A computational structural analysis of functional attributes of hypodermin A and B proteins: A way forward for vaccine development.

Hypodermosis is a parasitic disease of cattle. The pathogenicity of the disease is attributed to Hypodermin proteins (Hypodermin A, Hypodermin B and Hypodermin C). Studies suggest that Hypodermin proteins may be defined as Serine proteases and collagenases. The structure of both proteases Hypodermin A and Hypodermin B were modeled using the Swiss-model server followed by its validation using Procheck, Errat and Verify-3D. Afterwards, both Hypodermin A and Hypodermin B were docked against collagen in order to study its interaction with respective Hypodermin proteins. The structure of both Hypodermin A and Hypodermin B showed more bent towards hydrophobic nature as more beta sheets were present in them. Both structures were also superimposed to check out similarities and differences present between them. Serine, Aspartic acid, Histidine, Glutamic acid and Lysine are found as interacting residues that are involved in hydrogen bonding with collagen. The interactions are found in the active domain region of Hypodermin proteins. The interacting residues were present in the active region of the hypodermin proteins thus validating the docking studies. This study may help in the drug development against hypodermosis with least side effects.

Effect of reinfestations on systemic immune responses in cattle naturally infested by Hypoderma sp. (Diptera: Oestridae).

Systemic humoral and cellular immune responses were studied during natural infestations by Hypoderma lineatum in cattle at their first (G-1) and second exposure (G-2). Four out of seven animals in G-1 were palpation positive, with a mean intensity of 11.2 (12.81SD) warbles; the same proportion (4/7) presented warbles in G-2 but the intensity was 3.7 (2.21SD). The evolution of total IgG levels was characterized by a noticeable increment coinciding with the presence of warbles on the back, especially in G-2. The IgG1 isotype displayed a parallel evolution in both groups, with peak values prior to the appearance of first warbles. The IgG2 subclass followed an irregular pattern in both groups and IgM maintained low and constant levels throughout the study, mainly in G-1. CD4/CD8 ratios showed a predominance of CD4(+) throughout the infestation, principally in G-2 during the warble season. The evolution of IFN-γ in G-2 was constant, whereas in G-1 there was a gradual descent until warble emergence. The dynamics of the IL-10 differed between G-1 and G-2, although both groups showed a significant drop after the exit of the larvae that could be implicated in the termination of the inflammatory response. IL-4 and TNF-α levels did not show differences between groups. Our results suggest that the resistance mechanisms would become more apparent at the latest stages of the infestation by Hypoderma, supporting the hypothesis that considerable larval destruction in sensitized animals might take place after their arrival to the back.

Hidden antigens from third instar Hypoderma lineatum: impact of immunization on larval survival in artificial infestations.

Soluble fractions of Hypoderma lineatum third instar fat body, haemocytes and haemolymph were formulated with Quil A and used to immunize four groups of calves while a fifth group remained untreated. Calves received two subcutaneous injections of the soluble fractions, or adjuvant only delivered two weeks apart. Two weeks after the last injection the calves were exposed to 50 newly hatched larvae of H. lineatum which were placed on the skin and allowed to penetrate. Survival of larval stages was monitored by weekly palpation and collection of emergent third instars. Antibody responses to the immunogens were evaluated by immunoblots and following infestation antibody responses to first instar antigens were evaluated by an ELISA. Non-immunized calves and calves injected with adjuvant were all palpation positive for cattle grubs. In groups immunized with fat body, haemocyte and haemolymph components 100%, 33% and 33% were palpation positive for grubs respectively. First instar mortality, as reflected in palpable grubs, was high in the groups receiving injections with tissue components (99.3%, 95.1%, 95.8%, 83.9 and 80.4% mortality for those groups receiving fat body, haemocyte, haemolymph, adjuvant or control respectively). Second and third instar mortality was also higher in the immunized groups (100.0%, 91.7%, 91.7% for fat body, haemocyte, and haemolymph respectively) in comparison to the adjuvant only (14.0%) and unvaccinated (33.3%) groups. No viable flies emerged from pupae originating from larvae emergent from any of the immunized groups. Calves receiving the tissue extracts developed antibodies to several protein components following the second immunization which were still present 13 weeks post-infestation. Several proteins appeared to be common among the three tissue extracts and were recognized by antibodies from the immunized calves. All groups of calves became positive for antibodies to first instar antigens, although in some immunized calves the antibodies were transient.

A serological and molecular survey of cattle hypodermosis in east-Turkey.

The aim of this study was to determine the seroprevalence of hypodermosis in cattle in East-Turkey and to identify Hypoderma species using morphological and molecular methods. For this purpose, a total of 778 serum samples of cattle were collected from Erzurum provinces of east-Turkey from December 2008 to February 2009. The sera were analyzed using a Hypodermin C antigen by means of indirect ELISA. In addition, 10 Hypoderma spp. larvae were collected from slaughtered animals in the abattoir for morphological identification and molecular characterization on the basis of mitochondrial CO1 gene sequence analysis and PCR-RFLP. Two hundred and twenty three (28.6%) out of 778 cattle were seropositive for hypoderma antibodies. All positive cattle were female of a local breed. Seven out of 10 Hypoderma larvae were morphologically classified as third instar larvae (L3) of H. bovis and 3 were classified as L3 of H. lineatum. The TaqI restriction enzyme was used to differentiate the Hypoderma species on the basis of the 438 and 250 bp bands for H. bovis and the 488 and 200 bp bands for H. lineatum resulting from PCR-RFLP. According to the alignment of the mitochondrial CO1 sequences of the Hypoderma species and the PCR-RFLP results, all examined larva samples were classified as H. bovis.

Immunomodulatory effect of Hypoderma lineatum antigens: in vitro effect on bovine lymphocyte proliferation and cytokine production.

This study examines the immunomodulatory effect of a crude larval extract (CLE), obtained from first stage larvae (L1) of H. lineatum, and the purified fractions hypodermin A (HyA), HyB and HyC. Proliferative responses of peripheral blood mononuclear cells (PBMC) from uninfested and previously infested cattle and the production of the cytokines IL-10, IL-4 and IFN-gamma, in response to concanavalin A (Con A), were determined. The stimulation index of peripheral blood mononuclear cells from uninfested cattle was significantly lower than that from infested animals with the different antigens assayed. The HyA was the antigen that most inhibited the proliferative response, followed by the HyB, the HyC and the CLE. This hypodermin provoked an increase of IFN-gamma and a suppression of IL-10 production that would support a Th1-like cytokine response. The HyB reduced the production of IL-10 stimulated by the Con A in cultures from infested animals. The HyC did not modulate the production of cytokines. Finally, the CLE induced a marked suppression in the production of the different cytokines in cultures from naïve and previously sensitized animals. Our results indicate that Hypoderma larval secretions are comprised of different components (hypodermins) that individually induce distinct but partially overlapping modulatory responses.

Effect of treatment on the dynamics of circulating hypodermin C in cattle naturally infested with Hypoderma lineatum (Diptera: Oestridae).

An antigen capture ELISA, using a murine monoclonal antibody recognising recombinant hypodermin C (rHyC), was used to evaluate the influence of early treatment with eprinomectin (Eprinex) or fenthion (Spotton) on the kinetics of circulating hypodermin C in calves naturally infested with Hypoderma lineatum. No viable larvae were collected from treated animals, whereas a variable number of warbles were found in control animals. Treatment provoked a decrease in circulating HyC levels that was significant 9 days post-treatment (p.t.). Circulating antigen levels in the treated cattle remained detectable for approximately 99 days p.t. In contrast, control animals had no detectable antigen at 64 days p.t., 42 days earlier than in the treated animals. These results suggest that larvae were either gradually killed, resulting in slow release of antigen or they were encapsulated, leading to the slow liberation of antigen. Kinetics of circulating HyC did not differ among the two insecticide treatments. Antibodies persisted, in all groups, throughout the 120-day study. These results suggest that the antigen capture ELISA will be useful as a technique for detecting successful treatment of cattle grub infestations and for the detection of new infestations in previously infested cattle.

Stage specific mortality and humoral immune responses during pulse and trickle infestations of the common cattle grub, Hypoderma lineatum (Diptera: Oestridae).

Stage specific survival of larvae and pupae of the common cattle grub, Hypoderma lineatum de Villers, was compared in cattle artificially infested with larvae delivered by subcutaneous injection as a single infestation of 100 larvae, in trickle infestations of either two doses of 50 larvae, separated by 15 days, or in four doses of 25 larvae, separated by 9, 6, and 6 days, respectively. The duration of the migratory and 'warble' phases were also compared between treatments. The kinetics of the antibody response was monitored throughout the larval development by ELISA. No difference was noted in the level of antibodies among the three treatments. Mortality of first-instars differed among the infestation groups with mortality in the single pulse infestation group

Dynamics of the antibody response in cattle infested by Hypoderma (Diptera: Oestridae) after a treatment with injectable and pour-on ivermectin.

The dynamics of the anti-Hypoderma antibody response after a treatment with injectable and pour-on ivermectin against first instars of Hypoderma sp. were determined in naturally infested cows. Cattle were randomly assigned to three groups: group 1, which served as an untreated control; group 2, treated with pour-on ivermectin at a dose of 500 microg/kg of body weight, and group 3, which received 200 microg/kg of body weight of ivermectin by subcutaneous injection. Both ivermectins showed total efficacy against L-1 of Hypoderma sp. In untreated control animals, grubs became detectable in the 2 mo after treatment. Indirect enzyme-linked immunosorbent assay (ELISA) showed a significant increase in group 3 antibody levels 1 mo after treatment, whereas in group 2 the increase was not significant. Following the peak of anitibody activity in group 3, antibody levels declined at a faster rate than in those treated topically. In both treated groups, cattle remained serologically positive for 4 mo after ivermectin treatment. Thus, serological surveillance programs for cattle grubs conducted during this posttreatment period will not be influenced by treatment with ivermectin. The persistence of antibody levels after larval destruction makes the indirect ELISA unsuitable for the prediction of the efficacy of treatment.

Detection of antibodies to Hypoderma lineatum in cattle by Western blotting with recombinant hypodermin C antigen.

The cDNA encoding the entire mature hypodermin C (HC) of Hypoderma lineatum was cloned and expressed in Escherichia coli as a glutathione S-transferase fusion protein using pGEX vector. The recombinant HC protein (rHC) was tested by Western blotting to detect antibodies to H. lineatum in cattle. Western blotting with rHC as antigen clearly differentiated between H. lineatum-infested cattle sera and normal cattle sera. Forty-six out of forty-eight serum samples from cattle in Central Mongolia were positive, whereas all 30 serum samples from cows in Hokkaido, Japan, were negative by Western blotting. The result of Western blotting was identical to that of a previously developed enzyme-linked immunosorbent assay. These data demonstrated that Western blotting, with rHC expressed in E. coli, might be a useful method for the diagnosis of cattle hypodermosis.

[Development of the ELISA kit for the detection of Hypoderma bovis antibodies in cattle. I. Development of components]. / Opracowanie zestawu diagnostycznego do wykrywania przeciwcial anty-Hypoderma bovis u bydla testem ELISA. I. Uzyskanie komponentów.

The aim of these investigations was to prepare the antigen and control sera for the ELISA. Positive control sera were obtained from 163 cows showing clinical symptoms of H. bovis infection and negative control sera from 60 cows free from the infection. The antigen for the ELISA was obtained from H. bovis L1 larvae. Three basic fraction were isolated from whole excreted/secreted antigen by elution from gel and by preparative isoelectrofocusing. Using the Western-Blotting method it was demonstrated that Hy A (22-24 kDa and pI 4-5) had the best antigenic property. As can be seen from these investigations the developed ELISA kit for the detection of H. bovis antibodies in cows is highly specific and sensitive.

Comparative study of anti-Hypoderma antibody kinetics in sera, single and bulk milk samples of naturally infested cattle.

The investigation was carried out in Basilicata region (Southern Italy) from October 1997 to June 1998. Fifteen dairy cows bred in semiconfined conditions on a farm with a history of hypodermosis were sampled once a month for sera and milk; bulk milk from these animals was also collected monthly from the farm's tanker. Samples were tested for anti-Hypoderma spp. antibodies (Abs) with an ELISA technique and clinical parasitological examination was carried out monthly from January to July on all the animals in order to detect grubs. Blood and single and bulk milk samples yielded similar antibody kinetics and patterns in accordance with results obtained in previous immunological surveys in Italy. All animals were warbled in the spring time. November-January was confirmed to be the most suitable period for seroepidemiological survey for weather conditions in Southern Italy. The ELISA test proved once again to be very useful because it is simple to perform and cost effective. Either blood or milk samples may be used for epidemiological surveys; bulk milk may be very useful for the preliminary detection of hypodermosis on farms or in areas where there is no data available on the diffusion of the disease.

Serodiagnosis of goat warble fly infestation by Przhevalskiana silenus with a commercial ELISA kit.

Cross-reactivity between Hypoderma lineatum antigen and anti-Przhevalskiana silenus antibodies has been demonstrated by an ELISA technique. To evaluate the applicability of a commercial ELISA kit for the immunodiagnosis of goat warble fly infestation, different dilutions of serum and conjugate were tested, the development of antibody to P silenus in naturally infested goats was studied, and the results were compared with an ELISA technique using an antigen extracted from the first instar larvae of H lineatum. The best results were obtained with a serum dilution of 1:50; with both techniques the highest antibody concentration were recorded in October, November and December. In view of the confirmed cross-reactivity between H lineatum antigen and anti-P silenus antibodies, and the simplicity and rapidity of the assay, the commercial ELISA kit can be considered as a useful tool for the diagnosis of goat warble fly infestation.

Hypoderma lineatum antigen and anti-Przhevalskiana silenus antibodies: cross-reactivity and antibody kinetics in naturally infested goats.

To evaluate the cross-reactivity between Hypoderma lineatum antigen and anti-Przhevalskiana silenus antibodies six protocols with different concentrations of antigen and different dilutions of sera and conjugate were applied. The highest cross-reaction between the H. lineatum antigen and the anti-P. silenus antibodies is given by 2 micrograms/ml of antigen concentration, 1:400 of serum and 1:10,000 conjugate dilution. The study on the kinetic development of antibodies in goats naturally infested by P. silenus and the natural course of infestation pointed out the existence of a good correlation between individual antibody kinetics and the natural evolution of the cycle of infestation. The highest antibody concentration may be registered from October through November, coinciding with the end of the migration fo the larvae inside the animal's body. In our condition, this period can be considered as a favorable sampling period for immunodiagnosis and immunoepidemiological studies of goat warble fly infestation.

Serological prevalence of Hypoderma species in cattle in Great Britain (1995/96) and the relative value of serological surveillance over clinical observation.

Sera from 100,400 cattle on 2850 farms in England, Scotland and Wales were tested for the presence of antibodies to Hypoderma species between the end of November 1995 and the end of February 1996. Twelve animals were resampled because the initial results were equivocal but only one of them was confirmed as seropositive. This animal was a bull imported from Belgium which had been treated with an approved warble fly treatment within 24 hours of arrival at its destination in Great Britain. No seropositive animals were confirmed within the native British cattle population. Statistical analysis of these data indicates that the probable maximum number of infested cattle herds in Great Britain is 112. To detect the disease by direct clinical observation would require more than 500 herds to be infested, indicating that the serological testing of this number of cattle and farms is more than four times as sensitive as clinical observation for the detection of hypodermosis.

A competitive ELISA for the serodiagnosis of hypodermosis.

Hypodermosis is a parasitic disease of cattle caused by Hypoderma lineatum and H. bovis. It is an important health and welfare problem of infested cattle and a cause of considerable economic loss. In live animals, detection can be either by direct clinical examination of infested cattle and palpation of second and third stage larvae in the back or by the use of serological methods. This paper describes a competitive ELISA for the detection of antibodies to Hypoderma species in cattle sera. It has been validated using a small panel of 40 samples from clinically positive cattle and 200 samples from clinically negative (unexposed) cattle from a warble free area. Sensitivity and specificity calculated from this panel were 100% and 92% respectively at 2 SD from the mean or 92.5% and 98.5% at 3 SD from the mean. The use of serology and direct clinical examination of affected cattle, are discussed as methods for monitoring hypodermosis. The competitive ELISA should prove a useful adjunct to the standard sandwich ELISA, particularly in the resolution of samples which display non-specific binding properties.

Influence of parasiticide treatment on kinetics of antigen specific antibody response in cattle infested with Hypoderma lineatum (Diptera:Oestridae).

The effect of parasiticide treatment on dynamics of antigen specific antibody responses to Hypoderma lineatum (De Villers) was investigated in naturally infested calves. Parasiticides were applied to cattle 1 month prior to, 1 week prior to, and coincident with the appearance of warbles in untreated calves from the same source herd. When cattle were treated approximately 1 month before warbles appeared in untreated control animals, antigen specific antibody levels increased for 25-34 days then declined to undetectable levels by approximately 75 days. Antigen specific antibody levels sharply declined in cattle treated 1 week prior to appearance of warbles in untreated controls. Treatment applied coincident with arrival of grubs on the back did not alter the antigen specific antibody profile, but reduced the relative magnitude of antigen specific antibodies present. Persistence of the antigen specific antibody following treatment will permit conduct of serological surveillance programmes.

Effect of early treatment with ivermectin and doramectin on the dynamics of antibody response in cattle naturally infested by Hypoderma lineatum and H. bovis.

The field efficacy of two avermectins (ivermectin and doramectin) and the subsequent development of the antibody response were assessed in cows naturally infested with first-instar larvae (L-1) of Hypoderma sp. Twenty-eight Frisian cows were randomly divided into three groups while the first-instar larvae were still in migration: Group 1 (G-C) untreated control; Group 2 (G-Iv), treated with ivermectin injectable (0.2 mg kg-1 body weight) and Group 3 (G-Dor), which received doramectin injectable (0.2 mg kg-1 body weight). Serum antibody response was studied by an indirect ELISA test using, as antigen, the hypodermin C obtained from L-1 of H. lineatum. In treated animals no grubs were present on the back at any time during the trial, whereas a variable number of nodules were found in untreated animals. Both avermectins showed total efficacy against L-1 of Hypoderma sp., and there were no local or general reactions. In ivermectin-treated cattle serum antibody levels declined from one month p.t., while in those treated with doramectin they started to fall two weeks later, but no differences were found among both groups. On the other hand, G-C antibody levels progressively increased and remained high until December, when the first grubs became detectable on the back; after that, they began to decline. Early treatments against Hypoderma sp. have an influence on the results of ELISA, so they must be considered to determine the most suitable time for blood sampling.

Effect of the parasite enzyme, hypodermin A, on bovine lymphocyte proliferation and interleukin-2 production via the prostaglandin pathway.

The immune function of cattle infected with a primary infestation of Hypoderma lineatum is impaired during the first instar migration of the larvae. Hypodermin A (HA) is an enzyme secreted by the larvae that is implicated in immunosuppression. The response of bovine peripheral blood mononuclear cells (PBMC) to HA was examined in this study. HA blocked their proliferation in response to phytohaemagglutinin (PHA) and its effect was enhanced when cells were preincubated with HA before activation. This suggests that HA affects the lymphocyte commitment to blastogenesis during the early stages of their activation. HA also markly reduced the production of interleukin-2 (IL-2) in PHA-stimulated bovine PBMC cultures. Furthermore, indomethacin, which inhibits prostaglandin (PG) synthesis, blocked the immunosuppressive effect of HA on the PBMC proliferative response. The concentration of PGE2 in medium of PBMC or PMA-stimulated monocyte cultures was increased by incubation with HA. Thus, the HA appeared to act by reducing IL-2 production via a prostaglandin-dependent pathway.

Efficacy of doramectin against warble fly larvae (Hypoderma bovis).

The efficacy of doramectin in the treatment of cattle harbouring naturally acquired infestations of first, second and third instar larvae of Hypoderma bovis was determined in two studies carried out in the Burgundy region of France. In the first study, cattle on six farms with a history of H. bovis infestations were treated during October 1989 with either doramectin at a dose of 200 micrograms kg-1 liveweight (186 animals) or with an equivalent volume of saline (157 animals). During the following March and April, all animals were examined for the presence of warbles. In the second study, cattle on four farms with warbles present in their backs were treated during March 1990, with either doramectin or saline (as before). The viability of larvae within each warble on all the animals was then assessed every 2 days for 14 days. In Study 1, no warbles were present in any of the doramectin-treated cattle at any time, whereas warbles were found in 135 saline-treated animals. In Study 2, all larvae in warbles on the backs of the doramectin-treated cattle were dead by Day 14 after treatment, whereas viable larvae were still present in warbles in the backs of all saline-treated cattle. No adverse reaction to doramectin treatment was observed in any animal at any time. It was concluded that doramectin is both safe and 100% efficacious in the treatment of first, second and third instar H. bovis infections of cattle.