni in freshwater samples by using aquatic eDNA. True eDNA was detected in as few as 10 cercariae per liter of water in laboratory experiments. The field applicability of the method was tested at known transmission sites in Kenya, where comparison of schistosome detection by conventional snail surveys (snail collection and cercariae shedding) with eDNA (water samples) showed 71% agreement between the methods. The eDNA method furthermore detected schistosome presence at two additional sites where snail shedding failed, demonstrating a higher sensitivity of eDNA sampling. We conclude that eDNA provides a promising tool to substantially improve the environmental surveillance of S. mansoni. Given the proper method and guideline development, eDNA could become an essential future component of the schistosomiasis control tool box needed to achieve the goal of elimination.

BACKGROUND Soil transmitted helminths (STH) continue to be associated with high burdens of disease, with an estimated 1.45 billion people infected with STH globally. The promotion and construction of latrines is considered the first barrier to prevent transmission of STH. The absence of a reliable method to extract STH ova from soil makes it challenging to examine whether the use of latrines may or may not have an effect on environmental contamination with ova. The present study evaluated the recovery rate of a method developed to extract STH ova from soil. METHODS The adapted centrifugation and flotation technique was applied to 15 soil types, which were seeded with Ascaris suum ova. Soil type, soil moisture content, soil texture and organic matter content were assessed for each soil sample. RESULTS The average ova recovery rate was 28.2%, with the recovery rate of the method decreasing with increasing soil moisture content, particle size and organic matter content. The association between recovery rate and organic matter content was statistically significant. CONCLUSIONS The present study identified a low recovery rate for an adapted centrifugation-flotation method, although this was similar to the recovery rate demonstrated by other methods developed for soil. Soil organic matter content was significantly associated with ova recovery rates.

The exact impact of climate change on schistosomiasis, a disease caused by a blood fluke that affects more than 250 million people mainly in tropical and subtropical countries, is currently unknown, but likely to vary with the snail-parasite species' specific ecologies and the spatio-temporal scale of investigation. Here, by means of a systematic review to identify studies reporting on impacts of climate change on the agents of schistosomiasis, we provide an updated synthesis of the current knowledge about the climate change-schistosomiasis relation. We found that, despite a recent increase in scientific studies that discuss the potential impact of climate change on schistosomiasis, only a handful of reports have applied modelling and predictive forecasting that provide a quantitative estimate of potential outcomes. The volume and type of evidence associated with climate change responses were found to be variable across geographical regions and snail-parasite taxonomic groups. Indeed, the strongest evidence stems from the People's Republic of China pertaining to Schistosoma japonicum. Some evidence is also available from eastern Africa, mainly for Schistosoma mansoni. While studies focused on the northern and southern range margins for schistosomiasis indicate an increase in transmission range as the most likely outcome, there was less agreement about the direction of outcomes from the central and eastern parts of Africa. The current lack of consensus suggests that climate change is more likely to shift than to expand the geographic ranges of schistosomiasis. A comparison between the current geographical distributions and the thermo-physiological limitations of the two main African schistosome species (Schistosoma haematobium and S. mansoni) offered additional insights, and showed that both species already exist near their thermo-physiological niche boundaries. The African species both stand to move considerably out of their "thermal comfort zone" in a future, warmer Africa, but S. haematobium in particular is likely to experience less favourable climatic temperatures. The consequences for schistosomiasis transmission will, to a large extent, depend on the parasites and snails ability to adapt or move. Based on the identified geographical trends and knowledge gaps about the climate change-schistosomiasis relation, we propose to align efforts to close the current knowledge gaps and focus on areas considered to be the most vulnerable to climate change.

Water from wastewater-polluted streams and dug-outs is the most commonly used water source for irrigation in urban farming in Ghana, but helminth parasite eggs in the water represent health risks when used for crop production. Conventional water treatment is expensive, requires advanced technology and often breaks down in less developed countries so low cost interventions are needed. Field and laboratory based trials were carried out in order to investigate the effect of the natural coagulant Moringa oleifera (MO) seed extracts in reducing helminh eggs and turbidity in irrigation water, turbid water, wastewater and tap water. In medium to high turbid water MO extracts were effective in reducing the number of helminth eggs by 94–99.5% to 1–2 eggs per litre and the turbidity to 7–11 NTU which is an 85–96% reduction. MO is readily available in many tropical countries and can be used by farmers to treat high turbid water for irrigation, however, additional improvements of water quality, e.g. by sand filtration, is suggested to meet the guideline value of ≤1 helminth egg per litre and a turbidity of ≤2 NTU as recommended by the World Health Organization and the U.S. Environmental Protection Agency for water intended for irrigation. A positive correlation was established between reduction in turbidity and helminth eggs in irrigation water, turbid water and wastewater treated with MO. This indicates that helminth eggs attach to suspended particles and/or flocs facilitated by MO in the water, and that turbidity and helminth eggs are reduced with the settling flocs. However, more experiments with water samples containing naturally occurring helminth eggs are needed to establish whether turbidity can be used as a proxy for helminth eggs.

The freshwater gastropod family Viviparidae is nearly cosmopolitan, but absent from South America. On the African continent, two genera are recognized; the widespread Bellamya and the monotypic Neothauma , which is confined to Lake Tanganyika. Most of the African Bellamya species are confined to the major lakes of the Rift Valley area in Africa, i.e. Lake Albert, Lake Malawi, Lake Mweru, and Lake Victoria. The phylogenetic analyses of mitochondrial (COI and 16S) and nuclear (H3, 18S and 28S) DNA inferred three major lake-clades; i.e. Lake Victoria/Kyoga/Albert, Lake Malawi and Lake Mweru/Bangweulu. The endemic B. rubicunda from Lake Albert and B. unicolor from Lake Kyoga were inferred to be part of the Lake Victoria clade. Bellamya capillata as identified by shell characters was polyphyletic in gene trees. The monophyletic Bellamya species radiation in Lake Malawi was most nearly related to the Lake Victoria/Kyoga/Albert-clade. Taxa from the Zambian lakes, Mweru and Bangweulu, were inferred together and placed ancestral to the other lakes. Neothauma tanganyicense was inferred as the sister-group to the Zambian Bellamya . Within the lake-clades the endemic radiations show very low genetic diversities (0–4.1% in COI), suggesting much faster morphological divergence than molecular divergence. Alternatively, Bellamya in Africa constitutes only a few species with several sub-species or eco-phenotypic morphs. The African viviparids were inferred to be the sister-group to a clade comprising Asian species, and the relatively low genetic diversity between the clades (12.6–15.5% in COI) makes a recent Miocene dispersal event from Asia to Africa much more likely than an ancient Gondwana vicarience distribution.

Strongyle eggs of helminths of livestock usually hatch within a few hours or days after deposition with faeces. This poses a problem when faecal sampling is performed in the field. As oxygen is needed for embryonic development, it is recommended to reduce air supply during transport and refrigerate. The present study therefore investigated the combined effect of vacuum packing and temperature on survival of strongyle eggs and their subsequent ability to hatch and develop into L3. Fresh faecal samples were collected from calves infected with Cooperia oncophora, pigs infected with Oesophagostomum dentatum, and horses infected with Strongylus vulgaris and cyathostomins. The samples were allocated into four treatments: vacuum packing and storage at 5 °C or 20 °C (5 V and 20 V); normal packing in plastic gloves closed with a loose knot and storage at 5 °C or 20 °C (5 N and 20 N). The number of eggs per gram faeces (EPG) was estimated every fourth day until day 28 post set up (p.s.) by a concentration McMaster-method. Larval cultures were prepared on day 0, 12 and 28 p.s. and the larval yield determined. For C. oncophora, the EPG was significantly higher in vacuum packed samples after 28 days as compared to normal storage, regardless of temperature. However, O. dentatum EPG was significantly higher in samples kept at 5 °C as compared to 20 °C, irrespective of packing. For the horse strongyles, vacuum packed samples at 5 °C had a significantly higher EPG compared to the other treatments after 28 days. The highest larval yield of O. dentatum and horse strongyles were obtained from fresh faecal samples, however, if storage is necessary prior to setting up larval cultures O. dentatum should be kept at room temperature (aerobic or anaerobic). However, horse strongyle coprocultures should ideally be set up on the day of collection to ensure maximum yield. Eggs of C. oncophora should be kept vacuum packed at room temperature for the highest larval yield.

The importance of hands in the transmission of soil transmitted helminths, especially Ascaris and Trichuris infections, is under-researched. This is partly because of the absence of a reliable method to quantify the number of eggs on hands. Therefore, the aim of this study was to develop a method to assess the number of Ascaris eggs on hands and determine the egg recovery rate of the method. Under laboratory conditions, hands were seeded with a known number of Ascaris eggs, air dried and washed in a plastic bag retaining the washing water, in order to determine recovery rates of eggs for four different detergents (cationic [benzethonium chloride 0.1% and cetylpyridinium chloride CPC 0.1%], anionic [7X 1% - quadrafos, glycol ether, and dioctyl sulfoccinate sodium salt] and non-ionic [Tween80 0.1% -polyethylene glycol sorbitan monooleate]) and two egg detection methods (McMaster technique and FLOTAC). A modified concentration McMaster technique showed the highest egg recovery rate from bags. Two of the four diluted detergents (benzethonium chloride 0.1% and 7X 1%) also showed a higher egg recovery rate and were then compared with de-ionized water for recovery of helminth eggs from hands. The highest recovery rate (95.6%) was achieved with a hand rinse performed with 7X 1%. Washing hands with de-ionized water resulted in an egg recovery rate of 82.7%. This washing method performed with a low concentration of detergent offers potential for quantitative investigation of contamination of hands with Ascaris eggs and of their role in human infection. Follow-up studies are needed that validate the hand washing method under field conditions, e.g. including people of different age, lower levels of contamination and various levels of hand cleanliness.

Acute myocardial infarction (AMI) occurs due to a decrease of myocardial blood flow following a coronary arterial occlusion caused by an atherosclerotic plaque. This study aimed to determine the correlation between the hematocrit level and the occurence of AMI among patients with congestive heart failure (CHF). This was an observational analytic study with a cross sectional design. The population was both CHF patients with old myocardial infarction in the Cardiology Clinic and all AMI patients with CHF histories in the Intensive Cardiac Care Unit (ICCU) of Prof. Dr. R.D. Kandou Hospital, Manado, from November through December 2012. Samples were selected by using a purposive sampling method. Data were statistically analyzed by using a chi-square test. The results showed that the total samples were 41 patients. The chi-square test showed that there was a correlation between the hematocrit level and the occurence of AMI among CHF patients with a P -value of 0.008. Conclusion: Hematocrit levels were significantly correlated with the occurence of AMI among CHF patients in Prof. Dr. R.D. Kandou Hospital, Manado Key w ord s : CHF, AMI, hematocrit Abstrak: Infark miokard akut (IMA) terjadi oleh karena penurunan aliran darah miokard akibat oklusi arteri koroner oleh plak aterosklerotik. Penelitian ini bertujuan untuk mengetahui hubungan kadar hematokrit dengan kejadian infark miokard akut (IMA) pada pasien gagal jantung kongestif (CHF). Penelitian ini bersifat analitik observational dengan cross-sectional design . Populasi penelitian ialah semua pasien CHF di Poliklinik Jantung dan semua pasien IMA dengan riwayat CHF di Intensive Cardiac Care Unit (ICCU) BLU/RSUP Prof. Dr. R.D. Kandou, Manado, periode November-Desember 2012. Sampel penelitian diambil dengan teknik purposive sampling . Kriteria inklusi yaitu pasien CHF e t c ausa old myocardial infarction (OMI) dan pasien IMA dengan riwayat CHF, sedangkan kriteria eksklusi yaitu pasien IMA dengan penyakit infeksi dan pasien CHF dengan keganasan hematopoietik. Data hasil penelitian dianalisis dengan uji c hi- s quare . Hasil penelitian memperlihatkan jumlah sampel sebanyak 41 pasien. Uji chi-square terhadap hubungan hematokrit dan infark miokard akut pada pasien gagal jantung menunjukkan nilai P = 0.008. Simpulan: Terdapat hubungan bermakna antara kadar hematokrit dan infark miokard akut pada pasien gagal jantung kongestif di BLU/RSUP Prof Dr. R.D. Kandou Manado. Kata kunci: CHF, IMA, Hematokrit

Helminth parasite eggs in low quality water represent main food safety and health hazards and are therefore important indicators used to determine whether such water can be used for irrigation. Through sedimentation helminth eggs accumulate in the sediment, however resuspension of deposited helminth eggs will lead to increased concentration of suspended eggs in the water. Our study aimed to determine the erodibility (erosion rate and erosion threshold) and settling velocity of Ascaris and Trichuris eggs as well as cohesive sediment at different time points after incorporation into the sediment. Cohesive sediment collected from a freshwater stream was used to prepare a sediment bed onto which helminth eggs were allowed to settle. The erodibility of both sediment and helminth eggs was found to decrease over time indicating that the eggs were incorporated into the surface material of the bed and that this material was stabilized through time. This interaction between eggs and bulk sediment was further manifested in an increased settling velocity of suspended eggs when sediment was present in the suspension as compared to a situation with settling in clean water. The incorporation into the sediment bed and the aggregation with sediment particles decrease the mobility of both helminth egg types. Our findings document that helminth eggs should not be viewed as single entities in water systems when modelling the distribution of eggs since both erodibility and settling velocity of eggs are determined by mobility of the sediment present in the water stream. Recalculation of the erosion threshold for helminth eggs and sediment showed that even at relatively low current velocities i.e. 0.07-0.12ms(-1) newly deposited eggs will be mobile in open irrigation channels. These environmental factors affecting resuspension must be taken into account when developing models for sedimentation of helminth eggs in different water systems.

Helminth parasite eggs in low quality water represent health risks when used for irrigation of crops. The settling velocities of helminth eggs (Ascaris suum, Trichuris suis, and Oesophagostomum spp.) and wastewater particles were experimentally determined in tap water and in wastewater using Owen tubes. The settling velocities of eggs in tap water was compared with theoretical settling velocities calculated by Stoke's law using measurements of size and density of eggs as well as density and viscosity of tap water. The mean settling velocity in tap water of 0.0612 mm s(-1) found for A. suum eggs was significantly lower than the corresponding values of 0.1487 mm s(-1) for T. suis and 0.1262 mm s(-1) for Oesophagostomum spp. eggs. For T. suis and Oesophagostomum spp. eggs the theoretical settling velocities were comparable with the observed velocities in the Owen tubes, while it was three times higher for A. suum eggs. In wastewater, the mean settling velocity for A. suum eggs (0.1582 mm s(-1)) was found to be different from T. suis (0.0870 mm s(-1)), Oesophagostomum spp. (0.1051 mm s(-1)), and wastewater particles (0.0474 mm s(-1)). This strongly indicates that in low quality water the eggs are incorporated into particle flocs with different settling velocities and that the settling velocity of eggs and particles is closely associated. Our results document that there is a need to differentiate the sedimentation of different types of helminth eggs when assessing the quality of low quality water, e.g. for irrigation usage. The results can also be used to improve existing models for helminth egg removal.

Seasonal variation of the volatile terpenoids of Pinus pinea Ten and Pinus halepensis Mill, infested and uninfested by the caterpillar Marchalina hellenica, was followed by GC and GC-MS analyses of the pines cortical oleoresin, α-Pinene was found to be the dominant monoterpene in P. halepensis, while in P. pinea limonene was the most abundant compound. A significant decrease in the number of identified volatiles from winter to summer was observed and was more pronounced in the minor metabolites. Variation of the terpenoids according to the depth of the draining (drilling) holes in the trees was also determined. In addition, terpenoids were correlated with the results of cross feeding experiments designed for the study of the host preference of M. hellenica. All results revealed the dependence of M. hellenica on the secondary chemistry of the host. P. pinea, having a high content of limonene, appears to be more resistant to the caterpillar. Canonical discriminant analysis, in the discriminant space of the relative contribution of the ten major terpenes, separated P. pinea from P. halepensis, and infested from uninfested P. halepensis trees.