l'eterinar.v Microbiolog.l,, 29 ( 1991 ) 261-265 Elsevier Science Publishers B.V., Amsterdam
Abortion in Japanese cows caused by Chlamydia
psittaci M. Nabeya a, K. Kaneko a, H. Ogino a, D. Nakabayashi a, T. Watanabe a, J. Murayama a, K. HayashP, H. Fukushi b, T. Yamaguchi b, K. Hirai b, Y. Inaba c'' and M. Matumoto d aChuo Livestock Hygiene Service Center, Nishikawa-machi. Niigata 959-04, Japan bDepartment of ~'eterinao, Microbiology, Faculty of Agriculture. Gifu UniversiO,, G(fu. G~fu501-11. Japan ~Department of l'eterinar.v Epi:ootiology, College of Agriculture and Veterinao' A~dicine. Nihon UniversiO,, Fujisawa, Kanagawa 252, Japan aKitasato Institute, Minato-ku, Tokyo 108, Japan (Accepted 14 May, 1991 )
Nabeya, M., Kaneko, K., Ogino, H., Nakabayashi, D., Watanabe, T., Murayama, J., Hayashi, K., Fukushi, H., Yamaguchi, T., Hirai, K., Inaba, Y. and Matumoto, M., 1991. Abortion in Japanese cows caused by Chlamydia psittaci, l'et. MicrobioL 29:261-265 An outbreak of abortion in cows occurring in Niigata Prefecture was shown to be caused by Chlam rdia psittaci. Elementary bodies characteristic of Chlano'dia were found in the liver of aborted fetuses and C. psittaci antigen was demonstrated by indirect immunofluore~ence. C!,!aoo~ia was isolated from the liver of aborted fetuses by the yolk sac inoculation of developing chick embD'os and by the intraperitoneai inoculation of guinea pigs. Abortion occurred mostly in middle or late pregnancy. Aborted fetuses showed subcutaneous edema and gelatinous infiltration, enlarged liver and spleen, and dark red pleural and ascitic fluid. Focal necrosis was shown in the liver, spleen and lymph nodes. Serological findings and isolation of Chlano,dia from fecal specimens indicated a wide dissemination of C. psittaci among cows in the area.
Abortion in cattle has been a serious problem for cattle raisers in Japan. Chlamydia psittaci has been recognized as the cause of a variety of diseases in may species of animals and birds. The present report describes the etiological study of an outbreak of bovine abortion. The findings obtained suggest C. psittaci was the cause of the outbreak. ~Author for correspondence.
0378-1135/91/$03.50 © 1991 Elsevier Science Publishers B.V. All rights reserved.
M. NABEYA ET AL.
MATERIALS AND METHODS
Gimenez modification of Macchiavello staining (Schachter, 1988 ) was used to stain impression smears of animal tissue and yolk sac. The smears were airdried and heat-fixed. Indirect immunofluorescence was used to assay antibodies against C. psittaci in bovine sera. Impression smears of the spleen from infected guinea pigs were made on multitest slides (Flow Lab., USA), air-dried and fixed with acetone at - 20 °C for 15 rain. After three washings with phosphate buffered saline (0.15 M NaCl, 0.007 M phosphate buffer, pH 7.2)(PBS), the fixed preparations were treated with serial two-fold dilutions of the serum at 37 ° C for 60 min. After three washings with PBS, the preparations were treated with appropriately diluted fluorescein isothiocyanate conjugate of sheep antibody against bovine IgG (Binding Site, USA). The antibody titer was expressed as the reciprocal of the highest serum dilution showing positive fluorescence. Titers of l 0 or higher were taken as positive. Histological sections were examined for C. psittaci antigen by the indirect immunofluorescence method using a guinea pig antiserum against the Ov/ IPA strain of C. psittaci. For isolation of Clamydia the tissue material was ground to make a 10% emulsion in PBS and the fecal specimen was emulsified to make a 10% suspension in PBS containing 50/lg/ml gentamicin and 500/~g/ml streptomycin. The mixtures were allowed :o stand at 4°C for 24 h and clarified by centrifugation. The supernatant fluid was inoculated in 0.5 ml amounts into the yolk sac of embryonated eggs from a specific pathogen-free flock that had been incubated at 37°C for 6 or 7 days. The inoculated eggs were incubated at 37°C for 3 to l0 days, and yolk sac smears were microscopically examined for Chlamydia after staining by the Gimenez method. The material was also inoculated in 2 ml amounts intraperitoneally into guinea pigs weighing 700 to 900 g. Smears were prepared from the spleen 14 days later and microscopically examined for Chlamydia after staining by the Gimenez method. The fecal specimens were tested only in guinea pigs. For electron microscopy the tissue material was doubly fixed with 2.5% glutalaldehyde and 1% osmic acid and thin sections were made, stained doubly with uranium acetate and citrate, and examined using a Hitachi HO-I 2 electron microscope. Virus isolation was attempted in cultures of MDBK and BT-2 cells derived from bovine kidney and testicle, respectively. The supernatant of a 10% emulsion of the specimen in Eagle's minimum essential medium was inoculated in 0.3 ml amounts into five tube cultures of MDBK and BT. The inoculated cultures were incubated in a roller drum at 37°C for 7 days and examined for any cytopathic effect.
ABORTION IN JAPANESE COWS
Over the period from March 3 to May 23 in 1988 there occurred 17 cases of abnormal delivery in cows residing in five neighbouring towns and villages of Niigata Prefecture, where a total of I 125 milking cows were kept. Abortion occurred mostly in middle or late pregnancy (Table l ): One cow (No. 6) delivered at full term a weak staggering calf, which died three days later. Macroscopically, aborted fetuses showed subcutaneous edema and gelatinous infiltration, enlarged liver and spleen, and dark red pleural and ascitic fluid. Microscopically, focal necrosis was shown in the liver, spleen and lymph nodes. Chlamydial elementary bodies were detected in liver preparations stained by the Gimenez method in l 0 of 12 aborted fetuses and in a newborn calf (No. 6 ). The indirect immunofluorescence technique revealed C. psittaci antigen in necrotic regions of the liver in 10 of 13 aborted fetuses and in the newborn calf. Isolation of Chlamydia was attempted with liver tissues from nine aborted fetuses, and from the newborn calf, by the yolk sac inoculation of developing chick embryos and by the intraperitoneal inoculation of guinea pigs. All the materials gave positive results by both methods (Table l ). In some cases seTABLE !
Occurrence of abortion in cows, detection of elementary body and antigen, and isolation of C psittaci (Co) from aborted fetuses Cow No.
1 2 3 4 5 6 7 8 9 i0 !! 12 13 14 15 16 !7
Date of delivery
Mar. Mar. Mar. Mar. Mar. Mar. Mar. Mar. Mar. Mar. Mar. Mar. Apr. Apr. May
2i7 194 i83 144
+ NT + NT + + NT + + + + + + NT +
8 12 15 16 18 18 !8 18 19 i9 20 20 8 13 4 May 23
full term 129 55 144 143 147 184 219 221 150 193 193
+ + NT + NT + + NT + + + + + +
+ + NT** + NT + + NT + + + + + NT NT NT NT
*C. psittaci antigen was detected in necrotic regions of the liver by the indirect immunofluorescence technique. **Not tested.
M. NABEYAET AL.
rial passage was made and the multiplication of Chlamydia tended to increase. Chlamydia was isolated from the spleen, kidney, lung, heart, cerebrum stomach, and large and small intestines of three aborted fetuses Nos. 1, 7 and 11 ). Electron microscopic examination of infected yolk sacs revealed electron-dense elementary bodies, about 0.3 gm in diameter, and pleomorphic reticulate 0articles, 0.5-1.0 gm in diameter, in the cytoplasm of infected cells. No virus was isolated from the aborted fetuses in MDBK and BT cell cultures. Ten cows which had aborted were tested for antibodies reactive with one of the C. psittaci isolates by the indirect immunofluorescence method. One year before the outbreak eight of the ten cows were tested and two gave serum titers of 160 and 80 and the remaining six were negative ( ~