The impact of Ixodiphagus hookeri, a tick parasitoid, on Ambl yomma variegatum (Acari: Ixodidae) in a field trial in Kenya

In an experiment to investigate the effect of the tick parasitoid, Ixodiphagus hookeri, on tick numbers on cattle, 150 000 parasitoids were released over a period of 1 year in a field where ten cattle infested with multiple tick species were kept. Amblyomma variegatum was reduced from 44 to two ticks per animal while Rhipicephalus appendiculatus increased over the time of parasitoid release. During the time of release 51% of the nymphs of A. variegatum collected from the animals were parasitized. The recovery of the parasitoids after the releases were stopped was only 9%. The total numbers of A. variegatum remained low up to 1 year after the parasitoid release was stopped. This study gives an insight into how I. hookeri could be used strategically for the management of A. variegatum on small-scale farms.     

Borrelia Burgdorferi Sensu Lato in Ixodes Ricinus Ticks and Rodents in a Recreational Park in South-Western Ireland

Ixodes ricinus ticks infected with Borrelia burgdorferi sensu lato were numerous on the edges of paths and roads in a recreational park in south-western Ireland. The abundance of ticks at different sites was related to the presence of deer, but a negative relationship was shown between tick abundance and tick infection rates. This is thought to be due to the deposition of large numbers of uninfected ticks by deer, which are apparently not good reservoir hosts of B. burgdorferi s.l. Blood meal analysis only detected deer DNA in uninfected nymphs. Reservoir competent rodents, Apodemus sylvaticus and Clethrionomys glareolus, were abundant at all sites and a high proportion of captured specimens were infested with larval ticks. However, very few rodents were infected with B. burgdorferi s.l. and none of the unfed infected nymphs analysed for the identity of their larval blood meal had fed on rodents. The spirochaetes detected in I. ricinus in the study area may be poorly adapted to rodents or are not transmitted readily because of the absence of nymphal infestation. The majority of spirochaetes in these ticks were apparently acquired from non-rodent hosts, such as birds.     

Bodies of liquid water as a source of water gain for Ixodes ricinus ticks (Acari: Ixodidae)

It is well established that free-living unfed ticks can compensate for their inevitable body water losses by active water vapour absorption in periods of ambient relative humidity (RH) of greater than 75-90%. Whilst many species of terrestrial arthropods are known to be capable of locating liquid water and drinking when in need, the existing knowledge concerning the ecological significance of bulk water for rehydration in ticks is scarce. In the present laboratory study batches of unfed Ixodes ricinus (larvae, nymphs, and adults) in varying states of (de)hydration were supplied with tapwater either as droplets for 45 min in a Petri dish or in a small trough for 24 h. The body mass of each tick was gravimetrically determined before and after exposure. Though fully hydrated ticks were not usually attracted to liquid water, the response became increasingly positive with a growing body water deficit (p > 0.05). No tick was ever found with its mouthparts inserted into the water, nor had any tick residues of orange G in its alimentary tract when supplied with orange G-coloured water. Linear regression analysis demonstrated that water intake took place in a regulated manner in adult ticks, i.e. the more severely the ticks had been dehydrated the more net water they subsequently gained (p > 0.01). The maximum uptake rates were <20% of the original body mass per day. These findings indicate that unfed I. ricinus do not drink liquid water but are well able to use the high RH in the immediate vicinity of bulk water to actively take up vapour.     

Combined infection of<Emphasis Type="Italic">Ixodes ricinus</Emphasis> with three<Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> sensu lato genotypes

Ixodes ricinus ticks were collected by random collections from western and central Slovakia during the years 1996-98. The midgut content of 240 ticks was examined by dark-field microscopy and submitted for cultivation for the presence of borrelias. Spirochetes were found in 21 unfed and host-seeking adults and nymphs (8.8%). By the analysis of restriction fragment length polymorphism (RFLP) one sample from unfed I. ricinus male from western Slovakia was identified as a triple infection of Borrelia burgdorferi sensu stricto, B. garinii and B. afzelii. The simultaneous presence of different B. burgdorferi genospecies in one midgut sample (triple infection in the tick) could be observed only after the multipart amplification of denaturated DNA and subsequent pooling of the products for further analysis.     

A hidden beneficial: biology of the tick‐wasp Ixodiphagus hookeri in Germany

The parasitic wasp Ixodiphagus hookeri parasitizes hard ticks and is therefore considered as a potential candidate for biological control of ticks. However, there are still considerable gaps of knowledge about the biology of I. hookeri, especially for European populations. Thus, the present study was performed to assess important life‐history parameters of the parasitoid in Germany. Field studies accomplished in three successive years revealed that unfed Ixodes ricinus nymphs are infested by the parasitoid at a low but constant rate of 1.9–3.8% and that adult wasps are present only during a short period in late summer. The mean developmental time of wasps in I. ricinus nymphs ranged from 28 to 70 days under constant laboratory conditions and was prolonged in the second half of the year. Bioassays on parasitization and host preference behaviour showed that unfed nymphs of the host species I. ricinus are significantly preferred in experiments, in which unfed and engorged larvae as well as fully engorged nymphs were offered as alternatives. The marsh tick Dermacentor reticulatus was not accepted as an alternative host. Our data show that the investigated I. hookeri populations differ markedly from populations in other regions of the world in many aspects. The adaptation of different strains to local conditions explains the limited success of imported strains in earlier biological control attempts and highlights the importance of doing research to enhance the control potential of native strains.     

An empirical quantitative framework for the seasonal population dynamics of the tick Ixodes ricinus

The wide geographic and climatic range of the tick Ixodes ricinus, and the consequent marked variation in its seasonal population dynamics, have a direct impact on the transmission dynamics of the many pathogens vectored by this tick species. We use long-term observations on the seasonal abundance and fat contents (a marker of physiological ageing) of ticks, and contemporaneous microclimate at three field sites in the UK, to establish a simple quantitative framework for the phenology (i.e. seasonal cycle of development) of I. ricinus as a foundation for a generic population model. An hour-degree tick inter-stadial development model, driven by soil temperature and including diapause, predicts the recruitment (i.e. emergence from the previous stage) of a single cohort of each stage of ticks each year in the autumn. The timing of predicted emergence coincides exactly with the new appearance of high-fat nymphs and adults in the autumn. Thereafter, fat contents declined steadily until unfed ticks with very low energy reserves disappeared from the questing population within about 1 year from their recruitment. Very few newly emerged ticks were counted on the vegetation in the autumn, but they appeared in increasing numbers through the following spring. Larger ticks became active and subsequently left the questing population before smaller ones. Questing tick population dynamics are determined by seasonal patterns of tick behaviour, host-contact rates and mortality rates, superimposed on a basal phenology that is much less complex than has hitherto been portrayed.     

Diversity of Ixodes ricinus tick-associated bacterial communities from different forests

Nymphal Ixodes ricinus ticks (n=180) were collected from three different areas in the Netherlands to investigate the effect of forest composition on tick-associated microbial communities. Sampled habitats differed in thickness of leaf litter and humus layers and vegetation associations and were located near Amsterdam (Beech-Oak), Ede (Birch-Oak) and Veldhoven (Birch-Oak). Analysis of nine 16S rRNA gene clone libraries made from individual ticks showed nearest matches with presumed pathogens Candidatus Neoehrlichia mikurensis and Rickettsia australis and arthropod endosymbionts Wolbachia pipientis and Candidatus Midichloria mitochondrii. Total bacterial species diversity (Shannon index) and Borrelia species infections were determined in I. ricinus by, respectively, PCR-denaturing gradient gel-electrophoresis and PCR-reverse line blot with probes specific for Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii, Borrelia valaisiana, Borrelia lusitaniae and Borrelia ruski. Bacterial diversity differed significantly per area and was lowest in Ede. In contrast, Borrelia species-infected ticks were more abundant in Ede, Candidatus Neoehrlichia mikurensis-infected ticks in Ede and Veldhoven, and R. australis-infected ticks in Amsterdam. Borrelia afzelii was the most common Borrelia species found in all three areas. Bacterial tick diversity was influenced by local differences in forest structure, which is proposed to modulate animal populations that are commonly parasitized by I. ricinus.     

Decoupling of host-symbiont-phage coadaptations following transfer between insect species

Transferring endosymbiotic bacteria between different host species can perturb the coordinated regulation of the host and bacterial genomes. Here we use the most common maternally transmitted bacteria, Wolbachia pipientis, to test the consequences of host genetic background on infection densities and the processes underlying those changes in the parasitoid wasp genus Nasonia. Introgressing the genome of Nasonia giraulti into the infected cytoplasm of N. vitripennis causes a two-order-of-magnitude increase in bacterial loads in adults and a proliferation of the infection to somatic tissues. The host effect on W. pipientis distribution and densities is associated with a twofold decrease in densities of the temperate phage WO-B. Returning the bacteria from the new host species back to the resident host species restores the bacteria and phage to their native densities. To our knowledge, this is the first study to report a host-microbe genetic interaction that affects the densities of both W. pipientis and bacteriophage WO-B. The consequences of the increased bacterial density include a reduction in fecundity, an increase in levels of cytoplasmic incompatibility (CI), and unexpectedly, male-to-female transfer of the bacteria to uninfected females and an increased acceptance of densely infected females to interspecific mates. While paternal inheritance of the W. pipientis was not observed, the high incidence of male-to-female transfer in the introgressed background raises the possibility that paternal transmission could be more likely in hybrids where paternal leakage of other cytoplasmic elements is also known to occur. Taken together, these results establish a major change in W. pipientis densities and tissue tropism between closely related species and support a model in which phage WO, Wolbachia, and arthropods form a tripartite symbiotic association in which all three are integral to understanding the biology of this widespread endosymbiosis.     

A novel test system for detection of tick repellents

A bioassay was developed to examine the response of ticks towards potential repellents that may protect vertebrates against tick bites. Such tick repellents must be effective despite the unavoidable presence of various attractive host-derived stimuli. Therefore, a moving-object-bioassay (MO-bioassay) was developed that mimicks body warmth and movement of vertebrates by a rotating and heated drum. Compounds which were tested for their effects on ticks were applied onto a small elevated area of the drum. Ticks were allowed to approach the drum by walking on a glass rod which ended 1 mm away from the local elevation. Ticks could cling to this elevation that intermittently passed by, whereas the remaining drum surface was too far away from the tip of the rod to be contacted by the ticks.Without the presence of any repellents, 85.5% of 600 hungry, field-collected Ixodes ricinus nymphs moved to the heated, rotating drum within 2 min. Further experiments with unfed I. ricinus nymphs were performed to test whether one established and two potential tick repellents elicit an avoidance reaction in the ticks despite the proven attractiveness of the drum. Freshly applied DEET (N,N-diethyl-m-toluamide) at a concentration of 0.11 mg cm^–2 proved active as a repellent in the MO-bioassay over a distance of a few mm as well as by direct contact. A similar repellent effect was observed with (–)-myrtenal at 1 mg cm^–2, but not at 0.1 mg cm^–2, indicating that this terpenaldehyde is a weaker repellent for I. ricinus nymphs than DEET. No repellent effect was observed with camphor (0.1 mg cm^–2).The MO-bioassay thus is a rapid, simple and low-cost test method allowing the investigation of tick host-contact behaviour as well as the screening of candidate repellents which are either perceived as volatiles or via contact chemoreception.     

Infestation of mammals by Ixodes ricinus ticks (Acari: Ixodidae) in south-central Sweden

Infestation by Ixodes ricinus ticks on rodents, hares and cervids was examined at Bogesund, 10 km north of Stockholm, in south-central Sweden during 1991-1994 and on varying hares (Lepus timidus) at Stora Karlso and Gotska Sandon in the Baltic Sea during 1992-1993. At Bogesund, there were great differences between two consecutive years in the number of I. ricinus larvae infesting bank voles (Clethrionomys glareolus). The seasonal pattern of infestation by I. ricinus larvae and nymphs on bank voles was unimodal in 1991, with peaks in June-July and bimodal in 1992, with peaks in June and August. Male bank voles, compared to females and older voles, compared to young voles, harboured greater numbers of I. ricinus ticks. Apodemus mice, compared to bank voles, harboured greater numbers of I. ricinus ticks. Ixodes ricinus larvae engorged on Apodemus mice were heavier than larvae engorged on bank voles and resulted in larger nymphs. However, there was no difference in the proportions of viable nymphs resulting from larvae engorged on mice or voles. The ranges in the numbers of I. ricinus ticks infesting individual hosts were 1-451 for rodents, 16-2374 for hares and 428-2072 for roe deer (Capreolus capreolus). These ranges of tick numbers are estimated to represent potential blood losses from individual hosts of approximately 0.2-65% for rodents, 0.2-13% for hares and 0.3-9.0% for roe deer. Within the populations of all host species examined, the distributions of all stages of I. ricinus were clumped, with most host individuals harbouring few ticks and only a few individuals harbouring many ticks. The data suggest that, even though a small proportion of tick hosts may be severely affected, the direct effects of feeding by I. ricinus are unlikely to play an important role on mammal population dynamics.     

Ant predation on different life stages of two Australian ticks

Laboratory colonies of Rhytidoponera ants were allowed to prey on the fed and unfed stages of the Australian ticks Aponomma hydrosauri and Amblyomma limbatum. The unfed tick stages had a higher survival than the fed stages. The ants took longer to handle the adult ticks than the nymphs and longer to handle the nymphs than the larvae. The ants also took longer to handle the unfed than fed nymphs, but longer to handle the fed than unfed females. As well as the differences between the tick stages, there was a species effect, with the ants taking longer to handle A. limbatum, and with that tick species having a higher survival than A. hydrosauri after ant predation. These stage and species differences may influence the tick population dynamics. This revised version was published online in November 2006 with corrections to the Cover Date.     

Diversity and seasonal patterns of ticks parasitizing wild birds in western Portugal

The diversity and abundance of questing ticks and ticks parasitizing birds was assessed during 1?year in two recreational forests in western Portugal, a suburban forest and an enclosed game area. The aim of this study was to assess the distribution and seasonality of tick species and to understand the role of bird species as hosts for ticks. Ixodes ricinus was the most abundant questing tick collected in the enclosed game area, whereas in the suburban forest, only three ticks were collected by blanket dragging. Tick species parasitizing birds included I. ricinus, I. frontalis, I. arboricola, I. acuminatus, Haemaphysalis punctata, Hyalomma marginatum and H. lusitanicum. This is the first record of I. arboricola in Portugal. Tick prevalence and intensity of infestation differed between study areas and was higher in birds from the game area where a large population of deer and wild boar may support tick populations. Ground and shrub dwelling bird species such as Turdus merula, Erithacus rubecula and Sylvia melanocephala were the most heavily parasitized by ticks, but the importance of different bird species as hosts of larvae and nymphs of I. ricinus and I. frontalis differed. Therefore, different bird species may contribute differently for tick population maintenance.     

Risk factors in habitats of the tick Ixodes ricinus influencing human exposure to Ehrlichia phagocytophila bacteria

Ixodes ricinus L. (Acari: Ixodida) were sampled during 1996-99 in southern Scotland, on vegetation using cloth drags, on humans by removal from clothing and on roe deer (Capreolus capreolus L.) by searching legs of culled deer. Developmental microclimate was recorded by automatic recorders and questing microclimate by portable instruments during tick collections. Ticks and deer were examined for infection with Ehrlichia phagocytophila bacteria (Rickettsiales) using microscopy and polymerase chain reaction. This pathogen causes tick-borne fever of sheep in Europe and human granulocytic ehrlichiosis in North America, but in Europe human clinical ehrlichiosis due to E. phagocytophila has not been recorded despite serological evidence of exposure. Among three types of habitat, coniferous woodland was most infested with questing ticks (560 ticks/km of drag; mean numbers collected on long trousers: 24.3 larvae, 13.5 nymphs and 0.8 adult ticks/km walked), deciduous woodland had slightly lower infestation (426 ticks/km drag) and upland sheep pasture had much lower infestation (220 ticks/km drag). Of the three main vegetation types, bracken was least infested (360 ticks/km drag), ericas most (430 ticks/km drag) and grassland had intermediate infestation density (413 ticks/km drag). Questing and developmental microclimates were poor predictors of exposure within these habitats, except lower infestation of pastures was attributed to greater illumination there. Collectors who walked a total of 300 km through all habitats (taking 360 h in all seasons), wearing cotton trousers hanging outside rubber boots, were bitten by only four nymphs and 11 larvae of I. ricinus (but no adult ticks). There was a negative correlation between densities of deer and ticks collected, although presence of deer remains a major indicator of exposure. The proportion of infected ticks was fairly uniform at four sites studied. Overall prevalence of E. phagocytophila in I. ricinus was 3.3% in nymphs (40/1203) but only approximately 1.5% in adults of both sexes (although males do not bite). It was estimated that nymphs of I. ricinus gave 4.4% probability of one infected bite/person/year (for occupational exposure during this research) due to presence in all seasons and habitats, their human biting rate of 0.011 nymphs/h or 0.013 nymphs/km and widespread infection with E. phagocytophila. The frequency distribution of intensity of infection in ticks was approximately normal (mean 98 morulae/nymph infected), thus there is a high risk of receiving a high dose from any one infected tick bite.     

Distribution and molecular characterization of Wolbachia endosymbionts and filarial nematodes in Maryland populations of the lone star tick (Amblyomma americanum)

The lone star tick Amblyomma americanum is host to a wide diversity of endosymbiotic bacteria. We identified a novel Wolbachia symbiont infecting A. americanum. Multilocus sequence typing phylogenetically placed the endosymbiont in the increasingly diverse F supergroup. We assayed a total of 1031 ticks (119 females, 78 males and 834 nymphs in 89 pools) from 16 Maryland populations for infection. Infection frequencies in the natural populations were approximately 5% in females and <2% (minimum infection rate) in nymphs; infection was not detected in males. Infected populations were only observed in southern Maryland, suggesting the possibility that Wolbachia is currently invading Maryland A. americanum populations. Because F supergroup Wolbachia have been detected previously in filarial nematodes, tick samples were assayed for nematodes by PCR. Filarial nematodes were detected in 70% and 9% of Wolbachia-positive and Wolbachia-negative tick samples, respectively. While nematodes were more common in Wolbachia-positive tick samples, the lack of a strict infection concordance (Wolbachia-positive, nematode-negative and Wolbachia-negative, nematode-positive ticks) suggests that Wolbachia prevalence in ticks is not due to nematode infection. Supporting this hypothesis, phylogenetic analysis indicated that the nematodes were likely a novel species within the genus Acanthocheilonema, which has been previously shown to be Wolbachia-free.     

Biometrical analysis of the common tick, Ixodes ricinus, in the Sleza Massif (Lower Silesia, Poland).

The aim of this study was to biometrically analyze the behavioral activity of Ixodes ricinus ticks as influenced by season and trend in numbers, based on material collected during three years in the Sleza Landscape Park within the Sleza Massif (Lower Silesia, Poland) using a time series decomposition method. The effects of the abiotic factors of air temperature and humidity on this activity were also determined. A total of 2,745 nymphs and adults of I. ricinus was observed and collected from 2001 through 2003. It was the only collected species from the 17 representatives of the family Ixodidae recorded from Poland. The abundance of the common tick in the Sleza Landscape Park confirms the risk of tick-borne diseases in these nature areas that are under legal protection. The chance of being bitten by ticks increases in the spring and autumn when the behavioral activity of these arthropods reaches their highest levels. Predictions concerning the level of tick behavioral activity should take into account not only seasonality but also the effects of random components, which accounted for about half of the tick activity in our study. The method of time series decomposition employed in our research appears to be useful in making such prognoses. Humidity is particularly significant as it can determine the activity of I. ricinus to a large extent.     

Borrelia burgdorferi infection in Ixodes ricinus from habitats in Denmark

A total of 2647 ticks of the genus Ixodes was sampled by flagging the vegetation in thirty-one sites in eastern Jutland, Denmark. All ticks were identified as Ixodes ricinus Linnaeus. A total of 317 ticks (202 nymphs and 115 adults) from three different sites were examined for the spirochaete Borrelia burgdorferi Johnson et al. by indirect fluorescent antibody staining; the frequency of infection varied from 7% to 22%. It is concluded that I. ricinus, known to be the most common tick in Denmark, is the vector largely responsible for the transmission of B. burgdorferi in this country.     

Enhancement of tick-borne encephalitis virus transmission by tick salivary gland extracts

Abstract. To investigate the role of ticks in TBE virus transmission, salivary gland extract (SGE) was derived from partially fed female Ixodes ricinus, Dermacentor reticulatus and Rhipicephalus appendiculatus ticks. Guinea-pigs were infested with uninfected R.appendiculatus nymphs and inoculated with a mixture of TBE virus and SGE or with virus alone. The number of ticks which on average acquired virus from feeding on animals inoculated with TBE virus and SGE from partially fed ticks was 4-fold greater than the number that became infected by feeding on animals inoculated with virus alone or virus plus SGE from unfed I.ricinus. Viraemia was detected in 67% of guinea-pigs inoculated with virus plus SGE compared to 30% of guinea-pigs inoculated with virus alone. Virus titres in the blood were similar for both groups of animals [range 2.0-2.8 log₁₀ plaque-forming units (PFU)/ml of blood]; however, the number of ticks that became infected was significantly higher on animals inoculated with virus plus SGE from partially fed ticks. No significant difference was observed with respect to the tick species used to derive SGE. The results indicate that TBE virus transmission is enhanced by factor(s) associated with the salivary glands of feeding ticks, and that these factor(s) may facilitate efficient transmission of TBE virus between infected and uninfected ticks even when they feed on hosts that have no detectable viraemia.     

Immunomodulatory arsenal of nymphal ticks

Ticks have developed their own immunomodulatory mechanisms to inhibit the host inflammatory response. One of them involves the ability to subvert the cytokine network at the site of tick feeding by secreting cytokine binding molecules. Most studies have focused on the immunomodulatory prowess of adult female ticks. Here we describe anti-cytokine activity in salivary gland extracts (SGEs) prepared from 2-day-fed nymphs of Dermacentor reticulatus Fabricius, Ixodes ricinus L., Rhipicephalus appendiculatus Neumann and Amblyomma variegatum Fabricius. Anti-CXCL8 activity was detected in nymphs of all species. Relatively high activity against CCL2, CCL3 and CCL11 was observed in SGEs of R. appendiculatus and A. variegatum nymphs, whereas SGEs of I. ricinus nymphs showed comparatively high anti-interleukin-2 (-IL-2) and anti-IL-4 activities. These data show that nymphs, which epidemiologically are usually more important than adults as disease vectors, possess a range of anti-cytokine activities that may facilitate pathogen transmission.     

Regeneration of Haller's sensory organ in the tick, Ixodes rubicundus (Acari: Ixodidae)

A study of regeneration in nymphs and adults of the South African tick Ixodes (Afrixodes) rubicundus, from which the forelegs had been amputated during the previous instar, revealed that the structural changes in regenerated Haller's sensory organs resemble those observed in other ixodid ticks, in particular in another prostriate tick, Ixodes (Ixodes) ricinus. The adult regenerates re-establish their atavistic features in terms of the increased number of different sensilla on the distal knoll, in the anterior pit and the capsule. The nymphal regenerates, in contrast, re-establish the features of the previous instar through a reduction in the number of some sensilla on the distal knoll and in the post-capsular area. The structural changes in different compartments of the organ appear independent. The phenomenon of regenerative induction through the appearance of specific changes in Haller's organ of the contralateral non-treated foreleg is probably characteristic only of prostriate ticks. A unique modification in the regenerated Haller's organ as revealed by duplication of the Haller's organ capsule was discovered in both I. rubicundus nymphs and adult ticks.     

Absence of spirochaetes (Borrelia burgdorferi) and piroplasms (Babesia microti) in deer ticks (Ixodes dammini) parasitized by chalcid wasps (Hunterellus hookeri)

An entomophagous wasp (Hunterellus hookeri Howard) parasitizes about a third of the host-seeking nymphal Ixodes dammini Spielman et al. ticks on Naushon Island in Massachusetts (U.S.A.) where the agents of Lyme disease (Borrelia burgdorferi Johnson et al.) and human babesiosis (Babesia microti Franca) are enzootic. Following blood-feeding, wasp-parasitized ticks are destroyed by the developing wasp. The prevalence of either human pathogen in host-seeking ticks collected in wasp-infested sites is nearly 40% lower than that found in other sites. Nymphal ticks, collected early in their season of activity, are more frequently parasitized by the wasp and less frequently by the Lyme disease spirochaete than those collected later in the summer. Spirochaetes never infected wasp-infected ticks, and few wasp-infected ticks were concurrently infected by the Babesia piroplasm. Taken together, these correlations indicate that the wasp may render the tick inhospitable to both pathogens. The presence of the wasp may have reduced risk of human infection on the island by either pathogen by as much as a third.