Background

Only limited information is currently available on the prevalence of vector borne and zoonotic pathogens in dogs and ticks in Nigeria. The aim of this study was to use molecular techniques to detect and characterize vector borne pathogens in dogs and ticks from Nigeria.

Methodology/Principal Findings

Blood samples and ticks (Rhipicephalus sanguineus, Rhipicephalus turanicus and Heamaphysalis leachi) collected from 181 dogs from Nigeria were molecularly screened for human and animal vector-borne pathogens by PCR and sequencing. DNA of Hepatozoon canis (41.4%), Ehrlichia canis (12.7%), Rickettsia spp. (8.8%), Babesia rossi (6.6%), Anaplasma platys (6.6%), Babesia vogeli (0.6%) and Theileria sp. (0.6%) was detected in the blood samples. DNA of E. canis (23.7%), H. canis (21.1%), Rickettsia spp. (10.5%), Candidatus Neoehrlichia mikurensis (5.3%) and A. platys (1.9%) was detected in 258 ticks collected from 42 of the 181 dogs. Co- infections with two pathogens were present in 37% of the dogs examined and one dog was co-infected with 3 pathogens. DNA of Rickettsia conorii israelensis was detected in one dog and Rhipicephalus sanguineus tick. DNA of another human pathogen, Candidatus N. mikurensis was detected in Rhipicephalus sanguineus and Heamaphysalis leachi ticks, and is the first description of Candidatus N. mikurensis in Africa. The Theileria sp. DNA detected in a local dog in this study had 98% sequence identity to Theileria ovis from sheep.

Conclusions/Significance

The results of this study indicate that human and animal pathogens are abundant in dogs and their ticks in Nigeria and portray the potential high risk of human exposure to infection with these agents.

Author Summary

In Nigeria, dogs are not only kept as pets, but are also used for hunting as well as a source of animal protein among some ethnic groups. Ticks are known to infest dogs and serve as vectors for some pathogens of zoonotic and veterinary importance. There is limited information on the prevalence and distribution of vector borne pathogens in dogs and ticks in Nigeria. The aim of the study was to detect and characterize vector borne pathogens in dogs and ticks from Nigeria using molecular methods. The results of this study showed a high estimate of vector borne pathogens in Nigerian dogs (77.3%) and ticks (63.3%). DNA of Candidatus N. mikurensis, an emerging pathogen of humans was detected in Rhipicephalus sanguineus and Heamaphysalis leachi ticks. Another human pathogen, Rickettsia conorii israelensis the causative agent of Mediterranean spotted fever was detected in Rhipicephalus sanguineus ticks. This is the first description of Candidatus N. mikurensis in Africa and Rickettsia conorii israelensis in Nigeria. These results indicate that the use of molecular techniques for the diagnosis of emerging infections in developing countries is of utmost importance in assisting physicians and veterinarians in making accurate diagnoses and providing the appropriate treatment for their patients.

Anaplasma phagocytophilum is an emerging tick-borne pathogen that infects humans, domestic animals and wildlife throughout the Holarctic. In the far-western United States, multiple rodent species have been implicated as natural reservoirs for A. phagocytophilum. However, the presence of multiple A. phagocytophilum strains has made it difficult to determine which reservoir hosts pose the greatest risk to humans and domestic animals. Here we characterized three genetic markers (23S–5S rRNA intergenic spacer, ank and groESL) from 73 real-time TaqMan PCR-positive A. phagocytophilum strains infecting multiple rodent and reptile species, as well as a dog and a horse, from California. Bayesian and maximum-likelihood phylogenetic analyses of all three genetic markers consistently identified two major clades, one of which consisted of A. phagocytophilum strains infecting woodrats and the other consisting of strains infecting sciurids (chipmunks and squirrels) as well as the dog and horse strains. In addition, analysis of the 23S–5S rRNA spacer region identified two unique and highly dissimilar clades of A. phagocytophilum strains infecting several lizard species. Our findings indicate that multiple unique strains of A. phagocytophilum with distinct host tropisms exist in California. Future epidemiological studies evaluating human and domestic animal risk should incorporate these distinctions.

Background

In tropical Africa, where the spectrum of the bacterial pathogens that cause fevers is poorly understood and molecular-based diagnostic laboratories are rare, the time lag between test results and patient care is a critical point for treatment of disease.

Methodology/Principal Findings

We implemented POC laboratory in rural Senegal to resolve the time lag between test results and patient care. During the first year of the study (February 2011 to January 2012), 440 blood specimens from febrile patients were collected in Dielmo and Ndiop villages. All samples were screened for malaria, dengue fever, Borrelia spp., Coxiella burnetii, Tropheryma whipplei, Rickettsia conorii, R. africae, R. felis, and Bartonella spp.

Conclusions/Significance

We identified DNA from at least one pathogenic bacterium in 80/440 (18.2%) of the samples from febrile patients. B. crocidurae was identified in 35 cases (9.5%), and R. felis DNA was found in 30 cases (6.8%). The DNA of Bartonella spp. was identified in 23/440 cases (4.3%), and DNA of C. burnetii was identified in 2 cases (0.5%). T. whipplei (0.2%) was diagnosed in one patient. No DNA of R. africae or R. conorii was identified. Among the 7 patients co-infected by two different bacteria, we found R. felis and B. crocidurae in 4 cases, B. crocidurae and Bartonella spp. in 2 cases, and B. crocidurae and C. burnetii in 1 case. Malaria was diagnosed in 54 cases. In total, at least one pathogen (bacterium or protozoa) was identified in 127/440 (28.9%) of studied samples. Here, the authors report the proof of concept of POC in rural tropical Africa. Discovering that 18.2% of acute infections can be successfully treated with doxycycline should change the treatment strategy for acute fevers in West Africa.

Author Summary

In tropical Africa, clinical laboratories capable of performing complicated diagnostic studies like PCR are rare and are almost always found in large cities. Moreover, a number of infectious diseases, many of them are emerging and neglected, may be quickly and reliably diagnosed only by molecular biology. This is one of the reasons why the repertoire of bacterial infectious diseases in tropical Africa is poorly known. The laboratory based on the Point-of-Care (POC) principle has been designed in order to resolve the time lag between test results and patient care, which is the critical point for the treatment. We report here the first successful experience of the installation of POC laboratory in rural Senegal. During the first year of the study (February 2011 to January 2012) we identified DNA from at least one pathogenic bacterium in 80/440 (18.2%) of the samples from febrile patients. In most of the cases it was relapsing fever and rickettsiosis agents. Malaria was diagnosed in 54 cases. In total, at least one pathogen (bacterium or protozoa) was identified in 127/440 (28.9%) of studied samples. Discovering that at least 18.2% of acute infections can be successfully treated with doxycycline should change the treatment strategy for acute fevers in West Africa.

Anaplasma phagocytophilum is an obligately intracellular tick-transmitted bacterial pathogen of humans and other animals. During the course of infection, A. phagocytophilum utilizes gene conversion to shuffle ∼100 functional pseudogenes into a single expression cassette of the msp2(p44) gene, which codes for the major surface antigen and major surface protein 2 (MSP2). The role and extent of msp2(p44) recombination, particularly in hosts that only experience acute infections, is not clear. In the present study, we explored patterns of recombination and expression of the msp2(p44) gene of A. phagocytophilum in a serially infected mouse model. Even though the bacterium was passed rapidly among mice, minimizing the opportunities for the host to develop adaptive immunity, we detected the emergence of 34 unique msp2(p44) expression cassette variants. The expression of msp2(p44) pseudogenes did not follow a consistent pattern among different groups of mice, although some pseudogenes were expressed more frequently than others. In addition, among 263 expressed pseudogenes, 3 mosaic sequences each consisting of 2 different pseudogenes were identified. Population genetic analysis showed that genetic diversity and subpopulation differentiation tended to increase over time until stationarity was reached but that the variance that was observed in allele (expressed pseudogene) frequency could occur by drift alone only if a high variance in bacterial reproduction could be assumed. These findings suggest that evolutionary forces influencing antigen variation in A. phagocytophilum may comprise random genetic drift as well as some innate but apparently nonpurifying selection prior to the strong frequency-dependent selection that occurs cyclically after hosts develop strong adaptive immunity.

Background

Tick-borne relapsing fever spirochetes are maintained in endemic foci that involve a diversity of small mammals and argasid ticks in the genus Ornithodoros. Most epidemiological studies of tick-borne relapsing fever in West Africa caused by Borrelia crocidurae have been conducted in Senegal. The risk for humans to acquire relapsing fever in Mali is uncertain, as only a few human cases have been identified. Given the high incidence of malaria in Mali, and the potential to confuse the clinical diagnosis of these two diseases, we initiated studies to determine if there were endemic foci of relapsing fever spirochetes that could pose a risk for human infection.

Methodology/Principal Findings

We investigated 20 villages across southern Mali for the presence of relapsing fever spirochetes. Small mammals were captured, thin blood smears were examined microscopically for spirochetes, and serum samples were tested for antibodies to relapsing fever spirochetes. Ornithodoros sonrai ticks were collected and examined for spirochetal infection. In total, 11.0% of the 663 rodents and 14.3% of the 63 shrews tested were seropositive and 2.2% of the animals had active spirochete infections when captured. In the Bandiagara region, the prevalence of infection was higher with 35% of the animals seropositive and 10% infected. Here also Ornithodoros sonrai were abundant and 17.3% of 278 individual ticks tested were infected with Borrelia crocidurae. Fifteen isolates of B. crocidurae were established and characterized by multi-locus sequence typing.

Conclusions/Significance

The potential for human tick-borne relapsing fever exists in many areas of southern Mali.

Author Summary

Tick-borne relapsing fever is a spirochete-caused, recurrent illness acquired by the bite of fast-feeding ticks. In Mali, the potential for people to acquire relapsing fever is unknown although a few human cases have been reported there. Human malaria is also abundant in Mali, and could be complicating the diagnosis of relapsing fever. The relapsing fever spirochete, Borrelia crocidurae, is maintained in natural cycles involving small mammals and its tick vector Ornithodoros sonrai. Therefore, we investigated 20 villages across southern Mali to determine if relapsing fever spirochetes were circulating in small mammals and ticks that lived with people. We found that 11.3% of the 726 mammals tested showed evidence of prior infection, while 2.2% of the animals were actively infected. The tick vector was abundant in two villages we sampled, and overall 17.3% of the individual ticks tested were infected with spirochetes. We also isolated the spirochetes, Borrelia crocidurae, from rodents and ticks and compared their genetic makeup to other species of African spirochetes. We conclude that in some areas of Mali, people are at risk of acquiring tick-borne relapsing fever. Therefore, we recommend that blood smears from acutely ill patients be examined microscopically for spirochetes.

Background

The presence in Morocco of Argasid ticks of the Ornithodoros erraticus complex, the vector of tick-borne relapsing fever (TBRF) in North Africa, has been known since 1919, but the disease is rarely diagnosed and few epidemiological data are available.

Methodology/Principal Findings

Between 2006 and 2011, we investigated the presence of Ornithodoros ticks in rodent burrows in 34 sites distributed across Morocco. We also collected small mammals in 10 sites and we investigated TBRF in febrile patients in Kenitra district. The prevalence of Borrelia infections was assessed by nested PCR amplification in ticks and the brain tissue of small mammals, and by evaluation of thick blood films in patients. A high proportion of burrows were infested with ticks of the O. erraticus complex in all regions of Morocco, with a mean of 39.5% for the whole country. Borrelia infections were found in 39/382 (10.2%) of the ticks and 12/140 (8.6%) of the rodents and insectivores studied by PCR amplification, and 102 patients tested positive by thick blood film. Five small mammalian species were found infected: Dipodillus campestris, Meriones shawi, Gerbillus hoogstrali, Gerbillus occiduus and Atelerix algirus. Three Borrelia species were identified in ticks and/or rodents: B. hispanica, B. crocidurae and B. merionesi.

Conclusions/Significance

Tick populations belonging to O. erraticus complex are widely distributed in Morocco and a high proportion of ticks and small mammals are infected by Borrelia species. Although rarely diagnosed, TBRF may be a common cause of morbidity in all regions of Morocco.

Author Summary

In North Africa, Argasid ticks of the Ornithodoros erraticus complex are the only known vector of Borrelia infections causing tick-borne relapsing fever (TBRF) in humans. There is limited data on vector distribution, the animal reservoir of the disease has never been investigated, and there is no published data on TBRF patients. Our aim was to systematically investigate the distribution of O. erraticus s.l. in most regions of Morocco, to measure the proportion of infected ticks, to identify small mammalian species that act as potential reservoir, and to analyze data on TBRF patients fortuitously collected during a malaria eradication program. Our study shows that a high proportion of rodent burrows are colonized by vector ticks in all regions of Morocco from the Atlantic Sahara to the Mediterranean coast. We identified three Borrelia species in ticks and/or small mammals: B. hispanica, B. crocidurae and B. merionesi. We report five species of small mammals found infected for the first time. Our analysis of 102 TBRF patients shows that the disease is strictly seasonal in northwestern Morocco with a maximum incidence during summer. We believe that TBRF, although rarely diagnosed, is a common cause of morbidity in all regions of Morocco.

The redwood chipmunk contributes to the maintenance of tick-borne diseases in northern California. The range of redwood chipmunks overlaps that of western black-legged ticks and tick-borne disease, including granulocytic anaplasmosis and Lyme borreliosis. Chipmunks have high Anaplasma phagocytophilum PCR- and seroprevalence, are infested with a diversity of Ixodes spp. ticks, and are reservoir competent for Borrelia burgdorferi. We hypothesized that chipmunks could maintain tick-borne disease on the forest floor while also potentially bridging infection to arboreal sciurids as well. We used radio-telemetry to evaluate chipmunk movement and use of trees, characterized burrows, described prevalence of tick-borne disease, and identified ticks on these chipmunks. A total of 192 chipmunks from Hendy Woods, Mendocino County, California, USA, was evaluated between November 2005 and April 2009. The mean density was 2.26–5.8 chipmunks/ha. The longest detected life span was 3 years. Female weights ranged from 80–120 g and males from 80–180 g. The A. phagocytophilum and Borrelia spp. seroprevalence was 21.4% and 24.7%, respectively, and PCR prevalence for these pathogens was 10.6% and 0%, respectively. Ixodes spp. ticks included I. angustus, I. ochotonae, I. pacificus, and I. spinipalpis. The mean infestation level was 0.92 ticks/chipmunk. Based on telemetry of 11 chipmunks, the greatest distance traveled ranged from 0.14–0.63 km for females and 0.1–1.26 km for males. Areas occupied by chipmunks ranged from 0.005–0.24 km2 for females and 0.006–0.73 km2 for males. On 3 occasions, chipmunks were found in trees. Burrows were identified under a moss-covered redwood log, deep under a live redwood tree, under a Douglas fir log, in a clump of huckleberry, in a root collection from an overturned Douglas fir tree, and in a cluster of exposed huckleberry roots. The biology of the redwood chipmunk has multiple features that allow it to be an important reservoir host for tick-borne disease in northwestern California.

Ixodes spp. tick-borne zoonotic diseases are present across the Holarctic in humans, domestic animals, and wildlife. Small mammals are reservoirs for the rickettsial pathogen Anaplasma phagocytophilum and tick vectors may include catholic-feeding bridge vectors as well as host-specialist or nidicolous ticks. Far western North American communities in which A. phagocytophilum is maintained are complex ecologically, with multiple reservoir host and tick species, multiple strains of the bacterial pathogen A. phagocytophilum and differences in dynamics of hosts and vectors across heterogeneous landscapes. We evaluated sites in northern California in order to identify primarily nidicolous ticks and the hosts they infest. A total of 667 ticks was found in 11 study sites, including 288 on flags and 379 attached to small mammals. Larvae were over-represented among attached ticks and adults on flags. The most abundant species was I. pacificus. Two-hundred fourteen nidicolous ticks were found, most abundantly I. angustus and I. spinipalpis. All adult I. ochotonae, I. auritulus, I. angustus, I. jellisoni, and I. woodi were female, while the male:female ratio of I. spinipalpis was 1.2:1 and 1:1 for I. pacificus. The greatest number of ticks was obtained from Tamias ochrogenys, Peromyscus spp., and Neotoma fuscipes. Of 234 small mammal individuals that were infested with Ixodes spp., only 81 (34.6%) were infested with I. pacificus. The remaining infested small mammals hosted nidicolous tick species. Eight ticks were PCR-positive, including 6 I. pacificus (one adult, one larva, and 6 nymphs), and 2 adult I. ochotonae and high PCR prevalences of 18% and 9% were detected in woodrats and chipmunks, respectively. Nymphal I. angustus ticks were active year-long with a possible increase in August while larval activity was only observed in December and spring months and adults only during spring and fall. Overall, we show high tick species richness and year-round high levels of infestation on rodents by several different nidicolous ticks in areas where A. phagocytophilum is enzootic, including on reported reservoir species.

Anthropogenic environmental change is often implicated in the emergence of new zoonoses from wildlife; however, there is little mechanistic understanding of these causal links. Here, we examine the transmission dynamics of an emerging zoonotic paramyxovirus, Hendra virus (HeV), in its endemic host, Australian Pteropus bats (fruit bats or flying foxes). HeV is a biosecurity level 4 (BSL-4) pathogen, with a high case-fatality rate in humans and horses. With models parametrized from field and laboratory data, we explore a set of probable contributory mechanisms that explain the spatial and temporal pattern of HeV emergence; including urban habituation and decreased migration—two widely observed changes in flying fox ecology that result from anthropogenic transformation of bat habitat in Australia. Urban habituation increases the number of flying foxes in contact with human and domestic animal populations, and our models suggest that, in addition, decreased bat migratory behaviour could lead to a decline in population immunity, giving rise to more intense outbreaks after local viral reintroduction. Ten of the 14 known HeV outbreaks occurred near urbanized or sedentary flying fox populations, supporting these predictions. We also demonstrate that by incorporating waning maternal immunity into our models, the peak modelled prevalence coincides with the peak annual spill-over hazard for HeV. These results provide the first detailed mechanistic framework for understanding the sporadic temporal pattern of HeV emergence, and of the urban/peri-urban distribution of HeV outbreaks in horses and people.

Abstract

Granulocytic anaplasmosis (GA) is an emerging tick-transmitted disease that persists in rodent- Ixodes ricinus-complex tick cycles across the Holarctic. Although the putative reservoir for anaplasmosis in the western United States is the dusky-footed woodrat (Neotoma fuscipes), this rodent was not shown reservoir-competent because of failure of infection from woodrats to other animals via ticks. Redwood chipmunks are common in habitats where Anaplasma phagocytophilum is common, have high PCR- and seroprevalence, and are infested with a diversity of Ixodes spp. ticks. Experimental infection of seven wild-caught A. phagocytophilum-negative redwood chipmunks induced persistent periods of recurrent rickettsemia during the persistent phase of infection. Of three animals for which xenodiagnosis was attempted, all successfully infected pools of I. pacificus larvae during the primary rickettsemia. We show that chipmunks are reservoir-competent for GA and may be important for maintaining infection in nature.

Anaplasma phagocytophilum is a tick-transmitted bacterial pathogen of humans and other animals, and is an obligate intracellular parasite. Throughout the course of infection, hosts acquire temporary resistance to granulocytic anaplasmosis as they develop immunity specific for the major antigen, major surface protein 2 (Msp2). However, the bacterium then utilizes a novel recombination mechanism shuffling functional pseudogenes sequentially into an expression cassette with conserved 5′ and 3′ ends, bypassing host immunity. Approximately 100 pseudogenes are present in the only fully sequenced human-origin HZ genome, representing the possibility for almost unlimited antigenic diversity. In the present study, we identified a select group of 20% of the A. phagocytophilum HZ msp2 pseudogenes that have matched preferentially to human, canine, and equine expression cassettes. Pseudogenes cluster predominantly in one spatial run limited to a single genomic island in less than 50% of the genome but phylogenetically related pseudogenes are neither necessarily located in close proximity on the genome nor share similar percent identity with expression cassettes. Pseudogenes near the expression cassette (and the origin) are more likely to be expressed than those farther away. Taken together, these findings suggest that there may be natural selection pressure to retain pseudogenes in one cluster near the putative origin of replication, even though global recombination shuffles pseudogenes around the genome, separating pseudogenes that share genetic origins as well as those with similar identities.

We analyzed the structure of the expression site encoding the immunoprotective protein MSP2/P44 from multiple Anaplasma phagocytophilum strains in the United States. The sequence of p44ESup1 had diverged in Ap-variant 1 strains infecting ruminants. In contrast, no differences were detected between A. phagocytophilum strains infecting humans and domestic dogs.

Hendra virus (HeV) is a lethal paramyxovirus which emerged in humans in 1994. Poor understanding of HeV dynamics in Pteropus spp. (flying fox or fruit bat) reservoir hosts has limited our ability to determine factors driving its emergence. We initiated a longitudinal field study of HeV in little red flying foxes (LRFF; Pteropus scapulatus) and examined individual and population risk factors for infection, to determine probable modes of intraspecific transmission. We also investigated whether seasonal changes in host behaviour, physiology and demography affect host–pathogen dynamics. Data showed that pregnant and lactating females had significantly higher risk of infection, which may explain previously observed temporal associations between HeV outbreaks and flying fox birthing periods. Age-specific seroprevalence curves generated from field data imply that HeV is transmitted horizontally via faeces, urine or saliva. Rapidly declining seroprevalence between two field seasons suggests that immunity wanes faster in LRFF than in other flying fox species, and highlights the potentially critical role of this species in interspecific viral persistence. The highest seroprevalence was observed when animals showed evidence of nutritional stress, suggesting that environmental processes that alter flying fox food sources, such as habitat loss and climate change, may increase HeV infection and transmission. These insights into the ecology of HeV in flying fox populations suggest causal links between anthropogenic environmental change and HeV emergence.

Bartonella rochalimae was first isolated from the blood of a human who traveled to Peru and was exposed to multiple insect bites. Foxes and dogs are likely natural reservoirs for this bacterium. We report the results of experimental inoculation of two dogs, five cats and six guinea pigs with the only human isolate of this new Bartonella species. Both dogs became bacteremic for 5–7 weeks, with a peak of 103–104 colony forming units (CFU)/mL blood. Three cats had low bacteremia levels (< 200 CFU/mL) of 6–8 weeks’ duration. One cat that remained seronegative had two bacterial colonies isolated at a single culture time point. A fifth cat never became bacteremic, but seroconverted. None of the guinea pigs became bacteremic, but five seroconverted. These results suggest that dogs could be a reservoir of this strain of B. rochalimae, in contrast to cats and guinea pigs.

A total of 1,618 ticks [420 individual (adults) and pooled (larvae and nymphs) samples], 369 rodents (Apodemus agrarius, Rattus norvegicus, Tscherskia triton, Mus musculus, and Myodes regulus), and 34 shrews (Crocidura lasiura) that were collected in northern Gyeonggi-do near the Demilitarized Zone (DMZ) of Korea during 2004-2005, were assayed by PCR for selected zoonotic pathogens. From a total of 420 individual and pooled tick DNA samples, Anaplasma (A.) phagocytophilum (16), A. platys (16), Ehrlichia (E.) chaffeensis (63), Borrelia burgdorferi (16), and Rickettsia spp. (198) were detected using species-specific PCR assays. Out of 403 spleens from rodents and shrews, A. phagocytophilum (20), A. platys (34), E. chaffeensis (127), and Bartonella spp. (24) were detected with species-specific PCR assays. These results suggest that fevers of unknown causes in humans and animals in Korea should be evaluated for infections by these vector-borne microbial pathogens.

A total of 2,121 small mammals in California were assessed for Anaplasma phagocytophilum from 2006 through 2008. Odds ratios were >1 for 4 sciurids species and dusky-footed woodrats. High seroprevalence was observed in northern sites. Ten tick species were identified. Heavily infested rodent species included meadow voles, woodrats, deer mice, and redwood chipmunks.

Two species of Bartonella, a novel Bartonella clarridgeiae-like bacterium and B. vinsonii subsp. berkhoffii, were isolated from rural dogs and gray foxes in northern California. A novel B. clarridgeiae-like species was isolated from 3 (1.7%) of 182 dogs and 22 (42%) of 53 gray foxes, while B. vinsonii subsp. berkhoffii was isolated from 1 dog (0.5%) and 5 gray foxes (9.4%). PCR and DNA sequence analyses of the citrate synthase (gltA) gene and the 16S-23S intergenic spacer region suggested that strains infecting dogs and gray foxes were identical. Fifty-four dogs (29%) and 48 gray foxes (89%) had reciprocal titers of antibodies against Bartonella spp. of ≥64. The high prevalence of bacteremia and seroreactivity to Bartonella spp. in gray foxes suggests that they may act as a reservoir species for the B. clarridgeiae-like species in this region. Domestic dogs were also tested for other arthropod-borne infectious agents. Fifty-one dogs (28%) were positive for Dirofilaria immitis antigen, seventy-four (40%) were seroreactive to Anaplasma phagocytophilum, and five (2.7%) were seropositive for Yersinia pestis. Fourteen dogs (7.6%) were PCR positive for A. phagocytophilum. Polytomous logistic regression models were used to assess the association of Bartonella antibody titer categories with potential risk factors and the presence of other vector-borne agents in domestic dogs. Older dogs were more likely to be seroreactive to Bartonella spp. There was no association between the exposure of dogs to Bartonella and the exposure of dogs to A. phagocytophilum in this study.

There are only two reports in the literature demonstrating the presence of Campylobacter spp. in marine mammals. One report describes the isolation of a new species, Campylobacter insulaenigrae sp. nov., from three harbor seals (Phoca vitulina) and a harbor porpoise (Phocoena phocoena) in Scotland, and the other describes the isolation of Campylobacter jejuni, Campylobacter lari, and an unknown Campylobacter species from northern elephant seals (Mirounga angustirostris) in California. In this study, 72 presumptive C. lari and unknown Campylobacter species strains were characterized using standard phenotypic methods, 16S rRNA PCR, and multilocus sequence typing (MLST). Phenotypic characterization of these isolates showed them to be variable in their ability to grow either at 42°C or on agar containing 1% glycine and in their sensitivity to nalidixic acid and cephalothin. Based on both 16S rRNA PCR and MLST, all but 1 of the 72 isolates were C. insulaenigrae, with one isolate being similar to but distinct from both Campylobacter upsaliensis and Campylobacter helveticus. Phylogenetic analysis identified two C. insulaenigrae clades: the primary clade, containing exclusively California strains, and a secondary clade, containing some California strains and all of the original Scottish strains. This study demonstrates the inability of phenotypic characterization to correctly identify all Campylobacter species and emphasizes the importance of molecular characterization via 16S rRNA sequence analysis or MLST for the identification of Campylobacter isolates from marine mammals.

Anaplasma phagocytophilum, a recently reclassified bacteria in the order Rickettsiales, infects many different animal species and causes an emerging tick-borne disease of humans. The genome contains a large number of related genes and gene fragments encoding partial or apparently full-length outer membrane protein MSP2 (P44). Previous data using strains isolated from humans in the United States suggest that antigenic diversity results from RecF-mediated conversion of a single MSP2 (P44) expression site by partially homologous donor sequences. However, whether similar mechanisms operate in naturally infected animal species and the extent of global diversity in MSP2 (P44) are unknown. We analyzed the structure and diversity of the MSP2 (P44) expression site in strains derived from the United States and Europe and from infections of different animal species, including wildlife reservoirs. The results show that a syntenic expression site is present in all strains of A. phagocytophilum investigated. This genomic locus contained diverse MSP2 (P44) variants in all infected animals sampled, and variants also differed at different time points during infection. Although similar variants were found among different populations of U.S. origin, there was little sequence identity between U.S. strain variants (including genomic copies from a completely sequenced U.S. strain) and expression site variants infecting sheep and dogs in Norway and Sweden. Finally, the possibility that combinatorial mechanisms can generate additional diversity beyond the basic donor sequence repertoire is supported by the observation of shared sequence blocks throughout the MSP2 (P44) hypervariable region in reservoir hosts. These data suggest similar genetic mechanisms for A. phagocytophilum variation in all hosts but worldwide diversity of the MSP2 (P44) outer membrane protein.

In order to investigate the prevalence of tick-borne infectious agents among ticks, ticks comprising five species from two genera (Hemaphysalis spp. and Ixodes spp.) were screened using molecular techniques. Ticks (3,135) were collected from small wild-caught mammals or by dragging/flagging in the Republic of Korea (ROK) and were pooled into a total of 1,638 samples (1 to 27 ticks per pool). From the 1,638 tick samples, species-specific fragments of Anaplasma phagocytophilum (1 sample), Anaplasma platys (52 samples), Ehrlichia chaffeensis (29 samples), Ehrlichia ewingii (2 samples), Ehrlichia canis (18 samples), and Rickettsia rickettsii (28 samples) were amplified by PCR assay. Twenty-one pooled and individual tick samples had mixed infections of two (15 samples) or three (6 samples) pathogens. In addition, 424 spleen samples from small captured mammals (389 rodents, 33 insectivores, and 2 weasels) were screened for selected zoonotic pathogens. Species-specific DNA fragments of A. phagocytophilum (110 samples), A. platys (68 samples), E. chaffeensis (8 samples), E. ewingii (26 samples), E. canis (51 samples), and Rickettsia sp. (22 samples) were amplified by PCR assay. One hundred thirty small mammals had single infections, while 4, 14, and 21 striped field mice (Apodemus agrarius) had mixed infections of four, three, and two pathogens, respectively. Phylogenetic analysis based on nucleotide sequence comparison also revealed that Korean strains of E. chaffeensis clustered closely with those from China and the United States, while the Rickettsia (rOmpA) sequences clustered within a clade together with a Chinese strain. These results suggest that these agents should be considered in differential diagnosis while examining cases of acute febrile illnesses in humans as well as animals in the ROK.

Eschars can be used for genetic characterization of O. tsutsugamushi during the convalescent phase.

To verify the value of eschars for the diagnosis of scrub typhus and to characterize genotypes of Orientia tsutsugamushi in patients, we examined eschars and blood specimens of 7 patients from Shandong Province, People's Republic of China, for O. tsutsugamushi by polymerase chain reaction targeting the Sta56 gene. All 7 eschars and acute-phase blood samples were positive, while no specific DNA amplicons were obtained from the 7 convalescent-phase blood samples collected after antimicrobial drug therapy. The findings indicate that patients' eschars can be used for detection and genetic characterization of O. tsutsugamushi during the convalescent phase.

We report the first documented case of endocarditis associated with Bartonella clarridgeiae in any species. B. clarridgeiae was identified as a possible etiological agent of human cat scratch disease. Infective vegetative valvular aortic endocarditis was diagnosed in a 2.5-year-old male neutered boxer. Historically, the dog had been diagnosed with a systolic murmur at 16 months of age and underwent balloon valvuloplasty for severe valvular aortic stenosis. Six months later, the dog was brought to a veterinary hospital with an acute third-degree atrioventricular block and was diagnosed with infective endocarditis. The dog died of cardiopulmonary arrest prior to pacemaker implantation. Necropsy confirmed severe aortic vegetative endocarditis. Blood culture grew a fastidious, gram-negative organism 8 days after being plated. Phenotypic and genotypic characterization of the isolate, including partial sequencing of the citrate synthase (gltA) and 16S rRNA genes indicated that this organism was B. clarridgeiae. DNA extraction from the deformed aortic valve and the healthy pulmonic valve revealed the presence of B. clarridgeiae DNA only from the diseased valve. No Borrelia burgdorferi or Ehrlichia sp. DNA could be identified. Using indirect immunofluorescence tests, the dog was seropositive for B. clarridgeiae and had antibodies against Ehrlichia phagocytophila but not against Ehrlichia canis, Ehrlichia ewingii, B. burgdorferi, or Coxiella burnetii.

We examined 11 naturally occurring isolates of Ehrlichia equi in horses and two human granulocytic ehrlichiosis agent isolates in California for sequence diversity in three genes. Ehrlichia equi isolates were from Sierra (n = 6), Mendocino (n = 3), Sonoma (n = 1), and Marin (n = 1) counties, and human granulocytic ehrlichiosis (HGE) agent isolates were obtained from Humboldt county. PCR with specific primers for 16S rRNA, 444 Ep-ank and groESL heat shock operon genes successfully produced amplicons for all 13 clinical samples. The 444 Ep-ank gene of the HGE agent and E. equi isolates from northern California is different from the eastern U.S. isolates BDS and USG3. The translated amino acid sequence of the groESL heat shock operon gene fragment is identical among E. equi, the HGE agent, and E. phagocytophila, with the exception of the northern Californian equine CASOLJ isolate. Microheterogeneity was observed in the 16S rRNA gene sequences of HGE agent and E. equi isolates from northern California. These results suggest that E. equi and the HGE agent found in California are similar or identical but may differ from the isolates of equine and human origin found in the eastern United States.

On the basis of biochemical, phenotypic, and 16S rRNA analyses, Helicobacter canis was isolated from Bengal cats with and without chronic diarrhea. Because the cats were coinfected with other potential pathogens, including Campylobacter helveticus, and because H. canis was isolated from nondiarrheic cats, the causal role of H. canis in producing the diarrhea could not be proven. Histologically, the colons of the four affected cats were characterized by mild to moderate neutrophilic, plasmacytic, and histocytic infiltrates in the lamina propria. Rare crypt abscesses were also noted for three cats but were a more prominent feature of the colonic lesions noted for the fourth cat. This is the first observation of H. canis in cats and raises the possibility that H. canis, like H. hepaticus and H. bilis in mice, can cause inflammation of the colon, particularly in hosts with immune dysregulation. Further studies are needed to determine the importance of H. canis as a primary enteric pathogen in cats and the role of cats in the possible zoonotic spread of H. canis to humans.