Relative sensitivity of polymerase chain reaction assays used for detection of feline herpesvirus type 1 DNA in clinical samples and commercial vaccines

OBJECTIVE: To determine relative detection rates and detection limits for 6 published polymerase chain reaction (PCR) assays used for detection of feline herpesvirus type 1 (FHV-1) DNA. SAMPLE POPULATION: 5 vaccines licensed for use in preventing FHV-1-associated disease; 15 conjunctival biopsy specimens collected from cats with keratitis, conjunctivitis, or both; and a plaque-purified field isolate of FHV-1 cultured in vitro. PROCEDURE: Vaccines and clinical samples were assessed for FHV-1 DNA by use of all 6 assays. Detection rates were calculated by assuming that any sample in which FHV-1 DNA was detected was a true-positive result. Detection limits were estimated by use of serial dilutions of DNA extracted from cultured FHV-1 and 1 clinical sample. RESULTS: Testing by use of all 6 assays resulted in detection of FHV-1 DNA in all 5 vaccines. Testing by use of all 6 assays yielded concordant results for 9 of 15 conjunctival biopsy specimens (8 with negative results and 1 with a positive result). Calculated detection rates for clinical samples ranged from 29% to 86%. Assay sensitivity was ranked similarly by use of detection rate or detection limit. CONCLUSIONS AND CLINICAL RELEVANCE: Testing by use of all assays was equally likely to detect vaccine virus. Therefore, a positive PCR result in a cat may reflect vaccine virus rather than wild-type virus. Test sensitivity as assessed by detection limits and detection rates varied greatly. Because FHV-1 can be shed in clinically normal animals, high detection rate will not necessarily correlate with high diagnostic sensitivity.     

Assessment of viral presence in semen and reproductive function of frozen-thawed spermatozoa from Pallas' cats (Otocolobus manul) infected with feline herpesvirus.

Although herpesviruses are known to contaminate the semen of several mammalian species, the occurrence of feline herpesvirus type 1 (FHV-1) in semen of infected cats has not been reported. Our objectives in this study were to investigate the presence of FHV-1 DNA in seminal fluid and frozen-thawed spermatozoa from FHV-1 infected Pallas' cats (Otocolobus manul) and assess the functionality of their frozen-thawed spermatozoa in vitro. Over a 3-yr period, semen (n = 33 ejaculates) was collected periodically via electroejaculation from four Pallas' cats chronically infected with FHV-1. Spermic ejaculates were frozen by pelleting on dry ice and stored in liquid nitrogen. After thawing, sperm motility and acrosome status were assessed over time during in vitro culture. For vitro fertilization (IVF), viable domestic cat (Felis silvestris catus) oocytes were inseminated with frozen-thawed Pallas' cat spermatozoa and evaluated for embryo cleavage. For FHV-1 polymerase chain reaction (PCR) analysis, DNA was extracted from seminal fluid, frozen-thawed spermatozoa, inseminated oocytes, heterologous IVF embryos, and conjunctival biopsies and analyzed for presence of a 322-base pair region of the FHV-1 thymidine kinase gene. Immediately post-thaw, sperm motility and percentage of intact acrosomes were decreased (P < 0.05) compared to fresh samples, and declined further (P < 0.05) during culture. However, all frozen-thawed IVF samples were capable of fertilizing domestic cat oocytes (overall, 46.1 +/- 6.0% cleavage). PCR analysis did not identify FHV-1 DNA in any reproductive sample despite the repeated detection of FHV-1 DNA in conjunctival biopsies. These results suggest that semen collected from Pallas' cats infected with FHV-1 does not contain cell-associated or non-cell-associated virus and that frozen-thawed spermatozoa exhibit adequate function for potential genetic rescue with minimal risk of FHV-1 transmission.     

Cytologic findings, and feline herpesvirus DNA and Chlamydophila felis antigen detection rates in normal cats and cats with conjunctival and corneal lesions

Samples were collected from 36 cats with feline herpesvirus (FHV-1)-related ocular disease (conjunctivitis, epithelial or stromal keratitis, or corneal sequestration), and 17 cats without ocular changes. Corneoconjunctival swabs, scrapings and biopsies were tested in various combinations for presence of FHV-1 DNA using single round (sr) polymerase chain reaction (PCR) and nested PCR (nPCR). Additional swabs from the inferior conjunctival fornix were tested by enzyme-linked immunosorbent assay for Chlamydophila felis antigen. Cytologic evaluation was carried out on conjunctival (cats with conjunctivitis) and corneal (cats with keratitis) cytobrush preparations. FHV-1 DNA was detected by PCR in 14 (39%) cats with ocular disease and 1 (6%) of the control group. Agreement between srPCR and nPCR results was significant (P < 0.01). FHV-1 DNA was detected in 3/7 cats with conjunctivitis, 5/6 cats with epithelial keratitis, 3/11 cats with stromal keratitis, and 3/12 cats with corneal sequestration. There was a significant association (P = 0.0027) between viral presence and epithelial keratitis. However, no significant association was found between viral presence and conjunctivitis (P = 0.059), stromal keratitis (P = 0.15), or corneal sequestration (P = 0.18). With respect to FHV-1 DNA detection, intersample agreement was significant (P < 0.03). No sampling technique seemed more likely than another to harvest detectable viral DNA, except for cats with corneal sequestrum in which viral DNA was not detected using corneoconjunctival swabs. FHV-1 DNA was detected in 6/9 samples with intranuclear inclusion bodies and in 6/7 cats with eosinophils on cytologic examination. All samples tested negative for C. felis antigen.     

Establishment and Application of Quantitative Real-time PCR Detection Method for Feline Infectious Rhinotracheitis Virus

Feline herpesvirus type 1 (FHV-1) is the pathogeny of feline infectious rhinotracheitis.Based on the sequence of gD gene of FHV-1, one pair of primers(FHV-1F and FHV-1R) and FHV-1Probe were designed for the quantitative Real-time PCR detection method.The results showed that the method could detect FHV-1 DNA specifically and the sensitivity was 75 fg/μL.The reaction efficiency reached 107%, and the R² value was 0.9965.The coefficient of variance(CV) was below 2%.And the method was more sensitive than common PCR.It was specific, stable, and suitable for clinical diagnosis, detection and epidemiological investigation.Using this method, the Shanghai area pet clinic censorship suspected FHV-1 infected samples were detected, and the positive rate reached 27.78%, which indicated that the incidence of FHV-1 in Shanghai area was at a high level.     

猫传染性鼻气管炎病毒实时荧光定量PCR检测方法的建立及应用

猫传染性鼻气管炎(FIR)病毒是引起猫传染性鼻气管炎的病原,属于猫疱疹病毒1型(FHV-1)。选择FHV-1gD基因设计1对引物FHV-1F、FHV-1R和探针FHV-1Probe,建立FHV-1实时荧光定量PCR检测方法。结果表明,该方法可以特异地检测出FHV-1的DNA,敏感性达到75fg/μL,反应效率达到107%,R²值为0.9965,Ct值的变异系数低于2%。FHV-1实时荧光定量PCR检测方法敏感性高于普通PCR,特异性好、稳定性好,适用于FHV-1的临床诊断、检测和流行病学调查。利用该方法对上海地区宠物门诊送检的疑似FHV-1感染的样品进行检测,其阳性率达到27.78％,表明上海地区宠物猫FHV-1的发病率处于较高水平。     

Assessment of infectious organisms associated with chronic rhinosinusitis in cats

OBJECTIVE: To determine detection rates for feline herpesvirus type 1 (FHV-1), Mycoplasma spp, fungi, and bacteria in flush samples and biopsy specimens from the nasal cavities of cats with and without chronic rhinosinusitis (CRS). DESIGN: Prospective study. ANIMALS: 10 CRS-affected cats and 7 cats without signs of respiratory tract disease. PROCEDURES: Nasal flush samples and biopsy specimens were collected from all cats for bacterial (aerobic and anaerobic), fungal, and mycoplasmal cultures; additional biopsy specimens were collected for virus isolation and polymerase chain reaction (PCR) assay (to detect FHV-1 DNA). RESULTS: Aerobic bacteria were detected in flush samples from 5 of 7 control cats; culture of flush samples from CRS-affected cats yielded aerobic bacteria (9/10 cats), anaerobic bacteria (3/10), and Mycoplasma spp (2/10). No fungal organisms were isolated from any cat. Potential pathogens were isolated significantly more often from CRS-affected cats than from control cats. Bacterial culture of biopsy specimens yielded aerobic bacteria (2/7 control cats and 4/10 CRS-affected cats) and anaerobic bacteria (2/10 CRS-affected cats). Although FHV-1 was not detected in nasal biopsy specimens from control or CRS-affected cats, FHV-1 DNA was detected via PCR assay in specimens from 4 of 7 control cats and 3 of 10 CRS-affected cats. CONCLUSIONS AND CLINICAL RELEVANCE: Compared with findings in control cats, anaerobic bacteria, Mycoplasma spp, and a variety of potentially pathogenic organisms were detected more commonly in samples from cats with CRS. In both groups, FHV-1 was detected via PCR assay as a nonviable organism or in noncultivable amounts.     

Prevalence of feline herpesvirus 1, Chlamydophila felis, and Mycoplasma spp DNA in conjunctival cells collected from cats with and without conjunctivitis

OBJECTIVE: To use PCR assays to determine the prevalence of feline herpesvirus 1 (FHV-1), Chlamydophila felis, and Mycoplasma spp DNA in conjunctival cells collected from cats with and without conjunctivitis; to compare results of conventional and real-time fluorogenic PCR assays for amplification of FHV-1 DNA; and to determine whether copy numbers of FHV-1 DNA are correlated with conjunctivitis. ANIMALS: 55 cats with active conjunctivitis, 39 healthy cats that never had conjunctivitis, and 32 cats with a history of conjunctivitis that had been resolved for at least 3 months. PROCEDURES: Samples were obtained by rolling cotton-tipped applicators on the ventral conjunctiva of awake cats treated topically with proparacaine. The DNA was extracted from the swab specimens and assessed in PCR assays to detect DNA of FHV-1 (fluorogenic PCR assay and conventional PCR assay), Mycoplasma spp (conventional PCR assay), and C felis (conventional PCR assay). RESULTS: Overall prevalence rates of FHV-1, C felis, and Mycoplasma spp as assessed by the conventional PCR assays were 6.7%, 3.2%, and 9.6%, respectively. Percentage concordance between conventional PCR and fluorogenic PCR assays for FHV-1 was 92.5%. There were no significant differences among the 3 groups of cats for the mean copy number of FHV-1 divided by the copy number of glyceraldehyde-3-phosphate dehydrogenase. CONCLUSIONS AND CLINICAL RELEVANCE: Mycoplasma spp were the most prevalent organism detected and was associated with conjunctivitis. This study could not confirm that there are increased copy numbers of FHV-1 DNA in cats with conjunctivitis, compared with the copy numbers for cats without conjunctivitis.     

Evaluation of different sampling methods and results of real-time PCR for detection of feline herpes virus-1, Chlamydophila felis and Mycoplasma felis in cats

Objective To investigate how different sampling techniques affect detection of DNA from feline herpes virus Type 1 (FHV-1), Chlamydophila felis and Mycoplasma felis and to study the correlation between positive test results and clinical signs in cats. Animals Fifty-one cats; 24 with ocular signs and 27 healthy control cats. Procedures Samples were collected from all cats using cotton swabs, conjunctival and corneal biopsies, and corneal scrapings. Samples were analyzed for presence of FHV-1, C. felis, M. felis, and feline DNA, defined by 28S rDNA, by using real-time PCR. Results In affected cats, FHV-1 was detected in only one cat; C. felis and M. felis were not detected in any affected cats. None of the three organisms was detected in any control cats. Feline DNA was demonstrated in all conjunctival samples, in 82% of corneal swabs, 92% of corneal scrapings, and 100% of keratectomy samples. Conclusions Because of the generally low detection rate for FHV-1, C. felis, and M. felis DNA in this study, differences regarding sampling technique could not be determined and correlation between positive test results and degree of clinical signs could not be made. Detection of feline DNA in most samples irrespective of sampling technique, suggests a low prevalence of FHV-1, C. felis and M. felis in this population of cats.     

Results of molecular diagnostic assays targeting feline herpesvirus-1 and feline calicivirus in adult cats administered modified live vaccines

In this pilot study, 12 adult, gang-housed cats that were known to be previously exposed (n=12) to feline herpesvirus-1 (FHV-1) and/or vaccinated against (n=2) feline calicivirus (FCV) and FHV-1 were randomly assigned to one of two groups of six cats each. Nasal and pharyngeal samples were collected from each cat on days -7, -3, and 0 prior to vaccination and on days 3, 7, 10, 14, 17, 21, and 28 after vaccination with an FHV-1, FCV, and panleukopenia (FVRCP) vaccine developed for intranasal (six cats) or parenteral (six cats) use. FHV-1 DNA was amplified from 1/12 cats (1/69 samples; 1.4%) prior to vaccination and 2/12 cats after vaccination (2/154 samples; 1.3%). FCV RNA was amplified from 2/12 cats (2/69 samples; 2.9%) prior to vaccination and 7/12 cats (12/154 samples; 7.8%) after vaccination. Positive molecular diagnostic assay results for FHV-1 and FCV were uncommon prior to or after vaccination in these cats.     

山东省青岛市猫病毒性鼻气管炎流行情况调查

为了解山东省青岛市猫疱疹病毒Ⅰ型(FHV-1)的流行情况,2019年6月至2021年6月共采集猫眼鼻拭子样本218份,提取样本DNA后利用PCR方法进行FHV-1核酸检测,共检出阳性样本22份,阳性率为10.1%(22/218)。宠物救助站猫的FHV-1阳性率(16.7%)略高于其他来源猫,2~6月龄幼猫的FHV-1阳性率(13.6%)略高于成年猫(7.0%),成年公猫阳性率(7.3%)稍高于成年母猫(6.7%),但各组间均无显著性差异(P>0.05)。对2021年3月采集的60份猫血清进行了FHV-1中和抗体检测,共检出阳性样本40份,抗体阳性率为66.7%。结果表明,青岛市猫群中存在一定的FHV-1流行,抗体保护水平不高,应加快国产FHV-1相关疫苗的研制,及时对猫进行免疫预防。     

An optimised protocol for molecular identification of Eimeria from chickens

Molecular approaches supporting identification of Eimeria parasites infecting chickens have been available for more than 20 years, although they have largely failed to replace traditional measures such as microscopy and pathology. Limitations of microscopy-led diagnostics, including a requirement for specialist parasitological expertise and low sample throughput, are yet to be outweighed by the difficulties associated with accessing genomic DNA from environmental Eimeria samples. A key step towards the use of Eimeria species-specific PCR as a sensitive and reproducible discriminatory tool for use in the field is the production of a standardised protocol that includes sample collection and DNA template preparation, as well as primer selection from the numerous PCR assays now published. Such a protocol will facilitate development of valuable epidemiological datasets which may be easily compared between studies and laboratories. The outcome of an optimisation process undertaken in laboratories in India and the UK is described here, identifying four steps. First, samples were collected into a 2% (w/v) potassium dichromate solution. Second, oocysts were enriched by flotation in saturated saline. Third, genomic DNA was extracted using a QIAamp DNA Stool mini kit protocol including a mechanical homogenisation step. Finally, nested PCR was carried out using previously published primers targeting the internal transcribed spacer region 1 (ITS-1). Alternative methods tested included sample processing in the presence of faecal material, DNA extraction using a traditional phenol/chloroform protocol, the use of SCAR multiplex PCR (one tube and two tube versions) and speciation using the morphometric tool COCCIMORPH for the first time with field samples.     

Feline Herpesvirus 1 and Mycoplasma spp. Conventional PCR Assay Results From Conjunctival Samples From Cats in Shelters With Suspected Acute Ocular Infections

Signs of ocular infections like discharge and conjunctivitis occur commonly in cats in shelters and feline herpesvirus 1 (FHV-1), Chlamydia felis, Mycoplasma spp, and feline calicivirus (FCV) are thought to be the most common causes. While molecular assays are available to amplify nucleic acids of each of these agents as single tests or in panels, additional information is needed concerning whether the assay results can be used to predict response to treatment. The objectives of this study were to report results for conventional polymerase chain reaction (PCR) assays that amplify nucleic acids of FHV-1, Mycoplasma spp., C. felis, and FCV from cats with signs of acute ocular and upper respiratory infections in an animal shelter and to determine whether the results are associated with treatment responses to topical administration of cidofovir (anti-FHV-1) or oxytetracycline (anti-Mycoplasma spp. and C. felis). Conjunctival samples were collected from both eyes of 60 cats with ocular signs of disease. Total deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) were extracted from each sample and assayed for DNA of FHV-1, Mycoplasma spp., and C. felis and RNA of FCV by conventional PCR assays. Cats were randomized to be administered either oxytetracycline ointment or cidofovir drops in both eyes and a standardized ocular disease score system was used to determine a total ocular score for each cat prior to treatment on Day 0 and on Day 7. Nucleic acids of one or more agents were amplified from one or both eyes from 39 of 60 cats (65%). FHV-1 DNA (21 cats), Mycoplasma spp. DNA (25 cats) or FCV RNA (2 cats) were amplified most commonly. After treatment for 7 days, 32 of 60 cats (53.3%) were considered improved with 27 of 32 cats (84.4%) having ocular scores of 0 (21 cats) or 1 (6 cats). When the results of the FHV-1 PCR assay were compared to cidofovir treatment responses, the positive and negative predictive values of the assay were shown to be 29.4% and 60%, respectively. When the results of the Mycoplasma spp. PCR assay were compared to oxytetracycline treatment responses, the positive and negative predictive values of the assay were shown to be 40% and 38.5%, respectively. The predictive value of conventional PCR assay results for FHV-1 or Mycoplasma spp. DNA was low, suggesting that performing these tests to formulate a treatment protocol has minimal clinical utility in cats with suspected acute ocular infections.     

Detection of feline herpes virus 1 via polymerase chain reaction and immunohistochemistry in cats with ulcerative facial dermatitis, eosinophilic granuloma complex reaction patterns and mosquito bite hypersensitivity

Ulcerative dermatitis caused by feline herpes virus 1 (FHV-1) is an uncommon disease characterized by cutaneous ulcers secondary to epidermal, adnexal and dermal necrosis. Differential diagnoses for FHV-1 lesions include, but are not limited to, mosquito bite hypersensitivity and eosinophilic granuloma complex. Histopathological diagnosis of FHV-1 dermatitis is based on the detection of the intranuclear inclusion bodies. In cases where intranuclear inclusions are missing but clinical and histological findings are compatible with FHV-1 dermatitis, immunohistochemistry (IHC) and PCRs have been used. In this retrospective study, we evaluated the presence of FHV-1 by IHC and PCR in skin biopsies and compared the results of the two tests. Sixty-four skin biopsy specimens from cats with compatible lesions were reviewed and tested via PCR and IHC for evidence of FHV-1. Polymerase chain reaction was positive in 12 of 64 biopsies; PCR and IHC were positive only in two of 64 biopsies, and these cases were considered true positive cases. The higher number of PCR-positive cases was possibly attributed to amplification of viral DNA from a live attenuated vaccination, but a previous FHV-1 infection with subsequent amplification of latently inserted FHV-1 could not be excluded. If clinical signs and histopathology suggest FHV-1 infection in the absence of typical inclusion bodies, IHC is the preferred diagnostic test; PCR may be useful for initial screening, but due to false positives is not sufficient for a definitive diagnosis.     

Validation of nonnested and real-time PCR for diagnosis of sheep-associated malignant catarrhal fever in clinical samples.

Sheep-associated malignant catarrhal fever (SA-MCF), a frequently fatal disease primarily of certain ruminants, is caused by ovine herpesvirus 2 (OvHV-2). Molecular diagnosis of SA-MCF in affected animals has relied on detection of OvHV-2 DNA using a nested PCR, which has significant potential for amplicon contamination as a routine method in diagnostic laboratories. In this report, a nonnested and a previously developed real-time PCR were validated for detection of OvHV-2 DNA in samples from clinically affected animals. Three sets of blood or tissue samples were collected: 1) 97 samples from 97 naturally affected animals with evidence of clinical SA-MCF; 2) 200 samples from 8 animals with experimentally induced SA-MCF; and 3) 100 samples from 100 animals without any evidence of clinical SA-MCF. Among 97 positive samples defined by nested PCR from clinically affected animals, 95 (98%) were positive by nonnested PCR and 93 (96%) were positive by real-time PCR, respectively. One hundred percent of the samples from the animals with experimentally induced MCF were positive by real-time PCR, while 99% were positive by nonnested PCR. Neither nonnested PCR nor real-time PCR yielded a positive result on any of the 100 nested PCR-negative samples from animals without evidence of clinical MCF. The data confirmed that both nonnested and real-time PCR maintained high specificity and sensitivity for the detection of OvHV-2 DNA in clinical samples.     

猫疱疹病毒1型RPA-Cas12a-LFD检测方法的建立及初步应用

猫疱疹病毒1型(feline herpesvirus-1，FHV-1) 为水痘病毒属的双链DNA包膜病毒，是引起猫上呼吸道感染的主要病原之一。本研究以重组聚合酶扩增(RPA)、Cas12a反式酶切反应和侧流层析试纸条(LFD)显示技术为基础，旨在建立靶向FHV-1 TK基因的RPA-Cas12a-LFD快速可视化检测方法。根据FHV-1 TK基因的保守片段序列设计RPA引物和合成crRNA的引物，并通过反应体系验证、RPA-Cas12a-LFD检测灵敏度和特异性分析，以及临床样本的符合检测，评价FHV-1 RPA-Cas12a-LFD方法的有效性。结果显示，建立的FHV-1 RPA-Cas12a-LFD方法可以特异性地检出FHV-1(与其他猫相关病原无交叉反应)，灵敏度极高(最低检测限为2.35×10^-1 copies·μL^-1)，检测时间短，结果可视化。该方法对20份表现明显症状的患猫呼吸道样本的FHV-1检出率(35%，7/20)高于现有的TB Green qPCR方法(25%，5/20)，对其中5份阳性样本的检测符合率为100%。综上表明，成功建立的FHV-1 RPA-Cas12a-LFD检测方法的特异性好和敏感性极高，不需要特殊的检测设备，可以作为FHV-1现场快速检测的有效工具。     

Ultrastructural findings in feline corneal sequestra

OBJECTIVES: (1) To describe the ultrastructural features of corneal sequestra in cats; and (2) to enhance our understanding regarding the pathogenesis of feline corneal sequestration. METHODS: Nine corneal sequestra were harvested via keratectomy from globes of nine cats. The sequestra were routinely fixed then postfixed for high resolution light and transmission electron microscopy (HR-LM and TEM, respectively). The tissues were embedded in Epon/Araldite. Sections of 0.5-microm thickness were cut and stained with 1% toluidine blue in 1% sodium tetraborate solution for HR-LM. Ultrathin sections were collected on copper grids and stained with uranyl acetate and Sato's lead stain for TEM. Ultrathin sections were examined and the images were captured on an Advantage HR CCD camera using a Hitachi 7500 electron microscope operated at 80 kV. Two healthy corneas from two cats were harvested immediately following euthanasia. These corneal tissues (control samples) were processed in the same manner as the corneal sequestra for HR-LM and TEM. A portion of each sequestrum was also submitted for polymerase chain reaction (PCR) testing for infectious agents including feline herpesvirus-1 (FHV-1), Toxoplasma gondii, Chlamydophila felis and Mycoplasma spp. RESULTS: Ultrastructure of healthy corneal tissues revealed basal corneal epithelial cells aligned adjacent to a thin acellular layer similar to Bowman's layer with underlying tightly packed, regularly arranged, collagen fibrils oriented in different planes. Keratocytes were elongated and had long and irregularly shaped nuclei, and cytoplasm contained rough endoplasmic reticulum and abundant membrane-bound vesicles. In contrast, corneal sequestra contained varying amounts of an amorphous, electron-dense substance, continuous with intact basal epithelial basement membranes peripherally, and overlying corneal ulceration and loosely packed collagen fibrils. Remnants of necrotic keratocytes were seen in spaces between disarranged collagen layers. In all samples, occasional keratocytes exhibited morphology indicative of apoptosis including clumping and margination of chromatin, and shrunken cytoplasm. Varying degrees of inflammation were noted on HR-LM and TEM of affected corneas including peri- and intralesional neutrophils, lymphocytes, plasma cells, and macrophages. Corneal sequestra were FHV-1-positive (n = 3), FHV-1- and T. gondii-positive (n = 1), T. gondii-positive (n = 3), or negative for DNA of these infectious agents (n = 2) using PCR. All corneal sequestra were negative for DNA of Chlamydophila felis and Mycoplasma spp. using PCR. CONCLUSIONS: Apoptosis may play a role in the pathogenesis of feline corneal sequestration independent of the presence of DNA of these infectious organisms. Prospective clinical studies are warranted to further understand the significance of T. gondii in relation to feline corneal sequestration.     

Detection of feline herpesvirus 1 DNA in skin biopsy specimens from cats with or without dermatitis

OBJECTIVE: To compare detection rates of feline herpesvirus 1 (FHV-1) DNA in skin biopsy specimens from cats with herpetic dermatitis, cats with nonherpetic dermatitis, and cats without dermatitis. DESIGN: Prevalence survey. Animals-5 cats (9 biopsy specimens) with herpetic ulcerative dermatitis, 14 cats (17 biopsy specimens) with nonherpetic ulcerative dermatitis, and 8 cats (21 biopsy specimens) without clinically apparent skin lesions. PROCEDURES: A single-phase PCR assay was used to detect FHV-1 DNA in biopsy specimens. Assay results were compared with results of histologic examination. RESULTS: FHV-1 DNA was detected in all 9 biopsy specimens from the 5 cats with herpetic dermatitis and in 1 of 17 biopsy specimens from the 14 cats with nonherpetic dermatitis, but was not detected in any of the 21 biopsy specimens from the 8 cats without dermatitis. When results of histologic examination were used as the gold standard, sensitivity and specificity of the PCR assay were 100% and 95%, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Results confirmed that FHV-1 DNA can be detected in the skin of cats with herpetic dermatitis and suggest that the virus may play a causative role in the disease. In addition, the PCR assay may be useful in confirming a diagnosis of herpetic dermatitis.     

Detection of virulent feline herpesvirus-1 in the corneas of clinically normal cats

To evaluate the clinically normal feline cornea for the presence of virulent feline herpesvirus-1 (FHV-1), corneas from 31 cats (25 with normal eyes and six with active disease or corneal scarring) euthanased at a shelter were collected. Corneas from two specific pathogen-free cats were included as negative controls. Virus isolation (VI), fluorescent antibody (FA) staining and real-time polymerase chain reaction (rt-PCR) were performed on all samples. The presence or absence of dexamethasone in the media was evaluated for its effect on VI. VI was positive for FHV-1 in six corneas from five cats, all with clinically normal eyes. One cornea was positive for feline calicivirus (FCV) in addition to FHV-1, but only in media that included dexamethasone. Eight corneas were positive on rt-PCR for FHV-1, all from cats with clinically normal eyes. All positive VI samples were confirmed with FA staining. VI and rt-PCR were negative for FHV-1 and FCV in cats with active disease or corneal scarring. Data from this study indicate that virulent FHV-1 and FCV can be present in feline corneas that are clinically normal. Dexamethasone may enhance viral spread through a cell receptor mechanism.     

Schirmer tear test values and tear film break-up times in cats with conjunctivitis

OBJECTIVES: (1) To document tear film break-up time (TFBUT) in a group of cats with conjunctivitis; (2) to determine if TFBUTs from cats with conjunctivitis vary significantly from previously established normal values for TFBUT in young cats without ocular disease; (3) to determine if a correlation exists between Schirmer tear test (STT) values and TFBUTs in cats with conjunctivitis; (4) to determine if the TFBUTs in cats with conjunctivitis are influenced by the detection of DNA from feline herpes virus-type 1 (FHV-1), Chlamydophila felis, Mycoplasma spp., and feline calicivirus. ANIMALS STUDIED: Fourteen cats between the ages of 0.8 years to 12 years with active, untreated conjunctivitis and without active keratitis or other ocular or systemic abnormalities were included in this study. Procedures Complete ophthalmic examinations, including TFBUT, were performed on all cats. Polymerase chain reaction (PCR) screening for FHV-1, Chlamydophila felis, Mycoplasma spp., and feline calicivirus was performed on conjunctival swabs from affected eyes and blood samples from all cats. RESULTS: Mean TFBUT for cats in this study was 8.9 (+/- 4.8) s in the right eye (OD) and 8.1 (+/- 4.6) s in the left eye (OS). No correlation existed between mean TFBUTs and mean STT values OD or OS. Conjunctival swabs from seven cats (n = 9 eyes) tested positive via PCR for one of the above infectious agents. Blood samples from nine cats tested positive for FHV-1. Mean TFBUTs for cats from which the DNA from FHV-1 was isolated from the blood were significantly lower than mean TFBUTs for cats from which no such DNA was isolated from the blood. CONCLUSIONS: In this study, the mean TFBUT in cats with conjunctivitis was significantly lower than previously established values for clinically healthy cats. This supports the theory that qualitative tear film deficiency, and thus tear film instability, may play a role in the pathogenesis of feline conjunctivitis. Qualitative tear film deficiency may predispose to the development of conjunctivitis or may occur secondarily to this condition.