Effect of immunization route on mucosal and systemic immune response in Atlantic salmon (Salmo salar)

This study aimed to assess systemic and mucosal immune responses of Atlantic salmon (Salmo salar) exposed to two protein-hapten antigens – dinitrophenol (DNP) and fluorescein isothiocyanate (FITC) each conjugated with keyhole limpet haemocyanin (KLH) – administered using different delivery strategies. Fish were exposed to the antigens through different routes, and were given a booster 4 weeks post initial exposure. Both systemic and mucosal antibody responses were measured for a period of 12 weeks using an enzyme-linked immunosorbent assay (ELISA). Only fish exposed to both antigens via intraperitoneal (IP) injection showed increased systemic antibody response starting 6 weeks post immunization. No treatment was able to produce a mucosal antibody response; however there was an increase in antibody levels in the tissue supernatant from skin explants obtained 12 weeks post immunization from fish injected with FITC. Western blots probed with serum and culture supernatant from skin explants showed a specific response against the antigens. In conclusion, IP injection of hapten-antigen in Atlantic salmon was the best delivery route for inducing an antibody response against these antigens in this species. Even though IP injection did not induce an increase in antibody levels in the skin mucus, there was an increased systemic antibody response and an apparent increase of antibody production in mucosal tissues as demonstrated by the increased level of specific antibody levels in supernatants from the tissue explants.     

The survival and fecundity of buffalo flies after treatment of cattle with three anthelmintics

Two anthelmintics with known insecticidal action (ivermectin and closantel) and one with no recorded effect on insects (levamisole) were tested to evaluate their effects on buffalo fly (Haematobia irritans exigua). Blood from animals given closantel or levamisole had no significant effect on mortality of buffalo flies in an in-vitro assay. In contrast, blood from animals given ivermectin showed a dose-dependent effect on the mortality of buffalo flies. At 24 h after one injection of the recommended dose of ivermectin, 98% of the flies applied to cattle in an in-vivo assay are killed. Blood from cattle injected with ivermectin killed 95% of flies 8 d after injection and still killed 15% of flies at 18 days after injection. Surviving flies laid almost no eggs and this effect on flies was significant up to 33 d after injection. The results indicate that ivermectin may be useful to control buffalo fly populations in the field.     

Lesions and saliva-specific antibody responses in rabbits with immediate and delayed hypersensitivity reactions to the bites of Glossina morsitans centralis

Rabbits exposed to feeding tsetse flies developed cutaneous hypersensitivity responses to fly bites. These responses had characteristics of immediate and delayed type hypersensitivity. Saliva components from the tsetse fly Glossina morsitans centralis were electrophoretically separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Major salivary proteins of 160, 92, 66, 64, 55, 42, 33, 28, and 15 kilodaltons were identified. Separated salivary components were transferred to nitrocellulose filters and probed with lectins and with whole sera and purified IgG from rabbits which had been exposed, via fly feeding, to tsetse antigens for variable periods. Many of the salivary proteins were identified as glycoproteins. Several major salivary proteins were recognized by antibodies from sensitized rabbits.     

Humoral immune response of water buffalo monitored with three different antigens of Toxocara vitulorum

Humoral immune response of water buffalo naturally infected with Toxocara vitulorum was monitored using three different antigens of this parasite in serum and colostrum of buffalo cows and calves. Soluble extract (Ex) and excretory/secretory (ES) larval antigens and perienteric fluid antigen (Pe) of adult T. vitulorum were used to measure the antibody levels by an indirect ELISA. Serum of 7-12 buffalo cows for the first 365 days and colostrum of the same number of buffalo cows for the first 60 days of parturition, and serum of 8-10 buffalo calves for the first 365 days after birth were assayed. The ELISA detected antibodies against all three T. vitulorum antigens in the colostrum and serum of 100% of buffalo cows and calves examined. The highest antibody levels against Ex, ES and Pe antigens were detected in the buffalo cow sera during the perinatal period and were maintained at high levels through 300 days after parturition. On the other hand, colostrum antibody concentrations of all three antigens were highest on the first day post-parturition, but decreased sharply during the first 15 days. Concomitantly to the monitoring of immune response, the parasitic status of the calves was also evaluated. In calves, antibodies passively acquired were at the highest concentrations 24 h after birth and remained at high levels until 45 days coincidentally with the peak of T. vitulorum infection. The rejection of the worms by the calves occurred simultaneously with the decline of antibody levels, which reached their lowest levels between 76 and 150 days. Thereafter, probably because of the presence of adults/larvae stimulation, the calves acquired active immunity and the antibodies started to increase slightly in the serum and plateaued between the days 211 and 365. All three antigens were detected by the serum antibodies of buffalo calves; however, the concentration of anti-Pe antibody was higher than anti-EX and anti-ES, particularly after 90 days of age. By conclusion, the buffalo cows develop immunity and keep high levels of antibodies against T. vitulorum-Ex, ES and Pe antigens and these antibodies are transferred to their calves through the colostrum. This passively acquired immunity does not protect the calves against the acquisition of the infection, but these antibodies, passively or actively acquired, may have an important role during worm rejection by the calves and prevention of intestinal reinfection.     

Immunological and feeding studies on antigens derived from the biting fly, Stomoxys calcitrans.

Pairs of rabbits were immunised with three antigenic preparations derived from Stomoxys calcitrans gut, abdominal section and whole flies. Immunoblotting studies demonstrated that a humoral response was mounted against eight antigens from the gut preparation and 12 each from the abdominal and whole fly preparations. In vitro feeding experiments showed higher mortality between Days 4 and 7 in the group of flies which had fed upon blood from rabbits inoculated with the gut derived antigen. This group also produced the lowest percentage of viable eggs (15.5%).     

Mechanical transmission of turkey coronavirus by domestic houseflies (Musca domestica Linnaeaus)

Domestic houseflies (Musca domestica Linnaeaus) were examined for their ability to harbor and transmit turkey coronavirus (TCV). Laboratory-reared flies were experimentally exposed to TCV by allowing flies to imbibe an inoculum comprised of turkey embryo-propagated virus (NC95 strain). TCV was detected in dissected crops from exposed flies for up to 9 hr postexposure; no virus was detected in crops of sham-exposed flies. TCV was not detected in dissected intestinal tissues collected from exposed or sham-exposed flies at any time postexposure. The potential of the housefly to directly transmit TCV to live turkey poults was examined by placing 7-day-old turkey poults in contact with TCV-exposed houseflies 3 hr after flies consumed TCV inoculum. TCV infection was detected in turkeys placed in contact with TCV-exposed flies at densities as low as one fly/bird (TCV antigens detected at 3 days post fly contact in tissues of 3/12 turkeys); however, increased rates of infection were observed with higher fly densities (TCV antigens detected in 9/12 turkeys after contact with 10 flies/bird). This study demonstrates the potential of the housefly to serve as a mechanical vector of TCV.     

Serum antibody response of Castellana sheep to Haemonchus contortus infection and challenge: relationship to abomasal worm burdens

Primary and secondary serum antibody responses to Haemonchus contortus were studied in Castellana sheep. Ten-month-old sheep were infected (200 L3/kg live weight (lw)) and challenged (400 L3/kg lw) or uninfected and equally challenged with H. contortus. Primary infections induced a partially protective response upon challenge, characterized by higher serum protein levels, longer prepatent periods, lower fecal egg counts, and significant reduction in the establishment rate of the parasite and abomasal adult and L4 worm burdens. The resistant status of the infected and challenged sheep was not clearly related either to the serum specific antibody levels (IgG: IgG1, IgG2; IgM; IgA) estimated by ELISA or to immunodetection patterns in the Western blots.     

Immunogenicity in dogs of three recombinant antigens (TSA, LeIF and LmSTI1) potential vaccine candidates for canine visceral leishmaniasis

Control of canine visceral leishmaniasis (VL) remains a difficult and serious problem mostly because there is no reliable and effective vaccine available to prevent this disease. A mixture of three recombinant leishmanial antigens (TSA, LeIF and LmSTI1) encoded by three genes highly conserved in the Leishmania genus have been shown to induce excellent protection against infection in both murine and simian models of cutaneous leishmaniasis. A human clinical trial with these antigens is currently underway. Because of the high degree of conservation, these antigens might be useful vaccine candidates for VL as well. In the present study, using the dog model of the visceral disease, we evaluated the immunogenicity of these three antigens formulated with two different adjuvants, MPL-SE and AdjuPrime. The results were compared with a whole parasite vaccine formulated with BCG as the adjuvant. In order to investigate if sensitization with the recombinant antigens would result in recognition of the corresponding native parasite antigens upon infection, the animals were exposed for four weeks after the termination of the immunization protocol with the recombinant antigens to a low number of L. chagasi promastigotes, an etiological agent of VL. Immune response was evaluated by quantitative ELISA in the animal sera before and after exposure to the viable parasites. Both antigen specific IgG1 and IgG2 antibody levels were measured. Immunization of dogs with the recombinant antigens formulated in either MPL-SE or AdjuPrime resulted in high antibody levels particularly to LmSTI1. In addition, this immunization although to low levels, resulted in the development of antibody response to the whole parasite lysate. Importantly, experimental exposure with low numbers of culture forms of L. chagasi promastigotes caused a clear boost in the immune response to both the recombinant antigens and the corresponding native molecules. The boost response was predominantly of the IgG2 isotype in animals primed with the recombinant antigens plus MPL-SE. In contrast, animals primed with the recombinant antigens formulated in AdjuPrime as well as animals vaccinated with crude antigen preparation responded with mixed IgG1/IgG2 isotypes. These results point to the possible use of this antigen cocktail formulated with the adjuvant MPL-SE in efficacy field trials against canine VL.     

Transmission of bovine virus diarrhoea virus by blood feeding flies.

Three species of blood-feeding flies (Stomoxys calcitrans, Haematopota pluvialis and Hydrotaea irritans) were fed for five minutes on a bullock persistently infected with bovine virus diarrhoea virus (BVDV) containing 10(4.5)TCID50 non-cytopathic BVDV/ml serum, then subsequently fed on BVDV-free seronegative animals maintained in isolation. Virus was isolated from recipient animals between days 5 and 10 using H pluvialis, and up to 72 hours after transmission with S calcitrans; virus isolation was negative using H irritans. Positive seroconversion results obtained with H pluvialis gave a steadily increasing antibody titre. BVDV was recovered from flies 96 hours (four days) after an infective feed for H pluvialis and S calcitrans, but for two hours only for H irritans.     

Characterization of tick antigens inducing host immune resistance. II. Description of rabbit-acquired immunity to Amblyomma americanum ticks and identification of potential tick antigens by Western blot analysis

Feeding by adult Amblyomma americanum ticks induced a level of immunity in rabbits to subsequent tick feeding that resulted in a significant decrease in tick feeding success and fecundity. Histological analysis of tick feeding sites in hosts expressing resistance revealed a predominant eosinophil response, with weak basophil and neutrophil infiltrates. While the basophil was never the dominant granulocyte at the tick feeding sites in resistant hosts, this cell exhibited the greatest increase in density (tenfold) over levels observed in hosts experiencing their first infestation; eosinophils and neutrophils exhibited increases of five- and twofold, respectively. Serum from animals that expressed resistance was tested for the presence of anti-tick antibodies to tick-derived salivary gland substances (SGA) by Western blotting. Western blot analysis of female-derived SGA compared to male-derived SGA, using the Avidin/Biotin technique, resulted in the identification of approximately 25 proteins from the female preparation, but only seven from the male. The use of 125I labeled protein-A as the probe for anti-tick antibody in Western blot analysis resulted in fewer recognized proteins. Serum from rabbits immunized with A. americanum-derived SGA emulsified with complete (CFA) Freund's adjuvant recognized most of the proteins identified by active serum, whereas serum from animals immunized with SGA in incomplete (IFA) Freund's adjuvant did not. Furthermore, both sera recognized a multiplicity of proteins from extracts of larval A. americanum Dermacentor variabilis and Boophilus microplus ticks, suggesting the presence of common antigens between these distantly related ticks. The results from this study demonstrate that rabbits acquire a strong immunity to A. americanum ticks characterized by the production of antibody. Furthermore, ticks secrete a number of substances into rabbits during feeding, as seen by Western blot analysis but only three may be crucial to the induction of host immunity; proteins at 41, 40 and 39 kDa. The purified anti-tick antibody will be used for subsequent isolation and characterization of crucial antigens.     

Immune responses of chickens to dietary protein antigens. I. Induction of systemic and intestinal immune responses following oral administration of soluble proteins in the absence of adjuvant

Oral administration of protein antigens in solution leads to the development of oral tolerance in most mammals but rarely so in the chicken. As dietary proteins are not expected to be immunogenic, the present study was undertaken to evaluate immunological consequences following oral exposure to protein antigens in chicks, and to determine whether or not this form of antigen is ignored. Chicks and turkey poults were fed solutions containing bovine serum albumin (BSA), porcine serum albumin, beta-lactoglobulin or bovine hemoglobin over a period of 6 days (25mg/chick/day). At different time points after feeding serum and bile were examined for presence of specific antibodies by ELISA. Surprisingly, the fed antigens induced robust antibody responses in the absence of added adjuvant. This immune response was further characterised to show that (1) a daily feeding regimen was more immunogenic than single dose feedings, (2) by using a daily feeding regimen, as little as 2mg/chick/day was fully immunogenic, (3) effective immunization was attained in chicks older than 10 day of age, (4) the main antibody class in the serum was IgG, and (5) high IgA levels were detected in the bile after booster feedings. These observations are difficult to reconcile with current concepts on peripheral tolerance to innocuous antigens, and indicate that the bird regulates tolerance and response in a manner different from that described in mammals.     

Susceptibility of African buffalo and Boran cattle to Trypanosoma congolense transmitted by Glossina morsitans centralis

Four African buffalo (Syncerus caffer) and four Boran cattle (Bos indicus) were each exposed to the bites of 10 tsetse flies infected with Trypanosoma congolense. Although both groups of animals became infected, the buffalo showed no clinical signs of trypanosomiasis while the cattle suffered from the disease characterized by pronounced skin reactions, high parasitaemia and severe anaemia. The prepatent periods in the buffalo varied from 18 to 27 days in comparison with 11 to 14 days in the cattle. In the buffalo, skin reactions were only detectable by histological examination of skin biopsies, the peak of parasitaemia was at least a hundredfold below that in cattle and after 54 days parasites were no longer detected. In contrast, the cattle had a continuous high parasitaemia until they were treated with a trypanocidal drug 60 days after infection. Neutralizing antibody to metacyclic trypanosomes appeared in the buffalo during the prepatent period, 15-20 days after infection, whereas in cattle neutralizing antibody was not detected until 10 days after the first peak of the parasitaemia, 25-30 days after infection.     

Serological responses against the pathogenic dimorphic fungus Mucor amphibiorum in populations of platypus (Ornithorhynchus anatinus) with and without ulcerative mycotic dermatitis

Mucor amphibiorum, a dimorphic fungus, causes ulcerative dermatitis and systemic infections in the platypus Ornithorhynchus anatinus in some river systems in Tasmania but apparently not in other regions of Australia. As yet there are no suitable tests for population surveys, nor for detection of internal lesions in live animals. Consequently, immunoglobulins were purified from the serum of platypuses and anti-immunoglobulin antisera were prepared in rabbits in order to develop an enzyme-linked immunosorbent assay (ELISA) for anti-M. amphibiorum antibodies. Antigens from plate-grown cultures resulted in greater signal-to-noise ratios in indirect ELISA than those from broth-grown cultures. Platypuses with clinical ulcerative dermatitis had elevated anti-Mucor antibody levels compared to apparently unaffected individuals. Seroconversion was observed in one animal coincident with the development of cutaneous ulcers. The results suggested that platypuses in affected rivers were exposed to M. amphibiorum at a higher frequency than the occurrence of clinical disease. Some platypuses from New South Wales had elevated antibody levels but these increased significantly with age suggesting exposure to cross-reactive antigens, although exposure to M. amphibiorum cannot be excluded. Further studies are warranted to determine factors that result in progression from infection to disease, the occurrence of the fungus in areas where disease has not been observed and the specificity of antigen used in ELISA.     

Detection of IgG and IgE serum antibodies to Culicoides salivary gland antigens in horses with insect dermal hypersensitivity (sweet itch).

We postulated that all horses exposed to the bites of Culcoides (midges) would have an antibody response to the antigen secreted in Culcoides saliva, but that IgE antibody would be restricted to allergic individuals. Using immunohistology on sections of fixed Culicoides, we have demonstrated the presence of antibodies in horse serum which recognise Culicoides salivary glands. Antibodies were detected in the serum of horses with insect dermal hypersensitivity and in the serum of normal horses exposed to Culicoides bites. In contrast, no antibodies were detected in serum from native Icelandic ponies which had not been exposed to Culicoides. Anti-salivary gland IgG antibodies were detected in serum from both allergic and healthy horses exposed to Culicoides. IgE antibodies were only detected in horses with signs of insect dermal hypersensitivity, they were not found in serum of healthy controls nor in the serum of horses with a history of hypersensitivity but in remission at the time of sampling. Using western blotting we confirmed the presence of antibodies to Culicoides antigens and demonstrated that individual horses react to different numbers of antigens. This paper demonstrates the ability of serum from allergic horses to detect Culcoides antigens and will enable further studies to isolate and characterise the allergens.     

Molecular characterization of a myosin alkali light chain-like protein, a "concealed" antigen from the hard tick Haemaphysalis qinghaiensis

There should be some differences between antibodies generated by feeding ticks on animals and those derived by immunizing animals with tick extracts. Here, we found serum collected from sheep immunized with Haemaphysalis qinghaiensis salivary gland extracts could detect two more protein bands with molecular weights of 22 and 37 kDa (P22 and P37) on Western blots of extracts of tick salivary glands than serum from tick infected animals. Rabbit anti-H. qinghaiensis differential protein immune serum was then generated from P22 and P37 and was used to immunoscreen a cDNA library constructed from salivary glands, Malpighian tubules and ovaries of partially engorged H. qinghaiensis. A cDNA contains an open reading frame of 483 bp that codes for 160 amino acid residues with a coding capacity of 18 kDa was cloned and designated Hq02. Expression analysis by RT-PCR showed that this gene is expressed in salivary glands, midguts, other organs and different developmental stages of H. qinghaiensis. The predicted amino acid sequence of the Hq02 gene had high homology to some known myosin alkali light chain (MLC) proteins. A fusion protein consisting of 130 amino acids of Hq02 protein and 335 amino acids of T7 gene 10 protein was expressed in Escherichia coli and used to immunize sheep. Western blot showed that only rabbit anti-H. qinghaiensis differential protein immune serum could recognize the expressed Hq02 protein, while rabbit anti-H. qinghaiensis saliva immune could not. This proved Hq02 protein was a "concealed" antigen. Immunization with the recombinant Hq02 conferred a 21.8% reduction of engorgement weight for adult female ticks that fed on the immunized sheep. This is the first report of tick myosin alkali light chain and the function of this protein is discussed.     

Prevention of sand fly attack by topical application of a permethrin/pyriproxyfen combination on dogs

Dogs are the primary domestic reservoir of Leishmania infantum, the parasite responsible for most cases of human visceral leishmaniasis. A strategy for the control of leishmaniasis would be to inhibit the sand fly bite. A study was designed to measure the prevention of the sand fly attack by spraying a combination of permethrin and pyriproxyfen on dogs artificially exposed to the vector of leishmaniasis. Eight dogs were individually challenged with 100 female sand flies for 1 hour on Days -7, 0, 7, 14, 21, and 28. Four dogs were randomly assigned to a control group and four dogs were treated with topically applied permethrin/pyriproxyfen on Day 0. After each exposure, sand flies were collected, counted, and scored as fed or unfed. Efficacy of the combination for prevention of feeding was based on the number of unfed sand flies (dead or alive). The combination of permethrin/pyriproxyfen demonstrated a significant (P <.05) repellent effect against Phlebotomus perniciosus bites as soon as it was sprayed on the dogs, and its repellent efficacy lasted at least for 28 days. The combination product provided significant (P <.05) knockdown activity against challenge with sand flies for 21 days in adult dogs and 14 days in puppies. These findings indicate that adult animals in endemic areas should be sprayed with the permethrin/pyriproxyfen product at 3- to 4-week intervals, and young dogs should be sprayed at approximately 2-week intervals, to prevent sand fly attack.     

Partial purification of somatic and excretory-secretory products of adult Fasciola hepatica and their application for the serodiagnosis of experimental and natural fascioliasis using an ELISA

Six partially purified antigen fractions from adult Fasciola hepatica (three somatic tissues [Fhs] and three excretory products [Fhm]) were used in a micro-ELISA to monitor the serum antibody levels of an experimental rabbit F hepatica infection. Fhs 1 detected infection after 19 to 26 days and the titre remained significantly higher than that of the controls until day 103 of infection (end of experiment). Using Fhm 1 and Fhm 2, antibodies were detected between 12 and 19 days after infection. Fhm 2 distinguished infected from uninfected rabbits during the entire experimental period, whereas Fhm 1 did not. Excretory-secretory products of a low molecular weight were also antigenic and could differentiate between infected animals and controls. One hundred and nine sera from naturally infected cattle and uninfected controls were tested with the same antigens. Although antibody was detected, the results were inconsistent and further purification of the antigens may eventually improve the sensitivity of the method.     

The relationships among ecto- and endoparasite levels, class I antigens of the bovine major histocompatibility system, immunoglobulin E levels and weight gain

Natural infestations of the cattle tick Boophilus microplus, levels of the buffalo fly Haematobia irritants exigua and faecal nematode egg concentrations (Bunostomum phlebotomum, Cooperia spp., Haemonchus placei, Oesophagostomum radiatum and Trichostrongylus axei) were assessed in 221 Belmont Red calves during the post-weaning period, when the animals were between 9 and 18 months of age. In addition, the 98 males of this group were challenged with B. microplus larvae on two separate occasions. There were strong positive correlations among replicate assessments of the same parasite. Field tick counts and tick counts following deliberate challenge were strongly correlated, and both showed negative correlations with post-weaning weight gain. There was a weak positive correlation between buffalo fly counts and post-weaning weight gain. There was a negative correlation between total worm egg count and weight gain. Among worm species, only the effect of O. radiatum on weight gain was significant. Cattle with bovine major histocompatibility (BoLA) antigens W6.1 and W7 had significantly fewer ticks than cattle lacking these antigens. Cattle with BoLA antigens W7 and CA36 had lower concentrations of nematode eggs in their faeces than cattle lacking these BoLA antigens.     

Characterization of monoclonal antibodies against ovine Sarcocystis spp. antigens by immunoblotting and immuno-electron microscopy

Six monoclonal antibodies were raised in mice against purified cytozoite extracts of Sarcocystis gigantea and S. tenella from sheep. Each monoclonal antibody was evaluated for specificity by enzyme immunoassay, immunoblotting and immuno-electron microscopy using homologous and heterologous antigenic preparations. All six monoclonal antibodies exhibited good species-specificity when reacted against crude soluble cystozoite antigens in enzyme immunoassays. However, only two monoclonal antibodies (IgM and IgG2a) exhibited reactivity in Western blots against specific protein bands. Both reacted against S. gigantea antigens of 100,000, 43,000 and 39,000 molecular weight. Neither monoclonal antibody reacted against the heterologous species S. tenella. Ultrastructural studies performed with colloidal-gold conjugated antisera revealed that both monoclonal antibodies reacted against antigens located around micronemes and amylopectin granules in S. gigantea cystozoites. Another monoclonal antibody (IgGI) reacted only against microneme determinants in S. tenella cystozoites. In contrast, polyclonal sheep and rabbit immune sera cross-reacted against a wide range of cystozoite antigens.     

Cross-reactivity between antigens from Amblyomma cajennense and A. hebraeum (Acari: Ixodidae)

Laboratory animals exposed to feeding ticks develop resistance which is reflected by a decline in tick engorgement weight, egg-laying by adults and reduced egg viability. Serum antibodies from these hosts and their reaction with tick antigens have been detected by different methods, including precipitation techniques, immunofluorescent techniques, ELISA and Western blots. However, little is known about the effects of antibodies on ticks that engorge on resistant hosts, or which tissues of the tick body are possibly immunogenic. Some researchers, using immunohistochemistry, have detected host antibodies in the gut, salivary glands and haemolymph of ticks engorged on resistant animals. The same technique has helped considerably in determining antigenic sites or antibody targets in other arthropods. Consequently, immunohistochemistry techniques were used in this study to detect cross-reactivity between sera raised against Amblyomma cajennense (Fabricius, 1787) with Amblyomma hebraeum (Koch, 1844), and vice versa. The results show the existence of shared antigens between the 2 tick species. In general, our results point more to a 1-way cross-reactivity of A. hebraeum with A. cajennense than a reciprocal cross-reactivity, suggesting that A. hebraeum is more immunogenic than A. cajennense.