Identification of a novel herpesvirus in captive Eastern box turtles (Terrapene carolina carolina)

Herpesviruses are significant pathogens of chelonians which most commonly cause upper respiratory tract disease and necrotizing stomatitis. Herpesvirus infection was identified in two populations of captive Eastern box turtles (Terrapene carolina carolina) using histopathology and polymerase chain reaction (PCR) with DNA sequencing. Necrotizing lesions with eosinophilic to amphophilic intranuclear inclusion bodies were identified in the tissues of one hatch-year individual in January 2013, which was herpesvirus positive by PCR. A separate captive group of adults had an observed herpesvirus prevalence of 58% using PCR in July 2011. In these cases, a novel herpesvirus, Terrapene herpesvirus 1 (TerHV1), was identified and serves as the first herpesvirus sequenced in the genus Terrapene. Similar to the other herpesviruses of the Order Testudines, TerHV1 clusters with the genus Scutavirus of the subfamily Alphaherpesvirinae.     

Three novel herpesviruses associated with stomatitis in Sudan plated lizards (Gerrhosaurus major) and a black-lined plated lizard (Gerrhosaurus nigrolineatus).

Glossal stomatitis was observed in a Sudan plated lizard (Gerrhosaurus major) with severe dyspnea. On necropsy, intranuclear inclusion bodies were seen in the periglottal lingual epithelium. Labial stomatitis was seen in a second Sudan plated lizard and a black-lined plated lizard (G. nigrolineatus). Degenerate polymerase chain reaction (PCR) primers targeting a conserved region of herpesvirus DNA-dependent DNA polymerase gene were used to amplify products from lesions from each lizard. Nucleotide sequencing of the PCR products showed that the sequence from each lizard was unique. Phylogenetic and comparative sequence analyses suggest that these viruses are novel members of the subfamily Alphaherpesvirinae, and they are here termed gerrhosaurid herpesviruses 1-3. Results of our analyses suggest that the genus Gerrhosaurus can be infected by these novel herpesviruses.     

Varanid herpesvirus 1: a novel herpesvirus associated with proliferative stomatitis in green tree monitors (Varanus prasinus)

Stomatitis is a common problem in lizards, and the etiologies of stomatitis in lizards are not well understood. Four green tree monitor lizards (Varanus prasinus) from two different collections were evaluated because of proliferative stomatitis. Degenerate PCR primers targeting a conserved region of herpesvirus DNA-dependent DNA polymerase were used to amplify and sequence a product from gingival tissue of three of four lizards (cases 1, 3, and 4). DNA in situ hybridization of tissues from three lizards was positive for herpesvirus in the oral mucosa of all three lizards tested (cases 1-3) and the brain of two lizards (cases 1 and 3). Comparative sequence analysis suggests that this virus is a novel member of the subfamily alpha-herpesvirinae, and is here termed varanid herpesvirus 1.     

Identification and isolation of a novel herpesvirus in a captive mob of eastern grey kangaroos (Macropus giganteus)

A novel herpesvirus was detected in a captive mob of eastern grey kangaroos (Macropus giganteus) during diagnostic workup for individuals with ulcerative cloacitis. Virus was initially detected in tissues using a consensus herpesvirus PCR. No viral inclusions or particles had been evident in routine histologic or transmission electron microscopic sections of cloacal lesions. Virus was isolated from samples and transmission electron microscopy of the resulting isolates confirmed that the virus was morphologically consistent with a herpesvirus. Nucleotide sequencing of the PCR product from tissue samples and from the isolates revealed that the virus was in the subfamily Gammaherpesvirinae and was distinct from other known herpesviruses. The correlation between the lesions and the novel virus remains unknown. Two herpesviruses, both in the subfamily Alphaherpesvirinae, have previously been described in macropods and are known to cause systemic clinical disease. This is the first reported gammaherpesvirus within the order Marsupialia, and may provide valuable information regarding the evolution and phylogeny of this virus family. Based on current herpesvirus nomenclature convention, the authors propose the novel herpesvirus be named Macropodid herpesvirus 3 (MaHV-3).     

Detection and nucleotide sequencing of a DNA-packaging protein gene of equine gammaherpesviruses.

In previous studies, novel putative viral pathogens designated that asinine herpesvirus 4 (AsHV4) and asinine herpesvirus 5 (AsHV5) were associated with fatal interstitial pneumonia in donkeys (Equus asinus). Nucleotide sequence analysis of a portion of the DNA polymerase gene identified these putative pathogens as herpesviruses and possibly as members of the Gammaherpesvirinae subfamily. Although similar to equine herpesvirus 2 (EHV2) and equine herpesvirus 5 (EHV5), sequence diversity was observed among the detected viruses. In this study, novel sequence is reported for a DNA-packaging protein gene of EHV5 plus AsHV4, AsHV5, and a newly described putative pathogen herein designated asinine herpesvirus 6 (AsHV6). Phylogenetic analysis of these sequences suggested that the equine gammaherpesviruses may form a separate clade within the Gammaherpesvirinae subfamily. Based on the sequence of EHV2 and the novel sequences reported in this study, a PCR assay was developed to detect equine gammaherpesviruses. Products of the predicted size were produced after amplification of DNA from EHV2, EHV5, AsHV4, AsHV5, and AsHV6. This nonnested assay was shown to consistently amplify approximately 10 genomic copies of EHV2. Amplification products were not produced from DNA template of other alpha- and gammaherpesviruses. Because the role of gammaherpesviruses has not been well defined in equine disease, it is envisioned that a single, sensitive PCR assay to detect these potential pathogens will facilitate further assessment of their role in disease.     

Herpesvirus infection in tortoises (Malacochersus tornieri and Testudo horsfieldii)

Large numbers of pancake tortoises (Malacochersus tornieri) and Horsfield tortoises (Testudo horsfieldii) in three consignments imported into Japan died soon after arrival. Some tortoises in the first consignment were dead on arrival. Postmortem examination of two of the pancake tortoises and four of the Horsfield tortoises revealed necrotizing lesions of the oral mucosa in both species, primarily in the tongue. Eosinophilic to amphophilic inclusion bodies were visible in the nuclei of mucosal epithelial cells in the lesions. Similar inclusion bodies were observed in the liver, spleen, adrenal glands, stomach, lungs, kidneys, small and large intestines, pancreas, and cerebrum of the pancake tortoises and in the liver, spleen, and pancreas of the Horsfield tortoises. Electron microscopic examination of the cells containing inclusion bodies showed herpesvirus-like particles about 100 nm in diameter in the cytoplasm. Nested polymerase chain reaction analysis using a herpesvirus consensus primer method confirmed the presence of a characteristic herpesvirus base sequence in tissue from these lesions.     

Transmission and genetic diversity of Enterococcus faecalis among layer chickens during hatch

Background

Studies on transmission of Enterococcus faecalis among chickens during hatch have not been carried out so far. Information about vertical transmission and subsequent spreading and colonization of the cloacal mucosa through cloacal ''drinking'' during hatch are important to understand the epidemiology of E. faecalis infections. In the present investigation vertical transmission and subsequent spreading and colonization of the cloacal mucosa of chickens by E. faecalis through cloacal ''drinking'' were examined.

Methods

Two different batches of layer chickens originating from 45 weeks old Brown and White Lohmann parents, respectively from the same farm were sampled in the hatcher. Isolates were confirmed to be E. faecalis by polymerase chain reaction (PCR) and further by multilocus sequence typing (MLST) to state their population structure and comparison made to sequence types previously obtained from chicken.

Results

A total of 480 chickens were swabbed from the cloacae just after hatch and after 24 hours. A total of 101 isolates were confirmed as E. faecalis by a species specific PCR. The prevalence of E. faecalis increased from 14% at 0 h to 97% after 24 h for the Brown Lohmann chickens and from 0.5% to 23% for the White Lohmann flock. The 84 isolates analysed by MLST were distributed on 14 sequence types (ST). Three ST (401, 82 and 249) accounted for 64% of all isolates analysed by MLST after 24 h. ST 82 has previously been reported from amyloid arthropathy and other lesions in poultry.

Conclusions

The present findings demonstrated a high potential of a few contaminated eggs or embryos to rapidly facilitate the spread of E. faecalis to almost all chickens during hatch.     

Development and field validation of a Mycoplasma iowae real-time polymerase chain reaction assay

A Mycoplasma iowae real-time polymerase chain reaction (PCR) assay using primers and probes targeting the 16S rRNA gene was developed and field-validated in this study. The assay specifically identified M. iowae with a detection limit of 80 colony-forming units (cfu) per turkey cloacal swab sample (3.2 cfu per PCR reaction). It was validated by testing 154 field turkey cloacal swab samples in parallel with culture isolation. The diagnostic sensitivity of the PCR was 97.6%, and the specificity was 95.5%. The real-time PCR developed in this study is a rapid, sensitive, and cost-effective alternative to culture isolation for detecting M. iowae from cloacal swab samples.     

An attenuated herpes vaccine may protect Gyr hybrids from fatal inclusion body hepatitis. A preliminary report

Four Gyr hybrids were used for this falcon herpes vaccine experiment. Three falcons were given 1 ml of an attenuated falcon herpesvirus vaccine (DuFaHe) subcutaneously twice within 14 days, whereas the fourth falcon was used as a control. Eighteen days after the booster vaccination, all four Gyr hybrids were intranasally and ocularly challenged with a virulent low-passage falcon herpesvirus. The control falcon died 9 days after challenge with typical lesions of herpesvirus inclusion body hepatitis. The three vaccinated falcons seroconverted and did not show any symptoms. Following the challenge their antibody titres to falcon herpesvirus increased. No herpesvirus was isolated from any of the cloacal swabs taken during this experiment, indicating that there was no danger for any other birds from DuFaHe. This experiment shows that falcons can be protected from herpesvirus infection by an attenuated herpesvirus vaccine. However, it should be stressed that only four falcons were used for this experiment.     

Geographic variation in marine turtle fibropapillomatosis.

We document three examples of fibropapillomatosis by histology, quantitative polymerase chain reaction (qPCR), and sequence analysis from three different geographic areas. Tumors compatible in morphology with fibropapillomatosis were seen in green turtles from Puerto Rico and San Diego (California) and in a hybrid loggerhead/ hawksbill turtle from Florida Bay (Florida). Tumors were confirmed as fibropapillomas on histology, although severity of disease varied between cases. Polymerase chain reaction (PCR) analyses revealed infection with the fibropapilloma-associated turtle herpesvirus (FPTHV) in all cases, albeit at highly variable copy numbers per cell. Alignment of a portion of the polymerase gene from each fibropapilloma-associated turtle herpesvirus isolate demonstrated geographic variation in sequence. These cases illustrate geographic variation in both the pathology and the virology of fibropapillomatosis.     

Cloacal papillomatosis in the absence of herpesvirus and papillomavirus in a sulphur-crested cockatoo (Cacatua galerita)

CASE HISTORY A 21-year-old male sulphur-crested cockatoo (Cacatua galerita) was presented following the sudden appearance of blood associated with the passage of faeces and urates.

CLINICAL AND PATHOLOGICAL FINDINGS: There was fresh blood-staining of the feathers around the vent. The dorsal mucosal wall of the proctodeum was erythematous and roughened in appearance. An endoscopic biopsy was performed, and histological examination revealed multiple fronds of epithelium; the mucosa varied from simple to pseudostratified columnar epithelium, with diffuse hyperplasia of goblet cells. The underlying connective tissue stroma was well vascularised and was infiltrated with mixed inflammatory cells, comprising granulocytic cells and macrophages. PCR testing for both herpesvirus and papillomavirus, using consensus primers, was negative.

DIAGNOSIS: Cloacal papillomatosis.

CLINICAL RELEVANCE: This case manifested typical clinical signs and histological lesions of cloacal papillomatosis in the absence of demonstrable herpesvirus or papillomavirus. Veterinarians need to consider this disease in the differential diagnosis of blood in the droppings of parrots and cockatoos even in countries where psittacine herpesviruses are exotic diseases.     

Polymerase chain reaction (PCR) for the detection of herpesvirus in tortoises

A polymerase chain reaction (PCR)-based method using a herpesvirus consensus primer was assessed for the identification of herpesviral infections in tortoises. A single band of about 230 bp was detected in PCR products from two out of twenty swabs taken from the oral cavity, three out of three paraffin-embedded tissue sections from the liver (two cases) and oral mucosa (one case), and one out of two fresh tissue samples from the oral mucosa. Nucleotide sequencing of these PCR products indicated that the herpesvirus present in these tortoises might belong to the alphaherpesvirinae. PCR using swabs and biopsy tissues was a sensitive and highly specific method for the diagnosis of herpesviral infections in tortoises.     

Isolation of equine herpesvirus-5 from blood mononuclear cells of a gelding.

Horses are commonly infected by herpesviruses, but isolation of equine herpesvirus-5 (EHV-5) has only infrequently been reported. We describe the isolation and characterization of a strain of EHV-5 from the blood mononuclear cells of a healthy adult horse in California. The virus was initially identified by EHV-5 specific polymerase chain reaction (PCR), and it caused lytic infection of cultured rabbit kidney cells only after repeated serial passage. Virions with characteristic herpesvirus morphology were readily demonstrated in cell culture lysate by transmission electron microscopy. A portion of the glycoprotein B gene of this strain of EHV-5 had 99% identity to the published EHV-5 sequence, and it was clearly distinguishable from other EHV (1-4) by virus-specific PCR assays. Prevalence of EHV-5 infection in a group of young racehorses was estimated at 64% using the EHV-5 specific PCR on nasopharyngeal secretions.     

Morbidity and mortality associated with a new mycoplasma species from captive American alligators (Alligator mississippiensis).

Nine of 74 American alligators (Alligator mississippiensis) from a captive Florida herd of 3-4-m-long, 200-350-kg, adult males greater than 30 yr of age died within a 10-day period during 1995. Nonspecific clinical signs included anorexia, lethargy, muscle weakness, paraparesis, bilateral white ocular discharge, and various degrees of periocular, facial, cervical, and limb edema. Pneumonia, pericarditis, and arthritis were found on postmortem evaluation of the spontaneously dead and euthanatized alligators. Rapidly growing mycoplasmas were identified by culture, and mycoplasma nucleotide sequences were identified by polymerase chain reaction testing of fresh lung and synovial fluid from an affected alligator. Culture of banked frozen lung from necropsy specimens and fresh lung and fresh synovial fluid from newly affected alligators confirmed the presence of a new mycoplasma species in seven of eight individuals. Oxytetracycline was administered, but related deaths continued for 6 mo until only 14 of the initial alligators remained. An enzyme-linked immunosorbent assay to detect antibody was developed, and the organism was transmitted experimentally to naive juvenile alligators, although the source of the organism, Mycoplasma sp. (ATCC 700619), has not been identified. The alligator isolate is a novel species in the mycoplasma family because its nucleotide sequence does not match those of over 75 characterized mycoplasma species. Such factors as population density, animal age, and mycoplasmal virulence likely contributed to the course of disease.     

Development of species-specific PCR techniques for the detection of tortoise herpesvirus.

Previously, a nested polymerase chain reaction (PCR) was employed with consensus degenerate primers targeting highly conserved motifs within herpesviral DNA polymerase genes to detect a newly described tortoise herpesvirus. However, nucleotide sequence information obtained from the final amplified fragment was restricted to a small region of 181 bp. In the present study, additional sequences flanking this segment were determined from a PCR product successfully amplified using a set of known degenerate primers, which covered a 692-bp region within the tortoise herpesviral DNA polymerase gene. Polymerase chain reaction primers for specific amplification of the tortoise herpesviral DNA were designed on the basis of these nucleotide sequences and successfully amplified tortoise herpesviral DNA from the tissues of tortoises that were well characterized histopathologically with herpesviral infection. The lower limit of detection was 1,000 herpesviral DNA equivalents in the presence of normal tortoise genomic DNA. Furthermore, a more sensitive and specific PCR technique for the identification of herpesviral infections in tortoises was developed employing a heminested form, which will enable the detection of latent infections or herpesvirus carriers in tortoises.     

应用逆转录套式PCR检测新城疫病毒核酸

选择DNV融合蛋白保守的编码区域,设计并合成了一对外引物和一对内引物,建立并优化了检测新城疫病毒核酸的逆转录套式PCRI地,通过检测NDV感染的实验客观存在病料和临床病料,结果表明,逆转录套式PCR法最低能鉴别出约0.3pg的NDV RNA,攻毒后第8天还能从非免疫鸡和SPF鸡泄殖腔拭子中检出DNV,第8天非免疫鸡泄殖腔拭子中NDV的最大检出率为5/10,第8天SPF鸡泄殖腔拭子中NDV的最大检出率为6/10,对非免疫鸡和SPF鸡的泄殖腔中NDV最佳检出时间均在攻毒后第5天。逆转录套式PCRY应运地临床样品中DNV的最大检出率为6/7,经核酸杂交验证,该法具有很高的特异性和敏感性,也比较简便快速,为从分子水平探讨NDV的发病机理、临床早期快速诊断提供了新的研究手段。     

Isolation and characterisation of cervine herpesvirus-1 from red deer semen

AIM: This communication describes the isolation of herpesvirus during routine export examination of semen collected from red deer stags in New Zealand. METHODS: Virus isolation was carried out using bovine embryonic lung (BEL) cells and viruses were characterised by direct immunofluorescense, restriction-fragment-length polymorphism analysis (RFLP), polymerase chain reaction (PCR) analysis and nucleotide sequencing. RESULTS: Herpesvirus was isolated from red deer semen on 2 different occasions from different animals. In both cases the virus was identified as cervine herpesvirus-1 (CvHV-1), based on RFLP, PCR and sequence analysis. Nucleotide sequence analysis of the glycoprotein-D gene showed 99.7% homology to the Banffshire strain of CvHV-1 and 89.5%, 89.2%, 85.3% and 79.6% homology to bovine herpesvirus 1.2 (BoHV-1.2), bovine herpesvirus 1.1 (BoHV-1.1), cervine herpesvirus-2 (CvHV-2) and caprine herpesvirus-1 (CpHV-1), respectively. CONCLUSION: This is the first time that CvHV-1 has been isolated in New Zealand. Its inclusion in serological surveys will allow the prevalence of CvHV-1 in the red deer population to be assessed in this country. The clinical significance of CvHV1 infection in New Zealand red deer herds has yet to be determined.     

Analysis of porcine cytomegalovirus DNA polymerase by consensus primer PCR

We used a consensus primer PCR method to amplify a region of herpesviral DNA-directed DNA polymerase gene using degenerate primers for initial characterization of the porcine cytomegalovirus (PCMV) genome. The sequence of the PCR product from PCMV DNA template and its alignment with other herpesvirus DNA polymerase counterparts showed that both conserved amino acid residues and conservative amino acid substitutions are in parallel. Phylogenetic analysis revealed that PCMV should be included in the clade comprising human herpesvirus 6 and 7, rather than human and mouse cytomegaloviruses, in Betaherpesvirus subfamily.     

Keratitis and periocular lesions associated with equine herpesvirus‐3 in a 3‐month‐old filly

A 3‐month‐old Quarter Horse filly presented with corneal ulceration in the right eye with extensive coalescing periocular ulcerations, erosions, and cutaneous crusts. Similar periocular lesions were present around the left eye, on the gingival mucosa, and on the cutaneous and mucosal surfaces of the lips. Based on the severity of the filly's corneal lesions, expense and duration of treatment, euthanasia was elected. Histological post mortem examination revealed numerous hyperplastic and/or dysplastic epithelial cells adjacent to areas of ulceration and erosion with intranuclear viral inclusion bodies. Equine herpesvirus‐3 (EHV‐3) was identified by polymerase chain reaction from the right cornea and lip. The virus was isolated from the right cornea, right eyelid and lip. The dam presented with multifocal to coalescing perineal vesicles. EHV‐3 was confirmed by polymerase chain reaction from the vulvar lesions and the mare recovered spontaneously. This is the first case of EHV‐3 corneal infection reported in horses and emphasises that EHV‐3 should be included as a differential diagnosis for vesicular lesions involving the equine periocular and oronasal epithelium.