Molecular Identification of Encephalitazoon intestinalis and the Prevalence of Renal Microsporidiosis in Renal Transplant Recipients in Türkiye.

Objective: In patients with end-stage kidney disease, kidney transplantation is the kidney replacement therapy option that provides the most successful survival. However, immunosuppression agents administered after kidney transplantation can increase the risk of opportunistic infections. Microsporidia are obligate intracellular pathogens that can be fatal in immunosuppressed patients. The present study aimed to determine the prevalence of microsporidia in kidney transplantation recipients and the molecular characterization of the detected species. Methods: To evaluate the prevalence of renal microsporidiosis in kidney transplant recipients, the urine samples from a total of 325 patients were analyzed by real-time and nested polymerase chain reaction for Encephalitozoon spp. and Enterocytozoon bieneusi. Results: Only one (0.4%) sample from the adult patient was positive for the Encephalitozoon species, while no positivity was found in pediatric patients. It was determined as Encephalitozoon intestinalis by ITS rRNA gene region sequence analysis. A microsporidia species obtained from humans in Türkiye has been characterized for the first time and registered in GenBank. Conclusion: Our epidemiological results show that the prevalence of renal microsporidiosis in kidney transplant recipients is very low. In addition, as a result of the phylogenetic analysis of the detected isolate, it was observed that it was 100% identical to the isolates reported from dogs in Kayseri, Türkiye. This situation provided essential data regarding the zoonotic transmission dynamics of microsporidia.

Tick-borne microsporidiosis: ticks as a neglected source of human microsporidian infections?

We detected 24 Encephalitozoon cuniculi positive Ixodes ricinus ticks of 284 collected in the Czech Republic. Since the route of transmission of microsporidia is not fully understood, the presence of microsporidia in ticks raises the question of whether they may be involved in the transmission of these pathogens.

Prevalence of Encephalitozoon hellem among companion and exhibition birds in Japan.

Microsporidia can cause infection in various animals and humans. To determine the recent prevalence of Encephalitozoon in companion birds in Japan, 364 bird feces and 16 conjunctival exudates, as well as 28 exhibition bird feces, were examined using polymerase chain reaction (PCR). Thirty-five (9.6%) feces and 2 (12.5%) conjunctival exudates from companion birds were PCR positive, and sequence analysis revealed that all detected organisms were Encephalitozoon hellem genotype 1A. The prevalence by region varied from 4.5% in the Shikoku region to 14.3% in the Chugoku region. By age, the prevalence in birds younger than 6 months of age was 13.3%. We also discuss the threat of human infection as a zoonotic disease.

Otitis media/interna and encephalitozoonosis are the most common causes of head tilt in pet rabbits in the UK: 73 cases (2009‒2020).

BACKGROUND: There are limited studies that identify diseases associated with head tilt in pet rabbits. METHODS: This was an observational, retrospective, single-centre study of rabbits with head tilt presented between 2009 and 2020. Descriptive statistics were performed for all cases, whereas univariate and multivariate analyses were only performed for the 36 cases with a final diagnosis. RESULTS: Seventy-three rabbits met the inclusion criteria. The final diagnoses included Encephalitozoon cuniculi meningoencephalomyelitis (EC) (15/36; 41.7%), otitis media/interna (OMI) (8/36; 22.2%) and concurrent EC and OMI (13/38; 36.1%). Subacute-to-chronic onset was more common in rabbits with OMI than in those with EC (p = 0.018). Previous middle ear surgery (p = 0.046) and a diagnosis of otitis externa (p = 0.004) significantly increased the risk of OMI. Meloxicam was associated with improvement of clinical signs (p = 0.007). Upright ears (p = 0.013), recumbency (p = 0.037) and impaired mentation (p = 0.001) were associated with a higher risk of death/euthanasia. The proportions of residual head tilt (66.7%) and relapse of vestibular signs (42.1%) were high. LIMITATIONS: This was a retrospective study with cases varying in their investigation and conclusive final diagnoses. CONCLUSION: OMI and EC were the most common aetiologies of head tilt in pet rabbits in the UK. Meloxicam might be associated with a favourable outcome in affected rabbits. Paired EC serology and a CT scan of the head should be the baseline investigation for head tilt in rabbits.

CD8 T lymphocytes from B-1 cell-deficient mice down-regulates fungicidal activity of macrophages challenged with E. Cuniculi.

BACKGROUND: Encephalitozoon cuniculi is an opportunistic intracellular pathogen that establishes a balanced relationship with immunocompetent individuals depending on the activity of their CD8+ T cells lymphocytes. However, lower resistance to experimental infection with E. cuniculi was found in B-1 deficient mice (Xid), besides increased the number of CD8 T lymphocytes. Here, we evaluated the profile of CD8+ T lymphocytes from Balb/c wild-type (WT) or Balb/c Xid mice (with B-1 cell deficiency) on the microbicidal activity of macrophages challenged with E. cuniculi. METHODS: Naïve CD8 T lymphocytes from WT or Xid mice uninfected and primed CD8 T lymphocytes from WT or Xid mice infected with E cuniculi were co-cultured with macrophages previously challenged with E. cuniculi. We evaluated macrophages viability and microbicidal activity, and CD8 T lymphocytes viability and presence of activating molecules (CD62L, CD69, and CD107a). RESULTS: Macrophages co-cultured with naïve CD8 T lymphocytes from WT demonstrated high microbicidal activity. Naïve CD8 T lymphocytes obtained from WT mice had a higher expression of CD69 and LAMP-1-activating molecules compared to Xid CD8+ T lymphocytes. Primed CD8 T lymphocytes from Xid mice proliferated more than those from WT mice, however, when the expression of the activating molecule CD69 associated with the expression of CD62L was kept low. In conclusion, naïve CD8+ T lymphocytes from Xid mice, deficient in B-1 cells, they had reduced expression of activation molecules and cytotoxic activity.

Encephalitozoon cuniculi Microsporidia in Cerebrospinal Fluid from Immunocompetent Patients, Czech Republic.

We retrospectively analyzed of 211 frozen cerebrospinal fluid samples from immunocompetent persons in the Czech Republic and detected 6 Encephalitozoon cuniculi-positive samples. Microsporidiosis is generally underestimated and patients are not usually tested for microsporidia, but latent infection in immunodeficient and immunocompetent patients can cause serious complications if not detected and treated.

[Evaluation of Toll-Like Receptor Gene Expressions in Encephalitozoon intestinalis Infection]. / Encephalitozoon intestinalis Enfeksiyonunda Toll-Like Reseptör Gen Ekspresyonlarinin Degerlendirilmesi.

Microsporidia are obligate intracellular pathogens that can infect many vertebrate and invertebrate hosts. While the Microsporidia phylum was defined as protozoa until the 1990s, it has been associated with fungi in line with the data obtained as a result of phylogenetic and molecular analyzes in recent years. Although approximately 200 genera and 1400 Microsporidia species related to these genera have been reported to date, only 14 species are known to cause infection in humans. Encephalitozoon intestinalis is one of the most frequently detected species in humans and causes serious clinical conditions in immunosuppressed individuals. Little information is available about the immunology of this infection. This study was aimed to investigate the changes in Toll-Like receptor (TLR) gene expressions in Madin-Darby canine kidney (MDCK) cells treated with E.intestinalis spores. Three groups were formed in the study. In the first group, only the medium prepared for E.intestinalis was added to the MDCK cells. In the second group, 108 live spores waiting at +4 °C were added. In the third group, 108 heat-inactivated spores were added. All three groups were incubated at 37ºC with 5% CO2 . RNA isolation and cDNA synthesis were performed from samples taken from these groups at the 1st, 3rd, 6th, 12th and 24th hours. Expression of TLR1-10 genes from the obtained cDNAs was evaluated by real-time polymerase chain reaction (Rt-PCR). GAPDH and ACTB genes were used as housekeeping genes in the study. Target genes were normalized by taking the average of these two genes and statistical analysis was performed by applying the 2-ΔΔCt formula. Genes detected above the threshold value (threshold 1) were considered to have increased expression. Genes detected below the threshold value were considered to have decreased expression. The growth of the live and inactive spores were followed simultaneously with the experimental groups. Approximately two weeks after the start of the culture, it was observed that E.intestinalis grew in the culture with live spore, but did not grow in the culture with inactivated spores. No statistically significant change was observed in gene expressions in the inactivated spore group. In the live spore group, a significant increase was seen in the expression of only two genes. These genes were TLR3 and TLR4. It was observed that there was a significant increase in TLR3 gene expression at the first hour (1.6-fold of control group) but the expression level started to decrease at the third hour (1.4-fold of control group) and returned to the control level at the sixth hour. It was observed that TLR4 gene expression continued parallel to the control until the 24th hour and increased significantly (2.1-fold of control group) at the 24th hour. In conclusion, this study is the f irst report in which the changes in ten different TLR gene expressions were evaluated at different times in MDCK cells stimulated with E.intestinalis and the change in TLR3 gene expression.

Microsporidia (Encephalitozoon cuniculi) in Patients with Degenerative Hip and Knee Disease, Czech Republic.

Total joint arthroplasty is a commonly used surgical procedure in orthopedics. Revision surgeries are required in >10% of patients mainly because of prosthetic joint infection caused by bacteria or aseptic implant loosening caused by chronic inflammation. Encephalitozoon cuniculi is a microsporidium, an obligate intracellular parasite, capable of exploiting migrating proinflammatory immune cells for dissemination within the host. We used molecular detection methods to evaluate the incidence of E. cuniculi among patients who had total hip or knee arthroplasty revision. Out of 49 patients, E. cuniculi genotypes I, II, or III were confirmed in joint samples from 3 men and 2 women who had implant loosening. Understanding the risks associated with the presence of microsporidia in periprosthetic joint infections is essential for proper management of arthroplasty. Furthermore, E. cuniculi should be considered a potential contributing cause of joint inflammation and arthrosis.

Investigation of the presence of Enterocytozoon bieneusi and Encephalitozoon intestinalis in immunosuppressed patients with diarrhea by IFA and real time PCR methods.

PURPOSE: This study aimed to investigate the frequency of Enterocytozoon bieneusi and Encephalitozoon intestinalis in patients with diarrhea in the immunosuppressed. METHODS: Patients between the ages of 18-85 who applied to different clinics of Mus Bulanik and Bitlis State Hospitals and were referred to the microbiology or parasitology laboratory were selected for this study. A total of 200 individuals, including 88 immunosuppressed with diarrhea patients, 38 immunocompetent with diarrhea patients, 38 immunosuppressed without diarrhea patients, and 36 immunocompetent without diarrhea individuals, were included. Collected stool samples were evaluated using IFA-MAbs and real-time PCR methods to determine the frequency of E.intestinalis and E.bieneusi. RESULTS: E. intestinalis was detected in 59 (29.5%) of 200 samples and E. bieneusi was detected in 46 (23.0%) of them. Mixed infection was detected in 16 (8%) of the positive samples. While there was no statistically significant difference between E. intestinalis positivity and gender, age, diarrhea status and immune system status, a statistically significant relationship was determined between E. bieneusi positivity and diarrhea. When the real-time PCR method was accepted as the gold standard, the sensitivity of the IFA-MAbs method in the diagnosis of E. intestinalis was 94.54%, the specificity was 97.24, the sensitivity in the diagnosis of E. bieneusi was 95.45%, and the specificity was 98.72%. The overall accuracy of the IFA-MAbs method was 96.5% for the diagnosis of E. intestinalis and 98% for the diagnosis of E. bieneusi. CONCLUSIONS: The findings suggest that E. intestinalis and E. bieneusi should be considered in both immunosuppressed and healthy individuals with diarrhea. IFA-MAbs method can be used in addition to the real-time PCR method to diagnose E. intestinalis and E. bieneusi.

Renal pathology in wild European rabbits.

BACKGROUND: There is a relative paucity of data examining the prevalence of renal pathology in wild rabbits. METHODS: Sixty-two wild rabbits that had been shot for population control in Cambridgeshire, UK, underwent postmortem examination, including macroscopic and microscopic renal assessment. RESULTS: The majority (82%) of the animals had macroscopically and microscopically normal kidneys. One animal (1.6%) had severe perirenal abscessation. Pasteurella spp. was isolated from this lesion. Ten rabbits (16%) had microscopic renal pathology comprising minimal to mild renal inflammation or fibrosis. No Encephalitozoon cuniculi organisms were detected histologically. LIMITATIONS: The sample population was composed of shot rabbits, so the probability of detecting moribund individuals was reduced. Extrapolation of these data to the wider UK wild rabbit population may be limited as rabbits were shot at two sites within a 3 km radius of each other. CONCLUSION: Renal pathology is rare in the population examined.

[Ophthalmic findings caused by Encephalitozoon cuniculi (strain III) in a dog]. / Ophthalmologische Befunde durch Enzephalitozoon cuniculi (Stamm III) bei einem Hund.

A 2-year-old female mixed-breed canine patient from Namibia presented originally with chronic uveitis. A serum antibody titer and a PCR test performed on the aqueous humor were positive for encephalitozoon cuniculi. The left eye showed an immature anterior focal cortical cataract in the periphery with suspected lens capsule rupture and signs of chronic uveitis. An incipient anterior focal cortical cataract was also perceivable in the patient's right eye. Despite local treatment as well as systemic administration of carprofen, prednisolone, and fenbendazol recurrent uveitis occurred. The patient then underwent bilateral extracapsular lensextraction via phacoemulsification. A PCR test of the lens material was positive for encephalitozoon cuniculi strain III. Recurring uveitis and secondary glaucoma 10 months post-op resulted in permanent blindness of the left eye. The patient then continued to receive local anti-inflammatory treatment. The last recheck examination of both eyes, 31 month post-op, revealed no signs of uveitis. This is the first case reported of a cataract in a canine patient caused by encephalitozoon cuniculi strain III.

Distribution of M1 and M2 macrophages in cerebral granulomas caused by Encephalitozoon cuniculi.

Encephalitozoon cuniculi spores cause severe granulomatous inflammation in the brain where mononuclear cells and macrophages infiltrate. Here, we orally infected New Zealand white rabbits with 1 × 106E. cuniculi viable spores to study the recruitment and localization of macrophages in brain granulomas. At day 30 post-infection, the positive phenotype markers iNOS (M1) and Arg-1 (M2) were located in the periphery and center of granulomas, respectively. Live intracytoplasmic spores were found only in positive Arg-1 cells. This is the first work to describe the recruitment and distribution of M1 and M2 macrophages in the brain granulomas of rabbits infected with E. cuniculi.

A multidisciplinary review about Encephalitozoon cuniculi in a One Health perspective.

Encephalitozoon cuniculi is a microsporidian parasite mostly associated with its natural host, the rabbit (Oryctolagus cuniculus). However, other animals can be infected, like other mammals, birds, and even humans. Although it usually causes subclinical infection, it can also lead to encephalitozoonosis, a clinical disease characterized by neurological, ocular, and/or renal signs that can be even fatal, especially in immunocompromised individuals. Therefore, this multidisciplinary review contributes with updated information about the E. cuniculi, deepening in its molecular and genetic characterization, its mechanisms of infection and transmission, and its prevalence among different species and geographic locations, in a One Health perspective. Recent information about the diagnostic and therapeutic approach in the main host species and the prophylaxis and infection control measures currently suggested are also discussed.

Serological survey for antibodies to Encephalitozoon cuniculi and Toxoplasma gondii in pet rabbits in eastern coastal areas of China.

Encephalitozoon cuniculi (E. cuniculi) is a microsporidian parasite commonly detected in rabbits and can infect humans and cause encephalitozoonosis. And Toxoplasma gondii is a prevalent parasite distributed worldwide and can infect almost all warm-blooded animals, including humans. The aim of the current study was to investigate the seroprevalence of E. cuniculi and Toxoplasma gondii, and risk factors of infection in pet rabbits reared in eastern coastal areas of China (Tianjin, Shandong, Jiangsu, Zhejiang, Shanghai and Fujian). Total 222 blood samples of pet rabbits were collected from local veterinary hospitals. The seropositivity rates of E. cuniculi were 16.22% (36/222) according to an Enzyme-linked immunosorbent assay (ELISA). Female pet rabbits was significantly higher than that in males (P=0.002), Zhejiang were markedly higher than those in Jiangsu and Shanghai (P=0.017, P=0.022), and cross-breed rabbits were dramatically higher than those in Chinchilla, New Zealand white, Rex (P=0.02, P=0.006, P=0.008). The seroprevalence of T. gondii was 13.06% (29/222) by the method of ELISA. The seroprevalence in Zhejiang was significantly higher than that in Shanghai (P=0.017). No difference in seroprevalence was detected with respect to the gender, age, species, health status, or season. These findings show that E. cuniculi and T. gondii are present and spread in pet rabbits. Therefore, pet rabbits should be considered as an important reservoir of encephalitozoonosis for humans and maybe important implication for public health in eastern coastal areas of China.

Opportunistic pneumonia caused by E. cuniculi in mice immunosuppressed with cyclophosphamide.

Opportunistic fungal pneumonia is a cause of concern in immunocompromised patients due to its high morbidity and mortality rates. One such opportunistic agent affecting immunocompromised patients is the microsporidia called Encephalitozoon cuniculi. This study aimed to evaluate pneumonia caused by E. cuniculi in mice treated with the immunosuppressive agent cyclophosphamide (Cy). This study also aimed to describe the immune cells associated with the microsporidial pneumonia. C57BL/6 mice were infected intravenously with E. cuniculi spores and treated with Cy (75 mg/kg/week, intraperitoneally). Thirty days post-infection, the fungal burden (qPCR), histopathological lesions, cytokine production, and the phenotype of the immune cells in the lung parenchyma were evaluated. Histologically, interstitial pneumonia with lymphocytic infiltrate was observed in the infected animals. The infiltrate mainly consisted of CD8+ and CD4+ T lymphocytes, with reduced populations of B lymphocytes and macrophages. The production of tumor necrosis factor-alpha (TNF-α) was significant in the animals of the infected groups. Also, the fungal burden was higher in the Cy-treated animals, which was confirmed by the immunohistochemical observation of spores. These results demonstrated that E. cuniculi infection of C57BL/6 mice caused lymphocytic interstitial pneumonia (characterized by a predominant lymphocytic infiltrate), which was aggravated by Cy-induced immunosuppression. Thus, these results can be used to understand the different pathological, immunological, and therapeutic aspects of lymphocytic interstitial pneumonia.

Encephalitozoon cuniculi and Extraintestinal Microsporidiosis in Bird Owners.

We identified Encephalitozoon cuniculi genotype II parasites as a cause of extraintestinal microsporidiosis in 2 owners of birds also infected with E. cuniculi. Patients experienced long-lasting nonspecific symptoms; the disease course was more progressive in a patient with diabetes. Our findings suggest direct bird-to-human transmission of this pathogen.

Seroprevalence of Encephalitozoon cuniculi and Toxoplasma gondii antibodies and risk-factor assessment for Encephalitozoon cuniculi seroprevalence in Finnish pet rabbits (Oryctolagus cuniculus).

BACKGROUND: Neurological signs, such as head tilt, torticollis, paralysis, and seizures, are common in rabbits. Differential diagnoses include two zoonotic infections caused by the microsporidial fungi Encephalitozoon cuniculi and the apicomplexan protozoa Toxoplasma gondii. Both infections are mainly latent in rabbits but may cause severe or even fatal disease. Although several international studies have reported the seroprevalence of these pathogens in different commercial rabbit populations, similar prevalence studies and risk-factor analyses among family-owned pet rabbits are uncommon and lacking in Scandinavia. We sought to estimate the seroprevalence and possible risk factors for E. cuniculi and T. gondii among Finnish pet rabbits. We used ELISA to measure E. cuniculi IgG seroprevalence of 247 rabbits and modified direct agglutination test for T. gondii seroprevalence of 270 rabbits. Samples were collected as part of the Finnish Pet Rabbit Health Research project. Internet-based questionnaires (n = 231) completed by the rabbit owners were used for risk-factor analysis. RESULTS: The apparent seroprevalence of E. cuniculi was 29.2% and true seroprevalence of T. gondii 3.9%. Risk factors were analysed only for E. cuniculi due to the low T. gondii seroprevalence. The final multivariable logistic regression model revealed that rabbits spending the whole summer outdoors had a higher risk of being E. cuniculi seropositive than rabbits with limited outdoor access. Additionally, rabbits living in households with only one or two rabbits had higher risk of being E. cuniculi seropositive than those in multi-rabbit households. CONCLUSIONS: Nearly one third of Finnish pet rabbits participating in this study had E. cuniculi IgG antibodies, indicating previous exposure to this pathogen. The prevalence is similar to that reported previously in clinically healthy rabbit populations in UK and Korea. While the seroprevalence of T. gondii was low (3.9%), antibodies were detected. Therefore, these zoonotic parasitic infections should be considered as differential diagnoses when treating rabbits.

Mice with genetic and induced B-cell deficiency as a model for disseminated encephalitozoonosis.

Encephalitozoon cuniculi, an intracellular pathogen, lives in a balanced relationship with immunocompetent individuals based on the activity of T lymphocytes. We previously highlighted the greater susceptibility of B-1 cell-deficient mice (XID mice) to encephalitozoonosis. This study aimed to develop a model of disseminated and severe encephalitozoonosis in mice with combined immunodeficiency to elucidate the role of B cells. To address this objective, cyclophosphamide (Cy)-treated BALB/c and XID mice were inoculated with E. cuniculi, followed by the evaluation of the immune response and histopathological lesions. Immunosuppressed BALB/c mice manifested no clinical signs with an increase in the populations of T lymphocytes and macrophages in the spleen. Immunosuppressed and infected XID mice revealed elevated T cells, macrophages populations, and pro-inflammatory cytokines levels (IFN-γ, TNF-α, and IL-6) with the presence of abdominal effusion and lesions in multiple organs. These clinical characteristics are associated with extensive and severe encephalitozoonosis. The symptoms and lesion size were reduced, whereas B-2 and CD4+ T cells populations were increased in the spleen by transferring B-2 cells adoptive to XID mice. Moreover, B-1 cells adoptive transfer upregulated the peritoneal populations of B-2 cells and macrophages but not T lymphocytes and decreased the symptoms. Herein, we speculated the consistency in the development of severe and disseminated encephalitozoonosis in mice with genetic deficiency of Bruton's tyrosine kinase (Btk) associated with Cy immunosuppression develop with that of the models with T cell deficiency. Taken together, these data emphasized the crucial role of B cells in the protective immune response against encephalitozoonosis.

Molecular epidemiology and genotype/subtype distribution of Blastocystis sp., Enterocytozoon bieneusi, and Encephalitozoon spp. in livestock: concern for emerging zoonotic infections.

Intestinal parasitic infections have high prevalence rate in many regions especially in developing countries. The aim of this study was to determine the presence and genotype/subtype of some intestinal protozoa in livestock in Iran. Stool samples were collected from cattle, sheep, chickens, and horses. The presence of targeted parasites was evaluated using real-time PCR. Genotyping/subtyping of positive samples was characterized using sequencing of the ITS and barcoding region, respectively. Blastocystis sp., 27.7% (48/173) and Enterocytozoon bieneusi 26.0% (45/173) were the most frequent protozoa followed by Encephalitozoon spp., 0.57% (1/173). Cryptosporidium spp. were not detected among samples. Encephalitozoon spp., was detected only in chickens 2.2% (1/45). A statistically correlation was seen between animals and the prevalence of targeted protozoa. E. bieneusi genotypes I (9/38; 23.68%), BEB6 (22/38; 57.89%), D (6/38; 15.79%), and horse1 (1/38; 2.63%) were detected among samples. A statistically significant correlation was seen between the genotypes and animals (P ≤ 0.05). Blastocystis sp., ST1 (1/45; 2.22%), ST5 3/45; 6.66%), ST7 (1/45; 2.22%), ST10 (24/45; 53.33%), and ST14 (16/45; 35.55%) were characterized among samples. There was no significant correlation between certain subtypes and animals (P = 0.173). The presence of zoonotic potential genotypes of E. bieneusi in animals and zoonotic potential subtypes ST1 and ST7 among our samples provide a clue about the transmission dynamic of E. bieneusi and Blastocystis sp. between animals-animals and humans-animals.

Zoonotic Risk of Encephalitozoon cuniculi in Animal-Assisted Interventions: Laboratory Strategies for the Diagnosis of Infections in Humans and Animals.

The involvement of animals for therapeutic purposes has very ancient roots. To date, it is clear that animal-assisted interventions (AAIs), in addition to ensuring the replacement of missing or deficient affects, improves psychophysiological parameters connected to human health. However, AAI could potentially present risks related to the transmission of infectious agents from animals to humans. Among these microorganisms, E. cuniculi is a microspore which induces pathological effects (fever, headache, nausea, vomiting, diarrhea, breathlessness, respiratory symptoms, and weakness) in both humans and animals. Consequently, an accurate and fast diagnosis of E. cuniculi infection, as well as the identification of new diagnostic approaches, is of fundamental importance. This literature review was carried out to provide an extensive and comprehensive analysis of the most recent diagnostic techniques to prevent and care for E. cuniculi-associated risks in the AAI field.