Indicators of the molecular pathogenesis of virulent Newcastle disease virus in chickens revealed by transcriptomic profiling of spleen

Newcastle disease virus (NDV) has caused significant outbreaks in South-East Asia, particularly in Indonesia in recent years. Recently emerged genotype VII NDVs (NDV-GVII) have shifted their tropism from gastrointestinal/respiratory tropism to a lymphotropic virus, invading lymphoid organs including spleen and bursa of Fabricius to cause profound lymphoid depletion. In this study, we aimed to identify candidate genes and biological pathways that contribute to the disease caused by this velogenic NDV-GVII. A transcriptomic analysis based on RNA-Seq of spleen was performed in chickens challenged with NDV-GVII and a control group. In total, 6361 genes were differentially expressed that included 3506 up-regulated genes and 2855 down-regulated genes. Real-Time PCR of ten selected genes validated the RNA-Seq results as the correlation between them is 0.98. Functional and network analysis of Differentially Expressed Genes (DEGs) showed altered regulation of ElF2 signalling, mTOR signalling, proliferation of cells of the lymphoid system, signalling by Rho family GTPases and synaptogenesis signalling in spleen. We have also identified modified expression of IFIT5, PI3K, AGT and PLP1 genes in NDV-GVII infected chickens. Our findings in activation of autophagy-mediated cell death, lymphotropic and synaptogenesis signalling pathways provide new insights into the molecular pathogenesis of this newly emerged NDV-GVII.

Parvoviral enteritis and salmonellosis in raccoons with sudden death

Eight of 9 juvenile raccoons at a rehabilitation center died without obvious prior clinical signs. Gross changes were unremarkable except for mildly distended intestines. Microscopically, crypt loss, distension, necrosis, and regeneration with intranuclear viral inclusions were observed in the small intestine, with marked lymphoid depletion and necrosis in Peyer patches and mesenteric lymph nodes. Immunohistochemistry with a canine parvovirus antibody showed intensive signals of parvoviral antigens in the crypts and lymphoid germinal centers. Metagenomic sequencing allowed assembly of a complete parvoviral genome with >99% identity to canine parvovirus 2a, as well as Salmonella enterica subsp. enterica. Also, S. enterica subsp. enterica serovar Thompson with multiple antimicrobial resistance was isolated from the intestinal contents. Concurrent infection with parvovirus and Salmonella should be included as a differential diagnosis in raccoons with sudden death.

One Dose of a Novel Vaccine Containing Two Genotypes of Porcine Circovirus (PCV2a and PCV2b) and Mycoplasma hyopneumoniae Conferred a Duration of Immunity of 23 Weeks

Porcine circovirus type 2 (PCV2) and Mycoplasma hyopneumoniae (Mhyo) are important swine pathogens for which vaccination is a key control strategy. Three separate studies were performed to evaluate the duration of immunity (DOI) conferred by a novel vaccine combining PCV2a/PCV2b and Mhyo into a ready-to-use formulation. In each study, three-week-old naïve piglets were vaccinated (Day 0) and challenged 23-weeks later (Day 159) with either PCV2a, PCV2b or Mhyo. Pigs were euthanized three-to-four-weeks post-challenge. Vaccinated pigs had significantly lower PCV2 viremia from Day 168 until Day 175 (PCV2a study) or until euthanasia (PCV2b study), respectively. Fecal shedding was significantly lower for PCV2a-challenged from Day 171 until Day 178, and for PCV2b-challenged from Day 172 until euthanasia. In the PCV2a challenge study, there were no differences among vaccinates and controls in terms of percent of pigs positive for PCV2 immunohistochemistry, histiocytic replacement, or lymphoid depletion. However, significant differences for immunohistochemistry and histiocytic replacement, not lymphoid depletion, were observed among vaccinates and controls following PCV2b challenge. Vaccination supposed a significant reduction in the mean percentage of Mhyo-like lesions in the lung. Percentages of lung tissues positive for Mhyo via immunohistochemistry were 49.3% and 67.1% for vaccinated and control groups, respectively. One dose of the novel PCV2a/PCV2b/Mhyo vaccine conferred robust protection against challenge 23-weeks later for all three fractions.

Yellow Fever as Cause of Death of Titi Monkeys (Callicebus Spp.)

From 2016 to 2018, an epidemic wave of yellow fever (YF) occurred in Brazil, affecting a large number of Platyrrhini monkeys. Titi monkeys ( Callicebus spp.) were severely affected yet pathological characterizations are lacking. This study characterized epizootic YF in 43 titi monkeys ( Callicebus spp.) with respect to the microscopic lesions in liver, kidney, spleen, heart, brain, and lung, as well as the distribution of immunolabeling for YF virus antigen, and the flaviviral load in the liver. Of 43 titi monkeys examined, 18 (42%) were positive for yellow fever virus (YFV) by immunohistochemistry or reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). Affected livers had consistent marked panlobular necrotizing hepatitis, lipidosis, and mild inflammation, with intense immunolabeling for YFV mainly in centrilobular hepatocytes (zone 1; P = .05). In the spleen, consistent findings were variable lymphoid depletion (10/11), lymphoid necrosis (lymphocytolysis; 4/11), and immunolabeling for YFV in histiocytic cells (3/16). The main finding in the kidney was multifocal acute necrosis of tubular epithelium (5/7) that was occasionally associated with intracytoplasmic immunolabeling for YFV (6/15). These data indicate that titi monkeys are susceptible to YFV infection, developing severe hepatic lesions and high viral loads, comparable to humans and Alouatta spp. Thus, Callicebus spp. may be reliable sentinels for YF surveillance.

Indicators of the molecular pathogenesis of virulent Newcastle Disease Virus in chickens revealed by transcriptomic profiling of spleen

Newcastle disease virus (NDV) has caused significant outbreaks in South-East Asia, particularly in Indonesia in recent years. Recently emerged genotype VII NDVs (NDV-GVII) have shifted their tropism from gastrointestinal/respiratory tropism to a lymphotropic virus, invading lymphoid organs including spleen and bursa of Fabricius to cause profound lymphoid depletion. In this study, we aimed to identify candidate genes and biological pathways that contribute to the disease caused by this neurotropic velogenic NDV-GVII. A transcriptomic analysis based on RNA-Seq of spleen was performed in chickens challenged with NDV-GVII and a control group. In total, 6361 genes were differentially expressed that included 3506 up-regulated genes and 2855 down-regulated genes. Real-Time PCR of ten selected genes validated the RNA-Seq results as the correlation between them is 0.98. Functional and network analysis of DEGs showed altered regulation of ElF2 signalling, mTOR signalling, proliferation of lymphatic system cells, signalling by Rho family GTPases and synaptogenesis signalling in spleen. We have also identified modified expression of IFIT5, PI3K, AGT and PLP1 genes in NDV-GVII infected chickens. Our findings in activation of autophagy-mediated cell death, lymphotropic and synaptogenesis signalling pathways provide new insights into the molecular pathogenesis of this newly emerged NDV-GVII.

Molecular and pathological characterisation of genotype VII Newcastle disease virus on Egyptian chicken farms during 2016–2018

Newcastle disease virus (NDV) remains a constant threat to the poultry industry even with intensive vaccination programmes. In the present study, 40 samples were collected from farms showing high mortalities in some Egyptian governorates between 2016 and 2018. Tracheal samples were collected for virus isolation and confirmed by real-time RT-PCR. Molecular characterisation was performed by sequencing, followed by phylogenetic analysis of the novel sequences. Histopathological and immunohistochemical examinations were performed on different organs from NDV-infected broilers. The phylogenetic analysis revealed that the NDV isolates from different areas of Egypt were genetically closely related and all belonged to genotype VII. The histopathological hallmarks included haemorrhagic tracheitis, interstitial pneumonia with syncytia formation, haemorrhagic proventriculitis, necrotising pancreatitis, pan-lymphoid depletion, non-suppurative encephalitis and nephritis. Immunological detection of NDV antigen clarified the widespread presence of viral antigen in different organs with severe lesions. The present study confirmed that a virulent NDV of genotype VII became the predominant strain, causing severe outbreaks in poultry farms in Egypt. The presence of viral antigen in different organs indicates the pantropic nature of the virus. Immunohistochemistry was a very useful diagnostic tool for the detection of NDV antigen.

Postnatal toxicity of copper oxychloride in lactating female albino rats

Objective: To evaluate the postnatal toxicity of copper oxychloride (COC) in lactating female albino rats. Design: Randomized controlled experimental study. Animals: Eighteen pregnant female albino rats weight 150±10 g and 6-7 week old. Procedures: Eighteen pregnant female albino rats were divided into 3 groups treated orally with copper oxychloride 0, 73.5, 147 mg/kg (equivalent 1/20 and 1/10 of LD50) daily from first day of parturition for 21 days. Female rats and its offspring were euthanized at 21 days of treatment. The postnatal toxic effect in the neonates and dams were estimated through biochemical biomarkers as metabolic and oxidative stress biomarkers, histopathological and hematological evaluation. Results: There was a significant increase in liver enzymes activities as ALT and GGT and oxidative stress biomarker as MDA (P < 0.05) in both suckling pups and lactating dams beside decrease the level of metabolic biomarkers as glucose, total protein and cholesterol (P < 0.05). Additionally, antioxidant enzymes as SOD and CTA (P < 0.05) were reduced significantly in the treated groups in a dose dependent manner in comparison to control group. Moreover the histopathological revealed that Dams treated with COC at both doses shown degenerative changes and intralobular histiocytic infiltration with intralobular fibroblastic proliferation in the hepatic tissue. Neuronal necrosis, neuronophagia and astrocytosis in the brain tissue with degenerative changes of purkinje cells in cerebellum. Fibrous tissue proliferation of renal glomeruli with degenerative changes in renal tubular epithelium and marked lymphoid depletion in the splenic tissue in dose dependent manner. While Suckling pups of treated dams with different doses of COC shown focal histiocytic and lymphocytic infiltration besides congestion of portal vein and margination of leukocytes, focal edema in the neuropils with focal hemorrhagic areas in the brain tissue, degeneration in the renal glomeruli and severe lymphoid depletion with congestion of the splenic sinusoids in dose dependent manner. Conclusion and clinical relevance: The potential postnatal toxic effect of copper oxychloride in neonates and lactating female albino rats through transmammary transmission in milk clarified from biochemical and histopathological evaluation of dams and its pups.

The Pathology of Cetacean Morbillivirus Infection and Comorbidities in Guiana Dolphins During an Unusual Mortality Event (Brazil, 2017–2018)

Cetacean morbillivirus (CeMV; Paramyxoviridae) is the most significant pathogen of cetaceans worldwide. The novel “multi-host” Guiana dolphin ( Sotalia guianensis; GD)-CeMV strain is reported in South American waters and infects Guiana dolphins and southern right whales ( Eubalaena australis). This study aimed to describe the pathologic findings, GD-CeMV viral antigen distribution and detection by RT-PCR (reverse transcriptase polymerase chain reaction), and infectious comorbidities in 29 Guiana dolphins that succumbed during an unusual mass-mortality event in Rio de Janeiro state, Brazil, between November 2017 and March 2018. The main gross findings were lack of ingesta, pulmonary edema, ascites, icterus, hepatic lipidosis, multicentric lymphadenomegaly, as well as pneumonia, polyserositis, and multiorgan vasculitis caused by Halocercus brasiliensis. Microscopically, the primary lesions were bronchointerstitial pneumonia and multicentric lymphoid depletion. The severity and extent of the lesions paralleled the distribution and intensity of morbilliviral antigen. For the first time in cetaceans, morbilliviral antigen was detected in salivary gland, optic nerve, heart, diaphragm, parietal and visceral epithelium of glomeruli, vulva, and thyroid gland. Viral antigen within circulating leukocytes suggested this as a mechanism of dissemination within the host. Comorbidities included disseminated toxoplasmosis, mycosis, ciliated protozoosis, and bacterial disease including brucellosis. These results provide strong evidence for GD-CeMV as the main cause of this unusual mass-mortality event.

Sequential Pathology of a Genotype XIII Newcastle Disease Virus from Bangladesh in Chickens on Experimental Infection

The sequential pathology of a genotype XIII Bangladeshi strain of Newcastle disease virus (NDV) was studied in 5-weeks old chickens. Layer chickens of ISA Brown breed were inoculated through the intranasal and intraocular routes with the BD-C161/2010 strain of NDV and examined at different times post-infection (pi). NDV-infected chickens showed depression at 3 days pi (dpi) followed by dropped wings, paralysis and death starting at 4 dpi. Lungs of infected chickens showed hemorrhagic lesions starting at 24 hours pi (hpi) that was followed by pallor and slight contraction by 2 to 3 dpi and subsequently developed into severe hemorrhagic pneumonia with mononuclear cell infiltration. Hemorrhagic and necrotizing lesions were found in different visceral organs including proventriculus, intestine, gut-associated lymphoid tissues, liver and kidneys starting at 3 dpi that progressed rapidly. Severe lymphoid depletion was observed in the thymus, spleen and bursa of Fabricius starting at 1–3 dpi followed by hemorrhages, necrosis, inflammation and atrophy at 4–5 dpi. In the brain, mild neuronal lesions such as focal to diffuse encephalitis with encephalomalacia was observed at 2–3 dpi and moderate and diffuse meningoencephalitis with encephalomalacia at advanced stages. In conclusion, the BD-C161/2010 strain of NDV produced lesions typical of velogenic viscerotropic pathotype of NDV.

Natural Canine Distemper Virus Infection in Linnaeus’s 2-Toed Sloths (Choloepus didactylus)

An outbreak of canine distemper virus in a private zoo in eastern Tennessee in July 2016 led to fatal clinical disease in 5 adult, wild-caught Linnaeus’s 2-toed sloths ( Choloepus didactylus). Clinical signs included hyporexia, lethargy, mucopurulent nasal discharge, and oral and facial ulcers. At necropsy, affected animals had crusts and ulcers on the lips, nose, tongue, and oral cavity. Microscopically, all sloths had widespread, random, hepatic necrosis; lymphoid depletion; and bronchointerstitial pneumonia. The central nervous system did not contain gross or histopathologic lesions in any of the 5 sloths, although immunoreactivity for viral antigen was present within vessel walls. Epithelial cells and histiocytes within numerous organs contained intranuclear and intracytoplasmic inclusions and occasional syncytial cells. Canine distemper virus was confirmed with immunohistochemistry and virus isolation. Viral sequencing identified the novel American-4 strain prevalent in eastern Tennessee wildlife. This is the first pathologic characterization of canine distemper virus infection in sloths (family Choloepodidae, order Pilosa) and emphasizes the significant morbidity and mortality in this species.