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Arquivo Brasileiro de Medicina Veterinária e Zootecnia

Print version ISSN 0102-0935On-line version ISSN 1678-4162

Arq. Bras. Med. Vet. Zootec. vol.51 n.6 Belo Horizonte Dec. 1999 



Cryopreservation of an avian spirochete strain in liquid nitrogen

(Criopreservação de uma amostra de espiroqueta em nitrogênio líquido)


M.B. Labruna1, J.S. Resende2, N.R.S. Martins2, M.A. Jorge2

1Faculdade de Medicina Veterinária e Zootecnia – USP
Av. Prof. Orlando Marques de Paiva, 87
05508-900 - São Paulo, SP
2Escola de Veterinária da UFMG


Recebido para publicação, após modificações, em 3 de outubro de 1999.



Preservation of Borrelia anserina in laboratory media has not been used routinely, due to the bacterial fastidious nutritional requirements (Merchant & Packer, 1965; Holt et al., 1994). B. anserina infected Argas spp. colonies have been used to keep unchanged the bacterial viability and pathogenicity, however this is an expensive method as it requires a supply of susceptible chickens fed with antibiotic free diet and special housing. The purpose of this work was to test the infectivity of an avian spirochete strain subjected to liquid nitrogen refrigeration.

Argas sp. ticks were collected from a chicken house in a farm located on the rural area of Pedro Leopoldo, Minas Gerais, Brazil, where an endemic disease compatible with spirochetosis has been recorded. The ticks were transported to the Laboratory of Parasitic Diseases, Veterinary College, UFMG, and identified as Argas (Persicargas) miniatus Koch, 1844, according to Magalhães (1979). Susceptible chickens infested by these ticks showed clinical signs such as fever, depression and greenish diarrhea a few days later, and the presence of several spirochetes in the blood smear stained by Giemsa. Subsequently a spirochete infected A. (P.) miniatus colony was established under laboratory conditions. There was no serologic evidence indicating this spirochete was B. anserina. However the morphologic characteristics at microscopy and the epidemiological aspects of the disease in the chicken at the farm and experimentally are compatible with spirochetosis. Holt et al. (1994) reported that B. anserina is the only spirochete that infects birds.

Specific pathogen free (SPF) chickens (SPAFAS/Resende, Uberlândia, MG, Brazil), 20 to 30 week-old were used and fed with antibiotic free diet ad libitum. From the spirochete infected A. (P.) miniatus laboratory colony, eight chickens were obtained with clinical signs of spirochetosis according to Barnes (1991) and spirochetemia graded as "++++" according to the Dhawedkar & Dhanesar (1983) classification, indicating spirochetes with vigorous motility. Chickens were bled by the intracardiac route and the blood was allowed to clot individually in tubes at room temperature for two hours followed by four hours at 4°C and one hour at 37°C. Sera were pooled and kept for two hours at 4°C for erythrocytes and cellular residues decantation, then transferred to fresh tube and stored at 4°C without antibiotics and biologic preservatives until the final procedure 30 minutes later. Sera were examined under dark field microscope for the presence of spirochetes.

The pooled sera were divided into two aliquots in order to be subjected to different procedures. Sterilized glycerol (autoclaved glycerol / Merck) as stabilizer at a dilution of 1:2 (v/v) was added to a set of aliquots labeled as glycerol 50% sera (GS). To the other set of aliquots was added dimethylsulphoxyde (DMSO filtered by 0.22m filter) at a proportion of 1:10 (v/v) and labeled as DMSO 10% sera (DS). Thereafter the mixtures were distributed in cryotubes and immediately immersed in liquid nitrogen.

After 15 months of uninterrupted storage in liquid nitrogen, two cryotubes from each treatment (GS and DS) were removed and thawed in a water bath at 25°C. Motility was immediately observed under dark field microscopy. Eight chickens per treatment were used in the infectivity test. Each treated serum was used in groups of four chickens in two ways: a) undiluted (inoculated 0.25ml/chicken via brachial vein) and b) diluted 1:2 in sterile SPF chicken sera (inoculated 0.50ml/chicken via brachial vein). Four other chickens were inoculated via a wing vein with only 0.25ml of the sterile SPF sera, being the control group. After inoculation, chickens were examined daily for spirochetemia. Blood smears stained by Giemsa were observed under an immersion objective lens - 1000x. A minimum of 50 fields per blood smear were observed and when positive for spirochetes, a mean number of spirochetes per field was calculated. When a positive chicken showed a mean number per field higher than 10, no further blood smears would be observed.

In the freshly pooled sera from the spirochetemic chickens, numerous vigorously moving spirochetes per field were seen by dark field microscopy. When the samples were maintained for 15 months in liquid nitrogen, thawed and immediately observed under dark field microscopy, the GS samples had spirochetes showing "0" (zero) motility and the DS samples had spirochetes showing "++" motility, according to the Dhawedkar & Dhanesar (1983) classification. Results of chicken blood smears for the GS and DS treatments are showed on Tables 1 and 2. None of the control chickens showed spirochetes on the blood smears up to seven days post inoculation.





In this study, 10% DMSO in chicken sera used as a spirochete stabilizer was superior to 50% glicerol. Dhawedkar & Dhanesar (1983), using blood with 6 or 10% glycerol obtained a "++" motility and maintained the bacterial infectivity as long as six months in liquid nitrogen. Ginawi & Shommein (1980) and Dhawedkar & Dhanesar (1983) subjected B. anserina to different temperatures for different periods. Infectivity of the spirochete was reported as well as some motility. The reduced motility probably indicates lower infectivity. Although the GS spirochetes in this present work showed zero motility after thawing, three of eight inoculated chickens showed spirochetemia with more than 10 spirochetes per field after 96 hours. The dark field examination has lower sensitivity and apparently a small number of spirochetes, although not detectable in this method, remained viable. According to Prudovsky et al. (1978), cited by Dhawedkar & Dhanesar (1983), only a few viable organisms are sufficient to infect susceptible birds. This could explain the longer time that the GS infected chickens took to show more than 10 spirochetes per field when compared to the DS inoculated chickens. Thus, the DS infective dose administered to chickens was probably higher than the GS one. The "0" motility encountered in GS sample after thawing may be associated with the high glycerol concentration added to the sera before freezing. According to Cottrall (1978), B. anserina is extremely sensitive to the majority of chemical compounds including glycerol. Perhaps this could be the explanation for the "0" motility observed when 50% glycerol was used. However, it should not be considered 100% inactivity, as shown by the results on Table 1. Some GS chickens showed less than one spirochete per field at 48 or 72 hours post inoculation, but they did not develop the disease and their blood smears were negative at 96 hours. In this experiment there was either a partial loss of the spirochete virulence or inactivation of the majority by the treatment with glycerol 50% which induced an immune response and chicken recovery. It was not possible to report differences between the groups inoculated with stabilizer diluted in filtered sera and undiluted stabilizer since in the DS group, all eight chickens showed spirochetemia with more than 20 spirochetes per field at 72 hours post inoculation. At 48 hours post inoculation the eight DS inoculated chickens were already showing spirochetemia which demonstrated 10% DMSO as an excellent stabilizer considering that when infected fresh blood is inoculated from a spirochetemic chicken to a susceptible chicken intravenously, the incubation period is 24 to 48 hours.

Keywords: Chicken, spirochete, Borrelia anserina, cryopreservation



Soros de aves experimentalmente infectadas, contendo espiroquetas viáveis, foram submetidos a dois procedimentos antes da criopreservação: glicerol na diluição de 1/2 (v/v), designado como soro com glicerol a 50% (GS), e dimetilsulfóxido na proporção de 1/10 (v/v), designado como soro com DMSO a 10% (DS). Após 15 meses de estocagem em nitrogênio líquido, amostras dos tratamentos GS e DS foram descongeladas e suas infectividades foram testadas em frangos susceptíveis. Apesar de ambos os procedimentos terem mantidos a infectividade da bactéria, DMSO a 10% no soro de frango apresentou-se mais satisfatório como criopreservante.

Palavras-Chave: Frango, espiroqueta, Borrelia anserina, criopreservação.




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