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.     

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.     

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.     

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.     

The prevalence and pathogenicity of Chorioptes bovis (Hering, 1845) and Psoroptes cuniculi (Delafond, 1859) (Acari: Psoroptidae) infestations in feral goats in New Zealand

Between October 1976 and January 1980 a total of 368 feral goats was examined in New Zealand for the presence of Chorioptes bovis and 434 for the presence of Psoroptes cuniculi. The seasonal pattern of C. bovis infestation in feral goats was similar to that seen in sheep and cattle. The prevalence of infestation reached 100% in July and August (winter) and declined to 27% in February and March (summer). Fewer goats were infested with P. cuniculi and prevalence of infestation reached a maximum of 41% in July. No goats were found infested in the December and January samples. Chorioptes bovis infestation was independent of the age and sex of hosts. Infestation with P. cuniculi was independent of the sex of the hosts, but older goats were more frequently infested (16%) than younger goats (8.6%). Unilateral infestation with P. cuniculi was more common (74.5%) than bilateral infestation. No severe lesions were associated with C. bovis, but 21.3% of goats with P. cuniculi had the external auditory meatus blocked by waxy material and 12.8% had scabby encrustations on the ears. The importance of goats as a possible source of infestation to domestic animals is 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.     

First insight into Encephalitozoon cuniculi infection in laboratory and pet rabbits in Iran

Encephalitozoon cuniculi infects a wide variety of domestic and wild mammalian species including humans. Although the infection status has been studied in laboratory and pet rabbits worldwide, there is shortage of information regarding the disease in Iran. In the present study, the occurrence of infection in brains of 117 asymptomatic rabbits from six breeding and experimental units with highest population of rabbit colonies in the country (n = 60) as well as pet rabbits of pet stores in two cities (n = 57) were examined by nested-PCR. Histological sections of brains and kidneys were also studied by light microscopy. PCR results revealed that 3.3% of laboratory rabbits (2/60) and 59.6% of pet rabbits (34/57) harboured E. cuniculi in their brains. Histopathology on the other hand showed spores of the parasite in kidney and brain of one and kidney of another pet rabbit. As encephalitozoonosis may interfere with results of experiments performed on laboratory rabbits, routine screenings for identification and culling of infected animals is recommended. Furthermore, infected companion rabbits can transmit E. cuniculi to people in close contact with them, therefore, improving public knowledge of this zoonotic infection is suggested.     

The impact of COVID-19 on access to canine integrative medical care in Michigan,USA, and Ontario and British Columbia,Canada

ObjectiveTo determine the effects of the COVID-19 associated restrictions on the ability of owners in Michigan (MI), USA versus Ontario (ON) and British Columbia (BC), Canada, to obtain care for their chronically painful dogs.Study designCross-sectional survey.PopulationA total of 90 owners met the inclusion criteria for the study.MethodsAn anonymous electronic survey was distributed to owners at four veterinary integrative medicine (IM) clinics during July and August 2020. Two clinics in MI and one each in ON and BC were recruited. Owners were asked about availability of IM care preceding and during COVID-19 restrictions and their opinions of the impact of COVID-19 on their dog’s health. The survey asked where owners sought care for their dogs, types of chronic conditions treated, therapeutic modalities used, and if owners had a medical background. Comparisons were made within and between groups. Thematic analysis, Fisher’s exact test, chi-square analyses, McNemar’s and Wilcoxon signed-rank tests for paired comparisons were performed (p < 0.05).ResultsDuring COVID-19 restrictions, access to IM care was better for dogs in ON and BC than in MI (p < 0.001). The negative effect of the pandemic restrictions to IM care on quality of life was perceived greater by owners in MI than those in ON and BC (p < 0.001). The owners’ medical backgrounds had no effect on attempts to access care during this time (p = 0.76).Conclusions and clinical relevanceThe results suggest that a widespread disease in humans had an adverse impact on animal welfare. Providers of veterinary care should use this experience to establish protocols to ensure continuity of care for chronically painful animals in the event of a similar situation in the future.     

Detection of Encephalitozoon cuniculi in the feline cataractous lens

Purpose Identification of Encephalitozoon cuniculi (E. cuniculi) as a possible causative agent for cataracts and uveitis in cats. Methods Within a 12‐month study period, cats that were presented with focal anterior cortical or mature cataract and secondary uveitis underwent a complete ophthalmic examination, complete blood count, serum biochemistry, serologic tests for E. cuniculi and tests for feline immunodeficiency virus (FIV), feline infectious peritonitis (FIP), feline leukemia virus (FeLV) and Toxoplasma gondii (T. gondii). PCR for DNA detection of E. cuniculi and T. gondii as well as cytologic examination of aqueous humor after paracentesis and phacoemulsified lens material were also performed. In addition histopathologic examination of the resected anterior lens capsule and attached lens epithelial cells was performed. Serologic testing for antibodies against E. cuniculi was also performed in 100 ophthalmologically healthy cats. Results Eleven (19 eyes) European shorthair cats with a median age of 3.5 years were included. Nine of 11 cats had bilateral cataracts, with 12/19 eyes having focal anterior cortical cataracts and 7/19 eyes having mature cataracts. In 14/19 eyes anterior uveitis was present. All cats had a positive antibody titer (1:80–1:10 000) for E. cuniculi. Encephalitozoon cuniculi DNA was detected by PCR and sequencing in 18/19 lenses and in 10/19 aqueous samples. Five tentative positive results were detected by cytologic examination. Spores were detected in 15/19 samples of lens material with histopathologic staining. Only 2/100 ophthalmologically healthy cats showed a positive antibody titer for E. cuniculi. Conclusion Encephalitozoon cuniculi is a cause of focal anterior cortical cataract and anterior uveitis in cats.     

Encephalitozoon cuniculi–Associated Equine Encephalitis: A Case Report

A case of encephalitis of unknown origin in the horse was investigated. Postmortem examination findings revealed a nonsuppurative granulomatous meningoencephalitis in the right hemisphere of the cerebral cortex. Testing for West Nile virus, equine herpes virus, equine infectious anemia, Toxoplasma gondii, Neospora caninum, and Sarcocystis neurona were negative. The horse had a titer for Encephalitozoon cuniculi, and sections from the affected area of the brain tested positive for the organism using both polymerase chain reaction (PCR) and immunohistochemistry. Amplicons generated using PCR were sequenced, and E. cuniculi genotype II was identified. This is the first case of E. cuniculi genotype II associated with encephalitis in the horse.     

Inhibited development of Obeliscoides cuniculi in rabbits: the effects of active and passive immunization and resumption of larval development

Rabbits were either actively or passively immunized against Obeliscoides cuniculi and challenged with 5 000 infective larvae (IL). Worm development was inhibited in actively immunized animals and worm egg production was impaired by administration of immune serum. Inhibited fourth stage larvae (L₄) from high-level infections (200 000 IL) and normal L₄ from low-level infections (4 000 IL) were transferred to actively immunized and normal rabbits. In both instances, large numbers of L₄ developed to maturity; however, a significant number of inhibited L₄ failed to resume development. Inhibition of larvae resulted in the development of fewer adult male than female worms, and the change in sex ratio was further accentuated in actively immunized recipients. Worm egg production was also reduced in actively immunized rabbits. These data indicate that host immune responses contribute to inhibition of O. cuniculi in rabbits and that worm egg production is also responsive to immunologic control.     

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.     

Seroprevalence of Leptospira spp. in Working Horses Located in the Central Region of Chile

Urban working horses live in close contact with their owners. They are usually kept in periurban areas of big cities and cohabit with other animals under precarious sanitary conditions, whereas army horses are kept under controlled management and work. These characteristics leave urban working horses in higher risk of exposure to Leptospira spp. and could become a zoonotic risk for their owners. The aim of this study was to determine the frequency of seropositive working horses to diverse serovars of Leptospira spp. and compare them to a group of army horses. The microscopic agglutination test was used to assess the serum of 426 horses (160 working horses and 266 army horses) against two serovars of Leptospira borgpetersenii (Hardjo and Ballum) and four of Leptospira interrogans (Pomona, Canicola, Icterohaemorrhagiae, and Autumnalis). In the urban working horses group, 30.63% of horses were positive to at least one serovar at titers above 1:100, whereas 23.31% of the army horses were positive. The most frequent serovar in the working horse group was Ballum followed by Canicola, whereas in the army group was Autumnalis followed by Ballum. The serovars Hardjo, Pomona, and Icterohaemorrhagiae were not present in the army horses, whereas all serovars studied were detected in urban working horses. Although no horses studied presented clinical signs of leptospirosis, the study confirms exposure to Leptospira spp. and the importance of studying in more detail the livelihood conditions in which working horses are kept and possible risk of transmission to their owners.     

Owner assessment in judging the efficacy of airway disease treatment

Reasons for performing study: Efficacy of medications for recurrent airway obstruction is typically tested using clinical, cytological and lung function examinations of severely affected animals. These trials are technically challenging and may not adequately reflect the spectrum of disease and owner complaints encountered in clinical practice. Objective: To determine if owners of horses with chronic airway disease are better able to detect drug efficacy than a veterinarian who clinically examines horses infrequently. Method: In a double‐blinded randomised controlled trial, owners and a veterinarian compared the efficacy of dexamethasone (0.1 mg/kg bwt per os, q. 24 h, for 3 weeks; n = 9) to placebo (n = 8) in horses with chronic airway disease. Before and after treatment, owners scored performance, breathing effort, coughing and nasal discharge using a visual analogue scale (VAS). The clinician recorded vital parameters, respiratory distress, auscultation findings, cough and nasal discharge, airway mucus score, bronchoalveolar lavage fluid (BALF) cytology and arterial blood gases. Results: The VAS score improved significantly in dexamethasone‐ but not placebo‐treated horses. In contrast, the clinician failed to differentiate between dexamethasone‐ and placebo‐treated animals based on clinical observations, BALF cytology or endoscopic mucus score. Respiratory rate (RR) and arterial oxygen pressure (PaO₂) improved with dexamethasone but not placebo. Conclusions and clinical relevance: In the design of clinical trials of airway disease treatments, more emphasis should be placed on owner‐assessed VAS than on clinical, cytological and endoscopic observations made during brief examinations by a veterinarian. Quantifiable indicators reflecting lung function such as RR and PaO₂ provide a good assessment of drug efficacy.     

Salmon poisoning disease in dogs on southern vancouver island

Salmon poisoning disease in dogs has previously been reported in North America only along the western coast of the U.S.A. This paper presents the findings from eight affected dogs recently diagnosed on Vancouver Island, Canada. The clinical signs shown by these dogs were lethargy, anorexia, pyrexia and lymph node enlargement. The causative agent, Neorickettsia helminthoeca was observed in macrophages obtained from lymph node aspirates. This organism is transmitted to dogs in cysts of the fluke Nanophyetus salmincola salmincola within the tissues of the salmon or trout. The presence of fluke eggs in the feces of dogs showing typical signs is very suggestive of a diagnosis of salmon poisoning disease. Appropriate treatment, including chloramphenicol or oxytetracycline and fluid therapy, resulted in recovery. Prevention of salmon poisoning disease in endemic areas can be achieved by advising owners against allowing their dogs to eat raw salmon or trout. We suggest, based on the diagnoses made in these eight dogs, that Vancouver Island now be considered an endemic area for salmon poisoning disease.     

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.     

Detection of a novel clone of Acinetobacter baumannii isolated from a dog with otitis externa

In this study, the isolation of Acinetobacter baumannii in a dog with clinical bilateral otitis externa is described. Moreover, to investigate the zoonotic potential of the isolate, microbiological examinations on the family members were performed. An A. baumanniistrain was isolated from nasal swab in one of the dog owners. The identity of bacterial strains, either from dog and owner, was confirmed by phenotypic and molecular typing (wgMLST). Furthermore, to assess the pathogenic potential of the isolates a deep characterization of virulence and antibiotic resistance genes was done by Whole Genome Sequencing (WGS). Finally, the susceptibility towards a wide panel of antimicrobials was investigated. In our knowledge, this is the first recorded case of A. baumanniiisolation from canine auricular swabs in Italy. And interestingly, this study underlines the possible spread of this microorganism from human to animal.