Turkey ovarian tissue transplantation: effects of surgical technique on graft attachment and immunological status of the grafts, 6 days post-surgery.

Biobanked poultry ovaries can be revived via transplantation into a recipient female, which upon maturity will produce donor-derived progeny. Previously, a large portion of these recipients also produced recipient-derived progeny, making them gonadal chimeras. These were potentially created when portions of the recipient's ovary were inadvertently left behind. Completely removing the recipient ovary would solve this problem; however, leaving a portion of the recipient's ovary may have inadvertently increased the transplant attachment rate by providing a damaged area for attachment. To test this hypothesis in the turkey, we removed various portions (33-100%) of recipient ovarian tissue and determined the transplant attachment rate. Furthermore, the use of the abdominal air sac membrane as an additional anchoring point was tested. The overall attachment rate of transplants was 91% (27/30), while the average size of the transplants was 4.2 ± 0.6 mm2, 6 d postsurgery. There was no difference (P > 0.05) in the attachment rates, or transplant size between groups with varying amounts of recipent tissue removed, or by using the abdominal air sac membrane as an anchor. Finally, the immunological status of the grafts were evaluated by analyzing the presences of CD3 and MUM-1 (T and B cell markers). This showed that all transplants were infiltrated by large numbers of T and B cells. Shown by a high (P ≤ 0.001) percentage of CD3-positive immunostained cytoplasmic area (49.78 ± 3.90%) in transplants compared to remnant recipient tissue (0.30 ± 0.10%), as well as a high (P ≤ 0.001) percentage of MUM-1-positive immunostained nuclear area (9.85 ± 1.95%) in transplants over remnant recipient tissues (0.39 ± 0.12%). From this study we would recommend removing the entire recipient ovary, and not covering the transplants with the abdominal air sac membrane, to prevent gonadal chimeras. The high levels of lymphocytes within the grafts indicate possible tissue rejection, which could be overcome via immunosuppression with or without histocompatibility matching between donors and recipients.

Evaluation of chickens infected with a recombinant virulent NDV clone expressing chicken IL4.

BACKGROUND: There is evidence that chicken IL4 (chIL4) functions similarly to its mammalian analogue by enhancing type 2 T helper (Th2) humoral immunity and promoting protection against parasitic infections; however, no studies have been performed to assess the effect of chIL4 on the pathogenesis of Newcastle disease (ND). To assess the role of chIL4 in velogenic NDV pathogenesis we created a vNDV infectious clone expressing chIL4. We hypothesized that co-expression of chIL4 during virus replication would result in decreased inflammation caused by the Th1 response and thereby increasing survival to challenge with vNDV. METHODS: To evaluate the effect of chIL4 during early infection with velogenic Newcastle disease virus (NDV) in chickens, recombinant NDV clones expressing either chIL4 (rZJ1-IL4) or a control expressing green fluorescent protein (rZJ1-GFP) were created by inserting an expression cassette in an intergenic region of the NDV genome. The pathogenesis of rZJ1-IL4 was assessed in 4-week-old specific pathogen free chickens. The extent of virus replication was evaluated by titration in mucosal secretions and immunohistochemistry in multiple tissues. Expression of chIL4 was confirmed in tissues using immunohistochemistry. RESULTS: Infection of birds with the rZJ1-IL4 resulted in successful viral replication in vivo and in vitro and generation of the chIL4 in tissues. All birds were clinically normal 2 DPI, with one bird in each group showing conjunctival swelling and enlarged spleens grossly. At 5 DPI, moderate or severe depression was observed in birds infected with rZJ1-GFP or rZJ1-IL4, respectively. Neurological signs and thymic atrophy were observed in one bird infected with rZJ1-IL4. Grossly, conjunctival swelling, mottled spleen and proventricular hemorrhages were observed at 5 DPI in one bird from each group. At 5 DPI, severe necrosis in the spleen, bursa and cecal tonsils were observed in birds infected with rZJ1-GFP, along with minimal evidence of chIL4 expression. In contrast, splenic atrophy, and moderate necrosis in the bursa and cecal tonsils were observed in birds infected with rZJ1-IL4. In addition, chIL4 signal was found in all tissues of rZJ1-IL4 birds at 5DPI. CONCLUSIONS: The production of chIL4 by a recombinant NDV strain resulted in the activation of the positive feedback loop associated with IL4 production. Insertion of chIL4 into NDV may decrease necrosis to lymphoid organs while increasing the severity of lymphoid atrophy and prolonged disease. However, with a low number of birds it is difficult to determine whether these results are significant to disease outcome.

Captive Psittacine Birds in Ontario, Canada: a 19-Year Retrospective Study of the Causes of Morbidity and Mortality.

Psittacines (e.g. parrots, macaws and cockatoos) are common companion animals that are also kept in zoos and private breeding collections. Despite this popularity, long-term, comprehensive studies of diagnostic data from captive psittacines are rare. This study was conducted to assess trends in disease prevalence and to describe causes of morbidity and mortality in psittacines submitted for post-mortem examination to the veterinary hospital and diagnostic laboratory at the University of Guelph, Ontario, Canada. Post-mortem reports of 1,850 psittacines from 1998 to 2017 were assessed and included 110 species from 45 genera. Birds were often diagnosed with infectious disease processes (n = 823; 44.5%), including viral (n = 428; 23.1%), bacterial (n = 284; 15.4%) and fungal (n = 161; 8.7%). Non-infectious disease processes (n = 1,076; 58.2%) were most commonly degenerative (n = 465; 25.1%), metabolic (n = 392; 21.2%) or haemodynamic (n = 270; 14.6%). Exploratory statistical analyses, used to guide further research, revealed significant correlations and associations among disease processes and genera, age categories and sex. This 19-year retrospective study is the first to be conducted in Canada for psittacine birds and provides a broad overview of disease prevalence that can be used as a baseline to inform other studies addressing common and uncommon diseases affecting these birds in the future.

Pathogenesis of New Strains of Newcastle Disease Virus From Israel and Pakistan.

In the past few years, Newcastle disease virus (NDV) strains with epizootic characteristics belonging to subgenotypes VIIi and XIIIb emerged in the Middle East and Asia. In this study, 2 NDV strains-1 representative of subgenotype VIIi isolated in Israel (Kvuzat/13) and 1 representative of subgenotype XIIIb isolated in Pakistan (Karachi/07)-were characterized by intracerebral pathogenicity index and detailed clinicopathologic assessment. The intracerebral pathogenicity index values for Kvuzat/13 and Karachi/07 were 1.89 and 1.85, respectively, classifying these strains as virulent by international standards. In 4-week-old White Leghorn chickens, both strains caused 100% mortality within 4 (Kvuzat/13) and 5 (Karachi/07) days postinfection. Histopathology and immunohistochemistry for NDV nucleoprotein showed that both strains had wide systemic distribution, especially targeting lymphoid organs and mucosa-associated lymphoid tissues in the respiratory and intestinal tracts. Results of the animal experiment confirm that both Kvuzat/13 and Karachi/07 are highly virulent and behaved as velogenic viscerotropic NDV strains.

Neuropathogenic Capacity of Lentogenic, Mesogenic, and Velogenic Newcastle Disease Virus Strains in Day-Old Chickens.

Strains of Newcastle disease virus (NDV) have different abilities to elicit neurologic signs. To determine the capacity of different NDV strains to replicate and cause lesions in the brain, independently of their peripheral replication, 1-day-old chickens were inoculated in the subdural space with 7 NDV strains of different virulence (4 velogenic, 2 mesogenic, 1 lentogenic). Velogenic strains induced severe necrotizing and heterophilic ventriculitis and meningitis, as well as edema of the neuroparenchyma, and replicated extensively in the nervous tissue by day 2 postinfection, as demonstrated by immunohistochemistry, when all infected birds died. Clinical signs, microscopic lesions, and viral replication were delayed (days 3 and 4 postinfection) with mesogenic strains. Velogenic and mesogenic NDV strains replicated mainly in neurons, and immunolabeling was first detected in surface-oriented areas (periventricular and submeningeal), possibly as a reflection of the inoculation route. The lentogenic NDV strain did not cause death of infected birds; replication was confined to the epithelium of the ependyma and choroid plexuses; and lesions consisted of lymphoid aggregates limited to the choroid plexuses. Results show that extensive NDV replication in the brain is typical of velogenic and mesogenic, but not lentogenic, NDV strains. In addition, this study suggests that differences in the rate of NDV replication in nervous tissue, not differences in neurotropism, differentiate velogenic from mesogenic NDV strains. This study indicates that intracerebral inoculation might be used as an effective method to study the mechanisms of NDV neuropathogenesis.

Pathologic characterization of genotypes XIV and XVII Newcastle disease viruses and efficacy of classical vaccination on specific pathogen-free birds.

To characterize the clinicopathologic features of recently described genotypes of Newcastle disease virus (NDV), 1 representative strain of genotype XIV and 2 of genotype XVII, all isolated from West Africa, were used to infect groups of ten 4-week-old specific pathogen-free chickens. The pathobiology of these 3 strains was compared to a South African NDV strain classified within genotype VII. All chickens infected with the 4 viruses died or were euthanized by day 4 postinfection due to the severity of clinical signs. Gross and histologic lesions in all infected chickens included extensive necrosis of lymphoid tissues (thymus, spleen, bursa of Fabricius, cecal tonsils, gut-associated lymphoid tissue), gastrointestinal necrosis and hemorrhages, and severe hemorrhagic conjunctivitis. Immunohistochemical staining revealed systemic viral distribution, and the most intense staining was in the lymphoid organs. Results demonstrate that the 3 West African strains from the previously uncharacterized genotypes XIV and XVII are typical velogenic viscerotropic NDV strains with lesions similar to the South African strain. Under experimental conditions, QV4 and LaSota NDV vaccine strains successfully protected chickens from morbidity and mortality against the genotype VII and one genotype XVII NDV strain, with no significant differences in the amount of virus shed when 2 vaccine schemes were compared.

Synovial lesions in experimental canine Lyme borreliosis.

Borrelia burgdorferi is the causative agent of Lyme disease, which is mainly characterized by lameness in dogs. More than 95% of naturally infected dogs are asymptomatic or subclinical; however, in experimental studies, histologic synovial lesions are consistently observed in asymptomatic dogs inoculated with B. burdgorferi. This study investigates the ability of a synovial histopathologic scoring system, clinicopathologic data, and polymerase chain reaction (PCR) testing to differentiate between B. burgdorferi-infected and uninfected dogs. Eighteen 18-week-old beagles were subject to challenge with B. burgdorferi-infected wild-caught ticks (Ixodes scapularis), and 4 uninfected dogs served as controls. Infection was confirmed by serology (ELISA) and PCR amplification of B. burgdorferi-specific DNA of skin biopsies taken at the tick attachment site. A synovial scoring system from human medicine was adapted and implemented on postmortem synovial samples to discriminate infected and noninfected animals. Application of this system to elbows and stifles with a cumulative joint score cutoff  > 4 showed a sensitivity of 88.2% and a specificity of 100%, with a positive likelihood ratio of infinity and a negative likelihood ratio of 0.12. Complete blood count, serum biochemistry, urinalysis, urine protein:creatinine, urine PCR, synovial and lymph node cytology, and synovial PCR were evaluated but were not reliable indicators of clinical disease.

Early occurrence of apoptosis in lymphoid tissues from chickens infected with strains of Newcastle disease virus of varying virulence.

Newcastle disease virus (NDV), the causative agent of Newcastle disease, is a prevalent problem in the poultry industry and often the cause of severe economic loss. There are many strains of the virus and these have varying virulence. The most virulent strains cause systemic lesions of lymphoid tissues, with necrosis and severe lymphoid depletion. Less virulent strains do not cause as much necrosis, but may predispose to secondary infection with other pathogens. Apoptosis or programmed cell death, has been demonstrated to play a role in the pathogenesis of other paramyxovirus infections, notably those caused by measles and canine distemper viruses. To investigate the role of apoptosis in lymphoid organs during NDV infection, immunohistochemistry for determination of expression of caspase-3, a marker of imminent apoptosis, was performed on formalin-fixed paraffin wax-embedded tissues (spleen, thymus, caecal tonsils and bursa of Fabricius) from 4-week-old chickens infected with NDV strains of varying virulence 2 days previously. The amount of apoptosis was proportional to the severity of the clinical disease elicited by the strains.

Clinicopathological characterization in poultry of three strains of Newcastle disease virus isolated from recent outbreaks.

Newcastle disease is a severe threat to the poultry industry and is caused by Newcastle disease virus, a member of the genus Avulavirus, family Paramyxoviridae. The virus is rapidly evolving, and several new genotypes have been discovered in the past few years. Characterization of these strains is important to evaluate field changes, anticipate new outbreaks, and develop adequate control measures. Three Newcastle disease isolates (APMV-1/duck/Vietnam, Long Bien/78/2002, APMV-1/chicken/Australia/9809-19-1107/1998, and APMV-1/double-crested cormorant/USA, Nevada/19529-04/2005) from recent outbreaks were investigated via clinicopathological assessment, immunohistochemistry (IHC), in situ hybridization, virus isolation, and serology in experimentally infected 4-week-old chickens. Phylogenetic studies showed that Australia isolate belongs to class II genotype I, Long Bien to class II genotype VIId, and Nevada cormorant to class II genotype V. Even though all 3 viruses had a virulent fusion protein cleavage site and ICPI values greater than 1.5, they all differed in their ability to cause clinical signs, in their lesions, and in their viral distribution in body tissues. The Long Bien isolate showed the most severe clinicopathological picture and the most widespread viral distribution. The Australia and Nevada cormorant isolates had a milder pathological phenotype, with viral replication restricted to only a few organs. The variability in clinicopathological characteristics despite the similarity in ICPI suggests that full clinicopathological assessment is necessary to fully characterize new isolates and that there are differences in pathogenesis among viruses of different genotypes.

An in situ hybridization and immunohistochemical study of cytauxzoonosis in domestic cats.

Cytauxzoonosis, caused by the protozoan parasite, Cytauxzoon felis, is a tick-borne disease of domestic cats causing high mortality. The reservoir is wild felids. In this study, 7 archived cases of the disease were examined through in situ hybridization for localization of the parasite and by immunohistochemistry for various cell markers to characterize infected cells. The riboprobe used was specific for the 16S-like rRNA subunit of Babesia microti, which shares 91% identity with the same gene for C. felis. In situ hybridization highlighted the presence of the organism in several tissues, most prominently lung and spleen, and, in general, there were 2 to 10 times more infected cells seen with in situ hybridization than with HE. Parasite-laden cells were usually found within vessels. These cells were often tightly packed and frequently formed parasitic thrombi. Immunohistochemistry with an antilysozyme antibody confirmed the macrophage origin of the infected cells. Using an antibody specific for calprotectin (Mac387), parasitized cells were markedly devoid of this protein, which may explain the lack of diapedesis and vascular crowding of parasitized cells, providing more circulating parasites for the tick vector. Immunohistochemical labeling for 2 proliferation markers, proliferating cell nuclear antigen (PCNA) and p53, indicated that parasitized cells have a heightened replicative ability, which is probably an additional parasite-driven modification to facilitate survival and transmission.