Fluorescent tagging of VP22 in N-terminus reveals that VP22 favors Marek’s disease virus (MDV) virulence in chickens and allows morphogenesis study in MD tumor cells

Marek’s disease virus (MDV) is an alpha-herpesvirus causing Marek’s disease in chickens, mostly associated with T-cell lymphoma. VP22 is a tegument protein abundantly expressed in cells during the lytic cycle, which is essential for MDV spread in culture. Our aim was to generate a pathogenic MDV expressing a green fluorescent protein (EGFP) fused to the N-terminus of VP22 to better decipher the role of VP22 in vivo and monitor MDV morphogenesis in tumors cells. In culture, rRB-1B EGFP22 led to 1.6-fold smaller plaques than the parental virus. In chickens, the rRB-1B EGFP22 virus was impaired in its ability to induce lymphoma and to spread in contact birds. The MDV genome copy number in blood and feathers during the time course of infection indicated that rRB-1B EGFP22 reached its two major target cells, but had a growth defect in these two tissues. Therefore, the integrity of VP22 is critical for an efficient replication in vivo, for tumor formation and horizontal transmission. An examination of EGFP fluorescence in rRB-1B EGFP22-induced tumors showed that about 0.1% of the cells were in lytic phase. EGFP-positive tumor cells were selected by cytometry and analyzed for MDV morphogenesis by transmission electron microscopy. Only few particles were present per cell, and all types of virions (except mature enveloped virions) were detected unequivocally inside tumor lymphoid cells. These results indicate that MDV morphogenesis in tumor cells is more similar to the morphorgenesis in fibroblastic cells in culture, albeit poorly efficient, than in feather follicle epithelial cells.     

Differential quantification of cloned CVI988 vaccine strain and virulent RB-1B strain of Marek’s disease viruses in chicken tissues, using real-time PCR

The ‘gold standard’ vaccine against Marek’s disease in poultry is the CVI988/Rispens virus, which is not easily distinguishable, antigenically or genetically, from virulent Marek’s disease herpesvirus. Accurate differential measurement of the CVI988 vaccine and virulent viruses is important to investigate mechanisms of vaccinal protection. Minimal sequence differences between CVI988 and virulent MDV strains restrict the application of molecular diagnostic methods such as real-time PCR to distinguish between these viruses. The use of bacterial-artificial-chromosome (BAC) cloned CVI988 virus, which carries the BAC vector sequences in place of the U_s2 gene, allows its differential quantification from virulent strains using real-time PCR assays that target the BAC vector sequence and the U_S2 gene respectively. These novel assays allowed investigation of replication of both serotype-1 vaccine virus (cloned CVI988) and challenge virus (RB-1B strain) in tissues of individual chickens in an experimental vaccination-challenge model of Marek’s disease.     

Marek’s disease virus and skin interactions

Marek’s disease virus (MDV) is a highly contagious herpesvirus which induces T-cell lymphoma in the chicken. This virus is still spreading in flocks despite forty years of vaccination, with important economical losses worldwide. The feather follicles, which anchor feathers into the skin and allow their morphogenesis, are considered as the unique source of MDV excretion, causing environmental contamination and disease transmission. Epithelial cells from the feather follicles are the only known cells in which high levels of infectious mature virions have been observed by transmission electron microscopy and from which cell-free infectious virions have been purified. Finally, feathers harvested on animals and dust are today considered excellent materials to monitor vaccination, spread of pathogenic viruses, and environmental contamination. This article reviews the current knowledge on MDV-skin interactions and discusses new approaches that could solve important issues in the future.     

Effects of interferon-γ knockdown on vaccine-induced immunity against Marek’s disease in chickens

Interferon (IFN)-γ has been shown to be associated with immunity to Marek’s disease virus (MDV). The overall objective of this study was to investigate the causal relationship between IFN-γ and vaccine-conferred immunity against MDV in chickens. To this end, 3 small interfering RNAs (siRNAs) targeting chicken IFN-γ, which had previously been shown to reduce IFN-γ expression in vitro, and a control siRNA were selected to generate recombinant avian adeno-associated virus (rAAAV) expressing short-hairpin small interfering RNAs (shRNAs). An MDV challenge trial was then conducted: chickens were vaccinated with herpesvirus of turkey (HVT), administered the rAAAV expressing shRNA, and then challenged with MDV. Tumors were observed in 4 out of 10 birds that were vaccinated with HVT and challenged but did not receive any rAAAV, 5 out of 9 birds that were administered the rAAAV containing IFN-γ shRNA, and 2 out of 10 birds that were administered a control enhanced green fluorescent protein siRNA. There was no significant difference in MDV genome load in the feather follicle epithelium of the birds that were cotreated with the vaccine and the rAAAV compared with the vaccinated MDV-infected birds. These results suggest that AAAV-based vectors can be used for the delivery of shRNA into chicken cells. However, administration of the rAAAV expressing shRNA targeting chicken IFN-γ did not seem to fully abrogate vaccine-induced protection.     

Questionnaire study and postmortem findings in backyard chicken flocks in Finland

Background

Although modern commercial poultry production today is based on large farms and intensive husbandry, keeping backyard poultry has regained popularity in industrialized countries. However, the health status of backyard flocks is still relatively poorly documented. A questionnaire was sent to the owners of 376 backyard poultry flocks (<500 birds) in order to study health management procedures and characterize backyard poultry populations in Finland. Information was also collected on the postmortem findings from non-commercial flocks using necropsy data from the Finnish Food Safety Authority (Evira).

Results

Backyard flocks in Finland are small in size (<50 birds), comprising mainly chickens. Based on the results of the questionnaire, the health of such flocks is good, mortality low and vaccinations are not commonly used. Most of the flocks were registered in the national poultry register. The standard biosecurity practices are not generally applied and contact with wild birds, pets and farm animals is frequent, which can make the flocks more prone to infectious diseases. We conducted an 11-year retrospective study of the postmortem necropsy findings of the Evira in order to document the diseases, which caused mortality in backyard chickens in Finland. Necropsy was performed on a total of 132 non-commercial laying hens during 2000 – 2011. The most common postmortem findings were Marek’s disease (27%) and colibacillosis (17%).

Conclusions

This study is the first to report data on characteristics of and management practices for backyard chicken flocks in Finland. Close connections with commercial flocks are rare and farms are usually distantly located suggesting that the risk that these backyard flocks pose to commercial poultry is low.     

Recurrence rates and sites for grade II canine cutaneous mast cell tumors following complete surgical excision.

A retrospective study was performed on 31 dogs with completely excised, grade II, cutaneous mast cell tumors in order to determine recurrence rates and sites. Distant tumor recurrence developed in 22% of dogs, and local tumor recurrence developed in 11% of dogs; however, the vast majority of these animals were incompletely staged initially. Complete surgical excision of grade II mast cell tumors was associated with effective local control in 89% of these dogs. Therefore, adjuvant radiation therapy might not be indicated in the majority of dogs with complete surgical excision.     

Solitary dermal leiomyosarcomas in 12 ferrets.

Twelve 3-6-year-old ferrets (8 males, 3 females, 1 unknown) were presented with single cutaneous nodules. These dermal tumors were characterized histologically by nodular proliferation of neoplastic smooth muscle fibers with marked anisokaryosis and a mitotic rate of >2 mitoses per 10 high-power fields. Neoplastic cells stained strongly for vimentin in all tumors and for smooth muscle actin and desmin in all but 1 tumor. Histologic and immunohistochemical findings suggested a diagnosis of piloleiomyosarcoma for these tumors. Excision was curative in all animals available for follow-up. However, 3 of 5 animals developed adrenal disease within 7 months after removal of the dermal leiomyosarcoma.     

Morbidity,Mortality and Coronavirus Antigen in Previously Coronavirus Free Kittens Placed in Two Catteries with Feline Infectious Peritonitis

Serologically coronavirus free kittens were placed in 2 catteries with a history of feline infectious peritonitis (FIP), each cattery representing 1 of the 2 different predominant clinical characteristics of FIP - effusive and granulomatous. The kittens were clinically observed for 100 days. A 100% morbidity and a 90% mortality was observed. The first signs were observed after 14 and 27 days respectively. The clinical pattern of the disease was similar in all kittens and showed a pattern of recurrent periods of conjunctivitis, upper respiratory and gastrointestinal signs. Once developed, wasting and signs of CNS disturbances were consistent. The “effusive strain” had a 2 weeks earlier onset of signs and death, and a 40% outcome of effusive FIP. Mean survival times during the observation period were 57 ±26 and 57 ±16 (mean ±SD in days), respectively. The death rates were similar in both groups. Feline coronavirus (FCoV) antigen was immunohistochemically detected using indirect immunofluorescence and was present in all kittens and in 93% of the 5 investigated organs (lung, liver, spleen, kidney, and mesenteric lymph node).     

A possible role of capybaras (Hydrochoerus hydrochoeris hydrochoeris) in foot-and-mouth disease (FMD) endemicity

The susceptibility of capybaras exposed to foot-and-mouth disease (FMD) virus by the intramuscular route and the rodents' close coexistence with cattle in FMD endemic ecosystems suggested that the species might play an important role in the virus' survival in the field.In the present study 2 capybaras and 2 cattle were exposed by contact to a capybara inoculated intramuscularly with FMD virus. Both pairs of exposed animals were then used as a contact source with another 2 cattle and 2 capybaras, respectively. All the animals became infected prior to the appearance of clinical lesions in the respective donor animals and developed generalized FMD clinical lesions. Specific neutralizing antibodies and antibodies to virus-infection-associated antigen (VIA) were also developed.Virus was isolated from feces and from throat swabs of 1 of the capybaras up to 17, but not at 23 days post-contact. Virus was isolated from the remaining animals up to 7–14 days post-contact.The results indicate that these rodents might transmit virus over long distances due to their migratory movements, but probably do not act as natural virus reservoirs.     

Bovine leukemia virus: A herd-based control strategy

A study was conducted in order to develop a herd-based control strategy for bovine leukemia virus (BLV). Michigan State University's closed lactating dairy herd of 114 cows was utilized for the study. Ninety-five percent (95%) of the cows were positive for BLV antibodies, as determined by the agar gel immunodiffusion (AGID) test in November 1979. To develop the control strategy program, the following management practices were instituted: there was a complete physical separation of BLV positive from BLV negative animals for 3 years after which the two groups were physically mixed. Different and sterile supplies and equipment were utilized for any veterinary-medicine-related activity. Personnel working on the farm were constantly made aware of the transmission of the disease and how to minimize it. A constant vector control program and other miscellaneous management practices were implemented. The BLV seronegative animals were examined monthly for BLV antibodies starting at 6–7 months of age. The reactor animals were separated from the seronegative animals when the reactors had 2 positive AGID tests consecutively. A positive BLV serotest was not a criterion for culling. Following a 3-year complete separation of positive animals from negative animals, the overall point prevalence decreased from 95 to 34%. The results were further categorized according to 4 age groups of 6–15, 16–23, 24–47 and 48 months and older. The percentage of BLV-positive animals decreased from 19 to 17, 58 to 14, 90 to 33 and 100 to 90% for the respective age groups. Following a 10-month discontinuation of physical separation of positive animals from negative animals, a decrease in the overall point prevalence rate was still observed. The limitations and practical applications of the program are discussed.     

Evaluation of a 3A-truncated foot-and-mouth disease virus in pigs for its potential as a marker vaccine

Foot-and-mouth disease (FMD) is a highly contagious and economically devastating disease of cloven-hoofed animals in the world. The disease can be effectively controlled by vaccination of susceptible animals with the conventional inactivated vaccine. However, one major concern of the inactivated FMD virus (FMDV) vaccine is that it does not allow serological discrimination between infected and vaccinated animals, and therefore interferes with serologic surveillance and the epidemiology of disease. A marker vaccine has proven to be of great value in disease eradication and control programs. In this study, we constructed a marker FMDV containing a deletion of residues 93 to 143 in the nonstructural protein 3A using a recently developed FMDV infectious cDNA clone. The marker virus, r-HN/3A_93–143, had similar growth kinetics as the wild type virus in culture cell and caused a symptomatic infection in pigs. Pigs immunized with chemically inactivated marker vaccine were fully protected from the wild type virus challenge, and the potency of this marker vaccine was 10 PD₅₀ (50% pig protective dose) per dose, indicating it could be an efficacious vaccine against FMDV. In addition, we developed a blocking ELISA targeted to the deleted epitope that could clearly differentiate animals infected with the marker virus from those infected with the wild type virus. These results indicate that a marker FMDV vaccine can be potentially developed by deleting an immunodominant epitope in NSP 3A.     

Immunohistochemical Expression of Potential Therapeutic Targets in Canine Thyroid Carcinoma

Background

Thyroid carcinoma is a common endocrine tumor in the dog. Local invasive growth frequently precludes surgical excision and, in up to 38% of dogs, the tumor has already metastasized by the time of diagnosis. Therefore, it is important to investigate new treatment modalities that may be useful for the large number of dogs with inoperable tumors or metastatic disease.

Hypothesis/Objectives

To investigate the immunohistochemical expression of potential therapeutic targets in canine thyroid tumors.

Animals

74 dogs with thyroid neoplasia.

Methods

Immunohistochemistry was performed for thyroglobulin, calcitonin, vascular endothelial growth factor (VEGF), p53, cycloxygenase‐2 (cox‐2), and P‐glycoprotein (P‐gp).

Results

Fifty‐four (73%) tumors were classified as follicular cell thyroid carcinomas (FTCs) and 20 (27%) as medullary thyroid carcinomas (MTCs). Eighty percent of FTCs and all MTCs had a high percentage (76–100%) of neoplastic cells immunopositive for VEGF. Thirteen percent of FTCs and 50% of MTCs expressed cox‐2. Seven percent of FTCs and 70% of MTCs expressed P‐gp. No tumor was immunopositive for p53 expression. Expression of VEGF (P = .034), cox‐2 (P = .013), and P‐gp (P < .001) was significantly higher in MTCs compared to FTCs.

Conclusions and Clinical Importance

VEGF is a potential therapeutic target in both FTC and MTC in dogs. Cox‐2 and P‐gp may be useful molecular targets in canine MTC.     

Comparative efficacy of BAC-derived recombinant SB-1 vaccine and the parent wild type strain in preventing replication, shedding and disease induced by virulent Marek’s disease virus

A widely used vaccine against Marek’s disease (MD) in poultry is the virus SB-1, which is antigenically-related to the causative agent, Marek’s disease herpesvirus. We recently cloned the SB-1 genome as an infectious bacterial artificial chromosome, BAC, (pSB-1). The protective efficacies and replication kinetics of pSB-1 and the parent strain (SB-1) were compared in an experimental model of MD induced by a virulent strain, RB-1B. Although vaccine virus replication and shedding was lower for pSB-1 than for SB-1, both vaccines reduced replication and shedding of RB-1B, and were equally effective in protecting chickens against MD. With the cloning of pSB-1, we have now generated full length genomic clones of MD vaccine virus strains belonging to each of the three serotypes. Vaccine viruses derived from each of these clones demonstrated protective efficacies at levels similar to those produced by the respective parent viruses, demonstrating their suitability to be used as vaccine candidates.     

Clinical Features and Treatment Outcomes of 41 Dogs with Sublingual Ectopic Thyroid Neoplasia

Background

Thyroid neoplasia is common in dogs, but there are few reports of dogs with ectopic, sublingual thyroid tumors.

Objectives

To describe clinical features and outcomes of dogs with ectopic, sublingual thyroid neoplasia.

Animals

Five hundred and forty‐four dogs with thyroid neoplasia.

Methods

This retrospective study reviewed the medical records of dogs referred for thyroid neoplasia between 1995 and 2013. Data extracted included signalment, extent of thyroid disease (eutopic or ectopic; metastasis), serum thyroxine (T₄) concentration, treatment, and survival.

Results

Of 544 dogs with thyroid neoplasia, 41 (7.5%) dogs had ectopic sublingual thyroid tumors. The clinical features of these 41 dogs were similar to the cohort group of 503 dogs with eutopic or ectopic mediastinal thyroid tumors, but dogs with sublingual tumors were younger and less likely to have metastatic disease (15% versus 30%, P < .05). Of the 41 dogs, 28 received treatment: 21 with surgery (which included partial hyoidectomy in 13), 7 with radioiodine alone, and 13 with surgery followed by administration of radioiodine. Overall median survival was 562 days (range, 1‐1,850 days).

Conclusions and Clinical Importance

When compared with eutopic thyroid carcinomas, ectopic sublingual thyroid tumors generally have a less aggressive biologic behavior. Many dogs have prolonged survival, even without treatment, although death because of local tumor invasiveness or metastasis can develop in some dogs. Surgical thyroidectomy, including partial hyoidectomy, is generally effective for control of local disease. Administration of radioiodine, alone or in combination with surgical treatment, is recommended for multifocal disease or metastasis.     

Polycythemia in dogs with chronic hypoxic pulmonary disease

BackgroundProlonged tissue hypoxia caused by chronic pulmonary disease is commonly regarded as an important mechanism in the development of secondary polycythemia, but little clinical data are available to support this hypothesis.ObjectiveTo study the prevalence and severity of erythrocytosis accompanying chronic hypoxic pulmonary disease in dogs.AnimalsForty‐seven dogs with hypoxic chronic pulmonary disease, 27 dogs with nonhypoxic chronic pulmonary disease, and 60 healthy controls.MethodsDogs with chronic pulmonary disease and chronic hypoxemia (partial pressure of arterial oxygen [PaO₂] < 80 mm Hg on at least 2 arterial blood gas measurements a minimum of 1 month apart) were identified retrospectively from patient records. Association between arterial oxygen and red blood cell parameters was analyzed using Pearson''s correlation coefficients and multivariable linear regression analysis.ResultsRed blood cell parameters measured at the end of the hypoxemia period were within the laboratory reference range in most dogs. In chronically hypoxemic dogs, hematocrit (Hct) was increased in 4/47 (8.5%; 95% confidence interval [CI], 0‐17) dogs, erythrocyte count (Erytr) was increased in 12/47 (26%; 95%CI, 13‐38) dogs and hemoglobin concentration (Hb) was increased in 3/47 (6.4%; 95%CI, 0‐14) dogs. No marked polycythemia (Hct ≥65%) was noted in any of the dogs. Red blood cell parameters were not associated with the severity of hypoxemia (correlation to PaO₂: Erytr, r = −.14; Hb, r = −.21; Hct, r = −.14; P > .05 for all).Conclusions and Clinical ImportancePolycythemia is uncommon, and usually mild if present, in dogs with chronic hypoxia caused by pulmonary disease.     

Endocrine Changes after Mating in Pregnant and Non-Pregnant Llamas and Alpacas

Plasma concentrations of oestradiol-17ß, progesterone, 15-keto–dihydro–PGF_2α and luteinizing hormone (LH) were monitored in llamas and alpacas after mating with an intact male. Concentrations of LH and PGF_2α metabolite were high immediately after copulation. Ovulation occurred in 92% of the animals. The first significant increases in progesterone were recorded on day 4 after mating. In non-pregnant animals the lifespan of the corpus luteum was estimated to be 8–9 days. Luteolysis occurred in association with the release of PGF_2α. In pregnant animals, a transient decrease in progesterone concentrations was observed between days 8 and 18 in both species. No significant changes in PGF_2α secretion were registered during this period. Oes– tradiol–17ß concentrations were high on the day of mating, declined to low values on day 4, and started to increase again on day 8. Peak values after luteolysis in non-pregnant animals were significantly higher than those registered in pregnant ones. Furthermore, concentrations of oestradiol-17ß were elevated for a longer period in non–pregnant than in pregnant animals. The results suggest that progesterone from the corpus luteum exerts a negative influence on follicular activity in pregnant animals by reducing oes– tradiol-17ß secretion.     

Cystosonographic measurements of canine bladder tumours

The purpose of this study was to evaluate the variability of cystosonographic bladder tumor measurements with both operator and bladder volume changes. Ten dogs with bladder tumors were included. In each dog, three operators determined tumor dimensions for three different bladder volumes. Intraclass correlation coefficient was used to assess operator reliability. Response Evaluation Criteria in Solid Tumors (RECIST) and greater than or equal to 50% differences in tumor measurements were used as guidelines. Poor to fair correlations between operators were found for the different tumor dimensions (r_I = 0.4 – 0.7). The percent differences in tumor dimensions with operator and bladder volume changes were significant enough to misclassify the tumors into the categories of partial response (PR) or progressive disease (PD). These results suggest that cystosonographic measurements of bladder tumors are affected by both changes in operator and bladder volume, and the discrepancies are significant enough to change response classification.     

Granulosa cell tumor in 8 African pygmy hedgehogs (Atelerix albiventris)

A retrospective study involving eight African pygmy hedgehogs histopathologically diagnosed with granulosa cell tumors was conducted. The age at onset was 2.2–4.5 years, with a median age of 3.6 years. The most common clinical signs were hematuria and abdominal distension, which were observed in >50% cases. Exploratory laparotomy was performed in all cases, and ovariohysterectomy or excision of the abdominal mass was performed. Patients with only hematuria survived for >250 days after surgery, whereas those with initial ascites showed recurrence of ascites or tumor growth and survived for approximately 130 days after surgery. Intraperitoneal injection of carboplatin was performed in three recurrent cases. In one of these three cases, the tumor mass disappeared. Hence, carboplatin can be considered a potential antineoplastic drug for the treatment of granulosa cell tumors.     

INVESTIGATION OF GREEN TURTLE (CHELONIA MYDAS) CUTANEOUS FIBROPAPILLOMATOSIS RECURRENCE RATES FOLLOWING DIODE LASER SURGERY

A pilot study was performed to investigate fibropapillomatosis (FP) recurrence rates after 940 nm-diode laser surgeries. Fifteen animals had a total of 274 tumors removed by diode laser. Cutaneous FP recurrence rates were evaluated during a period of 1-year. According to tumor sizes classification, 166 tumors (60.6%) were <1 cm, 67 tumors (24.5%) between 1-4 cm, 31 tumors (11.3%) >4-10 cm and 10 tumors (3.6%) >10 cm. Seven animals (46.6%) were lightly afflicted, 4 (26.7%) moderately, and 4 (26.7%) heavily afflicted. Thirteen animals (86.7%) did not present tumor regrowth and 2 (13.3%) presented small tumors after 1-year.     

Effect of Vaccination in the Field with the Theileria annulata (Hisar) Cell Culture Vaccine on Young Calves Born during the Winter Season

The responses were monitored of young crossbred calves vaccinated against tropical theileriosis during the winter against a field tick challenge in the disease season. Thirty-eight calves below 2 months of age, born after the end of the disease season, were selected at an organized farm. Twenty-five animals were vaccinated with Theileria annulata (Hisar) cell culture vaccine (developed at CCS HAU Hisar laboratory) after the end of the disease season and 13 calves were kept as non-vaccinated controls. These calves were observed for their susceptibility to theileriosis in the new disease season. There was an increase in antibody titre in 18 of the 25 vaccinated animals one month after vaccination. The antibody titre then declined gradually, but remained higher than those of the non-vaccinated animals at month 0. No fever or other clinical signs of tropical theileriosis were observed in any of the vaccinated animals. Nine out of 25 (36%) vaccinated calves showed occasional piroplasms (<;0.5%) in blood smears. All the vaccinated animals withstood the field tick challenge. On the other hand, 9 of the 13 (69%) unvaccinated calves exhibited occasional piroplasms, and included three clinical cases of tropical theileriosis. These observations suggest that young crossbred calves vaccinated with the T. annulata (Hisar) cell culture vaccine at the end of the disease season were relatively resistant during the next disease season.