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Piroplasms, which include the agents of cattle fever and human and dog babesiosis, are a diverse group of blood parasites of significant veterinary and medical importance. The invasive Asian longhorned tick, Haemaphysalis longicornis , is a known vector of piroplasms in its native range in East Asia and invasive range in Australasia. In the USA, H. longicornis has been associated with Theileria orientalis Ikeda outbreaks that caused cattle mortality. To survey invasive populations of H. longicornis for a broad range of piroplasms, 667 questing H. longicornis collected in 2021 from 3 sites in New Jersey, USA, were tested with generalist piroplasm primers targeting the 18S small subunit rRNA (395–515 bp, depending on species) and the cytochrome b oxidase loci (1009 bp). Sequences matching Theileria cervi type F (1 adult, 5 nymphs), an unidentified Theileria species (in 1 nymph), an undescribed Babesia sensu stricto (‘true’ Babesia , 2 adults, 2 nymphs), a Babesia sp. Coco (also a ‘true Babesia ’, 1 adult, 1 nymph), as well as Babesia microti S837 (1 adult, 4 nymphs) were recovered. Babesia microti S837 is closely related to the human pathogen B. microti US-type. Additionally, a 132 bp sequence matching the cytochrome b locus of deer, Odocoileus virginanus , was obtained from 2 partially engorged H. longicornis. The diverse assemblage of piroplasms now associated with H. longicornis in the USA spans 3 clades in the piroplasm phylogeny and raises concerns of transmission amplification of veterinary pathogens as well as spillover of pathogens from wildlife to humans.

The escape kinetics from the anterior midgut (AM) of Trypanosoma cruzi during the initial steps of infection was assessed in Triatoma infestans, as well as its ability to survive migration in the digestive tract of the vector. All the four strains evaluated survived and reached variable parasite densities. After 49-50 days, YuYu [discrete typing units (DTU) I] strain reached the highest parasite numbers in the rectum followed by Bug (DTU V), CL-Brener (DTU VI) and Dm28c (DTU I). All strains accomplished metacyclogenesis. Bug strain reached the highest numbers of metacyclic trypomastigotes followed by YuYu and CL-Brener/Dm28c. A remarkable parasite reduction in the AM for Bug strain, but not Dm28c was noticed at 72 h of infection. In the posterior midgut + rectum high densities of parasites from both strains were detected at this period indicating the parasites crossed the AM. For Dm28c strain, in infections initiated with trypomastigotes, parasites left AM faster than those starting with epimastigotes. In conclusion, T. cruzi strains from different DTUs were able to infect T. infestans reaching variable parasite densities. The kinetics of migration in the digestive tract may be affected by strain and/or the evolutive form used for infection.

Trypanosoma cruzi, the etiological agent of Chagas disease, is ingested by triatomines during their bloodmeal on an infected mammal. Aiming to investigate the development and differentiation of T. cruzi inside the intestinal tract of Rhodnius prolixus at the beginning of infection we fed insects with cultured epimastigotes and blood trypomastigotes from infected mice to determine the amount of recovered parasites after ingestion. Approximately 20% of the ingested parasites was found in the insect anterior midgut (AM) 3 h after feeding. Interestingly, a significant reduction (80%) in the numbers of trypomastigotes was observed after 24 h of infection suggesting that parasites were killed in the AM. Moreover, few parasites were found in that intestinal portion after 96 h of infection. The evaluation of the numbers of parasites in the posterior midgut (PM) at the same periods showed a reduced parasite load, indicating that parasites were not moving from the AM. Additionally, incubation of blood trypomastigotes with extracts from R. prolixus AMs revealed that components of this tissue could induce significant death of T. cruzi. Finally, we observed that differentiation from trypomastigotes to epimastigotes is not completed in the AM; instead we suggest that trypomastigotes change to intermediary forms before their migration to the PM, where differentiation to epimastigotes takes place. The present work clarifies controversial points concerning T. cruzi development in insect vector, showing that parasite suffers a drastic decrease in population size before epimastigonesis accomplishment in PM.

Groups of nymphal Rhipicephalus appendiculatus Muguga, having a mean of 1 or 9 Theileria parva Muguga-infected salivary gland acini per tick, were kept under quasi-natural conditions at an altitude of 1950 m or 20 degrees C at a relative humidity of 85% in the laboratory and their survival and infection prevalence and abundance determined over time. Theileria parva infections for both categories of ticks survived in the nymphal ticks for 50 or 26 weeks post salivary gland infection under quasi-natural or laboratory conditions respectively. There was a distinct decline in infections in the more heavily infected nymphae under both conditions of exposure, reflecting an apparent density dependence in parasite survival. Nymphal ticks having an average infection level of 1 infected salivary gland acinus per tick, survived for up to 69 or 65 weeks post-repletion under quasi-natural or the laboratory conditions respectively. Nymphae having an average infection level of 9 infected salivary gland acini per tick survived for a similar duration under each of the 2 conditions. The infection level of 9 infected salivary gland acini per tick did not seem to significantly affect the survival of the tick vector compared to those having an average of 1 infected salivary gland acinus per tick.

The estimation of two parameters in the transmission of Babesia microti by the tick Ixodes trianguliceps amongst small mammals, (1) the duration of infectivity in natural hosts and (2) the probability of transmission from an infected to a susceptible vole, is described. When B. microti was maintained by direct tick transmission, the probability of a complete cycle of transmission via the larval-nymphal and nymphal-adult transstadial routes was 1.0 and 0.71 respectively, but only if the larvae or nymphs had engorged, as distinct from feeding slowly, while the source parasitaemia exceeded 2 or 0.2% respectively, but had not yet passed the peak level. The duration of this condition for infectivity in voles infected by nymphal bites was only 1-4 days, whilst infections delivered by adult ticks barely reached the threshold level necessary for successful transmission. When syringe passage was introduced into the parasite maintenance schedule (a) the probability of transmission declined markedly and (b) the time-course of the parasitaemia was altered. If these parameter values are put into a simple model, together with field data on tick and host survival rates, it becomes apparent that additional factors, such as the highly aggregated distribution of ticks on their hosts, must account for the maintenance of B. microti at the levels seen in wild small mammal populations.

An in vitro feeding method using rabbit or cattle skin membranes, applied successfully to all stages (larvae, nymphae and adults) of the ioxodid tick, Amblyomma variegatum, is described. The feeding apparatus consisted of a blood container with a membrane placed on top of a tick containment unit. A carbon dioxide atmosphere of between 5 and 10% and a temperature of 37 degrees C were used as stimulants for the attachment of the ticks. High CO2 concentrations in the atmosphere improved the feeding success of all instars. The effect of anticoagulation methods for the bloodmeal was investigated, and heparinized blood was found to be the most suitable for tick feeding. When the bloodmeal was replaced by tissue culture medium for feeding nymphs the subsequent moulting success was reduced. Adult ticks of both sexes remained attached for up to 16 days, until completion of their bloodmeals. All stages of the tick fed on whole blood in the artificial feeding system and all reached engorged weights less than those achieved by control ticks fed on experimental animals. A large proportion of ticks, fed artificially on whole blood, moulted or laid eggs successfully. The method was successfully applied for the transmission of Theileria mutans and Cowdria ruminantium to cattle.

A 2 hectare paddock on the Muguga Estate, Kiambu District of Kenya (altitude 2100 m) free of Theileria parva-infected ticks was seeded by applying Rhipicephalus appendiculatus nymphs to cattle infected with Theileria parva (Kiambu 4). It was estimated that over 50 000 engorged nymphs fell onto the pasture from 4 cattle with high parasitaemias during the cold season (June). Samples of these ticks were placed in plastic cylinders under the vegetation in the paddock and sexual stages of T. parva were detected in gut lumen smears in these samples up to 21 days after repletion. Zygotes were first observed to transform into kinetes on day 55 and parasites were first detected in salivary glands of adults on day 64. Moulting of the nymphs started on day 60 after repletion and was completed by day 87. Cattle introduced into the paddock showed their first infestation with adult R. appendiculatus on day 64 after repletion of the nymphs and the infestation level gradually increased. On day 76 after repletion, 17% of a sample of adult ticks infesting cattle showed salivary gland infections with T. parva and this increased to over 70% by day 150. From day 360 onwards, a decrease in both the percentage of ticks infected and the number of salivary gland acini infected/tick was noted. In addition, T. parva infections within the salivary glands required a longer period of feeding in the older ticks before they developed into sporozoites. Cycling of Theileria through ticks was prevented by the removal of female ticks before they completed repletion. Lethal challenge levels of T. parva for cattle existed in the paddock up to day 547 after repletion, after which introduced cattle showed no infection or only a sub-lethal infection. Cattle introduced into the paddock on day 808 after repletion showed no evidence of T. parva infection. The tick infestation of introduced cattle decreased markedly from day 368 and infestation had virtually ceased by day 808.

Theileria annulata was transmitted transtadially by an Israeli strain of Hyalomma excavatum from larvae to nymphs and from nymphs to adults. However, the adults developing from larvae which engorged on infected calves and fed during the nymphal stage on non-infected calves, rabbits or gerbils (Meriones tristrami), did not transmit theileriosis. Suspensions of infected unfed nymphs or adults did not produce theileriosis but both nymphs and adults of the same batch became infective to cattle after 2 days of feeding. Suspensions of salivary glands or of whole ticks derived from infected adults that remained attached to rabbits for more than 7 days, were not infective to cattle.

Transstadial transmission of Theileria annulata with Hyalomma anatolicum anatolicum, H. dromedarii and H. marginatum isaaci, and Haemaphysalis bispinosa, Rhipicephalus haemaphysaloides haemaphysaloides and Boophilus microplus was determined. It was found that the infection was successfully transmitted by H. a. anatolicum from larva to nymph and nymph to adult in all attempts. When larvae were fed on an infected calf the succeeding adults transmitted the infection when the intervening nymphs fed on a rabbit (non-susceptible host) but not when fed on a calf (susceptible host). Infective adult ticks transmitted the parasite during the first 24 h of feeding on a calf. When the feeding was interrupted after 24 h, and the tick transferred to another calf, the infection was transmitted to the latter as well. H. dromedarii successfully transmitted the infection from larva to nymph and from nymph to adult. Larvae of H. marginatum isaaci did not feed on calves but the infection was successfully transmitted from nymph to adult. Haemaphysalis bispinosa, Rhipicephalus h. haemaphysaloides and Boophilus microplus did not transmit Th. annulata from larva to nymph to adult.