| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
SHORT COMMUNICATION |
1 Norwegian School of Veterinary Science, Department of Production Animal Clinical Sciences, Kyrkjevegen 332/334, N-4325 Sandnes, Norway;
2 National Veterinary Institute, 0033 Oslo, Norway;
3 Swedish Institute for Infectious Disease Control, SE-17182 Solna, Sweden
4 Corresponding author (email: Snorre.Stuen{at}veths.no)
ABSTRACT:
This paper describes a case of Anaplasma phagocytophilum infection in a roe deer (Capreolus capreolus) calf in Norway. The calf was found deserted, paretic, and heavily infested with Ixodes ricinus ticks. It was euthanized and investigated postmortem. Anaplasma phagocytophilum was detected in several tissues by polymerase chain reaction (PCR) and 16S rRNA sequence analyses. Analyses for Borrelia burgdorferi sensu lato and tick-borne encephalitis (TBE) virus infections were negative. This is the first report of a possible paretic condition in A. phagocytophilum infected roe deer.
Key words: Anaplasma phagocytophilum, Capreolus capreolus, case report, granulocytic ehrlichiosis, paresis, roe deer.
Tick-borne fever (TBF), caused by Anaplasma phagocytophilum (formerly Ehrlichia phagocytophila), has for decades been considered a common disease in domestic ruminants along the coast of southern Norway (Øverås, 1972; Stuen, 1998). Several other mammalian species, including wild cervids and humans, have also been found infected with A. phagocytophilum (Bakken et al., 1994; Jenkins et al., 2001). In the present paper a paretic condition in an A. phagocytophilum infected roe deer (Capreolus capreolus) calf is described.
The calf was found alive, but unable to stand, on August 2004 in Vest-Agder County, southwestern Norway. The calf held its head in an upright position, but it was unable to move its limbs. The sheep tick Ixodes ricinus is abundant in the area and many ticks were seen on the head and body of the calf. It was euthanized for animal welfare reasons and the whole body was frozen at –20 C for later examination. The estimated age of the calf was about 3 mo (Østbye and Bjørnsen, 1990).
A routine necropsy including aerobic bacterial cultivation from brain, heart, kidney, liver, lungs, and spleen was conducted. Tissue samples were not processed for histopathology because of putrefaction. However, 5- to 10-g tissue samples from brain, heart, blood, kidney, liver, lungs, and spleen tissues were frozen at –20 C for polymerase chain reaction (PCR) analysis and DNA sequencing. All sampled tissues were tested for DNA from A. phagocytophilum and Borrelia burgdorferi sensu lato, while only brain tissue was tested for RNA from tick-borne encephalitis (TBE) virus. Serum was tested for neutralizing antibodies to TBE virus (Vene et al., 1998). The concentrations of cobalt, copper and selenium in the liver were determined on a wet weight (ww) basis by atomic absorption spectroscopy (Bernhoft et al., 2002).
Total genomic DNA was isolated from tissue and blood samples using a commercially available kit (DNeasy Tissue kit; QIAGEN, Valencia, California, USA) and the DNA content was measured spectrophotometrically. For A. phagocytophilum analysis, the samples were subjected to a seminested PCR strategy, using primers 16S-F5 (5'-AGTTTGATCATGGTTCAGA-3') and ANA-R4B (5'-CGAACAACGCTTGC-3') for initial amplification of a 507 bp fragment of the 16S rRNA gene in A. phagocytophilum. The subsequent seminested reaction with primers 16S-F5 and ANA-R5 (5'-TCCTCTCAGACCAGCTATA-3') produced a 282 bp fragment. The amplified products of the initial PCR were diluted at 1:100 in distilled water and 2 µl was used as a template in the second reaction. The PCR was performed in 25 µl reaction volumes containing 2.5 mM MgCl2, 0.2 mM dNTP, 0.5 µM of each primer, 0.7 U AmpliTaq Gold enzyme (Applied Biosystems, Foster City, California, USA), and approximately 100 ng of DNA. Cycling parameters were 95 C for 5 min, followed by three cycles of 94 C, 55–52 C (touchdown of 1.0 C per cycle), and 72 C for 30 sec each, another 35 cycles (25 cycles for the seminested reaction) of 94 C, 52 C, and 72 C for 30 sec each, and finally a 5 min incubation at 72 C.
Anaplasma phagocytophilum variants were detected by direct DNA sequence determination of PCR products. The PCR products were sequenced in both directions using Big Dye terminator cycle sequencing chemistry and capillary electrophoresis (ABI 310; Applied Biosystems). Sequences were visually inspected from chromatograms.
Tissue samples were also tested for Borrelia spp. infection using the SL primers of Demaerschalck et al. (1995). These primers were designed to target the ospA gene of all of the Borrelia burgdorferi sensu lato genospecies involved in Lyme disease. Cultured isolates of B. burgdorferi sensu stricto, B. garinii, and B. afzelii were kindly supplied by National Health Institute, Oslo, Norway (I. S. Aaberge) and they also provided known positive controls for the amplification reaction. PCR was performed on 150 ng genomic DNA in 25 µl reaction volumes with AmpliTaq Gold polymerase and the reaction buffer recommended by the supplier (Applied Biosystems). Cycling parameters were 95 C for 7 min, followed by 40 cycles of 94 C, 53 C, and 72 C for 30 sec each, and a final 5 min incubation at 72 C. To identify TBE virus in the brain tissue, a reverse transcription PCR (RT-PCR) was performed according to Schrader and Süss (1999).
The calf weighed only 6.5 kg and had signs of dehydration. More than 300 I. ricinus were found, mainly nymphs and larvae concentrated on the head. Body fat was not observed around the heart, intestines, and kidneys. Gross pathologic changes were not detected in the brain, spinal cord, vertebrae, or extremities. Petechial subendothelial hemorrhages were found in the heart. The spleen was enlarged and swollen with subcapsular petechiae. Little content was seen in the rumen, and there was no sign of diarrhea.
Bacterial cultivation was negative. However, all tissues examined by PCR were positive for A. phagocytophilum. Sequence analysis of the 16S rRNA gene revealed a genetic variant of A. phagocytophilum previously not identified in clinical cases of wildlife (GenBank accession number AJ242784; Table 1
). The PCR and RT-PCR analyses targeting B. burgdorferi s.l. and TBE virus, respectively, proved negative. Neutralizing antibodies to TBE virus were not detected. The concentrations of cobalt, copper, and selenium in the liver tissue were 0.11, 43.0, and 0.05 µg/g ww, respectively.
|
Recently, several heavily tick-infested roe deer have been found dead or severely ill in the same geographical area. A similar paretic condition has also earlier been observed in five roe deer calves heavily infested with ticks. Unfortunately, none of these calves were further examined (R. Moseid, pers. comm.).
A paretic condition may be caused by several factors, such as brain nematodes (e.g., Elaphostrongylus spp; Handeland et al., 2000), copper deficiency, exhaustion, spinal abscesses, infections, tick paralysis, and trauma. In the present case, brain nematodes, spinal abscesses, and trauma can be excluded. However, tick paralysis cannot be excluded, although toxins from I. ricinus are seldom mentioned in this context (Goethe and Neitz, 1991).
The knowledge of normal liver concentrations of cobalt, copper, and selenium in young roe deer is limited. The observed concentrations of cobalt and copper in this deer were within the normal ranges found in domestic and wild ruminants in Norway (Frøslie et al., 1987; A. Bernhoft, pers. inform.). Related to the knowledge of hepatic selenium in domestic ruminants, the measured selenium concentration in this roe deer must be regarded as deficient (Van Metre and Callan, 2001). The implication of low selenium status is unknown, but selenium deficiency may cause myodegeneration and impaired immunity. Unfortunately, histopathology was not performed. Because the present calf was cachectic, however, the paretic condition was most probably caused by exhaustion due to starvation and infestation/infection.
Blood loss due to heavy tick infestation may cause anemia and general weakness. A connection between tick infestation and starvation has already been mentioned as a cause of death in roe deer in Sweden (Alonso Aguirre et al., 1999). However, A. phagocytophilum infection may also cause severe illness, as observed in experimentally infected reindeer Rangifer tarandus tarandus (Stuen, 1996). In young lambs, the bacterium may cause lameness and pyemia (Brodie et al., 1986).
Experimental infection with A. phagocytophilum in red deer and reindeer showed that red deer were only subclinically infected while reindeer had a severe clinical reaction (Stuen, 1996; Stuen et al., 2001b). Thus, the agent may be more pathogenic in both roe deer and reindeer than in red deer. One possible reason for this could be an acquired resistance of red deer caused by a long-term exposure to I. ricinus and A. phagocytophilum (Wickel, 1996; Dumler and Brouqui, 1997). In Norway, red deer normally live in coastal lowland areas where I. ricinus is common, whereas the roe deer has expanded into tick-infested areas only during the last 40-50 yr (Østbye and Bjørnsen, 1990).
Six 16S rRNA gene variants of A. phagocytophilum have been identified in Norway. So far, two of these variants have been found in seriously infected roe deer (Stuen et al., in press). Although the variants can be distinguished on the basis of one or two nucleotide differences only, studies indicate that there may be biologic, ecologic, and pathologic differences between them (Massung et al., 2002; Stuen et al., 2003). However, whether all A. phagocytophilum variants cause clinical manifestations in cervids is unknown. In the present case, the same 16S rRNA gene variant has been found in diseased cattle (Table 1
).
Hunting statistics for the past decade indicate a reduced production of roe deer calves in the present area, while tick infestation has increased in the same period (R. Moseid, pers. comm.). Further investigations are needed to clarify whether ticks and A. phagocytophilum represent a health problem in roe deer populations.
AGUIRRE, A., C. BROJER, AND T. MORNER. 1999. Descriptive epidemiology of roe deer mortality in Sweden. Journal of Wildlife Diseases 35: 753–762.[Abstract]
BAKKEN, J. S., J. S. DUMLER, S.-M. CHEN, M. R. ECKMAN, L. L. VAN ETTA, AND D. H. WALKER. 1994. Human granulocytic ehrlichiosis in the upper Midwest United States. A new species emerging? Journal of the American Medical Association 272: 212–218.[Abstract]
BERNHOFT, A., T. WAALER, S. D. MATHIESEN, AND A. FLAOYEN. 2002. Trace elements in reindeer from Rybatsjij Ostrov, north western Russia. Rangifer 22: 67–73.
BJOERSDORFF, A., J. BERGLUND, B.-E. KRISTIANSEN, C. SODERSTROM, AND I. ELIASSON. 1999. Variable presentation and course in human granulocytic ehrlichiosis: 12 case reports of the new tick-borne zoonosis. Läkartidningen 96: 4200–4204. [In Swedish.][Medline]
BRODIE, T. A., P. H. HOLMES, AND G. M. URQUHART. 1986. Some aspects of tick-borne diseases of British sheep. Veterinary Record 118: 415–418.[Abstract]
DEMAERSCHALCK, I., A. B. MESSAOUD, M. DE KESEL, B. HOYOIS, Y. LOBET, P. HOET, G. BIGAIGNON, A. BOLLEN, AND E. GODFROID. 1995. Simultaneous presence of different Borrelia burgdorferi genospecies in biological fluids of Lyme Disease patients. Journal of Clinical Microbiology 33: 602–608.[Abstract]
DUMLER, J. S., AND P. BROUQUI. 1997. Human granulocytic ehrlichiosis. In Rickettsial infection and immunity, B. Anderson, H. Friedman, and M. Bendinelli (eds.). Plenum Press, New York, New York, pp. 149–162.
FROSLIE, A., G. HOLT, R. HOIE, AND A. HAUGEN. 1987. Levels of copper, selenium and zinc in liver of Norwegian moose (Alces alces), reindeer (Rangifer tarandus), roedeer (Capreolus capreolus) and hare (Lepus timidus). Norsk landbruks-forskning 1: 243–249. [In Norwegian.]
GOTHE, R., AND A. W. H. NEITZ. 1991. Tick paralyses: Pathogenesis and etiology. Advances in Disease Vector Research 8: 177–204.
HANDELAND, K., L. M. GIBBONS, AND A. SKORPING. 2000. Aspects of the life cycle and pathogenesis of Elaphostrongylus cervi in red deer (Cervus elaphus). Journal of Parasitology 86: 1061–1066.[Medline]
JENKINS, A., K. HANDELAND, S. STUEN, L. SCHOULS, I. VAN DE POL, R.-T. MEEN, AND B.-E. KRISTIANSEN. 2001. Ehrlichiosis in a moose calf. Journal of Wildlife Diseases 37: 201–203.[Abstract]
MASSUNG, R. F., M. J. MAUEL, J. H. OWENS, N. ALLAN, J. W. COURTNEY, K. C. STAFFORD, III, AND T. N. MATHER. 2002. Genetic variants of Ehrlichia phagocytophila,1 Rhode Island and Connecticut. Emerging Infectious Diseases 8: 467–472.[Medline]
SCHRADER, C., AND J. SUSS. 1999. A nested RT-PCR for the detection of tick-borne encephalitis virus (TBEV) in ticks in natural foci. Zentralblatt für Bakeriologie 289: 319–328.
STUEN, S. 1996. Experimental tick-borne fever infection in reindeer (Rangifer tarandus tarandus). Veterinary Record 138: 595–596.
———. 1998. Sjodogg (tick-borne fever)—a historical review. Norsk Veterinærtidsskrift 110: 703–706. [In Norwegian.]
———, AND E. OLSSON ENGVALL. 1999. Ehrlichia phagocytophila infection in lambs as a post mortem diagnosis. In Rickettsiae and rickettsial diseases at the turn of the third millenium, D. Raoult and P. Brouqui (eds.). Elsevier, Paris, France, pp. 406–411.
———, E. OLSSON ENGVALL, I. VAN DE POL, AND L. M. SCHOULS. 2001a. Granulocytic ehrlichiosis in a roe deer calf in Norway. Journal of Wildlife Diseases 37: 614–616.[Abstract]
———., K. HANDELAND, T. FRAMMARSVIK, AND K. BERGSTROM. 2001b. Experimental Ehrlichia phagocytophila infection in red deer (Cervus elaphus). Veterinary Record 149: 390–392.
———, K. BERGSTROM, M. PETROVEC, I. VAN DE POL, AND L. M. SCHOULS. 2003. Differences in clinical manifestations and hematological and serological responses after experimental infection with genetic variants of Anaplasma phagocytophilum in sheep. Clinical and Diagnostic Laboratory Immunology 10: 692–695.
———., T. MOUM, M. PETROVEC, AND L. M. SCHOULS. Genetic variants of Anaplasma phagocytophilum in Norway. International Journal of Medical Microbiology. In press.
VAN METRE, D. C., AND R. J. CALLAN. 2001. Selenium and vitamin E. Veterinary Clinics of North America: Food Animal Practice 17: 373–402.
VENE, S., M. HAGLUND, O. VAPALAHTI, AND Å. LUNDKVIST. 1998. A rapid fluorescent focus inhibition test (RFFIT) for detection of neutralizing antibodies to tick-borne encephalitis (TBE) virus. Journal of Virological Methods 73: 71–75.[Medline]
WIKEL, S. K. 1996. Immunology of the tick-host interface. In The immunology of host-ectoparasitic arthropod relationships, S. K. Wikel (ed.). CAB International, Wallingford, UK, pp. 204–231.
ØSTBYE, E., AND B. BJORNSEN. 1990. The roe deer. In Norges dyr. Pattedyrene 2, A. Semb-Johansson and R. Frislid (eds.). J. W. Cappelens Forlag AS, Oslo, Norway, pp. 128–147. [In Norwegian.]
ØVERAS, J. 1972. Diseases of sheep on Ixodes ricinus infested pasture. Norsk Veterinærtidsskrift 83: 561–567. [In Norwegian.]
Received for publication 3 February 2005.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
