A Study of Complement-fixation and Serum Agglutination Tests on Vibrio fetus Infected and Immunized Cattle

Complement-fixation (CF) and tube agglutination (TA) tests for demonstration of Vibrio fetus antibodies were conducted on the sera of three groups of ten heifers. One group was vaccinated subcutaneously with a commercial V. fetus var venerealis bacterin and challenged intra-utero, at the external os cervicus one month later; the second was infected only and the third vaccinated only.

The vaccinated cattle developed high CF serum titers, but no such increase was observed in animals infected only. A moderate increase in serum antibody titers was demonstrated by the TA test following either infection or vaccination; although titers observed were not higher than those observed in the sera of some apparently normal uninfected animals. The group receiving both vaccine and challenge was the only one in which significant serum antibody titers were demonstrable by the TA test. The sera of these animals also had significant titers in the CF tests.

The CF and TA tests detected serum antibodies produced by the parenteral inoculation of V. fetus antigen. These two tests were of limited value in detecting serum antibodies from animals with genital V. fetus var venerealis infection, although the formation of local antibodies was demonstrable by the vaginal mucus agglutination test.

     

Antibody recognition to secreted proteins of Mycobacterium avium subsp. paratuberculosis in sera from infected ruminants

Two liquid culture media to obtain secreted proteins of Mycobacterium avium subsp. paratuberculosis at different incubation periods were evaluated. Middlebrook 7H9-OADC (7H9) and Watson-Reid (WR) broths were inoculated with a field strain of M. paratuberculosis and growth curves determined using nonlinear regression analysis. Most culture filtrate (CF) proteins were of low molecular weight and reacted strongly against sera from cultured-positive cases of paratuberculosis. CF proteins obtained in WR yielded a higher number of bands and were detected earlier than those obtained from 7H9. A high degree of variability in CF protein immunoreactivity was seen among infected animals. Sera from cattle with clinical paratuberculosis or heavy fecal shedders of M. paratuberculosis reacted more intensively and to more CF proteins than did sera from other infected cattle. Immunoblots showed differences in antibody binding to CF proteins when sera were absorbed with M. avium but not with others environmental mycobacteria. Immunoblots with sera from infected goats and a sheep showed reactivity with proteins of 32, 33 and 46 kDa both before and after the sera were absorbed with M. phlei. Antibodies found in serum of infected deer reacted with CF proteins in a similar way as did for cattle. These results suggest that a pool of CF proteins of M. paratuberculosis could be good candidates as antigens for serodiagnosis of paratuberculosis.     

Complement-fixation and Hemagglutination Antigens from a Chlamydial (Ornithosis) Agent Grown in Cell Cultures

Methods are described for the preparation of complement-fixation (CF) and hemagglutination (HA) antigens from the Texas turkey ornithosis agent grown in McCoy cell culture monolayers. The particulate antigens prepared for this study were satisfactory for testing mammalian sera by direct CF tests and avian sera by indirect CF or modified direct CF tests. Comparison of titers were made on human, bovine and ovine sera using direct CF tests employing antigen prepared for this study, 6 BC yolk sac antigen, and a commercially available antigen.

The HA antigen agglutinated mouse erythrocytes, but it was not of value in hemagglutination inhibition tests because of “nonspecific” inhibitors in both mammalian and avian sera.

     

Complement-Fixing And Neutralizing Antibody Response To Bovine Viral Diarrhea And Hog Cholera Antigens

In calves inoculated with bovine viral diarrhea (BVD) viruses and soluble antigen, the complement-fixing (CF) antibodies appeared before serum-neutralizing (SN) antibodies and remained at high levels throughout the test period. A rapid rise in SN antibodies occurred after challenge with homologous virus with no apparent effect on CF antibody levels.

The CF antibody responses in calves infected with cytopathogenic NADL-MD and noncytopathogenic CG-1220 viruses were similar whereas SN antibody responses indicated strain specificity by reciprocal cross-neutralization tests.

The CF antibody levels in 5 hog cholera (HC) antisera were assayed using the soluble antigen of NADL-MD BVD virus. No demonstrable SN antibodies were present in four HC antisera tested against NADL-MD virus, but a significant titer was present in the commercially prepared antiserum.

Virus was reisolated from animals infected with BVD viruses by buffy coat culture technique during 3 weeks postinoculation, even when significant levels of CF and SN antibodies were present.

     

Detection and molecular characterization of Theileria sp. in fallow deer (Dama dama) and ticks from an Italian natural preserve

The prevalence of piroplasms in a closed population of fallow deer (Dama dama L.) living in the Italian preserve of “Bosco della Mesola” - Ferrara (Mesola wood) was investigated. Blood samples and ticks were collected from 62 fallow deer. On microscopic observation, 28 (45.0%) blood samples were positive for piroplasms while PCR provided evidence for piroplasms infection in 47 (75.8%) fallow deer. The 67 ticks, collected from positive and negative animals, were identified as Ixodesricinus L., 1758 (89.6%) and Haemaphysalisconcinna Koch, 1844 (10.4%). At the PCR, four samples of I. ricinus were positive for piroplasms. The sequences of the 18S rRNA gene from both blood and ticks were identical and showed high identity (99.6%) with Theileria sp. 3185/02 (DQ866842) and Theileria capreoli (AY726011) from roe deer. Interestingly, the phylogenetical analyses evidenced differences between the Theileria strain from Mesola wood and the ones isolated in fallow deer from other Italian areas.     

Theileriosis in a Missouri beef herd caused by Theileria buffeli: case report, herd investigation, ultrastructure, phylogenetic analysis, and experimental transmission

A 6-year-old Simmental cow infected with Theileria buffeli had a clinical disease characterized by theilerial parasitemia, macrocytic normochromic anemia with acanthocytosis and spherocytosis, lymphoid hyperplasia (lymphocytosis, edematous lymphadenomegaly), dysproteinemia, evidence of liver disease, and a low serum antibody titer against T. buffeli. The cow was in a herd in which all cattle originated in Missouri; 22/75 (29%) of cattle had a theilerial parasitemia and 26/75 (35%) had titers to T. buffeli of > or =1:160. Classification of the Missouri bovine organism as T. buffeli was based on DNA sequencing and comparison to sequences for T. buffeli and Theileria sp. type A obtained from GenBank. Intraerythrocytic veils and piroplasms were seen during transmission electron microscopy. The organism was successfully transmitted to two splenectomized calves, which developed mild anemias while parasitemic. Blood from the second calf was used as the source of T. buffeli antigen for an indirect immunofluorescence antibody test. Theilerial isolates from a Missouri white-tailed deer were also sequenced and resembled Theileria sp. types F and G and were not consistent with the bovine organism.     

Evaluation of electrophoretic immunoblotting for Brucella ovis infection in deer using ram and deer serum

AIMS: Recently the first case of natural infection of deer with Brucella ovis was discovered. The aim of this study was to develop and evaluate an electrophoretic immunoblotting method for testing deer serum for specific B. ovis antibodies. METHODS: An existing immunoblotting method for sheep serum was altered by using a recombinant protein G-alkaline phosphatase conjugate and Tris-buffered saline containing 3% non-fat dry milk powder for the blocking step and the serum and conjugate dilutions. The method was evaluated using 106 sheep sera from B. ovis - negative, accredited flocks, 69 sera from chronically infected rams shedding B. ovis in their semen, 110 sera from a B. ovis-infected flock, 18 sera from stags from which B. ovis was isolated, and 48 sera from deer flocks free from B. ovis infections. The immunoblotting method was applied to another 85 deer sera. RESULTS: The sensitivity of the new immunoblotting method was 98.6% for sheep and 94.4% for deer, and the specificity 99.1% for sheep and 100% for deer. Sixty-nine out of 97 deer sera, originating from the property from which the first B. ovis deer case had been reported, tested positive or suspicious in the complement fixation test. Of these, 53 sera exhibited staining patterns in blots typical for B. ovis infections and also one serum which was negative in the CFT. Only six out of 1498 deer sera. from throughout New Zealand had positive or suspicious reactions in the B. ovis complement fixation test. Of these, one exhibited a staining pattern in the blot suggestive of a B. ovis infection, while four showed patterns of suspicious reactions. CONCLUSION: The new immunoblotting technique is useful as a confirmatory serological test method for B. ovis infections in deer.     

Polyclonal antibody–based immunohistochemical detection of intraleukocytic Theileria parasites in roan and sable antelopes

Theileria parasites commonly infect African wild artiodactyls. In rare roan (Hippotragus equinus) and sable (H. niger) antelopes, Theileria sp. (sable)-associated calf mortalities constrain breeding programs. The pathogenicity of most leukocyte-transforming Theileria spp. originates in their invasion of and multiplication in various mononuclear leukocytes, the transformation of both infected and uninfected leukocytes, and their infiltration of multiple organs. Understanding the pathogenesis of theileriosis can be improved by the use of immunohistochemistry (IHC) to identify the localization of the parasites in tissue sections. Our aim was to develop a reproducible IHC assay to detect leukocyte-associated Theileria parasites in formalin-fixed, paraffin-embedded roan and sable tissues. Polyclonal antibodies were purified from the sera of 5 roans from an area endemic for Theileria sp. (sable) and tested for IHC reactivity in 55 infected and 39 control roan and sable antelopes, and for antigen and species cross-reactivity in an additional 58 cases. The 3 strongest antibodies consistently detected intraleukocytic theilerial antigens in known positive cases in roan and sable antelopes, and also detected other Theileria spp. in non-hippotraginid wild artiodactyl tissues. The antibodies did not cross-react with other apicomplexan protozoa, with the exception of Cryptosporidium. Given that PCR on its own cannot determine the significance of theilerial infection in wild ruminants, IHC is a useful laboratory test with which to confirm the diagnosis in these species.     

Preparation of monoclonal antibodies to Theileria sergenti and their reactivity to antigens in experimentally infected cattle

Two distinct monoclonal antibodies (3-H and 11-D) were produced against Theileria sergenti. These two new products, together with monoclonal antibody 1-G obtained in a previous study, were used to detect the parasites in experimentally infected cattle. During the first period of dexamethasone treatment, which was carried out to increase parasitemia in the infected cattle, the number of erythrocytes detected by 3-H, 11-D and 1-D increased in two experimentally infected calves. During the second period of dexamethasone treatment, the number of infected erythrocytes detected by 3-H and 11-D were similarly increased, but the number of infected erythrocytes detected by 1-G did not increase and infected erythrocytes in one calf were not detected by 1-G.     

Indirect fluorescent antibody and complement fixation tests in the diagnosis of bovine theileriosis and babesiosis in Japan

Antibodies against Theileria sergenti and Babesia ovata were detected by indirect fluorescent antibody (IFA) and complement fixation (CF) methods. Both of the antibodies against T. sergenti and B. ovata could be detected with a single IFA testing procedure. Complement fixing antibody tests for T. sergenti in experimentally infected cattle were positive for about 70 days after the parasites could no longer be detected in the blood smear, and were negative thereafter. B. ovata CF antibody titres were negative on the 270th day post-infection, while the IFA tests against both parasites were positive for longer periods. B. ovata IFA-test titres remained relatively high until the 420th day after inoculation. In a survey of sera from naturally infected animals, the rate of detecting antibodies against both parasites was higher using the IFA test than using the CF test. The IFA method was more effective for diagnosis of Babesia infection than the CF technique.     

Abnormality of osmotic fragility and morphological disorder of bovine erythrocytes infected with Theileria sergenti

The osmotic fragility and the surface structure of erythrocytes obtained from 3 calves infected with Theileria sergenti and from 3 phlebotomized ones were compared. As the parasitemia progressed, the osmotic fragility of the erythrocytes significantly increased in the infected calves. Particularly the hemolysis ratio in the isotonic area (21.5-94.1%) obviously increased. On the other hand, the percentage of parasitized cells in the erythrocytes did not show so much high values (16.1-21.3%). Similar phenomenon was found in each different percentage of erythrocytes suspension which was separated from density gradient centrifugation. No significant difference in the serum osmotic pressure between the infected calves and the phlebotomized calves was found. By scanning microscopy, the erythrocytes of infected calves, which were collected at the crisis period of parasitemia, were almost completely deformed and showed echinocyte form. Moreover, the appearance ratio of echinocyte form in the erythrocytes population was superior to the percentage of parasitized erythrocytes. Similar membranous alterations were also observed in the erythrocytes of grazing cattle in the crisis period of the theileriosis. It was proven that abnormality of osmotic fragility and morphological disorders of erythrocytes occurred not only in parasitized erythrocytes but also in non-parasitized ones in T. sergenti parasitemia.     

Serological relationship of a Japanese Babesia species and Babesia bigemina by the complement fixation and capillary-tube agglutination tests

The complement fixation (CF) test and the capillary-tube agglutination (CA) test were used to study the antigenic relationship between Babesia bigemina and the large Babesia species frequently infecting cattle in Japan. The CF antigen was prepared from parasitized erythrocytes by extraction with distilled water. The CA antigen was prepared from parasitized erythrocytes by mild sonification of mixtures of Babesia and erythrocyte stroma, following lysis of the erythrocytes with hypotonic saline solution. All the sera used were collected from experimentally-infected cattle. Cross reaction was demonstrated between the Japanese Babesia species and B. bigemina. There was, however, a difference of two dilutions in titer between homologous and heterologous antibody in the CF test, and a difference of more than three tubes in titer between both antibodies in the CA test. It was possible, therefore, to distinguish the Japanese Babesia species from B. bigemina by the CF and CA tests.     

Une étude expérimentale sur la variation du taux d'anticorps sériques suivant le cycle évolutif des oestres du cheval

The changes of antibody titers in the sera of colts infested naturally or artificially with Gasterophilus have been determined in relation to the life cycle of this arthropod using passive hemagglutination, complement fixation, double diffusion techniques and saline extracts of antigens from the third larval stages of Gasterophilus intestinalis and G. nasalis.

In the sera of the infected animals the hemagglutinating antibodies were present at low titers at the third week post-infestation by using somatic extract of G. intestinalis and at the seventh week in case of G. nasalis. At eight weeks post-infestation the antibody titers reached their maximum 1:8192 (G. intestinalis) and 1:4096 (G. nasalis), then dropped at 12 weeks post-infestation.

The complement fixing antibodies were present occasionally between the seventh and 11th weeks after infestation. Precipitating antibodies were absent in all sera.

     

Radioimmunoassay for Anaplasma marginale antibodies in cattle

A radioimmunoassay is described for use in the detection of Anaplasma marginale antibodies in cattle sera. Optimal sensitivity and specificity were obtained by using 2 antigens, an A marginale antigen and a RBC antigen (obtained before infection was established) from the same calf. In addition, sera were preabsorbed with RBC from healthy cattle and with sonicated Babesia bovis. Of 86 sera obtained from cattle with A marginale infection (as determined by blood smear examination or by results of subinoculation of blood from such infected cattle into splenectomized calves), 85 had positive results by use of this test. Of 100 sera obtained from cattle raised in an anaplasmosis-free area, 98 yielded negative results, and sera obtained from 35 cattle (97 sera) infected with B bigemina and from 18 cattle infected with Theileria orientalis yielded negative results. By use of this test, 99 of 100 sera obtained from cattle with B bovis infection were negative for A marginale. Anaplasma marginale antibodies were detected in 18 cattle that had been pastured in a Boophilus microplus-free area for 2 years after natural infection. After 3 years, 16 of these cattle were still seropositive for A marginale. Sixteen cattle pastured in a Bo microplus-infested area had detectable antibody against A marginale 27 months after initial infection with A marginale. Sensitivity and specificity of the test were assessed as 98.8% for each.     

Enzootic Pneumonia of Pigs: Complement-Fixation Tests for the Detection of Mycoplasma Antibodies in the Serum of Immunized Rabbits and Infected Swine

The direct, the modified direct and the indirect complement-fixation tests were investigated as methods for the detection of antibodies for the enzootic pneumonia mycoplasma and for Mycoplasma hyorhinis in the serum of infected pigs and of immunized rabbits.

Only the modified direct complement-fixation test in which the guinea-pig complement is supplemented with fresh, normal unheated calf serum was suitable for the detection of mycoplasma antibodies in sera of infected swine. Based on the close correlation between the production of typical lung lesions in experimentally infected pigs and the appearance of significant serum antibody titres, the modified direct complement-fixation test provides for the first time a sensitive, specific in vitro method for the detection of enzootic pneumonia in the live pig. This test also permitted the in vitro differentiation of the mycoplasma causing enzootic pneumonia from M. hyorhinis which causes polyserositis.

Antibodies in the sera of rabbits were demonstrable by the ordinary direct complement-fixation test. However, in contast to the observation made with swine sera, only a slight quantitative antigenic difference between the enzootic pneumonia mycoplasma and M. hyorhinis was seen when the tests were performed with rabbit serum antibodiies.

     

Comparison of two antigens for use in an enzyme-linked immunosorbent assay to detect African swine fever antibody

Two African swine fever virus (ASFV) antigens were tested for use in an ELISA to detect antibody to ASFV. Antigens used were the cytoplasmic soluble fraction (CS-P) of infected cells grown in the presence of porcine serum and the semipurified viral structural protein VP73 (SVP73). Both antigens were tested by ELISA against 72 sera obtained during several ASF field episodes and from ASFV-inapparent carriers. Of the 72 sera, only 2.8% has positive results by ELISA against CS-P antigen; 60% of positive-reacting sera (to both antigens) had higher ELISA values when the CS-P antigen was used. Samples (with positive results) that reacted only to CS-P antigen had results confirmed by immunoblot analysis. Such sera reacted against ASFV-infection proteins IP25, IP25.5, and IP30, but not against IP73. In time-course experiments to detect appearance of ASFV-antibodies in infected miniature pigs, antibodies were detected by immunoblot analysis on postinoculation day (PID) 8. At that time, only the polypeptides IP25, IP25.5 IP30, and IP31 were recognized; IP73 and IP12 were first detected 3 and 4 days later, respectively. In the same experiments, ASFV antibodies were detected by ELISA, using CS-P or SVP73 antigens, on PID 7 and 9, respectively. These results could explain the percentage of sera not having positive results by ELISA using SVP73 antigen, if the sera were obtained from ASFV-infected pigs during the first days of infection before induction of antibody response against the IP73 protein. This feature makes the use of CS-P antigen advantageous in early serologic detection of ASFV-infected pigs.     

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.     

Paratuberculosis in cattle and free-living exotic deer.

Paratuberculosis was studied among dairy cows and exotic deer that shared grazing areas at Point Reyes National Seashore, California. Of the 10 dairy herds tested, 5 (50%) were infected with Mycobacterium paratuberculosis (based on results of fecal culture). Mycobacterium paratuberculosis was cultured from 9 (8.7%) of the 103 bovine fecal samples and from 4 (3.9%) of the 103 bovine rectal mucosa scapings tested. Of 89 fecal samples from 52 axis deer (Axis axis) and 37 fallow deer (Dama dama), 5 (9.6%) and 3 (8.1%), respectively, contained M paratuberculosis. Culture of intestinal necropsy samples from the same deer indicated that 3 (5.8%) of the axis deer and 2 (5.4%) of the fallow deer were infected with M paratuberculosis. The cows were tested for serum antibodies by the complement-fixation test and by radioimmunoassay. Of 95 sera tested by complement fixation, 15 (15.8%) were positive, as were 15 (14.7%) of 102 sera tested by radioimmunoassay. Culture results and serologic test results were compared on a herd basis.     

Humoral Antibody Responses of Swine Infected Experimentally with Group E Streptococcus I. Whole Cell Agglutinin Response

Reference streptococcal antisera and sera collected from swine infected experimentally (by intranasal inoculation or contact exposure) with group E Streptococcus (GES) were studied in a tube agglutination system using whole GES cells.

Specificity studies revealed common group specific antigen among GES serotypes I and III, GES strains devoid of type specific antigen (untypable by ring precipitin testing) and group P and group U Streptococcus. The group specific antigens were not agglutinated by GES type specific antisera or by group specific antisera against Streptococcus groups A, B, C, D, F, G, H, K, L, M, N, or O. Results of the study suggested that GES serotypes I and III are invalid; i.e., they are devoid of type specific antigen.

Groug E Streptococcus type specific antigens II, IV, and V were agglutinated significantly only by their homologous antisera.

Experimentally infected swine developed significant titers against both the group and type specific antigen of GES. Antibodies appeared from three to eight weeks postexposure and persisted for the duration of the experiment (six months). The potential utilization of the whole cell agglutination (WCA) test for detection of GES carrier swine is discussed.

     

Bovine Vibriosis: The Distribution and Specificity of Antibodies Induced by Vaccination and Infection and the Immunofluorescent Localization of the Organism in infected Heifers

Two groups of three Holstein heifers were immunized respectively with Vibrio fetus venerealis and Vibrio fetus intestinalis incorporated in Freund's complete adjuvant. Both serum and vaginal mucus agglutination titers increased following immunization. Vaginal mucus samples were more frequently positive when the homologous cells were used as antigen in the agglutination test.

Ten non-immunized heifers were inoculated with another strain of V. fetus venerealis and slaughtered at periods of 30 to 40 and 60 to 70 days post-inoculation (DPI). Agglutinating antibodies were present in the vaginal mucus of some infected individuals by five weeks post-inoculation. In the course of the experiment 11 vaginal mucus samples were obtained which agglutinated heated cells of the infecting strain; one aggglutinated whole cells. Precipitins toward homologous antigens could not be demonstrated in vaginal mucus but four of six samples tested precipitated a heat stable extract from an intestinal strain of the same O-serotype. Bacterial antigen was detected by immunofluorescence on the surface, as well as within and beneath the epithelium at all levels of the reproductive tract regardless of time of slaughter. Lesions in infected animals consisted of focal and diffuse lymphocytosis, plasmacytosis, and epithelial vacuolation. Diffuse neutrophilic infiltration of the oviducts was observed.

Agglutinins appeared in the serum of each of nine heifers immunized with whole cells of same venereal strain. Group mean serum titers for whole and heated cells were 1/28,000 and 1/1,300 respectively. Vaginal mucus samples agglutinated whole cells in 48% of tests while 6.3% reacted with heated cells. Serum, but not vaginal mucus, of immunized animals precipitated soluble antigens of the immunizing strain. The immunizing strain of V. fetus did not infect the reproductive tract of any of six immunized heifers upon challenge.