Antigenic alteration in major piroplasm surface proteins of Theileria sergenti during infection

Theileria sergenti piroplasms were purified from different parasitemia peaks of cattle infected with parasitized erythrocytes or sporozoites during persistent infection. Their activities with monoclonal antibodies 13F5 and C9, which recognize 23 kDa and 32 kDa piroplasm surface proteins, respectively, were analyzed. Antigenic differences were observed among parasites from different parasitemia peaks during persistent infection when cattle were infected with sporozoites. Results of two-dimensional polyacrylamide gel electrophoresis showed that the 23 and 32 kDa proteins were expressed in all samples tested, regardless of their reactivities with the monoclonal antobodies. In contrast, parasites obtained from cattle inoculated with parasitized erythrocytes showed no antigenic alteration over a 2 month observation period. The results suggest that antigenic alteration of T. sergenti during persistent infection is related to whether the parasites proliferate through extraerythorocytic schizont stage in cattle or sporozoite and other sexual stages in tick vector.     

Development of an enzyme-linked immunosorbent assay for the detection of antibody to epizootic hemorrhagic disease of deer virus.

An enzyme-linked immunosorbent assay has been developed to detect antibodies to epizootic hemorrhagic disease of deer virus (EHDV). The assay incorporates a monoclonal antibody to EHDV serotype 2 (EHDV-2) that demonstrates specificity for the viral structural protein, VP7. The assay was evaluated with sequential sera collected from cattle experimentally infected with EHDV serotype 1 (EHDV-1) and EHDV-2, as well as the four serotypes of bluetongue virus (BTV), BTV-10, BTV-11, BTV-13, and BTV-17, that currently circulate in the US. A competitive and a blocking format as well as the use of antigen produced from both EHDV-1- and EHDV-2-infected cells were evaluated. The assay was able to detect specific antibody as early as 7 days after infection and could differentiate animals experimentally infected with EHDV from those experimentally infected with BTV. The diagnostic potential of this assay was demonstrated with field-collected serum samples from cattle, deer, and buffalo.     

Changes in plasma metabolites and muscle glycogen are correlated to bovine spongiform encephalopathy in infected dairy cattle

During the clinical phase of bovine spongiform encephalopathy (BSE), a significant decrease was observed in the ratio of muscle glycogen to plasma L-lactic acid concentrations in BSE infected field case and experimentally infected dairy cattle compared with healthy control cattle (P<0.001), this being due to changes in the concentration of both metabolites in the BSE infected cattle compared with the control group. Furthermore, the concentration of plasma alanine was significantly increased (P<0.05) in the infected animals. No significant difference was detected between these two groups in the ratio of hepatic glycogen to plasma lactate. We infer that BSE infected cattle exhibit signs of altered energy metabolism and when applied in conjunction with changes in other metabolite biomarkers these changes may be useful for discriminating BSE infected cattle from healthy cattle or those suffering with other disorders or diseases.     

Detection of antibodies to platelets and erythrocytes during infection with haemorrhage-causing Trypanosoma vivax in Ayrshire cattle

Ayrshire cattle, which were infected with a stock of Trypanosoma vivax from Galana, Kenya, which produced haemorrhagic disease, were examined for the presence of antibodies to erythrocytes and platelets. Antibodies to normal erythrocytes and platelets were detected in the plasma of infected animals using the enzyme-linked immunosorbent assay (ELISA). The antibodies were detectable following the first peak of parasitaemia (10-15 days after infection) and antibody activity was maximal 30-35 days after infection. Plasma from cattle, taken 32 days after infection, precipitated radiolabelled proteins from autologous platelets and, less efficiently, from autologous erythrocytes. Fluorescence-activated cell sorter (FACS) assays demonstrated that erythrocytes and platelets from infected cattle bound IgM and IgG in vivo, and that both normal blood cell types could adsorb these antibodies following incubation in plasma from infected animals. Complement (C3) was similarly adsorbed to erythrocytes during infection. Antibodies adsorbed to infected erythrocytes could be eluted and the eluted antibodies bound to normal erythrocytes, as detected by immunofluorescence, but they did not react with the infecting trypanosome. It is hypothesised that although anti-blood cell antibodies may not be the primary cause of the severe anaemia and thrombocytopaenia which accompany the haemorrhagic syndrome, they could play an important role in the maintenance of these signs of disease, adversely affecting the outcome of T. vivax-associated haemorrhagic disease in the field.     

A simpler, more sensitive competitive inhibition enzyme-linked immunosorbent assay for detection of antibody to malignant catarrhal fever viruses.

An earlier competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA) was developed for detection of specific antibody against malignant catarrhal fever (MCF) viruses (MCFV) in ruminants. In this study, the indirect CI-ELISA was improved by conjugating the monoclonal antibody 15-A directly with horseradish peroxidase and by developing a method of producing precoated, dried antigen plates. This new test is referred to as a direct CI-ELISA. The reformatted test yielded a significantly improved sensitivity, and the time required was reduced to about one-sixth of the previous time. Of 37 MCF cases in cattle that were confirmed by histopathology and polymerase chain reaction (PCR) assay, 37 (100%) were positive by the new test, whereas the indirect CI-ELISA detected only 23 (62%). The direct CI-ELISA detected antibody to MCFV in 100% of 48 sheep that had been defined as infected with ovine herpesvirus 2 (OvHV-2) by PCR, whereas the indirect CI-ELISA detected only 41 (85%). Comparison of antibody titers measured by the 2 assays for sera collected from OvHV-2-infected sheep and from cattle, bison, and deer with clinical sheep-associated MCF revealed that the direct CI-ELISA offered a 4-fold increase in analytical sensitivity over the indirect format. The number of seropositive animals detected by the direct CI-ELISA among apparently normal cattle and bison was 2-3 times greater than the number detected by the indirect CI-ELISA, indicating that a significant percentage of normal cattle and bison are subclincally infected with MCFV.     

Humoral immune response of sheep to infection with Eperythrozoon ovis

Circulating antibody was detected by an indirect fluorescent antibody test (IFAT) in the serum of sheep infected experimentally with Eperythrozoon ovis. Antibodies were first detected 15 to 32 days after infection with E ovis and titres peaked at 41 days. This antibody may be associated, at least in part, with protection against infection with E ovis since the initial increase in antibody titre coincided with a fall in the primary parasitaemia. A role for antibody is suggested further by the fact that the prepatent period of infection was prolonged by one day and the parasitaemia initially remained at low levels in infected sheep protected by passively transferred hyperimmune serum. Moreover, following primary infection, acquired immunity was manifest by a lack of parasitaemia following challenge infections while increased IFA titres were observed. No evidence of opsonic activity was observed in an in vitro erythrophagocytosis test in that neither mouse macrophages nor sheep monocytes phagocytosed E ovis infected or uninfected erythrocytes sensitised with hyperimmune serum.     

Analysis of the IgG antibody response against Paramphistomidae trematoda in naturally infected cattle. Application to serological surveys

The IgG antibody response to Calicophoron daubneyi (Digenea: Paramphistomidae) excretory/secretory antigens was evaluated in naturally infected cattle from Lugo (Galicia, NW Spain) by using an ELISA procedure. Two studies were conducted, first a survey in 524 cattle separated into three groups according to age, G-1 (0–2 years old), G-2 (3–5 years old) and G-3 (>6 years old). In the second study, three groups of cattle were employed: G-I, naturally infected; G-T, naturally infected and treated with oxyclozanide plus levamisole (Nilzan Plus^®); G-C, cattle maintained in a farm where C. daubneyi has never diagnosed. Variations on egg-output and haematic parameters (erythrocytes, haematocrite, leukocytes and lymphocytes) were also analyzed.

The ELISA procedure showed that 61.2% of the cattle in the first study had been exposed to the trematode, but only 10.1% passed eggs in the feces. Age-association with egg-output was shown but not with the IgG values. In the second experiment, the administration of the anthelmintic reduced significantly the IgG kinetic levels and the C. daubneyi-egg-output was suppressed during 12 weeks in the G-T group. The values of red cells, haematocrite, leukocytes and lymphocytes increased significantly in the treated cattle 5 weeks after chemotherapy; however, new reduction after week 5 was recorded, as results of the challenge of these cattle.

This is the first investigation in which evaluation of the IgG humoral response against C. daubneyi in cattle has been carried out. We proved that a notable IgG response in naturally infected cattle is induced, and can be detected by using an ELISA procedure. The IgG antibodies did not increase after challenge infection. Our results proved an important percentage of cattle were exposed to this trematode in the area of study and suitable measures for preventing this relationship must be considered.     

A microplate enzyme-linked immunorsorbent assay for measuring antibody to Anaplasma marginale in cattle serum

An enzyme-linked immunosorbent assay (ELISA) method is described for measuring antibody against Anaplasma marginale in cattle serum. This method was more sensitive and objective than a previously described ELISA method for A. marginale and possible reasons for this are discussed. All 83 cattle experimentally infected with A. marginale (81) or A. centrale (2) developed demonstrable specific antibody but the serums of 98.8% of 839 cattle from cattle tick-free areas did not react by ELISA; 378 serums containing antibody to Babesia bovis were tested for cross reactions in the A. marginale ELISA. There were no significant cross-reactions except when cattle had been inoculated at least twice with B. bovis-infected erythrocytes, presumably due to antibodies reacting with erythrocyte material in the ELISA antigen. The ELISA detected antibodies for more than 3 years after infection, at least 2 years longer than did a complement fixation test. When A. marginale infections in cattle were eliminated by long acting oxytetracycline, their serums ceased to react by ELISA. An ELISA score for serum antibody level was shown to have a statistically significant correlation with ELISA titre.     

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.     

BLUETONGUE: THE DISEASE IN CATTLE

Most researchers in South Africa found that although BT virus could be isolated from apparently healthy cattle and from inoculated cattle the virus did not produce overt clinical disease in cattle. However, when epizootics were reported outside Africa, clinical signs were observed in cattle in Israel, Palestine, Syria, Portugal, and Spain. Most natural BT infections in cattle in the United States do not result in overt clinical signs. However, in certain infected herds, approximately 5% of the cattle show from mild to severe disease. Except for severe cases, spontaneous recovery is usual. The clinical diagnosis of BT in cattle is difficult and requires laboratory assistance. Culicoides variipennis can serve as a vector of BT virus from cattle to cattle, cattle to sheep, sheep to cattle, and sheep to sheep. In utero transmission occurs in cattle and can result in abortion, hydraencephaly, congenital deformity, and birth of viraemic calves which may or may not develop BT antibody. Calves inoculated in utero or those born to infected dams may have a persistent viraemia with or without BT antibody. tone such animal has been held in insect-secure quarters and has continued to harbour virus for 3 years. Bluetongue virus was isolated from the semen of experimentally infected bulls. Calves inoculated with BT virus and also given an immuno-suppressant developed marked clinical disease in 8 to 12 days. Bluetongue virus is very closely associated with the erythrocytes of infected cattle, sheep, and goats. Cattle are considered important and relatively long-term virus reservoirs. In attempts to determine the maximum period of viraemia in cattle it is necessary to inoculate washed erythrocytes, rather than whole blood, and to use susceptible sheep as the assay system rather than embryonated chicken eggs.     

Inhibition of Babesia divergens in cattle by oxytetracycline

The effects of continuous oxytetracycline administration on the development of parasitaemia of Babesia divergens during both natural and artificial infections were studied. During natural exposure on grazing heavily infested with Ixodes ricinus, seven out of 42 cattle with no previous exposure to tick-borne diseases were injected every four days with a long acting preparation of oxytetracycline at a dose rate of 20 mg/kg. During the six week grazing period 21 untreated cattle developed a patent parasitaemia of B divergens and all became seropositive by the fluorescent antibody test. In contrast, no parasites were observed in treated cattle and antibody titres remained low. Artificial infections were studied with different dose levels of oxytetracycline and their effects on antibody stimulation noted. First, four groups of cows were infected with 10(8) erythrocytes infected with B divergens, three groups being injected every four days with the long acting oxytetracycline formulation at dose levels of 20, 10 and 5 mg/kg, respectively. The highest level completely inhibited parasite replication and antibody formation; the same was observed in one animal dosed at 10 mg/kg but the remainder, plus those treated at 5 mg/kg, developed both low parasitaemia and high antibody titres. The untreated cows developed severe babesiosis. A further untreated control group was added and three weeks after cessation of oxytetracycline treatment all were infected with 10(9) erythrocytes infected with a homologous isolate of B divergens. The controls, plus those in which the previous infection had been completely inhibited, developed severe clinical babesiosis but the remainder were refractory to parasite development.     

Evaluation of a 'dipstick' immunoassay to detect cysticercosis in experimentally infected cattle

A 'dipstick' immunoassay for bovine cysticercosis, using an antigen isolated from Taenia hydatigena cyst fluid, was evaluated in cattle experimentally infected with Taenia saginata. The assay correctly identified six out of seven infected cattle, including an animal in which only 12 living cysticerci were found. Cattle became seropositive as early as 3 weeks post-infection. A false-negative reaction was found for one very lightly infected animal, from which only four living cysticerci were recovered at necropsy. The assay was also used to detect circulating antibodies in experimentally infected cattle before and after therapeutic treatment with anthelminthics. The results suggest that praziquantel-treated animals gradually revert to being seronegative after the cysticerci are killed.     

The application of PCR-ELISA to the detection of Trypanosoma brucei and T. vivax infections in livestock

Teneral tsetse flies infected with either Trypanosoma brucei or T. vivax were fed on healthy cattle. Blood samples collected daily from the cattle were examined by microscopy for the presence of trypanosomes, in thick smear, thin smear and in the buffy coat (BC). All the cattle fed upon by infected tsetse developed a fluctuating parasitaemia. DNA was extracted from the blood of these cattle and subjected to polymerase chain reaction (PCR) using oligonucleotide primers specific for T. brucei or T. vivax. The PCR products unique to either T. brucei or T. vivax were identified following amplification of DNA from the blood samples of infected cattle, whereas none was detectable in the DNA from the blood of the cattle exposed to non-infected teneral tsetse. In a concurrent set of experiments, one of the oligonucleotide primers in each pair was biotinylated for use in PCR-ELISA to examine all the blood samples with this assay. Both the PCR and the PCR-ELISA revealed trypanosome DNA in 85% of blood samples serially collected from the cattle experimentally infected with T. brucei. In contrast, the parasitological assays showed trypanosomes in only 21% of the samples. In the blood samples from cattle experimentally infected with T. vivax, PCR and PCR-ELISA revealed trypanosome DNA in 93 and 94%, respectively. Microscopy revealed parasites in only 63% of the BCs prepared from these cattle. Neither PCR nor PCR-ELISA detected any trypanosome DNA in blood samples collected from the animals in the trypanosome-free areas. However, both assays revealed the presence of trypanosome DNA in a number of blood samples from cattle in trypanosomosis-endemic areas.     

Latency and persistence of bovine herpesvirus type 4, strain B11-41, in bovine nervous tissues

Three cattle were experimentally infected with bovine herpesvirus type 4 (BoHV-4), strain B11-41, isolated from the spinal cord of a cow, and monitored for clinical symptoms. None of them showed any clinical signs except increases of leukocyte numbers in two of them, and the body temperature remained normal throughout the experiment. Antibody titers against BoHV-4 continuously increased for one month and were maintained at a high level for more than 1 year by enzyme-linked immunosorbent assay (ELISA). The virus was isolated only from serum and peripheral blood leukocytes (PBL) of one cow in the early stage of infection, but the viral genome was detected in PBL continuously by PCR. When they were euthanized, the viral genome was detected in the lymph nodes and nervous tissues such as medulla, spinal cord, and trigeminal ganglion. These results indicate that cattle are infected with the virus latently and persistently, and the latency site would be in the tissues of the central nervous system as well as lymphoid tissues. When a seroepidemiological survey was performed on antibodies to BoHV-4 among cattle in Japan by ELISA, the rate of antibody-positive cattle was 8.9% and they were found irregularly on certain farms.     

鸡PD-1单克隆抗体的制备及生物学功能研究

试验旨在筛选并制备鸡PD-1单克隆抗体,对该单克隆抗体的免疫学特性、结合活性及其对鸡PD-1/PD-L1信号通路激活的阻断作用进行初步研究。运用杂交瘤细胞融合技术筛选杂交瘤细胞株,采用ELISA方法、Ig抗体亚型鉴定试剂盒、Western blotting鉴定抗体的免疫学特性,利用间接免疫荧光技术及流式细胞术检测筛选单抗与鸡PBMCs的结合情况,应用该单抗与IBDV感染7 d后的鸡PBMC细胞作用,利用实时荧光定量PCR技术检测IL-2、IL-6和IFN-γ等细胞因子的表达情况。结果显示,试验获得1株特异、稳定分泌鸡PD-1单克隆抗体的杂交瘤细胞株,命名为PD-1-D05。该单克隆抗体的亚型属于IgG1,杂交瘤细胞培养上清和腹水的效价分别为1：2¹¹和1：2.048×10⁵。ELISA和Western blotting检测结果表明,PD-1-D05单抗能与免疫原发生特异性反应,与pET-28a （+）、Rosetta菌株蛋白提取液上清及无关蛋白无交叉反应。间接免疫荧光及流式细胞术检测结果显示,PD-1-D05单克隆抗体能与鸡PBMC特异性结合,且IBDV感染7 d后的PBMC经单抗处理后,IL-2表达量显著升高（P<0.05）,IFN-γ转录水平显著下降（P<0.05）,IL-6表达水平较IBDV攻毒组细胞虽有所下降,但并无统计学差异（P>0.05）。结果表明,试验成功筛选并制备了能够稳定分泌鸡PD-1单克隆抗体的细胞株,所获得的PD-1单克隆抗体具有良好的免疫学特性,该单抗能够特异性识别鸡PD-1分子并与鸡PBMC细胞特异结合,并在一定程度上恢复由于IBDV感染导致的PD-1/PD-L1信号通路激活引发的免疫调节相关细胞因子IL-2、IFN-γ的异常表达。     

Vaccination of cattle with Anaplasma marginale derived from tick cell culture and bovine erythrocytes followed by challenge-exposure with infected ticks

Anaplasmosis, a hemolytic disease of cattle caused by the tick-borne pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae) has been controlled using killed vaccines made with antigen harvested from infected bovine erythrocytes. We recently developed a cell culture system for propagation of A. marginale in a continuous tick cell line. In this study, we performed a cattle trial to compare the bovine response to vaccination with A. marginale harvested from tick cell culture or bovine erythrocytes. All immunized and control cattle were then challenge-exposed by allowing male Dermacentor variabilis infected with A. marginale to feed and transmit the pathogen. Nine yearling cattle (three per group) were used for this study and were immunized with cell culture-derived A. marginale, erythrocyte-derived A. marginale or received adjuvant only to serve as controls. Each vaccine dose contained approximately 2 x 10(10) A. marginale and three immunizations were administered at weeks 1, 4 and 6. At week 8, cattle were challenge-exposed by allowing 60 D. variabilis male that were infected with A. marginale as adults to feed on the cattle. Antibody responses of cattle against major surface proteins (MSP) 1a, 1b and 5, as determined by ELISAs, peaked 2 weeks after the last immunization. Cattle immunized with infected IDE8 cell-derived antigens had a preferential recognition for MSP1b while cattle immunized with erythrocyte-derived antigens had a preferential recognition for MSP1a. Protection efficacy was evaluated using the percent infected erythrocytes (PPE), the packed cell volume (PCV), and the prepatent period. A. marginale-immunized cattle showed lower PPE and higher PCV values when compared to control animals and did not display clinical anaplasmosis. The cell culture-derived A. marginale shows promise for use as antigen in development of a new killed vaccine for anaplasmosis.     

The use of the enzyme-linked immunosorbent assay (ELISA) to detect the IgM and IgG antibody response to Leptospira interrogans serovars hardjo, pomona and tarassovi in cattle

Leptospira interrogans serovars pomona, hardjo and tarassovi were each used to inoculate 6 cattle. Three-hundred and ninety-nine sera collected from the inoculated animals and from a control group over a 3-month period were tested using the microscopic agglutination test (MAT) and the enzyme-linked immunosorbent assay (ELISA). Leptospiruria was monitored by microscopic examination and culture. The ELISA detected specific IgM antibody against the serovars in all infected cattle 1 week after inoculation. This IgM antibody persisted in most of the animals for 3-5 weeks. Specific IgG antibody appeared at the same time or just after IgM, but persisted for much longer. Levels of antibody detected by the ELISA and the MAT did not correlate with each other, nor with the periods of leptospiruria found in the infected cattle.     

Susceptibility of African buffalo and Boran cattle to Trypanosoma congolense transmitted by Glossina morsitans centralis

Four African buffalo (Syncerus caffer) and four Boran cattle (Bos indicus) were each exposed to the bites of 10 tsetse flies infected with Trypanosoma congolense. Although both groups of animals became infected, the buffalo showed no clinical signs of trypanosomiasis while the cattle suffered from the disease characterized by pronounced skin reactions, high parasitaemia and severe anaemia. The prepatent periods in the buffalo varied from 18 to 27 days in comparison with 11 to 14 days in the cattle. In the buffalo, skin reactions were only detectable by histological examination of skin biopsies, the peak of parasitaemia was at least a hundredfold below that in cattle and after 54 days parasites were no longer detected. In contrast, the cattle had a continuous high parasitaemia until they were treated with a trypanocidal drug 60 days after infection. Neutralizing antibody to metacyclic trypanosomes appeared in the buffalo during the prepatent period, 15-20 days after infection, whereas in cattle neutralizing antibody was not detected until 10 days after the first peak of the parasitaemia, 25-30 days after infection.     

Acquired methemoglobinemia in anemic cattle infected with Theileria sergenti.

To investigate the mechanism of anemia accompanying Japanese bovine theileriosis, we examined whether production of methemoglobin (MetHB), an indicator of erythrocyte oxidation, was associated with anemia in cattle experimentally infected with Theileria sergenti. The percentage of MetHB, which is an oxidized form of hemoglobin, increased according to the onset of anemia. During severe anemia, high levels of acquired methemoglobinemia were observed in all infected cattle. A significant correlation (r=-0.649; P<0.01) between an increase in MetHB concentration and a decrease in packed cell volume (PCV) was observed. It was considered that hemoglobin oxidation may be one of the aggravating factors of anemia in T. sergenti infection.     

Dominance of Chlamydia psittaci-specific IgG2 subclass in the humoral immune responses of naturally and experimentally infected cattle

Indirect enzyme-linked immunosorbent assays were applied to differentiate Chlamydia (C.) psittaci-specific IgG1 and IgG2 levels in 143 individual serum samples from cattle with naturally occurring chlamydial infections and in 190 sequential serum samples from 26 experimentally infected pregnant cows, calves, and a bull. The mean IgG1:IgG2 ratio of naturally infected cattle was 1:4 indicating a significant (p less than 0.001) IgG2 dominance. Similar ratios were detected in the experimentally infected cattle. The dominance of IgG2 was independent of breed, sex, and age. Twenty-nine cattle had significant immunoglobulin levels to both C. psittaci and Coxiella (C.) burnetii simultaneously. The predominance of C. psittaci-specific IgG2, in contrast to the predominance of C. burnetti-specific IgG1 detected in these same individual serum samples under identical conditions, indicates that the ability to preferentially produce either IgG1 or IgG2 was not limited in these individual cattle. A transient yet significant IgG1 response was also developed in cows following chlamydia-induced abortions (immunotype 1) or in cattle infected with the polyarthritis-serositis-encephalomyelitis agents (immunotype 2). IgG1 levels decreased faster than IgG2 levels. These findings have diagnostic implications and identify the need for determining the immunoglobulin classes and subclasses of the humoral immune responses of animals and man to chlamydial infections.