1000010325243

1000070263803

10325243

50379921

70263803

9000005461157

Hiraoka, Hiroko

Inokuma, Hisashi

Itamoto, Kazuhito

Miyama, Takako

Nakaichi, M

Okuda, Masaru

read0065882

read0138234

read0185156

Taura, Y

Une, S

Yoshizaki, Y

猪熊, 壽

English

奥田, 優

板本, 和仁

OAK Obihiro University Archives of Knowledge

1Short Communication Veterinary Record           C3620  Revised 02 March 2004  Clinical observation of Babesia gibsoni infection with low parasitemia confirmed by PCR Hisashi INOKUMA, Masaru OKUDA, Yukako YOSHIZAKI, Hiroko HIRAOKA, Takako MIYAMA, Kazuhito ITAMOTO, Satoshi UNE, Munekazu NAKAICHI, Yasuho TAURA  Faculty of Agriculture, Yamaguchi University,  1677-1 Yoshida, Yamaguchi, 753-8515, Japan.  * Address correspondence to: H. Inokuma,  Faculty of Agriculture, Yamaguchi University, 1677-1 Yoshida, Yamaguchi, 753-8515, Japan.  TEL/FAX  +81-83-933-5895  e-mail: inokuma@yamaguchi-u.ac.jp 2Babesia gibsoni is a hemoprotozoan parasite reported to cause clinically important hemolytic anemia in dogs.  This Babesia parasite distribute in many regions including Asia, Africa, Europe and America (Farwell and others 1982, Lobetti 1998).  Generally, the Babesia species is identified under a microscope by demonstrating the organisms in blood smears after Romanowsky staining (Lobetti 1998).  However, the detection of B. gibsoni in blood smears is sometimes very difficult, because of its low parasitemia.  Thus, B. gibsoni infection with low parasitemia is easily misdiagnosed as immune mediated hemolytic anemia (IMHA), and sometimes inappropriately treated with immune suppressive drugs.  Although clinical observation of canine B. gibsoni infection has been well documented in several reports (Farwell and others 1982, Lobetti 1998, Macintire and others 2002), most cases reported in previous papers were diagnosed by the demonstration of parasites on blood smear films.  Recently, polymerase chain reaction (PCR) has been developed to diagnose the B. gibsoni infection with higher sensitivity and specificity (Ano and others 2001, Fukumoto and others 2001, Macintire and others 2002, Stegeman and others 2003, Inokuma and others 2004).  After the introduction of PCR, clinically diagnosed animals with B. gibsoni infection showing low parasitemia have been easily diagnosed.  However, physical and laboratory findings of animals demonstrating low parasitemia have never been precisely documented.  Thus, clinical data from an animal with acute B. gibsoni infection demonstrating low parasitemia confirmed by PCR are evaluated and compared with those of animals with apparent B. gibsoni infection or IMHA using a retrospective case series.  A total of 35 animals with acute B. gibsoni infection and 8 with IMHA treated between 1998 and 2003 in the Veterinary Teaching Hospital, Yamaguchi University, were used in this study.  These animals showed symptoms suspected for acute B. gibsoni infection including anorexia, anemia, icterus, hematuria or fever at presentation.  In each animal, more than 100 fields of blood smear stained with Giemsa were observed under high magnification (x1000) to detect Babesia parasites.  Twenty-five dogs of 35 were easily diagnosed as having B. gibsoni infection by demonstration of parasites in the blood smears (B. gibsoni infection with moderate to severe parasitemia).  The remaining 10 dogs were not confirmed at presentation because apparent parasites could not be detected in the blood smear.  Positive reactions to diminazen aceturate, or the appearance of Babesia parasites in the blood films after treatment with immune suppressive drugs later caused these animals to be diagnosed with B. gibsoni infection (B. gibsoni infection with low parasitemia).  Total DNA was extracted from each canine sample and B. gibsoni-specific PCR amplification was performed using a primer set of Gib599F and Gib1270R (Inokuma and others 2004).  In our preliminary experiments, this PCR for B. gibsoni was able to detect approximately 10 infected red blood cells in 1 µl of peripheral blood.  To confirm the PCR findings, nucleotide sequences of randomly selected products of the B. gibsoni-specific PCR were determined by direct sequence analysis using the method previously described (Inokuma and others 2003).  All the analyzed sequences showed 100% homology with B. gibsoni Asia-1 (GenBank accession number AF175300).  Animals with IMHA showed acute onset of symptoms similar to those of B. gibsoni infection such as hemolytic and regenerative anemia, icterus, anorexia, or hematuria at presentation.  All 8 animals with IMHA were judged negative for B. gibsoni infection by PCR.  Information obtained from all 35 babesiosis and 8 IMHA cases included histories, findings of physical examination, CBC, serum biochemical analysis, the radiography of the 3abdomen.  To compare data from dogs with low parasitemia with those of B. gibsoni infection with moderate to severe parasitemia or IMHA cases, chi-square tests and one-factor ANOVA were performed by using StatView Ver.5.0 (Hulinks).  In the present study, all 35 animals with Babesia showed positive PCR reaction for B. gibsoni.  When the clinical and laboratory findings of Babesia infected animals showing low parasitemia were compared with those of dogs with moderate to severe parasitemia, nothing was different significantly among the 3 groups (Table 1).  Although the numbers of dogs that showed icterus and the mean total bilirubin level in dogs with more severe parasitemia were slightly higher than those of dogs with low parasitemia, the differences were not significant.  Oxidative damage to erythrocytes induced by B. gibsoni infection is thought to result in severe anemia even in the presence of low parasitemia (Otsuka and others 2002).  The degree of parasitemia in B. gibsoni infection did not relate to clinical and laboratory findings.  The clinical and laboratory findings of B. gibsoni infection with low parasitemia were also compared with those of IMHA (Table 1).  Some dogs with IMHA showed more severe clinical findings than those with B. gibsoni infection, and the mean RBC, PCV and hemoglobin value in IMHA were slightly lower than those in B. gibsoni infection without significant differences.  Although hematuria is not a specific finding for IMHA (Honeckman and others 1996, Miller 2000), the number of dogs with IMHA that showed hematuria was significantly higher than those with B. gibsoni infection with low parasitemia (Table1).  The mean body temperature of animals with Babesia infection was significantly higher than that of those with IMHA, but 2 of 8 dogs with IMHA showed fever (>39.5 C) at presentation.  Most animals with IMHA were also positive on direct Coomb’s-test showing significant numbers of spherocytes in blood smears; however, these results were also found in some animals with B. gibsoni infection.  Thus, it is very difficult for clinical veterinarians to confirm B. gibsoni infection and differentiate it from IMHA, if there were no evidence of Babesia parasites in blood smears.  In conclusion, B. gibsoni-specific PCR would be highly useful tool for diagnosing acute B. gibsoni infection in dogs.   Acknowledgements The authors would like to acknowledge the technical expertise of the DNA Core facility of the Center for Gene Research, Yamaguchi University, supported by a grant-in-aid from the Ministry of Education, Science, Sports and Culture of Japan.  This work was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (Grant No. 14360190).  References ANO, H., MAKIMURA, S. & HARASAWA, R. (2001) Detection of Babesia species from infected dog blood by polymerase chain reaction. Journal of Veterinary Medical Science. 63: 111-113. FARWELL, G.E., LEGRAND, E.K. & COBB, C.C. (1982) Clinical observation on Babesia gibsoni and Babesia canis infection in dogs. Journal of the American Veterinary Medical Association. 180: 507-511.  FUKUMOTO, S., KUAN, X., SHIGENO, S., KIMBITA, E., IGARASHI, I., NAGASAWA, H., FUJISAKI, K. & MIKAMI, T. (2001) Development of a polymerase chain reaction method for diagnosing 4Babesia gibsoni infection in dogs. Journal of Veterinary Medical Science. 63: 977-981. HONECKMAN, A.L., KNAPP, D.W. & REAGAN, W.J. (1996) Diagnosis of canine immune-mediated hematologic disease. Compendium. 18: 113-127.  INOKUMA, H., YOSHIZAKI, Y., MATSUMOTO, K., OKUDA, M., ONISHI, T., NAKAGOME, K., KOSUGI, R. & HIRAKAWA, M. (2004) Molecular survey of Babesia infection in dogs in Okinawa, Japan. Veterinary Parasitology (in press)  INOKUMA, H., YOSHIZAKI, Y., SHIMAD, Y., SAKATA, Y., OKUDA, M. & ONISHI, T. (2003) Epidemiological survey of Babesia species in Japan performed with specimens from ticks collected from dogs and detection of new Babesia DNA closely related to Babesia odocoilei and Babesia divergens. Journal of Clinical Microbiology 41: 3494-3498. LOBETTI, R.G. (1998) Canine Babesiosis. Compendium. 20: 418-430. MACINTIRE, D. K., BOUDREAUX, M. K., WEST, G. D., BOURNE, C., WRIGHT, J. C. & CONRAD, P. A. (2002) Babesia gibsoni infection among dogs in the southeastern United States. Journal of the American Veterinary Medical Association. 220: 325-329. MILLER, E. (2000) CVT Update: Diagnosis and treatment of immune-mediated hemolytic anemia. In Kirk’s Current Veterinary Therapy XIII. Ed. J. D. Bonagura, Philadelphia, W. S. Saunders. pp427-434.  OTSUKA, Y., YAMASAKI, M., YAMATO, O. & MAEDE, Y. (2002) The effect of macrophages on the erythrocyte oxidative damage and the pathogenesis of anemia in Babesia gibsoni-infected dogs with low parasitemia. Journal of Veterinary Medical Science. 64: 221-226. STEGEMAN, J. R., BIRKENHEUER, A.J., KRUGER, J. M. & BREITSCHWERDT, E. B. (2003) Transfusion- associated Babesia gibsoni infection in a dog. Journal of the American Veterinary Medical Association. 222: 959-963. 5Table 1. Comparison of physical examination and laboratory findings of B. gibsoni infection  with low parasitemia comfirmed by PCR with those of middle to high parasitemia cases and IMHA  Acute type of B.gibsoni infection   IMHA  Low parasitemia  Middle to high parasitemia        Numbers of dogs      10          25       8 a) Physical examination   Fever (>39.5 C)*      4      14        2  Anemia *       8      21       8  Icterus *       1       8         4  Splenomegaly *       7      16        3  Hematurea *       0       1       4 # Body temperature (C)**  39.6 (0.5)   39.7 (0.7)   38.8 (0.8) # b) Laboratory findings ** RBC (x104/µl)    334 (159)    303 (145)   204 (177) Hb (g/dl)    7.5 (3.4)    6.9 (3.0)    5.0 (2.0) PCV (%)   23.1 (9.2)  21.2 (6.8)    16.1 (5.1) Platelets (x104/µl)   7.0 (6.4)   4.9 (4.1)   15.5 (12.3) WBC (/µl)       13178 (10329)      15083 (10178)        31525 (30993) Total bilirubin (mg/dl)  0.57 (0.55)  1.89 (4.06)    8.1 (14.5) BUN (mg/dl)   15.5 (6.4)  22.4 (13.1)   37.7 (15.1) Creatinine (mg/dl)   0.6 (0.3)   0.8 (0.3)     0.7 (0.3) AST (U/l)    46 (24)    98 (216)   166 (226) ALT (U/l)   173 (262)  146 (256)    60 (29) ALP (U/l)  1697 (2119)  536 (622)    726 (421) CRP (mg/dl)    6.2 (2.2)    7.4 (3.6)    9.2 (6.5)  *: Numbers of dogs that showed the specific findings **: Mean (standard deviation)   #: significant differences (p<0.05) compared with the data of cases with low parasitemia 

Clinical observations of Babesia gibsoni infection with low parasitaemia confirmed by PCR in dogs

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Clinical observations of Babesia gibsoni infection with low parasitaemia confirmed by PCR in dogs

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