Serological evidence of African pygmy hedgehog adenovirus 1 in exotic companion animals

Abstract

African pygmy hedgehog adenovirus 1 (AhAdV-1) was first identified in 2020 from a colony of African pygmy hedgehogs that succumbed to severe bronchopneumonia in Japan. AhAdV-1 is closely related to Skunk adenovirus 1, which was isolated from a wild skunk with acute hepatitis and pneumonia in Canada in 2015. Similar viruses have been isolated or detected in a diverse range of animals across multiple countries. While adenoviruses are generally considered highly species-specific, the host range of AhAdV-1 remains unclear. This study aimed to investigate the potential host range of AhAdV-1 through serological surveillance among 17 exotic animal species that visited a veterinary hospital in Fukuoka, Japan. Neutralizing antibodies against AhAdV-1 were detected in several species, with particularly high seroprevalence observed in meerkats (41%), ferrets (62%), and African pygmy hedgehogs (63%). Notably, ferrets and hedgehogs with a history of respiratory symptoms exhibited significantly higher seroprevalence compared to asymptomatic individuals. These findings suggest a broad host range for AhAdV-1, with certain species showing high exposure rates. Further investigations are needed to determine the sources and transmission routes of AhAdV-1, as well as its potential for zoonotic infection.

INTRODUCTION

Adenoviruses have been isolated or detected in a wide range of vertebrate species. Adenoviruses are non-enveloped, icosahedral viruses, measure 70–100 nm in diameter and possess a linear, double-stranded DNA (dsDNA) genome of 26–48 kbp, encoding approximately 40 proteins. Morphologically, adenoviruses are characterized by one or two fibers at each icosahedral vertex, which play a crucial role in host cell attachment and entry [12, 29].

Adenoviruses are classified into five genera—Mastadenovirus, Aviadenovirus, Atadenovirus, Siadenovirus, and Ichtadenovirus—based on the biological classification of their hosts. Mastadenoviruses, which infect mammals, are further divided into seven species (A to G) according to hemagglutination properties, DNA homology, oncogenic potential in rodents, transformability, and associated clinical disease manifestations [12, 18]. While most adenoviruses are considered non-pathogenic, they can cause disease in immunocompromised hosts [5, 19].

Although adenoviruses are generally regarded as species-specific [2, 9], certain adenoviruses exhibit broader host ranges. Notably, canine adenovirus type 1 (CAdV-1) has been isolated from a variety of carnivorous species, including members of Canidae [4, 10, 23, 24], Mustelidae [11, 27, 28], Ursidae [8], and Procyonidae [13]. Similarly, canine adenovirus type 2 (CAdV-2) has been reported in multiple carnivorous species [30]. Additionally, a study suggests that bat adenoviruses (BtAdVs) may possess a broad host range in experimental systems using cultured cells [15].

African pygmy hedgehog adenovirus 1 (AhAdV-1) was first detected in African pygmy hedgehog in Japan in 2016 [20] followed by big outbreak in breeding facility in 2019 [21, 26]. The affected hedgehogs, housed in colonies, exhibited respiratory symptoms such as nasal discharge, sniffing, sneezing, coughing, and dyspnea, with some cases resulting in severe disease and mortality. The virus was successfully isolated from nasal swabs of deceased animals using Madin-Darby Canine Kidney (MDCK) cells, and sequencing analysis confirmed it as a novel adenovirus. Whole-genome sequencing revealed 99.88% homology with skunk adenovirus 1 (SkAdV-1), previously isolated from a wild skunk that died of acute necrotizing hepatitis and interstitial pneumonia in Canada in 2015 [17]. We have also successfully detected and isolated AhAdV-1 from African pygmy hedgehog [16] and meerkats exhibiting respiratory symptoms (manuscript in preparation), respectively. To date, AhAdV-1 has been isolated or detected in a wide range of phylogenetically diverse species, including skunks [17], raccoons [3], Grey fox [25] and ferrets [27] (Carnivora), common marmosets [6, 7] (Primates), and porcupines [1] (Rodentia). This suggests that AhAdV-1 is unique among adenoviruses in its ability to infect multiple species across different taxonomic orders. However, most reports of AhAdV-1 infections are based on incidental isolations from animals presenting with respiratory symptoms, and large-scale epidemiological studies have yet to be conducted.

In this study, we conducted serological surveillance of AhAdV-1 among various exotic companion mammals to assess its host range and potential cross-species transmission.

MATERIALS AND METHODS

Cells

Dog-derived MDCK cells (JCRB No. JCRB1957) were cultured in Dulbecco’s Modified Eagle Medium (DMEM; GIBCO, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; SERANA^®, Pessin, Germany), 100 U/mL of penicillin, and 100 µg/mL of streptomycin (FUJIFILM Wako, Osaka, Japan). Cells were maintained at 37°C in a humidified atmosphere containing 5% CO₂.

Virus

African pygmy hedgehog adenovirus 1 (AhAdV-1), originally isolated from the lung cells of a domestic meerkat with necrotizing bronchopneumonia in 2021 (manuscript in preparation), was used in this study. The hexon gene sequence of this isolate was 100% identical to that of AhAdV-1 isolate HO-2018 (Ochiai et al., 2019; GenBank accession no. MK937781), previously isolated from hedgehogs exhibiting respiratory clinical signs in Japan. Canine adenovirus 1 (CAdV-1) strain Utrecht (ATCC^® No. VR-293™) and Canine adenovirus 2 (CAdV-2) strain Tronto A 26/61 (ATCC^® No. VR-800™) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). All viruses were propagated in MDCK cells cultured in DMEM supplemented with 2% FBS at 37°C in 5% CO₂ until cytopathic effects (CPE) were observed. Supernatants were harvested and stored at −80°C until use.

Serum samples

Serum samples were collected from a total of 446 domestic exotic mammals including 17 species, 103 African pygmy hedgehogs (41 males and 62 females), 78 chinchillas (46 males and 32 females), 73 ferrets (34 males and 39 females), 70 rabbits (33 males and 37 females), 58 Guinea pigs (25 males and 33 females), 19 degus (11 males and 8 females), 17 meerkats (13 males and 4 females), 10 ground squirrels (2 males and 8 females), 4 sugar gliders (4 males), 3 rats (2 males, 1 female), 3 micro pigs (3 males), 2 common marmosets (2 males), 2 goats (1 male, 1 female), 1 fennec fox (1 male), 1 raccoon dog (1 female), 1 chipmunk (1 female) and 1 prairie dog (1 female) at a veterinary hospital in Fukuoka, Japan, between January 2020 and January 2024. The veterinarians broadly categorized the respiratory symptoms into upper and lower respiratory tract signs. Upper respiratory signs were primarily diagnosed as rhinitis, characterized by evident nasal discharge and sneezing. Lower respiratory signs were defined by abnormal breathing patterns in the absence of cardiac disease, as ruled out by radiographic and ultrasonographic examinations. To avoid a small sample size in each subgroup, these conditions were collectively referred to as “respiratory symptoms” in the manuscript. All serum samples were stored at −20°C until use. Prior to testing, sera were inactivated by incubation at 56°C for 30 min.

Plaque assay for virus titration

Virus titers were determined using a plaque assay. Serially diluted virus suspensions were inoculated onto MDCK cell monolayers in 6-well plates (Sumitomo Bakelite Co., Ltd., Tokyo, Japan). After incubation at 37°C in 5% CO₂ for 90 min, cells were washed twice with DMEM and overlaid with 0.8% agarose (SeaPlaque^® agarose, Lonza, Basel, Switzerland) in DMEM containing 10% FBS. Plates were incubated at 37°C in 5% CO₂ for 4 days. Cells were then fixed with 5% buffered formaldehyde for 1 hr, after which the agarose overlay was removed. Plaques were visualized by staining with crystal violet and subsequently counted.

Virus neutralization (VN) test

To assess the prevalence of neutralizing (VN) antibodies against AhAdV-1 in various mammalian species, an 80% plaque reduction VN test was performed using the collected serum samples. For initial screening, sera were diluted 1:5 in DMEM containing 2% FBS. To determine the VN titer, sera were serially two-fold diluted in DMEM containing 2% FBS. Equal volumes of diluted serum or control medium were mixed with 80 plaque-forming units (PFU) of AhAdV-1 and incubated at 37°C for 90 min. The virus-serum mixtures were then added to MDCK cell monolayers in 24-well plates and incubated at 37°C for 90 min. Cells were subsequently washed twice with DMEM and overlaid with 0.8% agarose in DMEM supplemented with 10% FBS. Plates were incubated at 37°C in 5% CO₂ for 4 days. After fixation with 5% buffered formaldehyde for 1 hr, the agarose layers were removed, and plaques were visualized by crystal violet staining. Sera that reduced the number of plaques by more than 80% compared to the mean number of plaques in control wells were considered seropositive. The same procedure was applied for the VN test against CAdV-1 and CAdV-2.

Statistical analysis

Statistical analyses were performed using Fisher’s exact probability test and the Wilcoxon rank-sum test. For multiple comparisons, P-values were corrected using the Benjamini-Hochberg method. A P-value of <0.05 was considered statistically significant.

RESULTS

Seroprevalence of AhAdV-1 among various exotic mammals

Of the 446 animals tested, 127 were seropositive for anti-AhAdV-1 antibodies. Specifically, positive cases were detected in 7 out of 17 meerkats, 45 out of 73 ferrets, 1 out of 78 chinchillas, 2 out of 58 guinea pigs, 1 out of 19 degus, 1 out of 10 ground squirrels, 3 out of 70 rabbits, 2 out of 3 micro pigs, and 65 out of 103 African pygmy hedgehogs. In contrast, no antibodies were detected in fennec foxes, raccoon dogs, common marmosets, chipmunks, prairie dogs, rats, goats, or sugar gliders. Notably, meerkats (41%), ferrets (62%), and African pygmy hedgehogs (67%) exhibited significantly higher seropositivity rates than other species (P<0.05) (Table 1).

Table 1. Seroprevalence of African pygmy hedgehog adneovirus-1 among various household exotic mammals.

Order	Species	Number of	Number of	% of positive animals
		examined animals	positive animals
Carnivora	Ferret	73	45	62
	Meerkat	17	7	41
	Fennec fox	1	0	0
	Raccoon dog	1	0	0
Primates	Common marmoset	2	0	0
Rodentia	Chinchilla	78	1	1
	Guinea pig	58	2	3
	Degu	19	1	5
	Ground squirrel	10	1	10
	Chipmunk	1	0	0
	Prairie dog	1	0	0
	Rat	3	0	0
Lagomorpha	Rabbit	70	3	4
Artiodactyla	Micro pig	3	2	67
	Goat	2	0	0
Diprotodontia	Sugar glider	4	0	0
Erinaceomorpha	African pigmy hedgehog	103	65	63
Total		446	127	28

Further analysis of anti-AhAdV-1 antibody titers among seropositive individuals revealed significantly higher titers in meerkats, ferrets, and African pygmy hedgehogs compared to other species, consistent with their higher seropositivity rates (Fig. 1). The mean antibody titers for meerkats, ferrets, and African pygmy hedgehogs were 1:55.4, 1:144.2, and 1:49.9, respectively.

Fig. 1.

Boxplot showing the distribution of anti-African pygmy hedgehog adenovirus-1 (AhAdV-1) antibody titers among various mammalian species. The central line within each box represents the median, while the box boundaries indicate the interquartile range (IQR; 25th to 75th percentiles). “<0” denotes samples that tested negative for anti-AhAdV-1 antibodies in the virus neutralization (VN) test screening. The “+” symbol represents the mean antibody titer for each group. Negative samples in the VN test screening were assigned a value of −1 log₂ × 10 (corresponding to an antibody titer of 1:5) for calculation purposes.

Among ferrets and African pygmy hedgehogs, seroprevalence was significantly higher in individuals with a history of respiratory clinical signs than in those without clinical symptoms (P<0.05). However, sex was not associated with either seropositivity or antibody titers (Tables 2, 3).

Table 2. Seroprevalence of ferrets regarding their sex and the history of respiratory clinical sign.

	Sex		Respiratory clinical sign		Total
	Male	Female	No	Yes
Number of examined animals	34	39	55	18	73
Number of positive animals	23	22	30	15	45
% of positive animals	68	56	55	83	62

Table 3. Seroprevalence of African pygmy hedgehogs regarding their sex and the history of respiratory clinical sign.

	Sex		Respiratory clinical sign		Total
	Male	Female	No	Yes
Number of examined animals	41	62	85	18	103
Number of positive animals	27	38	49	16	65
% of positive animals	66	61	58	89	63

Assessment of cross-reactivity with canine adenovirus

To evaluate the specificity of the AhAdV-1 VN test, anti-CAdV-1 antibody titers were assessed in 14 ferrets and 3 meerkats with anti-AhAdV-1 titers exceeding 1:2,560 (Table 4). Among them, 12 ferrets and all 3 meerkats tested negative for anti-CAdV-1 antibodies, suggesting that the VN test specifically detected anti-AhAdV-1 antibodies. However, two ferrets (KI-18, KI-513) were seropositive for anti-CAdV-1 antibodies.

Table 4. Assessment of cross-reaction of anti-African pygmy hedgehog adenovirus-1 antibodies against canine adenovirus-1 and canine adenovirus-2.

ID	Animal species	Anti-AhAdV-1 antibody titer	Anti-CAdV-1 antibody titer	Anti-CAdV-2 antibody titer	Cohabit with dogs	Other mammals living together
KI-42	Meerkat	1:2,560	<1:10	<1:10	-	Sugar gliders (2)
KI-170	Ferret	1:2,560	<1:10	<1:10	-	Ferret
KI-447	Ferret	1:2,560	<1:10	<1:10	-	Ferret
KI-692	Ferret	1:2,560	<1:10	<1:10	-	-
KI-766	Ferret	1:2,560	<1:10	<1:10	-	Ferret
KI-1009	Ferret	1:2,560	<1:10	<1:10	-	Ferrets (many)
KI-401	Ferret	1:5,120	<1:10	<1:10	-	Ferret
KI-529	Ferret	1:5,120	<1:10	<1:10	-	Ferret
KI-1013	Ferret	1:5,120	<1:10	<1:10	-	-
KI-1052	Meerkat	1:5,120	<1:10	<1:10	○	-
KI-308	Ferret	1:10,240	<1:10	<1:10	-	Ferret
KI-600	Meerkat	1:10,240	<1:10	<1:10	-	Meerkats (5)
KI-772	Ferret	1:10,240	<1:10	<1:10	-	-
KI-512	Ferret	1:20,480	<1:10	<1:10	-	-
KI-894	Ferret	1:81,920	<1:10	<1:10	-	-
KI-18	Ferret	1:81,920	1:320	1:5,120	○	Rabbit
KI-513	Ferret	1:81,920	1:5,120	1:5,120	○	-

DISCUSSION

This study provides evidence of AhAdV-1 exposure in a variety of exotic companion mammals in Japan. Notably, high seroprevalence was observed in meerkats (41%), ferrets (62%), and African pygmy hedgehogs (63%), whereas low seroprevalence was detected in chinchillas (1%), guinea pigs (3%), rabbits (4%), and degus (5%). These findings strongly indicate that AhAdV-1 infection occurs frequently in meerkats, ferrets, and African pygmy hedgehogs in Japan. Interestingly, regarding to their feeding habits, seroprevalence of carnivore and omnivore species, such as hedgehog, ferret and meerkat are higher than herbivore species such as rabbit, chinchilla, guinea pig. To further elucidate the factors contributing to this species-specific seroprevalence, future studies should explore viral susceptibility at the molecular level, including receptor identification and interspecies differences in viral affinity.

Interestingly, two ferrets exhibited anti-CAdV-1 antibodies alongside high anti-AhAdV-1 antibody titers. Notably, these ferrets cohabited with dogs, suggesting potential exposure to CAdV-1 via a live-attenuated canine adenovirus vaccine. In Japan, the commonly used CAdV-1 vaccine is a live-attenuated CAdV-2 vaccine, and vaccinated dogs may shed the virus, potentially leading to transmission regardless of field strain infection [14]. Additionally, previous studies have reported CAdV-1 infections in mink and otters [11, 27, 28], as well as CAdV-2 infections in otters [22], all of which belong to the Mustelidae family. This suggests that ferrets, also members of the Mustelidae family, may be susceptible to these viruses. Further investigations are warranted to evaluate the potential cross-reactivity between anti-AhAdV-1 and anti-CAdV antibodies in order to refine the interpretation of our serological data.

AhAdV-1 has previously been detected in marmosets, hedgehogs, porcupines, raccoons, meerkats, ferrets, and skunks, often in association with respiratory symptoms and necrotizing hepatitis—considered the primary clinical manifestations of AhAdV-1 infection [1, 3, 7, 17, 20, 25, 26]. However, a previous epidemiological study detected AhAdV-1 genes in nasal swabs from asymptomatic hedgehogs kept as companion animals, suggesting the possibility of subclinical infection [16]. In the present study, seroprevalence was significantly higher in ferrets and hedgehogs with a history of respiratory clinical signs than in those without (P<0.05), suggesting a potential link between AhAdV-1 infection and respiratory disease in these species. Further studies are required to clarify the pathogenicity and mechanisms of disease development associated with AhAdV-1 infection.

Given that the studied animals were primarily kept indoors with minimal exposure to wildlife or external sources of AhAdV-1, direct contact with seropositive animals within the household is unlikely to be the primary route of transmission. This raises the possibility that AhAdV-1 infection may occur via alternative sources, such as infected animals from the same household, pet owners, breeding facilities, or commercial pet trade networks. The high seroprevalence observed in meerkats, ferrets, and hedgehogs—species primarily imported from Southeast Asia and North America—contrasts with the low seroprevalence in guinea pigs and degus, which are predominantly bred and distributed within Japan. This suggests that some animals may have acquired AhAdV-1 infection in endemic regions prior to importation.

In conclusion, our findings indicate that AhAdV-1 has a broad host range and suggest the potential for zoonotic transmission. Future studies are required to elucidate the infection dynamics, transmission pathways, and clinical impact of AhAdV-1 in both animals and humans.

CONFLICT OF INTEREST

The authors declare no conflict of interest.