Zoo Animal Poxviruses: Veterinary Reference

Overview and Taxonomy of Zoo Animal Poxviruses: Veterinary Reference

The Poxviridae Family: A Taxonomic Framework for Zoo Animal Medicine

The family Poxviridae comprises a diverse assemblage of large, enveloped, double-stranded DNA viruses that are among the most structurally complex pathogens known to veterinary medicine. Within the context of zoological collections, these viruses present unique challenges owing to their broad host ranges, environmental persistence, and documented capacity for cross-species transmission events that can involve both exotic animal species and human caretakers. The taxonomic organization of poxviruses relevant to zoo animal medicine is anchored in two subfamilies: Chordopoxvirinae, which infects vertebrates, and Entomopoxvirinae, which infects insects. For the veterinary practitioner managing captive wildlife populations, the Chordopoxvirinae subfamily commands primary attention, particularly the genera Orthopoxvirus, Parapoxvirus, Capripoxvirus, Suipoxvirus, Avipoxvirus, and Molluscipoxvirus, each of which contains species that have been documented in zoo-housed animals or that pose significant zoonotic potential.

The genus Orthopoxvirus is arguably the most clinically consequential for zoo animal health, encompassing species such as Cowpox virus (CPXV), Monkeypox virus (MPXV), Vaccinia virus (VACV), Camelpox virus (CMLV), and Ectromelia virus (ECTV). The taxonomic boundaries within this genus have historically been challenging to delineate, as evidenced by molecular characterization studies of orthopoxvirus isolates from European zoonotic events. Source [9] describes two cases of cat-to-human orthopoxvirus transmission in northeastern Italy, wherein phylogenetic analysis of the hemagglutinin gene and the crmB gene revealed that the isolates formed a distinct cluster within the Orthopoxvirus genus, yet definitive species assignment proved elusive. The identity and similarity scores for the complete hemagglutinin nucleotide sequences from patient A, when compared to reference orthopoxviruses, ranged from 0.841 for Ectromelia virus to 0.940 for Camelpox virus, with Cowpox virus showing 0.927 similarity and Vaccinia virus 0.934 [9]. This genetic ambiguity underscores a critical reality for zoo veterinarians: the taxonomic classification of poxviruses encountered in captive wildlife may not always conform neatly to established species designations, particularly when dealing with emerging or geographically restricted viral lineages.

Epidemiological Context: Zoonotic and Anthropozoonotic Dynamics in Zoological Collections

The epidemiological significance of poxviruses in zoo settings is amplified by their documented capacity for bidirectional transmission between animals and humans. Source [5] provides a systematic review of health risks associated with zootherapeutic practices across Africa, explicitly identifying poxviruses among the pathogen groups of public health relevance that are transmitted through the use of animal products such as feces, fur, blood, and brain tissue. The review notes that certain practices involve species known to be reservoir hosts for poxviruses, including those within the Orthopoxvirus genus [5]. This finding is particularly salient for zoo veterinarians operating in or collaborating with institutions in regions where traditional medicine practices may intersect with captive wildlife management, as the potential for viral spillover from reservoir species to zoo animals, and subsequently to keepers or visitors, represents a One Health challenge that demands integrated surveillance strategies.

The cat-to-human orthopoxvirus transmission documented in Source [9] serves as a paradigmatic example of the zoonotic risks inherent in managing domestic and peridomestic species that may interface with zoo collections. In the Italian cases, two veterinary personnel developed cutaneous lesions, fever, and malaise after being scratched by cats with ulcerated skin lesions consistent with feline poxvirus infection [9]. The cats themselves likely acquired the virus from rodent reservoirs, a transmission pathway that mirrors the epizootiology of cowpoxvirus infections in European zoos, where elephants, felids, and other exotic mammals have been infected following contact with infected rats or other small mammals. The authors of Source [9] emphasize that the two cases, occurring more than a year apart in the same geographic region, indicate ongoing circulation of orthopoxvirus in domestic and wild fauna, underscoring the need for physicians and veterinarians to maintain clinical awareness of these zoonotic pathogens.

The potential for zoo animals to serve as sentinels for poxvirus activity in the broader environment is further supported by the detection of CTX-M-15-producing Escherichia coli in a bottlenose dolphin from a Portuguese zoo, as reported in Source [8]. While this study addresses antimicrobial resistance rather than poxvirus infection, the methodological approach, tracing the genetic relatedness of a zoo animal isolate to human clinical isolates using pulsed-field gel electrophoresis and multilocus sequence typing, provides a template for investigating poxvirus transmission linkages between captive wildlife and human populations. The dolphin isolate belonged to the pandemic ST131 clone and carried the blaCTX-M-15 gene, demonstrating that zoo animals can harbor clinically significant microbial lineages that are also circulating in human healthcare settings [8]. For poxviruses, analogous molecular epidemiological investigations would be invaluable for determining whether outbreaks in zoo collections originate from local wildlife reservoirs, infected human caretakers, or imported animal stock.

Taxonomic Challenges and Emerging Poxviruses in Zoo Animal Populations

The taxonomic complexity of poxviruses encountered in zoo animals is compounded by the phenomenon of host-range variants and the potential for recombination events that generate novel viral genotypes. Source [9] highlights that the orthopoxvirus isolates from Italy could not be assigned to a known species based on partial sequencing of the crmB gene and complete sequencing of the hemagglutinin gene, despite extensive comparison with reference sequences for Cowpox, Vaccinia, Camelpox, Monkeypox, Ectromelia, and Variola viruses. Preliminary sequence data on additional genes, including ATI, A27L, and CBP, supported the segregation of these Italian isolates from all known orthopoxvirus species, yet the available information was insufficient to determine whether they represented a novel species or a divergent strain of an existing one [9]. This taxonomic uncertainty has direct implications for veterinary reference work, as diagnostic assays, vaccine development, and biosecurity protocols are often species-specific. A zoo veterinarian confronted with a poxvirus-like disease in a felid or elephant must therefore consider the possibility that the etiological agent may not be captured by standard diagnostic panels designed for well-characterized orthopoxviruses.

The role of reservoir hosts in maintaining poxvirus diversity is critical to understanding the taxonomic landscape of these viruses in zoo environments. Source [5] notes that certain zootherapeutic practices involve species known to be reservoir hosts for poxviruses, including those that harbor filoviruses and coronaviruses, suggesting overlapping ecological niches that could facilitate co-infections and viral evolution. The systematic review identified that eastern Africa had significantly higher mean total risk scores for zoonotic pathogen spillover compared to western, central, and southern Africa, with physically sick children and pregnant or lactating women being at increased risk [5]. For zoo veterinarians, this geographic variation in risk underscores the importance of region-specific taxonomic knowledge: a poxvirus outbreak in a zoo in eastern Africa may involve viral lineages that are phylogenetically distinct from those circulating in European or North American collections, and the diagnostic reference materials available in commercial laboratories may not adequately represent these regional variants.

Diagnostic and Surveillance Considerations for Poxviruses in Zoo Settings

The accurate identification and taxonomic classification of poxviruses in zoo animals require a multi-modal diagnostic approach that integrates clinical pathology, molecular biology, and phylogenetic analysis. Source [6] provides a veterinary clinical pathology perspective on acute phase reactants (APRs) in nondomesticated mammals, emphasizing that serum amyloid A, haptoglobin, C-reactive protein, fibrinogen, albumin, and iron are the most commonly measured markers for identifying inflammatory disease, monitoring disease progression, and detecting preclinical or subclinical conditions. While APRs are not specific for poxvirus infection, they can serve as valuable screening tools in zoo animals that present with nonspecific signs such as lethargy, anorexia, or cutaneous lesions. The stepwise evaluation approach recommended by Source [6], beginning with assessment of analytical performance, followed by evaluation of overlap performance, clinical performance, and impact on patient outcomes, is directly applicable to the development of poxvirus diagnostic algorithms in zoo settings. The lack of species-specific standards and antibodies for nondomesticated mammals presents a particular challenge for APR interpretation, and Source [6] advises that more attention must be focused on assessing cross-reactivity and ensuring adequate analytical performance of assays before they are deployed for clinical decision-making.

Molecular diagnostic methods, including PCR amplification and sequencing of conserved poxvirus genes such as the DNA polymerase (E9L), hemagglutinin (HA), and crmB genes, are essential for definitive taxonomic assignment. Source [9] utilized both partial crmB sequencing and complete HA gene sequencing to characterize the Italian orthopoxvirus isolates, demonstrating the utility of these genetic targets for phylogenetic analysis. The use of whole-genome phylogenetic analysis, as exemplified in Source [3] for SARS-CoV-2 investigation in Malayan tigers at a Tennessee zoo, represents the gold standard for understanding transmission dynamics and evolutionary relationships. In that study, tiger sequences were compared with 30 geographically associated human cases and over 200 background sequences from Tennessee, revealing 3-6 single nucleotide polymorphism differences between one human tiger keeper and all three tiger sequences [3]. Although this investigation focused on coronavirus rather than poxvirus, the methodological framework, including environmental assessment, staff interviews, serial testing, and genomic sequencing, provides a template for poxvirus outbreak investigations in zoo settings.

The establishment of species-specific reference intervals for hematological and biochemical parameters, as described in Sources [1], [2], [4], and [7], is a prerequisite for interpreting clinical pathology data in zoo animals with suspected poxvirus infection. Source [1] established hematology and plasma biochemistry reference values for tree monitor lizards of the Hapturosaurus group, finding no significant differences between Varanus macraei, Varanus prasinus, and Varanus beccarii, which allowed the results to be combined for stronger statistical power. Similarly, Source [2] generated preliminary reference intervals for hematology variables in Iberian ribbed newts (Pleurodeles waltl), and Source [4] determined hematological reference values for tucúquere owls (Bubo magellanicus) in central Chile. These reference intervals, generated according to American Society for Veterinary Clinical Pathology (ASVCP) guidelines, enable veterinarians to identify abnormalities such as leukocytosis, thrombocytopenia, or anemia that may accompany poxvirus infection. Source [7] further demonstrated that seasonal variation, sex, and sex-season interactions can

Molecular Pathogenesis of Poxviruses in Zoo Animals

The molecular pathogenesis of poxviruses in zoo animals represents a complex interplay between viral virulence factors, host immune responses, and the unique physiological constraints of captive wildlife. Poxviruses, members of the family Poxviridae, are large, double-stranded DNA viruses that replicate exclusively within the cytoplasm of host cells, a feature that distinguishes them from most other DNA viruses and necessitates the encoding of their own DNA-dependent RNA polymerase and replication machinery. This cytoplasmic replication strategy has profound implications for viral pathogenesis, as it allows poxviruses to evade certain nuclear sensing pathways while simultaneously engaging in a sophisticated arms race with the host’s innate immune system. In the context of zoo animals, the molecular mechanisms underlying poxvirus infection are further complicated by species-specific variations in receptor usage, immune signaling cascades, and the presence of pre-existing or cross-reactive immunity from vaccination or prior exposure. Understanding these molecular events is critical for predicting host range, clinical outcomes, and zoonotic potential, particularly given the documented ability of poxviruses to cross species barriers in zoo settings.

Viral Entry and Cellular Tropism

The initial step in poxvirus pathogenesis involves viral attachment and entry into permissive host cells. Poxviruses, including orthopoxviruses such as cowpox virus (CPXV), monkeypox virus (MPXV), and vaccinia virus (VACV), utilize a multi-protein entry-fusion complex (EFC) that is conserved across the genus. This complex, composed of at least 11 viral proteins, mediates fusion with the plasma membrane or with endosomal membranes following macropinocytosis. The molecular determinants of host range are often linked to the ability of viral envelope proteins to bind to host cell surface glycosaminoglycans (GAGs) and extracellular matrix components. For instance, the viral proteins A27 and D8 bind to heparan sulfate and chondroitin sulfate, respectively, facilitating initial attachment. However, in zoo animals, the species-specific expression patterns of these GAGs can dramatically influence tropism. A Malayan tiger (Panthera tigris jacksoni) at a Tennessee zoo, for example, was found to be susceptible to SARS-CoV-2 infection [3], but the molecular basis for poxvirus susceptibility in felids is less well understood. Feline poxvirus infections, often caused by CPXV, have been documented in domestic cats and can serve as a source of zoonotic transmission to humans, as evidenced by cases in northeastern Italy where veterinary personnel were infected via scratches from cats with ulcerated skin lesions [9]. The partial sequence analysis of the crmB gene and hemagglutinin (HA) gene from these isolates revealed a distinct cluster within the orthopoxvirus genus, suggesting that the Italian isolates may represent a novel or divergent orthopoxvirus species [9]. This highlights the critical need for molecular characterization of poxvirus strains circulating in zoo animal populations, as sequence divergence in key entry proteins could alter host range and pathogenicity.

Once inside the cell, poxviruses undergo a tightly regulated replication cycle. The viral core is transported along microtubules to perinuclear sites where early gene transcription begins. The early genes encode proteins necessary for DNA replication, immune evasion, and intermediate transcription factors. The viral DNA polymerase, encoded by the E9L gene, is a target for antiviral drugs such as cidofovir, but its sequence conservation across poxviruses means that drug resistance can emerge through point mutations. In zoo animals, the use of such antivirals is often empirical, and the molecular basis for treatment failure is rarely investigated. The replication of viral DNA occurs in cytoplasmic factories, which are distinct from host cell nuclei and are surrounded by a membrane derived from the endoplasmic reticulum. This compartmentalization protects viral DNA from cytosolic DNA sensors such as cGAS (cyclic GMP-AMP synthase), which would otherwise trigger a potent type I interferon response. However, poxviruses have evolved multiple strategies to actively suppress this pathway, as discussed below.

Immune Evasion and Host Range Determinants

A hallmark of poxvirus pathogenesis is the extensive arsenal of immunomodulatory proteins encoded by the virus. These proteins are often non-essential for replication in cell culture but are critical for virulence in vivo. Poxviruses encode soluble decoy receptors for cytokines, chemokines, and interferons, as well as intracellular inhibitors of signaling pathways. For example, the CrmB gene, which was partially sequenced in the Italian feline poxvirus isolates [9], encodes a soluble tumor necrosis factor (TNF) receptor homolog. This protein binds to TNF-α and lymphotoxin-α, neutralizing their pro-inflammatory and apoptotic effects. The presence of a functional CrmB gene is a key virulence factor for CPXV and other orthopoxviruses, and its sequence variation can influence host range. In zoo animals, where TNF-α signaling may vary between species due to evolutionary divergence, the efficacy of this viral decoy receptor could be a determinant of disease severity. Similarly, poxviruses encode a viral interleukin-18 (IL-18) binding protein, which inhibits the induction of interferon-gamma (IFN-γ) and natural killer cell activation. The molecular interaction between viral IL-18BP and host IL-18 is species-specific; a viral protein that effectively neutralizes murine IL-18 may have reduced affinity for elephant or rhinoceros IL-18, potentially explaining why some poxvirus infections are subclinical in certain species but lethal in others.

The interferon signaling pathway is a primary target for poxvirus immune evasion. Poxviruses encode multiple proteins that inhibit the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway, including the viral IFN-γ receptor homolog (IFN-γR) and the viral double-stranded RNA (dsRNA) binding protein E3L. The E3L protein, which is essential for virulence, binds to dsRNA and prevents the activation of protein kinase R (PKR) and 2'-5'-oligoadenylate synthetase (OAS), thereby blocking the antiviral state. In zoo animals, the sequence of the host PKR gene can vary, and some species may have PKR variants that are less susceptible to E3L inhibition. This could explain the differential susceptibility of certain taxa to poxvirus infection. For instance, while CPXV is known to infect a wide range of mammals, including elephants, rodents, and felids, the molecular basis for the severe systemic disease observed in some zoo elephants versus the mild, localized lesions in others is not fully understood. A study of cat-to-human orthopoxvirus transmission in Italy [9] demonstrated that the virus could replicate efficiently in both feline and human cells, but the molecular determinants of this cross-species transmission likely involve the ability of viral proteins to interact with host factors in a species-independent manner. The hemagglutinin (HA) gene, which is involved in viral spread and is a target for serological diagnosis, showed high sequence identity between the Italian isolates and other orthopoxviruses (92.7% with cowpox, 93.4% with vaccinia) [9], but even small differences in the HA protein could affect receptor binding or fusion efficiency in different host species.

Systemic Dissemination and Pathology

Following local replication at the site of infection, poxviruses can disseminate via the lymphatic and hematogenous routes, leading to systemic disease. The molecular mechanisms of dissemination involve the production of extracellular enveloped virions (EEV), which are wrapped in an additional membrane derived from the trans-Golgi network. The EEV form is critical for long-range spread within the host and for evasion of antibody neutralization, as the outer membrane is more fragile and can be shed, revealing the intracellular mature virion (IMV) underneath. The viral proteins involved in EEV formation, such as B5R and F13L, are targets for neutralizing antibodies, and their sequence variation can influence the efficacy of the host antibody response. In zoo animals, the development of a robust antibody response is often delayed due to the immunosuppressive effects of stress, concurrent infections, or nutritional deficiencies. The acute phase response, as measured by serum amyloid A, haptoglobin, and C-reactive protein, is a valuable tool for monitoring inflammatory disease in nondomesticated mammals [6], but its utility in predicting poxvirus disease progression has not been systematically evaluated. The molecular pathogenesis of poxvirus-induced lesions involves direct viral cytopathic effects, including the formation of Guarnieri bodies (intracytoplasmic inclusion bodies) and the induction of apoptosis in infected cells. However, much of the tissue damage is immunopathological, resulting from the infiltration of neutrophils, macrophages, and cytotoxic T lymphocytes. The balance between viral immune evasion and host immune activation determines the extent of tissue necrosis, which is characteristic of poxvirus lesions.

The clinical presentation of poxvirus infection in zoo animals can range from mild, self-limiting skin lesions to fatal systemic disease. In the case of the Italian feline poxvirus outbreak [9], the infected cats presented with multiple cutaneous ulcerated lesions, and the virus was transmitted to humans via scratches. The molecular analysis of the crmB and HA genes indicated that the virus was closely related to cowpox virus but formed a distinct phylogenetic cluster [9]. This suggests that the virus may have adapted to a specific host or geographic region, with unique molecular determinants of pathogenicity. The ability of poxviruses to cause disease in a wide range of zoo animals, including elephants, rhinoceroses, and primates, underscores the importance of understanding the molecular basis of host range. For example, the elephantpox virus, which is closely related to cowpox virus, causes severe, often fatal disease in Asian and African elephants, with characteristic nodular skin lesions and systemic involvement. The molecular basis for this high virulence in elephants is unknown, but it may involve the inability of the elephant immune system to recognize or respond to specific viral immune evasion proteins. Similarly, outbreaks of monkeypox in zoo primates have been documented, with transmission from imported rodents to non-human primates and subsequently to humans. The molecular pathogenesis of monkeypox in non-human primates involves a similar repertoire of immune evasion genes, but the severity of disease is influenced by the host species, with New World monkeys often developing more severe disease than Old World monkeys.

Zoonotic Potential and One Health Implications

The molecular pathogenesis of poxviruses in zoo animals has direct implications for zoonotic transmission and public health. The documented transmission of orthopoxvirus from cats to veterinary personnel in Italy [9] and from rats to elephants to humans in Germany highlights the role of zoo animals as sentinels and amplifiers of poxvirus infection. The molecular characterization of these isolates is essential for understanding the evolutionary relationships between viruses circulating in different host species and geographic regions. The phylogenetic analysis of the Italian isolates, based on the complete HA gene sequence, placed them in a distinct cluster within the orthopoxvirus genus, separate from cowpox, vaccinia, and monkeypox [9]. This suggests that there may be a previously unrecognized orthopoxvirus species circulating in European wildlife, with the potential to cause disease in both animals and humans. The use of whole-genome sequencing and phylogenetic analysis, as demonstrated in the investigation of tiger-to-human transmission of SARS-CoV-2 [3], is a powerful tool for tracing the source and spread of zoonotic infections. In that study, genomic sequencing revealed that the tiger sequences were 3-6 single nucleotide polymorphisms (SNPs) different from the human tiger keeper, providing evidence of possible tiger-to-human transmission [3]. Similar approaches should be applied to poxvirus outbreaks in zoo animals to identify the molecular markers of host adaptation and virulence.

The risk of zoonotic poxvirus transmission from zoo animals is influenced by the molecular interactions between the virus and the human host. The ability of poxvirus immune evasion proteins to function across species barriers is a key determinant of zoonotic potential. For example, the viral TNF receptor homolog encoded by CrmB must bind to human TNF-α with sufficient affinity to neutralize its activity. Sequence analysis of the CrmB gene from the Italian isolates showed 93.3% identity with cowpox virus [9], suggesting that the protein is likely functional in humans. However, even minor amino acid substitutions could alter the binding affinity and affect the virulence of the virus in human hosts. The One Health approach, which recognizes the interconnectedness of human, animal, and environmental health, is essential for understanding and mitigating the risk of poxvirus zoonoses. The World Health Organization (WHO) and the World Organisation for Animal Health (WOAH) have emphasized the need for surveillance of poxviruses in wildlife and zoo animals, particularly in regions where traditional medicine practices involve the use of animal products that may harbor poxviruses [5]. A systematic review of zootherapeutic practices in Africa identified the use of species known to be reservoirs for poxviruses, highlighting the potential for spillover events [5]. The molecular detection and characterization of poxviruses in these contexts are critical for public health preparedness.

Molecular Diagnostics and Surveillance

The molecular diagnosis of poxvirus infection in zoo animals relies on PCR amplification of conserved viral genes, such as the DNA polymerase (E9L), the hemagglutinin (HA), and the crmB genes. The partial sequencing of the crmB and HA genes from the Italian isolates [9] allowed for phylogenetic classification and differentiation from other orthopoxviruses. However, the lack of species-specific standards and antibodies for nondomesticated mammals presents a challenge for serological diagnosis [6]. The use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification of fungal isolates in veterinary practice [10] demonstrates the potential for similar proteomic approaches to be applied to poxvirus identification. The development of comprehensive databases of viral protein spectra could enable rapid and accurate identification of poxviruses from clinical samples, even in the absence of species-specific reagents. Furthermore, the use of next-generation sequencing (NGS) for whole-genome characterization of poxvirus isolates from zoo animals is becoming increasingly feasible and affordable. The genomic data can provide insights into the molecular mechanisms of host adaptation, virulence, and transmission, as well as inform the development of vaccines and antiviral therapies. The investigation of the SARS-CoV-2 outbreak in tigers at a Tennessee zoo [3] demonstrated the power of genomic epidemiology in understanding cross-species transmission events, and similar approaches should be routinely applied to poxvirus outbreaks in zoo settings.

In conclusion, the molecular pathogenesis of poxviruses in zoo animals is a multifaceted process involving viral entry, replication, immune evasion, and dissemination. The species-specific interactions between viral proteins and host cellular factors determine the outcome of infection, ranging from subclinical to fatal disease. The documented zoonotic transmission of orthopoxviruses from zoo animals to humans underscores the importance of understanding these molecular mechanisms for public health. The use of advanced molecular techniques, including whole-genome sequencing and phylogenetic analysis, is essential for surveillance, diagnosis, and the development of control strategies. The integration of these molecular approaches with a One Health framework, as advocated by the WHO, WOAH, and the Centers for Disease Control and Prevention (CDC), will be critical for mitigating the risks posed by poxviruses in zoo animal populations and preventing future zoonotic spillover events.

Epidemiology and Transmission Dynamics in Zoological Collections

The epidemiology of poxviruses within zoological collections represents a uniquely complex intersection of wildlife virology, captive management practices, and anthropozoonotic interface dynamics. Unlike free-ranging populations, where transmission is governed by ecological density, seasonal breeding cycles, and natural barriers, the zoo environment creates an artificial, high-density, multispecies assemblage that fundamentally alters the transmission parameters of orthopoxviruses, parapoxviruses, and yatapoxviruses. Understanding these dynamics is not merely an academic exercise; it is a prerequisite for designing effective quarantine protocols, vaccination strategies, and outbreak containment measures that protect both animal collections and human personnel.

Reservoir Dynamics and the Role of Synanthropic Species

A foundational principle of poxvirus epidemiology in zoos is the critical role of synanthropic reservoir species, particularly rodents, which serve as the primary maintenance hosts for orthopoxviruses such as cowpox virus (CPXV) and monkeypox virus (MPXV). The zoo environment, with its abundant food sources, structural complexity, and climate-controlled buildings, often supports robust populations of commensal rodents (e.g., Rattus norvegicus, Mus musculus, and various vole species) that may be difficult to eradicate completely. These rodents typically exhibit subclinical or mild infections, yet they shed virus in high titers through respiratory secretions, urine, feces, and desquamated skin cells. The transmission chain from rodent to zoo mammal is well-documented; a seminal example involved CPXV transmission from a rat to an elephant in a German zoo, which subsequently led to human infection in a keeper [9]. This case illustrates the "bridge host" concept, where a non-reservoir species (the elephant) becomes an amplifier host, dramatically increasing the viral load in the environment and the probability of spillover to other species, including humans.

The risk posed by these reservoirs is compounded by the fact that many zoo exhibits are designed to mimic natural habitats, incorporating soil, mulch, logs, and water features that can harbor contaminated fomites for extended periods. Poxviruses are notoriously environmentally stable; CPXV, for instance, can remain infectious in dried scabs or contaminated bedding for months, particularly in cool, humid conditions. This environmental persistence creates a continuous source of exposure for naïve animals, even in the absence of active rodent ingress. Furthermore, the movement of animals between institutions, a routine practice for genetic management of endangered species, can inadvertently introduce novel poxvirus strains into collections that have no prior exposure history, leading to explosive outbreaks in immunologically naïve populations.

Intraspecific and Interspecific Transmission Pathways

Within a zoological collection, transmission occurs through multiple, often overlapping, routes. Direct contact transmission is the most efficient, occurring via bite wounds, scratches, or contact with exudative lesions. This is particularly relevant in social species housed in groups, such as felids, canids, and primates, where grooming, play, and agonistic interactions facilitate viral transfer. The Malayan tiger (Panthera tigris jacksoni) outbreak of SARS-CoV-2 at a Tennessee zoo, while not a poxvirus, provides a critical epidemiological parallel: the virus spread rapidly among all three tigers housed in the same enclosure, likely through direct contact and aerosolized respiratory droplets [3]. For poxviruses, the clinical presentation often includes severe, ulcerative dermatitis, which dramatically increases the infectiousness of affected animals. The high density of animals in zoo exhibits means that once a single individual becomes viremic and begins shedding, the basic reproductive number (R₀) can rapidly exceed 1, leading to an epidemic curve that peaks within days to weeks.

Aerosol and droplet transmission represent a second, and often underestimated, pathway. Many orthopoxviruses, including vaccinia virus (VACV) and MPXV, can be aerosolized from respiratory secretions, particularly in species that develop pneumonic involvement. The architectural design of many zoo buildings, with shared ventilation systems, open-air exhibits separated only by fencing, and keeper walkways that pass directly over enclosures, can facilitate airborne spread between taxonomically disparate species housed in different sections of the same building. The environmental assessment from the Tennessee tiger outbreak revealed that fencing between humans and animals allowed airflow, and an open outdoor exhibit observation point existed above the habitat, creating a plausible route for aerosol transmission from an infected visitor to the index tiger [3]. This finding has profound implications for poxvirus control, suggesting that physical barriers alone may be insufficient if airflow dynamics are not considered.

Fomite transmission is a third critical pathway, particularly in the context of veterinary and husbandry procedures. Shared equipment, nets, squeeze cages, feeding bowls, water hoses, and transport crates, can become heavily contaminated with viral particles. The role of human personnel as mechanical vectors cannot be overstated. Keepers and veterinarians moving between buildings, or even between different sections of the same building, can transfer virus on their clothing, boots, and hands. This is especially dangerous during the prodromal phase of infection, when animals may appear clinically normal but are already shedding virus. The use of operant conditioning and positive reinforcement training, while beneficial for welfare, can paradoxically increase transmission risk if animals are trained to present for examination or treatment while infectious, bringing them into close, unprotected contact with staff [11].

Zoonotic Spillover and the One Health Imperative

The zoonotic potential of poxviruses in zoos is a matter of grave concern, given the close and frequent contact between animal care staff and potentially infected animals. The cat-to-human orthopoxvirus transmission documented in northeastern Italy serves as a sentinel event, demonstrating that even in regions where poxvirus infections are considered rare, the virus circulates in domestic and peridomestic fauna and can spill over into humans who have occupational exposure [9]. In a zoo setting, the risk is magnified by the diversity of potential reservoir hosts and the intensity of exposure. Keepers, veterinarians, and veterinary technicians are at highest risk, particularly during procedures such as wound cleaning, lesion debridement, necropsy, or even routine physical examinations of animals with undiagnosed dermatological conditions.

The One Health approach, as exemplified by the CDC-led investigation of the Tennessee tiger outbreak, is essential for understanding and mitigating these risks [3]. This investigation employed whole-genome phylogenetic analysis to trace the direction of transmission, revealing that the tiger sequences were 3-6 single nucleotide polymorphisms (SNPs) different from the sequence obtained from a positive tiger keeper. This genetic relatedness, combined with the temporal sequence of symptom onset (keepers developed symptoms after the tigers), provided strong evidence for tiger-to-human transmission. This finding has significant implications for occupational health protocols: it suggests that personal protective equipment (PPE) protocols must be rigorously enforced, that staff should be vaccinated against relevant poxviruses (e.g., smallpox vaccine for MPXV), and that any animal with vesicular or ulcerative skin lesions should be considered a potential source of zoonotic infection until proven otherwise.

The risk is not limited to direct animal contact. The environmental assessment from the Tennessee outbreak also highlighted the potential for transmission from an ill visitor at an exhibit observation point [3]. This introduces a "reverse zoonosis" (anthroponosis) pathway, where humans infect zoo animals, which then become amplifiers that can re-infect humans. This bidirectional transmission dynamic is a hallmark of poxvirus epidemiology in zoos and underscores the need for visitor health screening, particularly during outbreaks of human poxvirus diseases (e.g., MPXV in non-endemic countries). The WHO and CDC have both emphasized that zoos must have active surveillance programs that include both animal health monitoring and occupational health monitoring for staff, with clear protocols for reporting and testing febrile or rash illnesses.

Environmental and Management Factors Influencing Transmission

The transmission dynamics of poxviruses in zoos are profoundly influenced by management practices and environmental design. The concept of "biosecurity" in a zoo context extends far beyond the simple quarantine of new arrivals. It encompasses the design of ventilation systems, the zoning of keeper work areas, the disinfection protocols for shared equipment, and the management of pest populations. The presence of free-ranging birds, for example, can serve as mechanical vectors, carrying virus-contaminated material from one exhibit to another. Similarly, the use of natural substrates like soil and mulch can create a persistent environmental reservoir that is difficult to decontaminate.

Seasonal factors also play a role. In temperate climates, rodent populations often peak in the fall, leading to increased virus circulation in reservoir populations. Concurrently, the stress of winter housing, with reduced ventilation and increased animal density, can exacerbate transmission. The stress of transport, introduction to new social groups, or concurrent disease (e.g., canine distemper in felids or mycobacterial infections in primates) can cause latent poxvirus infections to recrudesce, leading to unexpected outbreaks [12]. The use of acute phase reactants (APRs) such as serum amyloid A and haptoglobin has been proposed as a tool for detecting subclinical or preclinical disease in zoo mammals, which could allow for earlier intervention before an outbreak becomes established [6]. However, the lack of species-specific standards and antibodies for many nondomesticated mammals remains a significant challenge [6].

The role of the skin microbiome in modulating susceptibility to poxvirus infection is an emerging area of research. The skin is the primary physical barrier and the first site of contact for many poxviruses. Studies have shown that the skin microbiome varies significantly across mammalian orders, with host taxonomic order being the most significant factor influencing community composition [13]. Disruption of the skin microbiome, through antibiotic therapy, poor hygiene, or environmental stress, could potentially increase susceptibility to poxvirus infection by altering the local immune environment. This is a largely unexplored area in zoo medicine, but it has significant implications for understanding why some individuals within a collection become infected while others remain resistant.

Implications for Surveillance and Control

The epidemiological complexity of poxviruses in zoos demands a multi-layered surveillance strategy. Passive surveillance, relying on the observation of clinical signs, is insufficient, as many infections are subclinical or present with non-specific signs such as lethargy or anorexia. Active surveillance, involving regular serological screening of sentinel species (e.g., rodents trapped within the zoo grounds) and periodic PCR testing of environmental samples (e.g., swabs from ventilation ducts, water sources, and high-touch surfaces), is necessary to detect viral circulation before clinical cases emerge. The use of MALDI-TOF mass spectrometry for the rapid identification of fungal and bacterial co-infections that may complicate poxvirus lesions is also a valuable diagnostic adjunct [10].

When an outbreak does occur, the response must be rapid and decisive. Immediate isolation of affected animals, enhanced PPE for all staff, and closure of the affected area to visitors are essential first steps. The use of ring vaccination, with vaccinia-based vaccines, may be considered for at-risk species, although the safety of these vaccines in many exotic species has not been established. The development of recombinant vaccines, which are safer for use in wildlife, is a priority for the future [12]. The investigation must also include a thorough epidemiological investigation to identify the source of the outbreak, using tools such as whole-genome sequencing to trace transmission chains, as was done in the Tennessee tiger outbreak [3]. This approach, combined with a rigorous One Health framework that integrates human, animal, and environmental health data, offers the best chance of controlling poxvirus outbreaks in zoological collections and preventing the devastating consequences of these infections for both animal welfare and public health.

Clinical Manifestations and Pathological Findings in Susceptible Species

The clinical and pathological landscape of zoo animal poxvirus infections represents a formidable diagnostic challenge, characterized by extraordinary variability across taxonomic orders, viral strains, and husbandry contexts. Poxviruses, members of the family Poxviridae, subfamily Chordopoxvirinae, exhibit a remarkable capacity to breach species barriers within the controlled yet epidemiologically interconnected zoo environment. Understanding the nuanced manifestations and tissue-level alterations in susceptible zoo species is not merely an academic exercise; it is fundamental to early detection, containment, and the preservation of genetically valuable animal populations. The zoo setting, as highlighted by the behavioral and welfare frameworks described by Fernandez and Martin [11], creates unique interfaces for pathogen transmission that must be understood through the lens of both clinical medicine and population health management.

### Poxvirus Pathogenesis in Non-Human Primates and Proboscideans

Non-human primates (NHPs) and proboscideans are among the most clinically relevant and vulnerable taxa for poxvirus infections in zoological collections. The pathogenesis of monkeypox virus (MPXV) and cowpox virus (CPXV) in these species illustrates a spectrum ranging from subclinical seroconversion to fulminant, fatal disease. Following aerosol or direct contact exposure, the virus undergoes primary replication at the inoculation site, typically in the respiratory epithelium or through cutaneous abrasions. The incubation period, which generally spans 7 to 14 days, is followed by a prodromal phase characterized by lethargy, anorexia, and pyrexia, clinical signs that are notoriously nonspecific in zoo animals and easily attributed to stress, transport, or environmental change.

In great apes, particularly gorillas, chimpanzees, and orangutans, the progression to the eruptive phase is dramatic. A maculopapular rash emerges, often first noted on the face, palms of the hands, and soles of the feet, rapidly progressing through vesicular, pustular, and crusting stages over a 10- to 14-day period. The lesions, which can number in the hundreds, are umbilicated and firm upon palpation. In severe cases, confluence of pustules leads to extensive epidermal necrosis, secondary bacterial infection, and septicemia. The systemic inflammatory response, as measured through acute phase reactants like serum amyloid A (SAA) and haptoglobin, becomes markedly elevated, reflecting the profound cytokine dysregulation that characterizes orthopoxvirus disease [6]. The work of Hooijberg and Cray on acute phase reactants in nondomesticated mammals underscores the diagnostic utility of monitoring SAA and C-reactive protein in these species, as these biomarkers can rise days before overt clinical signs manifest, enabling earlier intervention [6].

Pathological examination of affected NHPs reveals a characteristic triad of findings: diffuse interstitial pneumonia with pulmonary edema, multifocal hepatic necrosis, and extensive lymphoid depletion within splenic and lymph node architecture. The pulmonary lesions, often the direct cause of mortality, exhibit marked alveolar septal thickening, type II pneumocyte hyperplasia, and the presence of intracytoplasmic eosinophilic inclusion bodies (Guarnieri bodies) within epithelial cells. These inclusions, pathognomonic for orthopoxvirus infection, are detected via hematoxylin and eosin staining and can be confirmed through immunohistochemistry for viral antigens. The gastrointestinal tract frequently shows erosive and ulcerative lesions throughout the oral mucosa, esophagus, and large intestine, contributing to dehydration and secondary malnutrition.

Proboscideans, Asian elephants (Elephas maximus) and African elephants (Loxodonta africana), exhibit a distinct clinical syndrome when infected with elephantpox virus or, in rare documented cases, cowpox virus transmitted from rodents. The transmission of cowpox virus from rat to elephant to human, as described in the German zoo outbreak referenced by the Italian orthopoxvirus investigators, illustrates the complex multi-host dynamics at play [9]. In elephants, the disease manifests as a severe, generalized exanthem with a predilection for the trunk, oral cavity, and perineal region. Vesicles and pustules form rapidly, often coalescing into large bullae that rupture, leaving painful, hemorrhagic erosions. The sheer mass of affected skin surface area predisposes these animals to fatal fluid and electrolyte imbalances, compounded by anorexia from oral pain. Systemic signs include profound lethargy, reluctance to move, and a characteristic drooping of the trunk.

Pathologically, the most striking finding in elephants is extensive epidermal necrosis with secondary bacterial colonization. The dermis shows severe edema, vascular thrombosis, and a mixed inflammatory infiltrate composed of neutrophils and macrophages. Inclusion bodies are readily identifiable within keratinocytes and endothelial cells. The lymph nodes are markedly enlarged and hemorrhagic, reflecting acute lymphoid necrosis. Pulmonary involvement, while less prominent than in NHPs, can manifest as interstitial pneumonia. The work of Manageiro et al. on CTX-M-15-producing Escherichia coli in a zoo dolphin serves as a critical reminder of the secondary bacterial infections that complicate poxvirus disease in immunocompromised or stressed zoo animals, with antimicrobial resistance posing an additional therapeutic hurdle [8].

### Avian and Reptile Poxvirus Syndromes

Avian poxviruses, belonging to the genus Avipoxvirus, are among the most common viral infections encountered in zoo bird collections worldwide. The clinical manifestations vary dramatically by species, viral strain, and route of infection, but two principal forms are recognized: cutaneous and diphtheritic. The cutaneous form, transmitted by arthropod vectors or through direct contact, presents as proliferative, nodular lesions on the unfeathered skin, including the legs, feet, beak, and around the eyes. These lesions, which begin as small papules and progress to wart-like growths, can become so extensive that they impair vision, prehension of food, and locomotion. In heavy infestations, particularly in passerines and psittacines, the lesions may become secondarily infected with bacteria or fungi, leading to septic arthritis and osteomyelitis. The identification of fungal isolates through MALDI-TOF mass spectrometry, as validated by Becker et al., offers a rapid and reliable method for characterizing these secondary invaders, which is essential for appropriate antimicrobial therapy [10].

The diphtheritic form, which carries a significantly higher mortality rate, involves the mucous membranes of the upper respiratory tract and oral cavity. Fibrinous, necrotic plaques form on the tongue, glottis, trachea, and esophagus, obstructing the airway and preventing feeding. Affected birds present with open-mouth breathing, dyspnea, audible respiratory sounds, and regurgitation. On postmortem examination, the tracheal lumen may be completely occluded by a caseous plug composed of fibrin, necrotic epithelium, and inflammatory cells. Histologically, these lesions exhibit ballooning degeneration of epithelial cells, intracytoplasmic eosinophilic inclusion bodies (Bollinger bodies), and a granulomatous inflammatory response. The liver and spleen may show focal necrosis, though systemic involvement is less common than in mammalian poxviruses.

In raptors, particularly owls, the clinical course can be more insidious. Hematological reference values for the tucúquere (Bubo magellanicus) established by Jimenez-Cortes et al. provide a critical baseline for identifying the leukocytosis, lymphopenia, and heterophilia that accompany systemic avipoxvirus infection [4]. These parameters, combined with plasma biochemistry and the assessment of acute phase reactants, enable clinicians to monitor disease progression and response to supportive care. The establishment of species-specific reference intervals, as emphasized by the American Society for Veterinary Clinical Pathology guidelines and exemplified by multiple studies in this compilation, is the cornerstone of accurate clinical interpretation in zoo species [1, 2, 4, 7, 16].

Reptile poxviruses, though less frequently documented, are emerging as significant pathogens in zoo herpetological collections. Affecting chelonians, crocodilians, and lizards, these viruses produce a chronic, debilitating syndrome characterized by multifocal, raised, caseous nodules on the skin and within the oral cavity. In green tree monitors (Varanus prasinus), black tree monitors (Varanus beccarii), and blue tree monitors (Varanus macraei), infection can precipitate severe anorexia, weight loss, and secondary infections. The hematological and plasma biochemistry reference values for these species, established by Sobrino-Yacobi et al., are invaluable for detecting the anemia, leukocytosis, and hypoalbuminemia that signal chronic inflammatory disease [1]. In tortoises and turtles, poxvirus infection manifests as a necrotizing dermatitis, often mistaken for shell rot. Histologically, these lesions show hyperkeratosis, acanthosis, and the presence of large, basophilic intracytoplasmic inclusion bodies within dermal fibroblasts. The virus may also target the respiratory epithelium, leading to caseous rhinitis and pneumonia.

### Carnivores, Perissodactyls, and the Spectrum of Disease in Artiodactyls

Felids, both large and small, are exquisitely sensitive to orthopoxvirus infections, particularly cowpox virus. The clinical syndrome in cheetahs, lions, tigers, leopards, and domestic cats begins with a localized, ulcerative lesion at the site of inoculation, often a scratch or bite wound from a rodent. Fever, depression, and anorexia ensue within 3 to 5 days. A generalized vesiculopustular rash develops, spreading from the face and paws to the entire body. In severe cases, particularly in immunologically naïve zoo felids, the disease progresses to severe pneumonia, with dyspnea, tachypnea, and hypoxemia. The cat-to-human orthopoxvirus transmission documented in northeastern Italy highlights the profound zoonotic risk associated with feline cases; veterinary personnel handling affected cats developed characteristic lesions on the hands, accompanied by fever and malaise [9]. The hemagglutinin gene sequences from these isolates clustered distinctly within the orthopoxvirus genus, underscoring the potential for novel or emergent strains to circulate within zoo-associated vector populations [9].

Pathologically, feline poxvirus infection is characterized by multifocal, coalescing areas of pulmonary consolidation, with histologic evidence of necrotizing bronchitis and bronchiolitis. The liver and spleen exhibit scattered foci of necrosis. Epidermal lesions show full-thickness necrosis with ballooning degeneration and intracytoplasmic inclusion bodies. In the Malayan tiger SARS-CoV-2 outbreak described by Grome et al., the potential for tiger-to-human transmission was documented through genomic sequencing, a finding that has profound implications for orthopoxvirus management in big cat facilities [3]. The environmental assessment revealing fencing that allowed airflow between exhibits and humans underscores the importance of facility design in zoonotic disease prevention. These architectural considerations, combined with the environmental enrichment and behavioral training frameworks outlined by Fernandez and Martin, must be integrated into comprehensive biosecurity plans [11].

Canids, including wolves, African wild dogs, and zoo-housed domestic dogs, are also susceptible to poxvirus infection, though the clinical picture is often less severe than in felids. The primary lesion is a solitary, ulcerated nodule, often on the face or limbs, which may be accompanied by mild fever and regional lymphadenopathy. Disseminated disease is rare but can occur in young or immunocompromised animals. The differential diagnosis must include canine distemper, which produces similar respiratory and gastrointestinal signs and, as noted by Leisewitz et al., is a common and frequently fatal disease in South African wildlife [12]. The neurological manifestations of distemper, including seizures and myoclonus, help differentiate it from poxvirus infection, though co-infections are possible.

In perissodactyls, particularly rhinoceroses, poxvirus infections are poorly documented but warrant vigilance. The reference intervals for ionized calcium and other biochemical parameters in southern white rhinoceroses (Ceratotherium simum simum) established by Trivedi et al. are essential for monitoring the systemic effects of any infectious process [16]. Artiodactyls, including giraffes, okapis, and antelope species, may develop poxvirus lesions resembling contagious ecthyma (orf virus), characterized by proliferative, scabby lesions on the lips, muzzle, and around the eyes. In severe cases, lesions may extend into the oral cavity, causing salivation and anorexia. Young animals are particularly susceptible, and mortality can approach 20% in naïve herds.

### Pathological Mechanisms and Diagnostic Challenges

The hallmark pathological feature of poxvirus infection across all susceptible species is the Guarnieri body, an eosinophilic, intracytoplasmic inclusion body representing viral replication factories within the host cell. These inclusions are most readily identified in epithelial cells of the skin, mucous membranes, and respiratory tract. The process of cell-to-cell spread via actin-based motility, combined with the release of extracellular enveloped virions, allows the virus to evade immune surveillance and disseminate systemically. The resulting tissue damage is mediated by both direct viral cytopathic effects and the host inflammatory response. Acute phase reactants, including SAA, haptoglobin, and fibrinogen, surge during the acute phase, and their measurement, as advocated by Hooijberg and Cray, provides a quantitative tool for assessing disease severity and monitoring therapeutic response [6].

The diagnostic challenge in zoo animals is compounded by the breadth of differential diagnoses, including bacterial pyoderma, mycotic dermatitis, herpesvirus infections, and papillomavirus lesions. The use of molecular diagnostics, including PCR and sequencing, is essential for definitive diagnosis. The successful application of rpoB gene sequencing for mycobacterial identification by Higgins et al. demonstrates the power of sequence-based diagnostics in a zoo setting and serves as a model for poxvirus diagnostics [15]. Similarly, the use of symmetric dimethylarginine as an early biomarker for kidney disease, as investigated in striped skunks by Jung et al., highlights the need for species-specific diagnostic markers that can detect end-organ damage before it becomes irreversible [14]. In animals that succumb to disease, full necropsy with histopathology, electron microscopy, and viral isolation is critical for confirming the diagnosis and identifying novel or emerging strains. The phylogenetic analysis of orthopoxvirus isolates, as demonstrated by the Italian investigators, enables the tracking of viral evolution and the identification of transmission chains within and between facilities [9].

A comprehensive understanding of clinical manifestations and pathological findings, grounded in robust reference data and advanced diagnostic techniques, is the foundation upon which effective prevention and control strategies for zoo animal poxviruses are built. The integration of clinical pathology, histopathology, molecular epidemiology, and a One Health approach, as exemplified by the tiger SARS-CoV-2 investigation [3], is essential for safeguarding the health of vulnerable zoo populations and protecting the humans who care for them.

Diagnostic Approaches: Molecular, Serological, and Histopathological Methods

The diagnosis of poxvirus infections in zoo animals presents a formidable challenge, demanding a multi-pronged diagnostic strategy that integrates molecular, serological, and histopathological modalities. The inherent diversity of poxvirus genera, from the Orthopoxvirus genus, which includes cowpox virus (CPXV) and monkeypox virus (MPXV), to the Capripoxvirus, Avipoxvirus, and Parapoxvirus genera, necessitates a diagnostic framework that is both broad in its capacity for initial detection and exquisitely specific for definitive characterization. The clinical context of a zoo environment, which often involves valuable, endangered, or behaviorally intractable animals, further complicates diagnostic efforts, often requiring non-invasive or minimally invasive sampling strategies combined with rapid, point-of-care testing. A failure to deploy a rigorous diagnostic algorithm risks delayed intervention, nosocomial spread within a collection, and, critically, unrecognized zoonotic transmission to keepers, veterinarians, or the visiting public [3, 9]. The World Organisation for Animal Health (WOAH) and the World Health Organization (WHO) both recognize the international significance of many poxviruses, reinforcing the need for standardized, validated diagnostic protocols in zoological institutions.

Molecular Diagnostics: The Gold Standard for Definitive Identification

Molecular techniques, particularly polymerase chain reaction (PCR) and its derivatives, have revolutionized the diagnosis of poxviral diseases, offering unparalleled sensitivity, specificity, and speed. For zoo animal poxviruses, the choice of genetic target is paramount. The hemagglutinin (HA) gene is a widely used target for orthopoxviruses due to its high degree of sequence variability, which allows for not only genus-level detection but also species-level differentiation. As demonstrated in the investigation of cat-to-human orthopoxvirus transmission in northeastern Italy, sequencing of the complete HA open reading frame (approximately 930 bp) allowed for the phylogenetic segregation of the isolated virus from other known orthopoxvirus species, including cowpox, vaccinia, and monkeypox [9]. This approach is critical for distinguishing between endemic zoonotic viruses (e.g., CPXV) and emerging or exotic threats (e.g., MPXV). Similarly, the crmB gene, encoding a cytokine response modifier, provides another target for phylogenetic analysis; partial sequencing of this gene in the Italian outbreak yielded identity scores of 0.933 with cowpox virus, confirming its position within the orthopoxvirus clade [9]. The selection of these specific, well-conserved yet variable genes enables the creation of robust phylogenetic trees that are essential for tracing the origin and transmission dynamics of an outbreak within a zoological collection. The application of whole-genome sequencing (WGS), as exemplified during the SARS-CoV-2 outbreak investigation in Malayan tigers at a Tennessee zoo, represents the apex of molecular resolution. While the cited investigation focused on a coronavirus, the methodological framework is directly transferable to poxvirus outbreaks. By sequencing the virus from all affected animals and comparing it to sequences from potentially infected humans (keepers and visitors), investigators were able to construct a phylogenetic tree showing single-nucleotide polymorphism (SNP) differences of only 3-6 between the tiger keeper and the tigers, providing strong evidence for tiger-to-human transmission [3]. For poxviruses, WGS can reveal recombination events, the acquisition of host-range genes, and the emergence of mutations that may confer resistance to antiviral compounds like tecovirimat. The application of gene expression assays, such as those developed for the diagnosis of Mycobacterium bovis in African lions, demonstrates how molecular diagnostics can be adapted to detect the host's response to infection rather than the pathogen itself [17]. While this specific example used quantitative reverse transcription PCR (RT-qPCR) to measure interferon-gamma (IFN-γ) and other gene transcripts from blood samples, a similar approach could theoretically be developed to detect poxvirus-specific immune signatures, offering a non-invasive diagnostic window in clinically suspect but PCR-negative animals.

Pan-poxvirus PCR assays, targeting conserved regions of the DNA polymerase (E9L) gene or the major core protein gene (A3L), are invaluable for initial screening. These assays can detect any member of the Chordopoxvirinae subfamily, providing a crucial first step in an outbreak investigation where the causative agent is unknown. When positive, the amplicon can be sequenced for genus and species identification. For definitive species assignment, sequencing of multiple genes (e.g., HA, crmB, and the ATI inclusion body protein gene) is often required. The Italian study highlighted that for their isolates, species assignment based on a single gene was not possible, underscoring the need for multi-locus sequence typing or WGS for accurate taxonomy [9]. The diagnostic workflow in a zoo setting must also consider sample quality. Vesicle swabs, scabs, and tissue biopsies are ideal for DNA extraction, but the quantity and quality of DNA can be severely compromised by formalin fixation and paraffin embedding (FFPE) of histopathological specimens. Despite this, validated PCR protocols exist for FFPE tissues, allowing for retrospective diagnosis when fresh tissue was not preserved. The use of internal amplification controls is mandatory to rule out PCR inhibition, which is particularly common in samples containing heme (from blood-contaminated swabs) or complex polysaccharides (from cutaneous scabs).

Serological Approaches: Detecting Past Exposure and Immune Status

Serological testing provides critical epidemiological data, revealing the history of infection within a zoo population and informing vaccination protocols. The primary challenge in serological diagnostics for zoo animal poxviruses is the lack of species-specific reagents. Most commercially available enzyme-linked immunosorbent assays (ELISAs) and virus neutralization tests (VNTs) are developed for human or domestic animal pathogens. Cross-reactivity between orthopoxviruses is extensive due to the presence of shared immunodominant antigens, such as the A27L and L1R proteins. This means that a positive serological result using a human-based vaccinia virus ELISA in a zoo elephant or lion indicates exposure to an orthopoxvirus, but it cannot definitively discriminate between a prior vaccinia vaccination, an inapparent cowpox infection, or a subclinical monkeypox infection. The need for thorough analytical validation of serological assays for non-domesticated mammals is paramount. As outlined in the context of acute phase reactants in zoo mammals, a stepwise approach to validation should include assessment of analytical performance (e.g., precision, linearity, and cross-reactivity), overlap performance (distinguishing healthy from diseased animals), and clinical performance (diagnostic sensitivity and specificity) [6]. For serology, this often involves demonstrating parallelism between the standard curve and serial dilutions of zoo animal sera, and confirming that the assay detects a specific immunoglobulin (e.g., IgG or IgM) from the target species.

Virus neutralization tests (VNTs) are the gold standard for serological diagnosis because they measure functional antibodies capable of preventing viral infection. A VNT involves incubating serial dilutions of heat-inactivated serum with a known titer of a reference poxvirus (often vaccinia virus or a specific zoo isolate) and then inoculating the mixture onto permissive cell cultures (e.g., Vero cells). The highest serum dilution that completely inhibits cytopathic effect (CPE) is the neutralizing titer. While VNTs are highly specific, they are labor-intensive, require live virus (posing a biosecurity risk), and take 3-5 days to complete. ELISAs, particularly those using whole-virus lysates or recombinant proteins, are more amenable to high-throughput screening. For example, an immunoglobulin M (IgM) capture ELISA can indicate recent or active infection, while an IgG ELISA indicates past exposure or vaccination. The application of these tests during the SARS-CoV-2 tiger outbreak demonstrated that despite clinical infection and confirmed viral shedding, all exposed staff were negative for anti-SARS-CoV-2 IgG, a finding that guided the conclusion that human infection was likely a recent event [3]. For poxviruses, IgM responses typically appear within 5-7 days of lesion onset and wane over several weeks, while IgG responses peak later and may persist for years. Paired serology (acute and convalescent sera collected 2-4 weeks apart) is the most powerful serological tool. A four-fold or greater rise in neutralizing antibody titer or IgG level between the acute and convalescent samples is considered diagnostic of recent infection.

Histopathological Examination and In Situ Detection

Histopathology remains a cornerstone of poxvirus diagnosis, providing immediate, low-cost evidence of viral cytopathic effect that can guide the selection of confirmatory molecular tests. The characteristic histopathological findings in poxviral lesions are well-described across a range of zoo species. Early lesions (macules and papules) show epidermal hyperplasia (acanthosis) and spongiosis. As the lesion progresses to a vesicle, there is marked ballooning degeneration of keratinocytes, leading to the formation of large, intra-cytoplasmic, eosinophilic inclusion bodies. These are the pathognomonic hallmark of poxvirus infection. In orthopoxvirus infections, the inclusion bodies are typically type B (or "Guarnieri bodies"), which are eosinophilic, granular, and often fill the entire cytoplasm, displacing the nucleus. These bodies are Feulgen-negative (indicating DNA is not the primary component) and are composed of viral proteins and cellular debris. In contrast, some poxviruses like Parapoxvirus and Molluscipoxvirus produce type A inclusion bodies, which are larger, more homogeneous, and are Feulgen-positive, containing mature virions embedded in a protein matrix. The presence of these inclusion bodies is highly suggestive but not pathognomonic; other viruses like herpesviruses can also cause cytoplasmic inclusions, though they are usually smaller and more basophilic. The differential diagnosis must be confirmed by molecular or immunohistochemical methods.

Immunohistochemistry (IHC) bridges the gap between morphology and molecular identification. Using polyclonal or monoclonal antibodies raised against a conserved poxvirus antigen (e.g., a vaccinia virus lysate), IHC can specifically label viral proteins within formalin-fixed, paraffin-embedded tissue sections. This technique is invaluable when fresh tissue is unavailable for PCR, or when the viral load is low and beyond the detection limit of standard histopathology. IHC can also be used to map the distribution of viral antigen within lesions, confirming the involvement of specific cell types (e.g., keratinocytes, endothelial cells, and macrophages). The major limitation of IHC is the requirement for species-specific or cross-reactive antibodies [6]. For many zoo animal poxviruses, antibodies validated for use in the species in question simply do not exist. Furthermore, the fixation process can mask or destroy certain epitopes, reducing sensitivity. The use of antigen retrieval techniques (e.g., heat-induced epitope retrieval) is essential to optimize IHC results.

Electron microscopy (EM) offers definitive, high-magnification visualization of poxvirus virions. Poxviruses are the largest known animal viruses, approximately 200-300 nm in diameter, and have a distinctive brick or ovoid shape with a complex surface structure. Negative staining of vesicle fluid or tissue homogenates allows for rapid (within hours) identification of the characteristic virion morphology. This technique was employed in the Italian orthopoxvirus outbreak to initially identify the agent [9]. EM does not require species-specific reagents and can provide a genus-level identification (e.g., distinguishing an orthopoxvirus from a herpesvirus) but cannot reliably differentiate between orthopoxvirus species. Its use in zoo diagnostics is largely restricted to specialist reference laboratories. Finally, in situ hybridization (ISH) using poxvirus-specific DNA or RNA probes offers another method for detecting viral nucleic acids within tissue sections, with the advantage over IHC of not being limited by antibody availability. Combined with histopathology, ISH can localize viral replication to specific cell types with high sensitivity and specificity. The integration of these histopathological and in situ techniques provides a spatial and temporal context for the molecular findings, creating a comprehensive diagnostic picture essential for managing poxvirus outbreaks in complex zoo environments.

Prevention, Biosecurity, and Vaccination Strategies in Zoo Settings

The prevention and control of poxvirus infections within zoological collections represent a formidable challenge that intersects virology, immunology, behavioral management, and architectural design. Unlike domestic animal agriculture, where standardized biosecurity protocols can be implemented across relatively uniform facilities, zoo settings present a kaleidoscope of species-specific susceptibilities, variable housing conditions, and complex human-animal interfaces that demand a bespoke, multi-layered approach. The fundamental objective is to prevent the introduction of poxviruses into naive collections, to contain outbreaks when they occur, and to mitigate the risk of zoonotic spillover to caretakers, veterinarians, and the visiting public. This requires a deep understanding of viral ecology, transmission dynamics, and the immunological peculiarities of diverse taxa, from marsupials to proboscideans.

Foundational Principles of Zoo Biosecurity for Poxviruses

Biosecurity in the context of zoo poxviruses must be conceptualized as a hierarchical defense system, where primary prevention targets the exclusion of viral pathogens from the facility, secondary prevention focuses on early detection and containment of incursions, and tertiary prevention aims to minimize morbidity and mortality within an affected population. The architectural and operational design of modern zoos must account for the fact that poxviruses, particularly orthopoxviruses like cowpox virus and monkeypox virus, possess a broad host range and can persist in the environment for extended periods within dried scabs or contaminated fomites. The SARS-CoV-2 outbreak in Malayan tigers at a Tennessee zoo, where genomic sequencing suggested transmission from an ill visitor at an open exhibit observation point, serves as a stark reminder that human-to-animal transmission pathways are not merely theoretical but represent a genuine and documented risk [3]. This incident underscores the necessity of physical barriers that prevent aerosol or droplet transmission between public viewing areas and animal habitats, a principle equally applicable to poxvirus prevention given the respiratory and contact transmission routes of many poxviruses.

The design of quarantine facilities for incoming animals is arguably the single most critical structural component of a poxvirus biosecurity plan. New arrivals, whether from other zoological institutions, rescue centers, or wild-caught specimens, represent the highest risk pathway for pathogen introduction. Quarantine should be physically isolated from the main collection, ideally in a separate building with dedicated ventilation systems that prevent air recirculation to other animal areas. The duration of quarantine must be informed by the incubation period of relevant poxviruses, which for orthopoxviruses can range from 5 to 21 days, though subclinical shedding may extend this window. During this period, animals should undergo baseline health assessments, including the collection of blood for serological banking and, where indicated, PCR-based screening of oral or conjunctival swabs. The establishment of species-specific reference intervals for hematological and biochemical parameters, as has been done for tree monitor lizards [1], Iberian ribbed newts [2], tucúquere owls [4], and eastern water dragons [7], provides a critical baseline for detecting the inflammatory responses characteristic of acute poxvirus infection. Acute phase reactants such as serum amyloid A and haptoglobin, which have been validated as biomarkers of inflammation in nondomesticated mammals [6], could serve as valuable screening tools during quarantine, prompting more specific diagnostic testing even in the absence of overt clinical signs.

Zoonotic Risk Mitigation and Personal Protective Equipment

The zoonotic potential of poxviruses in zoo settings cannot be overstated. Documented cases of cat-to-human orthopoxvirus transmission in northeastern Italy, where veterinary personnel developed lesions after being scratched by infected felines [9], illustrate the occupational hazard faced by animal care staff. Similarly, the potential for tiger-to-human transmission of SARS-CoV-2, as suggested by phylogenetic analysis showing 3-6 single nucleotide polymorphisms between tiger and human keeper sequences [3], highlights the bidirectional nature of zoonotic risks. For poxviruses, the risk is amplified by the fact that many species, including rodents, felids, and non-human primates, can serve as asymptomatic or mildly symptomatic reservoirs, shedding virus through respiratory secretions, skin lesions, or fecal material. The use of zootherapeutic practices in some regions, where animal products such as blood, brain tissue, or feces are used in traditional medicine, has been identified as a significant risk factor for poxvirus spillover, with certain practices involving species known to be reservoirs for poxviruses [5].

Consequently, a comprehensive personal protective equipment (PPE) protocol must be developed and rigorously enforced for all personnel with direct animal contact. This should include, at minimum, gloves, fluid-resistant gowns, and eye protection when handling animals with suspected or confirmed poxvirus infection. For high-risk procedures such as necropsy of suspected cases or handling of contaminated bedding, N95 respirators or higher-level respiratory protection is warranted, given the potential for aerosolization of viral particles. Hand hygiene stations should be strategically placed at all entry and exit points of animal areas, and staff should be trained in proper donning and doffing sequences to prevent self-contamination. The role of environmental contamination must also be addressed; poxviruses are enveloped viruses that are relatively susceptible to disinfectants, but they can survive in organic material. Effective disinfection protocols should employ EPA-registered disinfectants with demonstrated efficacy against orthopoxviruses, such as 0.5% sodium hypochlorite solution or quaternary ammonium compounds, with appropriate contact times. Regular environmental monitoring using validated techniques, such as MALDI-TOF mass spectrometry for fungal identification [10], could be adapted for viral surveillance in high-risk areas, though this remains an area for further development.

Vaccination Strategies: Balancing Efficacy and Safety Across Taxa

Vaccination against poxviruses in zoo animals presents a unique set of challenges that distinguish it from vaccination programs in domestic species or humans. The primary goal is to protect vulnerable species, particularly those that are endangered or have demonstrated high susceptibility to poxvirus-induced mortality. However, the use of live-attenuated vaccines, which have been instrumental in controlling poxvirus diseases in humans and domestic animals, carries significant risks in exotic species. The modified live virus (MLV) vaccines developed for canine distemper, for example, have been documented to cause mortality in some wild carnivores [12], a cautionary tale that applies directly to poxvirus vaccinology. The potential for vaccine virus to revert to virulence, cause disseminated disease in immunocompromised or naive hosts, or establish persistent infections in novel species necessitates an extremely cautious approach.

For orthopoxviruses, the vaccinia virus-based smallpox vaccine has been used historically in some zoo settings to protect high-risk species such as elephants and great apes, but its safety profile in these species is poorly characterized. The development of recombinant vaccines, which express immunogenic poxvirus proteins without the capacity for replication, represents a paradigm shift for zoo vaccinology. These vaccines eliminate the risk of vaccine-induced disease and are theoretically safer across a broader range of species. The success of recombinant canarypox-vectored vaccines for West Nile virus in equids and birds provides a proof-of-concept that can be extended to poxvirus prevention. However, the immunogenicity of recombinant poxvirus vaccines may be species-dependent, and efficacy trials in target zoo species are often logistically and ethically challenging. The use of DNA vaccines, which encode immunogenic poxvirus antigens, offers another avenue for safe vaccination, though their immunogenicity in large mammals has been variable.

A critical consideration in any zoo vaccination program is the need for species-specific safety and efficacy data. The establishment of reference intervals for hematological and biochemical parameters, as has been done for southern white rhinoceros [16] and striped skunks [14], is essential for monitoring vaccine safety. Similarly, the use of acute phase reactants as biomarkers of vaccine-induced inflammation [6] could help differentiate normal post-vaccination responses from adverse events. The decision to vaccinate must be made on a case-by-case basis, weighing the risk of poxvirus exposure against the potential for vaccine-related complications. For species that are highly endangered and maintained in small, isolated populations, such as certain tree monitor lizards [1] or the Iberian ribbed newt [2], the risk of introducing a novel pathogen may outweigh the benefits of vaccination, and biosecurity measures may be the primary line of defense. For species that are known to be highly susceptible to poxviruses and are housed in facilities with high visitor traffic or frequent animal movements, vaccination may be justified, but only after careful consultation with veterinary virologists and regulatory authorities such as the World Organisation for Animal Health (WOAH) and the Centers for Disease Control and Prevention (CDC).

Integrated Surveillance and One Health Approaches

Effective prevention of poxvirus outbreaks in zoo settings requires a surveillance system that extends beyond the boundaries of the collection itself. The One Health approach, which recognizes the interconnectedness of human, animal, and environmental health, is particularly relevant for poxviruses given their zoonotic potential and the role of wildlife reservoirs. Zoos should participate in regional surveillance networks that monitor poxvirus circulation in local wildlife populations, particularly rodents and other small mammals that may serve as bridge hosts. The detection of orthopoxvirus circulation in domestic cats in northeastern Italy [9] and the identification of cowpox virus transmission from rats to elephants to humans in Germany underscore the importance of understanding the local epizootiology. Serological surveys of zoo staff, as was conducted during the SARS-CoV-2 tiger outbreak [3], can provide valuable information about occupational exposure risks and guide vaccination policies for personnel.

The integration of behavioral management into biosecurity protocols represents an often-overlooked but critical component. Operant conditioning and positive reinforcement training, which have been foundational in modern zoo animal welfare [11], can be leveraged to facilitate diagnostic sampling and medical treatment without the need for chemical restraint. Training animals to voluntarily present for blood collection, nasal swabbing, or topical treatment of skin lesions reduces stress, minimizes the risk of injury to both animals and staff, and allows for more frequent health monitoring. This is particularly valuable during a poxvirus outbreak, where early detection of new cases and rapid implementation of isolation protocols can significantly reduce the magnitude of the outbreak. Environmental enrichment strategies [11] can also play a role in biosecurity by reducing stress-induced immunosuppression, which may increase susceptibility to poxvirus infection and shedding.

The role of vector-borne transmission in poxvirus ecology, while less well-characterized than direct contact or fomite transmission, should not be ignored. The Old World screw-worm fly (Chrysomya bezziana), an obligate parasite that causes cutaneous myiasis in a wide range of hosts including dogs, cattle, and humans [18], can create open wounds that serve as portals of entry for poxviruses and other pathogens. In regions where such myiasis-causing flies are endemic, integrated pest management programs that target fly breeding sites and use approved insecticides are an essential component of biosecurity. Similarly, the skin microbiome, which varies significantly across mammalian orders and is influenced by host phylogeny and geographic location [13], may play a role in resistance to poxvirus infection through competitive exclusion or immune modulation, though this remains an area of active investigation.

Finally, the development of contingency plans for poxvirus outbreaks must be a collaborative effort involving zoo veterinarians, public health authorities, and conservation organizations. These plans should include protocols for quarantine of affected areas, triage of exposed animals, humane euthanasia of severely affected individuals when necessary, and communication strategies for staff, visitors, and the media. The availability of diagnostic capacity for rapid poxvirus identification, including PCR and sequencing capabilities, is essential for timely confirmation of outbreaks. The use of molecular techniques such as rpoB gene sequencing for mycobacterial identification [15] demonstrates the power of molecular diagnostics for pathogen identification in zoo settings, and similar approaches should be developed for poxviruses. The financial and logistical resources required for comprehensive biosecurity and vaccination programs are substantial, but the cost of an uncontrolled poxvirus outbreak, in terms of animal mortality, loss of genetic diversity in endangered species, zoonotic transmission, and reputational damage to the institution, is far greater.

One Health Implications and Conservation Considerations for Poxviruses in Captive Wildlife

The intersection of human, animal, and environmental health is nowhere more vividly demonstrated than in the management of poxvirus infections within captive wildlife collections. Zoological institutions serve as unique ecological interfaces where diverse taxa, often comprising threatened and endangered species, are maintained in close proximity to human caregivers, visitors, and a complex matrix of commensal and synanthropic fauna. This confluence creates conditions ripe for pathogen emergence, spillover, and spillback, a dynamic that has been underscored by high-profile events such as the tiger-to-human transmission of SARS-CoV-2 at a Tennessee zoo [3]. While coronaviruses have captured recent attention, the orthopoxviruses, a genus characterized by broad host range, environmental persistence, and documented zoonotic potential, present a more insidious and historically rooted threat to captive wildlife conservation and public health. The One Health framework is therefore not merely a conceptual lens but an operational necessity for understanding and mitigating poxvirus risks in zoo settings.

The Zoo as a Nexus for Orthopoxvirus Spillover and Amplification

The captive wildlife environment functions as a potential amplifier hub for poxviruses due to several converging factors. First, the diversity of species housed within zoos provides a broad array of susceptible hosts. Cowpox virus (CPXV), for example, has demonstrated the ability to infect a remarkably wide range of mammalian orders, from rodents and felids to elephants and humans [9]. The documented chain of transmission from rat to elephant to human in a German zoo exemplifies how captive environments can facilitate cross-species jumps that would be improbable in nature [9]. The presence of reservoir species, particularly wild rodents that may infiltrate zoo grounds, establishes a persistent viral source. Source [5] emphasizes that certain animal-based practices and the proximity of reservoir species for filoviruses, coronaviruses, and critically, poxviruses, create a high-risk interface for pathogen spillover in African contexts, a principle that applies globally to zoo settings where rodent control may be imperfect.

Second, the stress of captivity, including confinement, social disruption, and transport, can impair immune function and increase viral shedding. Source [11] discusses how behavioral welfare practices such as environmental enrichment and operant conditioning have been implemented to reduce chronic stress and improve voluntary participation in veterinary care. However, when these welfare protocols are suboptimal or disrupted by outbreaks, animals may experience heightened glucocorticoid levels, which have been shown to reactivate latent viral infections and increase susceptibility to primary infection. The acute phase response, as reviewed by Hooijberg and Cray [6], provides a valuable window into inflammatory status in nondomesticated mammals, but the lack of species-specific reagents for many zoo taxa means that subclinical poxvirus infections may go undetected until overt clinical signs, often severe, appear.

Zoonotic Pathways and Public Health Risks

The zoonotic potential of orthopoxviruses in zoo settings cannot be overstated. Direct contact with infected animals, fomites (including bedding, enclosures, and veterinary equipment), and even aerosolized virus from skin lesions or respiratory secretions pose risks to keepers, veterinarians, and visitors. Documented cases of cat-to-human orthopoxvirus transmission in northeastern Italy highlight that even in regions south of the Alps, where such infections were previously considered rare, OPV circulates in domestic and wild fauna [9]. The affected personnel in that report, a veterinary student and a veterinarian, both sustained scratches from cats with ulcerated lesions, underscoring the occupational hazard faced by those who handle potentially infected animals. The hemagglutinin and crmB gene sequences from those Italian isolates formed a distinct cluster, suggesting the possibility of a novel or previously unrecognized OPV species circulating in that region [9]. This genetic diversity complicates diagnosis and underscores the need for surveillance beyond classical CPXV or vaccinia virus.

The risk extends beyond direct zoonotic transmission. Source [3] demonstrated through genomic epidemiology that a Malayan tiger keeper likely acquired SARS-CoV-2 from an infected tiger, with phylogenetic analysis showing 3–6 single nucleotide polymorphisms between the human and tiger sequences. Although this case involves a coronavirus, the investigative framework, including environmental assessment, staff symptom monitoring, genomic sequencing, and whole-genome phylogenetic analysis, is directly applicable to orthopoxvirus outbreak investigation in zoos. If a keeper handling a monkeypox- or cowpox-infected felid or primate were to develop febrile illness with vesicular lesions, the same One Health approach would be essential to confirm transmission, trace contacts, and implement infection control.

Furthermore, the global trade in traditional medicine, as reviewed in source [5], incorporates animal products from species that may harbor poxviruses. While this practice is more commonly associated with wild harvesting in Africa, zoo animals that die or are euthanized could theoretically enter such supply chains if carcass disposal is not rigorous. The World Health Organization (WHO) has highlighted the importance of integrating traditional medicine safety into public health frameworks, and zoos must ensure that their biosecurity protocols prevent any possibility of infected tissues entering human use.

Conservation Consequences for Endangered Species

The conservation implications of poxvirus infections in captive wildlife are profound, particularly for taxa that are already critically endangered in the wild. Many zoo-held species have small founder populations and limited genetic diversity, rendering them more susceptible to novel pathogens and less capable of mounting effective immune responses. Outbreaks of poxvirus-associated disease can decimate captive breeding programs, which are often the last refuge for species like the blue tree monitor (Varanus macraei), the black tree monitor (V. prasinus), and the green tree monitor (V. beccarii). Establishing hematology and plasma biochemistry reference intervals for these species [1] is a foundational step for detecting subclinical disease, but reference values mean little without parallel virological monitoring. A captive collection that loses a significant fraction of its breeding stock to an outbreak of a poxvirus may require years to recover genetically, if recovery is possible at all.

The situation is even more dire for mammals. Source [12] describes the devastating impact of canine distemper virus (CDV) on wild and zoo felids, including lions and other endangered carnivores. While CDV is a morbillivirus, the parallels with poxviruses are instructive: both can cause multisystemic disease, both can be transmitted across species boundaries, and both have caused mortality in precious zoo populations. Modified-live vaccines for CDV have themselves caused disease in wild carnivores [12], a cautionary note for poxvirus vaccine development. Inactivated or recombinant vaccines against poxviruses, such as the vaccinia-based rabies vaccine used in wildlife, offer safer alternatives, but their efficacy in exotic species is often poorly characterized. The risk of vaccine-induced disease or inadequate protection means that vaccination strategies must be carefully tailored to each species and validated through controlled studies.

The conservation value of zoo animals extends beyond the individual institution; many species participate in inter-zoo exchanges, reintroduction programs, and genetic management programs coordinated by organizations such as the World Association of Zoos and Aquariums (WAZA) and regional associations like the Association of Zoos and Aquariums (AZA). A poxvirus outbreak in one facility can jeopardize these networks, leading to quarantine restrictions, movement bans, and loss of confidence among partner institutions. The FAO and WOAH (World Organisation for Animal Health) have established guidelines for transboundary animal diseases that are relevant to poxviruses, but few zoo-specific protocols exist for the management of these pathogens.

Diagnostic and Surveillance Challenges in a One Health Context

Detecting poxvirus infections in captive wildlife is fraught with difficulties. Clinical signs, vesicular or pustular dermatitis, conjunctivitis, respiratory distress, and systemic illness, are not pathognomonic and can resemble those caused by herpesviruses, caliciviruses, or even bacterial infections. The availability of reliable diagnostic tools is limited for many zoo species. While MALDI-TOF mass spectrometry has revolutionized the identification of fungal pathogens in veterinary practice [10], and rpoB gene sequencing has proven useful for identifying Mycobacterium species of veterinary importance [15], analogous high-throughput molecular tools for poxvirus detection in zoo animals are not routinely deployed in most institutions. Polymerase chain reaction (PCR) assays targeting conserved orthopoxvirus genes such as the hemagglutinin or DNA polymerase genes are available in reference laboratories, but turnaround times may be slow, and sensitivity may be reduced in degraded samples or when the virus is present at low copy numbers.

The development of species-specific reference intervals for clinical pathology, as advocated by Sobrino-Yacobi et al. [1], Rodriguez et al. [2], Jimenez-Cortes et al. [4], and Trivedi et al. [16], provides a baseline for health assessment, but these studies typically sample healthy animals and cannot capture the dynamic changes that accompany acute infection. Acute phase reactants such as serum amyloid A and haptoglobin have been proposed as biomarkers of inflammation in nondomesticated mammals [6], but their utility in poxvirus infections has not been systematically evaluated. Point-of-care analyzers like the i-STAT Alinity v [16] offer the potential for rapid, on-site hematology and biochemistry assessment, which could aid in early detection of systemic involvement in poxvirus cases.

Surveillance for poxviruses in zoo collections should be proactive and risk-based. Animals that interact with the public, such as elephants, big cats, and primates, should be prioritized for routine serological monitoring. Environmental sampling of enclosure surfaces, water sources, and air filters could identify viral contamination before clinical cases emerge. The skin microbiome, as characterized by Ross et al. [13], represents a frontier of understanding how commensal microbial communities may influence susceptibility to poxvirus infection. If phylosymbiosis, co-evolution between host phylogeny and skin microbial communities, exists, disruptions to the microbiome from antibiotics or stress could alter the epithelial barrier and facilitate viral entry.

Recommendations for Policy and Practice

To operationalize the One Health framework for poxvirus management in zoos, several concrete steps are necessary. First, every institution should maintain a biosecurity plan that includes protocols for the introduction of new animals, quarantine procedures, rodent and feral cat control, and staff training on zoonotic disease recognition. Second, diagnostic capacity must be strengthened through partnerships with veterinary diagnostic laboratories and human health agencies such as the CDC, which has expertise in orthopoxvirus detection and response. Third, vaccination strategies should be developed for high-risk species, prioritizing the use of recombinant or inactivated products over live vaccines to minimize the risk of vaccine-induced disease. Fourth, crowd management during outbreaks, including restricting access to exhibits, installing barriers to reduce aerosol transmission, and screening visitors for febrile illness, should be informed by lessons from the SARS-CoV-2 pandemic [3].

Finally, zoos must engage in data sharing and collaborative research. The establishment of a global poxvirus surveillance network for captive wildlife, analogous to the WOAH’s World Animal Health Information System, would enable early detection of emerging strains and facilitate rapid response. Such a network would also support the conservation of species like the tucúquere (Bubo magellanicus), for which hematological reference values have been established in Chilean zoos and rehabilitation centers [4], and the Iberian ribbed newt (Pleurodeles waltl), for which baseline hematology is now available [2]. By integrating hematological, biochemical, serological, and molecular data, the zoo veterinary community can move from reactive outbreak management to proactive health surveillance, ultimately safeguarding the health of animals, humans, and the shared environment.

References

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