Effects of different immunomodulating liposome-based adjuvants and injection sites on immunogenicity in pigs.

Vaccine adjuvants, such as liposome-based cationic adjuvant formulations (CAFs), are able to boost immune responses and, by incorporation of distinct immunomodulators, steer immunity towards a desired direction in mice, non-human primates and humans, while less studied in pigs. Here we used commercial pigs to investigate polarizing adjuvant effects of CAFs with immunomodulators: C-type lectin receptor ligands trehalose-6,6'-dibehenate and monomycolyl glycerol, toll-like receptor 3 ligand Poly(I:C) or retinoic acid. Vaccines were formulated with a recombinant Chlamydia model protein antigen and administered via three injection routes. All adjuvants significantly increased antigen-specific IgG in serum, compared to non-adjuvanted antigen. Administering the vaccines through intramuscular and intraperitoneal routes induced significantly higher antigen-specific IgG and IgA serum antibodies, than the perirectal route. Although immunizations triggered cell-mediated immunity, no significant differences between adjuvants or injection sites were detected. Genes depicting T cell subtypes revealed only minor differences. Our findings suggest that specific signatures of the tested adjuvant immunomodulation do not translate well from mice to pigs in standard two-dose immunizations. This study provides new insights into immune responses to CAFs in pigs, and highlights that adjuvant development should ideally be carried out in the intended species of interest or in models with high predictive validity/translational value.

Experimental genetic crosses in tsetse flies of the livestock pathogen Trypanosoma congolense savannah.

BACKGROUND: In tropical Africa animal trypanosomiasis is a disease that has severe impacts on the health and productivity of livestock in tsetse fly-infested regions. Trypanosoma congolense savannah (TCS) is one of the main causative agents and is widely distributed across the sub-Saharan tsetse belt. Population genetics analysis has shown that TCS is genetically heterogeneous and there is evidence for genetic exchange, but to date Trypanosoma brucei is the only tsetse-transmitted trypanosome with experimentally proven capability to undergo sexual reproduction, with meiosis and production of haploid gametes. In T. brucei sex occurs in the fly salivary glands, so by analogy, sex in TCS should occur in the proboscis, where the corresponding portion of the developmental cycle takes place. Here we test this prediction using genetically modified red and green fluorescent clones of TCS. METHODS: Three fly-transmissible strains of TCS were transfected with genes for red or green fluorescent protein, linked to a gene for resistance to the antibiotic hygromycin, and experimental crosses were set up by co-transmitting red and green fluorescent lines in different combinations via tsetse flies, Glossina pallidipes. To test whether sex occurred in vitro, co-cultures of attached epimastigotes of one red and one green fluorescent TCS strain were set up and sampled at intervals for 28 days. RESULTS: All interclonal crosses of genetically modified trypanosomes produced hybrids containing both red and green fluorescent proteins, but yellow fluorescent hybrids were only present among trypanosomes from the fly proboscis, not from the midgut or proventriculus. It was not possible to identify the precise life cycle stage that undergoes mating, but it is probably attached epimastigotes in the food canal of the proboscis. Yellow hybrids were seen as early as 14 days post-infection. One intraclonal cross in tsetse and in vitro co-cultures of epimastigotes also produced yellow hybrids in small numbers. The hybrid nature of the yellow fluorescent trypanosomes observed was not confirmed by genetic analysis. CONCLUSIONS: Despite absence of genetic characterisation of hybrid trypanosomes, the fact that these were produced only in the proboscis and in several independent crosses suggests that they are products of mating rather than cell fusion. The three-way strain compatibility observed is similar to that demonstrated previously for T. brucei, indicating that a simple two mating type system does not apply for either trypanosome species.

In vitro generated antibodies guide thermostable ADDomer nanoparticle design for nasal vaccination and passive immunization against SARS-CoV-2.

Background: Due to COVID-19, pandemic preparedness emerges as a key imperative, necessitating new approaches to accelerate development of reagents against infectious pathogens. Methods: Here, we developed an integrated approach combining synthetic, computational and structural methods with in vitro antibody selection and in vivo immunization to design, produce and validate nature-inspired nanoparticle-based reagents against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Results: Our approach resulted in two innovations: (i) a thermostable nasal vaccine called ADDoCoV, displaying multiple copies of a SARS-CoV-2 receptor binding motif derived epitope and (ii) a multivalent nanoparticle superbinder, called Gigabody, against SARS-CoV-2 including immune-evasive variants of concern (VOCs). In vitro generated neutralizing nanobodies and electron cryo-microscopy established authenticity and accessibility of epitopes displayed by ADDoCoV. Gigabody comprising multimerized nanobodies prevented SARS-CoV-2 virion attachment with picomolar EC50. Vaccinating mice resulted in antibodies cross-reacting with VOCs including Delta and Omicron. Conclusion: Our study elucidates Adenovirus-derived dodecamer (ADDomer)-based nanoparticles for use in active and passive immunization and provides a blueprint for crafting reagents to combat respiratory viral infections.

Development of the livestock pathogen Trypanosoma (Nannomonas) simiae in the tsetse fly with description of putative sexual stages from the proboscis.

BACKGROUND: Tsetse-transmitted African animal trypanosomiasis is recognised as an important disease of ruminant livestock in sub-Saharan Africa, but also affects domestic pigs, with Trypanosoma simiae notable as a virulent suid pathogen that can rapidly cause death. Trypanosoma simiae is widespread in tsetse-infested regions, but its biology has been little studied compared to T. brucei and T. congolense. METHODS: Trypanosoma simiae procyclics were cultured in vitro and transfected using protocols developed for T. brucei. Genetically modified lines, as well as wild-type trypanosomes, were transmitted through tsetse flies, Glossina pallidipes, to study T. simiae development in the tsetse midgut, proventriculus and proboscis. The development of proventricular trypanosomes was also studied in vitro. Image and mensural data were collected and analysed. RESULTS: A PFR1::YFP line successfully completed development in tsetse, but a YFP::HOP1 line failed to progress beyond midgut infection. Analysis of image and mensural data confirmed that the vector developmental cycles of T. simiae and T. congolense are closely similar, but we also found putative sexual stages in T. simiae, as judged by morphological similarity to these stages in T. brucei. Putative meiotic dividers were abundant among T. simiae trypanosomes in the proboscis, characterised by a large posterior nucleus and two anterior kinetoplasts. Putative gametes and other meiotic intermediates were also identified by characteristic morphology. In vitro development of proventricular forms of T. simiae followed the pattern previously observed for T. congolense: long proventricular trypanosomes rapidly attached to the substrate and shortened markedly before commencing cell division. CONCLUSIONS: To date, T. brucei is the only tsetse-transmitted trypanosome with experimentally proven capability to undergo sexual reproduction, which occurs in the fly salivary glands. By analogy, sexual stages of T. simiae or T. congolense are predicted to occur in the proboscis, where the corresponding portion of the developmental cycle takes place. While no such stages have been observed in T. congolense, for T. simiae putative sexual stages were abundant in the tsetse proboscis. Although our initial attempt to demonstrate expression of a YFP-tagged, meiosis-specific protein was unsuccessful, the future application of transgenic approaches will facilitate the identification of meiotic stages and hybrids in T. simiae.

Evaluation and deployment of isotype-specific salivary antibody assays for detecting previous SARS-CoV-2 infection in children and adults.

BACKGROUND: Saliva is easily obtainable non-invasively and potentially suitable for detecting both current and previous SARS-CoV-2 infection, but there is limited evidence on the utility of salivary antibody testing for community surveillance. METHODS: We established 6 ELISAs detecting IgA and IgG antibodies to whole SARS-CoV-2 spike protein, to its receptor binding domain region and to nucleocapsid protein in saliva. We evaluated diagnostic performance, and using paired saliva and serum samples, correlated mucosal and systemic antibody responses. The best-performing assays were field-tested in 20 household outbreaks. RESULTS: We demonstrate in test accuracy (N = 320), spike IgG (ROC AUC: 95.0%, 92.8-97.3%) and spike IgA (ROC AUC: 89.9%, 86.5-93.2%) assays to discriminate best between pre-pandemic and post COVID-19 saliva samples. Specificity was 100% in younger age groups (0-19 years) for spike IgA and IgG. However, sensitivity was low for the best-performing assay (spike IgG: 50.6%, 39.8-61.4%). Using machine learning, diagnostic performance was improved when a combination of tests was used. As expected, salivary IgA was poorly correlated with serum, indicating an oral mucosal response whereas salivary IgG responses were predictive of those in serum. When deployed to household outbreaks, antibody responses were heterogeneous but remained a reliable indicator of recent infection. Intriguingly, unvaccinated children without confirmed infection showed evidence of exposure almost exclusively through specific IgA responses. CONCLUSIONS: Through robust standardisation, evaluation and field-testing, this work provides a platform for further studies investigating SARS-CoV-2 transmission and mucosal immunity with the potential for expanding salivo-surveillance to other respiratory infections in hard-to-reach settings.

If a person has been previously infected with SARS-CoV-2 they will produce specific proteins, called antibodies. These are present in the saliva and blood. Saliva is easier to obtain than blood, so we developed and evaluated six tests that detect SARS-CoV-2 antibodies in saliva in children and adults. Some tests detected antibodies to a particular protein made by SARS-CoV-2 called the spike protein, and these tests worked best. The most accurate results were obtained by using a combination of tests. Similar tests could also be developed to detect other respiratory infections which will enable easier identification of infected individuals.

Development and evaluation of low-volume tests to detect and characterize antibodies to SARS-CoV-2.

Low-volume antibody assays can be used to track SARS-CoV-2 infection rates in settings where active testing for virus is limited and remote sampling is optimal. We developed 12 ELISAs detecting total or antibody isotypes to SARS-CoV-2 nucleocapsid, spike protein or its receptor binding domain (RBD), 3 anti-RBD isotype specific luciferase immunoprecipitation system (LIPS) assays and a novel Spike-RBD bridging LIPS total-antibody assay. We utilized pre-pandemic (n=984) and confirmed/suspected recent COVID-19 sera taken pre-vaccination rollout in 2020 (n=269). Assays measuring total antibody discriminated best between pre-pandemic and COVID-19 sera and were selected for diagnostic evaluation. In the blind evaluation, two of these assays (Spike Pan ELISA and Spike-RBD Bridging LIPS assay) demonstrated >97% specificity and >92% sensitivity for samples from COVID-19 patients taken >21 days post symptom onset or PCR test. These assays offered better sensitivity for the detection of COVID-19 cases than a commercial assay which requires 100-fold larger serum volumes. This study demonstrates that low-volume in-house antibody assays can provide good diagnostic performance, and highlights the importance of using well-characterized samples and controls for all stages of assay development and evaluation. These cost-effective assays may be particularly useful for seroprevalence studies in low and middle-income countries.

Spatial, temporal and molecular dynamics of swine influenza virus-specific CD8 tissue resident memory T cells.

For the first time we have defined naïve, central memory, effector memory and differentiated effector porcine CD8 T cells and analyzed their distribution in lymphoid and respiratory tissues after influenza infection or immunization, using peptide-MHC tetramers of three influenza nucleoprotein (NP) epitopes. The hierarchy of response to the three epitopes changes during the response in different tissues. Most NP-specific CD8 T cells in broncho-alveolar lavage (BAL) and lung are tissue resident memory cells (TRM) that express CD69 and downregulate CD45RA and CCR7. NP-specific cells isolated from BAL express genes characteristic of TRM, but gene expression differs at 7, 21 and 63 days post infection. In all tissues the frequency of NP-specific CD8 cells declines over 63 days almost to background levels but is best maintained in BAL. The kinetic of influenza specific memory CD8 T cell in this natural host species differs from that in small animal models.

Closely related Lak megaphages replicate in the microbiomes of diverse animals.

Lak phages with alternatively coded ∼540 kbp genomes were recently reported to replicate in Prevotella in microbiomes of humans that consume a non-Western diet, baboons, and pigs. Here, we explore Lak phage diversity and broader distribution using diagnostic polymerase chain reaction and genome-resolved metagenomics. Lak phages were detected in 13 animal types, including reptiles, and are particularly prevalent in pigs. Tracking Lak through the pig gastrointestinal tract revealed significant enrichment in the hindgut compared to the foregut. We reconstructed 34 new Lak genomes, including six curated complete genomes, all of which are alternatively coded. An anomalously large (∼660 kbp) complete genome reconstructed for the most deeply branched Lak from a horse microbiome is also alternatively coded. From the Lak genomes, we identified proteins associated with specific animal species; notably, most have no functional predictions. The presence of closely related Lak phages in diverse animals indicates facile distribution coupled to host-specific adaptation.

Young infants exhibit robust functional antibody responses and restrained IFN-γ production to SARS-CoV-2.

Severe COVID-19 appears rare in children. This is unexpected, especially in young infants, who are vulnerable to severe disease caused by other respiratory viruses. We evaluate convalescent immune responses in 4 infants under 3 months old with confirmed COVID-19 who presented with mild febrile illness, alongside their parents, and adult controls recovered from confirmed COVID-19. Although not statistically significant, compared to seropositive adults, infants have high serum levels of IgG and IgA to SARS-CoV-2 spike protein, with a corresponding functional ability to block SARS-CoV-2 cellular entry. Infants also exhibit robust saliva anti-spike IgG and IgA responses. Spike-specific IFN-γ production by infant peripheral blood mononuclear cells appears restrained, but the frequency of spike-specific IFN-γ- and/or TNF-α-producing T cells is comparable between infants and adults. On principal-component analysis, infant immune responses appear distinct from their parents. Robust functional antibody responses alongside restrained IFN-γ production may help protect infants from severe COVID-19.

A spatial model of COVID-19 transmission in England and Wales: early spread, peak timing and the impact of seasonality.

An outbreak of a novel coronavirus was first reported in China on 31 December 2019. As of 9 February 2020, cases have been reported in 25 countries, including probable human-to-human transmission in England. We adapted an existing national-scale metapopulation model to capture the spread of COVID-19 in England and Wales. We used 2011 census data to inform population sizes and movements, together with parameter estimates from the outbreak in China. We predict that the epidemic will peak 126 to 147 days (approx. 4 months) after the start of person-to-person transmission in the absence of controls. Assuming biological parameters remain unchanged and transmission persists from February, we expect the peak to occur in June. Starting location and model stochasticity have a minimal impact on peak timing. However, realistic parameter uncertainty leads to peak time estimates ranging from 78 to 241 days following sustained transmission. Seasonal changes in transmission rate can substantially impact the timing and size of the epidemic. We provide initial estimates of the epidemic potential of COVID-19. These results can be refined with more precise parameters. Seasonal changes in transmission could shift the timing of the peak into winter, with important implications for healthcare capacity planning. This article is part of the theme issue 'Modelling that shaped the early COVID-19 pandemic response in the UK.

Sequential production of gametes during meiosis in trypanosomes.

Meiosis is a core feature of eukaryotes that occurs in all major groups, including the early diverging excavates. In this group, meiosis and production of haploid gametes have been described in the pathogenic protist, Trypanosoma brucei, and mating occurs in the salivary glands of the insect vector, the tsetse fly. Here, we searched for intermediate meiotic stages among trypanosomes from tsetse salivary glands. Many different cell types were recovered, including trypanosomes in Meiosis I and gametes. Significantly, we found trypanosomes containing three nuclei with a 1:2:1 ratio of DNA contents. Some of these cells were undergoing cytokinesis, yielding a mononucleate gamete and a binucleate cell with a nuclear DNA content ratio of 1:2. This cell subsequently produced three more gametes in two further rounds of division. Expression of the cell fusion protein HAP2 (GCS1) was not confined to gametes, but also extended to meiotic intermediates. We propose a model whereby the two nuclei resulting from Meiosis I undergo asynchronous Meiosis II divisions with sequential production of haploid gametes.

Magnitude and Kinetics of T Cell and Antibody Responses During H1N1pdm09 Infection in Inbred Babraham Pigs and Outbred Pigs.

We have used the pig, a large natural host animal for influenza with many physiological similarities to humans, to characterize αß, γδ T cell and antibody (Ab) immune responses to the 2009 pandemic H1N1 virus infection. We evaluated the kinetic of virus infection and associated response in inbred Babraham pigs with identical MHC (Swine Leucocyte Antigen) and compared them to commercial outbred animals. High level of nasal virus shedding continued up to days 4 to 5 post infection followed by a steep decline and clearance of virus by day 9. Adaptive T cell and Ab responses were detectable from days 5 to 6 post infection reaching a peak at 9 to 14 days. γδ T cells produced cytokines ex vivo at day 2 post infection, while virus reactive IFNγ producing γδ T cells were detected from day 7 post infection. Analysis of NP tetramer specific and virus specific CD8 and CD4 T cells in blood, lung, lung draining lymph nodes, and broncho-alveolar lavage (BAL) showed clear differences in cytokine production between these tissues. BAL contained the most highly activated CD8, CD4, and γδ T cells producing large amounts of cytokines, which likely contribute to elimination of virus. The weak response in blood did not reflect the powerful local lung immune responses. The immune response in the Babraham pig following H1N1pdm09 influenza infection was comparable to that of outbred animals. The ability to utilize these two swine models together will provide unparalleled power to analyze immune responses to influenza.

The dark side of technological advances in analysis of microbial ecosystems.

Recent technological advances mean that samples from animal experiments may be analysed more cheaply, more easily and with a much greater return of data than previously. Research groups are frequently faced with a choice of continuing to use established technology in which they may have made a significant investment of time and resources, and have significant amounts of reference data, or switching to new technology where reference data may be limited. Apart from cost, the choice needs to be based on a comparison between the increase in data available from future experiments by switching and the value of comparison with reference data from historical experiments analysed with earlier technology. One approach to this problem is to ensure that sufficient quantity and variety of samples are taken from each experiment and appropriately stored to allow re-establishment of a sufficiently large reference set and to avoid the need to repeat animal experiments. The establishment of 'biobanks' of experimental material will require funding for infrastructure, consistent storage of metadata and, importantly, horizon-scanning to ensure that samples are taken appropriately for techniques which will become accessible in future. Such biobanks are a recognised resource in human medicine, where the value of samples increases as more analysis is carried out and added to the metadata.

Induction of influenza-specific local CD8 T-cells in the respiratory tract after aerosol delivery of vaccine antigen or virus in the Babraham inbred pig.

There is increasing evidence that induction of local immune responses is a key component of effective vaccines. For respiratory pathogens, for example tuberculosis and influenza, aerosol delivery is being actively explored as a method to administer vaccine antigens. Current animal models used to study respiratory pathogens suffer from anatomical disparity with humans. The pig is a natural and important host of influenza viruses and is physiologically more comparable to humans than other animal models in terms of size, respiratory tract biology and volume. It may also be an important vector in the birds to human infection cycle. A major drawback of the current pig model is the inability to analyze antigen-specific CD8+ T-cell responses, which are critical to respiratory immunity. Here we address this knowledge gap using an established in-bred pig model with a high degree of genetic identity between individuals, including the MHC (Swine Leukocyte Antigen (SLA)) locus. We developed a toolset that included long-term in vitro pig T-cell culture and cloning and identification of novel immunodominant influenza-derived T-cell epitopes. We also generated structures of the two SLA class I molecules found in these animals presenting the immunodominant epitopes. These structures allowed definition of the primary anchor points for epitopes in the SLA binding groove and established SLA binding motifs that were used to successfully predict other influenza-derived peptide sequences capable of stimulating T-cells. Peptide-SLA tetramers were constructed and used to track influenza-specific T-cells ex vivo in blood, the lungs and draining lymph nodes. Aerosol immunization with attenuated single cycle influenza viruses (S-FLU) induced large numbers of CD8+ T-cells specific for conserved NP peptides in the respiratory tract. Collectively, these data substantially increase the utility of pigs as an effective model for studying protective local cellular immunity against respiratory pathogens.

Shape-shifting trypanosomes: Flagellar shortening followed by asymmetric division in Trypanosoma congolense from the tsetse proventriculus.

Trypanosomatids such as Leishmania and Trypanosoma are digenetic, single-celled, parasitic flagellates that undergo complex life cycles involving morphological and metabolic changes to fit them for survival in different environments within their mammalian and insect hosts. According to current consensus, asymmetric division enables trypanosomatids to achieve the major morphological rearrangements associated with transition between developmental stages. Contrary to this view, here we show that the African trypanosome Trypanosoma congolense, an important livestock pathogen, undergoes extensive cell remodelling, involving shortening of the cell body and flagellum, during its transition from free-swimming proventricular forms to attached epimastigotes in vitro. Shortening of the flagellum was associated with accumulation of PFR1, a major constituent of the paraflagellar rod, in the mid-region of the flagellum where it was attached to the substrate. However, the PFR1 depot was not essential for attachment, as it accumulated several hours after initial attachment of proventricular trypanosomes. Detergent and CaCl2 treatment failed to dislodge attached parasites, demonstrating the robust nature of flagellar attachment to the substrate; the PFR1 depot was also unaffected by these treatments. Division of the remodelled proventricular trypanosome was asymmetric, producing a small daughter cell. Each mother cell went on to produce at least one more daughter cell, while the daughter trypanosomes also proliferated, eventually resulting in a dense culture of epimastigotes. Here, by observing the synchronous development of the homogeneous population of trypanosomes in the tsetse proventriculus, we have been able to examine the transition from proventricular forms to attached epimastigotes in detail in T. congolense. This transition is difficult to observe in vivo as it happens inside the mouthparts of the tsetse fly. In T. brucei, this transition is achieved by asymmetric division of long trypomastigotes in the proventriculus, yielding short epimastigotes, which go on to colonise the salivary glands. Thus, despite their close evolutionary relationship and shared developmental route within the vector, T. brucei and T. congolense have evolved different ways of accomplishing the same developmental transition from proventricular form to attached epimastigote.

Early intervention with Bifidobacterium lactis NCC2818 modulates the host-microbe interface independent of the sustained changes induced by the neonatal environment.

Inflammatory and metabolic diseases can originate during early-life and have been correlated with shifts in intestinal microbial ecology. Here we demonstrate that minor environmental fluctuations during the early neonatal period had sustained effects on the developing porcine microbiota and host-microbe interface. These inter-replicate effects appear to originate during the first day of life, and are likely to reflect very early microbiota acquisition from the environment. We statistically link early systemic inflammation with later local increases in inflammatory cytokine (IL-17) production, which could have important enteric health implications. Immunity, intestinal barrier function, host metabolism and host-microbiota co-metabolism were further modified by Bifidobacterium lactis NCC2818 supplementation, although composition of the in situ microbiota remained unchanged. Finally, our robust model identified novel, strong correlations between urinary metabolites (eg malonate, phenylacetylglycine, alanine) and mucosal immunoglobulin (IgM) and cytokine (IL-10, IL-4) production, thus providing the possibility of the development of urinary 'dipstick' tests to assess non-accessible mucosal immune development and identify early precursors (biomarkers) of disease. These results have important implications for infants exposed to neonatal factors including caesarean delivery, antibiotic therapy and delayed discharge from hospital environments, which may predispose to the development of inflammatory and metabolic diseases in later life.

CD8+ T-cell specificity is compromised at a defined MHCI/CD8 affinity threshold.

The CD8 co-receptor engages peptide-major histocompatibility complex class I (pMHCI) molecules at a largely invariant site distinct from the T-cell receptor (TCR)-binding platform and enhances the sensitivity of antigen-driven activation to promote effective CD8+ T-cell immunity. A small increase in the strength of the pMHCI/CD8 interaction (~1.5-fold) can disproportionately amplify this effect, boosting antigen sensitivity by up to two orders of magnitude. However, recognition specificity is lost altogether with more substantial increases in pMHCI/CD8 affinity (~10-fold). In this study, we used a panel of MHCI mutants with altered CD8-binding properties to show that TCR-mediated antigen specificity is delimited by a pMHCI/CD8 affinity threshold. Our findings suggest that CD8 can be engineered within certain biophysical parameters to enhance the therapeutic efficacy of adoptive T-cell transfer irrespective of antigen specificity.

Distinct immune responses and virus shedding in pigs following aerosol, intra-nasal and contact infection with pandemic swine influenza A virus, A(H1N1)09.

Influenza virus infection in pigs is a major farming problem, causing considerable economic loss and posing a zoonotic threat. In addition the pig is an excellent model for understanding immunity to influenza viruses as this is a natural host pathogen system. Experimentally, influenza virus is delivered to pigs intra-nasally, by intra-tracheal instillation or by aerosol, but there is little data comparing the outcome of different methods. We evaluated the shedding pattern, cytokine responses in nasal swabs and immune responses following delivery of low or high dose swine influenza pdmH1N1 virus to the respiratory tract of pigs intra-nasally or by aerosol and compared them to those induced in naturally infected contact pigs. Our data shows that natural infection by contact induces remarkably high innate and adaptive immune response, although the animals were exposed to a very low virus dose. In contacts, the kinetics of virus shedding were slow and prolonged and more similar to the low dose directly infected animals. In contrast the cytokine profile in nasal swabs, antibody and cellular immune responses of contacts more closely resemble immune responses in high dose directly inoculated animals. Consideration of these differences is important for studies of disease pathogenesis and assessment of vaccine protective efficacy.

Dynamics of gamete production and mating in the parasitic protist Trypanosoma brucei.

BACKGROUND: Sexual reproduction in Plasmodium falciparum and Trypanosoma brucei occurs in the insect vector and is important in generating hybrid strains with different combinations of parental characteristics. Production of hybrid parasite genotypes depends on the likelihood of co-infection of the vector with multiple strains. In mosquitoes, existing infection with Plasmodium facilitates the establishment of a second infection, although the asynchronicity of gamete production subsequently prevents mating. In the trypanosome/tsetse system, flies become increasingly refractory to infection as they age, so the likelihood of a fly acquiring a second infection also decreases. This effectively restricts opportunities for trypanosome mating to co-infections picked up by the fly on its first feed, unless an existing infection increases the chance of successful second infection as in the Plasmodium/mosquito system. RESULTS: Using green and red fluorescent trypanosomes, we compared the rates of trypanosome infection and hybrid production in flies co-infected on the first feed, co-infected on a subsequent feed 18 days after emergence, or fed sequentially with each trypanosome clone 18 days apart. Infection rates were highest in the midguts and salivary glands (SG) of flies that received both trypanosome clones in their first feed, and were halved when the infected feed was delayed to day 18. In flies fed the two trypanosome clones sequentially, the second clone often failed to establish a midgut infection and consequently was not present in the SG. Nevertheless, hybrids were recovered from all three groups of infected flies. Meiotic stages and gametes were produced continuously from day 11 to 42 after the infective feed, and in sequentially infected flies, the co-occurrence of gametes led to hybrid formation. CONCLUSIONS: We found that a second trypanosome strain can establish infection in the tsetse SG 18 days after the first infected feed, with co-mingling of gametes and production of trypanosome hybrids. Establishment of the second strain was severely compromised by the strong immune response of the fly to the existing infection. Although sequential infection provides an opportunity for trypanosome mating, the easiest way for a tsetse fly to acquire a mixed infection is by feeding on a co-infected host.