Emergence of zoonotic sporotrichosis due to Sporothrix brasiliensis in Minas Gerais, Brazil: A molecular approach to the current animal disease.

Sporotrichosis is a neglected fungal zoonosis with significant impacts on human and animal health. Accurate diagnosis, treatment, and understanding of the transmission dynamics of Sporothrix species are essential for mitigating the spread of sporotrichosis. This study aimed to identify the Sporothrix species involved in the ongoing outbreaks of animal sporotrichosis in the metropolitan region of Belo Horizonte, Minas Gerais, Brazil, and analyse the phylogenetic relationships between pathogenic species to investigate the outbreak origin. Additionally, to better understand the evolution of the disease, we conducted a retrospective survey of positive feline and canine cases from November 2017 to July 2021 with proven cultures for Sporothrix. A significant increase in animal cases over the last 4 years was observed, with cats being the most affected host. Sporothrix brasiliensis was the predominant agent in 100% of the clinical isolates (n = 180) molecularly identified. Phylogenetic and haplotype analysis points towards the cases isolated from Minas Gerais sharing the haplotype originating from a long-lasting outbreak of cat-transmitted sporotrichosis in Rio de Janeiro, however, with a secondary contribution from genotypes circulating in other outbreaks in Brazil. Thus, we present clear evidence of the circulation of different S. brasiliensis genotypes associated with animal sporotrichosis in the metropolitan region of Belo Horizonte. Genetic monitoring can contribute to understanding the causal agent for zoonotic sporotrichosis in epidemiological processes and help to implement disease prevention and control measures.

Implementation of an Animal Sporotrichosis Surveillance and Control Program, Southeastern Brazil.

We report the implementation of an animal sporotrichosis surveillance and control program that evaluates strategies to identify suspected and infected cats in a municipality in southeastern Brazil. All adopted measures reinforced the program, although strategies had different abilities to detect the presence of infection.

Calf immunization protocols with low-virulence isolates of Anaplasma marginale: Analysis of post-inoculation effects and protection against natural challenge.

Bovine anaplasmosis is endemic and is of fundamental importance worldwide. Therefore, measures for controlling and preventing clinical diseases are warranted to ensure the reduction of associated economic losses. The objective of the present study was to assess the post-inoculation effects and protection conferred by three different protocols of inoculation of low-virulence live strains of Anaplasma marginale (UFMG1 and UFMG3) in field-challenged cattle. Sixty-eight Holstein calves with an average age of 17 days were randomly divided into four groups. The groups received two subcutaneous administrations spaced 40 days apart, at a dosage of 2 × 106 infected erythrocytes of the following A. marginale strains: G1 (UFMG1 + UFMG1); G2 (UFMG3 + UFMG3); G3 (UFMG1 + UFMG3); and G4 (control). Every two days, the animals were evaluated for rectal temperature, Packed Cell Volume (PCV), and blood smears. Blood samples were collected prior to inoculation, before the field challenge, and after the challenge period, nPCR and IFAT techniques were performed. There were no significant differences in rickettsemia levels, reduction in PCV, or antibody detection among the different inoculation strategies. Forty days after the second inoculation, 90 %, 84.6 %, and 90.9 % of the animals in G1, G2, and G3, respectively, tested positive using nPCR. After inoculation, the group G2, which received the UFMG3 inoculum, had a higher frequency of treatment (odds ratio of 6.7; 1.198-38.018 CI; p = 0.03), while groups G1 and G3 demonstrated similar treatment frequencies compared to the control. During the natural challenge phase, 13.3 % of animals in group G1 required treatment (odds ratio of 0.108; 0.018-0.635 CI; p = 0.014) compared to 58.8 % of the control group. Considering the results collectively, the protocol using the UFMG1 strain (G1) stands out for its potential to be safe and induce some degree of immunization against A. marginale, reducing the incidence of clinical disease and the need for treatment during natural challenge.

Hemopathogens in naturally infected bovine fetuses in Brazil.

The transplacental transmission of parasites and hemoparasites is crucial for understanding the epidemiology of diseases. This study aimed to assess the prevalence of hemopathogens in bovine fetuses at various gestational periods. Samples were obtained from a slaughterhouse in the state of Minas Gerais, Brazil, and a total of 236 fetuses were collected. DNA extracted from blood samples (145) and organ samples (a pool of brain and spleen) (236) underwent a nested PCR (nPCR) assay to detect Babesia spp., Theileria spp., Trypanosoma vivax, Anaplasma marginale, Anaplasma bovis, Anaplasma phagocytophilum, Ehrlichia minasensis, and hemotropic Mycoplasma spp. Additionally, serological analysis of 145 plasma samples was conducted using the indirect fluorescent antibody test-IFAT to detect IgG against Babesia bovis, Babesia bigemina, A. marginale, and Trypanosoma vivax. The observed prevalence of transplacental transmission was 19.3 %, 6.2 %, 42.7 % and 2.7 %, for A. marginale, B. bigemina, 'Candidatus M. haemobos', and Mycoplasma wenyonii, respectively. The prevalence of A. marginale by gestational trimester was 16 % (13/81) in the second trimester and 23 % (14/60) in the third trimester, with no positive samples in the first trimester. Regarding the species B. bovis and B. bigemina, all evaluated animals tested negative by nPCR, and no serological evidence for B. bovis was found by the IFAT. Babesia bigemina demonstrated an overall seroprevalence of 6.2 % (9/145), with 4.8 % (7/145) in the last trimester and 1.3 % (2/145) in the second trimester of pregnancy. In total, 42.7 % (62/145) of blood samples were positive for 'Candidatus M. haemobos', with 42 % (34/81) in the middle trimester, and 43 % (26/60) in the final trimester of pregnancy. Mycoplasma wenyonni was detected in 2.7 % (4/145) blood samples, all in coinfection with 'C. M. haemobos'. The prevalence by pregnancy trimester was 25 % (1/4) in the first trimester; 1.2 % (1/81) in the second trimester and 3.3 % (2/60) in the third trimester of pregnancy. Hemopathogen DNA was detected in fetus blood samples but not the brain or spleen samples. All the samples were negative for T. vivax, Theileria spp., Anaplasma spp. and Ehrlichia spp. Overall, in this study, approximately 70 % of fetuses were positive for one or more of the studied parasites. No significant associations were observed between pairs of pathogens, except 'C. M. haemobos' and A. marginale.

Realities, perceptions, and strategies for implementation of an ethical population management program for dogs and cats on university campuses.

Stray dogs and cats pose significant challenges for public health and animal welfare due to their potential involvement in zoonotic disease transmission, accidents, and aggressions. Large urban centers exacerbated challenges due to the presence of these animals in public areas with high human density. Ethical Population Management Programs (EPMP), rooted in the One Health approach, are crucial for addressing this issue comprehensively. This study aimed to demonstrate the approach on cats and dogs EPMP and evaluate the perceptions of academic community regarding EPMP implementation on a campus situated in urban territory. The study was conducted at the Pampulha campus of UFMG in Belo Horizonte, Brazil. In response to issues of animal abandonment and conflicts, the Permanent Commission for Animal Policies (CPPA-UFMG) was established in 2019 to manage the campus's dog, cat, and wildlife populations. The commission implemented the Trap-Neuter-Return (TNR) method, along with health assessments and vaccinations for animals. Interviews were conducted with campus staff to gauge their perception of animal management strategies. Retrospective and prospective analyses of the commission's actions were carried out to assess implementation processes and challenges. The animal population survey conducted on campus between July 2018 and September 2021 revealed a total of 266 animals recorded. Among these animals, 195 were cats (73.3%) and 71 were dogs (26.7%), with the majority being adults. Subsequent surveys in 2019 and 2021 showed a slight increase in the animal population, with measures such as sterilization contributing to population control. Perception analysis among campus users indicated strategies such as TNR were widely endorsed for population control. The employees perception questionnaire was applied to 115 individuals, representing 42 units/departments and five gates. Associations were found between these beliefs and support for institutional actions. The majority favored sterilization (92.17%) and agreed that TNR is an appropriate approach to population control. Overall, the study reflects a community concerned about animal welfare and supportive of measures to address population management and cruelty prevention. The continuous efforts of the university's CPPA have led to stability in the resident animal population, indicating success in achieving population control objectives.

Household outbreak of sporotrichosis: towards the One Health approach.

Although sporotrichosis requires a broad approach for control, few reports have described the relationship between the index case and secondary contacts. In the present work, we report an outbreak involving a woman, a dog, and two cats from the same household environment, including the clinical and epidemiological aspects and outcomes, and discuss the importance of a One Health approach to face this neglected disease. The joint efforts of professionals such as veterinarians and physicians are essential for early diagnosis and surveillance, which contributes to the rapid identification and control of zoonotic sporotrichosis outbreaks.

The health facility as a risk factor for multidrug-resistant gram-negative bacteria in critically ill patients with COVID-19.

BACKGROUND: The relationship between Multidrug Resistant-Gram Negative Bacteria (MDR-GNB) infection and colonization in critically ill COVID-19 patients has been observed, however, it is still poorly understood. This study evaluated the risk factors for acquiring MDR-GNB in patients with severe COVID-19 in Intensive Care Units (ICU). METHODS: This is a nested case-control study in a cohort of 400 adult patients (≥ 18 years old) with COVID-19, hospitalized in the ICU of 4 hospitals in the city of Curitiba, Brazil. Cases were critical COVID-19 patients with one or more MDR GNB from any surveillance and/or clinical cultures were taken during their ICU stay. Controls were patients from the same units with negative cultures for MDR-GNB. Bivariate and multivariate analyses were done. RESULTS: Sixty-seven cases and 143 controls were included. Independent risk factors for MDR bacteria were: male gender (OR = 2.6; 95% CI 1.28‒5.33; p = 0.008); the hospital of admission (OR = 3.24; 95% CI 1.39‒7.57; p = 0.006); mechanical ventilation (OR = 25.7; 95% CI 7.26‒91; p < 0.0001); and desaturation on admission (OR = 2.6; 95% CI 1.27‒5.74; p = 0.009). CONCLUSIONS: Male gender, desaturation, mechanical ventilation, and the hospital of admission were the independent factors associated with MDR-GNB in patients in the ICU with COVID-19. The only modifiable factor was the hospital of admission, where a newly opened hospital posed a higher risk. Therefore, coordinated actions toward a better quality of care for critically ill COVID-19 patients are essential.

Failure of the Amblyomma cajennense nymph to become infected by Theileria equi after feeding on acute or chronically infected horses.

Tick-borne diseases in horses are caused by the intraerythrocytic protozoan parasites Theileria equi and Babesia caballi. Although T. equi is highly endemic in Latin America, the New World vector of this important parasite is controversial. The aim of this study was to test the ability of nymph Amblyomma cajennense ticks acquire infection by T. equi following feeding on infected horses. Three experiments were performed: tick acquisition of T. equi from an experimentally infected horse, tick acquisition of T. equi from naturally infected foals and tick acquisition of T. equi from a chronically infected horse. A. cajennense adults were dissected and salivary glands were collected in aliquots. Methyl green pyronin staining of the salivary glands did not show the presence of hypertrophy of acini or cell nuclei normally suggestive of Theileria spp. infection. The pools of salivary glands were negative for Theileria DNA in nested PCR assays. Histopathological analysis failed to detect sporoblast and sporozoites of T. equi in salivary gland acini. This study was not able to observe infection of the A. cajennense by T. equi.

Report of Amblyomma sculptum in a House in a Rickettsia rickettsii Circulation Area.

Amblyomma sculptum is a tick that has medical and veterinary importance as, in Brazil, it is the main vector of Rickettsia rickettsii, a disease affecting humans. The presence of ticks was observed outside a residence in a peri-urban area of the Atlantic Forest region in Brazil, as well as on two dogs that lived there. Eighteen A. sculptum adults were seen walking on a cemented pillar at the porch of the house and sheltering inside the pillar's crevices; meanwhile on the dogs, only Rhipicephalus sanguineus sensu lato ticks were found. It is hypothesized that as the dogs circulated in the forest regions, they might have carried A. sculptum to the residence. This situation highlights the role of dogs as possible carriers of Brazilian spotted fever (BSF) tick vectors into human habitation. Strategies for the prevention and control of BSF should consider the hypothesis that ticks infected with R. rickettsii can be harbored in human dwellings in peri-urban areas.

Characterization of animal sporotrichosis in a highly urbanized area.

An epidemiological characterization of animal sporotrichosis was carried out between 2017 and 2018, in a highly urbanized area in Brazil, including outcomes and the spatial distribution of the cases according to the health vulnerability index (HVI) of the study territory. One hundred and sixty-five cats and four dogs suspected of sporotrichosis were identified by a surveillance and control program previously implemented in the study area. One hundred and five of these animals (62.1 %) were considered positive for Sporothrix spp., of which 103 were cats and two were dogs. Cases predominated among male cats (53.4 %), although gender did not present a statistically significant association (p > 0.05) with sporotrichosis cases. Considering the positive cats, 55.4 % were not neutered and free access to the street was reported as a habit for 86.4 % of them. Roaming free in the streets increased the chance of infection by 2.54 times. We observed a high frequency of euthanasia or death (48.8 %) among 86 infected cats available to be included in the follow-up stage of the study, even when they were treated (46.5 %) and a low cure rate (31 %). The disease spread, unrelated to the HVI in the territory. Data produced suggested that avoiding access to the street seems to have greater importance to sporotrichosis control in cats than neutering and reinforces the importance of health education, especially in relation to responsible feline ownership. The free offer of diagnosis and treatment also should be taken into consideration as important measures to control the disease.

Comparative PCR-based diagnosis for the detection of Leishmania infantum in naturally infected dogs.

The applicability of molecular biology/PCR for canine visceral leishmaniasis diagnosis presents challenges, mainly due to the diversity of targets described. The objectives of this study were to compare the sensitivities and reliability of five targets (kDNA/120, kDNA/145, ITS1, hsp70/234 and hsp70/1300) in four different tissue samples (bone marrow, popliteal lymph node, skin and conjunctival swab). Sixty-five dogs (32 males and 33 females) naturally infected with Leishmania infantum and ten dogs without infection were examined. Dogs were characterized by serological and parasitological methods. The parasitological test was considered the gold standard for analysis. All tests presented high specificity 100% (95% CI 0.72-1), and variable sensitivity. The targets kDNA/145, ITS1, kDNA/120, hsp70/234 and hsp70/1300 detected 100% (65/65), 93.4% (61/65), 92.3% (60/65), 84.61% (55/65) and 72.3% (77/65) of positive animals respectively. The performance of PCR methods was analyzed in two different scenarios. The highest sensitivity value identified in all scenarios studied was kDNA/145. Our results suggest that popliteal lymph node and conjunctival swab samples, besides being less invasive collections, represent a good substratum for PCR-based diagnosis, and the target kDNA/145 is the best choice for detecting L. infantum DNA in naturally infected dogs.

Spatial association between sporotrichosis in cats and in human during a Brazilian epidemics.

Sporotrichosis is a subcutaneous granulomatous disease caused by the fungus Sporothrix spp. In Brazil, S. brasiliensis is reported in regions of outbreaks and epidemics in the zoonotic form of the disease where cats play an important role in the transmission of the disease to humans. Therefore, it is important to assess how the presence of infected cats impacts the risk for sporotrichosis in humans. The objective of this study was to analyze the spatial association of sporotrichosis in cats and in humans from Belo Horizonte, a Brazilian city where an epidemics of sporotrichosis occurs since the first human case register in 2015, through an inhomogeneous Poisson process model. Feline and human cases of sporotrichosis recorded between January 2016 and June 2019 were georeferenced by address and spatial point patterns were generated. Feline case intensity and human demographic density were calculated using a kernel smoothed estimate. The distance to the nearest feline case was also compute. Model parameters were estimated by Maximum Likelihood Estimate. The model validation was performed by the evaluation of partial residual, leverage and influence measure. There were 343 cases of cats and 135 human cases of sporotrichosis. The average incidence of human sporotrichosis in the period was 1.343 per 100 thousand inhabitants, which is relatively low in relation to the population, but higher than that observed in other regions in zoonotic outbreak of the disease. The southern region of the municipality has a higher intensity of feline cases. According to the fitted model, the risk for human sporotrichosis is greater when at distances very close to a feline case, with a virtually stable effect for distances greater than 1 km. Regarding the intensity of feline cases there is a gradual increase in risk as the intensity of cases increases. From the leverage analysis it was observed that the model was particularly sensitive to the occurrence of human cases in the south and east regions, places with extreme values ​​of covariates. Poisson point process model seems to be a reasonable approach in spatial epidemiology when multiple sources of infection are involved, and there is a low incidence of the disease as long as it is reasonable to assume independence between cases. Interventions for disease prevention and control in humans are suggested to encompass disease control in cats and the search for feline cases, focused on diagnosis and control, close to reported human cases.

Associated factors and spatial patterns of the epidemic sporotrichosis in a high density human populated area: A cross-sectional study from 2016 to 2018.

An epidemiological characterization of human and feline sporotrichosis was carried out between 2016 and 2018, in a high density-populated area in Brazil. Professionals were trained to identify suspect cats and notify vets to interview the owners and collect swabs of the wounds from these animals. Mycological cultures were performed, and colonies identified as Sporothrix spp. Subsequently, data regarding the outcome from suspect animals were collected. Confirmed cases of human sporotrichosis (56) were also counted and analyzed for spatial distribution. 118 positive animals were observed. The prevalence of feline sporotrichosis was 8.36 ‰ (CI 95 %, 5.38-9.55 ‰). The odds for being positive in animals that lived only partially at home were 3.02 times greater than for those cats without access to the street (OR 3.02, CI 95 % 1,96-10,43). There was no statistically significant association between environmental variables and positive diagnosis, corroborating the hypothesis that direct transmission by infected cats plays a greater role in the occurrence and continuous outbreaks of sporotrichosis in Brazil. Among the positive animals, 61.90 % (CI 95 % 58.95-64.96) died, and they had an odds to die in the next six months 6.30 times greater than negative animals (p < 0.05, OR 6.30, CI 95 % 2,79-14,42). The case fatality rate was 55.08 % in cats (CI 95 % 49.20-51.15). The cause-specific death rate was 4.6 ‰ in cats (CI 95 % 3.4-6 ‰). Only 7.62 % (CI 95 % 7.12-8.16) positive cats were treated and cured. Among dead positive animals, 29.23 % were inappropriately discarded. In the study period 56 human cases were recorded in the Barreiro region. Regions with highest prevalence of feline sporotrichosis, had greater frequencies of both human and feline cases. This is the first report on the epidemic of sporotrichosis in Minas Gerais, Brazil. The free offer for treatment and veterinary care for these animals should be taken into consideration, as well as the collection and incineration of the dead ones, as measures of public health, followed by the guidance and care for the human patient.

Babesia bigemina: in vitro multiplication of sporokinetes in Ixodes scapularis (IDE8) cells.

This paper describes the in vitro multiplication process of Babesia bigemina sporokinetes in a cell line (IDE8) from Ixodes scapularis ticks. The inoculum was obtained from hemolymph of engorged females of Rhipicephalus (Boophilus) microplus ticks naturally infected with B. bigemina. These ticks had been fed on calves living in a tick endemic farm in Brazil. Microscopic morphological details are shown to describe the development of the parasite in the tick cells; the identity of the parasite was confirmed by a duplex PCR method.

In vitro establishment and propagation of a Brazilian strain of Anaplasma marginale with appendage in IDE8 (Ixodes scapularis) cells.

A Brazilian isolate of Anaplasma marginale with appendage was successfully established and maintained in vitro in a tick cell line (IDE8). Infection was confirmed by optical and transmission electron microscopy. In addition, primers MSP1aNF2 and MSP1aNR2 amplified products from DNA extracted from infected IDE8 cells. Comparisons with partial sequences of the msp1α gene and the complete genome of A. marginale confirmed that the sequences of amplified fragments were from the A. marginale genome. This is the first establishment of a Brazilian A. marginale isolate in tick cells, representing a new system for biological and molecular studies and also a new source of material for diagnosis and development of vaccines.

Rapid Monitoring of Vaccination to prevent measles in Ceará State, Brazil, 2015. / Monitoramento Rápido de Vacinação na prevenção do sarampo no estado do Ceará, em 2015.

OBJECTIVE: to describe the results of Rapid Monitoring Vaccination monitoring conducted with the aim of interrupting the measles outbreak in the State of Ceará, Brazil, in 2015. METHODS: this was a descriptive study using data taken from 52,216 vaccination cards of children aged from 6 months to less than 5 years and data on vaccination coverage, homogeneity, and reasons for non-vaccination extracted from the National Immunization Program Information System (SI-PNI). RESULTS: vaccination coverage against measles reached 96.7% in Ceará; of the 21 Regional Health Offices in the State, four did not reach minimum coverage of 95% for the first dose, and two for the second dose; 836 children (1.6%) were not vaccinated and 1,388 vaccine doses were not used. CONCLUSION: Measles vaccination campaigns enabled the immunization coverage goal in the State of Ceará to be surpassed, despite the considerable number of unvaccinated children found.

Detection and molecular characterization of Babesia caballi and Theileria equi isolates from endemic areas of Brazil.

Blood samples were collected from 487 adult horses, including 83 pregnant mares, at a slaughterhouse located in Araguari, Minas Gerais State, Brazil. For each blood sample, the packed cell volume (PCV) was determined, and Giemsa-stained smears were microscopically examined for the presence of hemoparasites. The plasma was examined by the indirect fluorescent antibody test for detection of antibodies against Babesia caballi and Theileria equi. In addition, DNA was extracted and analyzed by a multiplex real-time polymerase chain reaction (PCR), specific for B. caballi and T. equi. Products of PCR were sequenced and compared with each other and with known sequences. The serological results showed a total prevalence of 91.0% for T. equi and 83.0% for B. caballi, while by PCR, prevalences of 59.7% for T. equi and 12.5% for B. caballi were observed. However, no correlations were seen between positivity (neither by serology nor by PCR) and PCV values. As expected, the microscopic examination of blood smears showed low sensitivity in detecting the infections when compared to the PCR. Only 35 out of 570 blood smears were positive, with parasitemias below 0.1%. No congenital transmission was detectable. As far as sequencing is concerned, no differences were seen among the isolates of each species nor among them and known sequences available. These results confirm, by molecular methods, the high prevalence rates of T. equi and B. caballi infections in carrier horses in Brazil. However, no diversity was observed among the isolates within the studied regions.

Household outbreak of sporotrichosis: towards the One Health approach

ABSTRACT Although sporotrichosis requires a broad approach for control, few reports have described the relationship between the index case and secondary contacts. In the present work, we report an outbreak involving a woman, a dog, and two cats from the same household environment, including the clinical and epidemiological aspects and outcomes, and discuss the importance of a One Health approach to face this neglected disease. The joint efforts of professionals such as veterinarians and physicians are essential for early diagnosis and surveillance, which contributes to the rapid identification and control of zoonotic sporotrichosis outbreaks.

The health facility as a risk factor for multidrug-resistant gram-negative bacteria in critically ill patients with COVID-19

Abstract Background The relationship between Multidrug Resistant-Gram Negative Bacteria (MDR-GNB) infection and colonization in critically ill COVID-19 patients has been observed, however, it is still poorly understood. This study evaluated the risk factors for acquiring MDR-GNB in patients with severe COVID-19 in Intensive Care Units (ICU). Methods This is a nested case-control study in a cohort of 400 adult patients (≥ 18 years old) with COVID-19, hospitalized in the ICU of 4 hospitals in the city of Curitiba, Brazil. Cases were critical COVID-19 patients with one or more MDR GNB from any surveillance and/or clinical cultures were taken during their ICU stay. Controls were patients from the same units with negative cultures for MDR-GNB. Bivariate and multivariate analyses were done. Results Sixty-seven cases and 143 controls were included. Independent risk factors for MDR bacteria were: male gender (OR = 2.6; 95% CI 1.28‒5.33; p = 0.008); the hospital of admission (OR = 3.24; 95% CI 1.39‒7.57; p = 0.006); mechanical ventilation (OR = 25.7; 95% CI 7.26‒91; p < 0.0001); and desaturation on admission (OR = 2.6; 95% CI 1.27‒5.74; p = 0.009). Conclusions Male gender, desaturation, mechanical ventilation, and the hospital of admission were the independent factors associated with MDR-GNB in patients in the ICU with COVID-19. The only modifiable factor was the hospital of admission, where a newly opened hospital posed a higher risk. Therefore, coordinated actions toward a better quality of care for critically ill COVID-19 patients are essential.

Isolation and attempted cultivation of an Anaplasma marginale strain from Brazilian brown brocket deer (Mazama gouazoubira, Fisher, 1814) in the tick cell line IDE8.

The aim of the study was to isolate and establish an Anaplasma marginale strain from Brazilian brown brocket deer, Mazama gouazoubira, in the Ixodes scapularis cell line IDE8. Blood from a free-living adult female M. gouazoubira naturally infected with A. marginale (MGI5) was inoculated intravenously into a splenectomized calf. When A. marginale rickettsemia was 2.5%, blood was collected and cryopreserved in liquid nitrogen with dimethylsulfoxide (DMSO). IDE8 cell cultures were infected with calf blood inoculated with the A. marginale (MG15) isolate. The cultures were monitored by examination of Giemsa-stained cytocentrifuge smears. Light microscopy of stained IDE8 samples revealed the first inclusions of A. marginale (MGI5) at 48days post-inoculation (d.p.i). The IDE8-infected cells contained parasitophorous vacuoles with amorphous material and a few cocci-like organisms. A sample from IDE8-infected cells from the 16th subculture (336 d.p.i.) was analyzed by nPCR, nucleotide sequencing, electron microscopy, and an indirect fluorescent antibody test (IFAT). The IFAT highlighted some IDE8-infected cells with intense fluorescence in the parasitophorous vacuole, while in other cells, fluorescence was observed only at the periphery. DNA from a culture of the MG15 isolate was amplified with A. marginale msp4 gene primers, and nucleotide sequencing of the PCR product and BLAST software analysis further confirmed 100% identity with the MGI5 blood isolate (GenBank no. JN022558.1). Electron microscopy revealed increased numbers of lysosomes in the cytoplasm of IDE8 cells. Several cells exhibited large vacuoles containing cellular debris and amorphous material. After the 29th subculture, it was not possible to detect compatible Anaplasma structures by light microscopy, and subculture samples tested negative in nPCR. Despite the failure of the attempt to establish A. marginale (MGI5) in IDE8 cells, the results demonstrated the isolate's ability to infect, survive and multiply, although in limited numbers, in IDE8 cells.