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.     

The role of serum and biliary antibodies and cell-mediated immunity in the clearance of S. typhimurium from chickens

The development of three parameters of immunity in response to a non-lethal infection of $\text{Salmonella typhimurium}$ in adult chickens has been examined. Intravenous inoculation of 1 × 10⁶ organisms established infection in the liver, spleen and intestinal tract of all birds; the organism persisted in these sites until day 9 of the infection, after which it was cleared rapidly from all sites. High levels of agglutinating and haemagglutinating antibodies were found in serum and bile 5 days after infection; they peaked at days 7 to 10, and detectable antibody was still present in both fluids 6 weeks after infection. The presence of this antibody did not appear to cause a significant reduction in organism numbers in any of the sites examined. Cell mediated immunity was detected at day 14. It is suggested that cell mediated immunity is responsible for clearance of the organisms from the tissues.     

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.     

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 $\text{P. haemolytica}$.     

Immunoreactive protein antigens of Mycoplasma pulmonis in experimentally infected rats

Mycoplasma pulmonis was cultured in modified Hayflick's medium, washed in 0.25 M NaCl, and solubilized by 2.5% sodium dodecyl sulfate. Protein antigens of M pulmonis separated by polyacrylamide-gel electrophoresis were blotted onto nitrocellulose strips. Specific-pathogen-free rats were inoculated intranasally with M pulmonis. The serum samples of these rats were obtained periodically and used to react with fractionated M pulmonis antigens which were fixed on the nitrocellulose strips. The antigen-antibody reactions were further recognized by 125I-labeled antiglobulin. Detection of immunoreactive antigens was obtained by autoradiography. Antibody response was not detected in serum obtained 7 days after rats were inoculated, and by 14 days, a slight response to several proteins was found. At 28 days after rats were inoculated, many immunoreactive antigens were detected. Generally, antibodies against antigens of moderate to low molecular weight appeared early in the infection, and antibodies against antigens of high molecular weight appeared late. Important immunoreactive antigens thus identified can readily be distinguished from more than 58 different M pulmonis antigens detectable by sodium dodecyl sulfate polyacrylamide-gel electrophoresis. The humoral antibody response was measured by an enzyme-linked immunosorbent assay. The immunoglobulin G antibodies were initially detected at low level at 7 days after rats were inoculated. These humoral antibody responses reached maximum by 28 days. The increase in serum antibody titer corresponded with numbers of immunoreactive antigens detected by immunoradio-binding assay. The information gained by this investigation may improve our understanding of the antigenicity of M pulmonis and the immune response of rats exposed to M pulmonis.     

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.     

19s and 7s antibody response of Mastomys natalensis in experimental filarial (Litomosoides carinii, Acanthocheilonema viteae, Brugia malayi, B. pahangi) infections

Specific total antibody (ab), 19s and 7s ab levels in the serum of M. natalensis were investigated after infection with L. carinii, A. viteae, B. malayi and B. pahangi for period of about 500 days p.i., using ELISA (homologous adult antigen) and indirect immunofluorescence tests (IIFT: homologous adult and microfilariae antigen). Total ab levels in L. carinii infected animals rose moderately during prepatency Maximum levels occurred during patency. The response during prepatency was stronger in A. viteae and Brugia spp. infected hosts. Lateron ab levels increased continuously in Brugia infections; in A. viteae infection they decreased with decreasing parasitaemia. 19s abs were stimulated during prepatency and at the beginning of patency, or were found at moderate levels throughout to period of investigation (Brugia infections). 7s abs predominated beginning at the period of late prepatency (IIFT) or at the beginning of patency (ELISA). The time courses of 7s abs corresponded to those of total abs. As obvious by IIFT (adult worm antigen) total and 19s titres were higher against cuticle antigens, egg shell antigens and intrauterine amorphous material than against antigens located in the hypodermis and musculature. 7s abs showed best reactivity with cuticle antigens. Using microfilarial antigens 19s abs reacted predominantly with cuticle antigens whereas 7s abs often showed higher titres against antigens which were localized within the larvae than against cuticle antigens.     

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.     

Local antibody responses in the bile and faeces of sheep infected with Fasciola hepatica

The effect of infection with the liver fluke Fasciola hepatica on serum, bile and faecal immunoglobulin and antibody levels was studied in Scottish Blackface sheep. In the serum the immunoglobulins showing the most marked increase were IgG1 and IgG2 and their maximal values were reached at 16 weeks after infection. In the bile IgG2 rose to peak values at two weeks and IgG1, IgA and IgM were maximal at four weeks after infection. The levels of faecal IgG and IgA were low after primary infection but after reinfection a rapid increase in IgA concentration was observed within one to two weeks. Haemagglutinating antibody levels against egg antigens, juvenile and adult excretory-secretory antigens and adult fluke somatic antigens were evaluated. In the sera high titres were observed starting from two to four weeks after infection and persisting until 14 to 16 weeks. Bile haemagglutinating antibodies against excretory-secretory antigens showed the highest level at two and four weeks after infection while antibodies against adult somatic antigens reached maximal titres between four and eight weeks. Faecal antibody levels after primary infection were low but increased rapidly within two weeks after reinfection, coinciding with the elevation in faecal IgA concentration. However, there was no reduction in the number of flukes established in reinfected animals.     

Coccidiosis: T-lymphocyte-dependent effects of infection with Eimeria nieschulzi in rats

The intestinal pathology caused by infection with $\text{Eimeria nieschulzi}$ was investigated and comparisons were made between the effects in athymic nude (rnu/rnu) rats and their heterozygous (rnu/+) litter-mates. Most of the changes noted, i.e. increase in gut weight, partial villous atrophy and increased numbers of mast, goblet and pyroninophilic cells were shown to be largely or wholely thymus dependent. The numbers of intraepithelial lymphocytes were decreased in both groups during the period of study. The peripheral blood leucocyte response was similar in both groups of rats during a primary infection but differed after a challenge inoculum, indicating that the secondary type of response which occurred in the rnu/+ rats was thymus dependent, as is resistance to reinfection.     

The effect of oxfendazole terminated infections with Haemonchus contortus on the development of immunity in sheep

The relative contribution of third (L3), fourth (L4) or adult stages of Haemonchus contortus to the development of immunity was evaluated in three groups of sheep subjected to infections terminated by oxfendazole treatments at the L3, L4 or adult stage. A control group did not receive immunising infections. All the groups were challenged with 5000 L3, to evaluate the protection provided by the different protocols. All sheep were necropsied at the end of the experiment to count the abomasal worm burdens. A marked reduction in egg counts after challenge infection was only observed in sheep in which the infection was terminated in the adult stage (Group 4). A significant reduction in worm burden was also observed in Group 4. The immunising infections and/or the challenge infection resulted in moderately elevated IgG antibody levels against L3, L4 and adult somatic antigens in all the groups. In contrast, a strong IgG response against H. contortus excretory/secretory (ES) antigens was observed in the groups in which the immunising infection was terminated in the L4 and the adult stage. An elevated lymphocyte proliferation response against Haemonchus ES antigens was found only in the group that had their immunising infection terminated at the adult stage. The combined data suggest that exposure to and elicited immunological responses to ES antigens are important for the development of immunity against H. contortus.     

Humoral immune response of water buffalo monitored with three different antigens of Toxocara vitulorum

Humoral immune response of water buffalo naturally infected with Toxocara vitulorum was monitored using three different antigens of this parasite in serum and colostrum of buffalo cows and calves. Soluble extract (Ex) and excretory/secretory (ES) larval antigens and perienteric fluid antigen (Pe) of adult T. vitulorum were used to measure the antibody levels by an indirect ELISA. Serum of 7-12 buffalo cows for the first 365 days and colostrum of the same number of buffalo cows for the first 60 days of parturition, and serum of 8-10 buffalo calves for the first 365 days after birth were assayed. The ELISA detected antibodies against all three T. vitulorum antigens in the colostrum and serum of 100% of buffalo cows and calves examined. The highest antibody levels against Ex, ES and Pe antigens were detected in the buffalo cow sera during the perinatal period and were maintained at high levels through 300 days after parturition. On the other hand, colostrum antibody concentrations of all three antigens were highest on the first day post-parturition, but decreased sharply during the first 15 days. Concomitantly to the monitoring of immune response, the parasitic status of the calves was also evaluated. In calves, antibodies passively acquired were at the highest concentrations 24 h after birth and remained at high levels until 45 days coincidentally with the peak of T. vitulorum infection. The rejection of the worms by the calves occurred simultaneously with the decline of antibody levels, which reached their lowest levels between 76 and 150 days. Thereafter, probably because of the presence of adults/larvae stimulation, the calves acquired active immunity and the antibodies started to increase slightly in the serum and plateaued between the days 211 and 365. All three antigens were detected by the serum antibodies of buffalo calves; however, the concentration of anti-Pe antibody was higher than anti-EX and anti-ES, particularly after 90 days of age. By conclusion, the buffalo cows develop immunity and keep high levels of antibodies against T. vitulorum-Ex, ES and Pe antigens and these antibodies are transferred to their calves through the colostrum. This passively acquired immunity does not protect the calves against the acquisition of the infection, but these antibodies, passively or actively acquired, may have an important role during worm rejection by the calves and prevention of intestinal reinfection.     

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 $\text{1}\text{5}$.     

Immunodiagnosis of experimental Parelaphostrongylus tenuis infection in elk

Elk infected with the meningeal worm, Parelaphostrongylus tenuis (Protostrongylidae), do not consistently excrete larvae in feces, making the current method of diagnosing live animals using the Baermann fecal technique unreliable. Serological diagnosis could prove more useful in diagnosing field-infected animals but depends on the identification and availability of good quality antigen. To mimic field infections, 2 elk were inoculated with 6 infective L3 larvae of P. tenuis, and another 2 with 20 L3 larvae. Fecal samples were examined for nematode larvae using the Baermann technique and serum samples taken were tested for anti-P. tenuis antibody with ELISAs by using the excretory-secretory (ES) products of L3, and sonicated adult worms as antigens. One animal passed first-stage larvae in its feces 202 days postinoculation, but passed none thereafter. The remaining 3 inoculated animals did not pass larvae. In contrast to parasite detection, antibodies against larval ES products were detected in all animals starting from 14 to 28 days postinoculation and persisted until the termination of the experiment on day 243 in 2 animals that harbored adult worms. Antibodies against somatic antigens of the adult worm were not detected until day 56 but also persisted until the end of the experiment in the animals with adult worms. In 2 elk that had no adult worms at necropsy, anti-ES antibodies were detected transiently in both, while anti-adult worm antibodies were present transiently in one. These findings confirm the superiority of P. tenuis larval ES products over somatic adult worm antigens as serodiagnostic antigens, as previously observed in studies of infected white-tailed deer, and extend the application of the newly developed ELISA test in diagnosing and monitoring cervids experimentally infected with P. tenuis.     

Development of immune responsiveness to Ascarissuum antigens in pigs vaccinated with ultraviolet-attenuated eggs

Pigs fed $\text{Ascaris}$$\text{suum}$ eggs attenuated by short wave ultraviolet-radiation developed resistance to challenge infection. Per os inoculation of pigs on three successive weeks with 10,000 eggs irradiated to total exposures of 150,100 and 75 μW-min/cm², respectively, resulted in an 88% reduction in the number of larvae recovered from the lungs 7 days after challenge with 10,000 infective eggs. Peripheral blood lymphocytes (PBL) from vaccinated pigs were specifically stimulated in vitro to incorporate tritiated thymidine by egg hatching fluid (Ea) and by excretory-secretory products obtained from cultures in defined-media of second-stage larvae (L2) developing to third-stage (L2–3) and from cultures of third-stage larvae developing to fourth-stage (L3–4). PBL were also specifically stimulated by living L2. Ea and L2 stimulated pig PBL significantly at 7 days after the first inoculation; responses to L2–3 and L3–4 developed 7 days after a second inoculation. The antigen-responsive cells in the PBL population were non-immunoglobulin-bearing lymphocytes. Antibodies to Ea and L2–3 were not detected in the serum of vaccinated pigs, and only 3 of 7 pigs had low concentrations of serum antibodies to L3–4.     

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.     

Complement activation and fibrinolysis during infection with Theileria parva (East Coast fever) in cattle

The role played by complement and fibrinolysis in the pathogenesis of pulmonary oedema and the disseminated, subendothelial, petechial haemorrhages in cattle infected with $\text{Theileria parva}$ was investigated. There was a marked reduction in total haemolytic complement in animals that died of this disease whereas the survivors showed only transient changes. The reduction in C3 levels in animals with severe disease, however, was equivalent to that in the survivors. Neither circulating soluble immune complexes nor free antibody against macroschizonts were detected in sera from cattle with severe infection. In addition to the complement changes, high levels of fibrinogen degradation products (FDPs) were detected in sera from the infected animals. The rate of production of FDPs and reduction in total haemolytic complement paralleled the clinical course of the disease. It is, therefore, suggested that the pulmonary oedema and disseminated petechial haemorrhages seen in East Coast fever may be the direct result of the combined effects of complement activation and consumption of fibrinogen and fibrin.     

Serum and abomasal antibody response of sheep to infections with Haemonchus contortus

Serum and abomasal IgA, IgG and IgM antibody response against adult worm, L3 and egg antigens of Haemonchus contortus was monitored by the ELISA technique after one or two infections with this nematode. Following the first infection, antibody levels in serum did not change materially. After administration of a challenge dose of infective larvae, antibodies of the three immunoglobulin classes in infected animals rose slightly, but this rise appeared later than the fall in the faecal egg counts. In contrast, in abomasal mucosa, IgA anti-larval antibody levels, which did not increase materially after the primary infection, rose rapidly after a transient inhibition when sheep were challenged. A close temporal relationship was observed between the rise in local anti-worm IgA antibodies and the self-cure reaction, but antibody levels fell rapidly after worm diminution. The local antibody response was thus considered to be related to immunity of sheep to H. contortus.     

The anthelmintic efficacy of ivermectin in experimentally infected cattle

The 22,23-dihydro derivative (ivermectin) of the major B₁ components of the avermectins was evaluated, by oral and parenteral routes, for anthelmintic activity in cattle experimentally infected with 7 gastro-intestinal nematode species and lungworm. Treatment at three dosage levels was administered when the parasites were in the L₄ developmental stage or when adult. The dosage expected to remove 95% of the nematodes (ED₉₅) by each route of administration against the various developmental stages of each species of parasite was calculated by methods of linear regression to choose dose levels to be used in subsequent developmental field trials. Parenterally, efficacy against L₄ stages of Haemonchus placei, Ostertagia ostertagi, Trichostrongylus axei, T. colubriformis, Cooperia oncophora, C. punctata, Oesophagostomum radiatum and Dictyocaulus viviparus exceeded 99% with treatment at 0.2 mg ivermectin/kg. Parenteral treatment of the adult parasites resulted in at least 98% reduction of worm burdens, except $\text{? 90\%}$ for T. colubriformis, with 0.2 mg/kg. Oral treatment of L₄ stages of development with 0.2 mg/kg resulted in at least a 95% reduction in worm burdens of each of the 8 species of parasite. Oral treatment of the adult worms with 0.1 mg ivermectin/kg produced at least a 98% decrease in each parasite species, except for T. colubriformis (90% reduction) for which dose-response calculations suggest 95% efficacy with oral treatment at 0.2 mg/kg.     

Enhanced protection against the migratory phase,but defective protection against the intestinal phase of Strongyloides venezuelensis infection in cotton rats,Sigmodon hispidus

The protective capacity of the cotton rat, Sigmodon hispidus, against the migratory and intestinal phases of Strongyloides venezuelensis infection was examined. After subcutaneous infection with infective larvae (L(3)), adult worm recovery rates from male and female animals on Day 71 were only 0.10% and 0.06% of initial dose, respectively. To determine whether this enhanced protection was expressed during the migratory phase or the intestinal phase, larval recovery from the lungs of cotton rat was evaluated 3 days after subcutaneous L(3) infection. The larval recovery rate was only 0.5% of initial dose and about 40-fold lower than that from control mice. Protection in the intestine was also evaluated after intraduodenal implantation of adult worms. About 30% of implanted worms became established and worm burden remained constant until Day 28. Despite a high worm burden on Day 28, EPG was about 25-fold lower than the peak count. To evaluate expulsive capacity and monitor the cellular responses in the intestine of cotton rats, adult Nippostrongylus brasiliensis worms were implanted in addition to S. venezuelensis. Cotton rats were unable to expel adult S. venezuelensis worms, even after 21 days of observation. Although the number of mucosal mast cells increased significantly, the intraepithelial migration of mast cells was not observed. In contrast, N. brasiliensis was expelled by Day 6 in association with goblet cell hyperplasia. These results suggest that in cotton rats, the defective intestinal protection against adult S. venezuelensis worms results from dysfunction of mucosal mast cells.