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.

Seroprevalence of hypodermosis in cattle in some provinces of Turkey.

The aim of this study was to determine the seroprevalence of hypodermosis in cattle in the east and southeast of Turkey. For this purpose, a total of 634 sera samples of cattle were collected from Malatya, Elazig and Diyarbakir provinces of east and southeast of Turkey from November 2005 to February 2006. The sera were analyzed using a Hypodermin C antigen by means of indirect ELISA. One hundred and forty eight (23.3%) out of 634 cattle were seropositive for hypoderma antibodies. The highest percentage of seropositivity were detected at Elazig province (26.3%) followed by Malatya (22.3%) and Diyarbakir provinces (22.1%). The seropositivity rate was higher in female (31%) than male (14.1%). When the mean is considered by animal breed, the highest seropositivity was detected at local breed (27.7%) following crossbreed (26.8%) and purebreed (19.7%). There was a positive relation between age and seropositivity. Seropositivity rate was 15.9% in 2 and under ages while these rates were 38.1% and 30.4% in 3-4 ages and 5 and up ages, respectively.

Sero-epidemiological surveillance of hypodermosis in yaks and cattle in north China by ELISA.

A large-scale serological surveillance was conducted for hypodermosis in the north of China. A total of 4175 sera samples of yaks and cattle were collected from five provinces in north China that included Xinjiang, Inner Mongolia, Heilongjiang, Jiling and Gansu provinces, between 2001 and 2002, and were examined for anti-hypoderma antibody by ELISA. The results indicate that the naturally infested positive rates of Hypoderma spp. in the above provinces were 51.77%, 27.02%, 13.00%, 6.03% and 44.41%, respectively. These results suggest a significant warble fly population in the north of China. Positive sera were collected from infested cattle in Pingliang county of the Gansu province monthly between August 2001 and July 2002. These sera were used to evaluate the seasonal kinetics of anti-hypoderma antibody. The kinetics demonstrated that anti-hypoderma antibody was elevated from October to December. Thus, serological surveillances associated with low infestation rates and chemical therapy would best be performed from October to December in the region.

Cattle grub infestation by Hypoderma sp. in Albania and risks for European countries.

In order to estimate the presence and the seroprevalence of hypodermosis in Albania, 625 head of cattle were bled during two sampling seasons (i.e. from February to March 2003 and from November to December 2003). The cattle came from three collection sites in Northern (site A), Central (site B) and Southern (site C) Albania. Milk samples were collected monthly from four animals from October 2002 to May 2003 during the lactating period. The animals were also clinically examined for the presence of warbles by manual palpation from April to July 2003 and 2004 and third instar larvae were collected and morphologically identified. Serum and milk samples were processed by ELISA. One hundred and thirty-three (38.6%) out of 344 and 116 (41.3%) out of 281 animals were found to be seropositive for Hypoderma during the first and the second sampling season. In particular, the animals from site C presented the highest percentage of seropositive results (i.e. 72.8% and 97.8% in the first and in second year, respectively) followed by the animals from sites A (i.e. 35.8% and 23.8% in the first and in second year, respectively) and B (i.e. 17.8% and 3.4% in the first and in second year, respectively). The kinetics of anti-Hypoderma antibodies in milk samples showed the highest antibody titres from October to February 2003. All the seropositive animals in both the sampling periods showed the presence of one or more warbles under the skin during April and May 2003 and 2004 and the third-stage larvae collected were morphologically identified as Hypoderma bovis. The results of this survey indicate that hypodermosis is widespread in Albania and that early prophylactic treatments must be carried out accordingly. The hope is not only to reduce the parasitic intensity with obvious benefits for livestock production, but also to avoid the risk of spreading this parasitic disease to neighboring countries.

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.

[Development of the ELISA kit for the detection of Hypoderma bovis antibodies in cattle. II. Setting the best terms for collecting blood samples for the laboratory diagnosis of hypodermyiasis in different regions of Poland]. / Opracowanie zestawu diagnostycznego do wykrywania przeciwcial anty-Hypoderma bovis u bydla testem ELISA. II. Ustalenie optymalnych terminów dla pobierania próbek krwi do badan w kierunku hypodermozy w róznych rejonach Polski.

The experiments were carried out on 285 cows from 10 herds from different regions of Poland. Extensity of the H. bovis invasion in each herd varied from 10 to 86%. The highest extensity appeared in herds from eastern and north-eastern Poland and the lowest in herds from southern and central regions of the country. Seroconversion of Hypoderma bovis antibodies was calculated for animals in which warbles were detected by clinical examination. The highest serum densities were observed in herds in March, April and May. The results of these investigations demonstrated that the best term for collecting blood samples to examine cattle for hypodermosis is the winter-spring season.

[Development of the ELISA kit for the detection of Hypoderma bovis antibodies in cattle. III. Stability and usefulness of ELISA components preserved under different conditions]. / Opracowanie zestawu diagnostycznego do wykrywania przeciwcial anty-Hypoderma bovis u bydla testem ELISA. III. Badanie trwalosci i przydatnosci komponentów do testu ELISA przechowywanych w róznych warunkach.

The aim of investigations was to determine the stability of ELISA components preserved under different conditions. The following components for the ELISA were used: the secreted/excreted antigen prepared from H. bovis L1 larvae, its fraction containing hypodermine A, conjugat, positive and negative control sera. Freezing at -20 degrees C, lyophilization and preservation at 4 degrees C were applied. Seven kits for the ELISA containing components preserved using described methods, were prepared. The identical packets of sera were examined by the ELISA directly after preparation of the kits (day '0'), and next, at 3-4 week intervals for 9 months. The experiments showed that the best method of conservation of ELISA components for the detection of H. bovis antibodies was lyophilization. The kits prepared in this way did not lose their characteristics for 9 months and their specificity and sensitivity were close 100%.

Changes in the haemolytic activity of bovine serum complement by Hypoderma lineatum (insect oestridae) larval proteinases in naive and immune cattle.

Three serine proteinases (hypodermin A, B and C) of the first instar larvae of Hypoderma lineatum have been assayed for their ability to deplete seric complement of naive or immune cattle. In naive cattle complement consumption is initiated by hypodermin B through the sequence C1-C3 at a concentration of 5 micrograms/ml of serum, and by hypodermin A through the sequence of C3-C9 at a higher concentration of 150 micrograms. The third enzyme presenting a collagenolytic activity has no anti-complementary activity even on C1q. In immune cattle a 70% complement depletion through the classical pathway is observed with 15 micrograms of hypodermin B per ml of serum. The two enzymes A and C appear to play a minor role in the complement depletion via the classical pathway. The biological role of each of these enzymes in the parasite-host interrelationships is discussed. The participation of these enzymes in the immediate hypersensitive reactions following systemic treatment of cattle infested by this endoparasite is considered.