CF Test Research Articles

?Paramushir? virus, a new arbovirus, isolated from ixodid ticks in nesting sites of birds on the islands in the north-western part of the Pacific Ocean basin

From ticksIxodes signatus Birulya, 1895 andI. putus Pickard-Cambridge, 1878, collected at Paramushir (North Kuril isls), Tulenyi (Okhotsk sea), Bering (Commodore islands) at the nesting sites of colonial sea birds 4 isolates of a new virus were isolated. The CF test showed that these isolates were identical and did not have any antigenic relationships with 18 antigenic groups and 26 ungrouped viruses isolated from ticks.

Serologic studies in 11,460 pregnant women during the 1972 rubella epidemic in Israel.

A total of 11,460 women in the first 4 months of pregnancy, either exposed to or with suspected clinical rubella were tested for rubella antibody during an extensive epidemic of this disease in 1972. The proportion of women who were seronegative decreased from 25% at the beginning of the epidemic to 16% toward the end. In 542 (79%) of 682 cases with suspected clinical rubella, the laboratory findings were consistent with recent rubella infection, while in the remaining 140 cases of suspected clinical rubella recent infection could be excluded by serologic tests. CF tests were more useful than HI for confirmation of clinical rubella, and especially for retrospective diagnosis, since elevated titers (larger than or equal to 1:16) were suggestive of recent infection. Paired sera were tested from 4203 patients exposed to rubella: 1126 of the subjects were seronegative and the remaining 3077 seropositive (HI larger than or equal to 1:16) on first testing. In the seronegative group, 278 seroconversions were detected (24.6%): 247 cases of clinical rubella (21.9%) and 31 seroconversions without clinical symptoms (2.7%). Among the seropositive subjects in 2306 instances (74.9%) recent subclinical rubella could be excluded by low and stable HI or CF antibody titers in paired sera. In 32 (1.1%) an antibody rise (HI or CF) without clinical symptoms was detected, and in the remaining 739 (24%) high CF titers were found in paired sera, and these were classified as suspected subclinical rubella.

Serologic evidence of the postoperative persistence of hydatid cysts in man.

The sensitivity of the haemagglutination, complement-fixation and indirect fluorescent antibody tests in the diagnosis of primary and recurrent hydatid disease is presented. In the first, diagnostic titres were obtained in 90% of 50 patients with hepatic and 64%, 60%, and 68% of 25 patients with pulmonary cysts, respectively. In the postoperative study of 62 patients, elevated titres of HA, CF and IFA antibodies in 30 led to the presumptive diagnosis of recurrent disease, confirmed later by radiological and surgical follow-ups. In the 32 others the proportion of positive reactors was 29%, 6% and 28% in HA, CF and IFA tests. These patients had no detectable cysts. This indicates that in postoperative patients a negative CF test may be of a better prognostic value in indicating absence of cysts than the HA and IFA tests.

A Specific Complement-Fixation Test for Human Hepatitis A Employing CR326 Virus Antigen. Diagnosis and Epidemiology

A specific diagnostic complement-fixation test for hepatitis A antibody in human serum was described employing livers of marmosets infected with CR326 strain human hepatitis A virus. Persons with hepatitis A, but not hepatitis B, developed hepatitis A CF antibody shortly after the onset of illness and this persisted thereafter. Good agreement was noted in the development of CF and neutralizing antibodies in hepatitis A cases. Hepatitis A was shown to occur in a person with hepatitis B antigenemia and hepatitis B occurred in persons with hepatitis A antibody. Most persons with hepatitis A who were tested, but none of those with hepatitis B, developed increased anticomplementary activity in their sera at the time of onset of illness. At least one patient with hepatitis A developed antibody against normal liver that persisted. The possible inplications of this in relation to pathogenesis and to non-specific diagnostic tests in hepatitis were discussed. A limited epidemiologic study of a family outbreak of hepatitis in Costa Rica and of a group of young adults in our epidemic country acquire their infections at an early age and are immune thereafter; persons in areas of relatively low incidence may proceed into adulthood without experience with hepatitis A. The CF test should provide an excellent tool for diagnosis and for epidemiologic investigation of hepatitis A and should be of considerable value to detect hepatitis A virus in attempts to propagate the virus in cell culture.

SEROLOGICAL METHODS IN THE DIAGNOSIS OF BLUETONGUE

The viral antibodies in the serum of cattle, sheep and deer are not detectable in the ordinary direct CF test. We have shown that the so-called "non complement-fixing" viral antibodies in the serum of these animal species can be demonstrated by the MDCF test. The MDCF test, as well as the micro AGP test, are group reactive, with all BT isolates studied. However, it is possible with these tests to differentiate antigenically BT virus from EHD virus. The FAT was also useful in differentiating BT virus grown in TC cells from the EHD virus. In the PRN test, all the BT virus isolates studied cross reacted and although quantitative differences were frequently observed, no obvious antigenic classification was possible with the antiserums used in this work. Reactions between the BT viral isolates and the EHD virus were all within the limits of what is presently considered to be non-specific inhibition.

Distribution ofBlastomyces dermatitidisin dogs with skin test and serologic results following airborne infection

Reported is a short term (16 week) experiment to determine the distribution of B. dermatitidis in dogs when infected by the natural inhalation route by receiving about 250,000 viable particles during a 24h. exposure. Four dogs were positive to blastomycin and 1 to histoplasmin prior to exposure. Post exposure, all dogs developed positive skin tests to blastomycin by the 4th week with considerable cross reaction to histoplasmin. Serologic tests showed more poritive CF tests to Histoplasma than to Blastomyces so that the test appears of no practical value. Although 22 tissues were cultured on each dog, only 1 or 7 dogs was positive for B. dermatitidis by 2 weeks. At 4 weeks, 57% of the dogs were positive by culture and at 12, 15, and 16 weeks, all were positive. One dog developed eye lesions and became emaciated.

Immunochemical Studies on East Coast Fever: II. Partial Segregation and Characterization of the Theileria parva Piroplasm Antigen

The piroplasm antigens of Theileria parva were isolated from infected bovine erythrocytes either by sonication or by French pressure cell disruption. A soluble bovine erythrocyte antigen was also isolated from both infected and noninfected blood. Both piroplasm and erythrocyte antigens were readily detected by either complement fixation or immunodiffusion tests. Partial characterizations were achieved by Sephadex G200 and Sepharose 2B chromatography, density gradient centrifugation, and precipitation by ammonium sulfate solution. Results indicated that the piroplasm antigen occurred in a wide molecular weight range of 4 to 20 million with an approximate sedimentation coefficient range of 7 to 15S. The erythrocyte antigen had a molecular weight range of 20 to 40 million with sedimentation coefficient ranging from 3 to 9S. The piroplasm antigens were partially separable from the erythrocyte antigens by differential ammonium sulfate precipitations. The problems inherent in the preparation of soluble specific antigens from parasites that normally develop intracellularly have been recently reviewed by Fife (1972). These problems most often relate to the inability to separate completely the parasites from remnants of the host cell, particularly red cell stromata. Residual contaminants often give rise to preparations of low specificity and sensitivity, and if CF tests are used for assay, the antigens are often anticomplementary as well. Recent work by D'Antonio et al. (1966) on Plasmodium knowlesi-infected monkey blood, and Amerault and Roby (1968) on Anaplasma marginale-infected bovine blood has shown that passage of parasitized blood through a French pressure cell selectively fragments the erythrocyte membranes without apparent damage to the parasites. Resultant antigen preparations in both instances have Received for publication 29 October 1973. * Project supported by the United Nations Development Program, with the Food and Agriculture Organization of the United Naitons as the Executing Agency, in cooperation with the East African Community. The Project is also supported by the Overseas Development Administration of the United Kingdon (Research Projects R 2396 and R 2494), the United States Department of Agriculture, the Rockefeller Foundation, the International Atomic Energy Agency, and the Pfizer Corporation. been found highly specific and sensitive and without anticomplementary activity. Ultrasonication has also been used for the preparation of semipurified Babesia antigens (Goodger, 1971). Recently, work in this laboratory with the French pressure cell has resulted in the isolation and partial characterization of T. parva schizont antigens, using infected bovine lymphoblasts in cell culture as the source of material (Wagner et al., 1974). Similar techniques have been applied to T. parva-infected bovine blood (piroplasm stage). A comparison of the piroplasm antigens isolated from French pressure cell extracts and by sonication is the subject of this report. MATERIALS AND METHODS Steers of Bos taurus type were provided by the FAO project at EAVRO, Muguga, Kenya, and were infected with the Aitong strain of T. parva (Irvin et al., 1972) by the subcutaneous inoculation of a supernate extracted from infected adult Rhipicephalus appendiculatus ticks (Purnell et al., 1973). The inoculum used to infect the animals, popularly termed stabilate (Lumsden and Hardy, 1965) was preserved in liquid nitrogen (Cunningham et al., 1973). The animals were selected at a time when the level of parasitized erythrocytes rose above 50%. Five liters of infected blood were collected by venesection into 200 ml 0.1 MI phosphate-buffered 0.15 M saline solution (PBS) pH 7.2, containing 150,000 units of heparin. The parasitized erythrocytes were washed 4 times

MYOSITIS ASSOCIATED WITH INFLUENZA B INFECTIONS

An epidemic of acute myositis affecting children occurred in Seattle during March-May, 1973, with most pediatricians seeing several cases. The disease was characterized by a prodrome of upper respiratory symptoms, fever, and headache, followed in several days by sudden onset of refusal to walk or abnormal gait with toe-walking. Gastrocnemius-soleus muscles were chiefly affected, being tender, swollen and painful. Muscle symptoms, although dramatic, resolved completely within 3-7 days without special therapy. Transient elevations of serum CPK or SCOT were noted in 16 of 17 patients tested. Influenza B virus was isolated from throat swabs of 11 of the 17 patients studied. One of the isolates was characterized, and was antigenically “intermediate” between the 1967-71 isolates and B/Hong Kong/5/72. Paired sera from 12 patients were tested for HI antibodies to strain B/Hawaii/3/73 (an “intermediate”); (4 patients had a greater than 4-fold titer rise, and 4 had detectable but lesser titer rises. No titer rises by CF testing were detected for 8 other respiratory agents. This unusual complication of influenza has been previously reported in Stockholm (1955), Toronto (1969-70) and Boston (1972). The distinctive clinical picture of dramatic but self-limited calf muscle myositis following a flu-like illness makes this a clearly recognizable syndrome which should not be confused with dermatomyositis.

Serological and Immunological Studies of Pleuropneumonia of Swine Caused by Haemophilus Parahaemolyticus

The development of circulating antibodies for H. parahaemolyticus was studied in experimentally infected SPF pigs and in-contact SPF pigs. Blood serum titers were determined by a modified complement fixation test with normal SPF swine serum as a source of supplementary complement factor, and by an indirect haemagglutination test. CF and IHA titers became positive within the first 2 weeks following exposure to H. parahaemolyticus, and reached peak values after 2 to 7 weeks (Figs. 1 to 3). The exposed pigs proved immune, in that they showed no clinical symptoms on challenge after resp. 6, 9 and 11 weeks. While distinct titers were thus obtained with both tests in SPF swine experimentally exposed to H. parahaemolyticus, the CF test proved more specific than the IHA test when the 2 tests were compared in a field outbreak of polyserositis (Glassers disease) caused by H. parasuis. The CF test would therefore seem to be preferable to the IHA test in field diagnostic work (Table 1). A noticeable finding was that challenge did not elicit an anamnestic antibody response in any of the immune pigs (Figs. 1 to 3). This fact together with negative bacteriological findings in the animals in question would seem to suggest that the challenge dose was unable to establish a permanent infection in the respiratory tract of the immune pigs.

Yellow fever infection after recent convalescence from chikungunya fever: Report of a fatal case

An epidemic of chikungunya fever occurred in Luanda, Angola, at the end of 1970, being followed by, and partly concurrent with, an epidemic of yellow fever. In March 1971, a 16-year-old white boy, who had chikungunya fever 3 weeks before, was admitted to hospital with yellow fever. He had a severe illness ending in death on the 8th day. Serum taken on admission yielded YF virus designated as strain 47FA; it is essentially identical to YF (Asibi strain) virus. This serum had HI antibody only to CHIK virus, of 11 arbovirus antigens tested. A positive CF test for CHIK antibody in this serum confirmed the earlier infection with CHIK virus. The authors speculate that persons convalescent from chikungunya fever may be less resistant to infection with YF virus.

Antibodies To Mycoplasma pneumoniae: Correlation of Complement Fixation and Tetrazolium Reduction Inhibition Tests

SummaryThe role of lipoidal components of M. pneumoniae as antigenic determinants in vitro and in vivo is described.Sera with TRI or CF titers with lipid antigen of 8 or greater are usually positive in the ID test, suggesting that this test might be used as a screening test for antibody to M. pneumoniae.Since there is a high degree of correlation between TRI and CF titers when lipid extracts of M. pneumoniae are used as antigens, we feel that the lipid CF test will serve as an indicator of neutralizing antibody to M. pneumoniae.

Protein composition of coronavirus OC 43

A human coronavirus, strain OC 43, was propagated in suckling mouse brain and purified 5000-fold with, a 90% yield. Purity of the virus was confirmed by electrophoretic, ultracentrifugal, and electron microscopic procedures. Immunodiffusion and immunoelectrophoresis tests revealed one precipitin line with normal mouse brain, three with purified virus, and four with crude virus when tested against anti-pure virus or anti-crude virus animal serums. The association of a host cell antigen with the virion was confirmed by standard HI and CF tests. Polyacrylamide gel electrophoresis of solubilized purified virus revealed a minimum of six polypeptides with apparent molecular weights of 191,000 (No. 1), 104,000 (No. 2), 60,000 (No. 3), 47,000 (No. 4), 30,000 (No. 5), and 15,000 daltons (No. 6). A seventh band was occasionally found in the 165,000-dalton region of the gels. Four polypeptides contained carbohydrate and one contained lipid. Polypeptide No. 5 comprised 26% of the total viral protein and glycopolypeptide No. 3 comprised 23%. Three other components accounted for most of the remaining protein: polypeptide No. 4 (16%), glycopolypeptide No. 6 (14%), and glycolipopolypeptide No. 1 (13%). Glycopolypeptide No. 2 was 8% of the total protein. Bromelin digestion of the viral projections (spikes) removed glycopolypeptides No. 2 and No. 6. Association of the remaining polypeptides with structural components of the virion is only tentatively postulated. The buoyant density in potassium tartrate of the bromelin-treated virus was 1.15 g/cm 3 and of the intact OC 43 virion was 1.18 g/cm 3. By analytical ultracentrifugation the corrected sedimentation coefficient ( s 0 20w) of the OC 43 virion was determined to be 390 ± 16 S, and the apparent molecular weight ( MW a ) was calculated to be 112 ± 5 × 10 6 daltons.

THYROIDAL SUPPRESSIBILITY AND THYROGLOBULIN ANTIBODIES DURING TREATMENT OF THYROTOXICOSIS WITH ANTITHYROID DRUGS

In 71 thyrotoxic patients treated with antithyroid drugs two questions were examined: 1. When will the thyroid become suppressible and 2. which factor(s) may predict suppressibility. Besides clinical and recommended laboratory data, suppressibility of the thyroid gland and thyroidal antibodies (complement fixation test — CF test — and TRC test) were studied every three months.

Clinical reactivity and immunogenicity of hemagglutinin influenza vaccine

Subjects aged 3–6, 16–17 and 27–50 years were vaccinated with one dose of hemagglutinin influenza virus vaccine. Clinical reactions, hemagglutination-inhibiting (HI) and strain- and type-specific complement-fixing (CF-V and CF-S) antibodies were determined in sera taken before and four weeks after vaccine administration. The results indicated that the reactogenicity of the vaccine was very low. The HI antibody response differed with the age of the vaccinees, apparently being conditioned by prior exposure to the various influenza virus subtypes. The results of CF tests using strain-specific V antigens corresponded in general with HI tests, with two marked exceptions. In the youngest group nearly half of the subjects developed CF antibody to V-Swine, while all of them remained without antibody detectable in the HI test. However, when V antigen was used instead of intact virus as hemagglutinin, the post-vaccination sera of these subjects also reacted positively in the HI test. Secondly, a number of prevaccination sera from persons aged 27–50 years possessed CF antibody to A/PR 8 in the absence of homologous HI antibody. Among these subjects the antibody response to both A/PR 8 and Swine was more marked in the CF test than in the HI test. After vaccine administration most of the subjects developed antibody or responded by an antibody increase to the S antigens of both influenza A and B. No significant differences were found after intradermal (0.1 ml) and subcutaneous (0.5 ml) administration of one dose of vaccine.

Complement fixation kinetic studies with dengue viruses

A complement fixation kinetics test is described for dengue viruses employing antisera obtained from a variety of sources using the standard NCDC micro CF test with parameters that included various time periods of antigen-antibody-C' interaction and various concentrations of antigen.

Serum immunoglobulin D and malaria antibodies in South Vietnamese residents.

Serum immunoglobulin D concentrations were found to be elevated in Vietnamese and Montagnard but not in Cambodian residents of a Vietnam village which is highly endemic for malaria. Although a high frequency of positive malaria CF tests was demonstrated in all three groups, there was no significant correlation between the serum IgD levels and the malaria CF titres. Serum IgD levels in Vietnamese residents of a non-malarious community were similar to those of United States blood donors, however, in the former groups, a high frequency (i.e., 90%) of low titre CF reactions was observed, compared to a frequency of 8% low titre CF reactions in the U.S. donors.

Purification and further characterization of an "IBV-like" virus (coronavirus).

Examination of crude mouse brain harvest of coronavirus strain OC 43 by ultracentrifugation, sucrose density gradients, immunodiffusion, and immunoelectrophoresis demonstrated that the antigens active in the conventional CF and HA tests were associated with the virion. No soluble antigenic components were detected. Purification of the virus was achieved by adsorption and elution from human “O” erythrocytes followed by batch calcium phosphate chromatography. Studies with the purified virus confirmed our findings with the crude mouse brain harvest. Treatment of crude and purified antigen with a limited number of disrupting substances failed to yield soluble CF or HA subunits, but a systematic investigation of other techniques for releasing viral subunits is now made possible through the simple purification procedures described in this report.

Comparison of macrocomplement and microcomplement fixation techniques used in fungus serology.

A comparative evaluation was performed on the micro- and macrocomplement fixation (CF) tests that are used as standard procedures in the serodiagnosis of blastomycosis, coccidioidomycosis, and histoplasmosis. Tests with 937 sera from suspected and culturally proven cases of these diseases against yeast-form antigens of Blastomyces dermatitidis and Histoplasma capsulatum and against the soluble antigens, coccidioidin and histoplasmin, revealed that the microtiters were within +/- 1 dilution of the macrotiters in 83 to 93% of the sera. Tests on randomly coded quality control sera revealed the microform of the CF test to be highly reproducible. Our studies indicate that the results obtained by the two tests have similar diagnostic and prognostic interpretations. Because of the sensitivity, reproducibility, economy, and ease of performance, the microtest is highly recommended for use in fungus serology.

SV40-tumorigenesis in mouse.

SV40‐transformed cell lines from 8 kidneys and 2 embryos of inbread CBA mice were grown in vitro and repeatedly tested for oncogenicity in adult syngeneic hosts by subcutaneous inoculation of about 50 × 106 cells. In contrast to spontaneously developing permanent mouse embryo lines, these in vitro lines never induced any progressive tumours. After prolonged cultivation, one of the embryonic lines induced progressively growing tumours in irradiated mice, but not in normal ones. When the tumour was passed in vitro the oncogenic potential increased and after 3 passages, tumours were also obtained in untreated animals. The oncogenic potential was retained by in vitro cultivation and increased to a certain degree by in vivo passages. The oncogenic cell titre increased from a TPD50 of about 107 to 104. Corresponding increase in the oncogenic potential was also obtained when the same implantation procedure was carried out upon a population from a single cell derived from the mixed low oncogenic population and shown to be non‐malignant for normal mice. Attempts to correlate the increase in malignancy to other parameters studied failed except for the demonstration of a higher cloning ability of the malignant population. Tumour tissue contained 3 types of clones, 2 of which were present also in the original population otherwise found non‐malignant for normal mice. Irrespective of the type, 7 of 8 clones from the tumours and none of 5 clones from the latter population produced tumours in normal mice. The oncogenic cell titres of most clones were the same as those found for the mixed population. No virus or subviral infectivity could be recovered from cultures of transformed cells or tumours. All cells were positive both in CF tests and in immunofluorescence tests with sera from tumour‐bearing hamsters. No correlation between the amount of complement‐fixing neoantigen and malignancy was found. Tumour development could be prevented by SV40 immunization, indicating that the increase in oncogenicity was not due to loss of tumour specific transplantation antigens. Simultaneous inoculation of a malignant population and the one grown only in vitro did not support a growth of the latter or prevent a tumour production by the former. Tumour‐bearing, not pretreated mice and most of the irradiated ones with tumours had complement‐fixing antibodies against SV40 neoantigens and immunofluorescent antibodies to the nuclear antigen in their sera. High titres were found in animals carrying tumours for a longer time and low or no titres were recorded in animals which died early due to rapidly growing tumours.

Contagious agalactia of sheep and goats preliminary studies on vaccines

Vaccines against contagious agalactia were tested for their ability to protect goats against challenge by subcutaneous inoculation of a strain of M. agalactiae of known infectivity. The results were assessed on clinical signs, and ante and post-mortern isolation of the challenge organism. They were expressed quantitatively by means of a scoring system. Serological response to vaccination was measured by complement fixation and growth inhibition tests. Of a number of vaccines prepared from formalin killed M. agalactiae those containing concentrated suspensions of mycoplasmata cells in oil adjuvants gave the best protection. A vaccine prepared from a cell suspension emulsified in oil and Falba gave better results than aluminium hydroxide vaccines prepared by the method of Popovici and De Simon (1966). Before challenge, most of the goats vaccinated with oil and Falba vaccine became reactors to the CF test, but none reacted to the GI test. None of the dead vaccines prevented infection after challenge, but the resulting disease was in some instances less severe than in unvaccinated control goats. A live vaccine, attenuated by 40 passes on selective agar, gave complete protection against the challenge inoculation. The vaccine strain could be differentiated from the challenge strain by bacteriological methods. Goats inoculated with the live vaccine had a small residual infection with the vaccine strain when killed 3 to 4 months after vaccination and later work has shown that it can produce clinical contagious agalactia in lactating sheep. Before challenge, all the goats inoculated with the live vaccine became reactors to both the CF and GI tests.