In Ovo Models to Predict Virulence of Highly Pathogenic Avian Influenza H5-Viruses for Chickens and Ducks.

Highly pathogenic avian influenza (HPAI) H5-viruses are circulating in wild birds and are repeatedly introduced to poultry causing outbreaks in the Netherlands since 2014. The largest epizootic ever recorded in Europe was caused by HPAI H5N1 clade 2.3.4.4b viruses in the period 2021-2022. The recent H5-clade 2.3.4.4 viruses were found to differ in their virulence for chickens and ducks. Viruses causing only mild disease may remain undetected, increasing the risk of virus spread to other farms, wild birds and mammals. We developed in ovo models to determine the virulence of HPAI viruses for chickens and ducks, which are fast and have low costs. The virulence of five contemporary H5-viruses was compared studying replication rate, average time to death and virus spread in the embryo. Remarkable differences in virulence were observed between H5-viruses and between poultry species. The H5N1-2021 virus was found to have a fast replication rate in both the chicken and duck in ovo models, but a slower systemic virus dissemination compared to three other H5-clade 2.3.4.4b viruses. The results show the potential of in ovo models to quickly determine the virulence of novel HPAI viruses, and study potential virulence factors which can help to better guide the surveillance in poultry.

Immune Responses and Pathogenesis following Experimental SARS-CoV-2 Infection in Domestic Cats.

Several reports demonstrated the susceptibility of domestic cats to SARS-CoV-2 infection. Here, we describe a thorough investigation of the immune responses in cats after experimental SARS-CoV-2 inoculation, along with the characterization of infection kinetics and pathological lesions. Specific pathogen-free domestic cats (n = 12) were intranasally inoculated with SARS-CoV-2 and subsequently sacrificed on DPI (days post-inoculation) 2, 4, 7 and 14. None of the infected cats developed clinical signs. Only mild histopathologic lung changes associated with virus antigen expression were observed mainly on DPI 4 and 7. Viral RNA was present until DPI 7, predominantly in nasal and throat swabs. The infectious virus could be isolated from the nose, trachea and lungs until DPI 7. In the swab samples, no biologically relevant SARS-CoV-2 mutations were observed over time. From DPI 7 onwards, all cats developed a humoral immune response. The cellular immune responses were limited to DPI 7. Cats showed an increase in CD8+ cells, and the subsequent RNA sequence analysis of CD4+ and CD8+ subsets revealed a prominent upregulation of antiviral and inflammatory genes on DPI 2. In conclusion, infected domestic cats developed a strong antiviral response and cleared the virus within the first week after infection without overt clinical signs and relevant virus mutations.

Efficient Direct and Limited Environmental Transmission of SARS-CoV-2 Lineage B.1.22 in Domestic Cats.

The susceptibility of domestic cats to infection with SARS-CoV-2 has been demonstrated by several experimental studies and field observations. We performed an extensive study to further characterize the transmission of SARS-CoV-2 between cats, through both direct and indirect contact. To that end, we estimated the transmission rate parameter and the decay parameter for infectivity in the environment. Using four groups of pair-transmission experiment, all donor (inoculated) cats became infected, shed virus, and seroconverted, while three out of four direct contact cats got infected, shed virus, and two of those seroconverted. One out of eight cats exposed to a SARS-CoV-2-contaminated environment became infected but did not seroconvert. Statistical analysis of the transmission data gives a reproduction number R0 of 2.18 (95% CI = 0.92 to 4.08), a transmission rate parameter ß of 0.23 day-1 (95% CI = 0.06 to 0.54), and a virus decay rate parameter µ of 2.73 day-1 (95% CI = 0.77 to 15.82). These data indicate that transmission between cats is efficient and can be sustained (R0 > 1), however, the infectiousness of a contaminated environment decays rapidly (mean duration of infectiousness 1/2.73 days). Despite this, infections of cats via exposure to a SARS-CoV-2-contaminated environment cannot be discounted if cats are exposed shortly after contamination. IMPORTANCE This article provides additional insight into the risk of infection that could arise from cats infected with SARS-CoV-2 by using epidemiological models to determine transmission parameters. Considering that transmission parameters are not always provided in the literature describing transmission experiments in animals, we demonstrate that mathematical analysis of experimental data is crucial to estimate the likelihood of transmission. This article is also relevant to animal health professionals and authorities involved in risk assessments for zoonotic spill-overs of SARS-CoV-2. Last but not least, the mathematical models to calculate transmission parameters are applicable to analyze the experimental transmission of other pathogens between animals.

Zoonotic Mutation of Highly Pathogenic Avian Influenza H5N1 Virus Identified in the Brain of Multiple Wild Carnivore Species.

Wild carnivore species infected with highly pathogenic avian influenza (HPAI) virus subtype H5N1 during the 2021-2022 outbreak in the Netherlands included red fox (Vulpes vulpes), polecat (Mustela putorius), otter (Lutra lutra), and badger (Meles meles). Most of the animals were submitted for testing because they showed neurological signs. In this study, the HPAI H5N1 virus was detected by PCR and/or immunohistochemistry in 11 animals and was primarily present in brain tissue, often associated with a (meningo) encephalitis in the cerebrum. In contrast, the virus was rarely detected in the respiratory tract and intestinal tract and associated lesions were minimal. Full genome sequencing followed by phylogenetic analysis demonstrated that these carnivore viruses were related to viruses detected in wild birds in the Netherlands. The carnivore viruses themselves were not closely related, and the infected carnivores did not cluster geographically, suggesting that they were infected separately. The mutation PB2-E627K was identified in most carnivore virus genomes, providing evidence for mammalian adaptation. This study showed that brain samples should be included in wild life surveillance programs for the reliable detection of the HPAI H5N1 virus in mammals. Surveillance of the wild carnivore population and notification to the Veterinary Authority are important from a one-heath perspective, and instrumental to pandemic preparedness.

Highly Pathogenic Avian Influenza H5N1 Virus Infections in Wild Red Foxes (Vulpes vulpes) Show Neurotropism and Adaptive Virus Mutations.

During the 2020 to 2022 epizootic of highly pathogenic avian influenza virus (HPAI), several infections of mammalian species were reported in Europe. In the Netherlands, HPAI H5N1 virus infections were detected in three wild red foxes (Vulpes vulpes) that were submitted with neurological symptoms between December of 2021 and February of 2022. A histopathological analysis demonstrated that the virus was mainly present in the brain, with limited or no detection in the respiratory tract or other organs. Limited or no virus shedding was observed in throat and rectal swabs. A phylogenetic analysis showed that the three fox viruses were not closely related, but they were related to HPAI H5N1 clade 2.3.4.4b viruses that are found in wild birds. This suggests that the virus was not transmitted between the foxes. A genetic analysis demonstrated the presence of the mammalian adaptation E627K in the polymerase basic two (PB2) protein of the two fox viruses. In both foxes, the avian (PB2-627E) and the mammalian (PB2-627K) variants were present as a mixture in the virus population, which suggests that the mutation emerged in these specific animals. The two variant viruses were isolated, and virus replication and passaging experiments were performed. These experiments showed that the mutation PB2-627K increases the replication of the virus in mammalian cell lines, compared to the chicken cell line, and at the lower temperatures of the mammalian upper respiratory tract. This study showed that the HPAI H5N1 virus is capable of adaptation to mammals; however, more adaptive mutations are required to allow for efficient transmission between mammals. Therefore, surveillance in mammals should be expanded to closely monitor the emergence of zoonotic mutations for pandemic preparedness. IMPORTANCE Highly pathogenic avian influenza (HPAI) viruses caused high mortality among wild birds from 2021 to 2022 in the Netherlands. Recently, three wild foxes were found to be infected with HPAI H5N1 viruses, likely due to the foxes feeding on infected birds. Although HPAI is a respiratory virus, in these foxes, the viruses were mostly detected in the brain. Two viruses isolated from the foxes contained a mutation that is associated with adaptation to mammals. We show that the mutant virus replicates better in mammalian cells than in avian cells and at the lower body temperature of mammals. More mutations are required before viruses can transmit between mammals or can be transmitted to humans. However, infections in mammalian species should be closely monitored to swiftly detect mutations that may increase the zoonotic potential of HPAI H5N1 viruses, as these may threaten public health.

Mass Mortality Caused by Highly Pathogenic Influenza A(H5N1) Virus in Sandwich Terns, the Netherlands, 2022.

We collected data on mass mortality in Sandwich terns (Thalasseus sandvicensis) during the 2022 breeding season in the Netherlands. Mortality was associated with at least 2 variants of highly pathogenic avian influenza A(H5N1) virus clade 2.3.4.4b. We report on carcass removal efforts relative to survival in colonies. Mitigation strategies urgently require structured research.

Intranasal administration of a live-attenuated recombinant newcastle disease virus expressing the SARS-CoV-2 Spike protein induces high neutralizing antibody levels and protects from experimental challenge infection in hamsters.

The emergence of SARS-CoV-2 in December 2019 resulted in the COVID-19 pandemic. Recurring disease outbreaks repeatedly overloaded the public health sector and severely affected the global economy. We developed a candidate COVID-19 vaccine based on a recombinant Newcastle disease virus (NDV) vaccine vector, encoding a pre-fusion stabilized full-length Spike protein obtained from the original SARS-CoV-2 Wuhan isolate. Vaccination of hamsters by intra-muscular injection or intra-nasal instillation induced high neutralizing antibody responses. Intranasal challenge infection with SARS-CoV-2 strain Lelystad demonstrated that both vaccination routes provided partial protection in the upper respiratory tract, and almost complete protection in the lower respiratory tract, as measured by suppressed viral loads and absence of histological lung lesions. Activity wheel measurements demonstrated that animals vaccinated by intranasal inoculation rapidly recovered to normal activity. NDV constructs encoding the spike of SARS-CoV-2 may be attractive candidates for development of intra-nasal COVID-19 booster vaccines.

Tissue tropism and pathology of highly pathogenic avian influenza H5N6 virus in chickens and Pekin ducks.

The highly pathogenic avian influenza (HPAI) H5N6 virus caused outbreaks on commercial poultry farms in the Netherlands in 2017-2018, holding chickens and Pekin ducks. Intravenous pathogenicity index (IVPI) tests confirmed the high pathogenicity of the virus. Tissues derived from birds from infected farms (natural infection) and IVPI tests (experimental infection) were used to compare histopathology and virus distribution in both poultry species. After natural infection in chickens, histopathologic changes were present in the respiratory tract and several internal organs in both chickens and Pekin ducks. Viral antigen expression in the tissues of chickens varied from that in ducks. Virus expression was found in epithelial, mononuclear and endothelial cells in chickens. In contrast to the major role infected endothelial cells seem to play in systemic infections of chickens, in ducks the number of infected endothelial cells was very limited. Therefore, endothelial cell infection likely does not play a major role in systemic infection and disease progression in HPAI H5N6 virus infected Pekin ducks.

Differential susceptibility of SARS-CoV-2 in animals: Evidence of ACE2 host receptor distribution in companion animals, livestock and wildlife by immunohistochemical characterisation.

Angiotensin converting enzyme 2 (ACE2) is a host cell membrane protein (receptor) that mediates the binding of coronavirus, most notably SARS coronaviruses in the respiratory and gastrointestinal tracts. Although SARS-CoV-2 infection is mainly confined to humans, there have been numerous incidents of spillback (reverse zoonoses) to domestic and captive animals. An absence of information on the spatial distribution of ACE2 in animal tissues limits our understanding of host species susceptibility. Here, we describe the distribution of ACE2 using immunohistochemistry (IHC) on histological sections derived from carnivores, ungulates, primates and chiroptera. Comparison of mink (Neovison vison) and ferret (Mustela putorius furo) respiratory tracts showed substantial differences, demonstrating that ACE2 is present in the lower respiratory tract of mink but not ferrets. The presence of ACE2 in the respiratory tract in some species was much more restricted as indicated by limited immunolabelling in the nasal turbinate, trachea and lungs of cats (Felis catus) and only the nasal turbinate in the golden Syrian hamster (Mesocricetus auratus). In the lungs of other species, ACE2 could be detected on the bronchiolar epithelium of the sheep (Ovis aries), cattle (Bos taurus), European badger (Meles meles), cheetah (Acinonyx jubatus), tiger and lion (Panthera spp.). In addition, ACE2 was present in the nasal mucosa epithelium of the serotine bat (Eptesicus serotinus) but not in pig (Sus scrofa domestica), cattle or sheep. In the intestine, ACE2 immunolabelling was seen on the microvillus of enterocytes (surface of intestine) across various taxa. These results provide anatomical evidence of ACE2 expression in a number of species which will enable further understanding of host susceptibility and tissue tropism of ACE2 receptor-mediated viral infection.

Predictive Value of Precision-Cut Lung Slices for the Susceptibility of Three Animal Species for SARS-CoV-2 and Validation in a Refined Hamster Model.

In assessing species susceptibility for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and in the search for an appropriate animal model, multiple research groups around the world inoculated a broad range of animal species using various SARS-CoV-2 strains, doses and administration routes. Although in silico analyses based on receptor binding and diverse in vitro cell cultures were valuable, exact prediction of species susceptibility based on these tools proved challenging. Here, we assessed whether precision-cut lung slices (PCLS) could facilitate the selection of animal models, thereby reducing animal experimentation. Pig, hamster and cat PCLS were incubated with SARS-CoV-2 and virus replication was followed over time. Virus replicated efficiently in PCLS from hamsters and cats, while no evidence of replication was obtained for pig PCLS. These data corroborate the findings of many research groups that have investigated the susceptibility of hamsters, pigs and cats towards infection with SARS-CoV-2. Our findings suggest that PCLS can be used as convenient tool for the screening of different animal species for sensitivity to newly emerged viruses. To validate our results obtained in PCLS, we employed the hamster model. Hamsters were inoculated with SARS-CoV-2 via the intranasal route. Susceptibility to infection was evaluated by body weight loss, viral loads in oropharyngeal swabs and respiratory tissues and lung pathology. The broadly used hamster model was further refined by including activity tracking of the hamsters by an activity wheel as a very robust and sensitive parameter for clinical health. In addition, to facilitate the quantification of pathology in the lungs, we devised a semi-quantitative scoring system for evaluating the degree of histological changes in the lungs. The inclusion of these additional parameters refined and enriched the hamster model, allowing for the generation of more data from a single experiment.

Early immune responses in skin and lymph node after skin delivery of Toll-like receptor agonists in neonatal and adult pigs.

The skin is potentially an important vaccine delivery route facilitated by a high number of resident antigen presenting cells (APCs), which are known to be stimulated by different Toll-like receptor agonists (TLRa). In this study, neonatal and adult pigs were vaccinated in the skin using dissolving microneedle patches to investigate the immuno-stimulatory potential of different TLRa and possible age-dependent differences early after vaccination. These patches contained TLR1/2a (Pam3Cys), TLR7/8a (R848) or TLR9a (CpG ODN) combined with inactivated porcine reproductive and respiratory syndrome virus (PRRSV) or with an oil-in-water stable emulsion. Vaccinated skin and draining lymph nodes were analysed for immune response genes using microfluidic high-throughput qPCR to evaluate the early immune response and activation of APCs. Skin pathology and immunohistochemistry were used to evaluate the local immune responses and APCs in the vaccinated skin, respectively. In both neonatal and adult pigs, skin vaccination with TLR7/8a induced the most prominent early inflammatory and immune cell responses, particularly in the skin. Skin histopathology and immunohistochemistry of APCs showed comparable results for neonatal and adult pigs after vaccination with the different TLRa vaccines. However, in vaccinated neonatal pigs in the skin and draining lymph node more immune response related genes were upregulated compared to adult pigs. We showed that both neonatal and adult skin could be stimulated to develop an immune response, particularly after TLR7/8a vaccination, with age-dependent differences in regulation of immune genes. Therefore, age-dependent differences in local early immune responses should be considered when developing skin vaccines.

Immune responses induced by inactivated porcine reproductive and respiratory syndrome virus (PRRSV) vaccine in neonatal pigs using different adjuvants.

Vaccination of neonatal pigs could be supportive to prevent porcine reproductive and respiratory syndrome virus (PRRSV), which is an important porcine pathogen causing worldwide welfare and health problems in pigs of different age classes. However, neonatal immunity substantially differs to adults, thus different vaccines may be required in neonateal pigs. We examined if the immunogenicity and efficacy of inactivated PRRSV (iPRRSV) vaccines in neonatal pigs could be improved with adjuvants containing oil-in water (O/W) emulsions with or without Toll-like receptor (TLR) agonists and by altering the delivery route from intramuscular (i.m.) to the skin. Three-day-old PRRSV-naïve piglets (n = 54, divided in 6 groups) received a prime vaccination and a booster vaccination four weeks later. The vaccine formulations consisted of different O/W emulsions (Montanide™ ISA28RVG (ISA28)), a squalene in water emulsion (SWE) for i.m. or a Stable Emulsion (SE) with squalene for skin vaccination) and/or a mixture of TLR1/2, 7/8 and 9 agonists (TLRa) combined with iPRRSV strain 07V063. These vaccines were delivered either i.m. (ISA28, SWE, TLRa or SWE + TLRa) or into the skin (skiSE + TLRa) with dissolving microneedle (DMN)-patches. All animals received a challenge with homologous PRRSV three weeks after booster vaccination. Specific antibodies, IFN-γ production and viremia were measured at several time-points after vaccination and/or challenge, while lung pathology was studied at necropsy. After booster vaccination, only ISA28 induced a specific antibody response while a specific T-cell IFN-γ response was generated in the SWE group, that was lower for ISA28, and absent in the other groups. This suggests that prime vaccination in neonates induced a specific immune response after booster vaccination, dependent on the emulsion formulation, but not dependent on the presence of the TLRa or delivery route. Despite the measured immune responses none of the vaccines showed any efficacy. Further research focused on the early immune response in draining lymph nodes is needed to elucidate the potential of TLR agonists in vaccines for neonatal pigs.

Clinical and Pathological Findings in SARS-CoV-2 Disease Outbreaks in Farmed Mink (Neovison vison).

SARS-CoV-2, the causative agent of COVID-19, caused respiratory disease outbreaks with increased mortality in 4 mink farms in the Netherlands. The most striking postmortem finding was an acute interstitial pneumonia, which was found in nearly all examined mink that died at the peak of the outbreaks. Acute alveolar damage was a consistent histopathological finding in mink that died with pneumonia. SARS-CoV-2 infections were confirmed by detection of viral RNA in throat swabs and by immunohistochemical detection of viral antigen in nasal conchae, trachea, and lung. Clinically, the outbreaks lasted for about 4 weeks but some animals were still polymerase chain reaction-positive for SARS-CoV-2 in throat swabs after clinical signs had disappeared. This is the first report of the clinical and pathological characteristics of SARS-CoV-2 outbreaks in mink farms.

SARS-CoV-2 infection in farmed minks, the Netherlands, April and May 2020.

Respiratory disease and increased mortality occurred in minks on two farms in the Netherlands, with interstitial pneumonia and SARS-CoV-2 RNA in organ and swab samples. On both farms, at least one worker had coronavirus disease-associated symptoms before the outbreak. Variations in mink-derived viral genomes showed between-mink transmission and no infection link between the farms. Inhalable dust contained viral RNA, indicating possible exposure of workers. One worker is assumed to have attracted the virus from mink.

Two novel porcine teschovirus strains as the causative agents of encephalomyelitis in the Netherlands.

BACKGROUND: Porcine teschovirus (PTV) circulates among wild and domesticated pig populations without causing clinical disease, however neuroinvasive strains have caused high morbidity and mortality in the past. In recent years, several reports appeared with viral agents as a cause for neurologic signs in weanling and growing pigs among which PTV and new strains of PTV were described. CASE PRESENTATION: On two unrelated pig farms in the Netherlands the weanling pig population showed a staggering gate, which developed progressively to paresis or paralysis of the hind legs with a morbidity up to 5%. After necropsy we diagnosed a non-suppurative encephalomyelitis on both farms, which was most consistent with a viral infection. PTV was detected within the central nervous system by qPCR. From both farms PTV full-length genomes were sequenced, which clustered closely with PTV-3 (98%) or PTV-11 (85%). Other common swine viruses were excluded by qPCR and sequencing of the virus. CONCLUSION: Our results show that new neuroinvasive PTV strains still emerge in pigs in the Netherlands. Further research is needed to investigate the impact of PTV and other viral agents causing encephalomyelitis within wild and domestic pig populations supported by the awareness of veterinarians.

Toll-like receptor agonists as adjuvants for inactivated porcine reproductive and respiratory syndrome virus (PRRSV) vaccine.

Toll-like receptor (TLR) agonists can effectively stimulate antigen-presenting cells (APCs) and are anticipated to be promising adjuvants in combination with inactivated vaccines. In this study, the adjuvant potential of three different TLR-agonists were compared with an oil-in-water (O/W) adjuvant in combination with inactivated porcine reproductive and respiratory syndrome virus (iPRRSV) applied by different administration routes: intramuscular (i.m.) or into the skin using dissolving microneedle (DMN) patches. Pigs received a prime vaccination followed by a booster vaccination four weeks later. TLR1/2 (Pam3Cys), TLR7/8 (R848) or TLR9 (CpG ODN) agonists were used as adjuvant in combination with iPRRSV strain 07V063. O/W adjuvant (Montanide™) was used as reference control adjuvant and one group received a placebo vaccination containing diluent only. All animals received a homologous challenge with PRRSV three weeks after the booster vaccination. Antibody and IFN-γ production, serum cytokines and viremia were measured at several time-points after vaccination and/or challenge, and lung pathology at necropsy. Our results indicate that a TLR 1/2, 7/8 or 9 agonist as adjuvant with iPRRSV does not induce a detectable PRRSV-specific immune response, independent of the administration route. However, the i.m. TLR9 agonist group showed reduction of viremia upon challenge compared to the non-vaccinated animals, supported by a non-antigen-specific IFN-γ level after booster vaccination and an anamnestic antibody response after challenge. Montanide™-adjuvanted iPRRSV induced antigen-specific immunity after booster combined with reduction of vireamia. Skin application of TLR7/8 agonist, but not the other agonists, induced a local skin reaction. Further research is needed to explore the potential of TLR agonists as adjuvants for inactivated porcine vaccines with a preference for TLR9 agonists.

Neonatal porcine blood derived dendritic cell subsets show activation after TLR2 or TLR9 stimulation.

The present study investigated the innate immune response in vitro to determine porcine neonate responses with Toll-like receptor (TLR)2 ligand (Pam3Cys) or TLR9 ligand (CpG) and compared these with adults. We identified the same phenotypically defined dendritic cell (DC) subsets and DC proportions in porcine neonate and adult blood by flow cytometry, which were plasmacytoid DCs (pDCs): CD14-CD4+CD172a+CADM1-) and conventional DCs (cDCs), being further divided into a cDC1 (CD14-CD4-CD172alowCADM1+) and a cDC2 (CD14-CD4-CD172a+CADM1+) subset. With neonatal cells, the TLR2 ligand induced a stronger TNF expression in monocytes and pDCs, and a stronger CD80/86 upregulation in cDC1, when compared to adult cells. Furthermore, in neonatal mononuclear cells TLR9 ligand was more potent at inducing IL12p40 mRNA expression. These results indicate clear responses of porcine neonatal antigen presenting cells after TLR2 and TLR9 stimulation, suggesting that corresponding ligands could be promising candidates for neonatal adjuvant application.

Immunohistochemical evaluation of the activation of hepatic progenitor cells and their niche in feline lymphocytic cholangitis.

Objectives The aim of the study was to compare the hepatic progenitor cell niche in healthy feline livers and the liver tissue of cats with lymphocytic cholangitis. Methods Immunohistochemical stainings for vimentin, laminin, beta (ß)-catenin and Notch1 intracellular domain (NICD) were used on formalin-fixed liver biopsies from affected (n = 12) and unaffected cats (n = 2). Results All immunohistochemical markers used were expressed in more cells, or more intensely, in the liver tissue of cats with lymphocytic cholangitis than in the liver tissue of unaffected cats. Conclusions and relevance Enhanced expression of vimentin, laminin, cytoplasmic/nuclear ß-catenin and NICD in liver biopsies from cats with lymphocytic cholangitis indicates that the hepatic progenitor cell (HPC) niche is remodelled and activated. HPCs might provide insights into new regenerative treatment options for lymphocytic cholangitis in cats in the future.

A morphological and immunohistochemical study of the effects of prednisolone or ursodeoxycholic acid on liver histology in feline lymphocytic cholangitis.

Feline lymphocytic cholangitis (LC) has been commonly treated with prednisolone, and more recently with ursodeoxycholic acid (UDCA). Previously, we found that prednisolone treatment resulted in a statistically longer survival time than treatment with UDCA. In order to explain this difference, we compared the effects of prednisolone and UDCA treatment on hepatic tissue by evaluating consecutive liver biopsies. Archival serial biopsy materials from cats with LC treated with prednisolone (n = 5) or UDCA (n = 4) were evaluated. We employed haematoxylin and eosin staining to evaluate inflammation, and reticulin staining for fibrosis. Immunohistochemical stainings for Ki-67, K19 (Cytokeratin 19) and α-smooth muscle actin were used to evaluate cell type-specific proliferation and activation of hepatic stellate cells. Inflammation decreased more in the group treated with prednisolone, while the number of cholangiocytes, progenitor cells and fibroblasts did not differ between the treatment groups. Additionally, no difference was found for the amount of fibrosis in both treatment groups.