Molecular survey of Piroplasmida, Hepatozoon spp. and Anaplasmataceae in anemic and thrombocytopenic dogs from Uruguay.

Canine tick-borne diseases, such as babesiosis, rangeliosis, hepatozoonosis, anaplasmosis and ehrlichiosis, are of veterinarian relevance, causing mild or severe clinical cases that can lead to the death of the dog. The aim of this study was detecting tick-borne protozoan and rickettsial infections in dogs with anemia and/or thrombocytopenia in Uruguay. A total of 803 domestic dogs were evaluated, and 10% were found positive (detected by PCR) at least for one hemoparasite. Sequence analysis confirmed the presence of four hemoprotozoan species: Rangelia vitalii, Babesia vogeli, Hepatozoon canis and Hepatozoon americanum, and the rickettsial Anaplasma platys. The most detected hemoparasite was R. vitalii, followed by H. canis and A. platys. This is the first report of B. vogeli in Uruguay and the second report of H. americanum in dogs from South America. The results highlight the importance for veterinarians to include hemoparasitic diseases in their differential diagnosis of agents causing anemia and thrombocytopenia.

Posterior Quadratus Lumborum Block, A Novel Approach to Treat Chronic Hip Pain: A Case Series.

Osteoarthritis frequently results in chronic pain and has a major impact on patients' quality of life. We present a case series of 20 patients with chronic hip pain who received a posterior quadratus lumborum block with the aim of improving their pain and, consequently, their quality of life. The results showed global improvement in pain and quality of life. These preliminary results support our conclusion. Posterior quadratus lumborum block is a safe and minimally invasive option for refractory chronic hip pain.

Characterization of strains of Anaplasma marginale from clinical cases in bovine using major surface protein 1a in Uruguay.

The major surface protein 1a (MSP1a) gene has been used to characterize Anaplasma marginale genetic diversity. This pathogen causes significant productivity and economic losses to the cattle industry. The objective of the present study was to report the first characterization of A. marginale genetic diversity in Uruguay based on MSP1a genotypes and their putative relationship with Rhipicephalus microplus. This cross-sectional study was conducted between 2016 and 2020. The study included whole blood samples from clinical cases of bovine anaplasmosis obtained from 30 outbreaks located in six Uruguay territorial departments. Diagnosis was performed using Giemsa-stained smears and confirmed by nested Polymerase Chance Reaction (nPCR) targeting the A. marginale major surface protein 5 gene. The genetic diversity of A. marginale strains was characterized by analyzing the microsatellite and tandem repeats of MSP1a. Based on the microsatellite structure, four genotypes were identified. Genotype E was the most prevalent. Analysis of MSP1a tandem repeats showed 28 different strains from the combination of 31 repeats, with τ-10-15 and α-ß-ß-ß-Γ being the most common. Repeats Γ, ß, α, and γ were associated with the absence of R. microplus with statistical significance (p < 0.05). Molecular observations showed that 46.7% of the strains identified in our samples lacked the ability to bind to tick cells; therefore, they were probably transmitted by other vectors. Strain genetic diversity provides valuable information for understanding the epidemiological behavior of A. marginale and could contribute to the development of effective vaccines for the control of this disease.

Description of bovine babesiosis and anaplasmosis outbreaks in northern Uruguay between 2016 and 2018.

Bovine babesiosis and anaplasmosis cause important economic losses in livestock production. In Uruguay, the main aetiological agents of bovine babesiosis and anaplasmosis are Babesia bovis, B. bigemina and Anaplasma marginale. The aim of this work was to describe the outbreaks of bovine babesiosis and anaplasmosis in northern Uruguay between 2016 and 2018. Convenience sampling was carried out. We worked with blood and organ samples from bovines with clinical signs and autopsy findings compatible with babesiosis and anaplasmosis. A total of 140 presumptive outbreaks were studied. Epidemiological information such as place, date of occurrence, age, number of sick and dead animals, clinical signs, autopsy findings, the presence of ticks and health management that involved injectables were registered. The diagnoses were carried out by blood and organ smears stained with Giemsa and confirmed by multiplex PCR. There were 83 (59.2%) positive outbreaks, comprising 35 (42.2%) A. marginale, 19 (22.9%) B. bigemina, 18 (21.7%) B. bovis and 11 (13.2%) mixed infections (Babesia spp. + A. marginale). Cows were the most commonly affected category. The clinical signs and autopsy findings with a significant association (p ≤ 0.05) were anaemia, pale mucous membranes, fever, jaundice, ataxia and aggressiveness, splenomegaly, and orange discolouration of the liver. Babesiosis had a seasonal occurrence, mainly in autumn, while anaplasmosis cases were recorded throughout the year. The use of injectable agents was associated with A. marginale transmission. This work contributes updated information about epidemiological and clinical patterns of bovine babesiosis and anaplasmosis in northern Uruguay, which is important for implementing preventive measures and control.

An Hepatozoon americanum-like protozoan in crab-eating (Cerdocyon thous) and grey pampean (Lycalopex gymnocercus) foxes from Uruguay.

In South America, apicomplexan parasites of the genus Hepatozoon have been sporadically detected in mammals. Previous studies in wild canids from Brazil and Argentina demonstrated infections by species genetically related to Hepatozoon americanum. The aim of the present work was to detect the presence of Hepatozoon in road-killed foxes encountered in Uruguayan highways. Blood samples from 45 crab-eating (Cerdocyon thous) and 32 grey pampean (Lycalopex gymnocercus) foxes were analyzed by PCR for Hepatozoon 18S rRNA gene. Eight foxes (10.4%) were found to be infected with an H. americanum-like protozoan, an Hepatozoon closely related to H. americanum. Bayesian and maximum-likelihood phylogenetic analyses revealed that the sequences obtained in this study cluster with H. americanum from the United States, and with an H. americanum-like species from dog and foxes from Brazil and Argentina. In the Unites States, H. americanum causes severe disease in dogs. In addition to this, an increasing habitat overlap between dogs and foxes makes the presence of H. americanum-like protozoan in foxes acquires veterinary relevance. This work represents the first report of L. gymnocercus infected with an H. americanum-like protozoan, and of wild canids infected with Hepatozoon in Uruguay.

Rangelia vitalii in free-living crab-eating foxes (Cerdocyon thous) in Uruguay.

Rangelia vitalii is a protozoan parasite that causes a hemorrhagic and hemolytic disease in dogs known as rangeliosis. Current reports of the disease are concentrated in the southern and southeastern regions of Brazil, as well as in Uruguay, Argentina, and Paraguay, and mainly concern domestic dogs. South American wild canids, such as the crab-eating fox (Cerdocyon thous), the pampas fox (Lycalopex gymnocercus), and the maned wolf (Chrysocyon brachyurus) may also be affected, although existing reports are restricted to Brazil. The present study aimed to detect R. vitalii parasitism in the Uruguayan wild fox population. DNA extracted from the blood and/or spleen samples of road-killed C. thous and L. gymnocercus found in northern Uruguay were subjected to polymerase chain reaction (PCR) to amplify a 551-bp fragment of the Rangelia 18S rRNA gene. A total of 62 wild canids, including 38 C. thous and 24L. gymnocercus, were analyzed. Five crab-eating fox samples (13.2%) were positive for R. vitalii, with 99.5-100% identity between the sequences. All samples from pampas fox tested negative for R. vitalii. When compared with the R. vitalii sequences available in GenBank, a similarity of 98.9-100% was revealed. Molecular analysis results suggest that R. vitalii is circulating in the crab-eating fox population in Uruguay; however, its veterinary relevance for these foxes remains unknown.

Serratus intercostal interfascial plane block in supraumbilical surgery: a prospective randomized comparison.

BACKGROUND: Upper abdominal wall surgical incisions may lead to a severe postoperative pain. Therefore, adequate analgesia is important. Here we investigate whether the low serratus-intercostal interfascial plane block (SIPB) achieves an effective analgesia, considering opioids consumption, pain control and recovery quality in upper abdominal surgeries. METHODS: This blinded, randomized controlled study was conducted on 102 patients undergoing open upper abdominal wall surgery under general anesthesia. All patients who received serratus-intercostal plane block at the eighth rib as analgesic technique were included in SIPB group and in control group those who received continuous intravenous morphine analgesia. Pain scores in numeric verbal scale (NVS) and opioids consumption at 0, 6, 12, 24 and 48 hours postoperatively were assessed. The quality of the postoperative recovery was evaluated using the QoR-15 questionnaire at 24 hours. RESULTS: This study showed lower postoperative opioid consumption at 24 hours (P<0.0001; 4.17 mg vs. 41.52 mg of morphine) and better pain control (P<0.005) with mean pain scores (NVS 1.8±1.5 vs. 4.8±1.6) in group 0 (SIPB). The global QoR-15 scores 24 hours postoperatively were higher (better quality) in the SIPB group (122 vs. 100). CONCLUSIONS: The low serratus-intercostal interfascial plane block (SIPB) provides efficient analgesia leading to a saving of opioids and improvement of the recovery quality in patients undergoing upper abdominal wall surgeries.

Molecular characterization of novel Ehrlichia genotypes in Ixodes auritulus from Uruguay.

Ehrlichia are small intracellular Gram-negative bacteria transmitted by ticks. These microorganisms cause ehrlichiosis, a complex of life-threatening emerging zoonoses and diseases of global veterinary relevance. The aim of this study was to investigate the presence of Ehrlichia in free-living Ixodes auritulus collected in Uruguay. Ticks were collected from vegetation in five localities from the southeast and northeast of the country between 2014 and 2017. Detection of Ehrlichia DNA was performed in pools of adults or nymphs grouped according to the collection site and date. A total of 1,548 I. auritulus ticks were collected in four of the five locations sampled. Fragments of three loci (16S rRNA, dsb and groEL) were obtained by PCR, and phylogenies inferred using Bayesian inference analysis for each gene independently. DNA of Ehrlichia spp. was found in 15 out of 42 tick pools. Based on the topology of the phylogenetic trees, our sequences represent two novel genotypes for the genus named as Ehrlichia sp. Serrana and Ehrlichia sp. Laguna Negra. Both genotypes were closely related to Ehrlichia sp. Magellanica, a species detected in Ixodes uriae and Magellanic penguins. Considering that all stages of I. auritulus and I. uriae are parasites of birds, their phylogenetic relationships, and common eco-epidemiological profiles, it is reasonable to state that these genotypes of Ehrlichia spp. may represent a natural group likely associated with birds. Our results constitute the first characterization of Ehrlichia spp. in Uruguay. Future studies on birds reported as hosts for I. auritulus are needed to further understand the epidemiological cycles of both Ehrlichia genotypes in the country. Finally, I. auritulus does not feed on humans, so the two Ehrlichia species reported herein might have no implications in human health.

Validation of a multiplex PCR assay to detect Babesia spp. and Anaplasma marginale in cattle in Uruguay in the absence of a gold standard test.

Detection of bovine Babesia spp. and Anaplasma marginale is based on the reading of Giemsa-stained blood or organ smears, which can have low sensitivity. Our aim was to improve the detection of bovine Babesia spp. and A. marginale by validating a multiplex PCR (mPCR). We used 466 samples of blood and/or organs of animals with signs and presumptive autopsy findings of babesiosis or anaplasmosis. The primers in our mPCR amplified the rap-1a gene region of Babesia bovis and B. bigemina, and the msp-5 region of A. marginale. We used a Bayesian model with a non-informative priori distribution for the prevalence estimate and informative priori distribution for estimation of sensitivity and specificity. The sensitivity and specificity for smear detection of Babesia spp. were 68.6% and 99.1%, and for A. marginale 85.6% and 98.8%, respectively. Sensitivity and specificity for mPCR detection for Babesia spp. were 94.2% and 97.1%, and for A. marginale 95.2% and 92.7%, respectively. Our mPCR had good accuracy in detecting Babesia spp. and A. marginale, and would be a reliable test for veterinarians to choose the correct treatment for each agent.

Detection of a putative novel genotype of Anaplasma in gray-brocket deer (Mazama gouazoubira) from Uruguay.

Anaplasmataceae includes the genera Anaplasma, Ehrlichia, Neorickettsia and Wolbachia, comprising a group of obligate intracellular bacteria. The genus Anaplasma has pathogenic species transmitted by ticks of veterinary and human health importance. Wild ungulates such as deer represent important reservoirs and amplifiers of Anaplasmataceae. The interaction between deer and domestic ruminants represents a serious problem due to the transmission of these pathogens through their ectoparasites. In the present study, we investigated the presence of Anaplasmataceae organisms in blood, tissues and tick samples of a gray-brocket deer (Mazama gouazoubira). The specimen was found dead in a farm in northeast Uruguay. PCRs targeting partial regions of 16S rRNA and groESL genes were carried out for Anaplasmataceae DNA detection. Moreover, several ectoparasites were identified: the chewing louse Tricholipeurus albimarginatus, the Neotropical deer louse fly Lipoptena mazamae, and the ticks Haemaphysalis juxtakochi and Rhipicephalus microplus. A consensus sequence of 1274 bp of 16S rRNA was generated for Anaplasma sp. from the M. gouazoubira blood sample. All ticks analysed by PCR assays were negative. No band was detected in any of the samples after PCR targeting groESL gene. Phylogenetic analysis using 16S rRNA partial gene sequences, clustered the putative novel genotype sequence obtained in this study, named Anaplasma sp. genotype Mazama-Uruguay, along with Anaplasma sp. detected in Mazama sp., Mazama americana and Mazama bororo, all deer species from Brazil. Furthermore, this cluster showed to be closely related to Anaplasma bovis sequences obtained from various ruminants and other mammals from several parts of the world. The pathogenicity as well as its infecting potential to other cervids or domestic ruminants is currently unknown. Further studies should be performed in order to characterize this novel species, especially targeting other genes.

Mutant EZH2 Induces a Pre-malignant Lymphoma Niche by Reprogramming the Immune Response.

Follicular lymphomas (FLs) are slow-growing, indolent tumors containing extensive follicular dendritic cell (FDC) networks and recurrent EZH2 gain-of-function mutations. Paradoxically, FLs originate from highly proliferative germinal center (GC) B cells with proliferation strictly dependent on interactions with T follicular helper cells. Herein, we show that EZH2 mutations initiate FL by attenuating GC B cell requirement for T cell help and driving slow expansion of GC centrocytes that become enmeshed with and dependent on FDCs. By impairing T cell help, mutant EZH2 prevents induction of proliferative MYC programs. Thus, EZH2 mutation fosters malignant transformation by epigenetically reprograming B cells to form an aberrant immunological niche that reflects characteristic features of human FLs, explaining how indolent tumors arise from GC B cells.

Identification of free-living amoebas and amoeba-resistant bacteria accumulated in Dreissena polymorpha.

To identify the free-living amoeba (FLA) and amoeba-resistant bacteria (ARB) accumulated in zebra mussels and in the water in which they are found, mussels were collected at two locations in the Ebro river basin (North East Spain). FLAs and bacteria were isolated from mussel extracts and from natural water. PCR techniques were used to identify the FLAs and endosymbiont bacteria (Legionella, Mycobacterium, Pseudomonas and cyanobacteria), and to detect Giardia and Cryptosporidium. The most frequently found FLAs were Naegleria spp. The presence of Legionella, Mycobacterium and Pseudomonas inside the FLA was demonstrated, and in some cases both Legionella and Pseudomonas were found together. Differences between FLAs and ARB identified inside the mussels and in the water were detected. In addition, Escherichia coli, Clostridium perfringens, Salmonella spp. and Enterococcus spp. were accumulated in mussels in concentrations unconnected with those found in water. The results show the ability of the zebra mussel to act as a reservoir of potentially pathogenic FLAs, which are associated with potentially pathogenic ARB, although the lack of association between microorganisms inside the mussels and in the water suggests that they are not useful for monitoring microbiological contamination at a specific time.

Phylogenetic analysis of Hepatozoon spp. (Apicomplexa: Hepatozoidae) infecting Philodryas patagoniensis (Serpentes: Dipsadidae) in Uruguay.

The purpose of this study was to perform a phylogenetic analysis of Hepatozoon spp. infecting Philodryas patagoniensis in Uruguay. Twenty-five road-killed specimens of P. patagoniensis from ten departments were obtained. Samples of blood and/or heart tissue were taken. Polymerase chain reaction (PCR) assay was carried out amplifying a specific target region of the 18S rRNA gene of Hepatozoon spp. Eighteen out of twenty-five samples were positive to Hepatozoon spp., which gave an overall prevalence of 72%. Phylogenetic analyses with the obtained sequences were carried out to determine the relationship with closely related species found in the region. The results revealed that samples were split into two clades with a high bootstrap support. Clade I was formed with Hepatozoon spp. sequences obtained in this study from P. patagoniensis, Hepatozoon cuestensis from Crotalus durissus terrificus and Hepatozoon musa from Philodryas nattereri, and Hepatozoon spp. retrieved from Cerdocyon thous, Hemidactylus mabouia, and Phyllopezus pollicaris from Brazil, respectively. Clade II was grouped with Hepatozoon cevapii and Hepatozoon massardii, both species described for C. d. terrificus from Brazil. This is the first report of Hepatozoon spp. in snakes from Uruguay.

Borrelia burgdorferi sensu lato infecting Ixodes auritulus ticks in Uruguay.

In the southern cone of South America different haplotypes of Borrelia burgdorferi sensu lato (Bbsl) have been detected in Ixodes spp. from Argentina, southern Brazil, Chile, and Uruguay. So far, Lyme borreliosis has not been diagnosed in Uruguay and the medical relevance of the genus Ixodes in South America is uncertain. However, the growing number of new genospecies of Bbsl in the southern cone region and the scarce information about its pathogenicity, reservoirs and vectors, highlights the importance of further studies about spirochetes present in Uruguay and the region. The aim of this study was to determine the presence of Bbsl in Ixodes auritulus ticks collected from birds and vegetation in two localities of southeastern Uruguay. In total 306 I. auritulus were collected from 392 passerine birds sampled and 1110 ticks were collected by flagging in vegetation. Nymphs and females were analyzed for Borrelia spp. by PCR targeting the flagellin (fla) gene and the rrfA-rrlB intergenic spacer region (IGS). The phylogenetic analysis of Borrelia spp. positive samples from passerine birds and vegetation revealed the presence of four fla haplotypes that form a clade within the Bbsl complex. They were closely related to isolates of Borrelia sp. detected in I. auritulus from Argentina and Canada.

TET2 Deficiency Causes Germinal Center Hyperplasia, Impairs Plasma Cell Differentiation, and Promotes B-cell Lymphomagenesis.

TET2 somatic mutations occur in ∼10% of diffuse large B-cell lymphomas (DLBCL) but are of unknown significance. Herein, we show that TET2 is required for the humoral immune response and is a DLBCL tumor suppressor. TET2 loss of function disrupts transit of B cells through germinal centers (GC), causing GC hyperplasia, impaired class switch recombination, blockade of plasma cell differentiation, and a preneoplastic phenotype. TET2 loss was linked to focal loss of enhancer hydroxymethylation and transcriptional repression of genes that mediate GC exit, such as PRDM1. Notably, these enhancers and genes are also repressed in CREBBP-mutant DLBCLs. Accordingly, TET2 mutation in patients yields a CREBBP-mutant gene-expression signature, CREBBP and TET2 mutations are generally mutually exclusive, and hydroxymethylation loss caused by TET2 deficiency impairs enhancer H3K27 acetylation. Hence, TET2 plays a critical role in the GC reaction, and its loss of function results in lymphomagenesis through failure to activate genes linked to GC exit signals. SIGNIFICANCE: We show that TET2 is required for exit of the GC, B-cell differentiation, and is a tumor suppressor for mature B cells. Loss of TET2 phenocopies CREBBP somatic mutation. These results advocate for sequencing TET2 in patients with lymphoma and for the testing of epigenetic therapies to treat these tumors.See related commentary by Shingleton and Dave, p. 1515.This article is highlighted in the In This Issue feature, p. 1494.

A Novel Hypovirus Species From Xylariaceae Fungi Infecting Avocado.

The white rot root disease caused by Rosellinia necatrix is a major concern for avocado cultivation in Spain. Healthy escapes of avocado trees surrounded by diseased trees prompted us to hypothesize the presence of hypovirulent R. necatrix due to mycovirus infections. Recently, we reported the presence of another fungal species, Entoleuca sp., belonging to the Xylariaceae, that was also found in healthy avocado trees and frequently co-infecting the same roots than R. necatrix. We investigated the presence of mycoviruses that might explain the hypovirulence. For that, we performed deep sequencing of dsRNAs from two isolates of Entoleuca sp. that revealed the simultaneous infection of several mycoviruses, not described previously. In this work, we report a new member of the Hypoviridae, tentatively named Entoleuca hypovirus 1 (EnHV1). The complete genome sequence was obtained for two EnHV1 strains, which lengths resulted to be 14,958 and 14,984 nt, respectively, excluding the poly(A) tails. The genome shows two ORFs separated by a 32-nt inter-ORF, and both 5'- and 3'-UTRs longer than any other hypovirus reported to date. The analysis of virus-derived siRNA populations obtained from Entoleuca sp. demonstrated antiviral silencing activity in this fungus. We screened a collection of Entoleuca sp. and R. necatrix isolates and found that EnHV1 was present in both fungal species. A genetic population analysis of EnHV1 strains revealed the presence of two main clades, each of them including members from both Entoleuca sp. and R. necatrix, which suggests intra- and interspecific virus transmission in the field. Several attempts failed to cure Entoleuca sp. from EnHV1. However, all Entoleuca sp. isolates collected from avocado, whether harboring the virus or not, showed hypovirulence. Conversely, all R. necatrix isolates were pathogenic to that crop, regardless of being infected by EnHV1.

An autochthonous confirmed case of Rickettsia parkeri rickettsiosis in Uruguay.

Rickettsia parkeri, a member of the spotted fever group (SFG) rickettsiae, was first confirmed as an etiological agent of human rickettsiosis in 2004. Nearly all cases are characterized by an inoculation eschar, and no fatalities have been reported. In Uruguay, probable human cases of R. parkeri infection (confused initially with R. conorii infection) have been described since 1990 using the clinical name "cutaneous-ganglionar" rickettsiosis. This is the only tick-borne rickettsiosis reported in the country. A single case of R. parkeri rickettsiosis has been confirmed by molecular and serological testing in a Spanish traveler returning from Uruguay. We report the first autochthonous human R. parkeri infection, confirmed by molecular testing in Uruguay.

Molecular evidence of Babesia species in Procyon cancrivorus (Carnivora, Procyonidae) in Uruguay.

The crab-eating raccoon (Procyon cancrivorus) is a carnivore widely distributed from southern Central America to all South American countries except Chile. In the Southern cone of America, P. cancrivorus has been found parasitized by several Amblyomma spp. Particularly, in Uruguay, A. aureolatum is the only tick found in this wild carnivore. Piroplasmid hemoparasites were found in Procyon lotor from North America and Japan. In this work, molecular evidence Babesia sp. DNA was found in blood and tissues from road-killed P. cancrivorus from different locations in Uruguay. PCRs targeting 18S rRNA gene were carried out. Subsequently, the obtained amplicons were sequenced and full-length sequences was assembled. A phylogenetic tree was constructed and revealed that the Babesia sp. found in this work clustered with other 18sRNA sequences of Babesia spp. obtained from P. lotor from Japan and USA, along with Babesia spp. of maned wolf and I. ovatus. This is the first report of molecular evidence of Babesia sp. parasitizing P. cancrivorus.

EZH2 enables germinal centre formation through epigenetic silencing of CDKN1A and an Rb-E2F1 feedback loop.

The EZH2 histone methyltransferase is required for B cells to form germinal centers (GC). Here we show that EZH2 mediates GC formation through repression of cyclin-dependent kinase inhibitor CDKN1A (p21Cip1). Deletion of Cdkn1a rescues the GC reaction in Ezh2 -/- mice. Using a 3D B cell follicular organoid system that mimics the GC reaction, we show that depletion of EZH2 suppresses G1 to S phase transition of GC B cells in a Cdkn1a-dependent manner. GC B cells of Cdkn1a -/- Ezh2 -/- mice have high levels of phospho-Rb, indicating that loss of Cdkn1a enables progression of cell cycle. Moreover, the transcription factor E2F1 induces EZH2 during the GC reaction. E2f1 -/- mice manifest impaired GC responses, which is rescued by restoring EZH2 expression, thus defining a positive feedback loop in which EZH2 controls GC B cell proliferation by suppressing CDKN1A, enabling cell cycle progression with a concomitant phosphorylation of Rb and release of E2F1.The histone methyltransferase EZH2 silences genes by generating H3K27me3 marks. Here the authors use a 3D GC organoid and show EZH2 mediates germinal centre (GC) formation through epigenetic silencing of CDKN1A and release of cell cycle checkpoints.

A Rabbit Model of Acanthamoeba Keratitis: Use of Infected Soft Contact Lenses After Corneal Epithelium Debridement With a Diamond Burr.

Purpose: To develop a rabbit model of Acanthamoeba keratitis (AK) as the best method to reproduce the natural course of this disease. Methods: To induce AK, infected contact lenses (1000 amoebae/mm2, 90% trophozoites) were placed over the previously debrided corneal surface, in combination with a temporary tarsorrhaphy. Environmental and clinical strains of Acanthamoeba spp. (genotype T4) were used. Three groups (1L, n = 32; 2L-21d, n = 5; 2L-3d, n = 23) were established according to the number of contact lenses used (1L, 1 lens; 2L-21d and 2L-3d, 2 lenses) and the placement day of these (1L, day 1; 2L-21d, days 1 and 21; 2L-3d, days 1 and 3). The infection was quantified by a clinical score system and confirmed using corneal cytology and culture, polymerase chain reaction and histopathologic analysis. Results: The infection rate obtained was high (1L, 87.5%; 2L-21d, 100%; 2L-3d, 82.6%), although no clinical signs were observed in the 50% of the infected animals in group 1L. Among groups, group 2L-3d showed more cases of moderate and severe infection. Among strains, no statistically significant differences were found in the infection rate. In the control eyes, cross infection was confirmed when a sterile contact lens was placed in the previously debrided corneas but not if the eye remained intact. Conclusions: The combination of two infected contact lenses after corneal debridement seems to be an alternative model, clinically and histopathologically similar to its human counterpart, to induce the different AK stages and reproduce the course of the disease in rabbits.