A long-term bacteriological and immunological study in Holstein-Friesian cattle experimentally infected with Mycobacterium avium subsp. paratuberculosis and necropsy culture results for Holstein-Friesian cattle, Merino sheep and Angora goats.

The aims were to longitudinally evaluate the interferon-gamma (IFN-gamma) test in comparison to faecal culture and the absorbed ELISA in a cattle infection model for Johne's disease and to determine the adult infection status, by necropsy and tissue culture, of sheep, goats and cattle infected as young animals. Clinical disease, faecal culture results and immunological responses for Merino sheep [Stewart, D.J., Vaughan, J.A., Stiles, P.L., Noske, P.J., Tizard, M.L.V., Prowse, S.J., Michalski, W.P., Butler, K.L., Jones, S.L., 2004. A long-term study in Merino sheep experimentally infected with Mycobacterium avium subsp. paratuberculosis: clinical disease, faecal culture and immunological studies. Vet. Microbiol. 104, 165-178] and Angora goats [Stewart, D.J., Vaughan, J.A., Stiles, P.L., Noske, P.J., Tizard, M.L.V., Prowse, S.J., Michalski, W.P., Butler, K.L., Jones, S.L., 2006. A long-term study in Angora goats experimentally infected with Mycobacterium avium subsp. paratuberculosis: clinical disease, faecal culture and immunological studies. Vet. Microbiol. 113, 13-24], in the same experiments as the Holstein-Friesian cattle, have been described. Two longitudinal experiments involving Holstein-Friesian cattle challenged with either bovine or ovine strains of Mycobacterium avium subsp. paratuberculosis (Map) have been conducted over a period of 54 and 35 months, respectively. Blood samples for the IFN-gamma test and the absorbed ELISA and faecal samples for bacteriological culture were taken pre-challenge and monthly post-challenge. Cell-mediated (CMI) responses were substantially higher for the bovine Map strain during the 42-month period following dosing but then declined in the remaining 12 months. However, for the ovine Map challenge and control groups, CMI responses were not significantly different from each other. None of the cattle developed clinical disease and only one of the cattle in the bovine Map gut mucosal tissue challenged group was a persistent faecal shedder and also an ELISA antibody responder which developed after shedding commenced. Culture of tissues, following necropsy at the completion of the experiments, showed no evidence of infection in any of the challenged cattle and sheep for either the bovine or ovine Map strain in contrast to positive cultures for challenged goats in the same experiments. The tissues from the control cattle, sheep and goats were culture negative. The cattle were less susceptible to the bovine and ovine Map strains than goats and sheep with the goats being the least naturally resistant.

A long-term study in Angora goats experimentally infected with Mycobacterium avium subsp. paratuberculosis: clinical disease, faecal culture and immunological studies.

Two longitudinal experiments involving Angora goats challenged with either bovine or ovine strains of Mycobacterium avium subspecies paratuberculosis (Map) have been conducted over a period of 54 and 35 months, respectively. Blood samples for the interferon-gamma (IFN-gamma) test and the absorbed ELISA and faecal samples for bacteriological culture were taken pre-challenge and monthly post-challenge. Persistent shedding, IFN-gamma production, seroconversion and clinical disease occurred earlier with the bovine Map gut mucosal tissue challenge inoculum than with cultured bacteria. The IFN-gamma responses of the gut mucosal tissue and bacterial challenge groups were substantially and consistently higher than those of the control group. The in vivo and cultured cattle strains were much more pathogenic for goats than the sheep strains with persistent faecal shedding, seroconversion and clinical disease occurring in the majority of bovine Map challenged goats. With the ovine Map, 3 goats developed persistent antibody responses but only one of these goats developed persistent faecal shedding and clinical disease. However, there was no significant difference between the IFN-gamma responses of the tissue challenged, bacterial challenged and control groups. Compared with sheep, the ELISA appeared to have higher sensitivity and the IFN-gamma test lower specificity.

Development of a Johne's disease infection model in laboratory rabbits following oral administration of Mycobacterium avium subspecies paratuberculosis.

To assess the rabbit as a model for the study of Johne's disease pathogenesis, a breeding group of adult and juvenile New Zealand white rabbits were orally challenged with three doses of the Mycobacterium avium subspecies paratuberculosis wildtype bovine strain, CLIJ623, on three occasions. Faecal culture, post-mortem tissue bacteriological culture and histopathology were used to monitor the disease progression in the rabbits for more than 2 years. Of 4 adult and 16 juvenile orally dosed rabbits M. paratuberculosis organisms were recovered bacteriologically from two and three animals, respectively, using the BACTECtrade mark radiometric culture system. Tissue sites from which the bacteria were recovered included the mesenteric lymph nodes, ileocaecal valve, vermiform appendix, caecum, proximal colon and jejunum. Body weight loss, reduced abdominal fat and mild lesions were observed at necropsy in four infected rabbits. Diarrhoea and persistent faecal shedding of bacteria were not observed. Faecal culture did not yield any cultivable mycobacterial organisms on solid media.

Dual host infections: enhanced infectivity of eastern equine encephalitis virus to Aedes mosquitoes mediated by Brugia microfilariae.

When mosquitoes feed on a vertebrate host that is infected concurrently with virus and microfilariae (mf), both pathogens are ingested. If mf penetrate the mosquito midgut, a small portion of the ingested virus may disseminate directly into the mosquito hemocoel. This phenomenon, termed microfilarial enhancement of arboviral transmission, has the potential to enhance the infectivity of arboviruses to mosquitoes. We investigated whether concurrent ingestion of Brugia mf and eastern equine encephalitis virus would enhance the infectivity and subsequent transmissibility of the virus by Aedes mosquitoes. Trials with Ae. triseriatus and B. pahangi mf indicated that microfilarial enhancement was dose dependent. Both a sufficient number of penetrating mf and a sufficient viremia were required for enhancement to occur. Furthermore, studies with B. malayi and three species of Aedes indicated that under comparable conditions of host viremia and microfilaremia, microfilarial enhancement occurred in some mosquito species (i.e., Ae. aegypti and Ae. taeniorhynchus) but not in others (Ae. triseriatus). We suggest that certain key parameters determine whether dual virus/mf host infections will enhance arboviral infectivity to mosquitoes. These include species differences in the capacity of mf to penetrate the mosquito midgut, the amount of virus passing into the hemocoel during mf penetration, and the innate susceptibility of mosquitoes to hemocoelomically introduced virus.

Facilitation of Rift Valley fever virus transmission by Plasmodium berghei sporozoites in Anopheles stephensi mosquitoes.

Certain mosquito species are susceptible to viral infection but cannot transmit the virus due to a salivary gland barrier. We hypothesized that such species could transmit virus if the mosquito were infected with both virus and malaria parasites. Malaria sporozoites disrupt the integrity of mosquito salivary glands and, in so doing, may destroy salivary gland barriers to viral transmission. To examine this postulate, the model system of Rift Valley fever (RVF) virus and a rodent parasite, Plasmodium berghei, in Anopheles stephensi mosquitoes was used. Viral transmission rates for RVF virus-inoculated anophelines that were previously fed either gametocytemic blood (malaria-infected) or normal blood (control) were compared. Viral transmission rates for anophelines having concurrent sporozoite infection of the salivary glands were 32% (n = 25). None of the RVF virus-inoculated control anophelines (n = 55) transmitted virus. These studies confirm an earlier report that malaria sporozoites can disrupt salivary gland barriers and enhance mosquito transmission of arboviruses. Taken together with similar studies using microfilarial parasites, it is increasingly apparent that mosquito-borne parasites have the potential to enhance mosquito transmission of arboviruses.

Ookinete rates in Afrotropical anopheline mosquitoes as a measure of human malaria infectiousness.

Anopheles gambiae s.1. and An. funestus were sampled for Plasmodium spp. ookinetes in two P. falciparum-endemic sites in western Kenya. Since the ookinete is a transitional stage of short duration, occurring after fertilization and before oocyst development, only females in the half-gravid and gravid stages of blood digestion were examined. Preparations of homogenized midguts were spotted onto microslides and examined microscopically after staining with Giemsa. Overall, ookinetes were detected in 4.4% of 1,079 anophelines examined over an eight-month period. Anopheles funestus had higher ookinete rates than An. gambiae s.1., and ookinete rates were higher in half-gravid than in gravid An. gambiae s.1. Geometric mean numbers of ookinetes per infected female were less than five for each species at the two sites, and the maximum number observed was only 12. The low frequencies and numbers of ookinetes were sufficient to produce sporozoite rates of 4-18% in the vector populations. The intense transmission of P. falciparum in these two sites is maintained by anthropophilic vectors where only one in 23 blood meals initiates an infection of generally less than five ookinetes. Relationships between human malaria infectiousness and vector infectivity are dependent upon the high efficiency of the developmental transition from the ookinete to the subsequent oocyst and sporozoite stages.

Sporogonic development of cultured Plasmodium falciparum in six species of laboratory-reared Anopheles mosquitoes.

Sporogonic development of cultured Plasmodium falciparum was compared in six species of Anopheles mosquitoes. A reference species, A. gambiae, was selected as the standard for comparison. Estimates of absolute densities were determined for each lifestage. From these data, four aspects of parasite population dynamics were analyzed quantitatively: 1) successive losses in abundance as parasites developed from gametocyte to ookinete to oocyst stages, 2) oocyst production of sporozoites, 3) correlation between various lifestage parameters, and 4) parasite distribution. Parasite populations in A. gambiae incurred a 316-fold loss in abundance during the transition from macrogametocyte to ookinete stage, a 100-fold loss from ookinete to oocyst stage, yielding a total loss of approximately 31,600-fold (i.e., losses are multiplicative). Comparative susceptibilities in order were A. freeborni >> A. gambiae, A. arabiensis, A. dirus > A. stephensi, A. albimanus. The key transition(s) determining overall susceptibility differed among species. Despite species differences in oocyst densities and infection rates, salivary gland sporozoite production per oocyst (approximately 640) was the same among species. The most consistent association among lifestage parameters was a positive correlation between densities and infection rates of homologous lifestages. A curvilinear relationship between ookinete and oocyst densities in A. gambiae indicated a threshold density was required for ookinete conversion to oocysts (approximately 30 ookinetes per mosquito). The same relationship in A. freeborni was linear, with no distinct threshold. Ookinete and oocyst populations were negative binomially distributed in all species. Indices of heterogeneity in mosquito susceptibility to infection indicated that gene frequencies determining susceptibility fluctuated with time in all species, except A. freeborni where susceptibility remained homogenous throughout the study. This approach provides a framework for identifying mechanisms of susceptibility and evaluating Plasmodium sporogonic development in naturally occurring vector species in nature.

Quantitation of antisporozoite immunoglobulins in the hemolymph of Anopheles stephensi after bloodfeeding.

Passage of rat antibodies induced by Plasmodium falciparum circumsporozoite protein (anti-CS IgG) from the bloodmeal into the hemocoel of uninfected Anopheles stephensi mosquitoes was quantified using enzyme-linked immunosorbent assay (ELISA) techniques. Anti-CS IgG were present in hemolymph immediately upon cessation of mosquito feeding. Titers peaked at 3 hr post-ingestion then declined steadily, becoming negligible at 18 hr. Substantial titers were present in the bloodmeal at 24 hr post-ingestion. By 48 hr, anti-CS IgG in both mosquito hemolymph and bloodmeal were virtually absent. Estimated quantities of anti-CS IgG in the hemolymph at 3 hr post-ingestion were 905-958 ng/ml, representing approximately 0.5% of that present in the host serum. Rat IgG subclasses 1, 2a, and 2b passed into hemolymph more readily than did IgM and possibly IgG2c. Hemolymph volume of unengorged mosquitoes (0.53 microliters) increased after a bloodmeal (0.73 microliters at 3 hr post-ingestion), suggesting that anti-CS IgG may move into the hemocoel in an aqueous solution.

Quantitation of Plasmodium falciparum sporozoites transmitted in vitro by experimentally infected Anopheles gambiae and Anopheles stephensi.

The frequency and numbers of Plasmodium falciparum sporozoites transmitted in vitro and corresponding sporozoite loads were determined for experimentally infected Anopheles gambiae and An. stephensi. Geometric mean (GM) sporozoite loads in three experiments ranged from 808 to 13, 905 for An. gambiae and from 6, 608 to 17, 702 for An. stephensi. A total of 44.1% of 68 infected An. gambiae and 49.2% of 63 infected An. stephensi transmitted sporozoites in vitro. The GM number of sporozoites transmitted was 4.5 for An. gambiae and 5.4 for An. stephensi. Overall, 86.9% of the mosquitoes transmitted from one to 25 sporozoites, and only 6.6% transmitted over 100 sporozoites (maximum = 369). Sporozoite loads were not a useful predictor of potential sporozoite transmission. Despite higher sporozoite loads, the numbers of sporozoites transmitted in vitro by the experimentally infected mosquitoes were similar to estimates obtained, using the same techniques, for naturally infected An. gambiae in western Kenya. The low but highly variable numbers of sporozoites transmitted in vitro by mosquitoes used in malaria vaccine challenge studies appears to be a reasonable simulation of natural sporozoite transmission.

Plasmodium falciparum: administration of anti-sporozoite antibodies during sporogony results in production of sporozoites which are not neutralized by human anti-circumsporozoite protein vaccine sera.

Five days after receiving a Plasmodium falciparum NF54 infectious blood meal, Anopheles stephensi mosquitoes were fed rat anti-P, falciparum sporozoite or rabbit anti-R32tet32 antibodies. Sporozoites isolated from salivary glands were tested by the inhibition of sporozoite invasion (ISI) assay using monoclonal antibody (Mab) 2A10 to P. falciparum circumsporozoite protein or sera from human volunteers immunized with P. falciparum R32tet32 or (NANP)3-TT vaccines. Whereas sporozoites from control mosquitoes were neutralized by Mab 2A10 and vaccine sera, only the Mab and not the vaccine sera neutralized sporozoites from immune-fed mosquitoes. The implications of these results in vaccine design and the impact on transmission are discussed.

Plasmodium falciparum: the population structure of mature gametocyte cultures has little effect on their innate fertility.

In vitro cultured Plasmodium falciparum gametocytes were fed to Anopheles gambiae (G3) mosquitoes to identify parasite population characteristics useful for predicting successful mosquito infections. Parameters were collected from an initial study of 90 infections over a two year period and a second study of 55 infections over 12 weeks. Parasite isolate/clone was identified as the most reliable predictor of gametocyte infectiousness. Parameters such as gametocyte age structure (stage IV:V ratio), exflagellation rate and macrogametocyte maturity were not reliable for predicting infectiousness but were useful for monitoring overall culture maturity. Other variables such as gametocyte density, chronological age of the culture at the time of feed, gametocyte sex ratio, asexual parasitemia, and mixing cultures before mosquito feeding were not predictive. Thus, if a reliable parasite isolate or clone is used, there is no need to measure other characteristics of in vitro gametocyte populations because these will not significantly improve one's ability to predict oocyst infection rates.

Generation of immunity against Fusobacterium necrophorum in mice inoculated with extracts containing leucocidin.

The capacity of extracts from toxigenic and non-toxigenic ruminant strains of Fusobacterium necrophorum to protect against challenge with homologous and heterologous bacteria was examined in mice. The numbers of F. necrophorum which were infective or lethal for mice increased 5- to 8-fold in animals which had been previously inoculated with complete Freund's adjuvant (FCA). Although preparations containing lipopolysaccharide (LPS) and outer membrane proteins (OMP) from several strains gave protection against a non-toxigenic strain (FnB-3), they did not significantly immunize mice against a challenge infection with a toxigenic bovine strain, FnB-1. Only material which had been prepared by gel filtration of 18-h liquid culture supernates of toxigenic F. necrophorum elicited significant immunity against homologous challenge with FnB-1. This preparation contained LPS and the majority of the leucotoxic activity. However, passive protection was not afforded to mice inoculated with bovine or rabbit sera which possessed high neutralization titres against the leucocidin.

Evidence for phospholipase B activity in Fusobacterium necrophorum cultures and its association with hemolysin/leucocidin activities.

Phospholipase B (PLB) activity was present in Fusobacterium necrophorum cultures and it correlated closely with virulence and co-purified with the hemolysin/leucocidin activities. All three activities were associated with a large molecule or molecular complex (6 x 10(2)-2 x 10(3) kDa) exhibiting varying degrees of aggregation. These were present mainly in the culture medium and to a lesser extent in cell sonicates. The PLB and toxin activities were sensitive to heat, dissociating agents, proteolytic enzymes, prolonged purification regimes, freeze-drying and repeated freeze-thawing. The toxin(s) were stable over a broad range of pH, did not require divalent ions or reducing agents and could be kept for several months as an ammonium sulfate precipitate at 4 degrees C, or stored as a concentrated liquid in the presence of proteolytic inhibitors at - 20 degrees C.

An experimental ovine foot abscess model using a Fusobacterium necrophorum biotype AB.

An experimental procedure is described for the production of foot abscess in sheep that mimics the natural disease. Lesions were produced by the intradermal inoculation of suspensions of Fusobacterium necrophorum biotype AB containing from 5 x 10(2) to 5 x 10(8) bacteria, into interdigital skin devitalized by freezing with liquid nitrogen. A dose of 5 x 10(5) bacteria induced the development of foot abscess in 3 of 4 and 8 of 8 inoculated feet. It was found that to produce foot abscess in devitalized tissue required between 10(3) and 10(6) fewer bacteria than were necessary to produce similar lesions in healthy tissue.

A long-term study in Merino sheep experimentally infected with Mycobacterium avium subsp. paratuberculosis: clinical disease, faecal culture and immunological studies.

Two longitudinal experiments involving Merino sheep challenged with either bovine or ovine strains of Mycobacterium avium subsp. paratuberculosis (Map) have been conducted over a period of 54 and 35 months, respectively. Blood samples for the interferon-gamma test, the absorbed ELISA and faecal samples for bacteriological culture were taken pre-challenge and monthly post-challenge. Infections were induced with either a bovine or ovine strain of Map in separate experiments with infections being more easily established, in terms of faecal bacterial shedding and clinical disease when the challenge inoculum was prepared from gut mucosal tissue than cultured bacteria. The patterns of response for shedding and clinical disease were similar. Cell-mediated immune responses were proportionally elevated by at least an order of magnitude in all sheep dosed with either a bovine or ovine strain of Map. Conversely, antibody responses were only elevated in a relatively small proportion of infected sheep. Neither of the clinically affected tissue challenged sheep developed an antibody response despite the presence of persistent shedding and the development and decline in cell-mediated immunity. The results indicated that for sheep the interferon-gamma test may be useful for determining if a flock has been exposed to ovine Johne's disease.

Comparison of gene probe and conventional methods for the differentiation of ovine footrot isolates of Dichelobacter nodosus.

In a collaborative study that involved four Australian veterinary diagnostic laboratories a gene probe test based on the recombinant plasmids pJIR318, pJIR314B, and pJIR313, which contain genomic vap or vrl regions, was compared with conventional tests used for the differential diagnosis of ovine footrot. A total of 771 clinical dichelobacter nodosus isolates were tested and designated as belonging to one of several gene probe categories. The results showed that 87% of the virulent isolates belonged to gene probe category 1, compared to only 6% of the benign isolates. It was concluded that there was good correlation between the gene probe test and the virulence designation of these isolates as well as the results of elastase, gelatin-gel and protease isoenzyme tests. Furthermore, the gene probe test was converted to a polymerase chain reaction (PCR)-based test. It is suggested that diagnostic laboratories consider carrying out both this PCR test and tests based on the extracellular proteases of D. nodosus.

Patterns of erythrocyte digestion by bloodsucking insects: constraints on vector competence.

Two general patterns of erythrocyte digestion were observed in representative species from four insect orders. Ingested erythrocytes were hemolyzed rapidly, and blood meals remained liquefied within body lice, Pediculus humanus L. and the fleas Ctenocephalides felis (Bouché) and Xenopyslla cheopis (Rothschild). Peritrophic membrane was absent. In contrast, there was a lag time of 6-18 h before substantial degradation of erythrocytes within the blood meals of bed bugs, Cimex lectularius L.; the sand fly Phlebotomus papatasi Scopoli; and the mosquitoes Anopheles stephensi Liston and Culex pipiens L. Blood meals of sand flies and mosquitoes were clotted and surrounded by peritrophic membrane at 18-24 h after feeding. Clotting and peritrophic membrane were less pronounced in bed bugs. It is proposed that acquisition and maintenance of pathogen types (i.e., prokaryotic versus eukaryotic) within insects are constrained by the general pattern of bloodmeal processing.

Brugia malayi microfilariae (Nematoda: Filaridae) enhance the infectivity of Venezuelan equine encephalitis virus to Aedes mosquitoes (Diptera: Culicidae).

We examined the potentially conflicting effects that microfilarial (MF) enhancement of viral infectivity and MF-induced mortality in mosquitoes have on the vectorial capacity of Aedes aegypti (L.), Aedes triseriatus (Say), and Aedes taeniorhynchus (Wiedemann) for Venezuelan equine encephalitis virus (VEE) when mosquitoes feed on gerbils co-infected with Brugia malayi (Buckley). Groups of mosquitoes were fed on gerbils that were either dually infected (VEE plus B. malayi MF) or singly infected (VEE only). Mosquito mortality was recorded daily, and 5-8 d later, surviving mosquitoes were assayed for disseminated viral infection. The contrasting effects of MF enhancement and MF-induced mortality differed among mosquito species and were determined by the nature and consequences of MF penetration through the mosquito midgut, but not to differences in mosquito susceptibilities to parenterally introduced virus. In Ae. aegypti, MF-induced mortality was high and tended to eliminate any significant effect of MF enhancement. In Ae. triseriatus, MF-induced mortality was low, and feeding on dually infected hosts resulted in 9 times as many mosquitoes with disseminated viral infections as did feeding on singly-infected hosts. In Ae. taeniorhynchus, MF-induced mortality was extremely high, yet under our experimental conditions, feeding on a dually infected hosts resulted in nearly 30 times as many disseminated infections as did feeding on singly infected hosts. The final outcome on vectorial capacity depended on the specific combination of MF, virus, and mosquito species involved. Therefore, future efforts toward understanding MF enhancement should be directed toward mosquito-virus-parasite species combinations that occur together in nature.

Concentrations of human erythrocytes by anopheline mosquitoes (Diptera: Culicidae) during feeding.

Erythrocyte densities in the blood meals of six Anopheles mosquito species were compared with those of human host erythrocyte densities. During engorgement, An. gambiae Giles and An. stephensi Liston concentrated erythrocytes by factors of 1.8 and 1.7, respectively; An. freeborni Aitken did not concentrate; and An. arabiensis Patton and An. dirus Peyton & Harrison demonstrated an intermediate level of erythrocyte concentration (1.4 and 1.2, respectively). An. albimanus concentrated host hemoglobin, but hemolysis during engorgement decreased bloodmeal erythrocyte density below that of host blood. The degree to which anopheline species concentrated erythrocytes was related to the frequency and time spent undergoing prediuresis (anal excretion of fluid during feeding), suggesting that prediuresis is responsible for erythrocyte concentration and that the fluid produced represents efflux from the filtration of ingested blood. Differences observed in erythrocyte concentration by different anopheline species are consistent with species-specific patterns of host selection.

Prior blood feeding effects on susceptibility of Anopheles gambiae (Diptera: Culicidae) to infection with cultured Plasmodium falciparum (Haemosporida: Plasmodiidae).

We examined the relative susceptibilities of Anopheles gambiae Giles of different physiological ages to infection with cultured Plasmodium falciparum (Welch). Cohorts of mosquitoes were divided into three groups; one was fed uninfected blood on day 3 after emergence (i.e., one prior blood meal); another on days 3 and 7 after emergence (i.e., two prior blood meals); and a control group was maintained on sucrose. On days 10 to 12 after emergence, mosquitoes were fed human blood containing P. falciparum gametocytes. Prior blood feeding accelerated digestion of the infective blood meals and subtly altered susceptibility to infection with P. falciparum. When gametocyte cultures were highly fertile, all experimental groups were equally susceptible to infection. However, when gametocyte fertility was low, accelerated digestion had a detrimental effect on the transition of ookinetes to oocysts. Accelerated digestion may raise the threshold density of ookinetes required for the successful conversion of ookinetes to oocysts.