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Application of hybridoma technology to the diagnosis of parasitic disease.

Recent applications of hybridoma-derived monoclonal antibodies to the diagnosis of parasitic disease are reviewed. Diagnostic tests, utilizing monoclonal antibodies, have included radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) techniques to detect parasite antigen in host tissues and body fluids and circulating host antiparasite antibody. In general, the use of monoclonal-derived reagents has greatly increased the specificity of diagnosis by eliminating cross-reactions between closely related parasite species, without suffering a significant loss of sensitivity.

Animals↗

Amitraz in the control of non-ixodide ectoparasites of livestock.

Amitraz has been shown to be successful in controlling mange and lice infestations on pigs which had failed to respond to diazinon and gamma-BHC, respectively, and by the use of a prophylactic programme to maintain pig herds mange free. In addition, trials have indicated the elimination of psoroptic mange from sheep by applying the compound to the animal using either conventional or less conventional methods of treatment. Mixed infections of Chorioptes spp., Psoroptes spp. and Sarcoptes spp., in cattle have also been controlled using spray applications of amitraz, where in some cases organochlorine, organophosphorus and organotin compounds had failed. In a pilot study a heavy infestation of chorioptic mange was controlled on a calf using a pour-on formulation of amitraz.

Animals↗

Protective immunity to Ascaris suum: analysis of swine peripheral blood cell subsets using monoclonal antibodies and flow cytometry.

Outbred domestic swine or SLA inbred miniature swine were exposed to Ascaris suum either naturally on contaminated lots or by inoculation with UV-irradiated attenuated eggs. Both inbred and outbred swine developed virtually complete protection to a challenge of 10 000 eggs after natural exposure, but inbred swine were less resistant than outbred swine after UV-egg exposure. Flow cytometric analysis of peripheral blood mononuclear cells from these animals, performed to determine changes in cell subsets including helper T-cells, cytotoxic/suppressor T-cells, macrophages, and cells expressing class II major histocompatibility antigens, showed that both outbred and inbred swine had similar responses after parasite exposure. The levels of helper T-cells and cytotoxic/suppressor T-cells did not change after parasite exposure, while there was an appreciable but transient increase in macrophages only in those swine naturally exposed to A. suum. Swine exposed to A. suum, both naturally and by inoculation with UV-eggs, showed an increase in the amount of class II antigens detectable per cell. In a second set of experiments, outbred swine were exposed to A. suum naturally or by repeated experimental inoculation with different doses of normal eggs, and protective immunity and changes in blood cell subsets were determined. The greatest change in blood cell subsets was found at 3 and 5 weeks after initial parasite exposure, when macrophages were elevated moderately in a group of pigs inoculated every other day with 1000 eggs and markedly in a group that was naturally exposed; class II antigen expression was also increased during this period. These increases preceded peak serum antibody responses, which were lower in the naturally-exposed group relative to the experimentally-inoculated group. Both groups had high levels of protective immunity. This suggests than natural exposure to A. suum may activate cells and enhance specific immune responses to give high levels of protection.

Animals↗

Immunogenetic analysis of Trichinella spiralis infections in swine.

The immune responses of outbred swine, inoculated with several different low doses of Trichinella spiralis muscle larvae (ML), was followed over 5-6 weeks of primary infection, in order to determine an inoculation dose which could be used to identify genetic controls on the response to this helminth parasite. Reproducible infections were established when swine were inoculated with 100-300 ML. Humoral antibody responses to different larval stages were evident at 4 weeks using enzyme-linked immunosorbent assay (ELISA) of antibody-binding to excretory-secretory (ES) products of ML, and flow cytometric (FCM) analysis of antibody-binding to newborn larvae. T-cell blastogenesis to T. spiralis ML antigens was predominantly in the CD4+, class II restricted, T-cell subset. Having established an appropriate inoculation dose, swine leukocyte antigen (SLA) inbred miniature swine were then inoculated with this low dose of T. spiralis ML, to determine whether major histocompatibility complex (MHC) genes regulate swine immune responses to T. spiralis, as has been found in rodent models. Preliminary evidence indicated that swine of the SLA c/c haplotype may exhibit a lower burden of T. spiralis larvae in the tongue and diaphragm. This lower muscle burden correlated with the earlier development of a humoral antibody response in these genetically-defined swine.

Animals↗

Prevalence of Cryptosporidium oocysts in livestock in Trinidad and Tobago.

Faecal samples from 683 diarrhoeic and non-diarrhoeic piglets, calves, lambs and goat kids were examined for Cryptosporidium oocysts. The prevalence of infection in piglets and lambs was 19.6% and 20%, respectively, and these rates were significantly higher than those detected in calves (8.7%). Amongst the four animal species studied, the detection rates were higher in diarrhoeic than in non-diarrhoeic animals and in animals under extensive and semi-intensive husbandry systems. However, these differences were not statistically significant.

Animals↗

Detection of Cryptosporidium oocysts in extra-intestinal tissues of sheep and pigs.

Extra-intestinal infections by Cryptosporidium parvum have been detected in pigs and sheep. Detection was carried out by imprints of the mucosa of different organs and viscera in 55 sheep and 57 pigs slaughtered at three abattoirs in Zaragoza (northeast Spain). Imprints were stained by using a modified Ziehl-Neelsen technique. In addition to intestinal infections, cryptosporidial oocysts were found in the gall-bladders of two pigs which were 2 months old, and in some organs of sheep aged 5 days or more, including the gall-bladder (5), mesenteric lymph nodes (2), trachea (7), lung (3) and the uterus of one lamb.

Animals↗

Strategies to reduce transmission of Toxoplasma gondii to animals and humans.

Toxoplasma gondii is found in the tissues of food animals and is an important cause of abortion and mortality in sheep and goats throughout the world. It causes mental retardation and loss of vision in congenitally infected children and death in immunosuppressed patients. A live vaccine, using a nonpersistent strain of T. gondii, is available in New Zealand, the UK and Europe which prevents T. gondii abortion in sheep. A live vaccine using a mutant strain of T. gondii (T-263) is being developed in the USA to reduce oocyst shedding by cats. As yet, there are no drugs to kill T. gondii tissue cysts in human or animal tissues. Freezing to -12 degrees C, cooking to an internal temperature of 67 degrees C, or gamma irradiation (0.5 kGy) can kill tissue cysts in meat.

Abortion, Veterinary↗

Delayed-type skin hypersensitivity and in vitro lymphocyte immunostimulation responses of swine following inoculation with Mycobacterium avium cell walls and a mycobacterial immunopotentiating glycolipid.

Miniature swine (n = 5 per group) were inoculated intradermally with mineral oil-in-water emulsions containing either 150 micrograms of mycobacterial immunopotentiating glycolipid P3 (EP3), 150 micrograms of lyophilized Mycobacterium avium (serotype 8) cell walls (E-MaCW), or 150 micrograms P3 and 150 micrograms M. avium cell walls (EP3-MaCW). Swine vaccinated with E-MaCW and EP3-MaCW developed antigen-sensitive lymphocytes detectable with delayed-type hypersensitivity (DTH) skin tests and lymphocyte transformation assays. Swine injected with EP3 were not sensitized. In general EP3-MaCW evoked a more pronounced in vivo DTH tuberculin skin test and in vitro lymphocyte transformation responses than E-MaCW. Time-course studies indicated a more persistent response in swine injected with EP3-MaCW than in those given E-MaCW. Commercial type Yorkshire swine (n = 5) inoculated intradermally with EP3-MaCW developed cell-mediated immune (CMI) responses to avian tuberculin detectable in vivo with delayed-type skin hypersensitivity and in vitro with lymphocyte immunostimulation responses.

Adjuvants, Immunologic↗

Possible relationship of proliferative enteritis in pigs and hamsters.

Three- to six-week-old hamsters were orally inoculated with broths containing one of the following cultures: Campylobacter mucosalis; C. hyointestinalis; C. coli; C. jejuni, all of porcine proliferative enteritis origin, or else C. jejuni of hamster origin. Hamsters given the last of those organisms were shown to have colonisation of their intestines by C. jejuni and 36 of 40 developed an acute enteritis. Mild hyperplasia of enterocytes in ileal crypts was evident in one hamster 2 days after it was given C. coli. No other lesions were detected. Further 3-week-old hamsters were orally inoculated with homogenised intestinal mucosa collected from 4 pigs (A-D) affected by proliferative enteritis. Lesions of proliferative enteritis were detected in 7 of 41 hamsters necropsied 10-21 days after being dosed with mucosas B or D. Marked hyperplasia of ileal enterocytes, associated with numerous intracellular Campylobacter-like organisms, were invariably detected in experimentally affected hamsters. No particular Campylobacter sp. was consistently isolated. None of the controls had demonstrable lesions. The results suggested that cross-species transmission of proliferative enteritis was possible from pigs to hamsters. Therefore a common initiating or aetiological agent may be present. No specific organism was identified as filling this role by inoculation of hamsters with pure cultures.

Animals↗

Characters of Escherichia coli 078 isolated from septicaemic animals.

Twenty-one Escherichia coli isolates of serogroup 078 from animal septicaemia were obtained from laboratories in France, England and Canada. The bacteria were compared for outer membrane protein (OMP) patterns, lipopolysaccharide patterns, surface proteins of fimbrial types, biotypes, antibiotypes, colicin production, hydroxamate production and virulence in mice. Sixteen isolates from bovine, ovine, porcine and avian species in France and England had a similar OMP pattern. This characteristic associated with minor properties like surface proteins type, colicin V production and virulence in mice made these 16,078 E. coli isolates from 4 animal species, good candidates for the same clonal grouping. The five other bovine isolates with "Vir" or "31a" phenotypes were heterogeneous for most of the characteristics studied.

Animals↗

The pathogenesis of turbinate atrophy in pigs caused by Bordetella bronchiseptica.

The pathogenicity of 3 strains of Bordetella bronchiseptica designated B58, PV6 and B65 was compared by intranasal infection of gnotobiotic piglets. Strain B58 was a phase 1 isolate that produced haemolysin, an adhesin for calf erythrocytes, adenylate cyclase, mouse lethal factor, dermonecrotic factor and cytotoxin. B65 was a variant of B58 that produced no detectable haemolysin, adhesin or adenylate cyclase and 10-fold smaller amounts than B58 of mouse lethal factor, dermonecrotic factor and cytotoxin. Strain PV6 was a phase 1 isolate that produced only haemolysin, adhesin and adenylate cyclase. After nasal infection of gnotobiotic pigs, 10(3.2)-10(6.2) colony forming units ml-1 (cfu ml-1) of strains B58 and PV6 were cultured from nasal washings during the next 25 days. In contrast, only 10(1.0)-10(2.8) cfu ml-1 of strain B65 were recovered during the same period. Only pigs infected with strain B58 had turbinate atrophy when they were slaughtered 25 days after infection and neutralising antibody to cytotoxin was detected only in these pigs. These results suggested that the cytotoxin, which may be the same as the mouse lethal and dermonecrotic factors, was the cause of turbinate atrophy. They also support the view that the adhesin for calf erythrocytes is required for colonisation of the nasal cavity in vivo.

Animals↗

Horizontal transmission of Aujeszky's disease virus from sheep to pigs.

Eight 2-month-old merino lambs were inoculated intranasally with different (10(2.0)-10(5.0)TCID50) amounts of Aujeszky's disease virus (ADV). Electron microscopic studies indicated that ADV replicated in extra-neural sites, in the epithelial cells of the mucosa of the upper and lower respiratory tract. Although the virus was excreted continuously in nasal discharges, horizontal transmission to contact lambs failed. The surviving exposed and contact lambs had no demonstrable antibodies against ADV and they were susceptible when challenged by ADV. However, the virus was transmitted to susceptible pigs in contact with the exposed lambs. One of the five contact pigs showed characteristic clinical signs of Aujeszky's disease, developed a nonsuppurative meningoencephalomyelitis and ADV was recovered from the brain, nasal discharge and other organs. Restriction enzyme analysis of DNA from this virus confirmed the sheep origin of the isolate. The other 4 pigs seroconverted. ADV infection in sheep is therefore a possible source of infection for pigs, but the lack of horizontal transmission in sheep was confirmed.

Animals↗

A complex-trapping-blocking (CTB) ELISA, using monoclonal antibodies and detecting specifically antibodies directed against foot-and-mouth disease types A, O and C. II. Application.

The complex-trapping-blocking (CTB) enzyme-linked immunosorbent assay (ELISA) was evaluated to detect antibodies directed against foot-and-mouth disease virus (FMDV) strains A10 Holland, O1 BFS, and C1 Detmold. Log10 serum titres of uninfected, unvaccinated cattle (n = 100) were less than 1.80 in the CTB-ELISA. Sera from cattle vaccinated with either monovalent or trivalent vaccines were tested in both the CTB-ELISA and the serum neutralisation test (SNT); titres in both tests correlated positively (P less than 0.001). Titres of sera from cattle, sheep, and pigs vaccinated twice with FMDV A10 Holland also correlated positively in both tests. In another experiment, cattle vaccinated with FMDV strain C1 Detmold were intradermolingually challenged 3 weeks after primary vaccination; at the same time two controls were challenged. At 8 days after challenge, serum titres of the controls were distinctly higher in the CTB-ELISA than in the SNT, whereas serum titres of the vaccinated cattle were equally high in both tests. In potency tests for monovalent vaccines against FMDV strains A10 Holland, O1 BFS or C1 Detmold, serum titres correlated strongly in both tests with protection against the homologous FMDV strain. We concluded that the CTB-ELISA is not only sensitive, but easier to perform and more rapid and reproducible than the SNT. The CTB-ELISA may be useful in evaluating the immune response in cattle during FMD vaccine potency tests.

Animals↗

Experimental infections of pregnant sows with ovine Chlamydia psittaci strains.

Four sows were experimentally inoculated at 42 days pregnancy, with the BS isolate of Chlamydia psittaci from aborted ewes. Their body temperature and health were monitored until 30 days after farrowing. The sows seroconverted but their reproductive performance was not impaired. Chlamydiae were identified in the afterbirths from two sows with normal litters using stained smears, "Clearview" and "IDEIA" ELISA antigen detection tests. The presence of inclusion bodies was detected in sections of the infected fetal membranes after staining with haematoxylin and eosin, methylene blue and Streptavidin-Biotin using an anti-Chlamydia monoclonal antibody.

Animals↗

Phenotypic characterization of Zimbabwean isolates of Pasteurella multocida.

The phenotypic characteristics of 60 Zimbabwean isolates of Pasteurella multocida sensu stricto, from disease syndromes in different host species were studied. A number of representative strains were also serotyped. Consistent results were obtained in the tests for; catalase, oxidase, urease, indole, acid in glucose, inositol, salicin and sucrose. There was no obvious relationship between serotype, host or disease and the pattern of utilization of certain substrates by an isolate. This has been discussed in the context of recent proposals to reclassify Pasteurella and P. multocida on genotypic and phenotypic studies. It is suggested that notwithstanding the relevance of genetic studies in circumscribing P. multocida, the phenotype and disease significance of the taxon should not be ignored. A case of bronchitis in a dog which was simultaneously colonized by three different strains of Pasteurella is described. Also septicaemic pasteurellosis in a Nile crocodile (Crocodylus niloticus) is reported and for the first time prevalence of various serotypes in pasteurellosis of animals in Zimbabwe.

Alligators and Crocodiles↗

Development and use of a biotinylated 3ABC recombinant protein in a solid-phase competitive ELISA for the detection of antibodies against foot-and-mouth disease virus.

A biotinylated 3ABC recombinant protein was developed and used in a competitive ELISA (cELISA) to detect foot-and-mouth disease virus (FMDV) antibodies in cattle, sheep and pigs. In this report, we describe the cloning and expression of 3ABC protein in Escherichia coli cells as fusion protein with 6xHis and biotin. This cELISA uses streptavidin to capture bacterially expressed and in vivo biotinylated 3ABC antigen. The antigen capture strategy provides a simple and reliable method, which does not require purification of recombinant antigen before the serological assay. An hyperimmune guinea pig antiserum produced against purified 6xHis-3ABC was used as competitor in the test. The potential use of this cELISA for the identification of antibodies induced by FMD virus infection from those induced by vaccination is discussed.

Animals↗