Magnetic resonance imaging shows spinal curvature in Chinook salmon (Oncorhynchus tshawytscha) is associated with chronic inflammation of peri-vertebral soft tissues.

Chinook salmon (Oncorhynchus tshawytscha) farmed in New Zealand are known to develop abnormal spinal curvature late in seawater production. Its cause is presently unknown, but there is evidence to suggest a neuromuscular pathology. Using magnetic resonance imaging (MRI), we evaluated the relationship between soft tissue pathology and spinal curvature in farmed Chinook salmon. Regions of interest (ROIs) presenting as pathologic MRI signal hyper-intensity were identified from scans of 24 harvest-sized individuals: 13 with radiographically-detectable spinal curvature and 11 without. ROIs were excised from individuals using anatomical landmarks as reference points and histologically analysed. Pathologic MRI signal was observed more frequently in individuals with radiographic curvature (92%, n = 12) than those without (18%, n = 2), was localized to the peri-vertebral connective tissues and musculature, and presented as three forms: inflammation, fibrosis, or both. These pathologies are consistent with a chronic inflammatory process, such as that observed during recovery from a soft tissue injury, and suggest spinal curvature in farmed Chinook salmon may be associated with damage to and/or compromised integrity of the peri-vertebral soft tissues. Future research to ascertain the contributing factors is required.

Pathology of the peripheral neuropathy Charcot-Marie-Tooth disease type 4H in Holstein Friesian cattle with a splice site mutation in FGD4.

Charcot-Marie-Tooth disease (CMT) is a hereditary sensory and motor peripheral neuropathy that is one of the most common inherited neurological diseases of humans and may be caused by mutations in a number of different genes. The subtype Charcot-Marie-Tooth disease type 4H (CMT4H) is caused by homozygous mutations in the FGD4 (FYVE, RhoGEF, and PH domain-containing 4) gene. A previous genome-wide association study involving 130,783 dairy cows found 6 novel variants, one of which was a homozygous splice site mutation in the FGD4 gene. Descendants of carriers were genotyped to identify 9 homozygous Holstein Friesian calves that were raised to maturity, of which 5 were euthanized and sampled for histopathology and electron microscopy at 2 and 2.5 years of age. Three control Holstein Friesian animals were raised with the calves and euthanized at the same time points. No macroscopic lesions consistent with CMT4H were seen at necropsy. Microscopically, peripheral nerves were hypercellular due to hyperplasia of S100-positive Schwann cells, and there was onion bulb formation, axonal degeneration with demyelination, and increased thickness of the endoneurium. On electron microscopy, decreased axonal density, onion bulb formations, myelin outfoldings, and increased numbers of mitochondria were present. These changes are consistent with those described in mouse models and humans with CMT4H, making these cattle a potential large animal model for CMT.

Vertebral fusions in farmed Chinook salmon (Oncorhynchus tshawytscha) in New Zealand.

Vertebral fusions are an established economic concern in farmed Atlantic salmon, but have not been studied in detail in farmed Chinook salmon. Two radiographic studies of vertebral fusions were performed in farmed Chinook salmon. Sixteen of 1,301 (1.2%) smolt and 201 of 2,636 (7.6%) harvest fish had fusions. There were no significant differences in the number of fused vertebrae/fusion in smolt compared with harvest fish. Secondly, tagged fish were repeatedly radiographed to determine the progression of the fusions. Nineteen (4.4%), 23 (5.3%) and 39 (9.0%) fish had fusions as smolt, after 129 days in sea water, and at harvest, respectively. There were no significant differences in the average number of vertebra/fusion between the three time points. Of the fusions that were observed in smolt, additional vertebra did not become fused in 81% of the lesions. Within the rare fusions that did progress due to the involvement of adjacent vertebra, an average of 1.6 vertebrae were added per year. Fish with fusions were significantly lighter than non-affected fish at harvest. Fusions are common in farmed Chinook salmon; however, they are typically stable after development. As fish with fusions were lighter at harvest, reducing fusions may have an economic benefit.

Unilateral perivertebral fibrosis associated with lordosis, kyphosis and scoliosis (LKS) in farmed Chinook salmon in New Zealand.

Vertebral column lordosis, kyphosis and scoliosis (LKS) can result in downgrading of farmed Chinook salmon Oncorhynchus tshawytscha in New Zealand. No cause of LKS has been identified. Radiography and histology were used to quantify LKS and perivertebral fibrosis in 27 fish with LKS visible at harvest and 30 visually normal fish from 3 New Zealand farms. Radiographic LKS was present in all 27 fish with LKS and in 18 of 30 fish without visible LKS. Quantification of the radiographic severity revealed significantly higher radiographic severity scores in fish with visible LKS (mean ± SD = 5.89 ± 2.41) than in fish with no visible, but radiographic LKS (1.44 ± 0.86, p < 0.001). The most frequent histological finding was unilateral perivertebral fibrosis that often extended into the horizontal septum and adjacent myomeres resulting in separation or loss of myocytes. Fibrosis was visible in all fish with LKS and in 12 of 30 fish without visible LKS. Fibrosis scores were higher in fish with visible LKS (3.32 ± 1.71) than in fish without visible LKS (0.35 ± 0.57, p < 0.001). The radiographic LKS severity scores were significantly correlated to the fibrosis scores (R2 = 0.59 p < 0.001) in the fish. Histology of other tissues revealed multifocal inflammation within muscle, peripheral connective tissues and myocardium which were considered most likely incidental in these fish. In this study, LKS was consistently and significantly associated with perivertebral fibrosis, suggesting that perivertebral fibrosis is an important process in the development of LKS. Further research to determine the cause of the fibrosis is required.

Mucosal transmission and pathogenesis of chronic wasting disease in ferrets.

Chronic wasting disease (CWD) of cervids is almost certainly transmitted by mucosal contact with the causative prion, whether by direct (animal-to-animal) or indirect (environmental) means. Yet the sites and mechanisms of prion entry remain to be further understood. This study sought to extend this understanding by demonstrating that ferrets exposed to CWD via several mucosal routes developed infection, CWD prion protein (PrP(CWD)) amplification in lymphoid tissues, neural invasion and florid transmissible spongiform encephalopathy lesions resembling those in native cervid hosts. The ferrets developed extensive PrP(CWD) accumulation in the nervous system, retina and olfactory epithelium, with lesser deposition in tongue, muscle, salivary gland and the vomeronasal organ. PrP(CWD) accumulation in mucosal sites, including upper respiratory tract epithelium, olfactory epithelium and intestinal Peyer's patches, make the shedding of prions by infected ferrets plausible. It was also observed that regionally targeted exposure of the nasopharyngeal mucosa resulted in an increased attack rate when compared with oral exposure. The latter finding suggests that nasal exposure enhances permissiveness to CWD infection. The ferret model has further potential for investigation of portals for initiation of CWD infection.

Immunostaining for VEGF and Decorin Predicts Poor Survival and Recurrence in Canine Soft Tissue Sarcoma.

The aim of this study was to investigate whether using immunohistochemistry to detect the angiogenic proteins vascular endothelial growth factor (VEGF) and decorin can help predict the risk of local recurrence of, or death from, canine soft tissue sarcoma (STS). VEGF and decorin were detected using validated immunohistochemical methods on 100 formalin-fixed paraffin-embedded samples of canine STS. The tumours had been resected previously, with clinical outcome determined by questionnaire. Each slide was assessed by light microscopy and the pattern of immunostaining with VEGF and decorin determined. Patterns of immunostaining were then analysed to detect associations with outcome measures of local recurrence and tumour-related death. High VEGF immunostaining was significantly (p < 0.001) associated with both increased local recurrence and reduced survival time. The distribution of decorin immunostaining within the tumour was significantly associated with survival time (p = 0.04) and local tumour recurrence (p = 0.02). When VEGF and decorin scores were combined, STS with both high VEGF and low decorin immunostaining were more likely to recur or cause patient death (p < 0.001). The results of this study suggest that immunostaining of VEGF and decorin may help predict the risk of local recurrence of canine STS.

Wildlife nidoviruses: biology, epidemiology, and disease associations of selected nidoviruses of mammals and reptiles.

Wildlife is the source of many emerging infectious diseases. Several viruses from the order Nidovirales have recently emerged in wildlife, sometimes with severe consequences for endangered species. The order Nidovirales is currently classified into eight suborders, three of which contain viruses of vertebrates. Vertebrate coronaviruses (suborder Cornidovirineae) have been extensively studied, yet the other major suborders have received less attention. The aim of this minireview was to summarize the key findings from the published literature on nidoviruses of vertebrate wildlife from two suborders: Arnidovirineae and Tornidovirineae. These viruses were identified either during investigations of disease outbreaks or through molecular surveys of wildlife viromes, and include pathogens of reptiles and mammals. The available data on key biological features, disease associations, and pathology are presented, in addition to data on the frequency of infections among various host populations, and putative routes of transmission. While nidoviruses discussed here appear to have a restricted in vivo host range, little is known about their natural life cycle. Observational field-based studies outside of the mortality events are needed to facilitate an understanding of the virus-host-environment interactions that lead to the outbreaks. Laboratory-based studies are needed to understand the pathogenesis of diseases caused by novel nidoviruses and their evolutionary histories. Barriers preventing research progress include limited funding and the unavailability of virus- and host-specific reagents. To reduce mortalities in wildlife and further population declines, proactive development of expertise, technologies, and networks should be developed. These steps would enable effective management of future outbreaks and support wildlife conservation.

Reproductive biology of male common dolphins (Delphinus delphis) in New Zealand waters.

Reproductive parameters were assessed in 64 male common dolphins (Delphinus delphis) examined post-mortem from strandings and bycatch in New Zealand between 1999 and 2020. The stages of male sexual maturation were assessed using morphological measurements and histological examination of testicular tissue. Age was determined via growth layer groups (GLGs) in teeth. The average age (ASM) and length (LSM) at attainment of sexual maturity were estimated to be 8.8 years and 198.3 cm, respectively. Individual variation in ASM (7.5-10 years) and LSM (190-220 cm) was observed in New Zealand common dolphins. However, on average, sexual maturity was attained at a similar length but at a marginally younger age (< 1 year) in New Zealand compared to populations in the Northern Hemisphere. All testicular variables proved better predictors of sexual maturity compared to demographic variables (age and total body length), with combined testes weight the best outright predictor of sexual maturity. Reproductive seasonality was observed in male common dolphins, with a significant increase in combined testes weight in austral summer. This aligns with most other studied populations, where seasonality in reproduction is typically observed. Given the known anthropogenic impacts on New Zealand common dolphins, we recommend that these findings be used as a baseline from which to monitor population-level changes as part of conservation management efforts. Supplementary Information: The online version contains supplementary material available at 10.1007/s00227-023-04266-5.

Evidence for distinct chronic wasting disease (CWD) strains in experimental CWD in ferrets.

Chronic wasting disease (CWD) is an evolving prion disease of cervids (deer, elk and moose) that has been recognized in North America and Korea. Infection of non-cervid reservoir or transport species in nature is not reported. However, the ferret (Mustela putorius furo) is susceptible to CWD after experimental inoculation. Here, we report that infection of ferrets with either of two ferret CWD isolates by various routes of exposure has revealed biologically distinct strain-like properties distinguished by different clinical progression and survival period. The isolates of ferret CWD were also differentiated by the distribution of the infectious prion protein (PrP(CWD)) in the brain and periphery, and by the proteinase K sensitivity of PrP(CWD). These findings suggest that diversity in prion conformers exists in CWD-infected cervids.

Exogenous glucocorticoid treatment affects Sertoli cell load and epididymal sperm quality in domestic cats (Felis catus).

Elevated glucocorticoid (GC) concentrations associated with captivity-related stress have been linked to impaired testicular function and low sperm quality in felids, but direct physiological evidence is lacking. This study assessed the effects of exogenous GC treatment on felid testicular function using the domestic cat (Felis catus) as a model species. Sixteen intact male cats aged 2.4 ± 0.8 years (mean ± SEM) were divided randomly into treatment (n = 8) and control (n = 8) groups. Treatment cats were given 1 mg kg-1 oral prednisolone daily for 50 days. Blood samples were taken on Days 0 (first prednisolone treatment), 2, 4, 7, 10, 20, 30, 40, 50 (prior to neutering) and 60 of the trial. All cats were orchiectomised on day 50, epididymal sperm assessed, and the testes fixed for histological assessment. Testosterone concentrations did not differ between the two groups. While sperm motility was similar between the treatment and control groups, cats given prednisolone had a higher proportion of morphologically abnormal sperm in both the caput (72.5% vs. 59.6%, P < 0.001) and cauda (56.7% vs. 35.8%, P < 0.001) epididymis. Testicular histomorphometric data and total number of germ cells per seminiferous tubule cross section did not differ between groups, nor did the relative abundance of spermatogonia, spermatocytes, and spermatids. Cats given prednisolone had fewer Sertoli cells per tubule cross-section than those in the control group (17.1 ± 0.9 vs. 19.7 ± 0.8, P = 0.04), which was likely related to higher rates of Sertoli cell apoptosis in treatment versus control cats (0.25 ± 0.02 vs. 0.10 ± 0.02 apoptotic Sertoli cells per tubule, respectively; P < 0.001). Sertoli cell load (number of germ cells per Sertoli cell) was also higher in the treatment group than in the control group (11.5 ± 0.8 vs. 9.4 ± 1.2 germ cells per Sertoli cell, respectively; P < 0.001), and was positively correlated with the percentage of morphologically abnormal sperm in the epididymis (r2 = 0.78, P < 0.001). Prednisolone treatment resulted in an increase in the proportion of abnormal sperm in the epididymis, which may be explained by an increased nurturing demand on a reduced Sertoli cell population. These findings provide novel evidence to support the hypothesis that elevated GC concentrations, such as those resulting from captivity-related stress, have the potential to impair testicular function and sperm quality in felids.

Reproductive biology of female common dolphins (Delphinus delphis) in New Zealand waters.

Reproductive biology was assessed in 106 female common dolphins (Delphinus delphis) examined post-mortem from stranding and bycatch events along the New Zealand coastline between 1997 and 2019. The average age (ASM) and length (LSM) at sexual maturity was estimated at 7.5 years and 183.5 cm, respectively. The total number of corpora in mature individuals increased with age and appeared to persist throughout life. Ovarian asymmetry was apparent, with the left ovary displaying higher rates of ovulation, and a maximum of 19 corpora recorded for a 24-year-old female. The estimated ovulation and annual pregnancy rates for mature females were 0.39 year-1 and 30%, respectively. Conception and calving occurred year-round, with a weak seasonal increase observed in late austral spring and early austral summer. As these data did not clearly show whether seasonality was present, the gestation, lactation, and resting periods were calculated as either 12.6 or 12.8 months based on the presence/absence of seasonality, respectively. Similarly, calving interval ranged from 3.15 to 3.2 years, depending upon whether seasonality was considered. The estimated LSM of the New Zealand population aligns with other populations globally, although the estimated ASM is younger by approximately 6 months. Other reproductive parameters align with Northern Hemisphere populations, although demonstrate variation, which may reflect adaptations to local conditions such as water temperature and prey availability. As the species is subject to anthropogenic impacts including pollution and bycatch, we suggest our findings be used as a baseline with which to monitor trends in population parameters. Supplementary Information: The online version contains supplementary material available at 10.1007/s00227-022-04139-3.

Fourier transform infrared (FTIR) analysis identifies microplastics in stranded common dolphins (Delphinus delphis) from New Zealand waters.

Here we provide a first assessment of microplastics (MPs) in stomach contents of 15 common dolphins (Delphinus delphis) from both single and mass stranding events along the New Zealand coast between 2019 and 2020. MPs were observed in all examined individuals, with an average of 7.8 pieces per stomach. Most MPs were fragments (77%, n = 90) as opposed to fibres (23%, n = 27), with translucent/clear (46%) the most prevalent colour. Fourier transform infrared (FTIR) spectroscopy revealed polyethylene terephthalate (65%) as the most predominant polymer in fibres, whereas polypropylene (31%) and acrylonitrile butadiene styrene (20%) were more frequently recorded as fragments. Mean fragment and fibre size was 584 µm and 1567 µm, respectively. No correlation between total number of MPs and biological parameters (total body length, age, sexual maturity, axillary girth, or blubber thickness) was observed, with similar levels of MPs observed between each of the mass stranding events. Considering MPs are being increasingly linked to a wide range of deleterious effects across taxa, these findings in a typically pelagic marine sentinel species warrants further investigation.

Increased programmed death ligand (PD-L1) and cytotoxic T-lymphocyte antigen-4 (CTLA-4) expression is associated with metastasis and poor prognosis in malignant canine mammary gland tumours.

Aberrant expression of immune check point molecules, programmed death ligand (PD-L1) and cytotoxic T-lymphocyte antigen-4 (CTLA-4) has been reported in many human cancers with increased protein and gene expression correlated with an aggressive behaviour in some neoplasms. Additionally, PD-L1 blockade has been shown to be an effective therapy for some human cancers. Canine mammary gland tumours have previously been shown to produce PD-L1 protein, but there are no previous studies investigating CTLA-4 in these common canine neoplasms. The present study investigated protein and gene expression of PD-L1 and CTLA-4 using immunohistochemistry and RT-PCR in 41 histologically-malignant, outcome-known CMGTs. The PD-L1 and CTLA-4 immunostaining scores of the mammary gland tumours that subsequently metastasised were significantly higher than those of tumours which did not metastasise (PD-L1: p = 0.005, CTLA-4: p = 0.003). Gene expression of PD-L1 and CTLA-4 was also significantly higher in tumours which subsequently metastasised (PD-L1: p = 0.023, CTLA-4: p = 0.022). Further, higher PD-L1 or CTLA-4 immunostaining scores correlated with shorter survival times of dogs (PD-L1: rs = - 0.42, p = 0.008, CTLA-4: rs = - 0.4, p = 0.01) while PD-L1 immunostaining was independently prognostic of survival time (Δ F = 4.9, p = 0.035). These findings suggest that higher protein and gene expression of PD-L1 and CTLA-4 by tumour cells increases the chances of metastasis and measuring these proteins may predict likely neoplasm behaviour. Additionally, if increased expression of these proteins promotes metastasis, blocking PD-L1 or CTLA-4 may be beneficial to treat canine mammary gland tumours.

Papillomavirus DNA is not Amplifiable from Bladder, Lung, or Mammary Gland Cancers in Dogs or Cats.

Papillomaviruses (PVs) cause around 5% of all human cancers, including most cervical cancers and around a quarter of all oral cancers. Additionally, some studies have suggested that PVs could cause a proportion of human lung, breast, and bladder cancers. As PVs have been associated with skin cancer in cats and, more rarely, dogs, it was hypothesized that these viruses could also contribute to epithelial cancers of the lung, mammary gland, and bladder of dogs and cats. Formalin-fixed paraffin-embedded samples of 47 canine and 25 feline cancers were examined histologically for evidence of PV infection. Additionally, three sets of consensus PCR primers were used to amplify PV DNA from the samples. No histological evidence of PV infection was visible in any of the cancers. DNA from a bovine PV type was amplified from one sample, while two different samples were found to contain human PV DNA. However, these were considered to be contaminants, and no canine or feline PV types were amplified from any of the cancers. These results suggest that PVs do not frequently infect the lung, mammary gland, or bladder of dogs and cats and therefore are unlikely to be significant factors in the development of cancers in these tissues.

Temperature sensitive regions of the Chinook salmon vertebral column: Vestiges and meristic variation.

Variation of vertebral centra numbers is common in vertebrates. Likewise, the number of associated elements such as ribs and neural and haemal arches can vary and affect all regions of the vertebral column. In mammals, only the number of cervical vertebrae is invariable. Variation of total vertebral centra numbers is well documented in teleost fish, often related to temperature. Less information is available about which part of the vertebral column and which associated elements are liable to variation. Here, variation in number of vertebral centra and associated elements is studied in Chinook salmon in six distinct anatomical regions. Animals are raised at 8 and 12°C to ask whether the vertebral centrum numbers, the pattern, and the frequency of variation in particular regions are temperature dependent. No significant difference concerning the total number of vertebrae was found, but regional differences occurred between the 8 and 12°C groups. Twelve specimens out of 60 of the 12°C group had three postcranial vertebrae compared to only one specimen in the 8°C group. The number of transitional vertebrae is significantly different in 8 and 12°C specimens. Fewer transitional vertebrae occur in more anterior positions in 8°C specimens. Most specimens of both temperature groups had two ural centra; however 17 specimens out of 60 of the 12°C group had up to five ural centra. Specimens of the 12°C group show more variation in the presence of the vestigial ribs associated with transitional vertebrae. Clearly, the postcranial, transitional, and ural regions are temperature sensitive. This study shows that nonsignificant differences in the total number of vertebrae can mask significant regional variation. Variation of vertebral numbers could be the consequence of loss or gain of vertebral centra and/or a change in the identity of the associated element on the vertebral centrum.

Primary possum macrophage cultures support the growth of a nidovirus associated with wobbly possum disease.

The objective of the study was to establish a system for isolation of a recently described, thus far uncultured, marsupial nidovirus associated with a neurological disease of possums, termed wobbly possum disease (WPD). Primary cultures of possum macrophages were established from livers of adult Australian brushtail possums (Trichosurus vulpecula). High viral copy numbers (up to 6.9×10(8)/mL of cell lysate) were detected in infected cell culture lysates from up to the 5th passage of the virus, indicating that the putative WPD virus (WPDV) was replicating in cultured cells. A purified virus stock with a density of 1.09 g/mL was prepared using iodixanol density gradient ultracentrifugation. Virus-like particles approximately 60 nm in diameter were observed using electron microscopy in negatively stained preparations of the purified virus. The one-step growth curve of WPDV in macrophage cultures showed the highest increase in intracellular viral RNA between 6 and 12h post-infection. Maximum levels of cell-associated viral RNA were detected at 24h post-infection, followed by a decline. Levels of extracellular RNA increased starting at 9h post-infection, with maximum levels detected at 48 h post-infection. The establishment of the in vitro system to culture WPDV will facilitate further characterisation of this novel nidovirus.

Efficient in vitro amplification of chronic wasting disease PrPRES.

Chronic wasting disease (CWD) of cervids is associated with conversion of the normal cervid prion protein, PrP(C), to a protease-resistant conformer, PrP(CWD). Here we report the use of both nondenaturing amplification and protein-misfolding cyclic amplification (PMCA) to amplify PrP(CWD) in vitro. Normal brains from deer, transgenic mice expressing cervid PrP(C) [Tg(cerPrP)1536 mice], and ferrets supported amplification. PMCA using normal Tg(cerPrP)1536 brains as the PrP(C) substrate produced >6.5 x 10(9)-fold amplification after six rounds. Highly efficient in vitro amplification of PrP(CWD) is a significant step toward detection of PrP(CWD) in the body fluids or excreta of CWD-susceptible species.

The jaagsiekte sheep retrovirus envelope gene induces transformation of the avian fibroblast cell line DF-1 but does not require a conserved SH2 binding domain.

Ovine pulmonary adenocarcinoma, caused by jaagsiekte sheep retrovirus (JSRV), is a naturally occurring retrovirus-induced pulmonary neoplasm of sheep. We report here that expression of the JSRV env gene is sufficient to transform an avian embryo fibroblast cell line, DF-1. DF-1 cells transfected with an avian sarcoma-leukaemia retroviral expression vector containing the JSRV env gene [pRCASBP(A)-J:env] exhibited changes consistent with transformation, including contraction and rounding of cells with formation of dense foci. Transfection with a reporter construct expressing the green fluorescent protein did not induce morphological changes in DF-1 cells, eliminating the possibility that the vector, the transfection protocol or culturing techniques were responsible for the transformed phenotype. When pRCASBP(A)-J:env-transfected cells were inoculated into nude mice, tumours formed, verifying that the DF-1 cells were tumorigenic. Analysis of the JSRV env gene revealed a conserved tyrosine (597) and methionine (600) residue in the cytoplasmic tail within the transmembrane domain of the envelope, which creates a known binding site of SH2 domains in the p85 subunit of phosphatidylinositol 3-kinase. However, when this tyrosine residue was mutated to serine or alanine, transformation was not affected. Furthermore, mutation of the methionine residue to valine or leucine also failed to eliminate JSRV env-mediated transformation. These results are in contrast to mutational analysis performed in JSRV env-transformed murine NIH-3T3 cells in which both the tyrosine and methionine residues are necessary for transformation. These findings suggest that more than one mechanism may be involved in JSRV env-mediated transformation.