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Avian Spirochetosis in Chickens Following Experimental Transmission of Borrelia Raquel S. Lisboˆa, Rafaella C. Teixeira, Charles P. Rangel, Huarrisson A. Santos, Carlos L. Massard, and Adivaldo H. FonsecaA Curso de Po´s-graduac¸a˜o em Cieˆncias Veterina´rias, Universidade Federal Rural do Rio de Janeiro, BR 465, Km 7, Serope´dica, Received 2 July 2008; Accepted and published ahead of print 11 November 2008 SUMMARY. This study reports the experimental transmission of Borrelia anserina to domestic chickens by infected Argas (Persicargas) miniatus. Clinical alterations as well as prepatent and patent periods were evaluated. Twenty-seven 67-day-old birdswere divided into three groups in a randomized experimental design. The first group was exposed to ticks infected with B. anserina,the second group was exposed to noninfected ticks, and the third group wasn’t exposed to ticks. Blood smears from each bird ofgroups 1 and 2 were prepared daily and examined for 25 days postexposure (PE). Examination of the blood smears from birds ingroup 1 revealed large numbers of spirochetes from days 5 to 12 PE. In this group the prepatent and patent periods were 5–7 and4–7 days, respectively. Birds from group 1 presented ruffled feathers, pale combs, drowsiness, inappetence, loss of weight, andgreenish diarrhea after day 6 PE. The current study confirms the viability of experimental transmission of B. anserina to domesticchickens by A. (P.) miniatus.
RESUMEN. Espiroquetosis aviar en gallinas despue´s de la transmisio´n experimental de Borrelia anserina por Argas (Persicargas) El presente trabajo describe la transmisio´n experimental de Borrelia anserina a las gallinas dome´sticas por Argas (Persicargas) miniatus infectadas. Se evaluaron las alteraciones clı´nicas, los perı´odos prepatente y patente. Veintisiete aves con 67 dias de edadfueron divididas en tres grupos completamente aleatorizados. El primer grupo fue expuesto a garrapatas infectadas por B. anserina;el segundo a garrapatas no infectadas y el tercer grupo no fue expuesto a garrapatas. Se realizaron -frotis sanguı´neos de las aves de losgrupos 1 y 2 diariamente, hasta el dı´a 25 post-exposicio´n (PE). El examen de los frotis sanguı´neos de las aves infectadas (grupo 1)revelo´ gran nu´mero de espiroquetas del 5u al 12u dı´a PE. Los perı´odos prepatente y patente para este grupo fueron de 5–7 y 4–7dı´as respectivamente. A partir del sexto dı´a PE, las aves del grupo 1 presentaron plumas erizadas, cresta pa´lida, somnolencia,inapetencia, pe´rdida de peso y diarrea verdosa. El presente trabajo confirma la viabilidad de la transmisio´n experimental de B.
anserina en gallinas dome´sticas por A. (P.) miniatus.
Key words: Borrelia anserina, Argas miniatus, chickens, ticks, avian spirochetosis, spirochetemiaAbbreviations: PE 5 postexposure; UFRRJ 5 Universidade Federal Rural do Rio de Janeiro Avian spirochetosis is an acute septicemic disease of a variety of avian species caused by Borrelia anserina (16). The spirochete may be Place of the experiment and animals. Experiments were carried out found in the blood of infected birds during the beginning stages of from April to August 2005 in the Experimental Station for Parasitological Research W. O. Neitz, at Universidade Federal Rural Several species of Argas ticks transmit the pathogen. Ticks do Rio de Janeiro (UFRRJ), and in the Laboratory of Parasitological inoculate spirochetes by excretion of coxal fluid or by saliva (14).
Diseases, Department of Epidemiology and Public Health, Veterinary Colonies of Argas spp. infected with B. anserina have been used to Institute, UFRRJ/Projeto Sanidade Animal-Embrapa. Twenty-seven 67- keep bacteria viable and pathogenic (11) because ticks are natural day-old chickens, commercial lineage ‘‘Isa Brown’’ laying hens (Gallus reservoirs in which spirochetes survive for long periods (5).
gallus domesticus), were studied. The birds were held in hanging cages in In many countries in the past, avian spirochetosis was reported to a screened and ventilated house. They were provided water and balanced be one of the most severe diseases affecting poultry industry (1).
food with mineral-vitamin supplements. The food quantity was Nowadays the disease is practically confined to small flocks kept for controlled according to birds’ age and weight (6). No anticoccidialdrugs or growth promoter was provided.
subsistence or very limited local sale, where the tick vector remains Origin of B. anserina isolate and ticks. The B. anserina isolate was established (2). Ataliba et al. (2) reported that in addition to the obtained from naturally infected A. (P.) miniatus, collected from a small historical importance of avian spirochetosis, the pathogenic agent is group of hens from a rural area of the municipality of Pedro Leopoldo, prevalent worldwide. Clinically the disease is expressed by in Minas Gerais state, Brazil. The isolate was provided by Prof. Roma´rio hyperthermia, polidipsia, drowsiness, anorexia, inappetence, green- Cerqueira Leite. The isolate was molecularly characterized as B. anserina ish diarrhea, paralysis of the legs and wings, as well as sudden death and labeled as strain PL (2), cryopreserved with liquid nitrogen (2196 C) in experimentally infected hen blood. Dimethyl sulfoxide The current study aimed at evaluating the experimental 10% was used as a cryopreservative (11).
transmission of B. anserina to domestic chickens by the infected Argas (P.) miniatus ticks were collected from chickens in conventional tick Argas (Persicargas) miniatus. We investigated biological raises located in two different municipalities, Treˆs Rios and Rio de parameters such as prepatent period, patent period, and clinical Janeiro. Afterwards ticks were incubated at 27 6 1 C and 80% relativehumidity.
Infection of ticks. To obtain infected adult ticks, we used an adult bird that initially presented a blood smear negative to hemoparasites.
ACorresponding author. E-mail: [email protected] Afterwards this bird was immunosupressed by intramuscular adminis- Blood smears were taken daily, by a puncture in the wing vein with a sterile needle, from each bird from groups 1 and 2, and every 5 daysfrom group 3, until day 25 postexposure (PE), in order to examine thepresence of spirochetes and to calculate spirochetemia. Slides werestained with Giemsa. The number of spirochetes per field at 1000 3magnification was determined by computing the average count from 50random fields (16).
The birds were weighed on a Roberval balance at 36, 50, 62, 71, 77, and 85 days of age. Additionally, birds were also evaluated byconsidering clinical alterations.
Examination of the blood smears from birds in group 1 revealed large numbers of spirochetes (Fig. 1). The spirochetemia wasobserved from day 5 PE, with maximum peak between days 6 and9 PE, and from day 13 to day 25 PE all birds presented negativeblood smears (Table 1). Only one bird from group 1 was refractory Peripheral blood smear from chicken 2 showing erythrocytes to pathogen transmission and did not present spirochetes in the and spirochetes clumping observed on day 10 PE. 1000 3 magnifica- blood circulation or show clinical signs. Blood smears from birds in groups 2 and 3 remained negative during whole experimentalperiod.
The prepatent and patent periods in group 1 were 5–7 and 4– tration of a single dose (30 mg/kg) of methylprednisolone acetate 7 days, respectively. Birds from group 1 presented ruffled feathers, (Depo-MedrolH) (17). On the following day the bird was inoculated pale combs, drowsiness, and inappetence detected by the remaining with 0.5 ml of infected serum, previously submitted to one passage. Theinoculum was observed in dark field microscopy and presented ‘‘++’’ feed in the food container from day 6 PE. These signs continued viability according to a classification drawn up by Dhawedkar and until day 12 PE, corresponding exactly to the end of spirochetemia.
From day 7 to day 12 PE, greenish diarrhea was observed. At day 13 Blood smears were prepared to evaluate the development of PE infection signs disappeared, and birds began to eat again. The spirochetemia. On the fourth day after inoculation, the bird presented birds from groups 2 and 3 did not show any change in fecal color or peak of spirochetemia, and ticks were fed until complete engorgement.
The coxal fluid (excess fluid of the blood meal excreted to accomplish No significant differences were observed among the mean weights haemolymph volume and ion regulation) of some of these ticks was of birds in groups 1, 2, and 3 until the fourth weighing day analyzed by dark field microscopy, and spirochetes were detected.
(Table 2). On the fifth weighing day, executed at day 10 PE, group Experimental design. The experimental design was completely 1 was the only one that lost weight, and the mean weight of birds randomized in subdivided parcels, with three treatments in the parcels was significantly different from the other two groups (p , 0.05).
(groups) and six evaluations in different periods in the subparcels The refractory bird from group 1 was the only bird that gained (weight gain), including nine repetitions (chickens) per treatment. Thequantitative analysis of bird weight was calculated using ANOVA with weight. Groups 2 and 3 gained weight regularly. No dead birds were 5% significance (p , 0.05) using R software (7). Significant differences between group means were compare by Tukey’s test.
Group 1 was exposed to ticks infected with B. anserina, group 2 was exposed to noninfected ticks, and group 3 wasn’t exposed to ticks. Four adults of A. (P.) miniatus (two males and two females) were placed underthe wings of each bird from groups 1 and 2 to be fed until complete Chickens experimentally infected with B. anserina presented engorgement. The birds’ legs were held with a tape and an Elizabethan clinical alterations during the spirochetemia period, and they collar (a plastic sheet with a hole) was fixed around the neck to avoid the recovered afterwards. The prepatent period described in this study is in agreement with results reported by Hutyra et al. (10) and Bier Borrelia anserina spirochetemia in chickens experimentally infected by A. (P.) miniatus in group 1.
Number of spirochetes per blood smear fieldA in different days after exposure to ticksB AAverage of 50 blood smear fields. 1000 3 magnification.
B2 5 negative, + 5 small clumps, ++ 5 large clumps.
Experimental transmission of Borrelia anserina Mean and standard deviation of chickens’ weight. Group 1 (birds exposed to A. (P.) miniatus infected with B. anserina), Group 2 (birds exposed to A. (P.) miniatus not infected with B. anserina), and Group 3 (birds not exposed to ticks).A ASample mean (x¯) and standard deviation (SD) (n 5 9). Means followed by letters a and b are different (p , 0.05) by Tukey’s test.
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producing the balanced chicken food.
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