ZIKV was injected intravenously in the pregnant type 2 interferon (IFN)-deficient mouse at embryonic day (E) 12.5. The embryos were collected at E15.5 and postnatal day (P)2. Immunohistochemistry for cortical progenitors and neuronal markers at E15.5 showed the reduction of both populations as a result of ZIKV infection. Using confocal 3D imaging, we found that ZIKV infected brain sections displayed a reduction in the vasculature density and vessel branching compared to mocks at E15.5; altogether, cortical vessels presented a comparatively immature pattern in the infected tissue. These impaired vascular patterns were also apparent in the placenta and retina. Moreover, proteomic analysis has shown that angiogenesis proteins are deregulated in the infected brains compared to controls. At P2, the cortical size and brain weight were reduced in comparison to mock-infected animals. In sum, our results indicate that ZIKV impairs angiogenesis in addition to neurogenesis during development. The vasculature defects represent a limitation for general brain growth but also could regulate neurogenesis directly.Zika virus (ZIKV) is associated with brain development abnormalities such as primary microcephaly, a severe reduction in brain growth. Here we demonstrated  Toxoplasma gondii, others, Rubella, Cytomegalovirus, Herpes simplex virus and Syphilis). Recently, Zika virus (ZIKV) has been associated with primary microcephaly1, and it is considered a new TORCHS factor2. ZIKV infects human neural progenitors and impairs the growth of 3D differentiating cultures, such as neurospheres and brain organoids5. Moreover, in vitro, ZIKV alters the cell cycle and induces apoptosis in neural progenitor cells6. Studies using mouse models showed brain growth impairment as a result of ZIKV congenital infection9. However, the cellular and molecular mechanisms that underlie the brain growth failure in ZIKV congenital syndrome remain to be fully understood.Primary microcephaly is a severe brain malformation characterized by the reduction of the cephalic perimeter at birth. Microcephaly etiologies vary from genetic abnormalities to external factors such as the TORCHS pathogens  pregnant mice with ZIKV at E12.5 resulted in reduced brain size at birth. At E15.5, neurogenesis was affected by ZIKV infection, radial glia and Ctip2 neuronal population are reduced compared to control animals. Moreover, blood vessels were found to be immature, with fewer branches and reduced area at E15.5; key angiogenesis proteins were also found to be deregulated in the infected cerebral cortex at this time. The results reported here suggest a mechanism through which ZIKV infection of endothelial cells may contribute to the Zika congenital syndrome-like abnormalities demonstrated in this mouse model.Here, we describe the use of a congenital mouse model of ZIKV-induced microcephaly, to investigate the effects of ZIKV infection on neuro-vascular development. Infecting type 2 interferon (IFN)-deficient Ifng−/− pregnant females at E12.5. At P2, the primary microcephaly-like syndrome was characterized by the smaller brain size, lower brain weight, and smaller skull size. Brain width and length was decreased in ZIKV infected brains compared to MOCK brains  in ins Fig. . There wins Fig. . Similarins Fig. . Brain cins Fig.  was signins Fig. . Statistins Fig. , corticains Fig.  and areains Fig. , as wellins Fig.  in ZIKV To evaluate the impact of ZIKV congenital infection at E12.5 in neurogenesis, we harvested the brains three days after the infection. At E15.5, dividing cells in the VZ and SVZ were identified with the M-phase marker, phospho-histone H3  within ZIKV infected cerebral cortex compared to MOCK , reductions in neurogenesis and area of both the progenitor zone and developing cortical plate were associated with a pattern of delayed vascular development.This study characterizes a new model of ZIKV-induced microcephaly like syndrome in mouse and identifies changes in the vasculature as part of the disease aetiology. We demonstrated that ZIKV infection at E12.5 in 8. Indeed, microcephaly can result from defective neurogenesis, an essential part of cortical development. However, cortical development is a complex phenomenon, which is regulated by a multitude of parallel processes, signalling factors, and extrinsic factors all occurring in tightly defined timescales. Therefore, the dysregulation of any of these processes could result in defective neurogenesis leading to cortical malformation. Cortical vascular plexus development can start as early as E11 in the mouse and is essential for the delivery of nutrients and regulating the expression of signalling molecules, which has an impact on neurogenesis11. The cortical blood supply develops from two plexi, the ventricular plexus and the pial plexus13. This study, along with previous research17, showed that ZIKV also infects the endothelial cells of the cortical vasculature in addition to neural progenitors. In particular, the vascular structure is abnormal in ZIKV infected mouse brains, especially in the ventricular zone, where there is also evidence of reduction of neuronal proliferation. The blood-brain barrier, specialisation of the cerebral endothelial cells that limit paracellular diffusion and transcellular transport, have been suggested to regulate the entry of most flaviviruses18. However, blood vessels are the pathway of ZIKV entrance into the brain parenchyma19, suggesting that the virus might have direct effects on the vasculature as well as other neural tissues. Some disruption of the blood-brain barrier has also been reported following ZIKV infection20, which may facilitate viral entry. However, the barrier changes described to date have not been quantified and were only described in a model of pathogenesis with substantial lethality in the postnatal period. Mladinish and colleagues18 suggest that in their primary human brain endothelial cells, ZIKV does not result in death of the cells or altered permeability. Instead, endothelial cells appear to act as a reservoir for viral replication, as well as contributing to inflammatory signaling following infection.Previous studies have demonstrated that direct ZIKV infection and death of the NPCs led to the reduced brain size22. Following ZIKV injection, however, there is a substantial increase in Ang1, Ang3 and, to a lesser extent, endostatin and PEDF, which all suggest an anti-angiogenic status22. The exact effects and contribution of each of these proteins will need to be further elucidated. For instance, combined VEGF and Ang2 have a different effect on the development of vascular networks than either one individually23. The changes in protein concentration observed at E15.5 are likely to be a combination of the response of the blood vessels to ZIKV infection, as inflammation is a key regulator of angiogenesis24, and of the oxygen levels of the tissue.We found that following ZIKV injection, there was a general increase of the levels of angiogenic protein , within the developing cortex. During angiogenesis, the balance of these different molecules is of particular importance, and in this context, there is a clear interplay between ‘normal’ and ‘abnormal’ growth and stabilisation signals. In the MOCK animals, there is a high concentration of VEGF and PDGF-BB proteins in the brain, which would promote endothelial cell proliferation and the recruitment of endothelial precursor cells and pericytes to the extending vascular network26. Changes in vascular development can result in altered neural patterning12 and, in addition, endothelial cells produce trophic factors which have important roles in neurogenesis30. While there is no dispute that ZIKV has a direct impact on human NPCs in the brain19, our study shows that a vascular defect may also contribute to ZIKV induced microcephaly. ZIKV infection of the endothelial cells may induce cortical vasculature abnormalities, which may further add to the abnormal neurogenesis.Dividing progenitors are found in close proximity to blood vessels in the SVZ, and other neurovascular niches in the brain31, where the long-term consequences of the early disruption continue more subtly. The alterations in the vasculature in the cortical germinal zone might be just the “tip of the iceberg” in the ZIKV congenital infection. Our observations on the altered vascular development in the placenta and the retina suggest that ZIKV congenital infection might have a very general effect on the developing embryonic vasculature. It is important to note that the placenta produces several proinflammatory cytokines and expresses cytokine receptors. Several insults, such as LPS, bacterial and viral infection, including ZIKV, induce cytokine expression in the placenta impacting the fetus33.In our murine model of ZIKV infection, we observed a more severe vascular developmental alteration at E15.5 that was much less prominent by P2, in contrast to the microcephaly-like phenotype, that was strongest at P2. In spite of this recovery, there was still a reduced vessel branch length present at P2 in ZIKV infected brains at birth. This suggests that ZIKV congenital infection promotes an acute insult that delays the vascular development during critical stages of neurogenesis that was partially recovered by birth. Such a delayed developmental progress is common in developmental disorders5 PFU), injected intravenously (IV) into the Ifng−/− pregnant dam. In previous studies, a variety of ZIKV titres within the range of 103–1012 PFU administered through different methods of systemic administration were employed to generate mouse models for ZIKV infection34, but these methods have often led to death of the young mice or whole body size reduction38. Here, the mouse model produced pups with smaller brains mimicking microcephaly-like phenotype that survived birth, thus enabling the opportunity to perform postnatal studies. Our results indicate that in the Ifng−/− model, the embryos are not affected homogeneously by congenital ZIKV infection. Using PCR to detect viral loads in pup brains, we found that only some infected specimens were negative. Similarly, some morphological outcomes, such as skull size, also showed heterogeneous results. This non-uniform response to ZIKV infection in the production of microcephaly-like phenotypes has been reported by other authors, especially in models where the animals were not completely immunological deficient39. Therefore, Ifng−/− mice seem to be appropriate to model an effective but also realistic ZIKV congenital infection. It would be interesting to investigate whether Ifn gene polymorphism resulting in lower cytokine levels in humans is associated with an increased prevalence of ZIKV induced microcephaly. Clustering patterns of microcephaly following ZIKV infection in Brazil hints at the possible role of environmental and socio-economic issues like availability of pregnancy supplements, age at pregnancy, vaccinations, and sanitation40. Our study suggests that ZIKV may cause additional angiogenic defects that could contribute to the reduced neurogenesis in the ventricular zone and the later reduced cortical volume. While the link between ZIKV infection, disruption of vascular development and reduced proliferation of neural progenitors needs to be further investigated, this is a novel mechanism that has a potential for direct therapeutic targeting.The mouse model used in this study was produced by infection of ZIKV (10Ifng−/−) mice in the C57BL/6 background were obtained from the University of São Paulo (USP). Ifng−/− pregnant mice were injected with ZIKV strain 766/AfZIKV 105 plaque-forming units (PFU). Control Ifng−/− pregnant mice were injected with supernatant of Vero cells (MOCK). ZIKV injections were performed at a retro-orbital position intraveneously (IV) at embryonic day (E) 12.5. All animals were homozygous and culled using cervical dislocation (adults) or decapitation (embryos and pups). Samples were collected from embryos at three-days post-infection  or from pups at the 2nd postnatal day (P2) and fixed by immersion in 4% paraformaldehyde  for 72 hours, then washed in phosphate-buffered saline (PBS).All methods, protocols, and procedures are in accordance with the relevant guidelines and regulations and were approved by the institutional research committee of the Federal University of Rio de Janeiro, Brazil, under the protocol #01200.001568/2013-87. Interferon gamma knockout  as defined by Bookstein43.Images of whole brains obtained from three litters, n = 12 (3 mocks and 9 ZIKV), were taken with a light microscope  from a dorsal view. Length and width of the cortex were measured (in pixels) using ImageJ at the longest  and widest points of the cortex Fig. . To compBrains and placenta for immunohistochemistry were embedded in 5% agarose (Bioline), in 0.1 M PBS, cut coronally at 50 µm with a vibrating microtome , and stored in 0.01% PBS-azide. At least three brains or placentas from either MOCK or ZIKV-infected groups from different litters, and three sections per brain or placenta were used for all immunohistochemistry experiments. Sections were washed in 0.1M PBS, incubated in blocking solution  and 0.2% Triton-X100 (BDH) in 0.1M PBS), and then primary antibodies  at 4 °C overnight. Sections were washed and incubated with fluorescent-conjugated secondary antibody  and stained with 4′-6-diamino-2-phenylindole  nuclear counterstain. Retinas were removed at P7, flattened and fixed with cold (−20 °C) methanol. Free-floating retina, brain and placenta sections stained with Isolectin B4  were incubated with the stain for 2 hours before DAPI counterstain.Fluorescently labelled sections were imaged using a fluorescence microscope  and a confocal laser-scanning microscope . ImageJ was used to determine the area (in pixel units) covered by ventricular and subventricular zones and cortical layers on fluorescence microscopy images of DAPI counterstained samples. Pax6 and Ctip2 immunohistochemistry were used to define the radial glial cells and newly emerging cell layers of the cortical plate, and the numbers of immunoreactive cells were quantified. Pax6+, Ctip2+ and pH3+ number of cells were counted in three sections of at least three different brains of each experimental group. We used the field of view of 20X for Pax6 and Ctip2 and 40X for Ph3 analysis. Analysis of the cortical vasculature was carried out in three sections of at least three different brains from different litters stained with IB4. Images were acquired using a confocal microscope as described above. To determine vessel characteristics, the threshold tool (ImageJ) was used to produce binary images, and images were de-noised (using the ImageJ ‘Despeckle’ tool) to remove excessive non-specific stain signal. The width of vessels (µm) was determined using the histogram tool to measure the x-axis distance between the margins of each vessel, perpendicular to the pial surface. Vessel area was determined by the proportion of vascular staining within each cortical region. Skeletonised structure of the blood vessels was obtained using the ImageJ plugin ‘skeletonise’, to quantify vessel number, branch number, and branch length. All data were analysed with Excel or GraphPad Prism. Statistical evaluation was performed by Student’s unpaired t-test. Data in graphs are presented as mean ± SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.Cerebral cortex tissue extracts from E15.5 MOCK or ZIKV infected embryos were processed for analysis of angiogenesis-related proteins levels by using the Proteome Profiler™ Mouse Angiogenesis Antibody Array kit (R&D Systems) that identifies 53 angiogenesis-related proteins. Proteome assay and analysis were performed according to manufacturer’s instructions.44. ZIKV copies were calculated by interpolation onto an internal standard curve formed of eight 10-fold serial dilutions of a synthetic ZIKV RNA based on the Asian lineage. The ZIKV quantification was displayed in ZIKV RNA copies per gram of tissue.RNA was extracted from brain, eye and spleen tissue samples using RNeasy Plus Mini Kit (QIAgen), following the manufacturer’s recommendations. Viral RNA was quantified using One Step Taqman RT-qPCR (Thermo Fisher Scientific) on a 7500 Real-Time PCR System (Applied Biosystems) with primers and probe designed by Lanciotti and collaboratorsSupplementary Figure 1


Physeter macrocephalus) are rare in the Mediterranean Sea. Nevertheless, in 2014 a pod of 7 specimens stranded alive along the Italian coast of the Central Adriatic Sea: 3 individuals died on the beach after a few hours due to internal damages induced by prolonged recumbency; the remaining 4 whales were refloated after great efforts. All the dead animals were genetically related females; one was pregnant. All the animals were infected by dolphin morbillivirus (DMV) and the pregnant whale was also affected by a severe nephropathy due to a large kidney stone. Other analyses ruled out other possible relevant factors related to weather conditions or human activities. The results of multidisciplinary post-mortem analyses revealed that the 7 sperm whales entered the Adriatic Sea encountering adverse weather conditions and then kept heading northward following the pregnant but sick leader of the pod, thereby reaching the stranding site. DMV infection most likely played a crucial role in impairing the health condition and orientation abilities of the whales. They did not steer back towards deeper waters, but eventually stranded along the Central Adriatic Sea coastline, a real trap for sperm whales.Mass strandings of sperm whales ( Sonar is not the only known, anthropogenic sound-related factor in the oceans associated to these dramatic events: other military and industrial activities like seismic surveys  have been also linked to the phenomenon4. A study conducted by Madsen and colleagues5 has also suggested that acoustic disturbance due to the presence of wind turbines in the underwater environment could affect the behavior of marine mammals, similarly to naval sonar.In the last decades, cetacean mass strandings have attracted a growing attention from the media: these dramatic events induced an increase of the public concern towards the role of human activities and their effects on marine mammal conservation, with special emphasis on those related to anthropogenic sound sources. In particular, the use of naval sonar has been associated with both behavioral and pathological changes leading to lethal mass stranding of several cetacean species7, or the effects of lunar cycles8, meteorological and oceanographic factors like large-scale climatic events9, local disturbances and basin-related temperature variations that may influence prey distribution10. Furthermore, specific geographical and physical features of the coastlines could affect acoustic reflection and impair orientation and perception: these factors may influence mass strandings and thus explain the aggregation of such events in specific areas11. The Adriatic Sea is one of these “cul-de-sacs”, potentially fatal for sperm whales 12: in fact, despite this species not being considered a regular inhabitant of this narrow arm of the Mediterranean Sea13, 7 different mass stranding events have occurred since 155514, the latest of which in September 2014.However, mass strandings do not exclusively result from interaction with anthropic activities. Several natural factors are also considered equally responsible for similar events, including anomalies of the Earth geomagnetic field due to solar activitypost-mortem findings obtained from the sperm whales involved in this last stranding episode, thereby proposing a reasonable hypothesis based on our multidisciplinary studies, literature and previous experience15.The present work reports the A pod of 5 sperm whales was reported in Croatian waters by the Blue World Institute near the Vis Island  and later close to the Kornati Archipelagos . This sighting was communicated to the Italian Stranding Network, already experienced by a previous sperm whale mass stranding event occurred in 2009 along the Southern Adriatic coast of Italy. Consequently, the response was rapid when the 7 sperm whales were found beached alive on September 11th, early in the morning at Punta Penna beach, Nature reserve Punta Aderci, Vasto, Abruzzo Adriatic coastline (coordinates 42°10′28.0-N 14°42′02.4-E). The whales codenamed SW1, SW2 and SW3 did not survive the stranding. However, thanks to the efficient efforts of hundreds of volunteers coordinated by the personnel affiliated to the Centro Studi Cetacei Onlus, 4 animals were successfully refloated (https://www.youtube.com/watch?v=jExOUfEb1kc&t=279s). The outcome of refloating operation cannot be determined, as the released whales found the way to the high seas and were not seen again. However, a sperm whale carcass was found 14 days later along the Italian coast, about 130 nautical miles southward. Unfortunately, it cannot be assumed or excluded that the carcass belonged to one of the refloated specimens, since no further analysis was performed due to the advanced stage of decomposition.The databases consulted (see Methods) reported no anomalous weather and/or marine condition, no anomalies of the solar and lunar cycles or changes in the geomagnetic field. Our current opinion is that the stranding site was determined by the sum of different environmental co-factors, namely wind, waves and marine currents. No official military exercises nor seismic surveys were ongoing in the Adriatic Sea during the stranding events or in the previous days.16 and nutritional conditions, either evaluated during necropsies  or estimated during release efforts , are reported in Table The whale pod consisted of 2 adult females SW1 and SW2), a juvenile female (SW3), and 4 unsexed individuals. Data regarding total length, gender and age estimation, conservation code and SW2,Unfortunately, comparison of pictures of the stranded animals with photo-identification catalogues of sperm whales existing in the Mediterranean Sea (see Methods) showed no positive match.17).No genetic variability was detected in the mtDNA region of the analyzed samples. The haplotype detected in the mitochondrial control region of all samples was found to be the one reported in sperm whales of the Mediterranean Sea , demonstrated a good informativeness; therefore, the selected panel of STR markers showed adequate discrimination ability for this study.The genotypes were clustered by FCA and the graphical representation of the distribution of genetic distances between individuals showed that the samples were divided into two distinct clusters: the group including samples from the Mediterranean Marine Mammal Tissue Bank of the University of Padova, and that of the sperm whales included in the Vasto mass stranding event here reported . Samples SWB and SWC give rise to two separate clusters and do not share any parents. The samples from the Vasto mass stranding belong to a single cluster and result paternal sibs sharing the same candidate father (n. 4). Moreover, samples from SW2 and SW3 resulted full sib, indicating that the two whales share both the candidate father n. 4 and the candidate mother n. 6.Based on the genotypes supplied, the software calculated the probability of all full sib dyads considering four possible pairs Table . Only thet al.18, K = 2. Providing information on the sampling location did not influence the outcome of the analysis. Our results showed that the investigated samples were differentiated into two subpopulations, as specified here below: subpopulation A: SWA, SWB, SWC and SWD samples; subpopulation B: SW1, SW2 and SW3 samples.The Bayesian clustering analysis implemented in the STRUCTURE software indicated that the Ln probability of the genetic data, consistent among the five runs, was highest for k ≤ 2. The result agrees with the outcome of analysis according to Evanno st) was calculated according to Weir & Cockerham19 by FSTAT Software version 2.9.3.220. The Fst value was 0.03400, with a p-value < 0.05, which suggests a limited genetic differentiation.The measure of differentiation between the two populations due to genetic structure  for skin and blubber, as compared to skeletal muscle and liver tissues, which showed analogous values. Similar data were also obtained for δ13C values, showing less enrichment in 13C in the blubber and skin in comparison to the other tissues. The values of δ13C analysis of lipids extracted from the blubber (−19.37 ± 0.26‰) showed a depletion of δ13C (δ13Cbulk- δ13Clipid free) ranging at ∼3.4 in relation to the respective tissues. The variability of δ13C was lower in the liver (0.98) and muscle (0.44), considering that these tissues contain minimal amounts of lipids . The δ15N values conflicting results recommend caution in the δ15N correction. The variability reported in this study was <0.7‰.The carbon/nitrogen molar ratios (C:N) in the liver and muscle were similar for the 3 sperm whales. On the other hand, whale SW3 showed skin and blubber values higher than SW1 and SW2 , Schmorl’s and Danscher’s stains revealed the dual/associated occurrence of inorganic mercury and lipofuscins. A relevant and diffuse lymphocytic depletion, accompanied by histiocytic infiltration of the sinuses, was also observed in lymph nodes of all whales. Clumps of eosinophils were detected in superficial cervical and mesenteric lymph nodes. Microscopic analyses of cerebral tissue and samples from other organs revealed the existence of mild and non-specific degenerative changes, coupled with rare and/or occasional lymphocytic infiltrations around brain vessels.Systemic lymphadenopathy was noted in all the 3 whales, characterized by a diffuse greenish discoloration and the onset of a black pigmentation scattered on the cut surface. Histochemically, these dark pigments resulted strongly positive to Danscher’s staining methodRare fat emboli observed in the lung parenchyma from SW3 Fig. , along wpost-mortem overgrowth, rather than being considered as primary or secondary pathogens. The presence of any eventual ante mortem noise-induced hearing loss, wherever existent, could not be determined, since bacterial overgrowth obscured the organ of Corti, and decomposition had already set in among the few remaining hair cells of the small portion of the cochlear spiral  at of the cochlear spiral from SW2 revealed numerous extracellular bacilli overlying the apical aspect of the organ of Corti, the hearing organ Fig. . These bral Fig. .Apoptotic changes were revealed in brains by the positive immunoreaction of axons to a commercially available anti-caspases-3 antibody Fig. . Rhabdom22.Finally, clear-cut evidence of morbilliviral infection was found in circulating monocytes Fig. , splenic24. Molecular analyses carried out on SW1b showed evidence of herpes virus infection in the fetal liver and kidney with 100% homology of viral sequence with dolphin alpha-herpesvirus. (GenBank Acc No AY952779.1) No viral particles were observed with electron microscopy. Inoculated cell lines showed no cytopathic effect in three passages; cryolisated of cells lines were negative at molecular analysis for DMV.Biomolecular (RT-PCR) evidence of DMV infection was obtained from SW1–3 and the SW1b, as already reported elsewherePhotobacterium damselae subsp. damselae, Vibrio spp. and Aeromonas spp., which were recovered from the undamaged tissues and organs of all the three sperm whales. Interestingly, Clostridium perfrigens and its exotoxins and other clostridial alfa-toxins were detected from the kidneys of SW1b (beta-toxin), SW2 and SW3 . Brucella spp. and Salmonella spp. were not isolated from any of the four individuals under investigation.Microbiological examinations did not reveal the presence of any other significant pathogens, with the only exceptions of Neocyamus physeteris  were isolated from the skin of all the whales. Merocercoid larvae of Phyllobothrium delphini and Monorygma grimaldii  were found in the blubber and mesentery of SW1 and SW2, respectively. Lesions caused by copepods belonging to the genus Pennella  were detected in the blubber of all the animals. A sample of 150 nematodes were recognized as Anisakis spp. specimens and they were subsequently identified by diagnostic allozymes as A. physeteris. Molecular analyses carried out on mtDNA cox2 gene showed a 99% and 98% homology with two sequences previously deposited in Genbank26, confirming the results obtained with the allozymes. Pre-adults and adults of Anisakis physeteris  were isolated from the stomach chambers of all the animals. Some Anisakis specimens were also detected, mostly spoiled and dead, among the cephalopod beaks collected as stomach contents. Anisakis eggs were observed in feces. All the 3 sperm whales resulted infected  by A. physeteris, and mean abundance values (A) of infection resulted, respectively, A = 740 in SW1, A = 650 in SW2, and A = 730 in SW3. Fecal smears from SW1 and SW3 were also positive to MZN, but the molecular analyses did not confirm the presence of Cryptosporidium DNA. Finally, the tissue samples examined for Apicomplexa, including T. gondii, resulted negative on PCR analysis.Specimens of SW1–3 gastric contents resulted negative for all the tested biotoxins. In all the examined whales we digested food remains were found, along with a greenish-aqueous fluid. More in detail, squid beaks and lenses were found without any remain of soft tissues, thus suggesting no recent feeding activities . Prey residues were more digested in SW2 than in SW1 and SW3. Total numbers of upper and lower cephalopod beaks were 3969 and 4469 respectively in SW1; 3599 and 4200 in SW2; and 1605 and 1528 in SW3. SW1 had also foreign bodies in its stomachs .Histioteuthis bonnellii and 24% to H. reversa), followed by Ancistrocheirus lesueurii and Octopoteuthis sicula. Among these species, the largest one is H. bonnellii, which can attain a size of more than 6 kg in weight, while all the others are much smaller, rarely exceeding 1 kg in weight.Only eight cephalopod species were present; of the 7,539 recognizable lower beaks, over 95% were classified in the family Histioteuthidae  while PCDD and PCDF contributed for 21.7% , independently of the matrix. Spermaceti revealed greater dioxins concentrations than the bubbler.Analyses carried out on stomach contents of SW1–3 revealed neither harmful algal bloom (HAB)-related/associated hydrophilic nor lipohylic biotoxins.th that was recorded along the Adriatic coastline since 155514. Interestingly, another such event occurred as recently as 2009 along the Southern Adriatic coastline12. The causes of mass strandings are difficult to understand: viral infections and chemical pollution have been already considered to play a crucial role27. However, a complete investigation of the underlying causes requires a thorough multidisciplinary, multi-analytical effort to draw solid and reliable hypotheses15.The sperm whale mass stranding described in the present report is only the 728. This latter hypothesis, based on biological data, was supported also by genetic and stable isotope analyses. More in detail, sperm whales have a very low variability of the mitochondrial DNA control region compared to other cetacean species. Several studies detected no variability in the Mediterranean sperm whale population30. This means that mtDNA was not able to provide unequivocal information about matrilineal relatedness within our group of study. For these reasons, a parentage analysis was attempted, and the results suggested that the stranded whales likely belonged to a family pod, based on fullsib relationship between SW2 and SW3 and the kinship with SW1. Remarkably, SW1, SW2 and SW3 shared the same father. No relationship was noted with the pod stranded in 2009. However, here we note that 1 individual involved in the 2009 stranding shared the same father with a female stranded in 2008 along the Tuscany coastline.Seven animals were involved both in the 2009 and in the 2014 strandings. However, the social structure of the two stranded pods, inferred by biological and genetic information, was different. The 7 whales stranded in 2009 were all juvenile males forming a “bachelor’s school”, while in the most recent event the pod was composed by at least 3 females, with an older one, , one subadult (SW2) and one juvenile (SW3); the 4 refloated specimens could have been all juveniles, possibly both males and females, representing the typical composition of a “breeding school”, as defined by Whitehead and coworkers13C values determined in this study were similar to those reported by Pinzone and colleagues31 for sperm whales collected in the North Western Mediterranean Sea, whereas less negative δ13C values were observed by Ruiz-Cooley and colleagues32 for sperm whales stranded in the Gulf of California, due to a different diet. Sperm whales are teuthophageous, and in the Mediterranean feed preferentially on meso- and bathy- pelagic cephalopods of the family Histioteuthidae33. δ13C isotopic variations reflect differences of food sources and habitat distribution (nearshore vs. offshore). Nevertheless, a depletion of 13C and 15N may occur in tissues under fasting- and nutritional-induced stress, when adipose stores are used as energy source. Under these conditions, an increase of 15N may take place when the catabolism of protein prevails through the gluconeogenesis pathway28.Stable isotope analyses also support the Mediterranean origin of the stranded pod: the skin δpost-mortem examination. This female, in fact, showed a severe monolateral hydronephrosis, with concurrent fibrotic changes of the kidney parenchyma, caused by a large renal stone. Cetacean kidneys are essential not only for the osmoregulation, but also for hydration. Nevertheless, urinary tract lesions are infrequently reported in these mammals and, whenever diagnosed, they are often associated to systemic conditions34. In SW1, the features and the extension of the renal damage, associated with pregnancy and a reduced food intake (the latter meaning dehydration in cetaceans), could have led to a severe renal function impairment. The pathophysiology of the renal calculus  is largely unknown in marine mammals. Human uric acid calculi originate from oxalate concretions and account for a significant percentage of urinary stones with hyperuricosuria, low urinary volume and persistently low urinary pH as risk conditions. Etiological factors include genetic disorders, chronic diarrhea, dietary excess, insulin resistance, primary gout and metabolic disorders of uric acid. Possible symptoms and signs associated to this urinary stone include pain, malaise, nausea, lower urinary tract symptoms, and hematuria35.The above reported observations could be related either to SW1 pregnancy, or to the pathological changes observed at 24. The pathological changes typically associated with such infection were not observed in the stranded whales. However, a viremic condition was strongly suspected based on biomolecular and microscopic investigations. More in detail, the hypothesis of a DMV- related viremia was supported by a) positive immunolabeling of morbilliviral antigen in intravascular monocytes as well as in splenic macrophages and follicular-like dendritic cells; and b) by the absence of positive IHC staining in epithelial and nervous tissues. The viremia was likely characterized by an active viral replication in immune cells, being presumably associated to a condition of pronounced distress in SW1, SW2 and SW337. The concurrent finding of a herpesviral infection in the foetal individual indirectly confirms the poor health status of the oldest pregnant female, since herpesviruese are frequently detected in immunocompromised hosts and often reported in association with DMV infection39.Beside this potentially invalidating condition, SW1 and all the examined whales, including the foetus, were affected by DMV infection24 as well as in the debilitating condition affecting SW1. The different isotopic profiles reported for SW1 and the other 2 whales do not support a severe and prolonged fasting. Analyses of gastric contents with the consistent presence of pre-adults and adults of Anisakis physeteris nematodes in the stomachs of all the whales indicate that the whales fed in the recent past, but not in the days immediately preceding the stranding. Cephalopod beaks observed in their stomach belong to species living in the Southern Adriatic Sea: if the stranded pod is the same reported along the Croatian waters in the Central Adriatic basin on September 9th, a fasting period of at least 7 days could be hypothesized.The DMV infection reported in all the examined animals was probably an important co-factor in the mass strandingA. physeteris life cycle is embedded with the ecological characteristics of sperm whales, having squid rather than fish as suitable intermediate hosts40. The presence of Cryptosporidium oocysts on fecal smears was not confirmed by molecular methods. Therefore, further analyses would be recommended to precisely identify the nature of the observed parasites. Pinnipeds and sirenians are the marine mammals most exposed to the risk of Cryptosporidium infection, since they spend time on the mainland or in coastal waters. However, this parasite was found also in cetaceans, i.e. bowhead whales, North Atlantic right whales, minke whales, bottlenose dolphins and striped dolphins42. Here we emphasize that Cryptosporidium oocysts have never been observed in sperm whales43.These observations also confirm the specificity of this parasite species for this mammalian host and reveal how post-mortem, and on official communications from Governmental Institutions of Adriatic riparian States. More precisely, the “gas and fat embolic syndrome” was adequately ruled out based on the distribution and quantification of gas bubbles as well as of fat emboli in the circulatory system44. Lipid droplets were indeed found in pulmonary capillaries in SW3, but their amount and distribution rather suggest a damage related to the prolonged recumbency. The rolling movements of the stranded cetaceans due to the marine currents and waves injured the corresponding/ipsilateral adipose and soft tissues around the jaw, likely determining a subsequent lipid embolism. The distribution and quantification of the bubbles observed in the vessels are indicative of a peri-mortem change. No chemical analyses could be performed on the air samples collected during necropsies due to field conditions, but the evaluation of gas bubbles in the vessels according to Bernaldo de Quiros and colleagues46 excluded air embolism due to rapid ascent. Furthermore, researches and queries to Governmental Authorities, carried out in the two years after the event, indicate that no activity involving underwater sound sources was ongoing at the time of stranding, thus ruling out acoustic traumas and subsequent disorientation as causes of the stranding.Interactions with anthropic sound sources (i.e. military sonars and airguns used during seismic surveys) were excluded based on the absence of related pathological changes at 27 considered that methylmercury (MeHg) and other heavy metals could have been possible relevant co-factors. The Hg tissue concentrations measured in SW1, SW2 and SW3 are relatively high compared to those of terrestrial mammals. However, hepatic and renal total Hg and MeHg values are similar to those previously reported in Mediterranean sperm whales48, albeit higher than those measured in sperm whales involved in mass strandings that took place in the North Sea49. Even if MeHg may have reached the tissues of SW3 through blood or lymph, its relative percentages on total Hg, especially in the kidneys, are lower than those reported in the mass stranding occurred in 200912. In that situation, the body circulation of MeHg originating from diet and body storages was associated with a lower renal MeHg excretion caused by a lower food  intake, with subsequent modulation of glutathione complex and thiol activities, due to prolonged fasting12. Squadrone et al.50 reported that MeHg percentages in SW1–2 were lower than those reported in the liver and kidneys of the whales stranded in 200912. The only exception is SW3 , showing MeHg concentrations comparable to those of the 3 whales examined in 2009 .A previously published paper regarding this mass strandingAs stated above, the hypothesis of a prolonged fasting for the entire group was not supported by stable isotope and gastric contents analyses, as well as by the parasitic burdens in the gastric chambers. For these reasons, the higher amount of circulating MeHg in SW3 could be related to individual features like age, food intake and/or pathological conditions, but cannot be considered as a relevant cause of the mass stranding.51. Our present data suggest that exposure to MeHg prompts a preferential accumulation of inorganic Hg within lymphoid tissues in comparison to other non-lymphoid body districts of the whales52.Interestingly, the circulation of inorganic mercury, suggested by microscopic evidences of cytoplasmic accumulation of black pigments positive to Danscher’s staining in lymph node macrophages, hepatic Kupffer cells and neurons, was theorized in the 2009 stranding, and may apply also to the herein reported event. Since no hepatic degenerative changes were observed and fasting was not hypothesized, these findings support the theory that an increase of the inorganic Hg fraction in lymph nodes is likely due to the demethylation process of MeHg taking place in macrophage-rich lymphoid tissues53. In particular, PCB and DDT levels in the blubber were lower in the females stranded in 2014 than in the males stranded in 2009 , even if OC contents varied greatly among the individuals. In both stranding events, DDTs were more represented than PCBs, supporting the hypothesis of a more effective excretion of the latter ones but also of a preferential accumulation of DDTs through diet. Sperm whales feed on a narrow range of prey, but diet composition may differ with location and season, and bulls may feed on larger preys26. Squids are preferred preys in the Mediterranean Sea. Since the DDT levels in these cephalopods are normally higher than those of PCBs, the input of this xenobiotic through the food route is more relevant54. A very interesting result, emerging from the observation of DDT metabolites, is the relationship between pp’DDE and pp’DDT. A typical technical DDT is composed of pp’DDT (77.1%), op’DDT (14.9%), pp′DDD (0.3%), op’DDD (0.1%), pp’DDE (4.0%), op’DDE (0.1%) and unidentified compounds (3.5%)  and the pp’DDE/pp’DDT ratio is 0.05. A high pp’DDE/pp’DDT ratio states that most of the active substance (pp’DDT) has been degraded to pp’DDE, and therefore that no insecticide recently entered the ecosystem55. In all the examined specimens, pp’DDE/pp’DDT ratio resulted always higher than 10, reaching the value of 46 in the liver of SW2, and this ratio indicates an old DDT contamination. The pp’DDE/DDTs ratio, differently from the pp’DDE/pp’DDT ratio, can also indicate the efficiency of the metabolic processes of a population56. In the stranded whales the pp’DDE/DDTs ratio varied from 0.78 to 0.86, confirming a very high metabolism of this pesticide. The PCB and DDT burdens measured in our samples were higher than those documented in this species in previous non-Mediterranean studies57 but lower than those found in the sperm whales stranded along the coast of Apulia in 200953. However, differences in analytical techniques, or in the PCB congeners evaluated and/or DDT metabolites and tissue analyzed make any comparison challenging. It is also true that the sperm whales from the 2009 mass stranding were all males, while the specimens of this study were all females and a foetus. The fact that CH levels are normally higher in males than females is explained by the fact that females lose up to 90% of their total body burden of these substances during pregnancy and lactation, while males accumulate them throughout their lives59. A statistically significant difference of age and sex-related bubbler OC loads was also recently reported by Pinzone and colleagues31 in free-ranging Mediterranean sperm whales. The oldest female (SW1) showed the highest PCB and DDT levels among the whales stranded in Vasto, besides the OC content of her foetus. These results document a high level of these compounds in SW1 sperm whale, that match the highest values recorded in Mediterranean females of the same species31. The concentrations of PBDEs were also higher in the adult pregnant female than in the two younger individuals. Data on PBDEs in sperm whales are rarely reported, but our results indicate a relevant diaplacental transfer, as described in beluga whales (Delphinapterus leucas)60. Pathological changes observed in the ovary of SW2 further support the role of PBDEs as endocrine disruptors61.Nevertheless, we cannot definitively rule out that MeHg, a recognized immunotoxic pollutant, was responsible for the widespread lymphoid cell depletion observed in SW1–3. The same may apply also to the high concentrations of the organic pollutants  found in tissues, even if OC levels were lower than those reported in the previous sperm whale stranding of 200963.No differences were noted for HCBs, thus confirming the low concentrations already reported in other large Mediterranean marine mammals64 and mammals31 showed that the marine environment is not exempt from dioxin pollution. Even if their effects on the stranded whales could not be evaluated, data on dioxin loads in bubbler reveal a greater proportion of PCDD/F on total TEQ (21.7%) compared to the reference study by Pinzone et al.32 on individuals of the same species (13%) as well as to data obtained from bottlenose dolphins stranded in the Adriatic Sea in 2014 (1.3%).Dioxins and dioxins-like compounds are considered a possible threat for terrestrial environments during local environmental disasters . Some studies on Mediterranean fishpost-mortem examinations and by the success of the re-flotation attempts on the remaining 4 whales. SW1 showed relevant pathological changes in its urinary tract, which – in combination with the heavy metabolic demand of pregnancy and the dehydration related to fasting - likely led to a severe renal functional impairment in this animal. The tissue concentrations of MeHg and of the investigated OCs may have been not relevant enough to affect the health and behavior of SW1 and of the entire group. On the other hand, the DMV infection detected in all the examined whales likely played a pivotal role in changing the orientation capabilities of SW1, thus worsening its already poor health condition and favoring the entrance of the entire pod into the Adriatic Sea, with subsequent mass stranding along the Central Adriatic coastline.In conclusion, our data support the hypothesis of a “follow me”-related mass stranding, meaning that the pod followed a sick individual, likely the leader of this familiar group, towards an acoustically dead shore after spending debilitating days in the less-than-optimal Central Adriatic Sea, This “sick leader syndrome”-related hypothesis is supported by the life-threatening findings noted in the oldest female and by the good health conditions observed in the other individuals, documented by the results of the post-mortem and laboratory investigations, coupled with a correct and unbiased collection of data and information, to be interpreted using a multidisciplinary approach15.Finally, the hypothesis herein proposed underlines the importance of evaluating and discussing data using standardized protocols, as in the event described here. Evidences should result from thorough and detailed post-mortem investigations were carried out on the 3 sperm whales that died on the stranding . Other 4 individuals (SW4–7), belonging to the same pod, were successfully refloated after great efforts. During necropsies, a fetus was found in SW1 and it was labeled as SW1b.Detailed 12. Furthermore, detailed and exhaustive investigations were performed based on the previous experience to reach a thorough understanding of the event.Necropsy procedures and related analyses followed an already established protocol defined during a previous mass stranding of the same speciesTissues for the following analyses have been preserved and provided by the Mediterranean Marine Mammal Tissue Bank (CITES authorization no. IT020), Department of Comparative Biomedicine and Food Science, University of Padova, viale dell’Università 16, 35020 Legnaro - Agripolis PD, Italy.Re-floatation was achieved after evaluation of the vitality and size of each whale. Volunteers on the field dug furrows around the whales to allow careful swaying towards shallow waters. Coast guard vessels subsequently gently pulled the animals towards deeper waters (up to a depth of approx. 5 m) thanks to specific lifting bands passed around the head, behind the eyes, and across the abdomen. Other volunteers entered the waters and monitored the initial progress of each whale into the sea to remove any possible obstacle. The sperm whales were then freed from all harnesses and released in high waters, under kept continuous surveillance from a group of zodiacs. Another team maintained communication between operators on the shores and boats, thus ensuring overall coordination of all operations.Post-mortem examinations were conducted on the beach after consultation with the local Authorities and considering disposal options for the carcasses according to EU Directive 1069/09. A total number of 65 operators participated in the field operations, including veterinarians, biologists and student volunteers. The Cetacean stranding Emergency Response Team (CERT), established at the University of Padua in 2011 from the Italian Ministry for the Environment, was appointed as On-Scene Coordination Body (OSCB) according to ACCOBAMS resolution 4.16. Follow-up analyses, described in the specific sub-chapters below reported, involved 18 different Institutions.post-mortem operations, persons on the field were divided in different teams, each coordinated by an appointed team leader. The teams consisted of: a) 3 different necropsy teams of 4 persons each performed gross examination of the bodies - except the head - and collected photographic and written documentation; b) a specific team of 3 persons opened the heads and extracted the inner ears; c) a sampling groups of 10 persons was dedicated to receiving, labelling and processing samples for all ancillary analyses detailed in the following paragraphs; d) a logistic team composed of 2 persons took care of all disposable materials and sharpening of knives; e) a connection team of 9 persons ensured transportation of the samples and provided maintenance of the equipment; f) a team of 2 photographers documented the events; and, finally, g) 1 person took care of the communication between the OSCB and the different teams and 2 persons were selected to provide food and beverages. Furthermore, local NGOs informed the press and the general public on the nature and progress of the operations.During a) a logistic point where all equipment was available; b) a sampling point with a wet part and a clean area; c) a shadowed area dedicated to resting and storage of personal belongings.Cranes were used to move the bodies of the dead sperm whales to an area then limited to necropsy procedures. The setting included also 16. The heads were separated from the trunk and the brains were then removed cutting the occipital condyles with a chainsaw65. A comprehensive sampling of all organs and tissues was performed for later microscopic examinations. Given the importance of the inner ear, a special group was dedicated to remove the inner ear of SW2. Histopathological, immunohistochemical and ultrastructural studies were performed on tissues fixed by immersion in aldehydes. The eventual presence of Morbillivirus, Herpesvirus and Toxoplasma gondii was investigated by virological, microbiological and biomolecular techniques. Specific attention was payed to the presence of gas emboli by gross examination of subcutaneous, coronaric, mesenteric, renal and iliac veins, as well as of any gas collection in other organs. Gas bubbles were sampled from the coronary veins by vacutainer H tubes66 and findings were quantified and described according to de Bernaldo de Quiros and colleagues45. Lung tissue samples fixed in formalin were used to investigate the presence of lipid emboli. Furthermore, sampling for age determination as well as genetic, stable isotopes, diet and toxicological analyses were collected fresh or frozen. A complete list of all the samples collected during necropsies, their preservation and the subsequent exams carried out are included in supplementary material  were obtained. The second tooth was mounted in epoxy resin and cut in several sections 0.5 mm thick, using an ISOMET Low Speed Saw endowed with a diamond blade. Sections were later scanned with a high-resolution scanner and digitalized.The total number of GLGs in each of the tooth sections was determined in three separate sessions by three independent readers.Physeter Catalogue; the Mediterranean part of NAMSC 2004, North Atlantic & Mediterranean Sperm Whale Catalogue; the Tethys sperm whale catalogue 2008, TeSC). A non-automatic comparison was performed by visual inspection of each photo, since the total number (a few hundreds) of known individuals in the Mediterranean Sea is relatively small.Several photos of the flukes of each whale were taken to record any available characteristic that could result in individual identification, such as marks along the trailing edge , and/or pigmentation patterns on both surfaces. Additional photos of the dorsal fin and lower jaws were taken to aid the identification process. The best photo of each stranded whale was compared to the photos available in three photographic databases of free-ranging sperm whales from the Mediterranean Sea for the years 1990–2009  SWA: adult female, Piombino (LI), 2008122) SWB: 24 years old male, Cagnano Varano (FG), 2009123) SWC: 19 years old, male, Cagnano Varano (FG), 2009124) SWD: 19 years old, male, Cagnano Varano (FG), 200969. The sequences obtained were aligned with sequences previously obtained from Mediterranean sperm whales and deposited in the international GeneBank database.We performed the direct sequencing of a 399 bp portion of the mtDNA control region (Dloop)75 and resulted polymorphic in preliminary analyses carried out on samples from SW1–3 using the software CERVUS v. 3.0.776.Parentage analyses were performed on 16 microsatellite loci . These short tandem repeats (STR) have been characterized in different cetacean species77, to show eventual systematic relationships between the data obtained without entering a priori information about the nature of the data and possible relationships among them.An exploratory analysis was also conducted by Factorial Correspondence Analysis (FCA) implemented in GENETIX 4.052.278, to describe the relationship among the animals. The analyses were led using the Full likelihood method and simulations were performed with variable numbers of seeds, length and numbers of runs.Reference database or a group of candidate parents were not available, hence, we used the sibship reconstruction approach implemented in the COLONY software, version 2.0.5.9The age of the animals was used to provide paternity and maternity exclusion data. The analyses were then also performed without providing this information. The results relating to the best cluster, full sib and half sib were considered.79 was employed to assess the number of genetic groups in the sample. The posterior probability of the number of populations which best suits our genetic data (K) was calculated using the admixture model, which provides the opportunity for individuals to have common ancestors. The analysis was performed both by providing information on the location of sampling and without providing this information. Both the correlated allele frequencies model and the independent allele frequencies model were run80.Finally, the Bayesian clustering algorithm implemented in 2.3.4 STRUCTURE5 step of burn-in and 105 MCMC iterations, with 5 replicas for each K investigated. The optimal value of K was estimated based on the method described by Evanno and colleagues17.All runs were carried out by testing K (number of populations) from 1 to 5, with 5 × 10st differentiation factor between populations was calculated using the FSTAT Software version 2.9.3.219.The F13C), according to the Folch method modified by Boscolo et al.81, since lipids are depleted in 13C compared to the protein and carbohydrate fractions, thus giving rise to biased δ13C results. The isotope analyses were performed on bulk tissues and after extraction of lipids, to evaluate the data correction. The δ13C and δ15N signatures were determined at the ISPRA Laboratories of Chioggia with an Isotope Ratio Mass Spectrometer using a Delta V Advantage  interfaced with a Flash 2000 and Conflo IV . The analytical precision of measurements was 0.2%. Sucrose  and L-glutamic acid  were used as certified reference materials. The ratio of stable isotopes was expressed in delta notation (δ = [(Rsample/Rstandard) − 1)] × 103), where δ is the isotope ratio of the sample relative to the standards . Rsample and Rstandard are the fractions of heavy to light isotopes in the sample and standard, respectively. One is subtracted from the Rsample/Rstandard fraction so that samples with a ratio of heavy isotopes lower than the standard have a negative value and those with ratios of heavy isotopes higher than the standard have a positive value. This number is then multiplied by 1000 so that the δ notation is expressed in units of parts per thousand (‰).Lipids from blubber samples (0.02 mg) were extracted before analyzing carbon isotopes  equipped with a fluorescence X-ray scanning system. Schmorl’s stain was used to evaluate the presence of lipofuscin pigments. Finally, lung samples stored in buffered formalin were post-fixed with osmium tetroxide (OsO4) to detect fat emboli82.Tissues samples were collected from a) body organs and b) macroscopically evident lesion sites of the 3 whales which underwent a full necropsy. Specimens were formalin-fixed, paraffin-embedded, cut into 5 µm-thick sections and then stained with haematoxylin and eosin for routine light microscopic evaluation. Periodic acid Schiff (PAS), Gram and Giemsa stains were also performed, to assess the presence of non-viral pathogens within the tissues. The presence of inorganic mercury deposits within tissues and cells was investigated by auto-metallographic techniques, including Danscher’s stainingMorbillivirus nucleoprotein (N) antigen by means of a suitable immunohistochemical technique (IHC)21. IHC analyses were performed to detect any stress-related changes linked/associated to stranding, and to confirm that the animals stranded alive. Two different antibodies (Abs) were used for this purpose on skeletal and cardiac muscles, namely a polyclonal rabbit Ab raised against human fibrinogen , which was used at a dilution of 1:200 and incubated for 2 hours at 37 °C, and a polyclonal rabbit Ab against human myoglobin  that was employed at a dilution of 1:800 and incubated for 16 minutes at 42 °C. The anti-human myoglobin Ab was used also on sections of the kidneys of SW1–3, to detect the occurrence, if any, of intra-renal myoglobin.Selected tissue sections obtained from a) lungs and lymph nodes of all the 3 animals, b) the brains of SW2 and SW3 and c) the spleen of SW1, were additionally submitted to a detailed search for 23. Further analyses were conducted to determine the eventual presence of any additional viral and/or bacterial pathogen that may have influenced the mass stranding. Investigations for Herpesvirus were performed using a consensus PCR method which amplifies a region of herpesviral DNA-directed DNA polymerase83. For electron microscopy, tissue samples were prepared following routine procedure84. Moreover, multiple organs were homogenated and inoculated on marmoset lymphocyte cell line (B95a) for viral isolation; after three passages the cryolisate of the cells lines were submitted for molecular analysis.Methodology to reveal molecular evidences of DMV infection have been already reported elsewhereMycoplasma spp. were carried out on specific tissues; furthermore, both viable and PCR products of Brucella spp. were carried out according to OIE Terrestrial Manual85.Investigations for standard microbiology and 86. The foreign bodies of anthropogenic origin were also weighed and sorted.The organic fraction of the stomachs contents was represented exclusively by cephalopod beaks. The beaks were washed, weighed and sorted according to three stages of digestion: complete, damaged and highly digested beaks. Only the first two stages were taken into account and divided into upper and lower beaks for counting. The lower beaks were identified to the lowest taxonomic level possible, according to Clarke87.The right ear of SW2 was extracted and fixed in 10% neutral buffered formalin within 30 hours after death. The periotic bone surrounding the cochlea was decalcified for 9 days and 17.5 hours by using different dilutions of a specific rapid decalcifying agent RDO® . Precisely, the samples were immersed in 50% RDO for the first 3 days and then transferred to 25% RDO for the remaining time. Fresh batch of the solution were prepared every 24 hours and substituted, according to a previously optimized protocolstria vascularis of the cochlea were exposed. Subsequently, the cochlea was dissected, dehydrated with increasing concentrations of ethanol, critical point dried with CO2, and then coated with gold-palladium89. The sample was evaluated with a Hitachi S-4700 scanning electron microscope (SEM) at the University of British Columbia Bioimaging Facility for evidence of acoustic trauma.The decalcification of the periotic bone was stopped when the vestibular scala and the 2 mesh to separate and collect helminths. Portions of the small and large intestine  were removed and the sediment, obtained after washing on sieve (1 mm2 mesh) and decantation on conical glass, were examined for parasites using a stereomicroscope. The stomach content, composed mainly of cephalopods beaks, was removed and stored for further prey identification. All the parasites recovered during post-mortem examination were washed and counted; most of them were stored in ethanol 70%, while anisakid nematodes were frozen at −80 °C. The morphometric identification was based on Oliver and Trilles90 methodology for crustacean parasites while for the collected helminths on Delyamure91 and on Mattiucci et al.92.The animals were surveyed for ectoparasites and endoparasites. The three stomach compartments were opened and washed on a sieve with 1 mmAnisakis spp. collected during the parasitological survey was first undertaken using multilocus allozyme electrophoresis (MAE) on the frozen samples, according to the methodology reported in detail by Mattiucci et al.92. Furthermore, a subsample (N = 100) of the same specimens was sequenced at the mtDNA cox2 gene, according to the procedures described by Valentini et al.24 and by Mattiucci et al.25.Genetic identification of the adults and pre-adults of Cryptosporidium spp. Molecular analyses were performed using a PCR93 and a real-time PCR95.Fecal samples were collected and immersed in a high-density solution (sodium nitrate and sodium thiosulphate/1.450) prior to microscopic examination. Stool smears were stained by a modified Ziehl Neelsen (MZN) technique to detect Toxoplasma gondii using a PCR technique amplifying a conserved region of the nss-rRNA gene of Apicomplexa96. DNA extraction was performed on the tissues using NucleoSpin® Tissue kit . The reaction was carried out in 30 µl volume containing 1 X PCR buffer, 2 mM MgCl2, 200 µM each of the dNTPs, 2 U Platinum® Taq DNA Polymerase , 1 µM of each primer, and 1–3 µl of DNA extract. The reaction mixture was first treated at 95 °C for 5 min, followed by 12 cycles at 94 °C for 30 sec and 58 °C for 30 sec (decreasing each step −0.5 °C from 58 °C to 52 °C); 23 cycles at 94 °C for 30 sec, 52 °C for 30 sec and 72 °C for 30 sec and a final extension step at 72 °C for 7 min. DNA of T. gondii oocysts (GenBankTM accession no. AY663792), isolated from a domestic cat, were used as positive control. PCR products were analyzed by electrophoresis in SYBR Safe stained  2% agarose gel, visualized with Geldoc XR  under UV light, subsequently purified and sequenced at BMR-Genomics .Tissue samples were analyzed for the detection of The gastric contents of the 3 sperm whales were analyzed for the presence of saxitoxins, domoic acid and lipophilic biotoxins .The search for saxitoxins (SXTs) was carried out according to the AOAC method 959.08. This is a biological method based on an acid extraction of the toxins that is then inoculated in mice.The search for domoic acid (DA) was carried out according to the European Reference Laboratory Method, EURL SOP for determination of domoic acid version June 1, 2008. DA was extracted by a mixture of methanol and water, then filtered through a membrane filter and analyzed by HPLC with UV detection.The search for lipophilic toxins (DSP) was performed according to the European Reference Laboratory Method. This method was carried out for four chemical groups of toxins: okadaic acid (OA), including dinophysistoxins (DTX), pectenotoxins (PTX), azaspiracids (AZA) and yessotoxins (YTX). The method is based on the extraction of the toxins with 100% methanol from homogenized tissue. The extracts were filtered and analyzed by liquid chromatography coupled mass spectrometry. To determine the total content of OA group toxins, an alkaline hydrolysis is necessary from ethanolic extract to convert any acylated esters to the parent toxin.49. In the present article, the results of metal analyses were compared with all findings herein reported, and considered in the Discussion section. Further, previously unreported, analyses were performed on tissues sampled during necropsies of the three whales, and detailed here below.Data regarding heavy metal analyses have been published separatelyBlubber samples were freeze-dried and extracted with n-hexane in a Soxhlet apparatus. The lipid content of the extracted organic material was determined by gravimetry.97. Tissue samples (about 2 g) were lyophilized in an Edwards freeze drier for 2 days, and extracted with n-hexane for gas chromatography (Merck) in a Soxhlet apparatus for analysis of organochlorine compounds. Whatman-H cellulose thimbles  to be used for extraction of the samples were preheated for about 30 min at 110 °C and pre-extracted for 9 h in a Soxhlet apparatus with n-hexane, to remove organochlorine contamination. Each sample was spiked with surrogate compound 98 prior to extraction. This compound was quantified and its recovery calculated. Surrogate recovery was reported with the sample results. The samples were then purified with sulphuric acid to obtain a first lipid sedimentation. The extract then underwent liquid chromatography on a column containing Florisil that had been dried for 1 h in an oven at 110 °C. This further purified the apolar phase of lipids that could not be saponified, including steroids like cholesterol. Decachlorobiphenyl (DCBP - IUPAC number 209) was used as an internal standard, added to each sample extract prior to analysis, and included in the calibration standard, constituted by a mixture of specific compounds . The analytical method used was High Resolution Capillary Gas Chromatography with an Agilent 6890 N and a 63Ni ECD and an SBP-5 bonded phase capillary column . The carrier gas was nitrogen with a head pressure of 15.5 psi [ = 0.1068687 MPa] (splitting ratio 50/1). The scavenger gas was argon/methane (95/5) at 40 ml/min. Oven temperature was 100 °C for the first 10 min, after which it was increased to 280 °C at 5 °C/min. Injector and detector temperatures were 200 °C and 280 °C respectively. The extracted organic material (EOM%) from freeze-dried samples was calculated in all samples. Capillary gas chromatography revealed op9- and pp9- isomers of DDT and its derivatives DDD and DDE, and about 30 PCB congeners. Total PCBs were quantified as the sum of all congeners. These congeners constituted 80% of the total peak area of PCBs in the samples. Total DDT was calculated as the sum of op9DDT, pp9DDT, op9DDD, pp9DDD, op9DDE and pp9DDE. The results were expressed in ng/g lipidic weight (ng/g lipidic weight).Analyses for HCB, DDTs and PCBs were performed according to methods recommended by the U.S. Environmental Protection Agency (EPA) 8081/8082 with modifications99. The same specimens were analyzed also for the presence of the six non dioxin-like-polychlorbiphenyl (NLD-PCB) indicators (ICES-6), and their value was calculated as sum of the congeners. All values were expressed as “upper bound” concentration in pg/g whole weight (NL-PCBs in ng/g whole weight), so that values below of the limit of quantification (LOQ) were considered equal to the LOQ itself. All analyses were performed according to U.S. Environmental Protection Agency method 1613 rev. B, and method 1668 rev. B, respectively for Dioxins (PCDD and PCDF) and PCBs (LD-PCB and NLD-PCB), in-house modified to adapt the procedures to specific matrix101.The toxicologically relevant 7 Polychlordibenzo-p-dioxins (PCDD); the 10 Polychlordibenzofurans (PCDF) congeners; and the 12 dioxin-like polychlorbiphenyl congeners (DL-PCB), were investigated in the blubber of the three adult sperm whales and in the spermaceti of SW3. The overall toxicity of analytical products was calculated as Toxicity Equivalent Quantity (TEQ) by multiplying the effective concentration of each congener for the Factor of Toxicity Equivalent (TEF) to the toxicity value of 2,3,7,8-tetraclorodibenzodioxin, according to Van den Berg and colleagues102, as used in other studies on these species63.The non-parametric Kruskal-Wallis test was used to compare the levels in the biological matrixes of the three considered xenobiotics. The Kolmogorov-Smirnov test was employed to evaluate any significant difference (p < 0.1)www.idromare.it), the Marine Forecasting Systems National Group of Operational Oceanography of the National Institute for Geophysics and Vulcanology , the European Ocean Observatory Network  and the “Poseidon System” of the Hellenic Center for Marine Research (www.poseidon.hcmr.gr). The INGV also provided information on past seaquakes and geomagnetic field abnormalities (www.earthquake.rm.ingv.it).A series of multidisciplinary and wide range investigations were performed to assess any possible environmental cause temporally and spatially associated to the mass stranding. Weather and marine data from August 1, 2014 were obtained from different national and international meteorological archives, namely the Institute for the Protection of the Environment and Research .Since previous studies reported that solar activity and lunar cycles are among the natural factors that potentially cause and/or influence sperm whale mass stranding events, specific information was obtained from the US National Oceanic and Atmospheric Administration .Finally, historical data sets on cetacean strandings along the Central and Southern Adriatic Sea coastline, and specifically those concerning sperm whales in Italy, were obtained from the Italian Cetacean Stranding Database (Supplementary Dataset 1


Altered gene expression in pathways relevant to leukaemogenesis, as well as reduced levels of circulating lymphocytes, have been reported in workers that were exposed to benzene concentrations below 1 ppm. In this study, we analysed whole blood global gene expression patterns in a worker cohort with altered levels of T cells and immunoglobulins IgM and IgA at three time points; pre-shift, post-shift (after three days), and post-recovery (12 hours later). Eight benzene exposed tank workers performing maintenance work in crude oil cargo tanks with a mean benzene exposure of 0.3 ppm (range 0.1–0.5 ppm) and five referents considered to be unexposed were examined by gene expression arrays. By using our data as independent validation, we reanalysed selected genes that were reported to be altered from previous studies of workers being exposed to sub-ppm benzene levels Four out of six genes previously proposed as marker genes in chronically exposed workers separated benzene exposed workers from unexposed referents . Even better separation of benzene exposed workers and referents was observed for short-term exposure for genes in the Jak-STAT pathway, particularly elevated expression of IL6 and reduced expression of IL19. Benzene, a haematotoxic and leukaemogenic agent ,2, is a + T cell count after a period of three consecutive work shifts [In a longitudinal study of a small group of healthy petroleum workers that were exposed to benzene concentrations well below 1 ppm , we found a dose-dependent decline in the immune parameters IgA, IgM, and CD4k shifts . In the k shifts . The Schk shifts . This pak shifts ,14, and k shifts . We testWe conducted the study with the approval of the Regional Committee for Medical Research Ethics of Western Norway (REC West no. 102.04) and the Norwegian Social Science Data Services (project no. 11187). The Directorate for Health and Social Affairs gave permission to establish a biobank. Participation was voluntary, and informed written consent was obtained from all participants. All of the subjects were informed about their own test results on exposure and haematological parameters both in personal letters, and if follow-up was recommended, the participants were also informed by oral communication. 3). Before maintenance work started, the tanks were cleaned with hot crude oil and sea water and purged with inert gases and fresh air. The empty storage vessels ready for inspection and maintenance had sludge lining the inside of the tank. Hence, the tanks were continuously ventilated with forced air. Use of protective suits (Tyvek®) and half-face air-purifying respirators with a combination of particle filter and organic vapor cartridge were mandatory during cleaning and inspection, and the chemical protective suits were worn over the cotton coveralls and were discarded twice during each shift. During scaffold building and welding, the use of respirators varied, but was not systematically recorded. Catering (reference population) included work in the canteen and housekeeping (cleaning of the living quarter and some laundry). The referents spent the whole work period inside the living quarters, and although one of them was exposed to cooking fumes and one was potentially exposed to crude oil residues during cleaning of dirty suits, none of the referents were in the processing area where crude oil or benzene-containing produced water was present. During the study period, all subjects lived on board the production vessel and were exposed to the same environment in the living areas and had similar work schedules and diet. Smoking was prohibited in the processing area, but the living quarter had a limited number of designated smoking rooms. Since alcohol was banned on the installation, no participant consumed alcohol during the study period.The study population consisted of nine men that were exposed to benzene during maintenance work in crude oil cargo tanks on a production vessel on the Norwegian continental shelf and six referents recruited from the catering section on the same vessel, who were considered to be unexposed to benzene . The tan0) and it was considered a baseline measurement. The second sample was collected at the end of the work shift (1800–2100) on the third day of tank work , and a third sample was collected in the morning (0700–0900) the following day . Blood and urine samples were obtained from the referents on the same days following the same time protocol. The participants completed a self-administrated questionnaire including questions on age, sex, medication use, and whether they were current smokers during the study period (1) and the following morning (t2) as a ratio of the time point prior to expose (t0), which gave two time-points of fold change data for each person. Secondly, the data were analysed without fold change at each time point.The sampling period was three consecutive 12-h work shifts . The firy period . Time spThe original study population ,15 comprAverage age was found to be significantly different between the two groups. An age balanced dataset was therefore generated by excluding workers at age < 35 and >55 see . The ageTM) attached in the worker’s breathing zone (the worker’s lapel). Personal exposure to BTEX was not measured for the reference group as they were assumed not to be exposed to benzene above the background concentration in the indoor environment. The dosimeter badges were desorbed with CS2, and were analysed quantitatively and qualitatively by gas chromatography with mass spectrometry . The limits of detection were 0.001 ppm for benzene and 0.01 ppm for toluene, ethylbenzene and xylene. Blood samples for determining benzene and toluene were collected by venipuncture into Venoject II® tubes (hard plastic) with heparin, and the urine samples for the determination of benzene and toluene, t,t-muconic acid , and 1-hydroxypyrene  were collected in glass bottles (Pyrex®) with polypropylene stoppers. The samples were stored at 4 °C until they were transported to the Biomonitoring Laboratory at the Finnish Institute of Occupational Health in Helsinki, Finland  and frozen at −20 °C to the University of Cincinnati, Cincinnati, OH. USA (urinary 1-HP), for analysis. The benzene and toluene concentrations in blood were analysed by a head-space sampler (Perkin Elmer Headspace sampler HS40) and a gas chromatograph (Perkin Elmer Autosystem Gas Chromatograph) using photoionization detection according to the method described by Pekari et al. [The workers’ environmental and biological concentrations of relevant petroleum-derived hydrocarbons were sampled according to the protocol given in i et al. . Samplesi et al. . Urinaryi et al. . Urinaryi et al. . The limy NIOSH, 03. The l® and total RNA from the human whole blood were extracted by using the PAXgene Blood RNA Kit from PreAnalytiX/Qiagen. Amount (9.7–97 µg) and quality of the extracted RNA was verified by the NanoDrop® ND-1000 spectrophotometer  and the Agilent 2100 Bioanalyzer . Paxgene tubes were stored at −20 °C prior to extraction of RNA, but had an acceptable RNA quality with Integrity Number (RIN)-values of RNA ≤ 7.2.Blood samples for collection of RNA were drawn by venipuncture into PAXgene Blood RNA tubesAll microarray experiments were performed using the Illumina BeadStation 500GX, which is based upon fluorescence detection of biotin labelled complimentary RNA (cRNA). 500 ng of total RNA from each sample was reversely transcribed, amplified, and Biotin-16-UTP –labelled, using the Illumina TotalPrep RNA Amplification Kit .750 ng of biotin labelled cRNA was hybridized to the HumanRef-8 v3 Illumina Sentrix BeadChip according to manufacturer’s instructions. The HumanRef-8 v3 BeadChip targets approximately 24,500 well annotated RefSeq transcripts and covers 18,631 unique curated genes. The samples were labelled and hybridised in two batches, but care was taken to ensure that all the samples from the same individual were handled in the same batch in each step. The samples were randomly distributed on the slides, 8 samples on each slide.The data from the Illumina BeadStudio v.3.3.7 software was quality controlled using R version 2.8.1 . Prior t1) and pre-next shift  over pre-shift . As the fold change ratio was log transformed for normalisation fold change becomes a difference as: FCt1 = log2(t1/t0) = log2(t1) − log2(t0), and likewise for FCt2. The microarray data are available in ArrayExpress, access number E-MTAB-5331, where they have been described according to MIAME principles [The gene expression data was log transformed, and changes in the gene expression data from prior exposure to after exposure were calculated as fold change (FC) data post-shift  and “the China benzene exposure study” ,8,9 (Ill1)  were presented and used without any transformations  and measures of exposure at post shift (trmations . The obsrmations .https://www.camo.com/unscrambler).PCA was used to visualize the relationship between chemical exposure and immunoglobulin levels and leukocyte counts. PCA identifies the relationships that explain as much of the variance in the data as possible and separates these into orthogonal principal components . The anaNext, supervised multivariate analysis was performed using gene expression data as input and benzene exposure vs. referents as response . The first analysis was performed on the six genes Schiffmann and coworkers  selectedλ, into the algorithm to make the estimated regression coefficients more stable. Adding the penalty term may reduce overfitting and improve the prediction error. RR does not perform variable selection since none of the input terms are downscaled to zero, whereas LASSO performs both variable selection and regularization in order to enhance the prediction accuracy and improve the interpretability of the statistical model. Elastic Net employs a predefined tuning parameter, α, to define the balance between the RR and LASSO methods. Elastic Nets produce sparse models aiming for optimal prediction [α = 0.5, and λ was set to “λ minimum”. The model was performed within each time point and validated using full cross validation where one patient at the time is omitted from the calibration and used for validation. The analyses were run with fold change on each of the two time points; immediately after three consecutive work shifts (time 1) and again after recovery of 12 h (time 2) over time point 0, prior to benzene exposure, and without fold change at each time point.Elastic Net (R package glmnet version 2.0-16) were used for the analysis, and genes selected in each model were recorded ,29. Elasediction . ElasticPCA  was alsoTechnical verification of individual genes ) was not included since the Illumina array used in the present study has previously been shown to be highly reliable . PCR is Characteristics of the study population and descriptive statistics of environmental hydrocarbon exposure on the third day of study and concentrations in blood and urine post shift are given in PCA on the descriptor variables  on data with fold change performed on the age balanced data set is displayed in Elastic Net analysis selected four of the six genes identified as markers predicting low benzene exposure (below 1 ppm) from “the China benzene exposure study”  on data For comparison, we also ran all 30 genes selected for testing by Schiffman , and thiThe Elastic Net analysis performed with genes belonging to the Jak-STAT pathway on the age-balanced data set selected 16 out of 162 genes reported to predict exposure to benzene  Supplem. As seenTo provide a more in-depth understanding of the responses, we repeated our analyses on genes without fold change at each time point separately . As before, Elastic Net analyses were performed using exposure group as the response (referents vs. exposed workers), followed by PCA and gene plotting. No models were selected at any time point when just the six China marker genes were used (not shown). When all 30 genes were used, eight were selected at time 0 , ZNF703, TTC9B), and two at time 1 . see  and at tTo inspect the expression of individual genes without fold change selected by Elastic Net, time series plots of average gene expression were chosen. The time trends in the six marker genes that were proposed by Schiffman and coworkers  Figure  show tha+ T cell count [Benzene exposed workers, which were previously reported to have a dose-dependent decline in several immune parameters, including CD4ll count , deviatell count ,8,9. We ll count , may addll count ,32. Hencll count ,8,9.2). Interleukins IL-6 and IL-19, in particular, showed a strong ability to separate the workers . The finding that four out of the six proposed marker genes were selected by Elastic Net suggests that, in our data set, all four are required for prediction. C-type lectin domain family 5A (CLEC5A) was the best individual predictor in this study among the previously proposed marker genes, but also worked well together with acyl-CoA synthetase long chain family member 1 (ACSL1). CLEC5A is a myeloid differentiation gene known to participate in inflammatory responses . Althoug workers . IL-6 waOur data included three time points of measurements, one prior to (pre-shift) and two time points after the start of benzene exposure (post-shift and pre-next shift). Most of the genes change their expression over time, suggesting that longitudinal data collection with repeated measures can detect effects that add more information than only one time point. In the “The China benzene exposure” study ,8,9 the Although this small and exploratory study was not designed to explain a possible correlation between low level benzene exposure and reported excess risk of hematopoietic malignancies in this group of workers ,37,38, oAnalysis of age at exposure in a cohort of workers producing rubberized cloth, the so-called Pliofilm cohort, suggested that the effect of benzene on leukaemia mortality is of greater magnitude for workers exposed at older ages (>45) than for those exposed at younger ages . HoweverCigarette smoke contains benzene; hence, smoking is a potential confounder for occupational exposure to benzene . Most ofBenzene and toluene are simultaneously present in crude tank; hence, co-exposure occurred in the present cohort. Although the mean toluene exposure was as low as 3% of the Norwegian occupational exposure limit (25 ppm), the internal concentration of unmetabolized toluene was significantly elevated in exposed workers as compared to referents. A few studies reporting on benzene’s haematotoxic effects in workers also report co-exposure with toluene ,43. Whil®) is 0.5 ppm [The Norwegian and European occupational exposure limit of benzene (OEL), both averaged over an 8-h shift, is 1 ppm, while the American Conference of Governmental Industrial Hygienists (ACGIH) Threshold Limit Value (TLV 0.5 ppm . However 0.5 ppm . Hence,  0.5 ppm ,8,25,26, 0.5 ppm .Gene expression patterns in peripheral blood cells from workers that were exposed to benzene at concentrations well below 0.5 ppm benzene differed significantly as compared to non-exposed referents. Changes in expression of the gene pairs CLEC5/ACSL1 and IL6/IL19 clearly separated exposed workers from referents following a three-day work shift. The results were consistent with other reports and they suggest that gene expression analyses in the study of subjects exposed to low benzene exposure are feasible. Multivariate selection of individual genes facilitates analysis in small datasets.