Serological diagnosis of human hypodermosis: A preliminary report

In France, farm workers are occasionally infested with the larvae of Hypoderma lineatum and H. bovis, which are primirily parasites of cattle. A specific serological test for diagnosing human cases is described here.The immunological responses in cattle are directed mainly against the proteolytic secretion of the first instar larvae. This secretion was used as an antigen for the diagnostic immuno-electrophoretic test in man. The antigen was highly specific and produced negative results to human sera from 108 patients with other forms of parasitoses and 23 non- parasitized controls.We report here on 13 cases of Hypoderma infestation in man. Ten of these were positive and three negative to the test. In the negative cases, the serum was collected 1–2 months after the emergence of the larvae. The immunological response disappeared rapidly after larval emergence; in one case within 9 days.     

Immunization and Parenteral Chemotherapy for the Control of Cattle Grubs Hypoderma Lineatum

Immunization with two types of warble larvae antigens A and B, the latter treated with tannic acid, and injections of dimethoate, an organic phosphate, were tried for the control of prehypodermic larvae of Hypoderma lineatum De Vill., and H. bovis L., in range calves.

In test groups of 20 calves, given intramuscularly, antigen A had no effect, but combined treatment with antigens A and B reduced the number of H. bovis L., larvae by 81 per cent (P<.001), and proved as effective as dimethoate subcutaneously or intramuscularly. H. lineatum De Vill., did not respond to any treatment.

Antigen B and dimethoate were free from harmful effects on the host, but antigen A caused anaphylaxis and irritation at the site of injection.

     

Colostral transfer of antibodies specific for Hypoderma lineatum proteins

Antibodies to Hypoderma lineatum were transferred to calves via colostrum. The antibodies transferred in the colostrum demonstrated specificity to all the H. lineatum first-instar proteins which were resolved by non-denaturing polyacrylamide gel electrophoresis and were capable of mediating a Type I hypersensitivity reaction. The kinetic decline of colostrum-acquired antibodies, the effect upon development of an autologous humoral response to H. lineatum and the host or parasite protective role of these antibodies are discussed.     

Hypodermosis in cattle slaughtered in Nigde province,Turkey<Superscript>*</Superscript>

This study was carried out to investigate the prevalence of hypodermosis in cattle between January and June 2005 in Nigde province, which is located in the middle of Turkey. A total of 1336 cattle, which were slaughtered in Nigde, were investigated for Hypoderma larvae. The 68 out of 1336 cattle (%5.08) were found positive for Hypoderma larvae. A total of 536 Hypoderma larvae were found in the skin and subcutaneous tissue of the back of infested cattle. The 489 out of 536 larvae (%91.23) were identified as Hypoderma bovis and 47 out of 536 (%8.77) as H. lineatum. Number of Hypoderma larvae counted on single infested cattle varied between 1–45 and the mean number of Hypoderma larvae per cattle was 7.88 (536/68). Hypodermosis was recorded for the first time in cattle from Nigde. ^*This study was supported by the Scientific Research Projects Unit of Nigde University (FEB 2004/07).     

皮蝇幼虫在牦牛体内移行动态与伊维菌素最佳驱除时间

2004年经剖检60头幼年牦牛,对牛皮蝇幼虫在牦牛体内移行动态进行了观察。8月份,第一期幼虫在牦牛体内出现,10月中旬牦牛体内第一期幼虫呈高峰。从11月中旬到第二年2月中旬第二期幼虫出现,元月中旬二期幼虫出现高峰,三期幼虫从元月中旬到6月初出现,3月中旬出现高峰。用伊维菌素给45头幼年牦牛投药:9月中旬,10月中旬和11月中旬各15头。9月中旬和11月中旬的投药群,在12月中旬均出现瘤疱,而10月中旬的投药群未发现瘤疱,表明10月中旬对第一期幼虫为最佳驱除时间。     

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.     

The efficacy of eprinomectin extended-release injection against Hypoderma spp. (Diptera: Oestridae) in cattle

The efficacy of eprinomectin in an extended-release injection (ERI) formulation was determined in cattle harboring naturally acquired infestations of first- or second- and third-stage larvae of Hypoderma spp. in three studies conducted according to the same protocol in the USA (two studies) and Germany (one study). Thirty cattle sourced from herds with a history of Hypoderma infestation were included in each study. Cattle were formed into replicates of three animals each on the basis of pre-treatment anti-Hypoderma antibody titers. Within replicates each animal was randomly allocated to one of the following treatments: ERI vehicle (control) at 1 mL/50 kg bodyweight, administered once on Day 0; Eprinomectin 5% ERI at 1 mL/50 kg bodyweight (1.0 mg eprinomectin/kg), administered once on Day 0 (when larvae were expected to be first instars); or Eprinomectin 5% ERI at 1 mL/50 kg bodyweight (1.0 mg eprinomectin/kg), administered once when larvae were second or third instars (study dependent, Day 73, 119, or 140). Treatments were administered by subcutaneous injection in front of the shoulder. In all studies, emerging and/or expressed Hypoderma larvae were recovered, speciated, and counted and viability was determined. Eprinomectin LAI treatment was 100% (p < 0.05) efficacious against first- and second- or third-stage larvae of Hypoderma bovis (two studies) and Hypoderma lineatum (one study). All animals accepted the treatment well. No adverse reaction to treatments was observed in any animal in any study.     

Optimization of in-house ELISA based on recombinant major sperm protein (rMSP) of Dictyocaulus viviparus for the detection of lungworm infection in cattle

The aim of this study was to optimize an in-house ELISA based on a recombinant version of the major sperm protein (MSP) of Dictyocaulus viviparus for routine diagnosis of lungworm infection in cattle. A recombinant MSP (rMSP) was cloned into pGEX-6P-1 vector and expressed as a glutathione-S-transferase (GST) fusion protein in Escherichia coli BL21 (DE3) chemically competent cells. The product was then employed as capture antigen in an ELISA, and validated against 304 samples of known status (216 negative and 88 positive) in which the antibody levels in sera had also been measured earlier with a commercial ELISA kit (Ceditest® lungworm ELISA). The receiver operating characteristic (ROC) curve analysis of the ELISA results estimated the optimized diagnostic sensitivity and specificity as 97.7% (95% confidence interval [CI]: 91.9–99.7%) and 98.1% (CI: 95.3–99.5%), respectively. The results from the in-house rMSP-based ELISA were compared with results obtained on both fecal examination and the Ceditest® lungworm ELISA. Rising antibody levels in sera of experimentally infected calves were observed between 21 and 28 days post infection, when patency was also confirmed by the presence of larvae in feces. Notably, using the in-house rMSP-based ELISA infection was confirmed in calves shedding larvae approximately 3–4 weeks post inoculation, while using the Ceditest® lungworm ELISA those animals remained negative. Additionally, 251 sera samples from calves naturally exposed to the parasites on pasture were used to evaluate the test. In in-house rMSP-based ELISA no cross-reactions were observed with sera from calves infected with the gastrointestinal nematodes (Ostertagia ostertagi and Cooperia oncophora), even though the presence of eggs in the feces was confirmed. Overall, the in-house rMSP-based ELISA optimized in this study showed excellent diagnostic performance for detection of lungworm infection in cattle.     

Toxicity of Alpholate to Cattle

Although Apholate is used as a sexual sterilant of both sexes of houseflies (Musca domestica L.) it can not be used for `systemic' chemosterilization of the prehypodermic larvae of the warble flies Hypoderma bovis (L.) and H. lineatum (De Vill.) because of its high toxicity to 4- to 5-month-old calves.

An intramuscular dose of 2.5 mg./Kg. killed the calves in 5 to 7 days. The pathognomonic clinical signs were impaction of the rumen, anorexia, depression, and general weakness. The hematopoietic system was affected. There was marked leukocytopenia characterized by lymphocytopenia within 24 hours of Apholate injections.

     

Differentiation of resistance in cattle to larval Hypoderma lineatum

Cattle were successively infested for 4 years with larval cattle grubs, Hypoderma lineatum, to demonstrate the acquisition of resistance. The effects of host age and dietary-induced state with Vitamin A on susceptibility to infestation and their interactions with effects from previous infestations were also determined.Cattle acquired resistance to infestation after a single infestation. This resistance was not affected by host age for at least 4 years, was not affected by a deficiency of vitamin A, and was effective in the skin and connective tissues of the host. The effects of this acquired resistance were highly variable in degree, were produced primarily during the first 90 days of infestation and increased after repeated provocation to provide nearly complete protection in some animals.Calves and yearlings had innate resistance that decreased with age, was localized in the skin, was impaired by a deficiency of Vitamin A, and resulted in a mean loss of 65–75% of the larvae in an infestation dose of up to 150.The combined effects of the 2 types of resistance reduced the grub-burden in cattle throughout their life.     

The feasibility of exterminating warble flies (Hypoderma spp.) on a regional basis

A program to exterminate warble flies (Hypoderma lineatum and H. bovis) was begun in Wetaskiwin County, Alberta, Canada, in 1968 and has since been adopted by nearly all the local authorities (countries, municipalities, etc.) in Alberta, which has an area of about 650 000 km². Except for lactating cows, which were treated with rotenone, all the cattle in the program were treated annually with systemic insecticides.The program has been under continuous evaluation in Wetaskiwin County where the mean numbers of warble grubs in untreated cattle were 9.0, 6.9, 3.4, 0.2, 2.5 and 1.4 in 1969–1974, respectively. In 1973 and 1974, the infestation was found mainly in one of the seven divisions of the county. That division did not receive adequate surveillance.The extermination of warble flies would be feasible in other areas if the procedures adopted in the Alberta program were carried out for a minimum period of 3 years and were followed by spring treatment of infested herds for several years. The recommended spring treatment is usually required for the control of cattle lice anyway.     

Persistent activity of topical ivermectin against artificial infestations with Hypoderma lineatum (Diptera: Oestridae)

In controlled experiments utilizing artificial infestations, a topical formulation of ivermectin (IVOMEC Pour-On for Cattle) was 100% effective against migrating first instar Hypoderma lineatum for 3 weeks following treatment. Larvae were apparently killed early in the infestation as no significant level of specific antibodies was found in the treated calves. At 4 weeks post-treatment the efficacy remained high at 96%; mortality of larvae in the one calf in which warbles were observed and from which mature third instars were collected, was higher than that seen in the untreated calves, indicating some level of treatment induced effect. No specific antibodies were detected in calves that did not develop palpable warbles. Antibody kinetics in those calves from which viable larvae emerged were typical. The length of activity of this product against early stages of the cattle grub makes it practical to apply treatment up to 3 weeks before the end of fly activity.     

Isolation of Theileria mutans from Kenyan buffalo,and transmission by Amblyomma gemma

Blood from 2 buffalo harbouring Theileria organisms was inoculated into a splenectomized Ayrshire calf. The calf developed an infection which extended over a long period. The infection was transmitted to two cattle with Amblyomma gemma by transstadial transmission between the larvae and nymphs. Severe anaemia developed in these cattle and correlated with the parasitaemia. Schizonts morphologically characteristic of T. mutans were detected for short periods in the lymphoid cells of cattle infected by the ticks. The antigens and sera prepared from the cattle reacted with T. mutans sera and antigens in the indirect fluorescent antibody test. After recovery from the primary parasitaemia, the cattle had detectable organisms and antibodies to T. mutans for more than two years.     

Cross-transmission studies with Hypoderma lineatum de Vill. (Diptera: Oestridae): attempted infestation of goats (Capra hircus)

The potential for cross-transmission of Hypoderma lineatum from cattle to domestic goats (Capra hircus) was examined using artificial infestation techniques. Two routes of infestation, subcutaneous injection and dermal penetration, were used to expose goats to newly hatched first instars. Presence of antibodies and appearance of circulating antigen (hypodermin C) were evaluated at selected intervals for up to 40 weeks post-infestation. In addition, immunoblots against H. lineatum first-instar proteins were conducted using sera taken at 10 weeks post-infestation. Goats were palpated for the presence of developing larvae at sub-dermal sites beginning at week 30 pi. No developing larvae were palpated at any time, regardless of the route of infestation nor was circulating antigen detected in any infested goats. Antibodies were present at weeks 6 and 10 and week 27 pi in both infested groups. Immunoblots indicated all infested goats produced antibodies to first instar H. lineatum antigens. H. lineatum appears to be incapable of completing development in domestic goats although the transient appearance of ELISA detectable antibodies and the presence of bands on immunoblots suggests that at least some larvae survive long-enough to engender a humoural response. The host specificity of H. lineatum is discussed in light of the general concepts of host-parasite relationships of oestrids.     

Evaluation of an antigen capture ELISA for the early diagnosis of Hypoderma lineatum in cattle under field conditions

An antigen capture or sandwich ELISA (sELISA) was evaluated for the diagnosis of Hypoderma lineatum in cattle under field conditions in northwestern Spain. The kinetics of circulating hypodermin C (HyC) and specific antibodies during the course of natural infestation were determined in a group of 10 Frisian calves. In addition, oesophagi and blood samples were taken from 105 cows at a slaughterhouse in order to compare three methods for the diagnosis of H. lineatum: sandwich ELISA for the detection of the antigen HyC (sELISA), indirect ELISA for the detection of antibodies anti-HyC (iELISA) and the detection of first instars (L1) in the oesophagus. In naturally infested cattle, HyC was present in circulation at low levels during the early and late phases of the infestation. However, in the middle phase, coinciding with the presence of L1 in the oesophagus, two peaks of increased HyC concentration were observed. Specific antibodies increased progressively until the first appearance of larvae in warbles on the back. There was no correlation between antigen or antibody levels and the number of grubs in the back. Prevalence of first instars in the oesophagi of slaughtered cows was 21.9% (23/105). The percentage of cattle that were positive for circulating antigen was slightly higher (24.8%), suggesting the recent destruction of migrating larvae in some animals. However, there was no correlation between the number of L1 and HyC levels. With the iELISA, 79% of the animals were positive to Hypoderma, which means that a high percentage of those animals have been exposed to the parasite but they had no apparent current infestation. The sELISA is a good tool to follow larval development within the host; however, the episodic elevation of HyC levels limits the usefulness of this test for the early diagnosis of Hypoderma under field conditions.     

Prevalence and economic significance of bovine hypodermosis in Afyonkarahisar province of Turkey

This study was carried out to investigate the prevalence and economic losses due to in cattle of hypodermosis between May 2008 and April 2009 in Afyonkarahisar province of Turkey. A total of 6,598 cattle—3,498 in pasture and 3,100 in slaughterhouse—were examined for hypodermosis. One hundred ninety-five out of 6,598 cattle (2.96%) were found positive. Concerning this infestation rate, the prevalence of hypodermosis in field was 5.3%, whereas the prevalence was 0.3% in slaughter house. Hypoderma bovis and Hypoderma lineatum were recorded in slaughter house. First-stage Hypoderma spp. larvae weren’t found in cattle organs in slaughterhouse. The annual economic loss on skins is estimated to be about 18,288 TL (currency exchange rate is 1 USD:1.499 TL).     

Use of enoyl coenzyme A hydratase of Mycobacterium avium subsp. paratuberculosis for the serological diagnosis of Johne's disease

Johne's disease (JD), caused by Mycobacterium avium subspecies paratuberculosis (MAP), remains difficult to control because of the lack of specific and sensitive diagnostic tests. In order to improve the specificity of sero-diagnosis for JD, the phage display library derived from genomic DNA of MAP was immunoscreened to identify novel antigenic targets. We selected a clone using antibodies from MAP experimentally infected cattle, and annotated its coding sequence as MAP1197 in the MAP genome, which encoded “echA12_2” in the MAP protein (Map-echA) belonging to Enoyl-CoA hydratase, known as a crotonase enzyme. The Map-echA was expressed in Esherichia coli and purified as a histidine-tag recombinant protein (rMap-echA), and the diagnostic potential of the protein was further evaluated by enzyme-linked immunosorbent assays (ELISA). Antibody responses to rMap-echA were higher in MAP-infected cattle than in uninfected cattle. The specificity of the Map-echA ELISA was also confirmed by evaluation with hyper-immune sera against various kinds of Mycobacterium species. Furthermore, in all experimentally infected cattle the antibody against rMap-echA was detected 2–7 months earlier than by a commercially available ELISA kit. These results suggested that Map-echA can be used as a specific and sensitive serological diagnostic antigen for the detection of MAP infection.     

Serodiagnosis of Metastrongylus apri infection of pigs by immunofluorescence

The lyophilised first stage larvae of Metastrongylus apri were used as antigen in the indirect fluorescent antibody test for the diagnosis of the infection of pigs. The larvae were recovered from the embryonated eggs discharged by the gravid females of the nematode in vitro. Ten pigs were used in the test. Serial serum samples were taken of seven pigs, experimentally infected with varying doses of M. apri larvae, all of which were found to discharge eggs of the nematode in their faeces after infection was established. The other three pigs were uninfected and used as controls.Positive cuticular fluorescence of the larvae was first detected with the serum samples obtained between 14 and 33 days post-infection. This fluorescence persisted with subsequent serum samples up to 85 days post-infection. Pronounced and uniform cuticular fluorescence was generally observed with the serum dilutions of up to $\text{1}\text{10}$. Earlier post-infection serum samples either exhibited no cuticular fluorescence or gave non-brilliant fluorescence. The pre-infection serum of these pigs and also serum samples obtained from the three uninfected control pigs did not give cuticular fluorescence even at the initial serum dilution of $\text{1}\text{5}$.     

Using indirect ELISA to assess different antigens for the serodiagnosis of Fasciola gigantica infection in cattle, sheep and donkeys

An indirect enzyme-linked immunosorbent assay (iELISA) was evaluated for its diagnostic capability in detecting antibodies against Fasciola gigantica infection in cattle, sheep and donkeys sera using crude worm, excretory–secretory and glutathione S-transferase antigens prepared from adult liver fluke. Presence of F. gigantica worms at post-mortem examination of cattle, sheep and donkey’s livers was taken as a gold standard for the evaluation of the assay. The diagnostic sensitivity, specificity and accuracy percentages of iELISA were determined for each antigen. Excretory–secretory antigen gave the best results for the serodiagnosis of F. gigantica infection in cattle, sheep and donkeys using iELISA with diagnostic sensitivity percentages of 93.3%, 94.9% and 93.3%, respectively, while the specificity percentages were 96.7%, 97.2% and 96.3%, respectively, whereas the accuracy percentages were 95%, 96% and 95.7%, respectively. The diagnostic sensitivity percentages of iELISA using crude worm antigen were 96.7%, 100% and 93.3%, respectively, while the specificity percentages were 80%, 83.3% and 85.2%, respectively, whereas the accuracy percentages were 88.3%, 86.7% and 87%, respectively. The diagnostic sensitivity percentages of iELISA using glutathione S-transferase antigen were 66.7%, 71.8% and 60%, respectively, while the specificity percentages were 70%, 77.8% and 77.8%, respectively, whereas the accuracy percentages were 68.3%, 74.7% and 73.9%, respectively. Conclusively, excretory–secretory antigen dependent iELISA can be used as a reliable serodiagnostic test for F. gigantica infection in cattle, sheep and donkeys.     

Feline gingivitis

AIM: To discover whether cross infection between red deer (Cervus elaphus) and cattle is possible with either a bovine isolate of the cattle lungworm, Dictyocaulus viviparus, or with a cervine isolate of the lungworm, Dictyocaulus eckerti.

METHOD: Twelve cattle and 12 red deer were reared parasitefree from birth. At 3–4 months of age, half of each group (n=6) were experimentally infected with D. viviparus and the other half with D. eckerti. The course of infection was monitored for 34 days, after which the animals were slaughtered and the lungs removed to assess levels of infection.

RESULTS: Faecal larval counts demonstrated that patent Dictyocaulus infections occurred in all groups. At necropsy, adult worms were found in the lungs in all groups except the cattle that were infected with D. eckerti. The largest numbers of adult worms were found in the lungs of the red deer infected with D. eckerti.

CONCLUSION: It was demonstrated that both cattle and red deer could be infected with either D. viviparus or D. eckerti. However, D. eckerti larvae that originated from deer established more successfully in deer and D. viviparus larvae that originated from cattle established more successfully in cattle.