Autochthonous canine babesiosis in the Netherlands

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Pathogenesis of Ehrlichiosis and / or Anaplasmosis

Disease manifestations caused by members of the E. canis genogroup (genogroup III) infecting dogs can be indistinguishable (Breitschwerdt et al., 1998) and there can be strain variation in pathogenecity (Hegarty, Levy, Gager and Breitschwerdt, 1997).
Monocytic ehrlichiosis in dogs and humans is caused by E. canis and E. chaffeensis, respectively. Canine monocytic ehrlichiosis, the disease caused by E. canis, can be differentiated into three stages characterized by thrombocytopenia, leukopenia and anaemia (Van Heerden, 1982). The first, acute phase may be manifested by fever, dyspnoea, anorexia, and slight weight loss (Van Heerden, 1982). Haematological results often indicate thrombocytopenia, leukopenia, mild anaemia, and hypergammaglobulinaemia. The second phase is subclinical and follows the acute phase. During the subclinical phase, dogs can remain persistently infected for years without clinical signs but with mild thrombocytopenia (Codner and Farris-Smith, 1986).
The chronic phase is the third stage, characterized by haemorrhages, epistaxis and oedema. Haematology results resemble those during the first phase of the disease. The course of the third phase is often complicated by co-infections by other microorganisms (Rikihisa, 1991; Rikihisa, Ewing, Fox, Siregar, Pasaribu and Malole, 1992; Iqbal, Chaichanasiriwithaya and Rikihisa, 1994). Dogs infected with E. canis become life-long carriers, even after treatment (Wen, Rikihisa, Mott, Greene, Kim, Zhi, Couto, Unver and Bartsch, 1997).
Canine granulocytic ehrlichiosis (recently renamed canine granulocytic anaplasmosis by Dumler et al. [2001]) caused by Anaplasma phagocytophilum, is associated with two distinct clinical syndromes, which include chronic, moderate to severe anaemia and polyarthritis (Goldman, Breitschwerdt, Grindem, Hegarty, Walls and Dumler, 1998). Clinical signs are nonspecific and include fever, lethargy, anorexia, vomiting and diarrhoea (Goldman et al., 1998; Kohn, Galke, Beelitz and Pfister, 2008). Most often blood abnormalities are normocytic, normochromic non-regenerative, moderate thrombocytopenia with large platelets, lymphopenia and eosinopenia (Goldman et al., 1998, Kohn et al., 2008).


Canine hepatozoonosis is a disease caused by intraleukocytic Hepatozoon species (MacIntire, Vincent-Johnson, Dillon, Blagburn, Lindsay, Whitley and Banfield, 1997). Unlike most other tick-borne infections, Hepatozoon is transmitted by ingestion of an infected tick by dogs, rather than by the tick biting (Ewing and Panciera, 2003). There are currently two known species causing hepatozoonosis, H. canis and H. americanum (MacIntire et al., 1997; Ewing and Panciera, 2003). Hepatozoon canis, whose major vector is R. sanguineus, is endemic in Africa, southern Europe, the Middle East and Asia (MacIntire et al., 1997; Mathew, Ewing, Panciera and Woods, 1998; Panciera, Ewing, Mathew, Cummings, Kocan, Breshears and Fox, 1998; Panciera, Ewing, Mathew, Lehenbauer, Cummings and Woods, 1999), whereas H. americanum, whose major vector is Amblyomma maculatum, is endemic in the southern USA (Vincent-Johnson, Macintire, Lindsay, Lenz, Baneth, Shkap and Blagburn, 1997). Hepatozoonosis caused by H. canis is often a subclinical infection whereas H. americanum causes a more severe disease (Baneth et al., 2003). Dogs infected with H. americanum are often febrile, stiff, lethargic, and depressed (Ewing and Panciera, 2003). Gait abnormalities and muscle wasting are usually obvious, as is copious mucopurulent ocular discharge. Atrophy of head muscles is especially noticeable (Ewing and Panciera, 2003). Dogs may eat readily when food is placed immediately in front of them, but they often refuse to move to food and water, presumably owing to intense pain, which derives in part from periosteal bone proliferation and inflamed muscles (Vincent-Johnson et al., 1997; MacIntire et al., 1997; Ewing, Mathew, Lehenbauer, Cummings and Woods, 1999).


The only Theileria species known to cause disease in dogs is Theileria annae, which has been reported only in Spain (Garcia, 2006). Ixodes hexagonus is suspected to be the tick vector responsible for the transmission of Theileria annae (Camacho et al., 2003). The disease caused by T. annae is characterized by severe regenerative anaemia and thrombocytopenia. Azotaemia is found in many cases (Camacho, Guitian, Pallas, Gestal, Olmeda, Goethert, Telford and Spielman, 2004). Abnormally high serum concentrations of urea and creatinin, together with elevated concentrations of inorganic phosphorus, hypoalbuminaemia, hypercholesterolaemia, proteinuria, high protein / creatinin and presence of hyaline and granular casts in the microscopic examination of urine sediment suggest a glomerular component of the disease (Garcia, 2006).

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Molecular detection and analysis

Detection and identification of tick-borne pathogens has largely relied on morphological and biological observations. Previously, parasitological (blood smear) examinations used in conjunctions with serology (immunoflourescent antibody test, IFAT) were methods of choice in diagnosing tick-borne infections. Parasitological examinations have limited specificity, however, and there is often antigenic cross-reactivity in the use of serology. Molecular diagnosis is increasingly being used as a reliable tool for the detection and characterization of blood-parasite infection in the host (Caccio, Antunovic, Moretti, Mangili, Marinculic, Baric, Slemenda and Pieniazek, 2002; Birkenheuer et al., 2004) and in the vector (Inokuma, Beppu, Okuda, Shimada and Sakata, 2003). Advances in molecular methodology, particularly automated DNA sequencing, have made it possible to ascertain the evolutionary relationships of species from genes (Stevens, Noyes, Schofield and Gibson, 2001). Within the piroplasmids (Babesiidae and Theileriidae) the 18S rRNA gene proves to be highly conserved in demonstrating genotypic diversity between the species. Phylogenetic analysis of the 18S rRNA gene proved to be useful in proving that multiple small canine piroplasm species exist (Kjemtrup and Conrad, 2006a). A more complete phylogenetic analysis of the 18S rRNA gene that included canine Babesia species from Asia, the Midwestern United States, California, Africa, Africa and Spain confirmed that there are three genotypical distinct small Babesia species of canines (Kjemtrup et al., 2000b). A recent and complete analysis of the 18S rRNA gene, suggests that piroplasms may be divided into five clades: (1) B. microti group, containing Babesia rodhaini, Babesia felis, Babesia leo, B. microti and B. microti-type canine isolate; (2) western USA Theileria-like group, containing B. conradae; (3) Theileria group, containing all Theileria species from bovines; (4) a first group of Babesia species including B. canis and B. gibsoni from canines together with Babesia divergens and Babesia odocoilei; and (5) a second group composed mainly of Babesia species from ungulates: Babesia caballi, Babesia bigemina, Babesia ovis, Babesia bovis and Babesia sp. from cattle (Criado-Fornelio, Martinez-Marcos, Buling-Sarana and Barba-Carretero, 2003). Phylogenetic analysis of the 18S rRNA gene was also used to reinforce the designation of Hepatozoon americanum as a new species separate from Hepatozoon canis (Baneth, Mathew, Shkap, Macintire, Barta and Ewing, 2000). There is sufficient evidence to suggest that studies of the 18S rRNA gene have added important information in understanding the taxonomic position of many piroplasm species, particularly those from canines (Kjemtrup and Conrad, 2006a).

Chapter 1: General Introduction 
1.1. Background
1.2. Tick-borne pathogens
1.3. Canine babesiosis
1.4. Ehrlichiosis and Anaplasmosis
1.5. Hepatozoonosis
1.6. Theileriosis
1.7. Molecular detection and analysis
1.8. Objectives of the study
1.9. Overview of the thesis
1.10. References
Chapter 2: Confirmation of occurrence of Babesia vogeli in domestic dogs in South Africa 
2.1. Abstract
2.2. Introduction
2.3. Materials and Methods
2.4. Results
2.5. Discussion
2.6. Conclusion
2.7. Tables
2.8. References
Chapter 3: Molecular detection of tick-borne protozoal and ehrlichial infections in domestic dogs in South Africa 
3.1. Abstract
3.2. Introduction
3.3. Materials and Methods
3.4. Results
3.5. Discussion
3.6. Conclusion
3.7. Figures and Tables
3.8. References
Chapter 4: Preliminary evaluation of the BrEMA1 gene as a tool for correlating Babesia rossi genotypes and clinical manifestation of canine babesiosis 
4.1. Abstract
4.2. Introduction
4.3. Materials and Methods
4.4. Results
4.5. Discussion
4.6. Conclusion
4.7. Figures and Tables
4.8. References
Chapter 5: Autochthonous canine babesiosis in the Netherlands 
5.1. Abstract
5.2. Introduction
5.3. Materials and Methods
5.4. Results
5.5. Discussion
5.6. Conclusion
5.7. Figures and Tables
5.8. References
Chapter 6: Detection of Theileria sp. infections in dogs in South Africa 
6.1. Abstract
6.2. Introduction
6.3. Materials and Methods
6.4. Results
6.5. Discussion
6.6. Conclusion
6.7. Figures and Tables
6.8. References
Chapter 7: Molecular characterization of Babesia gibsoni infection from a pit-bull terrier pup recently imported into South Africa 
7.1. Abstract
7.2. Introduction
7.3. Materials and Methods
7.4. Results
7.5. Discussion
7.6. Conclusion
7.7. Figures and Tables
7.8. References
Chapter 8: General discussion 
8.1. General discussion
8.2. Conclusion
8.3. References
Scientific publications connected with this thesis


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