In this study, an overall C. piliforme prevalence of 40% was recorded in the faeces of clinically normal rabbits. Very high prevalence rates have been reported in healthy mice (83.01%) and rats (90.9%) by PCR in a laboratory animal facility in Turkey.¹¹ In contrast, a study in three provinces of China detected C. piliforme antibodies in only 5.08% of clean-grade animals and 9.96% of specific pathogen-free animals using LAMP-LFD analysis.¹²

Fingerhood et al.¹³ detected C. piliforme by PCR in colon (5/10), liver (5/10), and heart (1/10) samples from pre-weaned orphaned kittens in the USA. Oliveira et al.¹⁴ detected C. piliforme by PCR in skin, liver, heart, intestines, and brain samples from a kitten that died after exhibiting nervous signs and diarrhoea. Similarly, Brooks et al.¹⁵ demonstrated the C. piliforme genome by PCR in liver samples of two farm-raised white-tailed deer fawns (Odocoileus virginianus), suggesting a probable association between copper toxicity and C. piliforme infection. Garcia et al.¹⁶ detected C. piliforme in all 24 liver samples (100%), 20 of 25 colon samples (83.33%), and 5 of 25 heart samples (20%) from foals suspected to have died of Tyzzer’s disease by PCR. These findings suggest that the sensitivity of PCR depends on the type of sample collected from infected animals.

Furukawa et al.⁶ detected C. piliforme DNA by PCR in 3 of 5 (60%) faecal samples collected from naturally infected rabbits. The authors explained that bile salts and bilirubin in faecal extracts reduced the sensitivity of PCR, and the detection limit of their assay was 10 bacteria per reaction.

Pritt et al.¹⁷ were unable to detect C. piliforme genomic DNA by PCR in caecum samples from 20 randomly selected seropositive rabbits without clinical signs. This indicates that serological tests cannot be considered confirmatory for the presence of C. piliforme in laboratory or commercial farm animals, as antigens used in these tests may cross-react with antibodies produced against non-pathogenic bacteria.¹⁸ Kirtland et al.¹⁹ evaluated the utility of plasma sorbitol dehydrogenase measurement for identifying subclinical or early Tyzzer’s disease in farm populations and potential carrier animals but concluded that it lacked sensitivity and specificity.

Disparity in the prevalence of C. piliforme reported in previous studies may be attributed to the health status of the animals (diseased vs. healthy) and the type of samples analyzed (affected organs vs. faeces). These studies also demonstrated that C. piliforme infections are often subclinical and cleared by immunocompetent hosts following seroconversion.²⁰

The significant prevalence difference observed between two farms: Mohammadabad (70%) and Chengalpattu (19.2%), even after Bonferroni correction, may be due to environmental and management-related differences such as farming system (backyard vs. organized) and feeding practices (wet mash vs. vegetables).

Garcia et al.¹⁶ reported that all but one foal suspected to have died of C. piliforme were younger than 45 days. Artukovic et al.²¹ also observed that weaned rabbits were more predisposed to C. piliforme infection than adult rabbits. Tyzzer’s disease was confirmed in a 45-day-old kitten and in 4 to 5-day-old white-tailed deer fawns by Oliveira et al.¹⁴ and Brooks et al.,¹⁵ respectively. Tyzzer’s disease primarily affects well-nourished young animals, particularly those on protein-rich diets.²² In contrast, in the present study, although not statistically significant, C. piliforme was detected by nPCR in 42% of faecal samples collected from rabbits older than 5 months, while only 25% of samples from rabbits younger than 5 months were positive. Similarly, Ulker et al.¹¹ reported a higher prevalence of C. piliforme in adult mice and rats compared with young animals. The differences in prevalence rates between age groups in various studies may be due to sample selection from healthy versus diseased animals.

Garcia et al.,¹⁶ Fingerhood et al.,¹³ and Brooks et al.¹⁵ studied foals, kittens, and deer fawns, respectively, that were suspected to have died of C. piliforme. In contrast, Ulker et al.¹¹ and the present study examined faecal samples collected from clinically normal animals. Brooks et al.¹⁵ suggested that C. piliforme infection in deer fawns was probably associated with liver damage caused by copper toxicity. Co-infections of C. piliforme with canine distemper virus were also reported in two domestic dog puppies and a grey fox kit.²³ Fingerhood et al.¹³ observed that 15 pre-weaned kittens that died of Tyzzer’s disease had co-morbidities such as sepsis, coccidiosis, and feline parvovirus (FPV). Oliveira et al.¹⁴ similarly reported co-infection with FPV in the ileum of a kitten that died of Tyzzer’s disease. In their studies, adult animals (deer and cats) were clinically healthy, suggesting that C. piliforme infection causes mortality in animals that are either co-infected or immunosuppressed. The high positivity rate in healthy adult rabbits in the present study suggests that they may act as carriers, as Tyzzer’s disease is generally transmitted through the faeco-oral route²⁴ and C. piliforme spores can survive in the environment for at least 5 years.²⁵

In the present study, breed, sex, weight, and type of rearing or feeding did not have a statistically significant association with C. piliforme infection in rabbits. It has been recorded that Tyzzer’s disease in animals is mainly predisposed by immunosuppression, stress, high environmental temperatures, overcrowding, poor sanitation, dietary changes, and the administration of sulfonamides or corticosteroids.^25,26

In this study, C. piliforme DNA was amplified as an 850 bp amplicon by nested PCR. A larger-sized band of around 1500 bp was also observed. A similar larger band was reported by Niepceron and Licois,⁷ who explained that this was probably due to the presence of outer primers from the first-round PCR. They attempted the second-round PCR with a higher annealing temperature to achieve a single 850 bp band without by-products, but this resulted in a tenfold reduction in sensitivity in caecal samples.

A few non-specific amplification products were also observed in the second-round PCR. Such non-specific amplicons have been reported in mouse, rat, and hamster faeces,⁶ in rabbit caecum,⁷ in deer fawn liver samples,¹⁵ and in DNA extracted from cell-culture–propagated C. piliforme isolates.¹⁸ This may be due to the use of 16S rRNA primers. Because faeces contain diverse bacteria, cross-reactions between primers and the 16S rRNA regions of closely related clostridial organisms in the gastrointestinal tract may generate erroneous products. Furukawa et al.⁶ noted that diagnosis becomes challenging if obscure bands appear at the specific region when using faecal samples. Thus, although detection of C. piliforme in faeces by PCR is simple, rapid, and non-invasive, the lack of sensitivity and specificity cautions against its use as a sole confirmatory test in live animals.

Primers targeting specific genes of C. piliforme could not be designed because no complete genome sequence is currently available. Confirmation of C. piliforme infection in suspected cases is therefore achieved not by a single test but by a combination of methods. Recent studies have used clinical signs, gross lesions, histopathological changes, detection of clostridia in tissues, immunohistochemistry, and 16S rRNA gene PCR for confirmation in dead animals.^13,14,23 However, no foolproof test exists for screening or diagnosis of C. piliforme in live animals, and only a combination of serological testing and PCR is recommended. Hence, the presence of C. piliforme in the present study should be further confirmed by sequencing the PCR products, and results should always be interpreted in correlation with serology and clinical signs/gross lesions.

Recently, Uprety et al.⁴ used shotgun metagenomics to obtain partial genome sequences of C. piliforme from the liver of a foal that died of Tyzzer’s disease. They identified partial sequences of virulence factor genes such as alveolysin, exo-α-sialidase, and those involved in flagellar and spore formation, thereby providing the first genetic evidence of virulence factors for C. piliforme. They also showed that flagellin protein sequences had low identity with those of C. colinum, the next closest phylogenetic species, indicating their potential utility in serological assays and vaccine development.

Immunocompromised humans are susceptible to C. piliforme infection, as demonstrated by a case detected in an HIV patient in the USA.³ A serological study detected antibodies against C. piliforme in 85.7% of laboratory animal handlers, 40.5% of personnel involved in laboratory animal care, and 22.0% of unrelated personnel.²⁷ The drastic decline of North American muskrats over the last 50 years may be attributed to Tyzzer’s disease,²⁸ and the susceptibility of a wide range of mammals to C. piliforme infection has been documented, especially in animals with co-morbidities.^13-15,23 These studies highlight the potential public health risk of C. piliforme infection for laboratory animal handlers and immunocompromised individuals.