Molecular detection and genotype identification of E. cuniculi from pet rabbits

Encephalitozoon cuniculi is a microsporidian which is frequently reported from rabbits. This microorganism can either ravage rabbit farms or transmit to humans from pet rabbits. This study aimed to investigate the prevalence and the genotype distribution of E. cuniculi among pet rabbits. In this study urine samples were collected from 50 pet rabbits, aged 2 months to 3 years, admitted to teaching veterinary hospital. Four races Lop, Dutch, Mix, and Angora were screened for E. cuniculi. The clinical symptoms were recorded and total DNA was extracted from urine samples. E. cuniculi was identified using amplification of the small subunit ribosomal RNA (ssu rRNA) gene and its genotypes were characterized using PCR/sequencing of the polar tube protein (PTP) gene. Phylogenetic tree was drawn to confirm the characterized genotypes. Out of 50 samples, 41 (82 %) of rabbits were asymptomatic, while nine (18 %) had at least one of symptoms including head-tilt, circling, and ataxia. A statistical correlation was seen between mean age + SD and symptoms (P-value = 0.039). The presence of E. cuniculi was confirmed in 16/50 (32 %) rabbits and all of them were identified as the genotype I. Our findings represented no consistency between E. cuniculi PCR – positive and the presence of symptoms (P-value = 0.318). Our results showed positive correlation between symptoms and age; however, the lack of correlation between PCR results with age may signify the latent infection in younger rabbits. All identified E. cuniculi were the genotype I, which is reported from rabbits and humans, highlighting the zoonotic concern for this genotype, particularly among subjects who keep pet rabbits.     

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.     

Serological survey of Encephalitozoon cuniculi in farm rabbits in Italy

Rabbit sera (n = 1600) from 40 commercial farms were submitted to a serological screening for Encephalitozoon cuniculi by an enzyme-linked immunosorbent assay (ELISA) and a carbon immunoassay (CIA test). Antibodies anti-Encephalitozoon cuniculi were found in 505/1600 (31.6%) sera analysed, and all the farms (100%) resulted positive. Rabbits older than 4 months showed a significantly higher seropositivity for E. cuniculi (chi-squared test: p < 0.0001) than rabbits under 4 months, E. cuniculi sero-prevalence showed an increasing trend in rabbits within the farm along with the increase in the “number of rabbits on the farm”; however, this trend was not significant (Spearman’s correlation: p = 0.073).The findings of the present study confirm that rabbit is the main reservoir of E. cuniculi; they are of epidemiological relevance and immediate public health importance because of the recognized infectivity in humans by the microsporidium.     

Encephalitozoon cuniculi infection in rabbits

The microsporidian parasite Encephalitozoon cuniculi commonly infects rabbits. Most infections are initially asymptomatic, but for reasons yet to be explained, many rabbits subsequently develop disease as a result of infection with this organism. Three common forms of this disease are recognized, and they can occur individually or in combination. The ocular form is associated with cataracts and when there is extensive damage to the lens, uveitis. The neurological form can vary from a mild change in the rabbit’s behavior to severe vestibular disease. The signs associated with the renal form of the disease are those of chronic progressive renal disease. Definitive diagnosis of encephalitozoonosis in the rabbit is difficult. Animals with encephalitozoonosis are expected to be seropositive, but many apparently healthy rabbits are also seropositive, so this assay is not specific and its results must be considered in the light of other diagnostic findings. The absence of antibody, however, should cause the practitioner to consider other differentials. Drugs proven to be efficacious for E. cuniculi infections include albendazole and fenbendazole. Supportive care and treatment with antiinflammatory medications may also be necessary in some forms of encephalitozoonosis. Lens removal or removal of the lens contents is indicated in some rabbits with E. cuniculi-induced ocular disease.     

Variation of the CD4, CD8, and MHC II cell population in granulomas of immunocompetent and immunosuppressed rabbits in Encephalitozoon cuniculi infection

Encephalitozoon cuniculi (E. cuniculi) is a fungi-related, obligate, zoonotic, spore-forming intracellular eukaryotic microorganism. This emerging pathogen causes granulomas in brain and kidneys of infected individuals. The objective of this study was to detect the distribution of CD4, CD8 and MHCII-positive cells within granulomas in these organs in infected immunocompetent (group A) and infected immunosuppressed (group B) New Zealand white rabbits using immunohistochemistry. In brain, labeled CD4 immune cells were mainly located in the periphery of granulomas in group B. Kidneys of groups A and B, displayed CD4-positive in granulomas and were significant different when compared to brain. CD8 immune cells in brain and kidneys were disseminated in the granulomas in groups A and B; however, no significant difference was observed. MHCII-positive cells were more numerous in brain sections of group B and were significantly different when compared to kidney sections. Granulomas were not observed in control animals of group C and D. In conclusion, we identified CD4-positive cells in both the brain and kidneys of immunocompetent and immunosuppressed animals; CD8-positive cells were more numerous in brain of immunosuppressed rabbits and MHCII cells were more predominant in brain of immunocompetent rabbits. Apparently, the immunosuppression stimulated a change in the cellular phenotype of Th1- to Th2-like granulomas in brain and kidneys by an unknown mechanism. These results increase our understanding of CD4, CD8 and MHCII positive cells within the E. cuniculi granuloma microenvironment and will help in future microsporidian granulomas studies of both immunocompetent and immunosuppressed individuals.     

Comparison of enzyme-linked immunosorbent assay and immunofluorescence test for determination of anti-Encephalitozoon cuniculi antibodies in sera from rabbits with different clinical and histopathological presentations

Encephalitozoon cuniculi is a microsporidian that infects rabbits resulting in a varied pathology of the nervous, renal, and ocular tissues. Serology based studies using different methods have been conducted indicating a high seroprevalence worldwide in pet rabbits. While qualitative results are not especially helpful in the diagnosis of this infection, quantitative titers can have increased predictive value. In the current study, results of enzyme-linked immunosorbent assay (ELISA) and immunofluorescence test methods were compared using banked samples from rabbits with confirmed infection and clinically normal rabbits. Pearson's analysis found good and significant correlation (R = 0.56, P = 0.0002). Bland Altman analysis demonstrated the methods were similar but not equivalent. The sensitivity ranged from 0.79 to 0.84 and the specificity was 1.00 for both assays. Anti-E. cuniculi IgM titers were also determined by ELISA to be an independent measure from IgG (R = 0.31, P = 0.06) with a sensitivity of 0.24 and specificity of 1.00. In total, these results confirm an agreement in the detection of IgG antibody by two different methods conducted at laboratories in the United States and Austria that are involved in research and clinical diagnostic testing of this agent.     

Acute Phase Protein Levels in Rabbits with Suspected Encephalitozoon cuniculi Infection

The objective of this study was to evaluate the application of acute phase protein assays for C-reactive protein (CRP), haptoglobin (HP), and serum amyloid A (SAA) in the diagnosis of Encephalitozoon cuniculi (ECUN) infection in pet rabbits. Serum samples from 48 pet rabbits were submitted from veterinary clinics within the United States. Participating veterinarians completed a questionnaire that was used to classify rabbits as either non-ECUN suspect (n = 19) or suspected of having ECUN infection (n = 29). A previously described enzyme-linked immunosorbent assay diagnostic test was used to detect immunoglobulin G (IgG) titers against ECUN. Samples were additionally tested for levels of CRP, HP, and SAA. A nearly 10-fold mean increase in CRP levels was observed in the ECUN-suspect group. This increase was significant (P < 0.05). There was no significant difference in HP or SAA levels between the clinical groups. These data support the use of CRP as an adjunct test in the diagnosis of ECUN infection in pet rabbits.     

Rabbit Clinical Pathology

With rabbit patients, as in other species, analyzing blood and urine samples can be useful and informative, although interpretation of the results is sometimes challenging. This article summarizes the interpretation of laboratory results from rabbits. Hematological parameters can yield information about the red blood cell population and leukocyte response to stress and pathogens. Biochemistry evaluation can be used to investigate liver, kidney, and other organ function, and urinalysis results may yield additional information about kidney function and electrolyte imbalances. Serological tests are available for several pathogens of rabbits, including Encephalitozoon cuniculi, although the significance of positive results and antibody titers is not clear. Serum protein electrophoresis aids the understanding of protein disorders and the immune response to acute and chronic inflammation.     

Clinical Update on Testing Modalities for Encephalitozoon cuniculi in Clinically Sick Rabbits

This article describes the clinical presentation of rabbit patients infected with Encephalitozoon cuniculi that present to veterinary hospitals. The difficulty in obtaining a definitive diagnosis with this disease, even in animals with severe clinical signs, is frustrating to veterinarians and owners alike. A detailed review of current diagnostic testing methodologies and recent advances should help with the interpretation of E. cuniculi test results to communicate and inform owners on treatment and prevention of this devastating disease.     

Microsporidia (Encephalitozoon cuniculi) in wild rabbits in Australia

Objective To determine the prevalence of infection with Encephalitozoon cuniculi i n wild rabbit populations in Western Australia, and to isolate the organism from seropositive rabbits.
Design Serological screening of wild and clinically affected domestic rabbit populations.
Sample population Eighty-one wild rabbits from southwestern Western Australia and 29 laboratory rabbits.
Procedure Indirect immunofluorescence antibody technique and in-vitro amplification of parasite isolates in fibroblast cultures.
Results Of the 81 wild rabbits and 29 laboratory rabbits, 20 and 22 respectively, had antibodies to E cuniculi . E cuniculi from the urine of one seropositive laboratory rabbit and from brain and kidney tissues of eight and five seropositive laboratory and wild rabbits respectively were isolated in fibroblast cultures.
Conclusion E cuniculi infection has been shown for the first time to be prevalent in wild rabbits in Australia. Techniques have been developed for the isolation and culture of the causative agent. Comparative studies can now be undertaken to determine risk factors for clinical disease in domestic rabbits and the relationship among E cuniculi isolates from wild and domestic rabbits.     

Serological survey for antibodies to Encephalitozoon cuniculi in rabbits in Taiwan

Encephalitozoon cuniculi (E. cuniculi) is a microsporidian parasite commonly found in rabbits that can infect humans, causing encephalitozoonosis. Our laboratory recently confirmed the first case of encephalitozoonosis in a rabbit in Taiwan; the prevalence of encephalitozoonosis is not well documented, even when many clinics suspect pet rabbits as being infected. This study surveys the seropositivity of E. cuniculi using carbon immunoassay (CIA) and enzyme-linked immunosorbent assay (ELISA). Serological examination of 171 rabbits using CIA and ELISA showed that 63.2% (108/171) and 67.8% (116/171) were seropositive against E. cuniculi, respectively. Thirteen of the 14 rabbits (92.9%) with neurological symptoms were seropositive. Except for gender, health status and location had a significant effect on E. cuniculi seropositivity (p<0.05). Adult rabbits aged older than 4 months exhibited significantly higher seropositivity for E. cuniculi than young rabbits (p<0.05). In conclusion, this study shows that E. cuniculi is present and widespread among healthy rabbits in Taiwan. Therefore, the fields of veterinary and human medicine in Taiwan should be aware of this zoonotic issue and the resulting public health concern of encephalitozoonosis.     

Isolation of Streptococcus cuniculi from corneal lesion in laboratory-raised mice

Corneal lesions appearing as white mass beneath intact epithelium, with ocular discharge in one mouse, was observed in a batch of laboratory-raised BALB/c mice (n=9 of 56). The affected mice remained active, well-groomed and had normal appetite. Isolates recovered from swab cultures of the external and internal contents of the eye had partial 16S rRNA gene sequence of 99.1% similarity to Streptococcus cuniculi. No previous report of S. cuniculi infection in laboratory rodents has been presented. The isolate was susceptible to all antibiotics tested. We suggest S. cuniculi is an opportunistic bacteria in laboratory mice but are uncertain of its source. Our findings revealed that S. cuniculi is able to colonize laboratory mice and should be considered when mice present with eye lesion or ocular discharge.     

Species-specificity of equine and porcine Lawsonia intracellularis isolates in laboratory animals

Lawsonia intracellularis infection causes proliferative enteropathy (PE) in many mammalian species, with porcine and equine proliferative enteropathy (PPE and EPE) known worldwide. Hamsters are a well-published animal model for PPE infection studies in pigs. There is no laboratory animal model for EPE infection studies and it is not known whether there is species-specificity for equine or porcine isolates of L. intracellularis in animal models. The objective of this study was to determine whether it is possible to generate typical EPE lesions in hamsters after inoculation with an equine strain of L. intracellularis (EPE strain) and whether it is comparatively possible to generate PPE lesions in rabbits after inoculation with a porcine strain of L. intracellularis (PPE strain). In 2 separate trials, 4-week-old and 3-week-old weanling golden Syrian hamsters were challenged with EPE strains and compared to uninfected (both trials) and PPE-infected controls (Trial 2 only). Concurrently, 6 female New Zealand white juvenile rabbits were infected with PPE strain and observed concomitantly to 8 similar rabbits infected with EPE strain for a different experiment. Hamsters and rabbits were observed for 21 to 24 days post-infection (DPI), depending on the experiment. Neither infected species developed clinical signs. The presence of disease was assessed with diagnostic techniques classically used for pigs and horses: immune-peroxidase monolayer assay on sera; quantitative polymerase chain reaction (qPCR) detection of molecular DNA in feces; and hematoxylin and eosin (H&E) stain and immunohistochemistry (IHC) on intestinal tissues. Our results showed that EPE-challenged hamsters do not develop infection when compared with PPE controls (IHC, P = 0.009; qPCR, P = 0.0003). Conversely, PPE-challenged rabbits do not develop typical intestinal lesions in comparison to EPE-challenged rabbits, with serological response at 14 DPI being significantly lower (P = 0.0023). In conclusion, PPE and EPE strains appear to have different host-specificities for hamsters and rabbits, respectively.     

Large-scale management systems and parasite populations: Coccidia in rabbits

Broiler rabbit production has become an important branch of animal protein and fur production, not only in the traditionally rabbit breeding and consuming countries, but recently in many other countries of the world.The profitability of the rabbit industry is dependent primarily on the good feed conversion. Aspects of this include factors such as morbidity and mortality of infections, adequate knowledge and fulfilment of both accommodation and nutritional requirements, appropriate breeding systems, etc.There is much controversy about the role coccidia in the losses of intensified rabbit breeding enterprises. There is no doubt about the significance of Eimeria stiedai which may cause condemnation of large amounts of large amounts of liver as a result of infection in rabbit colonies of small holders. However, this parasite is rare in large scale rabbit farms. In the latter, intestinal coccidiosis is most frequently — although not invariably — incriminated either as a primary or more often as a predisposing factor of intestinal enteropathies causing severe mortality, mainly in the early post-weaning period.The ability to isolate and maintain species of Eimeria in specifc pathogen-free (SPF) rabbits has made it possible to characterize them, such as the most pathogenic E. intestinalis and E. flavescens: the pathogenic E. magna, E. irresidua and E. piriformis; and the least pathogenic E. perforans, E. neoleporis (syn o E. coecicola and E. media species.Prophylactic measures are dealth with briefly, and laboratory and field trials with the currently most promising rabbit anti-coccidial drugs (Lerbek, robenidine and salinomyciin) are dealt with in more detail.     

Diet is a main source of vitamin D in Finnish pet rabbits (Oryctolagus cuniculus)

During the winter time in Finland, sunlight is inadequate for vitamin D synthesis. Many pet rabbits live as house rabbits with limited outdoor access even during summer and may therefore be dependent on dietary sources of vitamin D. The aims of this study were to report the serum 25‐hydroxyvitamin D concentrations in Finnish pet rabbits and to identify factors that influence vitamin D status. Serum 25‐hydroxyvitamin D concentrations from 140 pet rabbits were determined using a vitamin D enzyme immunoassay (EIA) kit. Eleven rabbits were excluded from the statistical analysis because of unclear dietary data. The remaining 129 rabbits were divided into groups depending on outdoor access during summer (no access n = 26, periodic n = 57, regular n = 46) as well as daily diet: little or no hay and commercial rabbit food ≤1/2 dl (n = 12); a lot of hay and no commercial food daily (n = 23); a lot of hay and commercial food <1 dl (n = 59); a lot of hay and commercial food ≥1 dl (n = 35). The range of serum 25‐hydroxyvitamin D concentration was from 4.5 to 67.5 ng/ml with a mean of 26.1 ng/ml. Statistical general linear model adjusted for weight, age and season indicated that diet was associated with vitamin D concentrations (p = 0.001), but outdoor access during summer was not (p = 0.41). Mean 25‐hydroxyvitamin D concentration was significantly higher in the rabbits receiving a lot of hay and commercial food ≥1 dl (33.9 ± 13.2 ng/ml) than in rabbits in other diet groups (24.0 ± 8.5 ng/ml, 21.7 ± 8.1 ng/ml, and 22.2 ± 18.0 ng/ml, respectively). This investigation showed wide variation in 25‐hydroxyvitamin D concentrations among Finnish pet rabbits. Diet remains a main source since outdoor access seems to be too limited to provide adequate vitamin D synthesis for most of them, and the use of vitamin D supplements is rare.     

Eimeria stiedae causes most of the white-spotted liver lesions in wild European rabbits in Cambridgeshire,United Kingdom

In rabbits, a white-spotted liver can be indicative of one of several disease processes, frequently caused by parasites. To date, the prevalence of white-spotted liver in wild rabbits, Oryctolagus cuniculus, in the United Kingdom is undetermined. We evaluated the prevalence and main parasitic etiologies of this entity in a U.K. population of wild rabbits. Wild rabbits (n = 87) were shot in Cambridgeshire for population control, and cadavers were donated for research. Postmortem examination was undertaken, including gross and histologic hepatic examination. Macroscopic lesions consistent with white-spotted liver were found in 46 of 87 (53%) rabbits examined; most of these lesions were considered to be mild. For 28 of 46 (59%) rabbits with gross hepatic lesions, an etiologic agent was apparent histologically. Eimeria stiedae was detected in 21 of 87 (24%) rabbits, and Calodium hepaticum (syn. Capillaria hepatica) was detected in 7 of 87 (8%). In the subset of rabbits killed in the summer, there was a significant association between white-spotted liver and juvenile age class. There was also an association between white-spotted liver caused by E. stiedae and juvenile age class. When restricting analysis to rabbits with white-spotted liver caused by E. stiedae and submitted in the summer, both juvenile age class and female had significant effects. E. stiedae and C. hepaticum can be transmitted to pet lagomorphs via contaminated vegetation, and to humans in the case of the latter, which demonstrates the importance of monitoring the prevalence of these parasitic diseases in wild rabbits.     

Comparative effects of Herba Cox®, a commercial herbal extract,on rabbits (Oryctolagus cuniculus) experimentally infected with Eimeria stiedae

The objective of this study was to evaluate the efficacy of Herba Cox®, a commercial herbal compound containing extracts from Bombax malabaricum, Aegle marmelos, Anethum foeniculum, Resina salvia, Ferula asafoetida and Papaver somniferum, for the treatment of rabbit hepatic coccidiosis. Thirty rabbits were allocated into three groups (10 × 3), the G1 group served as a negative control group, G2 group (positive control group) was infected with 5 × 10⁴ sporulated E. stiedaeoocysts and served as infected-untreated group, and G3 group was infected with 5 × 10⁴ sporulated E. stiedaeoocysts and treated with Herba Cox®, 1 ml/liter of drinking water, starting 7 days before infection and continuing for 4 weeks post-infection. When compared to the infected group (G2), body weight and weight gain were significantly (P ≤ 0.05) increased, the feed conversion rate was improved and no mortality was detected in infected treated group (G3) and similar to negative control group (G1). In addition, faecal oocyst output and liver enzymes were significantly decreased. Malondialdehyde, nitric oxide, and glutathione concentrations observed in G3 were similar to those in G1. In infected-untreated rabbits (G2), the haemoglobin, lymphocytes, and CD4+/ CD8+ ratio were significantly decreased, while the total leukocyte count, percentage of heterophils, and heterophil/lymphocyte ratio were increased. Significantly more severe histopathological hepatic lesions were observed in G2 when compared to G1 and G3. In conclusion, the obtained results showed that Herba Cox® should be considered a safe and novel effective compound for the treatment of E. stiedae infection in rabbits.     

Immunological diagnosis of encephalitozoonosis from post-mortem specimens

Antibody to $\text{Encephalitozoon cuniculi}$ for immunopathological diagnosis was obtained from organs and fluids from rabbit carcasses. By the use of the india-ink immunoreaction antibodies to $\text{E. cuniculi}$ were detected in lungs, kidneys and pleural fluid of seropositive animals. Mean values of the antibody titres in the lungs were $\text{1}\text{10}$ to $\text{1}\text{2}$ of that in serum. Antibody titres from other organs were inconsistent. No titres were found in organs or fluids from seronegative rabbits.