Echinococcus granulosus,Taenia hydatigena,T. ovis: Evaluation of cyst fluids as antigen for serodiagnosis of larval cestodes in sheep

In order to monitor the progress of New Zealand's hydatids eradication campaign, a specific, serological, diagnostic test is required to identify infected sheep. An indirect haemagglutination test, using pyruvic aldehyde-stabilized sheep erythrocytes as the antigen carrier, was developed for the serodiagnosis of larval cestode infections of sheep. Using cyst fluid from Echinococcus granulosus, Taenia hydatigena and T. ovis as the antigens in this test, it was shown that the larval cestode species, responsible for an infection in sheep harbouring a single specific infection, could be identified by the higher titre given with the homologous antigen, in comparison to that given with the heterologous antigens. Sera of sheep infected with two or more species were also tested by this method, and the only specific infections to be diagnosed by differential titres were those due to the presence of live E. granulosus cysts. These antigens cannot be used for the diagnosis of specific larval cestode infections in the field because of the cross-reactivity between cyst fluids. However, the test did show that infection with larval cestodes could be diagnosed on a non-specific basis.     

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 $\text{1}\text{5}$.     

Lymphocyte stimulation in swine dysentery

In lymphocyte stimulation studies of pigs affected with swine dysentery (SD) all of the pigs gave significant response (P < 0.05) to soluble antigen from $\text{Treponema}$$\text{hyodysenteriae}$. Swine infected with virulent or attenuated $\text{T.}$$\text{hyodysenteriae}$ gave significant lymphocytic response 3 or 6 weeks after infection; uninfected pigs did not give a similar lymphocytic response. The delayed hypersensitivity (DH) skin test, in which soluble $\text{T.}$$\text{hyodysenteriae}$ antigen preparation was used, detected only 3 out of 14 SD-affected swine. The lymphocyte stimulation assay by detection of protein synthesis may offer a rapid, reliable test for the diagnosis of SD within herds suspected of being affected.     

Hemolytic complement measurement in eleven species of nonhuman primates

A microtiter system was used to measure hemolytic complement levels in serum from eleven nonhuman primate species. The species studied were $\text{Macaca mulatta}$ (rhesus macaque), $\text{Macaca radiata}$ (bonnet macaque), $\text{Macaca nemestrina}$ (pig-tailed macaque), $\text{Macaca fascicularis}$ (crab-eating macaque), $\text{Macaca speciosa}$ (stumptailed macaque), $\text{Papio cynocephalus}$ (yellow baboon), $\text{Papio anubis}$ (olive baboon), $\text{Cercopithecus aethiops}$ (African green monkey), $\text{Aotus trivirgatus}$ (owl monkey), $\text{Ateles fusceps robustus}$ (spider monkey), and $\text{Galago crassicaudatus panganiensis}$ (thick-tailed galago).The optimal hemolytic complement titer of the various nonhuman primate species was found to vary with different species sources of erythrocytes and anti-erythrocyte reagents used in the assay. No single erythrocyte and anti-erythrocyte test reagent produced optimal titers for all of the primate species examined. Sera from several species was found to have high spontaneous lytic activity towards non-sensitized sheep erythrocytes which for six species ($\text{M. mulatta, M. radiata, M. speciosa, P. cynocephalus, P. anubis}$ and $\text{A. trivirgatus}$) was equal to the titer for antibody sensitized erythrocytes. Evidence of alternate pathway complement activation as a possible reason for the high titer of lytic activity towards unsensitized erythrocytes could not be demonstrated for any nonhuman primate species. In one species, $\text{M. mulatta}$, the sensitizing activity of normal serum for sheep erythrocytes was shown to be in the IgM containing fraction obtained with gel filtration and to be absorbed by boiled sheep erythrocyte stroma which contains Forssman antigen.     

Lack of reactivity of sera from Theileriaparva-infected and recovered cattle against cell membrane antigens of Theileria parva transformed cell lines

Sera from $\text{Theileria}$$\text{parva}$ infected, recovered and rechallenged cattle were tested in complement-dependent cytotoxicity, membrane immunofluorescence and antibody-dependent cellular cytotoxicity assays for the presence of antibodies against cell membrane antigens of $\text{T}$. $\text{parva}$ transformed cell lines.In the complement-dependent antibody-mediated cytotoxicity assay, sera from lethally infected animals were negative. Some recovered cattle showed a positive reaction, but such reactions were also observed when an eland cell line infected with $\text{T}$. $\text{taurotragi}$, and bovine lymphoblastoid cells were used as targets. Reaction was less against Ig-negative peripheral blood lymphocytes.Evidence is presented that these reactions could be evoked by attachment of immune complexes to Fc-receptors. It is concluded that cattle exposed to $\text{T}$. $\text{parva}$ infection do not develop antibodies against specific $\text{T}$. $\text{parva}$ (or $\text{T}$. $\text{parva}$-induced) cell surface antigens.     

Echinococcosis in pigs and intestinal infection with Echinococcus spp. in dogs in southwestern Lithuania

Cystic echinococcosis is a major emerging zoonosis in many Eastern European and Asian countries. Post slaughter examinations of 684 pig livers in Lithuania revealed significantly higher numbers of Echinococcus granulosus infections in animals from family farms (13.2%; 95% CI 10.7–16.2) as compared with those from industrial farms (4.1%; 95% CI 0.8–11.5). The prevalence was also significantly higher in pigs older than 1 year than in younger ones. In addition, in 0.5% of the pigs from the family farms, infertile and calcified E. multilocularis lesions were identified by PCR. Faecal samples from rural dogs (n = 240) originating from 177 family farms in 12 villages were investigated for taeniid eggs with two methods. Significantly more dogs excreting taeniid eggs were diagnosed with the flotation/sieving method (n = 34) as compared to the modified McMaster method (n = 12). Multiplex PCR performed with DNA from taeniid eggs isolated from faeces of 34 dogs revealed 26 infections with Taenia spp., 9 with E. granulosus and 2 with E. multilocularis (4 cases with concurrent Taenia spp. and E. granulosus or E. multilocularis infections). Genotyping of E. granulosus cyst tissues from 7 pigs, 1 head of cattle and from E. granulosus eggs from 8 dog faeces revealed the genotype G6/7 (‘pig/camel strain’) in all cases. The high infection pressure with Echinococcus spp. in family farms necessitates initiating control programs.     

Equine ehrlichiosis: A comparison between E. equi and other pathogenic species of Ehrlichia

Ehrlichia equi, etiologic agent of equine ehrlichiosis, is a rickettsia which morphologically closely resembles the agents of bovine petechial fever, tick-borne fever, and canine ehrlichiosis (tropical canine pancytopenia). Natural infections of E. equi have been reported only in horses; however, the experimental host range of E. equi has been broadened to include burros, sheep, goats, dogs, cats, monkeys and baboons. Infection of primates indicates that E. equi may be a zoonotic agent. An indirect fluorescent antibody test employing E. equi-infected granulocytes as antigen has been developed and used to show that infected horses develop a prolonged antibody response to E. equi. Cell-mediated immune responses measured by the leukocyte migration inhibition test were also detected in horses recovered from acute illness. Protective immunity in horses, monkeys and baboons to reinfection is long-lasting. In contrast to the blood of dogs recovered from clinical E. canis infection, the blood of horses and dogs recovered from clinical infection with E. equi is not infectious for susceptible animals. Infection of dogs with E. equi does not provide protection against subsequent infection with E. canis.     

Bovine babesiosis: The immunization of cattle with fractions of erythrocytes infected with Babesia bovis (syn B. argentina)

Soluble antigen which protected susceptible cattle against challenge with $\text{Babesia bovis}$ was extracted from $\text{B. bovis}$-infected erythrocytes by sonic disintegration and separation of the soluble from the insoluble matter by ultracentrifugation. The material was then fractionated by the precipitation of fibrinogen-like proteins. The precipitate contained the babesial antigens that were located on the stroma of the infected erythrocytes. Antigen originally located on the parasite remained in solution. Both fractions conferred protection on splenectomized calves against challenge with $\text{B. bovis}$. However, the fraction containing the parasite antigens appeared to have more potential for development as a killed vaccine because it was not heavily contaminated with antigenic material from bovine erythrocytes.     

Application of indomethacin as a potentiator of lymphocyte blastogenesis in Brucella abortus exposed cattle

Lymphocytes from $\text{Brucella abortus}$ field strain infected, strain 19 vaccinated, non-exposed and field strain infected, but immunologically unresponsive cattle were incubated with $\text{B. abortus}$ antigen and indomethacin. There were significant increases (P < 0.005) in the blastogenic responses, as measured by [³H] thymidine uptake, in cultures with indomethacin as compared to cultures without indomethacin. Lymphocyte blastogenic responses to $\text{B. abortus}$ antigen were potentiated by indomethacin in both $\text{B. abortus}$ exposed and non-exposed cultures. However, potentiation of sensitized lymphocyte blastogenic responses by indomethacin was significantly greater (P < 0.005) than that in non-exposed lymphocytes. Additionally, indomethacin significantly potentiated Brucella-induced lymphocyte blastogenic responses in lymphocytes from anergic cattle.     

mRNA from porcine colostral and milk cells coding for specific antibodies in response to orally administered Escherichia coli strains

The effect of feeding live $\text{Escherichia coli}$ bacteria to sows on the lymphocyte populations in colostrum and milk was studied by $\text{in ovo}$ translation of the mRNA of these cells (Kortbeek-Jacobs and van der Donk, 1978). Four $\text{E. coli}$ strains were tested: one wild type K88 producing strain, two conjugated K12 strains producing K88 antigen and one wild type K12 strain. Only after feeding the wild type K88 bacteria, colostral lymphocytes contained mRNA coding for anti-K88 antibodies, predominantly of the IgA and IgM isotypes. Milk lymphocytes of the sows receiving one of the other strains were capable of producing more anti-K88 antibodies than the sows that were fed the wild type K88 strain. Antibodies were predominantly of the IgM class. The reactivity of the milk lymphocytes is ascribed to the strain with which the piglets were challenged. The results support the phenomenon of sensitized gut lymphocytes that home to the mammary gland.     

Serological responses of dogs to cell wall and internal antigens of Brucella canis (B. canis)

The serological responses of dogs to cell wall and internal antigens of B. canis were studied in experimentally infected specific-pathogen-free (SPF) Beagles. Sera from infected and false positive field dogs also were examined. Cell wall antigens were extracted from B. canis by two procedures that employed either hot phosphate buffered saline (PBS) or sodium desoxycholate (SDC). Agar gel immunodiffusion (AGID) tests employing sera from experimentally infected SPF dogs were used to evaluate antigenic extracts. Extraction with PBS yielded two antigens; SDC extracted an antigen complex and sonication of PBS extracted cells liberated four internal antigens.Sera from field dogs that were negative for B. canis infection in repeated tests often had heterospecific antibodies. Such cross-reactive sere commonly gave “spur” (partial fusion) reactions with a positive reference serum when tested against the SDC cell wall antigen. In addition, false positive dogs did not have antibody to one of the cell wall antigens or to the internal antigens. In contrast, sera from infected field dogs commonly gave “identity” (fusion) reactions in the AGID test with two antigens in the SDC extract, and produced precipitin lines to one to four internal antigens.Examination of a library of sera obtained from experimentally infected SPF dogs over a period spanning 4$\text{1}\text{2}$ years revealed that none of the serodiagnostic tests employed (tube agglutination, slide agglutination, AGID) was accurate during the inital 12 weeks of infection; hemocultures were the most sensitive during this period. Tube and slide agglutination tests were initially sensitive, but they showed a lack of sensitivity and specificity after the bacteremic period ceased, as well as in their failure to exclude false positive reactions in field animals. Immunodiffusion tests that employed SDC or PBS extracts of B. canis cell walls were sensitive and accurate in identifying most infected dogs. After the bacteremia had ceased, however, AGID tests that employed cell wall antigens gave equivocal results. Immunodiffusion tests that employed sonicated (internal) antigens were sensitive shortly after the onset of bacteremia, and they had the advantage of detecting infected animals for at least 6 months following the cessation of bacteremia, a time when other serological tests gave equivocal results.     

Solid-phase enzyme immunoassay of bovine antibody responses following immunization against and natural infection with Pasteurella haemolytica

A solid-phase enzyme immunoassay (EIA) was developed in order to monitor bovine antibody responses following immunization against and natural infection with $\text{Pasteurella haemolytica}$ serotype A:1. Non-ionic surfactants, used in many antibody EIAs to reduce non-specific immunoglobulin binding, had to be avoided because they inhibited specific binding of bovine antibodies to $\text{P. haemolytica}$ antigens. Calves were immunized with a KSCN extract of $\text{P. haemolytica}$. Subcutaneously immunized animals developed a significantly higher humoral antibody response than did intranasally vaccinated animals. Intranasally immunized calves developed a slightly, but not significantly higher nasal antibody response than did calves vaccinated subcutaneously. Field study results based on bacterial isolation and EIA detection of antibodies to $\text{P. haemolytica}$ indicate that cattle can generate carrier states where bacteria are present in the upper respiratory tract, yet no humoral antibody response is induced. The converse was also found where cattle were free from $\text{P. haemolytica}$ in the upper respiratory tract, yet possessed a good humoral antibody response to $\text{P. haemolytica}$.     

Nippostrongylus brasiliensis infection in the rat: 1. Production of bile lgA and serum lgG antibodies by adult rats

Bile lgA and serum lgG antibody responses were assayed by a micro ELISA technique. Antibodies of both classes were produced by 14 days after infection against whole worm antigens and worm metabolic product antigens, by adult rats infected once with $\text{Nippostrongylus brasiliensis}$. Rats were reinfected on day 28 and titres of both classes of antibody against both antigens were greater 7 and 14 days after reinfection than after the initial infection. Bile lgA antibodies were produced against phosphorylcholine after a single infection with the parasite but no increased response occurred after reinfection. The possible significance of lgA and lgG antibody responses in relation to immunity to intestinal nematode infections is discussed.     

Thymus atrophy during early pregnancy and its effect on a Trichinella spiralis infection in mice,including intestinal pathology and blood eosinophilia

The effects of pregnancy on the course of a $\text{Trichinella spiralis}$ infection and associated histopathological changes in thymus and small intestine were studied in outbred Swiss mice. For this purpose pregnant mice were orally infected with $\text{T. spiralis}$ on various days (1 to 8) $\text{post coitum}$. Virgin, age-matched infected and non-infected mice served as controls.Pregnancy induced a severe but reversible thymus atrophy, which was even more marked during a $\text{T. spiralis}$ infection. Thymus atrophy was most dramatic during mid and late pregnancy. During the involution phase a distinct increase in mast cells was observed.In animals infected during early pregnancy a not statistically significant inhibitory effect on worm expulsion was observed, whereas no effect was seen on the yield of muscle larvae, small intestine pathology (numbers of eosinophils, intestinal mast cells and globule leucocytes), blood eosinophilia and antibody production.Infection given during mid-pregnancy exerted an inhibitory effect on blood eosinophilia.On the basis of these results, it is concluded that although a severe atrophy of the thymus occurs during mid and late pregnancy, no effect on worm expulsion and intestinal pathology was observed when the infectious agent was given before thymus depletion started.     

Serodiagnosis of haemonchosis with a somatic antigen (Hc26) in several breeds of sheep.

Sera from 53 sheep belonging to Castellano, Churro, Manchego, and Merino breeds were analyzed to test the diagnostic value of a 26-kD antigen from adult Haemonchus contortus at prepatency and early and late patency of experimental haemonchosis. Animals that received zero, 1, or 2 infections with the parasite were tested. In addition, sera from 20 experimentally infected and 10 noninfected Texel sheep were used to test the antigen. Sera from 37 infected animals at prepatency as well as at patency in primary and secondary infection were found positive with the 26-kD antigen. However, sera from 10 animals with the lowest worm burdens (second infection) did not recognize the antigen during early patency (day 28 postinfection). IgG1 was the only isotype implicated in antigen recognition because IgG2, IgA, and IgM, in the same sera, showed no reactivity with the peptide. Antigen specificity was confirmed because hyperimmune sera against infective larvae and adult stages of the most common gastrointestinal nematodes found in natural infections in sheep (Trichostrongylus colubriformis and Teladorsagia circumcincta) did not recognize this peptide. The antigen was recognized only by anti-adult H. contortus hyperimmune sera and appeared to be absent in the L3 parasite stage. In addition, the partial N-terminal amino acid sequence of the diagnostic peptide is reported.     

An ELISA test to detect antibody to Bordetella bronchiseptica

An enzyme-linked immunosorbant assay (ELISA) was developed to measure antibody to $\text{Bordetella bronchiseptica}$ in dogs. The ELISA test was more rapid and sensitive and required 50 to 150 times less antigen than the amount of antigen required for the conventional tube agglutination test. A survey of 50 canine serum samples using ELISA suggested that 8% of all sera had titers greater than 1:64, 56% had titers of 1:8 to 1:64, and 36% had titers of less than 1:8. The mean titer of survey sera was 1:46 and the median titer was 1:16. Serum antibody responses in dogs inoculated with a commercially available bacterin were compared with responses in dogs inoculated with experimental endotoxin depleted bacterin.     

Studies on degradation and outflow rate of protein supplements in the rumen of sheep and cattle

The rates of disappearance (p) of protein from fish-meal, meat-and-bone meal, soya-bean meal, cottonseed meal, linseed-meal and groundnut-meal were described when nylon bags were incubated in the rumens of sheep and cattle given either barley or dried grass. The exponential equation p = a + b (1 ? e^?ct),where a, b and c are constants, e is the natural logarithm, and p the amount disappearing in time, t, was used. There was no consistent difference between the degradabilities determined with sheep and cattle. Degradation was finally combined with different outflow rate (k) and the effective degradability (P) was described as the equation $\text{P}\text{=}\text{a + (bc)}\text{(c + k)}$.     

Distribution and intensity of <Emphasis Type="BoldItalic">Echinococcus granulosus</Emphasis> infections in dogs in Moroto District,Uganda

This study was carried out during August 2007–March 2008 in pastoral areas of Moroto District, Uganda. It investigated the distribution and infection intensity of Echinococcus granulosus in the dog population and involved the postmortem examination of 327 dogs (106 domesticated; 80 semi-domesticated, and 141 strays; comprised of 163 females and 164 males). The overall prevalence of E. granulosus was 66.3% (95% CI = 60.8–71.4) with parasite burdens of 6–5,213 among the infected dogs. The prevalence of E. granulosus was primarily associated with the season. While the dogs were more likely to have high parasite burdens during the rainy as opposed to the dry season, the parasite burden of E. granulosus infection was also highly associated with age and husbandry. Young dogs were at greater risk of carrying a heavy burden of E. granulosus infection than adults. Likewise, stray dogs were more likely to have a heavy parasite burden. As the study documented a high prevalence and intensity of E. granulosus infection in the dog population in Moroto District of Uganda, further studies need to be carried out in human and intermediate hosts to elucidate the cycle of transmission that could help to design appropriate controlling measures.     

Expansion of intracellular IFN-γ positive lymphocytes during Mycoplasma agalactiae infection in sheep

A method to assess the expansion of antigen-specific intracellular IFN-γ positive T cell subsets during the infection will be helpful for a better understanding of mycoplasmal infections physiopathology in the sheep. We analysed the percentage of antigen-specific lymphocytes positive for intracellular IFN-γ during the infection of sheep with Mycoplasma agalactiae by culturing peripheral blood mononuclear cells of infected or uninfected animals with irradiated M. agalactiae. The expansion of antigen-specific IFN-γ positive lymphocytes in infected sheep was initially sustained by CD4⁺ T cells at day 15 after infection, when antigen specific IgG start to be detectable, followed by CD8/IFN-γ double positive cells. γδ T-cells were not expanded at any time point analysed. IFNγ⁺ T cells disappear 60 days after infection, suggesting that antigen specific IFNγ⁺ T cells, mainly detected in the early phase of the disease, could be useful to understand the role of cell-mediated immunity during M. agalactiae infection.     

Brucella abortus antigen-induced lymphocyte reactivity in guinea pigs

A whole blood lymphyocyte transformation (WBLT) assay was used to detect anti-brucella lymphocyte reactivity in guinea pigs. Brucella antigens stimulated an antigen-specific lymphoproliferative response in WBLT assays from $\text{Brucella abortus}$ infected guinea pigs. The response was best detected from 6 to 16 weeks after challenge inoculation with viable $\text{B. abortus}$ 2308. Lymphocytes were not stimulated by unrelated bacterial antigens and control animals did not respond to the Brucella antigens. The responding cell population was characterized as mostly T lymphocytes. The WBLT assay was found to be specific for the detection of anti-brucella lymphocyte reactivity. However, a negative response was not definitive, which indicated a need for repeated testing to establish that a guinea pig did not have anti-brucella lymphocyte sensitization.