Meat Juice and Oral Fluid as Alternatives to Serum for Aujeszky Disease Monitoring in Pigs.

Aujeszky Disease Virus (ADV) is a double-stranded DNA virus with a lipoprotein envelope. The natural hosts of the infection are Suidae, but the virus can infect many other mammals. The gold-standard method identified by the WOAH for the diagnosis of Aujeszky disease is the ELISA method. The objective of this study was to compare the performance of meat juice and oral fluid matrices using a commercial ELISA kit designed for serum. A total of 80 blood and oral fluid samples were collected from four pig farms selected for this study. Diaphragm muscle samples of about 100 g and blood samples were collected from 213 animals at the abattoir. These biological matrices were collected from the same animals and tested using a competitive ELISA kit to detect antibodies against ADV. The relative accuracy of the meat juice compared to that of the serum was 96.7% (95% CI: 93.3-98.7%), with 206 correct results out of 213. The relative accuracy of the oral fluid compared to that of the serum was 61.3% (95% CI: 49.7-71.9%), with 58 correct results out of 80. Meat juice has a better combination of sensitivity and specificity than oral fluid. The usage of meat juice in routine diagnostic examinations could be achieved after further investigations to optimize the procedure.

A Qualitative PCR Assay for the Discrimination of Bubaline Herpesvirus 1, Bovine Herpesvirus 1 and Bovine Herpesvirus 5.

Bubaline herpesvirus 1 (BuHV-1), Bovine herpesvirus 1 (BoHV-1) and Bovine herpesvirus 5 (BoHV-5) are classified in the genus Varicellovirus, subfamily Alphaherpesvirinae. BoHV-1 is the causative agent of infectious bovine rhinotracheitis, BoHV-5 induces moderate disease in adult cattle while BuHV-1 has instead been associated with a decline in livestock production of water buffaloes. The aim of this study was to develop a qualitative PCR assay that allows the discrimination of BuHV-1, BoHV-1 and BoHV-5. The alignment of homologous genes identified specific nucleotide sequences of BuHV- 1, BoHV-1 and BoHV-5. The design of the primers and the optimization of the PCR assay were focused on the target sequences located on the portions of gD, gE and gG genes. This assay involved the use of three different PCR end-points: the PCR of a portion of the gD gene identified only the presence of BoHV-1; the PCR of a portion of the gE gene confirmed the presence of both BoHV-5 and BuHV-1; the PCR of a portion of the gG gene discriminated between BoHV-5 and BuHV-1, as the amplification product was observed only for BoHV-5. This qualitative PCR assay allowed the differentiation of BoHV-1 and BoHV-5 infections both in cattle and water buffaloes and heterologous BuHV-1 infections in bovine.

Molecular and serological investigation of Hepatitis E virus in pigs slaughtered in Northwestern Italy.

BACKGROUND: Hepatitis E Virus (HEV) is recently considered an emerging public health concern. HEV genotypes 1 and 2 are widely distributed and pathogenic only for humans. In contrast, HEV, genotypes 3 and 4 are observed in swine, deer, wild boars and rabbits and can also be transmitted to humans. The presence of HEV in the liver, muscle, faeces, blood, and bile was detected by real-time RT-PCR in 156 pigs belonging to twenty different farms, ranging from 1 to 8 months of age. The phylogenetic analysis was performed on the viral strain present in the positive biological matrix, with the lowest Ct. HEV-IgG and HEV-IgM in the sera were analysed by two different ELISA kits. RESULTS: Twenty-one pigs, i.e., 13.46% of them (21/156, 95% CI: 8.53%-19.84%), tested positive for HEV in at least one biological matrix by real-time RT-PCR, while phylogenetic analysis revealed the presence of HEV subtypes 3f and 3c. Pig serums analysed by ELISA showed an overall prevalence of 26.92% (42/156, 95% CI: 20.14%-34.60%) for HEV-IgG, whereas the 28.95% (33/114, 95% CI: 20.84%-38.19%) of them tested negative resulted positive for the HEV-IgM. CONCLUSIONS: The faeces are the biological matrix with the highest probability of detecting HEV. The best concordance value (Kappa Kohen index) and the highest positive correlation (Phi index) were observed for the correlation between bile and liver, even when the number of positive liver samples was lower than the positive bile samples. This finding may suggest that a higher probability of HEV occurs in the bile, when the virus is present in the liver, during the stages of infection. Finally, the presence of HEV in muscle was observed in 11 pigs, usually used for the preparation of some dishes, typical of the Italian tradition, based on raw or undercooked meat. Therefore, their consumption is a possible source of infection for final consumer.

The "Queen of the Moors". Paleopathological investigation of a natural mummy from Scicli, South-Eastern Sicily.

A natural, well-preserved mummy belonging to a 45-55 year old female was found in the Church of Santa Maria della Consolazione in Scicli, south-eastern Sicily. The body was submitted to external examination, digital radiology, and computed tomography scanning. Paleopathological investigation allowed us to detect pulmonary pathology related to tuberculosis, atherosclerosis, and phleboliths. The presence of the latter, along with good dental condition with focal caries and obesity indicates a subject belonging to a high social class in good nutritional status. Along with other examples, this case allows to infer that tuberculosis was a common disease in that area, if not in the whole island, prior to the antibiotic era. Mummies need to be properly surveyed and protected, but also adequately studied by multidisciplinary teams of experts. The presence in such a team of at least one skilled anatomic/surgical pathologist, as long as well trained in the study of ancient human remains, represents an undeniable condition.

Feline morbillivirus in northwestern Italy: first detection of genotype 1-B.

OBJECTIVES: A novel morbillivirus was recently described in stray and domestic cats in Asia, the USA and Europe. Most cats infected with feline morbillivirus (FeMV) showed lower urinary tract or kidney disease. Although the association of FeMV infection and kidney diseases has been suggested, the virus pathogenicity remains unclear. The present study aimed to investigate the distribution of FeMV infection, as well as the relationship between FeMV infection and kidney diseases in cats from northwestern Italy. METHODS: A total of 153 urine samples (150 individuals and three pools) and 50 kidney samples were collected and included in the study; total RNA was extracted and a reverse transcription quantitative PCR (RT-qPCR) was performed in order to identify FeMV. Kidneys were also submitted to anatomopathological examination. Phylogenetic analysis and isolation attempts were carried out on positive samples. In FeMV-positive cats, urinalysis and blood analysis were performed. RESULTS: FeMV RNA was detected in 7.3% of urine samples and in 8% of kidney samples, both in healthy cats and in cats with clinical signs/post-mortem lesions compatible with kidney disease. At histopathological examination, tubulointerstitial nephritis (TIN) was shown in 3/4 positive kidney samples, but a clear relationship between FeMV and TIN was not observed. Isolation attempts were unsuccessful, although the urine sample of one castrated male cat hosted in a cattery showed a positive signal in RT-qPCR until the fourth cell passage. Phylogenetic analysis revealed that this FeMV strain belonged to genotype 1-B. In the same cattery, a second genotype 1-B variant was detected from a urine pool. Urinalysis showed proteinuria in three cats, while at blood analysis three cats presented altered creatinine levels. CONCLUSIONS AND RELEVANCE: Data reported suggest the presence of a FeMV sub-cluster distinct from the strain previously isolated in Italy, whose role in renal disorders remains uncertain.

CANINE DISTEMPER VIRUS AS AN EMERGING MULTIHOST PATHOGEN IN WILD CARNIVORES IN NORTHWEST ITALY.

Canine distemper (CD) may pose a serious threat to Alpine wild carnivores and affect their population dynamics. Since 2006, the strain Europe Wildlife 2006-09, a distinct CD virus subgroup within viral lineage Europe 1 (EU1) characterized by increased virulence and host range expansion, has been linked to multiple CD outbreaks in Alpine wild carnivores. The aim of this study was to fill knowledge gaps about ongoing Alpine outbreaks of CD. To do this, we report on the circulation of canine distemper virus (CDV) and outbreaks of CD in Alpine wild carnivores in northwest Italy. A specific diagnostic protocol applied to a sample of 548 wild carnivores collected between January 2013 and December 2015 revealed the circulation of CDV belonging to the EU1 lineage. All isolates were carriers of amino-acid mutations defining the cluster Europe Wildlife 2006-09. A self-maintained multihost pathogen system may have developed in northwest Italy in which interspecies transmission from red foxes (Vulpes vulpes) to other noncanid species enhanced pathogen maintenance in the system.

Mortality outbreak by perch rhabdovirus in European perch (Perca fluviatilis) farmed in Italy: Clinical presentation and phylogenetic analysis.

This work reports a mortality outbreak, occurred in 2015 and affecting juveniles of European perch (Perca fluviatilis L.) farmed in Italy. Perch rhabdovirus (PRV) was detected by viral isolation and biomolecular investigations. Phylogenetic analysis clustered our isolate into genogroup B, which also includes PRV isolates from Perca fluviatilis identified in France (2004-2009); diagnostic investigations also revealed opportunistic bacteria (Aeromonas hydrophila) and parasites (Chilodonella piscicola). Since, occasionally, PRV has been reported in the natural environment, which is often a source of eggs and broodstock for farms, it could be possible that both similar France and Italian isolate were imported from a same place elsewhere and have a common origin. Improving biosecurity measures (batch control) and disinfection of egg strings with an iodine-based solution helps prevent apparent vertical transmission of PRV.

The genome of Border disease virus genotype 8 from chamois by next generation sequencing.

Border disease virus (BDV) is widespread both in domestic small ruminants and wildlife. Here we report the genome of BDV genotype 8 from chamois, strain Italy­58987, obtained by next generation sequencing and the comparison with other pestiviruses. The sequence of 12,245 bp long was aligned to 22 pestivirus genomes and it showed a nt/aa similarity of 81.3/89.4% with BDV genotypes, and 65.9/67.8% with the other  pestiviruses. The genome showed a mean nt/aa similarity of 91.2/95.0% with three Swiss genomes closely related to the BDV­8 5'­UTR and Npro sequences. The identification of divergent BDV­8 isolates in North­Western Italy and in Switzerland suggests that this genotype may have been circulating in a wider area than previously supposed, and may have a high host adaptability.

A comparative study on subacute toxicity of arsenic trioxide and dimethylarsinic acid on antioxidant status in Crandell Rees feline kidney (CRFK), human hepatocellular carcinoma (PLC/PRF/5), and epithelioma papulosum cyprini (EPC) cell lines.

Arsenic (As) is a global contaminant of terrestrial and aquatic environments posing concern for environmental and human health. The effects of subacute concentrations of arsenic trioxide (AsIII) and dimethylarsinic acid (DMAV) were examined using Crandell Rees feline kidney (CRFK), human hepatocellular carcinoma (PLC/PRF/5), and epithelioma papulosum cyprini (EPC). Whole monolayer with suffering cells (confluence 100%, pyknosis and refractive cells; value scale = 2) led to identification of subacute As concentrations for the three cell lines. The selected AsIII concentrations were 1.33 µM for CRFK and 33.37 µM for PLC/PRF/5 and EPC, at 48 hr time point. The selected DMAV concentrations were 0.67 mM for PLC/PRF/5, 1.33 mM for CRFK, and 2.67 mM for EPC for 48 hr. Unlike the AsIII test, the three cell lines did not exhibit marked susceptibility to DMAV-mediated toxicity. Several oxidative stress biomarker levels, directly or indirectly associated with reactive oxygen species (ROS) elimination including superoxide dismutase, catalase, glutathione peroxidases, glutathione reductase, glutathione S-transferase, glyoxalase I, glyoxalase II, and total glutathione, were determined in the three cell lines at 24 and 48 hr. Antioxidant responses in metal-treated cells were significantly altered compared to controls, suggesting a perturbation of redox state. The weakening of antioxidant pathway in either healthy or tumoral cells was greater using AsIII than DMAV. Differences in level of several oxidative stress biomarkers suggest that the oxidative stress mechanism induced by AsIII is distinctly different from DMAV. Multifaceted mechanisms of action underlying ROS generation in tumor and nontumor cells versus AsIII and DMAV exposure are thus involved. Since As-mediated toxicity is quite complex, more data regarding both oxidant-enhancement and oxidant-lowering strategies may be useful to improve knowledge regarding the influence of As on human and animal cells.

Highlighting priority areas for bovine viral diarrhea control in Italy: A phylogeographic approach.

The prevalence and genetic diversity of bovine viral diarrhea virus (BVDV) in a geographic area are largely influenced by live animal trade and management practices. Despite control and eradication programs currently underway in several European countries, the risk of BVDV spread within and among countries is still present. BVDV-1 is the predominant type circulating in European cattle population. In this study, a phylogeographic analysis was applied to the BVDV-1 highest prevalent subtypes in Italy to reconstruct the origin and spatial-temporal distribution and to trace main viral flows between different locations to highlight priority areas for BVDV control. A comprehensive dataset of BVDV-1b (n = 173) and 1e (n = 172) 5' UTR sequences was analysed, including both novel and published sequences from Italy and from European countries bordering and/or with commercial cattle flows with Italy. A common phylogeographic pattern was observed for BVDV-1b and 1e subtypes: interspersion from multiple Italian areas and European countries was widespread until the end of the last century, whereas significant local clusters were observed starting from 2000. These findings support a continuous viral flow among different areas over long time scales with no evidence of significant geographical structure, while local transmission networks are limited to more recent years. Northern Italy has been confirmed as the area of origin of the main clades of both BVDV subtypes at national level, acting both as a crucial area for introduction and a maintenance source for other areas. Piedmont, Central and Southern Italian regions contributed to limited geographical distribution and local BVDV-1b and 1e persistence. On the whole, priority control measures for BVDV-1b and 1e in Italy should be focused on: i) implementation of BVDV systematic control in all Northern Italian regions to break the viral flow from larger to smaller animal populations; and ii) breaking the dynamics of infections in regions with self-maintenance of BVDV by voluntary control programs.

Efficient isolation on Vero.DogSLAMtag cells and full genome characterization of Dolphin Morbillivirus (DMV) by next generation sequencing.

The Dolphin Morbillivirus (DMV) genome from the first Mediterranean epidemic (1990-'92) is the only cetacean Morbillivirus that has been completely sequenced. Here, we report the first application of next generation sequencing (NGS) to morbillivirus infection of aquatic mammals. A viral isolate, representative of the 2006-'08 Mediterranean epidemic (DMV_IZSPLV_2008), efficiently grew on Vero.DogSLAMtag cells and was submitted to whole genome characterization by NGS. The final genome length was 15,673 nucleotides, covering 99.82% of the DMV reference genome. Comparison of DMV_IZSPLV_2008 and 1990-'92 DMV strain sequences revealed 157 nucleotide mutations and 47 amino acid changes. The sequence similarity was 98.7% at the full genome level. Whole-genome phylogeny suggested that the DMV strain circulating during the 2006-'08 epidemics emerged from the 1990-'92 DMV strain. Viral isolation is considered the "gold standard" for morbillivirus diagnostics but efficient propagation of infectious virus is difficult to achieve. The successful cell replication of this strain allowed performing NGS directly from the viral RNA, without prior PCR amplification. We therefore provide to the scientific community a second DMV genome, representative of another major outbreak. Interestingly, genome comparison revealed that the neglected L gene encompasses 74% of the genetic diversity and might serve as "hypervariable" target for strain characterization.

Pseudorabies virus in North-West Italian wild boar (Sus scrofa) populations: prevalence and risk factors to support a territorial risk-based surveillance.

Although  the  eradication  of  Pseudorabies  virus  (PrV)  in  domestic  pigs  is  ongoing,  the circulation  of  this  virus  in  wild  boars  remains  a  threat  in  the  currently  unprotected, 'low prevalence', pig population. In this study, we reported PrV prevalence data and the influence of  possible  risk  factors  in  2  North-West  Italian  wild  boar  populations  (free  and  enclosed) with the goal of supporting the implementation of a risk-based AD surveillance system. Sera from  1,425  wild  boars  were  collected  between  2011  and  2015  and  tested  by  ELISA  for  the presence  of  PrV  antibodies;  the  overall  raw  seroprevalence  was  30.39%  (433/1,425;  CI  95% 28.01-32.85%).  A  significant  difference  was  however  observed  between  the  prevalence rates  of  the  free  range  population  (9.98%;  90/902;  CI  95%;  8.10-12.12%)  and  the  enclosed  population of La Mandria park (Piedmont, Italy) (65.58%; 343/523; CI 95%; 61.51-69.65%). In both populations a significantly higher number of adults and females were found positive to PrV  ELISA.  Specific  territorial  data  on  PrV  circulation  in  wild  boars  should  be  acquired  from other regions for guiding risk-based measures in order to reduce the threat of AD re-infection in a more cost-effective manner.

Microbial agents in macroscopically healthy mammary gland tissues of small ruminants.

BACKGROUND: Health of mammary glands is fundamental for milk and dairy products hygiene and quality, with huge impacts on consumers welfare. METHODS: This study aims to investigate the microbial agents (bacteria, fungi and lentiviruses) isolated from 89 macroscopically healthy udders of regularly slaughtered small ruminants (41 sheep, 48 goats), also correlating their presence with the histological findings. Multinomial logistic regression was applied to evaluate the association between lesions and positivity for different microbial isolates, animal age and bacteria. RESULTS: Twenty-five samples were microbiologically negative; 138 different bacteria were isolated in 64 positive udders. Coagulase-negative staphylococci were the most prevalent bacteria isolated (46.42%), followed by environmental opportunists (34.76%), others (10.14%) and pathogens (8.68%). Most mammary glands showed coinfections (75%). Lentiviruses were detected in 39.3% of samples. Histologically, chronic non-suppurative mastitis was observed in 45/89 glands, followed by chronic mixed mastitis (12/89) and acute suppurative mastitis (4/89). Only 28 udders were normal. Histological lesions were significantly associated with the animal species and lentiviruses and coagulase-negative staphylococci infections. Goats had significantly higher risk to show chronic mixed mastitis compared to sheep. Goats showed a significantly lower risk (OR = 0.26; 95% CI [0.06-0.71]) of being infected by environmental opportunists compared to sheep, but higher risk (OR = 10.87; 95% CI [3.69-37.77]) of being infected with lentiviruses. DISCUSSION: The results of the present study suggest that macroscopically healthy glands of small ruminants could act as a reservoir of microbial agents for susceptible animals, representing a potential risk factor for the widespread of acute or chronic infection in the flock.

Surveillance of Infectious Bovine Rhinotracheitis in marker-vaccinated dairy herds: Application of a recombinant gE ELISA on bulk milk samples.

Infectious Bovine Rhinotracheitis (IBR) occurs worldwide, requiring significant resources for eradication programs or surveillance purposes. The status of infection is usually detected by serological methods using the virus neutralization test (VNT) or enzyme-linked immunosorbent assay (ELISA) on individual sera. The gE DIVA (Differentiating Infected from Vaccinated Animals) vaccines approach, adopted in order to reduce the virus circulation and prevent clinical signs, have tightened the range of available methods for the serological diagnosis. Different gE blocking ELISA could be performed to detect specific antibodies in sera of infected or whole virus-vaccinated animals but with less sensitivity if applied to bulk milk samples, especially in marker-vaccinated herds. A new rec-gE ELISA was recently developed in Italy and applied with good performances on blood serum samples. The present paper focuses on the application of a rapid protocol for purification/concentration of immunoglobulin G (IgG) from bulk milk and on the use of the new rec-gE indirect ELISA. The study involved three different partners and 225 herds (12,800 lactating cows) with different official IBR diagnostic statuses. The diagnostic specificity of the method was demonstrated closed to 100% while the diagnostic sensitivity was strictly related to the herd-seroprevalence. Considering 2.5% as the limit of detection of within-herd seropositivity prevalence, the diagnostic sensitivity showed by the proposed method was equal to 100%. A single reactivation of a whole strain vaccine in an old cow was detected inside a group of 67 lactating cows, showing the field applicability of the method.

Evidence of circulation of the novel border disease virus genotype 8 in chamois.

Evidence of association between the novel putative border disease virus genotype 8 (BDV-8) and fatal disease in an Alpine chamois (Rupicapra rupicapra) is reported. Diagnostically, we also demonstrated, as already previously reported, the failure of BDV-specific primers (PDB1 and PDB2) to detect BDV-8.

Phylogeography, phylodynamics and transmission chains of bovine viral diarrhea virus subtype 1f in Northern Italy.

Bovine viral diarrhea virus (BVDV) type 1 in Italy is characterized by high genetic diversity, with at least 20 subtypes. Subtype 1f is endemic in a restricted geographic area, meaning that it has local distribution. We investigated the population dynamics of BVDV-1f in Northern Italy and characterized the transmission chains of a subset of samples from Piedmont and Aosta Valley regions. A total of 51 samples from 1966 to 2013 were considered and 5' UTR sequences were used for phylogeography. A subset of 12 samples was selected for Npro gene sequencing and further characterization of the transmission chains using both molecular and epidemiological data. Phylogeography estimated the root of BVDV-1f tree in Veneto in 1965. Four significant subclades included sequences clustering by region: Lombardy (n=3), Lombardy and Emilia-Romagna (n=7), Piedmont (n=17), Piedmont and Aosta Valley (n=21). The Piedmont-only subclade has a ladder-like branching structure, while the Piedmont and Aosta Valley subclade has a nearly complete binary structure. In the subset, the outbreak reconstruction identified one sample from Piedmont as the most probable source of infection for the Aosta Valley cases. An ad hoc questionnaire submitted to public veterinarians revealed connections between sampled and non-sampled farms by means of trades, exhibitions and markets. According to the phylogeography, BVDV-1f moved westward, entering from Veneto, and spreading to Lombardy and Emilia-Romagna in the early 1990s, and finally to Piedmont and Aosta Valley in the first decade of 2000s. Both phylogeographic analyses on the whole dataset and on the selection of Npro dataset pointed out that subtype 1f entered Aosta Valley from Piedmont. The integration of molecular and epidemiological data revealed connections between farms, and such approach should be considered in any control plan. In Aosta Valley, the study showed that BVDV1f can be controlled only monitoring the introduction of cattle from Piedmont region.

A new genotype of border disease virus with implications for molecular diagnostics.

Border disease virus (BDV) is a (+) single-stranded RNA pestivirus affecting mainly sheep and goats worldwide. Genetic typing of BDV has led to the identification of at least seven major genotypes. This study reports the detection of a BDV strain from a goat in northwestern Italy during routine investigations. Sequence analysis revealed mutations in the 5'-UTR of the virus with implications for BDV molecular diagnostics. Moreover, subsequent phylogenetic analysis based on the combined 5'-UTR and Npro/partial C genes, showed divergence from known BDV genotypes, revealing the detection of a novel pestivirus group, for which we propose the name BDV genotype 8.

Development and validation of an indirect ELISA as a confirmatory test for surveillance of infectious bovine rhinotracheitis in vaccinated herds.

BACKGROUND: Bovine herpesvirus 1 (BoHV1) is a member of the viral subfamily of Alphaherpesvirinae that infects various species, including cattle, sheep, and goats. The virus causes infectious bovine rhinotracheitis (IBR), which is included in a European list of diseases that may require control and eradication programs. The lack of confirmatory tests affects the validity of diagnostic tools, especially those used for vaccinated herds. In this study, we report the development and validation of an indirect enzyme-linked immunosorbent assay (ELISA) based on BoHV1 glycoprotein E, which was expressed as a secreted recombinant antigen in a mammalian cell system. The performance of the new rec-gE ELISA was compared with that of commercially available indirect and/or blocking ELISAs. RESULTS: The sample set included blood sera from animals from IBR-positive farms, IBR-free farms, and marker-vaccinated farms. The indirect ELISA proposed in this study is based on antibody reactivity against BoHV1 gE, and showed high sensitivity and specificity (98.41 and 99.76 %, respectively). CONCLUSIONS: The ELISA performed well, in terms of both its diagnostic sensitivity and specificity, and as a confirmatory methodology, and therefore should improve the diagnostic protocols used for IBR surveillance.

Potential Pathogenetic Role of Bovine Herpesvirus 4 in Two Dairy Cows with Dermatitis-Pyrexia-Hemorrhagic Syndrome.

Dermatitis, pyrexia, and hemorrhagic syndrome (DPHS) is a rare bovine syndrome of unclear etiology. We describe two DPHS cases, the first to occur in Italy, with clinicopathological findings suggesting a potential pathogenetic role of bovine herpesvirus-4 (BoHV-4).

First report of malignant catarrhal fever in a captive pudu (Pudu puda).

A male, six-year-old pudu (Pudu puda) from an Italian zoo was submitted for postmortem examination after sudden death. Necroscopy revealed non-suppurative bronchopneumonia and degeneration of the liver and haemorrhagic lesions of the thymus, pericardium and spleen. Microscopically, multifocal perivascular mononuclear cell infiltrates were observed in the kidneys, lungs, spleen, and the portal triads of the liver. Histological examination of the brain showed meningitis, vasculitis and perivascular cuffs of mononuclear inflammatory cells. A region of the DNA polymerase gene of malignant catarrhal fever viruses was amplified by real-time PCR and nested PCR. PCR products from the tissue samples were sequenced and analysed. The sequences showed 99% similarity with a portion of the caprine herpesvirus 2 DNA polymerase gene. This is the first report of malignant catarrhal fever in a captive pudu.