Defining the immune response to Ehrlichia species using murine models

Pathogenic bacteria belonging to the family Anaplasmataceae include species of the genera Ehrlichia and Anaplasma. Ehrlichia chaffeensis, first known as the causative agent of human monocytic ehrlichiosis, also infects several vertebrate hosts including white-tailed deer, dogs, coyotes and goats. E. chaffeensis is transmitted from the bite of an infected hard tick, such as Amblyomma americanum. E. chaffeensis and other tick-transmitted pathogens have adapted to both the tick and vertebrate host cell environments. Although E. chaffeensis persists in both vertebrate and tick hosts for long periods of time, little is known about that process. Immunological studies will be valuable in assessing how the pathogen persists in nature in both vertebrate and invertebrate hosts. Understanding the host immune response to the pathogen originating from dual host backgrounds is also important to develop effective methods of diagnosis, control and treatment. In this paper, we provide our perspective of the current understanding of the immune response against E. chaffeensis in relation to other related Anaplasmataceae pathogens.     

Persistent Ehrlichia ewingii Infection in Dogs after Natural Tick Infestation

Background

Ehrlichia ewingii, which causes disease in dogs and people, is the most common Ehrlichia spp. infecting dogs in the United States, but little is known about how long E. ewingii infection persists in dogs.

Hypothesis/Objectives

To evaluate the persistence of natural infection with E. ewingii in dogs.

Animals

Four Class A Beagles; no previous exposure to ticks or tick‐borne infectious agents.

Methods

Dogs were exposed to ticks by weekly walks through tick habitat in north central Oklahoma; dogs positive for infection with Ehrlichia spp. by sequence‐confirmed PCR and peptide‐specific serology were evaluated for 733 days (D). Whole blood was collected once weekly for PCR, and serum was collected once monthly for detection of antibodies to Ehrlichia canis (peptide p16), Ehrlichia chaffeensis (indirect fluorescence antibody [IFA] and variable‐length PCR target [VLPT]), and E. ewingii (peptide p28).

Results

All dogs (4/4) became infected with Ehrlichia spp. as evidenced by seroconversion on IFA to E. chaffeensis (4/4); PCR detection of E. ewingii (4/4) and E. chaffeensis (2/4) DNA using both nested and real‐time assays; and presence of specific antibodies to E. ewingii (4/4) and E. chaffeensis (2/4). Infection with E. chaffeensis was not detected after D55. Intermittent E. ewingii rickettsemia persisted in 3 of 4 dogs for as long as 733 days.

Conclusions and Clinical Importance

Our data demonstrate that dogs infected with E. ewingii from tick feeding are capable of maintaining infection with this pathogen long‐term, and may serve as a reservoir host for the maintenance of E. ewingii in nature.     

The transmission of Cowdria ruminantium by Amblyomma sparsum

Transmission of Cowdria ruminantium (heartwater) by nymphs of Amblyomma sparsum has been demonstrated. Adults of the same tick species failed to transmit the disease after feeding on infected sheep in either the larval or the nymphal stage, and there was no transovarian transmission. A. sparsum seldom parasitizes domestic livestock but could be of importance in maintaining reservoirs of heartwater infection in wildlife populations.     

Babesiosis in dogs and cats--expanding parasitological and clinical spectra

Canine babesiosis caused by different Babesia species is a protozoal tick-borne disease with worldwide distribution and global significance. Historically, Babesia infection in dogs was identified based on the morphologic appearance of the parasite in the erythrocyte. All large forms of Babesia were designated Babesia canis, whereas all small forms of Babesia were considered to be Babesia gibsoni. However, the development of molecular methods has demonstrated that other Babesia species such as Babesia conradae, Babesia microti like piroplasm, Theileria spp. and a yet unnamed large form Babesia spp. infect dogs and cause distinct diseases. Babesia rossi, B. canis and Babesia vogeli previously considered as subspecies are identical morphologically but differ in the severity of clinical manifestations which they induce, their tick vectors, genetic characteristics, and geographic distributions, and are therefore currently considered separate species. The geographic distribution of the causative agent and thus the occurrence of babesiosis are largely dependent on the habitat of relevant tick vector species, with the exception of B. gibsoni where evidence for dog to dog transmission indicates that infection can be transmitted among fighting dog breeds independently of the limitations of vector tick infestation. Knowledge of the prevalence and clinicopathological aspects of Babesia species infecting dogs around the world is of epidemiologic and medical interest. Babesiosis in domestic cats is less common and has mostly been reported from South Africa where infection is mainly due to Babesia felis, a small Babesia that causes anemia and icterus. In addition, Babesia cati was reported from India and sporadic cases of B. canis infection in domestic cats have been reported in Europe, B. canis presentii in Israel and B. vogeli in Thailand. Babesiosis caused by large Babesia spp. is commonly treated with imidocarb dipropionate with good clinical response while small Babesia spp. are more resistant to anti-babesial therapy. Clinical and parasitological cure are often not achieved in the treatment of small Babesia species infections and clinical relapses are frequent. The spectrum of Babesia pathogens that infect dogs and cats is gradually being elucidated with the aid of molecular techniques and meticulous clinical investigation. Accurate detection and species recognition are important for the selection of the correct therapy and prediction of the course of disease.     

Molecular events involved in cellular invasion by Ehrlichia chaffeensis and Anaplasma phagocytophilum

Ehrlichia chaffeensis and Anaplasma phagocytophilum are obligatory intracellular bacteria that preferentially replicate inside leukocytes by utilizing biological compounds and processes of these primary host defensive cells. These bacteria incorporate cholesterol from the host for their survival. Upon interaction with host monocytes and granulocytes, respectively, these bacteria usurp the lipid raft domain containing GPI-anchored protein to induce a series of signaling events that result in internalization of the bacteria. Monocytes and neutrophils usually kill invading microorganisms by fusion of the phagosomes containing the bacteria with granules containing both antimicrobial peptides and lysosomal hydrolytic enzymes and/or through sequestering vital nutrients. However, E. chaffeensis and A. phagocytophilum alter vesicular traffic to create a unique intracellular membrane-bound compartment that allows their replication in seclusion from lysosomal killing. These bacteria are quite sensitive to reactive oxygen species (ROS), so in order to survive in host cells that are primary mediators of ROS-induced killing, they inhibit activation of NADPH oxidase and assembly of this enzyme in their inclusion compartments. Moreover, host phagocyte activation and differentiation, apoptosis, and IFN-γ signaling pathways are inhibited by these bacteria. Through reductive evolution, lipopolysaccharide and peptidoglycan that activate the innate immune response, have been eliminated from these gram-negative bacteria at the genomic level. Upon interaction with new host cells, bacterial genes encoding the Type IV secretion apparatus and the two-component regulatory system are up-regulated to sense and adapt to the host environment. Thus dynamic signal transduction events concurrently proceed both in the host cells and in the invading E. chaffeensis and A. phagocytophilum bacteria for successful establishment of intracellular infection. Several bacterial surface-exposed proteins and porins are recently identified. Further functional studies on Ehrlichia and Anaplasma effector or ligand molecules and cognate host cell receptors will undoubtedly advance our understanding of the complex interplay between obligatory intracellular pathogens and their hosts. Such data can be applied towards treatment, diagnosis, and control of ehrlichiosis and anaplasmosis.     

Microbial pathogens in ticks, rodents and a shrew in northern Gyeonggi-do near the DMZ, Korea

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.     

Erysipelothrix rhusiopathiae

Erysipelothrix rhusiopathiae is a facultative, non-spore-forming, non-acid-fast, small, Gram-positive bacillus. The organism was first established as a human pathogen late in the nineteenth century. Three forms of human disease have been recognised since then. These include a localised cutaneous lesion form, erysipeloid, a generalised cutaneous form and a septicaemic form often associated with endocarditis. The organism is ubiquitous and able to persist for a long period of time in the environment, including marine locations. It is a pathogen or a commensal in a wide variety of wild and domestic animals, birds and fish. Swine erysipelas caused by E. rhusiopathiae is the disease of greatest prevalence and economic importance. Diseases in other animals include erysipelas of farmed turkeys, chickens, ducks and emus, and polyarthritis in sheep and lambs. Infection due to E. rhusiopathiae in humans is occupationally related, principally occurring as a result of contact with contaminated animals, their products or wastes, or soil. Erysipeloid is the most common form of infections in humans. While it has been suggested that the incidence of human infection could be declining due to technological advances in animal industries, infection still occurs in specific environments. Additionally, infection by the organism is possibly under-diagnosed due to the resemblance it bears to other infections, and problems encountered in isolation and identification. Various virulence factors have been suggested as being involved in the pathogenicity of E. rhusiopathiae. The presence of a hyaluronidase and neuraminidase has been recognised, and it was shown that neuraminidase plays a significant role in bacterial attachment and subsequent invasion into host cells. The role of hyaluronidase in the disease process is controversial. The presence of a heat labile capsule has been reported as important in virulence. Control of animal disease by sound husbandry, herd management, good sanitation and immunization procedures is recommended.     

Molecular evidence of Anaplasma infection in naturally affected domestic cats of Pakistan

Feline anaplasmosis is considered as an emerging tick-borne disease of zoonotic potential. The aim of current study was to investigate the molecular prevalence of anaplasmosis, associated risk factors, and alterations in hematological parameters of domestic cats from Lahore, Pakistan. Blood samples of 100 domestic cats from district Lahore were examined microscopically and the extracted genomic DNA from each sample was processed for the amplification of 16 S rRNA gene of Anaplasma. PCR confirmed isolates were purified for sequencing. The data regarding the risk factors was collected in a predesigned questionnaire and statistically analyzed by logistic regression analysis. The study found a molecular prevalence of 13% (13/100) among analyzed blood samples. The nucleotide analysis of Anaplasmataceae species sequences amplified by PCR showed high resemblance (99%) with isolates from Korea, Japan, Malaysia, Philippines, and India. The potential risk factors found to be significantly associated (p < 0.05) with disease dynamics based on the Chi-Square test were tick infestation on studied animals, previous tick history, tick control status, house hygiene, and housing type. A significant (p < 0.05) decrease in the number of platelets, erythrocytes, hemoglobin level, and pack cell volume was observed in cats suffering from anaplasmosis compared to the healthy ones. The current study is the first report of Anaplasma infection in domestic cats of Pakistan. This study will be effectual in designing the control strategies for this disease.     

New defensins from hard and soft ticks: Similarities,differences, and phylogenetic analyses

Despite the importance of ticks as vectors of disease very little is known about their immune system. Antimicrobial peptides, including defensins (phylogenetically ancient antibacterial peptides) are major components of innate immunity in ticks that have been shown to provide protection against gram-negative and gram-positive bacteria, fungi, viruses and protozoan parasites. With the aim of studying the evolution of the genes involved in tick defense, we identified the preprodefensin genes from four Ornithodoros tick species (O. papillipes: isoforms A, B, and D; O. tartakovskyi and O. puertoricensis: isoforms A and B; O. rostratus: isoform A) and from two Dermacentor tick species (D. reticulatus and D. marginatus: one isoform) not previously described. Phylogenetic analyses revealed that Ornithodoros defensin isoforms (A, B, C, and D) form 4 separate clades, while hard tick defensins are divided into several branches based on particular tick species.     

Disease at the wildlife-livestock interface: Acaricide use on domestic cattle does not prevent transmission of a tick-borne pathogen with multiple hosts

Several prominent and economically important diseases of livestock in East Africa are caused by multi-host pathogens that also infect wildlife species, but management strategies are generally livestock focused and models of these diseases tend to ignore the role of wildlife. We investigate the dynamics of a multi-host tick-borne disease in order to assess the efficacy of tick control from an ecological perspective. We examined the efficacy of a widespread measure of tick control and developed a model to explore how changes in the population of ticks due to control measures on cattle impact dynamics of Theileria parva infection in a system with two primary host species, cattle and Cape buffalo (Syncerus caffer). We show that the frequency of acaricide application has a significant impact on the tick population both on the host and in the environment, which can greatly reduce the pathogen load in cattle. We also demonstrate that reducing the tick population through cattle-related control measures is not sufficient to diminish disease transmission in buffalo. Our results suggest that under current control strategies, which target ticks on cattle only, T. parva is likely to remain a significant problem in East Africa, and require the continued use of acaricides, which has significant economic and ecological consequences.     

Natural history of Ehrlichia ruminantium

Ehrlichia ruminantium is an obligately intracellular proteobacterium which causes a disease known as heartwater or cowdriosis in some wild, and all domestic, ruminants. The organism is transmitted by ticks of the genus Amblyomma, and it is of serious economic importance wherever the natural vectors occur, an area which includes all of sub-Saharan Africa, and several islands in the Caribbean. The disease was first recognized in South Africa in the 19th century, where its tick-borne nature was determined in 1900, but the organism itself was not demonstrated until 1925, when it was recognized to be a rickettsia, initially named Rickettsia ruminantium. It was thus the first species of what are now known as Ehrlichia to be discovered, and most of the early work to elucidate the nature of the organisms, and its reservoirs and vectors, was performed in South Africa. The next milestone was the development, in 1945, of an infection and treatment regimen to immunize livestock, and this is still the only commercially available “vaccine” against the disease. Then in 1985, after fruitless attempts over many years, the organism was propagated reliably in tissue culture, opening the way for the first application of the newly developed techniques of molecular genetics. From 1990 onwards the pace of heartwater research accelerated rapidly, with notable advances in phylogeny, diagnosis, epidemiology, immunology, and vaccine development. The complete genome sequence was published in 2005, and during the last two years a new understanding has arisen of the remarkable genetic variability of the organism and new experimental vaccines have been developed. Despite all this the goal of producing an effective vaccine against the disease in the field still remains frustratingly just beyond reach. This article summarises our current understanding of the nature of E. ruminantium, at a time when the prospects for the development of an effective vaccine against the organism seem better than at any time since its discovery 83 years ago.     

Comparative Evaluation of 2 In-Clinic Assays for Vector-Borne Disease Testing in Dogs

Vector-borne agents comprise medically important infections affecting dogs throughout much of the world. Sensitive detection of antibodies directed at tick-borne disease-causing organisms in dogs is diagnostically important for veterinarians, pets and their owners, and epidemiologically important for public health surveillance. The SNAP 4Dx Plus Test (IDEXX Laboratories, Inc., Westbrook, ME) identifies antibodies to or infection with multiple tick-borne pathogens and canine heartworm antigen in a single assay. Recently, VetScan FLEX4 Rapid Test (Abaxis, Inc., Union City, CA) was launched as a new assay to detect tick-borne pathogen antibodies and heartworm antigen. In the present study, we evaluated the comparative performance of SNAP 4Dx Plus (SNAP) and FLEX4 Rapid Test (FLEX4) using samples selected based on geographic distributions for canine vector borne diseases, including Borrelia burgdorferi (n = 105), Anaplasma phagocytophilum (160), Anaplasma platys (115), Ehrlichia canis (154), Ehrlichia ewingii (163), Ehrlichia chaffeensis (151) and Dirofilaria immitis (105). Canine vector borne diseases infection status was established for each sample by a combination of reference methods that included necropsy (D. immitis, heartworm disease), Western immunoblotting (B. burgdorferi), immunofluorescence assays (A. phagocytophilum and E. canis) and species-specific ELISAs (A. platys, E. canis, E. ewingii and E. chaffeensis). For comparisons among the 2 assays, samples were evaluated per the manufacturers’ instructions for each test kit.By testing each same sample set compared to the defined reference results, sensitivities differed substantially between SNAP and FLEX4, at 95.5 vs. 40.9%, respectively for B. burgdorferi, 97.1% vs. 61.4% for E. canis, 98.2% vs. 59.3% for E. ewingii, 64.3% vs. 35.7% for E. chaffeensis, 84.5% vs. 12.7% for A. phagocytophilum, 83.3% vs. 33.3% for A. platys, and 94.1% vs. 88.2% for D. immitis. Specificities for both rapid assay tests ranged from 98% to 100%.Based upon the comparative results derived from this study, the SNAP test was more sensitive than the FLEX4 test for detection of antibodies to all tick-borne pathogens and heartworm disease (Dirofilaria immitis) antigen in dogs.     

Perspectives on canine and feline hepatozoonosis

Two species of Hepatozoon are currently known to infect dogs and cause distinct diseases. Hepatozoon canis prevalent in Africa, Asia, southern Europe, South America and recently shown to be present also in the USA causes infection mainly of hemolymphoid organs, whereas Hepatozoon americanum prevalent in the southeastern USA causes myositis and severe lameness. H. americanum is transmitted by ingestion of the Gulf Coast tick Amblyomma maculatum and also by predation on infected prey. H. canis is transmitted by Rhipicephalus sanguineus, in South America also by Amblyomma ovale, and has also been shown to be transmitted transplacentally. Hepatozoonosis of domestic cats has been described mostly from the same areas where canine infection is present and the exact identity of the species which infect cats, their pathogenicity and vectors have not been elucidated. The diagnosis of hepatozoonosis is made by observation of gamonts in blood smears, histopathology, PCR or serology. The main treatment for H. canis is with imidocarb dipropionate whereas H. americanum infection is treated with an initial combination of trimethoprim-sulfadiazine, pyrimethamine and clindamycin followed by maintenance with decoquinate. Treatment for both diseases has not been reported to facilitate complete parasite elimination and new effective drugs are needed for the management of these infections. Prevention of hepatozoonosis should be based on avoidance of oral ingestion of infected tick vectors and infected prey.     

First isolation and molecular characterization of Ehrlichia canis in Costa Rica, Central America

The present study investigated Ehrlichia species in blood samples from dogs suspected of clinical ehrlichiosis, using molecular and isolation techniques in cell culture. From a total of 310 canine blood samples analyzed by 16S rRNA nested PCR, 148 (47.7%) were positive for Ehrlichia canis. DNA from Ehrlichia chaffeensis or Ehrlichia ewingii was not detected in any sample using species-specific primers in separated reactions. Leukocytes from five PCR-positive dogs were inoculated into DH82 cells; successful isolation of E. canis was obtained in four samples. Partial sequence of the dsb gene of eight canine blood samples (including the five samples for in vitro isolation) was obtained by PCR and their analyses through BLAST showed 100% of identity with the corresponding sequence of E. canis in GenBank. This study represents the first molecular diagnosis, isolation, and molecular characterization of E. canis in dogs from Costa Rica.     

The first survey and molecular identification of Entamoeba spp. in farm animals on Qinghai-Tibetan Plateau of China

Protozoans of Entamoeba spp. are globally distributed zoonotic parasites that infect diverse animal hosts and humans. Prevalence and species/genotypes distribution of Entamoeba spp. in domestic animals are not fully investigated on Qinghai-Tibetan Plateau (QTP), an animal husbandry and agriculture region of China. In a survey, 528 fecal samples were collected from 7 species of domestic animals on multiple locations across QTP region and analyzed by PCR and sequencing analysis. The overall prevalence of Entamoeba spp. infection in all examined animals was 97.9 %. Four Entamoeba species, E. bovis, E. moshkovskii, E. ecuadoriensis and E. histolytica were found, and majority (94.2 %) of Entamoeba-infected animals harbored E. bovis. Six Entamoeba genotypes, Entamoeba ribosomal lineages (RL) 1, 2, 3, 4, 8 and 9 were identified by sequencing analysis. Two zoonotic species, E. moshkovskii and E. histolytica, were present in horses, while E. ecuadoriensis and E. bovis were found in horses and all species of seven farm animals, respectively. It was also observed that six Entamoeba genotypes were distributed in animals in specific pattern. The results revealed high prevalence of Entamoeba spp. infection in livestock, broad range of hosts as well as diversity and species/genotype distribution of Entamoeba spp. in farm animals inhabiting on the high altitude QTP region.     

The spreading process of Ehrlichia canis in macrophages is dependent on actin cytoskeleton,calcium and iron influx and lysosomal evasion

Ehrlichia canis is an obligate intracellular microorganism and the etiologic agent of canine monocytic ehrlichiosis. The invasion process has already been described for some bacteria in this genus, such as E. muris and E. chaffeensis, and consists of four stages: adhesion, internalisation, intracellular proliferation and intercellular spreading. However, little is known about the spreading process of E. canis. The aim of this study was to analyse the role of the actin cytoskeleton, calcium, iron and lysosomes from the host cell in the spreading of E. canis in dog macrophages in vitro. Different inhibitory drugs were used: cytochalasin D (actin polymerisation inhibitor), verapamil (calcium channel blocker) and deferoxamine (iron chelator). Our results showed a decrease in the number of bacteria in infected cells treated with all drugs when compared to controls. Lysosomes in infected cells were cytochemically labelled with acid phosphatase to allow the visualisation of phagosome-lysosome fusion and were further analysed by transmission electron microscopy. Phagosome-lysosome fusion was rarely observed in vacuoles containing viable E. canis. These data suggest that the spreading process of E. canis in vitro is dependent on cellular components analysed and lysosomal evasion.     

Anaplasmosis in Cattle in Italy

Bovine anaplasmosis caused by Anaplasma marginale is a disease transmitted by ticks belonging to the Ixodidae family. Southern Italy is considered an endemic zone but environmental and social factors are changing the epidemiology of the disease to expand to previously anaplasmosis-free regions. The available data of published reports of anaplasmosis in Italy together with the data obtained by the National Centre of Reference for Anaplasma, Babesia, Rickettsia and Theileria (C.R.A.Ba.R.T.), allowed to report A. marginale infection in different Italian regions (Lazio, Marche, Campania, Puglia, Basilicata, Calabria, Lombardy, Tuscany, Umbria and Sicily). Cattle are also subject to infection with the related Ixodes ricinus-transmitted pathogen, Anaplasma phagocytophilum that results in reduced milk production in cattle. A. phagocytophilum infect also small ruminants, domestic and wild animals and causes the human granulocytic anaplasmosis. Different studies have been conducted on the presence of A. phagocytophilum in Italy both in the tick vectors and in the wild and domestic reservoirs. Contrary to A. marginale, the prevalence of A. phagocytophilum embraces the whole Italian territory from the Alps to the southern and insular regions.     

A preliminary investigation of Ehrlichia species in ticks, humans, dogs, and capybaras from Brazil

A molecular epidemiologic investigation in two Brazilian states (Rondônia and São Paulo) was undertaken to determine if Ehrlichia species responsible for human and animal ehrlichioses in North America could be found in Brazilian vectors, potential natural mammalian reservoirs and febrile human patients with a tick bite history. Samples, including 376 ticks comprising 9 Amblyomma species, 29 capybara (Hydrochaeris hydrochaeris) spleens, 5 canine blood, and 75 human blood samples from febrile patients with history of tick bites were tested by a real-time PCR assay targeting a fragment of the Ehrlichia dsb gene. Ehrlichia DNA was not detected in any tick, capybara or human samples. In contrast, 4 out of 5 dogs contained Ehrlichia canis DNA in their blood, which were sequenced, representing the first report of E. canis infecting dogs in the Amazon region of Brazil. Further studies are needed to evaluate the presence of other agents of human and animal ehrlichioses in Brazil.     

Bartonella bovis and Candidatus Bartonella davousti in cattle from Senegal

In Senegal, domestic ruminants play a vital role in the economy and agriculture and as a food source for people. Bartonellosis in animals is a neglected disease in the tropical regions, and little information is available about the occurrence of this disease in African ruminants. Human bartonellosis due to Bartonella quintana has been previously reported in Senegal. In this study, 199 domestic ruminants, including 104 cattle, 43 sheep, and 52 goats were sampled in villages from the Senegalese regions of Sine Saloum and Casamance. We isolated 29 Bartonella strains, all exclusively from cattle. Molecular and genetic characterization of isolated strains identified 27 strains as Bartonella bovis and two strains as potentially new species. The strains described here represent the first Bartonella strains isolated from domestic ruminants in Senegal and the first putative new Bartonella sp. isolated from cattle in Africa.     

Association between cattle herd Mycobacterium avium subsp. paratuberculosis (MAP) infection and infection of a hare population

Paratuberculosis has long been considered a disease of domestic and wild ruminants only. The known host range of Mycobacterium avium subsp. paratuberculosis (MAP) was recently extended to include non-ruminant wildlife species believed to be exposed to spillover of MAP from infected domestic cattle herds. The aim of the present study was to assess the association between cattle herd MAP infection pressure level and the infection level of a hare population in two dairy farms of southern Chile. Fifty hares from a herd A and 42 hares from herd B were captured and sampled for MAP culture. The results showed a statistically significant association between the cattle herds’ infection prevalence and the hare infection prevalence.