The occurrence of Dirofilaria immitis, Borrelia burgdorferi, Ehrlichia canis and Anaplasma phagocytophium in dogs in China

A survey of the occurrence of Dirofilaria immitis, Borrelia burgdorferi, Ehrlichia canis and Anaplasma phagocytophium in dogs was undertaken in the People's Republic of China between October 2008 and October 2009. A total of 600 blood samples were taken from dogs in four cities in China: 300 in Beijing, 150 in Shenzhen, 30 in Shanghai and 120 in Zhengzhou. All samples were tested for the heartworm antigen and antibodies of canine B. burgdorferi, E. canis and A. phagocytophium by using the canine SNAP? 4Dx? test kit. The occurrence of D. immitis, B. burgdorferi, E. canis and A. phagocytophium was 1.17% (7/600), 0.17% (1/600), 2.17% (13/600) and 0.5% (3/600), respectively. In Shenzhen city 2% (3/150), 8.67% (13/150) and 2% (3/150) of samples were positive for D. immitis, E. canis and A. phagocytophium, respectively. The occurrence of heartworm antigen was 0.33% (1/300) in Beijing, 2.00% (3/150) in Shenzhen, 3.33% (1/30) in Shanghai and 1.67% (2/120) in Zhengzhou. We found E. canis and A. phagocytophium only at one site, Shenzhen, while the only occurrence of B. burgdorferi was at Beijing. In conclusion, the dog population in China is at potential risk for D. immitis, B. burgdorferi, E. canis and A. phagocytophium infection, the risk being especially high in southern China.     

Dog tick-borne diseases in Sicily

In Sicily many tick borne diseases are endemic, in particular way those that see like main carrier ticks that prefer, for their vital cycle, climatic conditions characterized by high temperatures and a warmth-humid atmosphere. The more important pathologies transmitted by ticks causing diseases in dogs are babesiosis and ehrlichiosis. Borrelia burgdorferi, Anaplasma phagocytophilum, Rickettsia conorii, Coxiella burnetii and tick transmitted encephalitis virus assume particular relevance because they are agents of zoonosis. Our centre, C.R.A.Ba.R.T, have conducted many researches and carried out many tests for diagnostic aim in order to estimate the spread of the main tick borne diseases in Sicilians' dogs. A study lead on 342 dogs has evidenced seroprevalence for Babesia canis, Ehrlichia canis and Rickettsia respective of 5.17%, 21.70% and 53.43%. A study on zoonotic agent seroprevalences in dogs gave the following percentages: C. burnetii 31.50%, R. conorii 73.60% and A. phagocytophilum 32.80%. The data carried out from IZS Sicily diagnostic service on 5,634 tests done in 2004-2005, confirm the experimental results on the presence of B. canis, E. canis, R. conorii, A. phagocytophilum, C. burnetii, Rickettsia spp., Anaplasma spp. and Ehrlichia spp. in all the Sicilian areas.     

Ehrlichia ewingii sp. nov., the etiologic agent of canine granulocytic ehrlichiosis.

The 16S rRNA gene was amplified, cloned, and sequenced from the blood of two dogs that were experimentally infected with the etiologic agent of canine granulocytic ehrlichiosis. The 16S rRNA sequence was found to be unique when it was compared with the sequences of other members of the genus Ehrlichia. The most closely related species were Ehrlichia canis (98.0% related) and the human ehrlichiosis agent (Ehrlichia chaffeensis) (98.1% related); all other species in the genus were found to be phylogenetically much more distant. Our results, coupled with previous serologic data, provide conclusive evidence that the canine granulocytic ehrlichiosis agent is a new species of the genus Ehrlichia that is related to, but is distinct from, E. canis and all other members of the genus. We propose the name Ehrlichia ewingii sp. nov.; the Stillwater strain is the type strain.     

Unique macrophage and tick cell-specific protein expression from the p28/p30-outer membrane protein multigene locus in Ehrlichia chaffeensis and Ehrlichia canis

Ehrlichia chaffeensis and Ehrlichia canis are tick-transmitted rickettsial pathogens that cause human and canine monocytic ehrlichiosis respectively. We tested the hypothesis that these pathogens express unique proteins in response to their growth in vertebrate and tick host cells and that this differential expression is similar in closely related Ehrlichia species. Evaluation of nine E. chaffeensis isolates and one E. canis isolate demonstrated that protein expression was host cell-dependent. The differentially expressed proteins included those from the p28/30-Omp multigene locus. E. chaffeensis and E. canis proteins expressed in infected macrophages were primarily the products of the p28-Omp 19 and 20 genes or their orthologues. In cultured tick cells, E. canis expressed only the p30-10 protein, an orthologue of the E. chaffeensis p28-Omp 14 protein which is the only protein expressed by E. chaffeensis propagated in cultured tick cells. The expressed Omp proteins were post-translationally modified to generate multiple molecular forms. E. chaffeensis gene expression from the p28/30-Omp locus was similar in tick cell lines derived from both vector (Amblyomma americanum) and non-vector (Ixodes scapularis) ticks. Differential expression of proteins within the p28/p30-Omp locus may therefore be vital for adaptation of Ehrlichia species to their dual host life cycle.     

Serologic and molecular evidence of Ehrlichia spp. in coyotes in California

In order to determine the role of coyotes in the epidemiology of granulocytic and monocytic ehrlichial agents in California (USA), we tested 149 serum samples for antibodies against Ehrlichia equi, E. risticii, and E. canis, using an indirect immunofluorescent antibody test. Polymerase chain reaction (PCR) assay was used to survey for the presence of members of the E. phagocytophila genogroup, E. risticii and E. canis in blood samples of 95 coyotes. Sixty-eight (46%) samples were seropositive for E. equi, two (1%) for E. risticii and none of the samples had antibodies reactive to E. canis. Two and one coyote were positive for E. risticii and members of the E. phagocytophila genogroup by PCR assay, respectively. In contrast, the 95 samples were negative for E. canis by PCR. Ninety-five percent of the 68 E. equi seropositive coyotes and the one coyote PCR positive for members of the E. phagocytophila genogroup originated from a coastal area. However, the two E. risticii seropositive coyotes and the two coyotes PCR positive for E. risticii were from northern California. Sequence analysis of the three amplified PCR products revealed the agent to be similar in two coyotes to the sequences of E. risticii from horses originating from northern California and identical in one coyote to the agent of human granulocytic ehrlichiosis and E. equi from California. Thus, coyotes are exposed to granulocytic ehrlichiae and E. risticii and may play a role in the epidemiology of these ehrlichial agents in California.     

Canine ehrlichiosis caused simultaneously by Ehrlichia canis and Ehrlichia platys

To identify the causative agent of canine ehrlichiosis that has occurred in the suburbs of Guangzhou, China, since 1998, the 16S rRNA gene was amplified and sequenced. Two sequences of 1,482 and 1,483 base pairs were obtained and named as Gzh981 and Gzh982, respectively. The level of similarity of these two was 91.50%, and Gzh981 closely resembled the 16S rRNA gene of Ehrlichia canis, whereas Gzh982 resembled Ehrlichia platys. We therefore conclude that E. canis and E. platys together caused recent outbreaks of canine ehrlichiosis in China.     

Molecular survey of Anaplasma phagocytophilum and Ehrlichia canis in red foxes (Vulpes vulpes) from central Italy

During the 2007-2008 hunting season, 150 spleen samples were collected from free-ranging red foxes (Vulpes vulpes) in central Italy. The specimens were tested by two nested PCR assays to detect DNA of Anaplasma phagocytophilum, etiologic agent of granulocytic ehrlichiosis of animals and humans, and DNA of Ehrlichia canis, which causes the monocytic ehrlichiosis in canids. None of the foxes were PCR-positive for E. canis; 25 (16.6%) were positive for A. phagocytophilum. No specific gross alterations were detected at necropsy, and no histopathologic lesions found on PCR-positive spleen samples.     

Detection of antibodies reactive with Ehrlichia chaffeensis in the raccoon

Antibodies reactive with Ehrlichia chaffeensis were detected in raccoon (Procyon lotor) serum samples by using an indirect immunofluorescence assay. Samples from 411 raccoons trapped in the southeastern United States from 1977 to 1999 were tested. Serologically reactive samples with reciprocal titers of > or =16 were detected from 83 raccoons (20%) from 13 of 16 counties in eight states, indicating that raccoons are commonly exposed to E. chaffeensis. Samples collected as early as 1977 were positive. A polymerase chain reaction assay specific for E. chaffeensis failed to detect the presence of ehrlichial DNA in serum samples from 20 representative seroreactive raccoons. Because of serologic cross-reactivity among antigens derived from different Ehrlichia spp., additional immunologic, molecular, or culture-based studies will be required to confirm E. chaffeensis infections of raccoons in the southeastern United States.     

Molecular Detection of Ehrlichia canis in Dogs in Malaysia

An epidemiological study of Ehrlichia canis infection in dogs in Peninsular Malaysia was carried out using molecular detection techniques. A total of 500 canine blood samples were collected from veterinary clinics and dog shelters. Molecular screening by polymerase chain reaction (PCR) was performed using genus-specific primers followed by PCR using E. canis species-specific primers. Ten out of 500 dogs were positive for E. canis. A phylogenetic analysis of the E. canis Malaysia strain showed that it was grouped tightly with other E. canis strains from different geographic regions. The present study revealed for the first time, the presence of genetically confirmed E. canis with a prevalence rate of 2.0% in naturally infected dogs in Malaysia.     

Tick-transmitted diseases in dogs: clinicopathological findings

In this article we describe the main clinicopathological findings of some tick-transmitted diseases observed in Italy, due to Ehrlichia canis and Babesia canis, and most rarely Anaplasma phagocytophilum and Anaplasma platys. Canine monocytic ehrlichiosis (CME) is a multisystemic disorder that is characterized by various clinical signs. Acutely-infected dogs show various clinical and haematological abnormalities including fever, lymphadenopathy, anorexia, lethargy, depression and thrombocytopenia. Many dogs with CME evolve in to an asymptomatic or chronically symptomatic carrier states. In Italy there are very few cases of Canine Granulocytic Ehrlichiosis (CGE) and all are attributed to A. phagocytophilum. The early manifestations of CGE are usually mild and consist in acute onset of fever and depression with or without thrombocytopenia. Lameness due to polyarthritys is also possible. Other clinical manifestations most rarely described are very similar to those reported in chronic form of E. canis infections. There are very few studies about clinicopathological findings of canine babesiosis in Italy. In our country this infection is caused by Babesia canis (large form of parasite) subspecies B. canis canis and B. canis vogeli. These two subspecies are morphologically indistinguishable. Clinical signs reflect the intravascular and extravascular haemolysis due to the life cycle of the parasite. The most common haematological abnormalities found in canine babesiosis are anaemia and thrombocytopenia. It is important to point out that co-infection between two or more agents is possible. In this case it is very difficult to attribute the clinical signs and haematological and/or biochemical abnormalities to a single specific agent.     

Phylogenetic position of Cowdria ruminantium (Rickettsiales) determined by analysis of amplified 16S ribosomal DNA sequences.

The 16S ribosomal DNA sequence of Cowdria ruminantium, the causative agent of heartwater disease in ruminants, was determined. An analysis of this sequence showed that C. ruminantium forms a tight phylogenetic cluster with the canine pathogen Ehrlichia canis and the human pathogen Ehrlichia chaffeensis. Although a close relationship between the genus Cowdria and several members of the tribe Ehrlichieae has been suspected previously, the tight phylogenetic cluster with E. canis and E. chaffeensis is surprising in view of known differences in host preference and target cells.     

Predominance of <Emphasis Type="Italic">Ehrlichia chaffeensis</Emphasis> in <Emphasis Type="Italic">Rhipicephalus sanguineus</Emphasis> ticks from kennel-confined dogs in Limbe,Cameroon

Rhipicephalus sanguineus ticks (n = 63) collected from five dogs (two adults and three puppies) housed in a kennel were screened for Ehrlichial agents (Ehrlichia canis, E. chaffeensis, and E. ewingii) using a species-specific multicolor real-time TaqMan PCR amplification of the disulphide bond formation protein (dsb) gene. Ehrlichia chaffeensis DNA was detected in 33 (56%) ticks, E. canis DNA was detected in four (6%) ticks, and one tick was coinfected. The E. chaffeensis and E. canis nucleotide sequences of the amplified dsb gene (374 bp) obtained from the Cameroonian R. sanguineus ticks were identical to the North American genotypes.     

Intestinal nematode infections in Turkish military dogs with special reference to Toxocara canis

The prevalence and potential zoonotic risk factors of intestinal nematodes of military working dogs, which are used for different military purposes, were assessed. Faecal samples from 352 defined-breed Turkish military dogs were investigated and 107 (30.4%) dogs were found to be infected with one or two nematode species. The following nematodes, with their respective prevalences, were diagnosed in the faecal samples: Toxascaris leonina (21.8%), Toxocara canis (13.3%), Trichuris vulpis (2.9%) and Uncinaria stenocephala (1.2%). Toxocara canis infections were more frequently seen in puppies (0-6 months old). The prevalence of T. canis was significantly higher in male than in female dogs and also higher in dogs which were exercised daily than in those without exercise. The highest prevalence was found in Belgian malinois breed dogs. Toxocara canis infections were not influenced by the floor type of the kennels (i.e. concrete or soil floor). There was no difference in the occurrence of T. canis infection when the last anthelmintic treatment was carried out less or more than 3 months prior to sampling. It is suggested that T. canis infected military dogs would be a threat not only for dog trainers but also for military personnel, notably during national and international operations.     

Antigenic and genomic relatedness among Ehrlichia risticii, Ehrlichia sennetsu, and Ehrlichia canis.

Antigenic and genomic relatedness among Ehrlichia risticii, E. sennetsu, and E. canis was analyzed by enzyme-linked immunosorbent assay, Western blotting (immunoblotting) and DNA-DNA hybridization. E. risticii and E. sennetsu were serologically related, and their Western blot antigen profiles were nearly identical. Two antigens of E. sennetsu corresponding to the 28- and 51-kDa antigens of E. risticii were apparently larger than the E. risticii antigens, and the 55-kDa antigen of E. risticii appeared to be unique to this species. The 110-, 70-, and 44-kDa antigens of these two species were identical, as determined by the use of monospecific antibodies. DNA homology between these two species was high. On the other hand, E. canis was antigenically least reactive with the antisera to E. risticii and E. sennetsu. However, a dog convalescent-stage E. canis antiserum recognized antigens in the other two species which were different from those recognized by their homologous antisera. Similarly, homology between the DNA of E. canis and the DNAs of the other two species was very minimal. These results indicate that E. risticii and E. sennetsu are closely related both at the genomic and antigenic levels and that the relationship of these two species with E. canis is minimal.     

Incidence and seroprevalence of canine ehrlichiosis in the Medjez El Bab region (northwestern Tunisia during 1994, 1995 and 1996

A sero-epidemiological survey, realized in the Medjez El Bab region (North-West of Tunisia), has concerned 180 dogs which status has been determined during the study. The animals were identified, then underwent an annual blood sampling during three successive years, in order to search for antibodies against E. canis and E. chaffeensis by indirect immunofluorescence. The results show that, in all sero-positive dogs, the levels of antibodies against E. canis were higher than those against E. chaffeensis. The sero-prevalence of E. canis was 42.8%, 50% and 48.9%, in 1994, 1995 and 1996, respectively, and was higher than that against E. chaffeensis during the three year studies. The incidence of E. canis infection was 12.6% during the three years whereas E. chaffeensis infection did not exceed 4.7%.     

Prevalence and intensity of Toxocara canis (Werner, 1782) in dogs and its potential public health significance in Ile-Ife, Nigeria

A study on the prevalence and intensity of Toxocara canis (Werner, 1782) in dogs was carried out in Ile-Ife, Nigeria. Faecal samples were collected from 269 dogs between January and December 2004, processed by the Kato-Katz technique and then examined for T. canis eggs. The prevalence of T. canis obtained was 33.8%. The intensity of infection, measured as mean egg count per gram of faeces ( +/- SEM) was 393.8 +/- 83.4. The prevalence and intensity of T. canis in dogs aged 0-6 months were significantly higher (P < 0.05) than older age groups. The prevalence and intensity of T. canis infection were significantly higher in males than in female dogs (P < 0.05). Since T. canis is known to cause visceral larva migrans (VLM) in young children, there is the possibility that the high prevalence of T. canis infection obtained in this study might constitute an important risk factor for transmission to humans. Therefore, there is the need to educate the residents of Ile-Ife on the danger of close association of their children with household pets.     

Molecular survey of canine vector-borne diseases in stray dogs in Thailand

Despite the large population of stray dogs in Thailand, there is limited information on the prevalence of canine vector-borne diseases (CVBDs). In this study, a molecular survey was conducted to determine the prevalence of Babesia spp., Ehrlichia canis, Hepatozoon spp., Anaplasma platys and Mycoplasma spp. in dogs in Thailand. Of the 181 dog blood samples tested by PCR, 78/181 (43.1%) were found to be infected with one or more pathogens. The overall prevalence rates of Mycoplasma spp., Hepatozoon spp., Babesia spp., A. platys and E. canis infections were 19.9%, 18.8%, 9.4%, 4.4% and 3.9%, respectively. To the authors' knowledge, this is the first report of Mycoplasma infection in Thailand in dogs. The current findings are important for future surveillance of CVBDs and designing appropriate approaches for diagnosis and control for the diseases in Thailand.     

The map1 gene of Cowdria ruminantium is a member of a multigene family containing both conserved and variable genes

Heartwater is an economically important disease of ruminants caused by the tick-transmitted rickettsia Cowdria ruminantium. The disease is present in Africa and the Caribbean and there is a risk of spread to the Americas, particularly because of a clinically asymptomatic carrier state in infected livestock and imported wild animals. The causative agent is closely related taxonomically to the human and animal pathogens Ehrlichia chaffeensis and Ehrlichia canis. A dominant immune response of infected animals or people is directed against variable outer membrane proteins of these agents known, in E. chaffeensis and E. canis, to be encoded by polymorphic multigene families. We demonstrate, by sequence analysis, that map1 encoding the major outer membrane protein of C. ruminantium is also encoded by a polymorphic multigene family. Two members of the gene family are located in tandem in the genome. The upstream member, orf2, is conserved, encoding only 2 amino acid substitutions among six different rickettsial strains from diverse locations in Africa and the Caribbean. In contrast, the downstream member, map1, contains variable and conserved regions between strains. Interestingly, orf2 is more closely related in sequence to omp1b of E. chaffeensis than to map1 of C. ruminantium. The regions that differ among orf2, map1, and omp1b correspond to previously identified variable sequences in outer membrane protein genes of E. chaffeensis and E. canis. These data suggest that diversity in these outer membrane proteins may arise by recombination among gene family members and offer a potential mechanism for persistence of infection in carrier animals.     

Longitudinal analysis of tick densities and Borrelia, Anaplasma, and Ehrlichia infections of Ixodes ricinus ticks in different habitat areas in The Netherlands

From 2000 to 2004, ticks were collected by dragging a blanket in four habitat areas in The Netherlands: dunes, heather, forest, and a city park. Tick densities were calculated, and infection with Borrelia burgdorferi and Anaplasma and Ehrlichia species was investigated by reverse line blot analysis. The lowest tick density was observed in the heather area (1 to 8/100 m2). In the oak forest and city park, the tick densities ranged from 26 to 45/100 m2. The highest tick density was found in the dune area (139 to 551/100 m2). The infection rates varied significantly for the four study areas and years, ranging from 0.8 to 11. 5% for Borrelia spp. and 1 to 16% for Ehrlichia or Anaplasma (Ehrlichia/Anaplasma) spp. Borrelia infection rates were highest in the dunes, followed by the forest, the city park, and heather area. In contrast, Ehrlichia/Anaplasma was found most often in the forest and less often in the city park. The following Borrelia species were found: Borrelia sensu lato strains not identified to the species level (2.5%), B. afzelii (2.5%), B. valaisiana (0.9%), B. burgdorferi sensu stricto (0.13%), and B. garinii (0.13%). For Ehrlichia/Anaplasma species, Ehrlichia and Anaplasma spp. not identified to the species level (2.5%), Anaplasma schotti variant (3.5%), Anaplasma phagocytophilum variant (0.3%), and Ehrlichia canis (0.19%) were found. E. canis is reported for the first time in ticks in The Netherlands in this study. Borrelia lusitaniae, Ehrlichia chaffeensis, and the human granylocytic anaplasmosis agent were not detected. About 1.6% of the ticks were infected with both Borrelia and Ehrlichia/Anaplasma, which was higher than the frequency predicted from the individual infection rates, suggesting hosts with multiple infections or a possible selective advantage of coinfection.