Seasonal and spatial dynamics of ectoparasite infestation of a threatened reptile, the tuatara (Sphenodon punctatus)

Abstract The conservation of threatened vertebrate species and their threatened parasites requires an understanding of the factors influencing their distribution and dynamics. This is particularly important for species maintained in conservation reserves at high densities, where increased contact among hosts could lead to increased rates of parasitism. The tuatara (Sphenodon punctatus) (Reptilia: Sphenodontia) is a threatened reptile that persists at high densities in forests (～ 2700 tuatara/ha) and lower densities in pastures and shrubland (< 200 tuatara/ha) on Stephens Island, New Zealand. We investigated the lifecycles and seasonal dynamics of infestation of two ectoparasites (the tuatara tick, Amblyomma sphenodonti, and trombiculid mites, Neotrombicula sp.) in a mark‐recapture study in three forest study plots from November 2004 to March 2007, and compared infestation levels among habitat types in March 2006. Tick loads were lowest over summer and peaked from late autumn (May) until early spring (September). Mating and engorgement of female ticks was highest over spring, and larval tick loads subsequently increased in early autumn (March). Nymphal tick loads increased in September, and adult tick loads increased in May. Our findings suggest the tuatara tick has a 2‐ or 3‐year lifecycle. Mite loads were highest over summer and autumn, and peaked in March. Prevalences (proportion of hosts infected) and densities (estimated number of parasites per hectare) of ticks were similar among habitats, but tick loads (parasites per host) were higher in pastures than in forests and shrub. The prevalence and density of mites was higher in forests than in pasture or shrub, but mite loads were similar among habitats. We suggest that a higher density of tuatara in forests may reduce the ectoparasite loads of individuals through a dilution effect. Understanding host–parasite dynamics will help in the conservation management of both the host and its parasites.     

Spatial and temporal factors affecting parasite genotypes encountered by hosts: Empirical data from American dog ticks (Dermacentor variabilis) parasitising raccoons (Procyon lotor)

The American dog tick (Dermacentor variabilis) is an important vector of numerous pathogens of humans and animals. In this study, we analysed population genetic patterns in D. variabilis at scales of the host individual (infrapopulation) and population (component population) to elucidate fine-scale spatial and temporal factors influencing transmission dynamics. We genotyped D. variabilis collected from raccoons (Procyon lotor) trapped in two habitat patches (located in Indiana, USA) which were spatially proximate (5.9 km) and limited in size (10.48 Ha and 25.47 Ha, respectively). Despite the fine spatial sampling scale, our analyses revealed significant genetic differentiation amongst component populations and infrapopulations (within each component population), indicating a non-random pattern of encountering tick genotypes by raccoons at both scales evaluated. We found evidence for male-biased dispersal in the ticks themselves (in one component population) and an age-bias in spatial scales at which raccoons encountered ticks in the environment. At the scale of the component population, our analyses revealed that raccoons encountered ticks from a limited number of D. variabilis family groups, likely due to high reproductive variance amongst individual ticks. Finally, we found evidence for a temporal effect with raccoons encountering ticks in the environment as “clumps” of related individuals. While the genetic structure of parasite populations are increasingly being investigated at small spatial scales (e.g. the infrapopulation), our data reveal that genetic structuring can originate at scales below that of the infrapopulation, due to the interaction between temporal and biological factors affecting the encounter of parasites by individual hosts. Ultimately, our data indicate that genetic structure in parasites must be viewed as a consequence of both spatial and temporal variance in host-parasite interactions, which in turn are driven by demographic factors related to both the host and parasite.     

Potential Parasite Transmission in Multi-Host Networks Based on Parasite Sharing

Epidemiological networks are commonly used to explore dynamics of parasite transmission among individuals in a population of a given host species. However, many parasites infect multiple host species, and thus multi-host networks may offer a better framework for investigating parasite dynamics. We investigated the factors that influence parasite sharing – and thus potential transmission pathways – among rodent hosts in Southeast Asia. We focused on differences between networks of a single host species and networks that involve multiple host species. In host-parasite networks, modularity (the extent to which the network is divided into subgroups of rodents that interact with similar parasites) was higher in the multi-species than in the single-species networks. This suggests that phylogeny affects patterns of parasite sharing, which was confirmed in analyses showing that it predicted affiliation of individuals to modules. We then constructed “potential transmission networks” based on the host-parasite networks, in which edges depict the similarity between a pair of individuals in the parasites they share. The centrality of individuals in these networks differed between multi- and single-species networks, with species identity and individual characteristics influencing their position in the networks. Simulations further revealed that parasite dynamics differed between multi- and single-species networks. We conclude that multi-host networks based on parasite sharing can provide new insights into the potential for transmission among hosts in an ecological community. In addition, the factors that determine the nature of parasite sharing (i.e. structure of the host-parasite network) may impact transmission patterns.     

Developmental stages and molecular phylogeny of Hepatozoon tuatarae, a parasite infecting the New Zealand tuatara, Sphenodon punctatus and the tick, Amblyomma sphenodonti

The developmental stages of Hepatozoon tuatarae were elucidated in both the tuatara host, Sphenodon punctatus and the tick, Amblyomma sphenodonti. PCR amplicons from A. sphenodonti samples identified DNA matching H. tuatarae. Dissection of tick samples showed oogenesis and sporogony occurring in the haemocoel of A. sphenodonti with the average mature oocyst size being 236 × 228 μm. Partial sequence data of the parasite’s small subunit ribosomal gene, obtained by PCR, was used for phylogenetic comparison. Characterisation of the H. tuatarae lifecycle will help in conservation management of the tuatara.     

Buffering role of the intertidal anemone Anthopleura aureoradiata in cercarial transmission from snails to crabs

In nature, parasite transmission from one host to the next takes place within complex biotic communities where non-host organisms can reduce transmission rates, for instance by preying on infective stages. We experimentally investigated the impact of four very different non-host organisms on the transmission of the microphallid trematode Maritrema novaezealandensis from its snail first intermediate host to its crustacean second intermediate host. We show that in laboratory mesocosms, accumulation of parasites in juvenile stalk-eyed mud crabs, Macrophthalmus hirtipes (Ocypodidae), was not reduced in the presence of cockles, Austrovenus stutchburyi, barnacles, Balanus sp., or the algae Enteromorpha spp., three organisms whose feeding mode or general abundance could negatively impact the parasite's infective stages (cercariae). In contrast, the presence of the anemone Anthopleura aureoradiata in the mesocosms caused a more than 4-fold reduction in the number of parasites acquired by crabs when compared to control mesocosms. Observations on fluorescent-dyed cercariae confirmed that they are ingested by anemones. Given the often high densities of anemones on mudflats, they may represent an important regulator of the abundance of M. novaezealandensis, and thus of the impact of this parasite on its hosts. These anemones may decrease cercarial transmission for many other trematode species as well. Our results stress the need for studies of parasite transmission in natural contexts rather than under simplified laboratory conditions.     

Feeder use predicts both acquisition and transmission of a contagious pathogen in a North American songbird

Individual heterogeneity can influence the dynamics of infectious diseases in wildlife and humans alike. Thus, recent work has sought to identify behavioural characteristics that contribute disproportionately to individual variation in pathogen acquisition (super-receiving) or transmission (super-spreading). However, it remains unknown whether the same behaviours enhance both acquisition and transmission, a scenario likely to result in explosive epidemics. Here, we examined this possibility in an ecologically relevant host–pathogen system: house finches and their bacterial pathogen, Mycoplasma gallisepticum, which causes severe conjunctivitis. We examined behaviours likely to influence disease acquisition (feeder use, aggression, social network affiliations) in an observational field study, finding that the time an individual spends on bird feeders best predicted the risk of conjunctivitis. To test whether this behaviour also influences the likelihood of transmitting M. gallisepticum, we experimentally inoculated individuals based on feeding behaviour and tracked epidemics within captive flocks. As predicted, transmission was fastest when birds that spent the most time on feeders initiated the epidemic. Our results suggest that the same behaviour underlies both pathogen acquisition and transmission in this system and potentially others. Identifying individuals that exhibit such behaviours is critical for disease management.     

Computational prediction of protein interactions related to the invasion of erythrocytes by malarial parasites

Background

The invasion of red blood cells (RBCs) by malarial parasites is an essential step in the life cycle of Plasmodium falciparum. Human-parasite surface protein interactions play a critical role in this process. Although several interactions between human and parasite proteins have been discovered, the mechanism related to invasion remains poorly understood because numerous human-parasite protein interactions have not yet been identified. High-throughput screening experiments are not feasible for malarial parasites due to difficulty in expressing the parasite proteins. Here, we performed computational prediction of the PPIs involved in malaria parasite invasion to elucidate the mechanism by which invasion occurs.

Results

In this study, an expectation maximization algorithm was used to estimate the probabilities of domain-domain interactions (DDIs). Estimates of DDI probabilities were then used to infer PPI probabilities. We found that our prediction performance was better than that based on the information of D. melanogaster alone when information related to the six species was used. Prediction performance was assessed using protein interaction data from S. cerevisiae, indicating that the predicted results were reliable. We then used the estimates of DDI probabilities to infer interactions between 490 parasite and 3,787 human membrane proteins. A small-scale dataset was used to illustrate the usability of our method in predicting interactions between human and parasite proteins. The positive predictive value (PPV) was lower than that observed in S. cerevisiae. We integrated gene expression data to improve prediction accuracy and to reduce false positives. We identified 80 membrane proteins highly expressed in the schizont stage by fast Fourier transform method. Approximately 221 erythrocyte membrane proteins were identified using published mass spectral datasets. A network consisting of 205 interactions was predicted. Results of network analysis suggest that SNARE proteins of parasites and APP of humans may function in the invasion of RBCs by parasites.

Conclusions

We predicted a small-scale PPI network that may be involved in parasite invasion of RBCs by integrating DDI information and expression profiles. Experimental studies should be conducted to validate the predicted interactions. The predicted PPIs help elucidate the mechanism of parasite invasion and provide directions for future experimental investigations.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-014-0393-z) contains supplementary material, which is available to authorized users.     

Social interactions modulate the virulence of avian malaria infection

There is an increasing understanding of the context-dependent nature of parasite virulence. Variation in parasite virulence can occur when infected individuals compete with conspecifics that vary in infection status; virulence may be higher when competing with uninfected competitors. In vertebrates with social hierarchies, we propose that these competition-mediated costs of infection may also vary with social status. Dominant individuals have greater competitive ability than competing subordinates, and consequently may pay a lower prevalence-mediated cost of infection. In this study we investigated whether costs of malarial infection were affected by the occurrence of the parasite in competitors and social status in domestic canaries (Serinus canaria). We predicted that infected subordinates competing with non-infected dominants would pay higher costs than infected subordinates competing with infected dominants. We also predicted that these occurrence-mediated costs of infection would be ameliorated in infected dominant birds. We found that social status and the occurrence of parasites in competitors significantly interacted to change haematocrit in infected birds. Namely, subordinate and dominant infected birds differed in haematocrit depending on the infection status of their competitors. However, in contrast to our prediction, dominants fared better with infected subordinates, whereas subordinates fared better with uninfected dominants. Moreover, we found additional effects of parasite occurrence on mortality in canaries. Ultimately, we provide evidence for costs of parasitism mediated by social rank and the occurrence of parasites in competitors in a vertebrate species. This has important implications for our understanding of the evolutionary processes that shape parasite virulence and group living.     

Parasite age-intensity relationships in red-spotted newts: does immune memory influence salamander disease dynamics?

Acquired immune memory in vertebrates influences transmission and persistence of infections, with consequences for parasite dynamics at both the individual and population levels. The potential impact of acquired immunity is of particular interest for salamanders, whose acquired immune systems are thought to be less effective than those of frogs and other tetrapods. One way to examine the importance of acquired immunity to parasite dynamics at the population level is by examining the relationship between host age and parasite infection intensity. Acquired immunity reduces infection rates in older animals, causing decreased parasite intensity in older age classes and leading to curvilinear age-intensity relationships for persistent parasites and convex age-intensity relationships for transient parasites. We used age-intensity relationships to look for the signature of acquired immunity for 12 parasite taxa of red-spotted newts (Notophthalmus viridescens), using data from a 2-year parasitological survey of six newt populations. We estimated ages from snout-vent length (SVL) based on the relationship between SVL and skeletochronologically-derived ages in a subset of newts. We found evidence of acquired immunity to two parasite taxa, bacterial pathogens and the protist Amphibiocystidium viridescens, whose convex age-intensity relationships could not be easily explained by alternative mechanisms. Our results suggest that the acquired immune response of newts is sufficient to influence the dynamics of at least some parasites.     

Effect of sexual segregation on host-parasite interaction: Model simulation for abomasal parasite dynamics in alpine ibex (Capraibex)

We investigated whether sexual segregation might affect parasite transmission and host dynamics, hypothesising that if males are the more heavily infected sex and more responsible for the transmission of parasite infections, female avoidance of males and the space they occupy could reduce infection rates. A mathematical model, simulating the interaction between abomasal parasites and a hypothetical alpine ibex (Capraibex) host population composed of its two sexes, was developed to predict the effect of different degrees of sexual segregation on parasite intensity and on host abundance. The results showed that when females tended to be segregated from males, and males were distributed randomly across space, the impact of parasites was the lowest, resulting in the highest host abundance, with each sex having the lowest parasite intensity. The predicted condition that minimises the impact of parasites in our model was the one closest to that observed in nature where females actively seek out the more segregated sites while males are less selective in their ranging behaviour. The overlapping of field observation with the predicted optimal strategy lends support to our idea that there might be a connection between parasite transmission and sexual segregation. Our simulations provide the biological boundaries of host-parasite interaction needed to determine a parasite-mediated effect on sexual segregation and a formalised null hypothesis against which to test future field experiments.     

Endemic infection reduces transmission potential of an epidemic parasite during co-infection

Endemic, low-virulence parasitic infections are common in nature. Such infections may deplete host resources, which in turn could affect the reproduction of other parasites during co-infection. We aimed to determine whether the reproduction, and therefore transmission potential, of an epidemic parasite was limited by energy costs imposed on the host by an endemic infection. Total lipids, triacylglycerols (TAG) and polar lipids were measured in cockroaches (Blattella germanica) that were fed ad libitum, starved or infected with an endemic parasite, Gregarina blattarum. Reproductive output of an epidemic parasite, Steinernema carpocapsae, was then assessed by counting the number of infective stages emerging from these three host groups. We found both starvation and gregarine infection reduced cockroach lipids, mainly through depletion of TAG. Further, both starvation and G. blattarum infection resulted in reduced emergence of nematode transmission stages. This is, to our knowledge, the first study to demonstrate directly that host resource depletion caused by endemic infection could affect epidemic disease transmission. In view of the ubiquity of endemic infections in nature, future studies of epidemic transmission should take greater account of endemic co-infections.     

Parasite-mediated selection drives an immunogenetic trade-off in plains zebras (Equus quagga)

Pathogen evasion of the host immune system is a key force driving extreme polymorphism in genes of the major histocompatibility complex (MHC). Although this gene family is well characterized in structure and function, there is still much debate surrounding the mechanisms by which MHC diversity is selectively maintained. Many studies have investigated relationships between MHC variation and specific pathogens, and have found mixed support for and against the hypotheses of heterozygote advantage, frequency-dependent or fluctuating selection. Few, however, have focused on the selective effects of multiple parasite types on host immunogenetic patterns. Here, we examined relationships between variation in the equine MHC gene, ELA-DRA, and both gastrointestinal (GI) and ectoparasitism in plains zebras (Equus quagga). Specific alleles present at opposing population frequencies had antagonistic effects, with rare alleles associated with increased GI parasitism and common alleles with increased tick burdens. These results support a frequency-dependent mechanism, but are also consistent with fluctuating selection. Maladaptive GI parasite ‘susceptibility alleles’ were reduced in frequency, suggesting that these parasites may play a greater selective role at this locus. Heterozygote advantage, in terms of allele mutational divergence, also predicted decreased GI parasite burden in genotypes with a common allele. We conclude that an immunogenetic trade-off affects resistance/susceptibility to parasites in this system. Because GI and ectoparasites do not directly interact within hosts, our results uniquely show that antagonistic parasite interactions can be indirectly modulated through the host immune system. This study highlights the importance of investigating the role of multiple parasites in shaping patterns of host immunogenetic variation.     

A new ovine Babesia species transmitted by Hyalomma anatolicum anatolicum

The pathogenicity and morphology of a large Babesia species, Babesia sp. Xinjiang, are described here. The parasite has very low virulence for sheep, and caused no detectable clinical symptoms. Splenectomized sheep infected with the parasite showed mild fever and low parasitemia and would recover gradually. If splenectomized sheep were immuno-suppressed with dexamethasone, the parasitemia could reach 8.5%, and death occurred. A splenectomized calf could not be infected with the Babesia species. Paired parasites were the typical form of the Babesia species in erythrocytes and the average size of a pair of parasites was 2.42 (±0.35) μm × 1.06 (±0.22) μm. Merozoites were found in the gut, salivary gland, haemolymph, ovary and eggs of female Hyalomma anatolicum anatolicum engorged on sheep infected with the parasites. The results of experimental transmission showed that the larval, nymph and adult stages of H. a. anatolicum could transmit the Babesia species to sheep.     

Presidential address: rediscovering parasites using molecular tools--towards revising the taxonomy of Echinococcus, Giardia and Cryptosporidium

Molecular biology has provided parasitologists with a fantastic variety of techniques that have had a major impact on research into parasites and parasitism. Molecular tools have revealed the extent and nature of genetic diversity in parasites and this information has made a significant contribution to studies on the population genetics and evolutionary biology of parasites. Similarly, epidemiology has benefited enormously from the application of molecular tools in terms of studying parasite life cycles and transmission, and in the development of specific and sensitive methods for diagnosis and surveillance. However, the theme I wish to develop in this paper is concerned with the contribution molecular tools have made to parasite taxonomy and systematics, and in particular, the fact that in many cases molecular tools are validating the proposals made many years ago by taxonomists and biologists which were discounted or not fully accepted at the time. To do this I have chosen four examples (Echinococcus, Entamoeba, Giardia, Cryptosporidium) where recent research involving molecular characterisation has confirmed observations made many years ago and has resulted in a need to revise the taxonomy of different groups of parasites.     

Secondary metabolites in floral nectar reduce parasite infections in bumblebees

The synthesis of secondary metabolites is a hallmark of plant defence against herbivores. These compounds may be detrimental to consumers, but can also protect herbivores against parasites. Floral nectar commonly contains secondary metabolites, but little is known about the impacts of nectar chemistry on pollinators, including bees. We hypothesized that nectar secondary metabolites could reduce bee parasite infection. We inoculated individual bumblebees with Crithidia bombi, an intestinal parasite, and tested effects of eight naturally occurring nectar chemicals on parasite population growth. Secondary metabolites strongly reduced parasite load, with significant effects of alkaloids, terpenoids and iridoid glycosides ranging from 61 to 81%. Using microcolonies, we also investigated costs and benefits of consuming anabasine, the compound with the strongest effect on parasites, in infected and uninfected bees. Anabasine increased time to egg laying, and Crithidia reduced bee survival. However, anabasine consumption did not mitigate the negative effects of Crithidia, and Crithidia infection did not alter anabasine consumption. Our novel results highlight that although secondary metabolites may not rescue survival in infected bees, they may play a vital role in mediating Crithidia transmission within and between colonies by reducing Crithidia infection intensities.     

Population genetic structure and history of a generalist parasite infecting multiple sympatric host species

Host specificity is predicted to shape patterns of parasite gene flow between host species; specialist parasites should have low gene flow between host species, while generalists are predicted to have high gene flow between species. However, even for generalist parasites external forces, including ecological differences between host species may sometimes intervene to limit gene flow and create genetic structure. To investigate the potential for cryptic parasite genetic structure to arise under such circumstances, we examined the population genetic structure and history of the generalist nematode, Trichostrongylus axei, infecting six sympatric wild ungulate species in North America. Using genotypes for 186 T. axei larvae at two mitochondrial genes, cox1 and nad4, we found that T. axei was completely panmictic across host species, with 0% of genetic variation structured between host species and 97% within individual hosts. In addition, T. axei showed no evidence of recent genetic bottlenecks, had high nucleotide diversities (above 2%), and an effective population size estimated to be in the tens of millions. Our result that T. axei maintains high rates of gene flow between multiple sympatric host species adds to a growing body of information on trichostrongylid population genetic structure in different ecological contexts. Furthermore, the high rates of gene flow, coupled with high levels of genetic diversity and large effective population size which we observed in T. axei, point to a potentially broad capacity for rapid evolutionary change in this parasite.     

Spatial proximity and asynchronous refuge sharing networks both explain patterns of tick genetic relatedness among lizards,but in different years

A major question for understanding the ecology of parasite infections and diseases in wildlife populations concerns the transmission pathways among hosts. Network models are increasingly used to model the transmission of infections among hosts – however, few studies have integrated host behaviour and genetic relatedness of the parasites transmitted between hosts. In a study of the Australian sleepy lizard Tiliqua rugosa and its three‐host ixodid tick (Bothriocroton hydrosauri), we asked if patterns of genetic relatedness among ticks were best explained by spatial proximity or the host transmission network. Using synchronous GPS locations of over 50 adult lizards at 10 min intervals across the three‐month activity period, over 2 years, we developed two alternative parasite transmission networks. One alternative was based on the spatial proximity of lizards (at the centre of their home ranges), and the other was based on the frequency of asynchronous shared refuge use between pairs of lizards. In each year, adult ticks were removed from lizards and their genotypes were determined at four polymorphic microsatellite loci. Adult ticks collected from the same host were more related to each other than ticks from different hosts. Similarly, adult ticks collected from different lizards had a higher relatedness if those lizards had a shorter path length connecting them on each of the two networks we explored. The predictors of tick relatedness differed between years. In the first year, the asynchronous shared refuges network was the stronger predictor of tick relatedness, whereas in year two, the spatial proximity‐based network was the stronger predictor of tick relatedness. We speculate on how changing environmental conditions might change the relative importance of alternative processes driving the transmission of parasites.     

Intracellular survival of apicomplexan parasites and host cell modification

The intracellular stages of apicomplexan parasites are known to extensively modify their host cells to ensure their own survival. Recently, considerable progress has been made in understanding the molecular details of these parasite-dependent effects for Plasmodium-, Toxoplasma- and Theileria-infected cells. We have begun to understand how Plasmodium liver stage parasites protect their host hepatocytes from apoptosis during parasite development and how they induce an ordered cell death at the end of the liver stage. Toxoplasma parasites are also known to regulate host cell survival pathways and it has been convincingly demonstrated that they block host cell major histocompatibility complex (MHC)-dependent antigen presentation of parasite epitopes to avoid cell-mediated immune responses. Theileria parasites are the masters of host cell modulation because their presence immortalises the infected cell. It is now accepted that multiple pathways are activated to induce Theileria-dependent host cell transformation. Although it is now known that similar host cell pathways are affected by the different parasites, the outcome for the infected cell varies considerably. Improved imaging techniques and new methods to control expression of parasite and host cell proteins will help us to analyse the molecular details of parasite-dependent host cell modifications.     

Sex-biased parasitism,host mass and mutualistic bat flies: an antagonistic individual-based network of bat-bat fly interactions

Individual-based networks provide the building blocks for community-level networks. However, network studies of bats and their parasites have focused only on the species level. Intrapopulation variation may allow certain host individuals to play important roles in the dynamics of the parasites. Therefore, we evaluated how the variation in host sex, body size, ectoparasite abundance and co-occurrence configure individual-based networks of the lesser bulldog bat Noctilio albiventris and bat flies. We expected bat individuals with greater body mass and forearms acting as the core in the network. We also expected males to play a more important role in the network. We sampled a network of N. albiventris bat individuals and their bat flies to describe the structure of an antagonistic individual-based network. We aimed to identify the most relevant bat individuals in the network, focusing on the implications inherent to each of the following approaches: (i) core-periphery organization; (ii) modularity; (iii) species level metrics; and (iv) the main ecological driver of bat individual roles in the network, using niche-based predictors (body mass, forearm and sex). We showed that a network of N. albiventris individuals and their bat flies had low modularity containing a persistent nucleus of individuals and bat flies with well-established interactions. Male individuals with greater body mass played an important role in the network, while for females neither mass nor forearm length were important predictors of their role in the network. Finally, individuals with a high abundance of Paradyschiria parvula played a core role. These results provide an alternative perspective to understand the patterns and mechanisms of interspecific interactions between parasites on the host, as well as sex-biased parasitism.     

Babesia bigemina: In vitro multiplication of sporokinetes in Ixodes scapularis (IDE8) cells

This paper describes the in vitro multiplication process of Babesia bigemina sporokinetes in a cell line (IDE8) from Ixodes scapularis ticks. The inoculum was obtained from hemolymph of engorged females of Rhipicephalus (Boophilus) microplus ticks naturally infected with B. bigemina. These ticks had been fed on calves living in a tick endemic farm in Brazil. Microscopic morphological details are shown to describe the development of the parasite in the tick cells; the identity of the parasite was confirmed by a duplex PCR method.