A simplified Barbervax® vaccination regimen in lambs to evoke immunological protection to Haemonchus contortus.

This study assessed the feasibility of altering the pre-weaning vaccination schedule of the commercially available Barbervax® vaccine directed against Haemonchus contortus, to avoid the 2nd priming vaccination which typically falls between lamb marking and weaning. Merino lambs (n = 175) born to maiden ewes, located in the Northern Tablelands, New South Wales, were randomly allocated to treatment groups (n = 35) and subjected to five different vaccination regimes. One group remained as unvaccinated controls and another had the full set of three priming doses. The other three groups were vaccinated only at marking and weaning receiving a double dose of vaccine at marking and/or weaning. The hypothesis tested was that reducing the interval between lamb marking and weaning to 6 weeks, and providing a double dose of vaccine at marking, weaning or both would remove the need for the second priming vaccination between lamb marking and weaning. This priming vaccination in the vaccination protocol necessitates an additional mustering of ewes with young lambs at foot and is a time consuming and costly exercise which increases the risk of mis-mothering. Blood and faecal samples were collected at frequent intervals for worm egg count (WEC), larval differentiation and H. contortus vaccine specific ELISA antibody analysis. Overall, the results supported the hypothesis, and it was found that antibody titres and WEC reductions equivalent to the registered vaccine regimen were achieved by the alternative regimens. This finding requires further investigation under a wider range of conditions. Deviation from the registered vaccination protocol would constitute off-label usage, and at this time and until further evaluations are done these deviations are not recommended.

Trials with the Haemonchus vaccine, Barbervax®, in ewes and lambs in a tropical environment: Nutrient supplementation improves protection in periparturient ewes.

Haemonchus contortus is an economic problem in sheep farms worldwide, mainly in the tropics and subtropics. A vaccine against haemonchosis, called Barbervax®, was evaluated in ewes under two nutritional status, naturally infected with gastrointestinal nematodes. Ewes were divided into four groups: Supplemented Diet - Vaccine; Supplemented Diet - No vaccine; Basal Diet - Vaccine and Basal Diet - No vaccine. Their lambs were divided in Vaccinated and No vaccine. Ewes were immunised six times starting about 1 month of pregnancy with the first three doses at 3 week intervals and the last three shots at 4 week intervals. Supplemented ewes had higher body weight, body score and packed cell volume compared with those fed a basal diet. Both groups of vaccinated ewes showed a similar response in circulating anti-vaccine antibodies but the vaccine had no discernible effect on either body weight, body score and packed cell volume. There was a marked group difference in the number of ewes that received precautionary treatments with anthelmintic. All 14 Basal Diet - No vaccine ewes required treatment. In contrast only 7 ewes, in the Supplemented Diet - Vaccine group required anthelmintic treatment. In the Basal Diet - Vaccine and in the Supplemented Diet - No Vaccine groups, 12 and 13 ewes needed anthelmintic treatment, respectively. Vaccinated lambs showed much higher antibody titres resulting in 80% less Haemonchus spp. egg counts comparing with no vaccine lambs. Taken together these results clearly suggest that in pregnant and lactating ewes a combined protective effect between vaccination and improved nutrition resulted in fewer precautionary anthelmintic treatments. Thus, it was possible to achieve a more sustainable level of control of the haemonchosis, less dependent on anthelmintic drugs.

Attempts to vaccinate ewes and their lambs against natural infection with Haemonchus contortus in a tropical environment.

A vaccine containing integral membrane glycoproteins from the intestine of Haemonchus contortus was evaluated in three groups of grazing sheep each containing 13 ewes and their 16 lambs naturally infected with gastrointestinal nematodes. Two groups were vaccinated with either 5 or 50µg of the antigen per immunisation, while the third, the control group, received adjuvant alone. The sheep were immunised six times at 3week intervals, partly because the vaccine antigens are hidden and thus no immunological boost would be delivered by subsequent infection and partly because the level of Haemonchus spp. challenge was expected to be high. The vaccinated ewes, first immunised approximately 1month before lambing, showed a circulating antibody response but no signs of reduced anaemia or Haemonchus spp. egg counts, compared with control ewes. Several ewes with severe haemonchosis in all three groups had to be given precautionary treatment with anthelmintic drugs. In contrast, vaccinating their lambs with either 5 or 50µg of the antigen per immunisation resulted in 10 fold higher antibody titres. In the case of the lower antigen dose this was associated with significantly less anaemia, 72% reduction in the overall number of Haemonchus spp. eggs produced and significantly fewer worms compared with control lambs. It is hypothesised that the heavily pregnant or lactating ewes did not have sufficient physiological reserves to mount a protective response following vaccination in the tropical weather and high challenge conditions that prevailed. Nevertheless, the vaccine could afford useful protection for lambs against H. contortus.

Vaccination of grazing calves with antigens from the intestinal membranes of Haemonchus contortus: effects against natural challenge with Haemonchus placei and Haemonchus similis.

A vaccine containing integral membrane glycoproteins from the intestine of Haemonchus contortus was evaluated in three groups of eight 5 months old grazing calves, naturally infected by Haemonchus similis, Haemonchus placei and other gastrointestinal nematodes. Vaccinated calves received 5 or 50µg of the antigen and 1mg of saponin adjuvant, while the controls received adjuvant alone, initially three times, 3 weeks apart and then four more times at 6 weeks intervals. Three weeks after the last immunisation all of the calves were euthanised for worm counts. Immunisation stimulated high titre antibodies against the vaccine antigens, reduced the egg output of Haemonchus spp. by 85% and the numbers of H. placei and H. similis by 63% and 32%, respectively, compared with control calves. It was concluded that vaccination with intestinal membrane glycoproteins from H. contortus could substantially reduce the transmission of H. placei and H. similis, thus providing protective benefit downstream. This appears to be the first known successful demonstration of a vaccine protective for cattle naturally exposed to infection with any gastrointestinal nematode parasite.

Streptococcus pyogenes infection of tonsil explants is associated with a human ß-defensin 1 response from control but not recurrent acute tonsillitis patients.

Host defence peptides (HDP), including the defensins and hCAP-18, function as part of the innate immune defences, protecting the host epithelia from microbial attachment and invasion. Recurrent acute tonsillitis (RAT), in which patients suffer repeated symptomatic tonsil infections, is linked to Streptococcus pyogenes, a group A streptococcus, and may reflect the impaired expression of such peptides. To address this, the defensin and hCAP-18 messenger RNA expression profiles of 54 tonsils excised from control and RAT patients undergoing tonsillectomy were quantified and compared. Marked variation in expression was observed between individuals from the two groups, but statistically no significant differences were identified, suggesting that at the time of surgery the tonsil epithelial HDP barrier was not compromised in RAT subjects. Surgical removal of the tonsils occurs in a quiescent phase of disease, and so to assess the effects of an active bacterial infection, HaCaT cells an in vitro model of the tonsil epithelium, and explants of patient tonsils maintained in vitro were challenged with S. pyogenes. The HaCaT data supported the reduced expression of hCAP-18/LL-37, human ß-defensin 1 (HBD1;P < 0.01) and HBD2 (P < 0.05), consistent with decreased protection of the epithelial barrier. The tonsil explant data, although not as definitive, showed similar trends apart from HBD1 expression, which in the control tonsils but not the RAT patient tonsils was characterized by increased expression (P < 0.01). These data suggest that in vivo HBD1 may play a critical role in protecting the tonsil epithelia from S. pyogenes.

Attempts to immunize sheep against Teladorsagia circumcincta using fourth-stage larval extracts.

A ConcanavilinA (ConA)-binding fraction of a detergent-soluble membrane extract from Teladorsagia circumcincta (formerly Ostertagia circumcincta) fourth-stage larvae was isolated, and two vaccine trials were conducted with this preparation in groups of 7 worm-free sheep. All groups were challenged with a total of 5000 T. circumcincta larvae from 1 week after the final immunization and protection assessed by comparing the egg and worm counts, and length of developing worms, of the immunized groups with their respective controls. Immunization with the ConA-binding antigen induced high-titre serum antibody responses in both trials. However, no significant reduction in either egg count or worm burdens was observed in the vaccinated groups in either trial. It was concluded that detergent-soluble, ConA-binding extracts prepared from T. circumcincta fourth-stage larvae did not contain significantly protective antigens, despite the fact that an extract prepared in a similar manner from Ostertagia ostertagi had previously significantly protected calves against homologous challenge.

Protection of calves against Haemonchus placei and Haemonchus contortus after immunization with gut membrane proteins from H. contortus.

A vaccine containing integral membrane glycoproteins from the intestine of Haemonchus contortus was evaluated in four groups of nine worm-free calves challenged with either 8000 H. contortus or Haemonchus placei infective larvae. Vaccinates received 50 µg of the antigen and 1 mg QuilA adjuvant three times 21 days apart, while the controls got adjuvant alone. The calves were challenged 7 days after the last immunization and killed for worm counts 43 days later. Immunization resulted in high titre antibodies against the vaccine antigens and significant reduction in egg output and worm numbers of both challenge species, compared with control calves. It was concluded that vaccination of calves with native parasite gut membrane glycoproteins obtained from H. contortus conferred protection against both H. placei and H. contortus.

Antibodies from sheep immunized against Haemonchus contortus with H-gal-GP inhibit the haemoglobinase activity of this protease complex.

Highly protective intestinal cell membrane antigens have been prepared from Haemonchus contortus, an important blood feeding nematode which parasitizes sheep and goats. One such antigen, H-gal-GP, is a glycoprotein complex containing predominantly digestive proteases. This study showed that H-gal-GP readily digested ovine haemoglobin and albumin, the two most abundant proteins in the parasite's blood meal. It was found that adding protective antibodies from H-gal-GP immunized sheep to the H-gal-GP catalysed haemoglobin digestion reaction, reduced the rate by 70-90% at pH 5·0. This reduction was only 30% when nonprotective IgG from sheep immunized with denatured H-gal-GP was added and IgG from worm-free sheep had no effect. These results support the theory that the mechanism of protection in sheep vaccinated with H-gal-GP is by specific antibodies impairing the parasites ability to digest its blood meal.

Protective immunization of calves against Ostertagia ostertagi using fourth stage larval extracts.

ConA lectin was used to isolate glycoproteins from detergent extracts of fourth stage Ostertagia ostertagi larvae. This preparation contained proteins additional to those observed in a similar fraction prepared from adult O. ostertagi. Two vaccine trials were conducted with this preparation, and sub-fractions thereof, in groups of 6-8 worm-free calves. All groups were challenged with 50,000 O. ostertagi larvae 1 week after the final immunization, and protection was assessed by comparing the egg and worm counts of the immunized groups with their respective controls. Immunization with the ConA-binding antigen or its sub-fractions induced high titre serum antibody responses. In the first trial, the cumulative egg count of the group immunized with unfractionated antigen was 60% lower than the corresponding control value, and worm counts were 47% lower. In the second trial, the cumulative egg counts of the vaccinated groups ranged from 70% to 85% lower than the corresponding controls, with worm counts up to 64% lower. It was concluded that detergent-soluble, ConA-binding extracts prepared from O. ostertagi fourth stage larvae contained protective immunogens that were as effective as the best antigens published for O. ostertagi to date.

Attempts to immunize sheep against Haemonchus contortus using a cocktail of recombinant proteases derived from the protective antigen, H-gal-GP.

The objective of this study was to determine whether an antigen cocktail containing recombinantly expressed versions of most of the protective proteases of H-gal-GP, a known protective antigen from Haemonchus contortus, would confer any protection to lambs in a vaccine-challenge trial. Haemonchus contortus metalloendopeptidases, MEP1, MEP3 and MEP4, were expressed as soluble recombinant proteins in insect cells, but attempts to express the H. contortus aspartyl proteases, PEP1 and PEP2, by the same techniques were not successful. Recombinant H. contortus PEP1 was therefore expressed in Escherichia coli and refolded. Groups of sheep were immunized thrice with either native H-gal-GP, a cocktail of recombinantly expressed proteins (rMEP1, rMEP3, rMEP4 and rPep1), or adjuvant only (QuilA in PBS). All sheep were challenged with 5000 infective larvae 1 week after the final vaccination. High levels of serum antibodies that recognized H-gal-GP were detected in both the native antigen and recombinant cocktail-immunized groups by the time of challenge, but protective immunity was only observed in the group immunized with native H-gal-GP.

Kinetics of the local immune response in the gastric lymph of lambs after primary and challenge infection with Teladorsagia circumcincta.

Groups of 5-month-old lambs which had been trickle infected with Teladorsagia circumcincta for 8 weeks then drenched, and worm-free control lambs were challenged with 50 000 T. circumcincta L3s. From 10 days later fewer parasites were recovered from the previously infected sheep, and secondary cellular and humoral responses were observed in the gastric lymph. Increases in CD4+ and CD25+ T lymphoblast traffic on day 3, followed by CD21+ and IgA+ lymphoblasts on day 5, and an increase in total and parasite specific IgA concentrations peaking on day 6 were observed in previously infected lambs. Similar peaks in lymphoblast output were not observed until days 10-12 in the control lambs. This data was highly comparable with that obtained recently from yearling sheep subjected to an identical infection-challenge regime, and contrasted with that obtained from similar experiments in the 1980s when 4(1/2)-month-old previously infected lambs were more susceptible to and had much weaker immune responses to challenge than 10-month-old sheep. The fact that 40% fewer larvae were given during the trickle infection regime in the four recent trials is offered as an explanation for this difference.

Kinetics of the local cellular response in the gastric lymph of immune and susceptible sheep to infection with Teladorsagia circumcincta.

Groups of yearling sheep were trickle infected with Teladorsagia circumcincta for 8 weeks, then the infection cleared with anthelmintic and both these animals and a group of parasite naïve sheep were challenged with 50 000 infective T. circumcincta larvae. The previously infected sheep demonstrated acquired immunity to the parasite, manifested by reduced worm burdens which were evident as early as 2 days after challenge. Cannulation of the common efferent gastric lymph duct allowed the kinetics of their local cell traffic to be monitored, and the phenotype of these lymphocytes was analysed. A blast cell response, consisting of both T and B lymphocytes, was observed in both groups of sheep, however this occurred more rapidly in the previously infected, immune animals. CD4+, CD8+ and CD25+ blast cell output peaked at day 3 in the previously infected animals, whereas CD21+ blast cell output peaked slightly later at day 5. In the control group the peak output of all phenotypes of blast cells occurred more slowly, peaking 10 days after infection.

Morphometric convergence and molecular divergence: the taxonomic status and evolutionary history of Gymnura crebripunctata and Gymnura marmorata in the eastern Pacific Ocean.

To clarify the taxonomic status of Gymnura crebripunctata and Gymnura marmorata, the extent of morphological and nucleotide variation between these nominal species was examined using multivariate morphological and mitochondrial DNA comparisons of the same characters with congeneric species. Discriminant analysis of 21 morphometric variables from four species (G. crebripunctata, G. marmorata, Gymnura micrura and Gymnura poecilura) successfully distinguished species groupings. Classification success of eastern Pacific species improved further when specimens were grouped by species and sex. Discriminant analysis of size-corrected data generated species assignments that were consistently accurate in separating the two species (100% jackknifed assignment success). Nasal curtain length was identified as the character which contributed the most to discrimination of the two species. Sexual dimorphism was evident in several characters that have previously been relied upon to distinguish G. crebripunctata from G. marmorata. A previously unreported feature, the absence of a tail spine in G. crebripunctata, provides an improved method of field identification between these species. Phylogenetic and genetic distance analyses based on 698 base pairs of the mitochondrial cytochrome b gene indicate that G. crebripunctata and G. marmorata form highly divergent lineages, supporting their validity as distinct species. The closely related batoid Aetoplatea zonura clustered within the Gymnura clade, indicating that it may not represent a valid genus. Strong population structuring (overall Phi(ST) = 0.81, P < 0.01) was evident between G. marmorata from the Pacific coast of the Baja California peninsula and the Gulf of California, supporting the designation of distinct management units in these regions.

Recent vaccine related studies with economically important gastrointestinal nematode parasites of ruminants.

Although several native antigens of sufficient efficacy to be commercially useful have now been discovered for Haemonchus and Ostertagia, all will have to be synthesised artificially to be economically viable. Despite numerous attempts, recombinant DNA technology has not yet yielded the solution, but the effort continues, spurred on by the successes with cestodes and certain ticks. Teladorsagia and Trichostrongylus lag behind in the vaccine research stakes: here, the first and probably most difficult step of identifying a reliably protective native antigen extract does not seem to have been achieved yet. It may be necessary to stimulate elements of the mucosal response to induce protection, a subject still in its infancy as far as ruminants are concerned. Several laboratories have started to work on antigen delivery methods with this in mind. It is easy to understand why intestinal antigens protect against blood feeding Haemonchus rather than mucosal browsers, but quite why conventional immunisation works for Ostertagia, but not apparently for Teladorsagia or Trichostrongylus remains a mystery.

Spatio-temporal analysis of Xyleborus glabratus (Coleoptera: Curculionidae [corrected] Scolytinae) invasion in eastern U.S. forests.

The non-native redbay ambrosia beetle, Xyleborus glabratus Eichhoff (Coleoptera: Curculionidae: Scolytinae), has recently emerged as a significant pest of southeastern U.S. coastal forests. Specifically, a fungal symbiont (Raffaelea sp.) of X. glabratus has caused mortality of redbay (Persea borbonia) and sassafras (Sassafras albidum) trees in the region; several other Lauraceae species also seem susceptible. Although the range of X. glabratus continues to expand rapidly, little is known about the species' biology and behavior. In turn, there has been no broad-scale assessment of the threat it poses to eastern U.S. forests. To provide a basic information framework, we performed analyses exploiting relevant spatio-temporal data available for X. glabratus. First, we mapped the densities of redbay and sassafras from forest inventory data. Second, we used climate matching to delineate potential geographic limits for X. glabratus. Third, we used county infestation data to estimate the rate of spread and modeled spread through time, incorporating host density as a weighting factor. Our results suggest that (1) key areas with high concentrations of redbay have yet to be invaded, but some are immediately threatened; (2) climatic conditions may serve to constrain X. glabratus to the southeastern U.S. coastal region; and (3) if unchecked, X. glabratus may spread throughout the range of redbay in <40 yr. Disruption of anthropogenic, long-distance dispersal could reduce the likelihood of this outcome.

Vaccination against Haemonchus contortus: performance of native parasite gut membrane glycoproteins in Merino lambs grazing contaminated pasture.

In a replicated trial, parasitological and antibody responses of grazing weaner Merino sheep were assessed following vaccination with gut membrane proteins prepared from adult worms of the gastrointestinal nematode, Haemonchus contortus. Each vaccinated animal received 100 microg native H11 and 100 microg native H-gal-GP combined together in 5mg Quil A administered intramuscularly on days 0, 34, 80 and 127. Control animals received 5mg Quil A alone on the same days. Vaccinated and unvaccinated control animals grazed pastures contaminated with the parasite from day 34 of the trial, and levels of parasitism were monitored by worm-egg counts (WECs) in faeces and packed cell volumes (PCVs) in blood. The level of larval contamination on pasture was estimated from the worm counts of tracer sheep introduced monthly to the paddocks. WECs and anaemia were significantly reduced in vaccinated animals, and, in contrast to vaccinates, all control sheep required salvage treatment with anthelmintic. By the last 2 months of the trial, pastures grazed by vaccinated animals had significantly lower contamination with H. contortus larvae. Vaccinated animals had high levels of vaccine antigen-specific IgG1 and IgG2 antibodies in plasma, whereas those responses in the control sheep were very low. IgG1 titres in the vaccinated group, but not IgG2 titres, were inversely correlated with worm-egg counts. The levels of systemic IgA and IgE remained low but increased in both groups towards the end of the experiment most probably from exposure to the natural infection from pasture. The results showed that H11 and H-gal-GP behaved like "hidden" antigens producing high levels of protection that were probably mediated through mechanisms involving antibodies, and in particular, IgG1. It was concluded that if similar protective effects could be obtained with recombinant versions of the proteins present in either H11 or H-gal-GP, then the prospects for a commercial Haemonchus vaccine were real.

Cytokine expression in naïve and previously infected lambs after challenge with Teladorsagia circumcincta.

Infection of sheep with Teladorsagia circumcincta triggers an immune response with predominantly type-2 (Th2) characteristics, including local eosinophila, mastocytosis and increased mucus production. In order to better understand the protective immune responses elicited, we used RT-PCR assays to define the changes in expression levels of a range of cytokine transcripts in lymph nodes draining the ovine abomasum following a challenge infection with T. circumcincta. This study compared the changes in cytokine expression in the abomasal lymph node following challenge with T. circumcincta in naïve sheep (Group 2) and sheep immunised by a previous trickle infection (Group 3), in comparison to unchallenged naive sheep (Group 1). There was a significant up-regulation of interleukin-4 (IL-4), IL-5 and IL-13 in both the challenged groups compared to naïve individuals. There was also an up-regulation of IL-1beta, IL-6, IL-10, IL-18, transforming growth factor-beta1 (TGFbeta1) and tumour necrosis factor-alpha (TNFalpha) by day 5 after infection. IL-12p40 was found to be increased in the previously infected Group 3 animals by day 5 following challenge. By contrast, transcription of this cytokine was found to be reduced by day 10 following infection of Group 2 animals. Expression of IL-2 and Interferon-gamma (IFNgamma) did not significantly differ between the three groups.

An ovine chitinase-like molecule, chitinase-3 like-1 (YKL-40), is upregulated in the abomasum in response to challenge with the gastrointestinal nematode, Teladorsagia circumcincta.

Mammalian chitinases and chitinase-like proteins are a group of molecules known to be upregulated and secreted in Th2-induced inflammatory responses, such as asthma, allergy and nematode infection. As part of an investigation of potential components of the innate immune response to Teladorsagia circumcincta, a gastrointestinal nematode that colonises the abomasum in sheep, we carried out RT-PCR analysis of two members of the mammalian chitinase family of molecules, acidic chitinase (ChiA) and chitinase-3 like 1 (Chi3L1) using primers to homologous bovine/human sequences. Both sets of primers detected transcripts in the abomasum which were confirmed to be ovine ChiA and Chi3L1 by sequence analysis. Chi3L1 transcripts were found to be significantly upregulated in both the abomasum and gastric lymph nodes in response to T. circumcincta challenge of previously infected animals.

Parasite loss and inhibited development of Teladorsagia circumcincta in relation to the kinetics of the local IgA response in sheep.

Groups of yearling sheep, which had been trickle infected with Teladorsagia circumcincta for 8 weeks and then drenched, were challenged with 50 000 T. circumcincta larvae together with groups of worm-free controls. Fewer parasites and a greater proportion of early fourth stage larvae were recovered from previously infected sheep compared to controls. Worm loss and arrested development were evident by 5 days after challenge whereas growth retardation of developing worms was observed by day 10. In the previously infected sheep a secondary IgA response was observed in the efferent gastric lymph from 5 days post-infection. Western blot analysis showed the lymph IgA to be predominantly dimeric and nonsecretory in nature and that the somatic antigens recognized were predominantly in the 100-250 kDa range. The concentration of IgA in lymph was always higher than in blood and in the previously infected sheep increased fivefold 8 days post-challenge in contrast to blood where IgA levels were unchanged. The timing of the response suggested that it occurred too late to have been the cause of worm loss or arrested development, though it may have retarded the growth of developing parasites.

Attempts to detect synergy between vaccination and anthelmintic against a drug resistant isolate of Haemonchus contortus.

An experiment was conducted to determine whether any synergistic activity could be detected between an experimental vaccine and anthelmintic treatment against a drug resistant strain of Haemonchus contortus, i.e. would the combined effect of both interventions be greater than the sum of either alone. Two groups of 14 worm-free sheep were immunised twice, either with Haemonchus galactose containing glycoprotein complex (H-gal-GP) in QuilA as adjuvant or with adjuvant alone. All were challenged with 5000 Haemonchus L3 from the White River isolate which were resistant to ivermectin and fenbendazole. By 26 days post-challenge the H-gal-GP vaccinated sheep had shed 89% fewer nematode eggs than the adjuvant only controls, indicating that this antigen did indeed protect against the anthelmintic resistant isolate. Twenty six days after challenge seven vaccinates and seven control sheep were drenched with ivermectin, but over the next 11 days the mean egg count of either group did not differ significantly from those of the corresponding untreated groups of sheep. On day 37 the drenched sheep were treated with fenbendazole. This time the egg counts of both treated groups fell significantly compared to those of the corresponding untreated groups, but again there was no suggestion that the drug was more effective in the vaccinated sheep. It was concluded that there was no evidence for synergy between a gut membrane protein vaccine and ivermectin or fenbendazole against an anthelmintic resistant isolate of H. contortus.