Rabbit Brain Protozoa

Encephalitozoonosis, a zoonotic parasitic disease caused by Encephalitozoon cuniculi, primarily affects rabbits, guinea pigs, and mice. These animals typically harbor latent or chronic infections that remain asymptomatic until the host’s immune system weakens, at which point the infection can become life-threatening. As a result, it may interfere with the interpretation of experimental research outcomes and cause significant economic losses in the livestock industry.

Rabbit encephalitozoon belongs to the phylum Protozoa, class Microsporea, order Encephalitozoonida, family Encephalitozoonidae, and genus Encephalitozoon. The spores of different species of rabbit encephalitozoon vary in morphology, typically appearing oval or rod-shaped, with dimensions ranging from approximately 1.5 μm to 2.5 μm. Inside each spore are a nucleus and a few small vacuoles, while the spore wall is thick and features a polar body at one end. From this polar body, a polar filament emerges, spiraling along the inner wall before extending outward naturally. When stained with Giemsa stain, the spores turn blue; when treated with peroxidase, they exhibit a deep red color. They also test positive with Gram staining and show a weakly positive reaction with the PAS (Periodic Acid-Schiff) method. Specimens of the parasite must be observed under a phase-contrast microscope.

A defining feature of their infectivity is the presence of polar tubes—helical, filamentous, hollow tubes composed of proteins, which are embedded within their spores (Xu and Weiss, 2005). In 1995, Didier et al. (1995) characterized the existence of at least three distinct strains of E. cuniculi (I, II, and III), based on pronounced differences identified in the internal transcribed spacer (ITS) region of the ribosomal RNA gene. These strain types were eventually linked to the animal species from which they were originally isolated: strain I was designated as the "rabbit lineage," strain II as the "mouse lineage," strain III as the "dog lineage," and strain IV as the "human lineage" (Mathis et al., 2005; Tarabani et al., 2010). However, while each strain exhibits a preference for its primary host species, in practice, E. cuniculi has been shown to display relatively low host specificity. In humans, in addition to strain IV, the other three strains (I, II, and III) have also been detected, highlighting the pathogen's zoonotic potential (Sak et al., 2011a).

Tularemia, caused by *Francisella tularensis*, is widely distributed across the globe and has also been reported in China. Both infected rabbits and other susceptible animals serve as sources of infection. Transmission primarily occurs via the oral route, though inhalation or direct contact within shared environments can also lead to horizontal spread. Additionally, vertical transmission through the placenta is possible. After infection, the pathogen can be detected in multiple tissues within a short period. During the first month post-infection, it mainly accumulates in the kidneys, lungs, and liver; thereafter, it becomes more prevalent in the kidneys, brain, and heart. By three weeks post-infection, specific antibodies begin to appear in the serum, peaking around 60 days before gradually declining. Infectious spores are excreted from the body via the urinary and reproductive systems. Remarkably, the parasite can persist within its host for an extended duration—some strains even survive throughout the host’s lifetime. Although these spores do not grow or develop outside the host, they exhibit remarkable resilience against external environmental conditions. Notably, Kváč et al. (2016) were the first to report the presence of *F. tularensis* in milk, demonstrating that even after high-temperature short-time (HTST) pasteurization—specifically at 72°C for 15 seconds or 85°C for 5 seconds—the spores retained their infectious potential.

In 2015, a foreign study used serological (IFAT and ELISA) and molecular (PCR) assays to investigate the prevalence of the disease among different animal hosts across various provinces in Egypt. A total of 324 serum samples and 274 urine samples were collected from seven different animal species: cattle, water buffalo, sheep, goats, rabbits, dogs, and rats. Serological tests revealed that 38.9% of the animals tested positive for antibodies. Among the urine samples, only 5 (1.82%) were confirmed positive by PCR testing.

Most animals infected with Toxoplasma gondii do not show clinical symptoms. However, young dogs born with congenital infections often exhibit neurological signs, including depression, ataxia, blindness, and seizures—severe cases can even lead to death. Occasionally, rabbits may also develop neurological symptoms, typically as chronic or latent infections that become apparent under stress, resulting in progressive weakness, weight loss, and uremia. Individuals with compromised immune systems, such as AIDS patients, are particularly susceptible to Toxoplasma gondii. Moreover, Toxoplasma gondii and its metabolic byproducts significantly impair kidney function, leading to impaired reabsorption of water and sodium, which in turn causes polyuria. In advanced stages of the disease, these patients may also experience massive proteinuria.

The rabbit brain parasite primarily infects the brain and kidneys, leading to the formation of granulomas in brain tissue, non-purulent encephalitis, interstitial nephritis, and, in severe cases, interstitial myocarditis. Affected kidneys become enlarged, with hemorrhagic spots on their capsule, accompanied by irregular grayish-white depressions that extend deep into the cortical region. Under the microscope, extensive granulomas are observed in the renal cortex, each centered around the parasite and surrounded by a dense infiltrate of lymphocytes, plasma cells, and macrophages. Meanwhile, the interstitium shows abundant accumulations of lymphocytes and plasma cells. Minimal lesions can also be detected in the medulla, where many renal tubules exhibit dilation, and tubular epithelial cells often undergo necrosis and sloughing. In the brain, characteristic granulomatous lesions are evident, with parasites at their centers and surrounded by a mixed inflammatory response involving lymphocytes, plasma cells, microglial cells, and epithelial elements. Notably, some small blood vessels display macrophage infiltration, while others show prominent reactive gliosis around the lesion perimeter. Ocular involvement includes uveitis and cataracts.

Pathological histological examination involves inoculating samples into mice; after 2–3 weeks, ascites can be effectively detected. Alternatively, inoculating monolayer cells from the choroid plexus of rabbits allows detection within infected cells as early as 4 to 21 days post-inoculation.

Immunological testing reveals that specific antibodies appear within 3 to 4 weeks, preceding both pathological changes and the presence of parasites in urine. Newborn animals can acquire maternal antibodies, which persist for up to 4 weeks. Serological tests have been widely used to detect antibodies against the rabbit brain parasite, helping to confirm or rule out infection in suspected animals—and even identify asymptomatic carrier individuals.

Nucleic acid testing has detected the pathogen in multiple tissues and organs, with the brain considered the most suitable tissue due to its potentially highest spore concentration among infected individuals (Csokai et al., 2009; Repique et al., 2013). The pathogen can also be identified in urine, feces, and cerebrospinal fluid. According to Cox et al. (1979), during the acute phase of infection—up to day 63—large numbers of spores were excreted in the urine of infected rabbits. However, after this period, spore excretion became intermittent and occurred in much smaller quantities, eventually ceasing altogether around day 98 post-infection, which may lead to false-negative results. Additionally, aqueous humor from uveitis cases or liquefied lens samples can serve as diagnostic materials and are also capable of detecting the pathogen.

The parasitic site of *Encephalitozoon cuniculi* is a critical organ for toxicological and related studies. Infected animals often exhibit asymptomatic symptoms, yet their immune function remains compromised, potentially affecting experimental outcomes. Moreover, the granulomatous lesions induced by the parasite can obscure the evaluation of treatment efficacy. In severe cases, this may lead to renal failure or neurological symptoms. Therefore, infected animals are not suitable for experimental research.

Cryprosporidium parvum oocysts in rabbits exhibit a certain level of environmental resistance. Dried oocysts can survive for at least 4 weeks at 22°C, while in cool, moist conditions, they may persist for several months. Importantly, oocysts can be effectively eliminated through high-pressure sterilization, oxidative disinfection, and the use of bactericidal agents. Currently, the most effective preventive measures include strengthening livestock management practices, rigorously implementing disease-control protocols, maintaining excellent hygiene conditions, and promptly culling breeding animals that have already been infected. Establishing pathogen-free herds remains the most fundamental approach to preventing this disease. Given that Cryptosporidium infections are often subclinical, it is crucial to conduct thorough pathogen testing when introducing new animals to prevent the introduction of the disease. Regular health monitoring is also essential for early detection and control.

According to testing data from 2021–2022 provided by Suzhou Xishan Bio, a third-party testing institution in China, the positive rate for rabbit brain protozoa antibodies among rabbits was 20.6%. In the 2020 edition of the Pharmacopoeia, specifically in Part III titled "Specific Biological Raw Materials/Animals and Excipients," the section on "Quality Testing of Experimental Animals Used in Biopharmaceutical Production and Assays" lists laboratory mice, laboratory rabbits, and laboratory long-clawed gerbils as optional testing items under certain circumstances. Additionally, GB 14922-2022, the National Standard for Laboratory Animals—Microbiological and Parasitological Classification and Monitoring, explicitly designates laboratory rabbits as an essential testing item when required. Both the data and the requirements outlined in the Pharmacopoeia and national standards underscore the need for experimental animal institutions and professionals to pay closer attention to this pathogen.

References

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2. Gu Youfang, Shen Yonglin, Wang Zhikai. Coccidiosis in Rabbit Brains[J]. Animal Husbandry & Veterinary Medicine, 1997, 29(1):35-37

3. Laboratory Animals: Microbiology and Parasitology Standards and Monitoring—GB 14922-2022

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7. Magalhães, T.R., Pinto, F.F. & Queiroga, F.L. A multidisciplinary review on Encephalitozoon cuniculi from a One Health perspective. Parasitol Res 121, 2463–2479 (2022).