Major macroscopic lesions of abortion-associated C. jejuni-inoculated ewes included placentitis and endometritis, regardless of the ovine or bovine strains used. The affected placentomes and uterus appeared edematous, hemorrhagic, and fibrinopurulent (Figure 1B). Enlarged placentomes were more prominent in the G3 ewes orally or i/v infected with the ovine abortion-II isolate. Liver and spleen also exhibited petechial hemorrhages on their surface in ewes that were i/v inoculated with the bovine abortion strain (G2-V-1) as well as in ewes orally or i/v inoculated with the ovine abortion strain (G3-V-1; G3-O-1; G3-O-2). Additionally, hydrothorax and subcutaneous hemorrhage as potential signs of acute septicemia were detected in the dead ewe at 20–24 h after i/v inoculation (Figure 1C and D); however, pneumonia was not accompanied with the former lesion. No gross lesions were noted in G1 or the control ewes.

In line with macroscopic observations, histological lesions mainly consisted of suppurative placentitis or endometritis or both. Placentitis was commonly observed widespread throughout placenta, comprising placentomes and chorioallantoic membranes. In the placentomes of inoculated ewes (G2 and G3), trophoblasts lining the placental villi were necrotic with extensive neutrophilic infiltration (Figure 2A and B). Occasionally, small bacterial colonies were found within the necrotic lesions (Figure 2C). Concomitantly, chorioallantoic membranes exhibited moderate to severe necrosis and suppurative inflammation of the epithelium (Figure 2D). In IHC analysis, large amounts of C. jejuni antigens were evident in trophoblastic epithelial cells lining the chorioallantoic membrane (Figure 2E and F); however, it was notable that under the IHC conditions, including the use of a single detection antibody, bacterial antigens were rarely detected in the placentomes and in the stroma of chorioallantoic membranes regardless of the ovine or bovine strains used and the severity of the lesions observed. These data indicated a possible tissue tropism of the abortion-associated C. jejuni in the chorioallantoic placenta of sheep.

Moderate to severe endometritis observed in abortion associated C. jejuni-inoculated ewes was characterized by suppurative inflammation in the endometrium and in the lumen of uterus (Figure 3A), and occasional severe necrosis of endometrium accompanied by severe suppurative inflammation (Figure 3B). In G2 ewes infected i/v with the bovine abortion isolate, endometrial glands were filled with large numbers of neutrophils (Figure 3C). In addition, 2 ewes from G2 which were orally inoculated with the bovine isolate showed mild diffuse suppurative lymphadenitis (Figure 3D). Three ewes from G2 and G3 i/v inoculated with bovine or ovine strains, respectively, exhibited periportal suppurative hepatitis (Figure 3E). In G1 and the control group, no histological lesions were detected in cotyledons, endometrium, liver, spleen, and other major organs. Histological examination of intestinal tissues was not performed as these tissues underwent spontaneous autolysis.

Using the in situ TUNEL assay, relative to the negative control or 81–176 inoculated ewes, the placentomes of the ewes infected with bovine or ovine abortion C. jejuni strains showed in situ TUNEL-positive cells (red staining) (Figure 4). Most of TUNEL-positive cells appeared to be trophoblasts exfoliated from placental villi. The in situ TUNEL-positive signals were also detected in trophoblasts lining the chorioallantoic membrane of the infected ewes (data not shown).

The ovine and bovine abortion-associated C. jejuni strains induced abortion or stillbirth. One of the ewes orally inoculated with the ovine isolate (G3-O-2) aborted at PID 15 (Figure 1A). A month later, another ewe (G3-O-1) from the same group delivered a dead fetus. Furthermore, one ewe (G3-V-1) i/v inoculated with the ovine isolate died 20 h after inoculation (Table 1). Rectal temperatures of inoculated ewes were elevated (105–105.5°F) 3 to 4 days post i/v inoculation and at PIDs 10-12 in orally inoculated ewes. Stillbirths were observed in 2 cases in G2 (G2-V-1 and G2-V-2), which were inoculated i/v with the bovine isolate (Table 1), whereas the two ewes orally inoculated in G2 group delivered normally. No clinical signs were observed in the G1 ewes orally or i/v inoculated with 81–176 strain, and they delivered normally. Feces changed from pasty to diarrheic in most of the inoculated ewes, which was profoundly seen in the G2 and G3 groups. No abortions or clinical signs were observed in control non-inoculated ewes (Table 1).

In total 51 diarrheic and 50 non-diarrheic piglets aged 3–7 days were included in this study. The piglets were selected from four commercial Danish swine herds presenting high standards of management and housing. Diarrhea of unknown etiology (not caused by either enterotoxigenic E. coli, C. perfringens type C, rotavirus A, coronavirus or parasites) and poorly responding to antibiotic therapy was present in at least 30 % litters in each herd for a period of minimum six months. From each herd 11–14 diarrheic and 12–13 non-diarrheic piglets (age matched) from several litters (maximum two piglets per litter) were selected. The diarrheic piglets had diarrhea for at least 2 days prior to euthanasia and were selected from the litters with the highest prevalence of diarrhea. The non-diarrheic piglets did not have diarrhea at any time and were selected from the litters with no diarrhea or very low prevalence of diarrhea. For further details on the selection of herds and piglets the reader is referred to Kongsted et al.; 2013.

The objective of this study was to elucidate the role of E. coli, Enterococcus spp., C. perfringens and C. difficile in neonatal porcine diarrhea with no previously established etiology. We used FISH method for investigation of the prevalence, abundance and location of these potentially pathogenic bacteria in the intestinal tissue because a direct visualization of microorganisms helps to determine their association with the mucosa surface and may therefore have a potential value in elucidating their role in the disease. Sensitivity of FISH method for detection of bacteria depends on many factors including metabolic activity of microbial cells and availability of target sequences in the tissue samples. It has been shown that in natural samples the fluorescence signal intensity may be too low for identification of microorganisms. However, because the intestinal microbiota are expected to express high metabolic activity and possess high content of rRNA, the FISH method seems to be an appropriate approach for in situ detection of enteric bacteria. In order to better control for eventual false negative results due to methodological problems we decided to apply a general bacterial probe simultaneously with the specific probe on all tissue specimens. As the FISH results of hybridization with the general probe were satisfactory, we consider sensitivity of FISH performed in this investigation to be high.

According to informations provided at probeBase, three oligonucleotide probes used in this study: Enterococcus spp, C. perfringens and C. difficile probes, were shown to be highly specific and reliable for detection of these particular bacteria. However, the E. coli probe used in this investigation was noted to be unable to target all E. coli strains. Additionally, the target sequence of this probe was shown to match other enterobacteria; however these were not relevant as swine pathogens.

Quantification of microbial community detected by FISH in the intestinal tissue samples is not possible when the bacteria are present in high concentration as it hinders distinction of fluorescence signals from single bacterial cell. Therefore, evaluation of the amount of bacteria was done in a semi-quantitative manner, based on the subjective judgment of the investigator.

Adhesion of bacteria to the epithelial cells is believed to be an initial and pivotal event in the pathogenesis of most bacterial enteric infections and is necessary for allowing bacteria to survive and persist in a continuously moving environment and to defeat host defense mechanisms. In this study adherent bacteria were seen in 37 % of diarrheic and 14 % of non-diarrheic piglets. The non-diarrheic piglets were collected from the same herds as the diarrheic ones. Therefore, it cannot be excluded that some of the non-diarrheic piglets were in the initial phase of infection, thus expressed similar composition of eventual pathogens as the diarrheic piglets. In situ hybridization with the specific probes identified these bacteria as E. coli and/or Enterococcus spp. and there was a significant positive correlation between adherence of these bacteria and diarrhea. Furthermore, large amounts of E. coli were seen in significantly higher number of diarrheic piglets compared to non-diarrheic animals. Overgrowth and colonization of the mucosal surface by E. coli suggest its involvement in diarrhea. The piglets involved in this study were negative for enterotoxigenic E. coli (ETEC), which are considered to be the most common cause of pig neonatal diarrhea and for which the ability to attach to and colonize the intestinal epithelium is believed to be a hallmark virulence trait. However, E. coli strains other than ETEC have also been shown to be able to adhere to the intestinal mucosa surface and cause diarrhea. In addition, attaching and effacing E. coli (AEEC) that have ability to cause attaching and effacing lesions in the gut mucosa, have been associated with diarrhea in domestic animals including pigs. Therefore, further work will be done towards identifying and defining the pathogenicity of adherent E. coli found in this study.

We observed a significant positive correlation between the presence of E. coli and histomorphological changes in the intestinal mucosa. Villous atrophy is a common condition in diarrheal diseases and in suckling piglets this is primarily associated with viral or parasitic infections. However, some reports have shown that shortening of villi and epithelial lesions can follow colonization of the mucosa by E. coli[22,23]. Since the piglets included in this study were thought to be free from infection with commonly known pathogens, at this stage of investigation it is difficult to conclude whether overgrowth and colonization of the mucosa by E. coli was a primary event in the pathogenesis of villous atrophy or was secondary to infection with other, yet unidentified microorganisms, which cause alteration in the intestinal villi. Further studies are currently being conducted in order to determine the etiology of the presently described diarrhea.

Enterococci are commensal bacteria in the intestinal tract. However, it has been reported that certain members of enterococci can sporadically cause diarrhea in neonatal animals including piglets. The pathogenic potential of enterococci seems to be associated with their ability to intimately adhere to the intestinal epithelium but the mechanisms by which these bacteria cause diarrhea remain unclear. So far, no evident mucosal damage has been reported in association with enterococci infection. In this study we also observed adhesion of Enterococcus spp. to the intestinal epithelial cells in the diarrheic piglets, which suggests pathogenic ability of these bacteria. A significant positive correlation between the presence of enterococci and histological lesions (villous atrophy and mild epithelial lesions) can be explained by the fact that the positive fluorescence signals for adherent Enterococcus spp. were seen in the small intestine of piglets that also had adherent E. coli. If these lesions were a consequence of a bacterial infection, they should be associated with E. coli rather than Enterococcus spp. as discussed above. Nevertheless, simultaneous colonization of the intestinal mucosa surface by these bacteria is an interesting finding and suggests their close interactions. Previously, a virulent synergistic effect between E. coli and E. faecalis has been described in relation to experimental polymicrobial infections and it has been suggested that E. faecalis may inhibit phagocytosis of other pathogens including E. coli, and prevent them from intracellular death.

The majority of the piglets positive for adherent E. coli and adherent enterococci belonged to the same herd, which indicates that environmental factors influence composition of intestinal microbiota and eventual pathogens. This finding emphasizes the complexity of pathogenesis of porcine neonatal diarrhea and suggests that consideration of herd related aspects may be crucial for diagnosis and control of diarrheic conditions in piglets.

C. perfringens type A and C. difficile are nowadays regarded as ones of the most common bacterial species involved in pig neonatal diarrhea worldwide. In this study, the occurrence of C. perfringens and its amount detected by FISH were similar in diarrheic and non-diarrheic piglets. Pathologically, degenerative and necrotic changes in the intestinal mucosa are commonly associated with clostridial enteritis and the bacteria are usually present among the necrotic tissue. Such lesions were observed only in one diarrheic piglet in this study and it has been confirmed by microbiological testing that this piglet was positive for C. perfringens type C, which in that case can be regarded as a cause of enteritis. However, the mechanisms that could be involved in C. perfringens type A infection, remain unclear and there is no certain evidence for an adhesion of this bacterium to not destroyed intestinal tissue. Only few studies have investigated C. perfringens type A adhesive properties, but their results were inconclusive and to date, it is generally believed that C. perfringens does not have the ability to adhere to healthy intestinal epithelium. In agreement with this, the presence of C. perfringens in close proximity to the mucosal surface was seen in similar prevalence in both groups of piglets in this study (20 % diarrheic vs. 30 % non-diarrheic) and did not correlate with histological lesions, suggesting that the localization of C. perfringens cells in the intestinal mucosa is not linked to its pathogenicity. However, the pathogenesis of clostridial enteritis is commonly associated with the ability to produce toxins and diagnosis of the infection is based on the detection of large numbers of toxigenic bacteria. Therefore, the role of C. perfringens type A should not be definitely ruled out and the determination of the importance of this bacterium in neonatal diarrhea should be supported by thorough investigation on clostridial toxins.

C. difficile infections are currently reported as one of the most common causes of pig neonatal diarrhea in some countries and whenever diagnosed, the culturing reveals heavy growth of this bacterium. Microscopically, C. difficile infection is characterized by catarrhal, fibrinous or purulent colitis, however such lesions were not observed in this study. Furthermore, there was no association between the presence of this bacteria and pathological changes in the colon. Moreover, the occurrence of C. difficile and its amount did not differ significantly between diarrheic and non-diarrheic piglets. Therefore the presence of this bacterium seems not to be linked to the investigated diarrhea and these results are in agreement with other reports. Additional studies with focus on clostridial toxins are being conducted to determine the role of both Clostridia species in the pathogenesis of presently reported neonatal diarrhea.

To establish a novel mouse model for ZIKV infection, we compared the clinical, histological, and virological findings of male (group 1) and female (group 2) mice with dexamethasone immunosuppression and ZIKV infection with those of the appropriate controls (groups 3 to 8) (Table 1). In terms of the clinical parameters, the dexamethasone-immunosuppressed mice developed mild (~ 5%) weight loss (Fig. 1A) and no mortality at 5 dpi (Fig. 1B and C). The weight loss of the dexamethasone-immunosuppressed mice with ZIKV inoculation (groups 1 and 2) was consistently more significant than those of their comparators, including the ZIKV-inoculated male mice without dexamethasone immunosuppression (groups 3 and 4) and mock-infected mice without dexamethasone immunosuppression (groups 7 and 8) starting at 1 dpi (P < 0.05). Minimal histological changes and inflammatory infiltrates were seen in the tissues of the male and female mice with dexamethasone immunosuppression and ZIKV inoculation (groups 1 and 2). On the other hand, ZIKV-NS1 protein expression was detected by immunohistochemical staining in most tissues of these mice, but not in dexamethasone-immunosuppressed mice with mock infection, suggesting that the viral protein expression was specific and not related to dexamethasone effects (Fig. 2). The dexamethasone-immunosuppressed mice with ZIKV inoculation (groups 1 and 2) also had high mean viral loads in blood and most tissues at 5 dpi, especially in the testis/epididymis, ovary/uterus, prostate, spleen, and pancreas (Figs 3A and B and S1A and B). These findings at 5 dpi were suggestive of disseminated but non-lethal ZIKV infection involving different organs with minimal inflammatory response due to dexamethasone immunosuppression.

A novel duck reovirus (NDRV) disease, called “spleen necrosis disease,” “new liver disease in Muscovy ducks” or “duck hemorrhagic-necrotic hepatitis,” was recently found among several duckling species, including shelducks, Pekins, wild mallards and Muscovy in China1. Similarly, avian reovirus (ARV) infection was recorded in Muscovy ducks (Cairina moschata) in south western Poland during the summer 20122. NDRV is a member of the genus Orthoreovirus in the family Reoviridae3.

The disease can be distinguished from Muscovy duck reovirus (MDRV) infections by clinical presentation of hemorrhagic and necrotic lesions in the liver and spleen and 5–50% mortality rates. Furthermore, the existing commercial vaccines against ARVs or MDRVs fail to prevent NDRV infection and transmission. A reverse transcription loop mediated isothermal amplification (RT-LAMP) provides a rapid and precise detection assay for viral pathogens with high sensitivity and specificity4. This technique is profitable and convenient, only requires a constant temperature water bath and averts some deficiencies, including high necessity for equipment, tremendous cost, lengthened examination period and low sensitivity5. In comparison with the equipment required for traditional polymerase chain reaction (PCR) and quantitative polymerase chain reaction (qPCR) assays, RT-LAMP is easy to conduct in resource-limited laboratory settings in underdeveloped countries6. RT-LAMP has been widely exploited in clinical diagnosis of various viral pathogens including porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), classic swine fever virus (CSFV), H10N8 subtype of influenza A virus, porcine deltacoronavirus (PDCoV), Zika virus (ZIKV), chikungunya virus (CHIKV), Rift Valley fever virus (RVFV), St. Louis encephalitis virus (SLEV), yellow fever virus (YFV), dengue virus serotypes 1–4, Japanese encephalitis virus (DENV1–4) and West Nile virus (WNV)7–10. The ARV structural and molecular compositions are generally similar to those of mammalian reovirus (MRV). However, some of the ARV biological properties differ from mammalian reovirus, which have been shown elsewhere11,12. The S3 segment encoding the σB protein of duck and MRVs is structurally similar to ARV σB gene13. Reverse transcription PCR (RT-PCR) on both sigma C (σC) and sigma B (σB)-encoding genes followed by restriction fragment length polymorphism (RFLP) analyses were employed to characterize Tunisian ARV isolates14. Furthermore, the highly variable sequences of the S3 and M3 have been reported to differentiate between novel duck reovirus (NDR), MDRV and avian reovirus15. Thus, a simple, rapid and sensitive diagnostic technique for detection of NDRV is required. The potential application of RT-LAMP assay using the S3 gene of NDRV-NPO3 strain for specific diagnosis of NDRV infection with limited sensitivity and without its utilization in the detection of NDRV in naturally- and experimentally-infected ducks have been reported16. In this study, we developed and evaluated a RT-LAMP assay targeting the gene encoding the σB major outer-capsid protein to detect NDRV in naturally suspected NDRV-infected ducks and experimentally infected ducklings with NDRV.

We next evaluated the effects of recombinant type I interferon treatment in our mouse model. We used male mice as they had earlier onset of weight loss and clinical symptoms requiring necropsy at 12 dpi. The mice were treated with pegylated interferon-α2b (PegIntron®, Merck & Co., Inc., Whitehouse Station, NJ, USA) 1920 IU/dose every 96 h subcutaneously at 1 dpi, 5 dpi, and 9 dpi (group 10) or interferon-β1b (Betaferon®, Bayer Schering Pharma AG, Berlin, Germany) 160,000 IU/dose every 48 h intraperitoneally at 1 dpi, 3 dpi, 5 dpi, 7 dpi, and 9 dpi (group 11). As shown in Fig. 6A, the mice treated with pegylated interferon-α2b (group 10) or interferon-β1b (group 11) had < 10% weight loss with spontaneous recovery at 14 dpi. The weight loss of the untreated group became significantly more than those of the mice treated with either interferon-α2 or interferon-β1b starting at 10 dpi (P < 0.05). All of these mice remained asymptomatic and survived through the study period (Fig. 6B and C). None of their tissues showed prominent inflammatory reactions in H&E staining at 5 dpi or 14 dpi. ZIKV-NS1 protein expression was only rarely seen in the immunohistochemical staining of the testis/epididymis, kidney, spleen, small intestine, lung, and pancreas collected at 5 dpi, and testis, epididymis, kidney, and spleen at 14 dpi. They had reduced mean viral loads in blood and all the tissues (↓ 2–4 log10copies/106 β-actin) as compared with those of the untreated mice at 5 dpi and 14 dpi (Fig. 7A and B). The reductions were most significant in the tissues with high viral loads, such as the spleen, testis, pancreas, and prostate (P < 0.05). Overall, these findings suggested that early use of systemic recombinant type I interferons improved the clinical, histological, and virological parameters of mice with disseminated ZIKV infection.

The mouse rectum, in contrast to the human, is extremely short and is susceptible to prolapse. Rectal prolapse (Fig. 1) in mice may be caused by inflammation in the colon due to infectious agents, such as pinworms or Helicobacter species (Fig. 1C–F), with increased abdominal pressure caused by pregnancy, masses, stranguria, or diarrhea. With age, it is plausible that the pelvic musculature may weaken allowing for prolapses to occur more readily – although this has not been investigated extensively (24). In many cases, the definitive cause for rectal prolapse is not determined; however, known causes may be excluded. In the UW aging colonies, pinworms and Citrobacter rodentium have not been diagnosed. The colonies are not tested for Helicobacter species, which can cause colitis and rectal prolapse in certain susceptible GEMs such as the 129-Smad3

tm/Par/J model of bacterial-driven colon cancer (14). In immune competent mice, such as B6, Helicobacter species are considered commensal and should not cause disease. Rectal prolapses vary in clinical appearance (Fig. 1B–D) and, in female mice, must be distinguished from uterine or vaginal prolapse. Mild prolapse is a protrusion of the rectum extending only 1–2 mm from the base of the tail and perineum and the prolapsed mucosa is light pink and moist, with little adherent material (Fig. 1B). Mild prolapse often cause little detriment to the animal, which can survive and thrive with these lesions for some time. Severe rectal prolapse is a protrusion of both distal colon (DC) and rectal mucosa extending >3 mm from the base of the tail and perineum (Fig. 1C,D). The severely prolapsed mucosa is often reddened and edematous to necrotic with adhered fecal or bedding material (Fig. 1C). Often, feces inside the cage will be sticky, an indication of diarrhea. In long-standing cases, the mucosa may become proliferative (Fig. 1D). Exposure of the delicate colorectal mucosa to the cage environment provides a nidus of chronic inflammation and entry of bacteria in the systemic circulation. There is no completely effective treatment for rectal prolapse. Clinical recommendations for mice with rectal prolapses vary between facilities and experimental protocols; in general, mild rectal prolapses with no other clinical signs of systemic illness are often monitored, while euthanasia is recommended for mice with moderate to severe rectal prolapses.

Histologically, the prolapsed mucosa may be ulcerated to necrotic or, with chronicity, proliferative with squamous metaplasia (Fig. 1E,F). Adherent bacterial colonies, plant material and fecal matter may also be present. In typical cases, the underlying vasculature and submucosa (SM) is edematous and inflamed and may be congested. Inflammation may be acute, primarily neutrophilic when there are ulcers and bacteria, or lymphohistiocytic when chronic. Chronic prolapse can lead to severe mucosal hyperplasia with subsequent mucosal herniation due to the thin SM. The herniated glands must be differentiated from invasive carcinoma (25).

A total of ten 1-day-old ducklings were randomly allocated into two groups (five ducklings each). Group 1 animals were received intraperitoneal inoculation of NDRV allantoic fluid (Table 4). Ducklings from group 2 were intraperitoneally inoculated with physiological saline and served as the control group. Ducklings were followed hourly for 72 hours with their corresponding uninfected-control ones. All ducklings in the infected group died with 72 hours post-infection (hpi). The gross anatomical lesions of the ducklings showed an enlarged liver (hepatomegaly) and pleural exudates with yellow discoloration 48 hpi and hepatomegaly with brittle texture, plaque bleeding and necrosis, red darken splenomegaly, patchy hemorrhagic necrosis, bursal necrosis, renal bleeding, enlarged heart and inflated intestine 72 hpi (Fig. 6).

Twenty eight samples were collected at 24, 48 and 72 h, respectively from different affected organs, including heart, liver, spleen, lung and brain as well as anal swab (sticky stool) and serum were collected. Among the 21 NDRV LAMP-positive specimens, 18 were positive RT-PCR (Table 4). The RT-LAMP rapid detection assay was 10.7% (3/28) higher than that of the traditional RT-PCR method. Thus, the conventional RT-PCR method had low sensitivity and is inappropriate for use in diagnosis of NDRV.

Only puppies between 1 week to 4 months of age submitted for routine autopsy evaluation at the Laboratory of Animal Pathology, Veterinary Teaching Hospital, Universidade Estadual de Londrina, Southern Brazil to determine the cause of death by their owners between January 2013 to December 2017 were included. All autopsies and histopathologic findings were done by veterinary pathology residents (n = 7) under the supervision of two animal pathologists. These animals originated from several cities within the State of Paraná, Southern Brazil. The biological data of the puppies are given in Table 1; all data relative to breed, gender, age, clinical manifestation, and pathologic findings from autopsy reports were reviewed and tabulated. The owners of all dogs agreed to have the death of these animals investigated and consented to the usage of the results for scientific purposes.

Rabbit hemorrhagic disease (RHD), characterized by severe necrotizing hepatitis and disseminated intravascular coagulation in the liver, spleen, kidney and other solid organs, is a highly contagious and lethal infection in rabbits. RHD is peracute and often lethal hepatitis caused by the rabbit caliciviruses Lagovirus europaeus GI.1 (previously called rabbit hemorrhagic disease virus—RHDV) and Lagovirus europaeus GI.2 (previously called RHDV2 or RHDVb). This disease was first reported in China in 1984, and has, since, spread rapidly around the world in less than 10 years, causing considerable economic losses in the rabbit industry and impacting the ecology of wild rabbit populations. In 1989, OIE designated this illness as a viral hemorrhagic disease and added it to List B of the International Animal Health Code. Subsequently, a novel lagovirus, GI.2, emerged in France in 2010. GI.2 is now endemic in Europe and Australia, and appears to be replacing GI.1 strains in these regions.

RHDV (rabbit hemorrhagic disease virus) is the etiologic factor of RHD. RHDV is a calicivirus in the genus Lagovirus, family Caliciviridae. RHDV is a non-enveloped icosahedral virus possessing a single-stranded positive sense RNA genome that is approximately 7.4 kb in length. The genome comprises a 5′ untranslated region (UTR), a 3′ UTR, and two overlapping open reading frames (ORFs): ORF1 and ORF2. ORF1 encodes a polyprotein that is cleaved by the viral protease into seven nonstructural proteins (NSP1-7) and a major structural capsid protein VP60 (presently VP1) at its C-terminus. VP10 (presently VP2) is a minor structural protein that is encoded by ORF2.

Vaccination is the main approach for controlling RHDV because no effective treatment is available for this disease. Inactivated vaccines against RHDV were introduced in the early 1990s, improving the survival of rabbits on rabbit farms. However, RHDV inactivated vaccines are manufactured using the livers of rabbits infected with RHDV. This is because RHDV cannot grow in any continuous cell lines. Therefore, biological risks, animal-welfare concerns, and high costs are the major bottleneck problems in the production and usage of tissue-inactivated vaccines.

RHDV spreads mainly through the upper respiratory and digestive tracts. The initial steps leading to RHDV infection take place on mucosal surfaces. It is generally believed that mucosal immunization is an effective approach for preventing systemic infection by pathogens present on mucosal surfaces. The gastrointestinal (GI) tract is the largest mucosal surface accessible via oral administration. Oral vaccination can trigger a response involving neutralizing mucosal antibodies (IgA) and cell-mediated immunity, and does not interfere with IgG-based responses. Additionally, oral vaccines show better safety and compliance profiles, and are simpler to manufacture and administer, than traditional injectable preparations. However, the delivery of antigens for oral vaccination of the GI tract is hindered by multiple physicochemical and biological barriers; antigens can be subjected to early disintegration and advanced degradation by low pH and proteases present in the GI tract. L. casei is a probiotic that is well known for its health-promoting properties, such as maintaining homeostasis and suppressing pathogens in humans and animals. L. casei has shown a good safety profile, can colonize the intestine, and exerts a nonspecific immunoadjuvant effect. For those reasons, oral vaccines using L. casei as a delivery system for pathogenic antigens have garnered much interest in vaccine development. Currently, there is increasing interest in the development of L. casei oral vaccines, and this approach is significant for the effective induction of a mucosal immune response. The results to date have been confirmed that the safety and the effectiveness of L. casei were used as the oral vaccine vehicle, which were extensively used in protecting individuals against a variety of pathogens.

Developing an efficient and safe oral vaccine that can induce strong mucosal and systemic immune responses is desirable for effective prevention of RHDV. Therefore, in our current study, we developed a recombinant L. casei expressing the major structural capsid protein VP60(VP1)-eGFP fusion protein of RHDV. Then we evaluated the humoral and mucosal immune responses to this recombinant L. casei, and assessed its immunogenic properties upon administration as an oral vaccine.

The FFPE tissue blocks and/or glass slides of all selected cases were reviewed; when necessary additional glass slides were prepared from the FFPE blocks and routinely processed for histopathology with the Haematoxylin and Eosin stain (H&E). Only sections of the cerebrum, cerebellum, brainstem, lung, small intestine, eye, and liver were evaluated during this study. These organs were selected for analyses due to: 1) all were present in each puppy; 2) the infectious disease agents investigated are known to produce specific histopathologic pattern(s) in these organs; and 3) to maintain the uniformity of the pathologic investigation. In addition, when available, the target organs (e.g., eye, thymus, palatine tonsils, and urinary bladder) of these viral agents were also revised. Furthermore, the eye of a puppy with the “blue eye” phenomenon from a previous study in which five infectious disease agents were identified10 was evaluated for histopathologic findings associated with CAdV-1, since in that previous study10 the histopathologic features of the ocular lesions were not described.

In specific cases, histochemical stains were used to assist in the identification of infectious disease pathogens; these included Gram, PAS, and GMS. The principal histopathologic pattern observed in each organ was reviewed, tabulated, and then related to specific infectious disease agent(s) due to the intralesional presence of these by IHC. Furthermore, all puppies were screened for CDV by RT-PCR, since this is the most prevalent and endemic infectious disease agent of dogs in urban cities of Brazil15,16.

Bacteriophages (or phages), which specifically infect bacteria, are the most abundant organisms on the earth. Based on their life cycles, phages can be classified as virulent and temperate phages. Upon infection, the virulent phages take over the machinery of the host cell to produce and finally lyse the host cell to release their progenies. In addition to the lytic cycle, temperate phages also have a lysogenic cycle, in which phages incorporate their genomes into a host chromosome (or maintain their genome extrachromosomally) and replicate with it without lysing their host cells (Howard-Varona et al., 2017). Therefore, temperate phages are considered as natural vectors for gene transmission among bacteria and play important roles in virulence of bacterial pathogens (Boyd, 2012; Cuenca Mdel et al., 2016).

Many studies have indicated that the infection of temperate phages might lead to the enhancement of host virulence as many virulent genes of pathogenic bacteria were identified in the phage genome (Boyd, 2012). For instance, lambdoid phages encode both subunits, Stx1 and Stx2, of Shiga toxins, which are the major virulent factor of Shiga toxin-producing Escherichia coli (Herold et al., 2004). Many effector proteins, which are bacterial virulent factors and are injected into host cells by bacterial type III secretion system to help bacterial invasion and survival, were identified in Salmonella typhimurium phages (Figueroa-Bossi et al., 2001). Infection with such temperate phages might lead to the transmission of the virulent genes, thus increases the virulence of host bacteria (Cuenca Mdel et al., 2016). Additionally, it was reported that temperate staphylococcal phage 80α could efficiently encapsidate and transfer bacterial pathogenicity island, SaPI1, to a recipient strain (Ruzin et al., 2001). In contrast to the enhancement of virulence, however, the roles of temperate phages in attenuation of bacterial virulence are largely unknown.

Bordetella bronchiseptica is a common pathogen colonizing the upper respiratory tract of a variety of animals (Goodnow, 1980). Meanwhile, it was reported that B. bronchiseptica can cause severe pulmonary infections in immunosuppressed patients, such as people with lymphoproliferative disorders (Stoll et al., 1981), HIV infection (Rampelotto et al., 2016), cystic fibrosis (Register et al., 2012), and lung transplantation patients (Ner et al., 2003). In addition, B. bronchiseptica infection causes atrophic rhinitis and bronchopneumonia in pigs, leading to a huge loss to the pig industry (Brockmeier, 2004; Brockmeier et al., 2008). Recently, drug-resistant B. bronchiseptica strains were frequently reported, which brings an urgency of developing new strategies to treat or prevent the infection (Kadlec et al., 2004; Zhao et al., 2011; Pruller et al., 2015a). Phage therapy could be an alternative strategy against drug-resistant B. bronchiseptica infection. In fact, several B. bronchiseptica phages have been isolated (Liu et al., 2004; Petrovic et al., 2017; Chen et al., 2019a), and some of those were tested against B. bronchiseptica infection in our previous study (Chen et al., 2019a).

We have been working on isolation and characterization of B. bronchiseptica phages since we reported the use of phages to treat B. bronchiseptica infection (Chen et al., 2019a). An interesting temperate phage, PHB09, which has a specific integration site in the host genome, was found from our B. bronchiseptica phage collections. The aim of this study was to investigate the effects of PHB09 integration on its host. Interestingly, we found that the specific integration of PHB09 significant decreased the virulence of parental strain B. bronchiseptica Bb01 in mice. Furthermore, mice intranasally inoculated with 1.8 × 108 colony-forming unit (CFU) lysogenic strain were completely protected when challenged with 3.0 × 108 CFU B. bronchiseptica Bb01, indicating the vaccine potential of the lysogenic strain. To our knowledge, this is the first report that the integration of temperate phage reduces the virulence of host bacteria in the mammal, indicating the complicated roles of temperate phages in bacterial virulence other than the simple delivery of virulent genes. Our study also provides a new strategy to design avirulent strains for developing bacterial vaccines.

The liver, spleen, and small intestine, collected from the rabbits in different treatment groups, were subjected to histopathological evaluation. Lesions on liver, spleen, and small intestine were more severe in the PBS-treated group than in the vaccinated groups. In the PBS-treated group, the hepatocytes in the liver presented a disordered appearance and clearly manifested degeneration and necrosis. Splenic corpuscles appeared destroyed, and some had disappeared entirely, splenic sinus was congested, and intestine villi were necrotic and severely desquamated. There are almost no differences between the pPG-eGFP-VP60/LC393-immunized group and inactivated vaccine-immunized group (Figure 8).

All FFPE tissues from the three necropsied cats were subjected to IHC analysis using the 1.B.450 mouse monoclonal anti-canine parvovirus antibody (Abcam AB59832, Cambridge, UK) as the primary antibody. Briefly, after deparaffinization and rehydration, tissue sections were pretreated by 0.1% (w/v) trypsin at 37 °C for 25 min, followed by blocking endogenous peroxidase activity with 5% (w/v) skim milk at 37 °C for 40 min. After washing three times with PBS, the sections were incubated with primary antibody (1:200 dilution) at 4 °C overnight followed by detection using the Dako REAL EnVision Detection System (Dako, Glostrup, Denmark) at RT for 45 min. After triplicate washings with PBS, a positive antigen-antibody reaction was observed by labeling with DAB and counterstained with Mayer’s hematoxylin. The FPLV-positive intestinal tissue from a PCR-positive FPLV-infected cat served as a positive control. Samples treated with distilled water (DW) instead of the primary antibody and non FPLV-infected samples served as negative controls.

Porcine hemagglutinating encephalomyelitis coronavirus (PHEV) is a member of the Coronaviridae family, which causes porcine encephalomyelitis. PHEV predominantly affects 1-3 week-old piglets[1], with clinical piglets vomiting, exhaustion and obvious neurological symptoms as the main feature. The mortality rate is up to 20-100%[2]. Since 1958, when the disease broke out in the Canadian province of Ontario for the first time[3], many countries have reported about it. Serological test results proved that it is common for the pigs to be infected by PHEV[4,5], and the disease may have spread worldwide. In August 2006, the disease broke out in part of the pig farms in Argentina, resulting to 1226 deaths, with the morbidity rate up to 52.6%[1]. In China, an PHEV infection has been reported occurring in a pig farm in Beijing as early as in 1985, followed with reports from Jilin, Liaoning, Shandong, Taiwan, etc. The large-scale epidemics of HEV occurred in Taiwan in 1994 had a fatality rate of almost 100%[6], resulting to serious economic losses. Serological survey conducted by foreign scholars revealed that PHEV infection in pigs is very common, with a worldwide distribution[4,5].

Coronaviruses are usually divided into three groups based on genetic and serological relationship. HEV, togather with murine hepatitis virus (MHV), bovine coronavirus (BCoV), human coronavirus OC43 (HcoV-OC43), rat coronavirus (RCoV), belongs to group 2[8].

In this report, the clinical and neuropathologic feathers of spontaneous PHEV infection had been reported. Microscopically, coronavirus-like particles were detected in the supernatant of the brain samples by electron microscopy. One coronavirus strain (isolate PHEV-JLsp09) was isolated from the piglets. The Hemagglutinin- esterase (HE) gene of PHEV strains was amplified by reverse transcription- polymerase chain reaction (RT-PCR) and sequenced. The homology and phylogenetic analyses were done between the group 2 coronaviruses and influenza C virus strains downloaded from Genbank, based on the sequence of HE gene.

The diseased pig cases came from a pig farm in Siping, China. The Tu San Yuan pigs were naturally propagated and raised. The sows mainly showed elevated body temperature and loss of appetite. Their early clinical manifestations were sneezing, coughing, regurgitating, vomiting and spitting up milk. Sick piglets often stayed together, had arched backs and did not like to walk. During late stages of the disease, they suffered from physical deterioration. Piglets with poor resistance would suffer from severe dehydration. Exhibiting difficulty in breathing and cyanosis, they would sink into comas and die. Piglets with better resistance would lose their appetite and soon became emaciated and died. In the current study, 28 piglets died within 20 days of birth. The only 10 surviving piglets were sent to the animal facility of our lab to be raised separately. Their clinical signs were significant increase of body temperature, lethargy, crowding together, eyes half-closed or lying on the ground showing sleepiness and loss of appetite. Nervous system signs of the diseased pigs included nervous whole body muscle convulsions, taking a dog-sitting position and weakness/fainting. During the late stage, diseased pigs wobbled and could not stand, their noses and feet were cyanotic and they discharged watery feces. Some of them lost their eyesight or had opisthotonos and nystagmus. Finally, they experienced breathing problems, lay on their sides exhausted and vomited. The only 10 surviving pigs sent to our lab for examination died within 3 days; the death rate of the piglets was 100%.

Barbering, alopecia, dermatitis, and scarring are commonly seen in mice (Fig. 2). Dermatitis may have a variety of causes including infectious agents, such as fur mites, or fighting with secondary bacterial infections. These will often respond to treatments such as removal of aggressive mice, topic and oral antibiotic, or corticosteroids (26). In contrast, idiopathic ulcerative dermatitis in the B6 mouse does not respond to treatment. Affected B6 mice are usually pruritic leading to self-mutilation, disease progression and, often, euthanasia for humane reasons. The B6 chronic ulcerative dermatitis has been attributed to many causes (27, 28) most recently primary follicular dystrophy (29). The clinical disease is variable, waxes and wanes, appears to have sex, and season predilections. As seen with other strain-specific diseases, the use of F1 hybrids has a decreased incidence of B6 dermatitis (1, 30); however, skin lesions remain a significant background factor in aging colonies (29). Collectively, skin lesions in aging mouse colonies have variable presentations ranging from the very mild alopecia to severe ulcerative disease requiring euthanasia. Mild changes, such as ill-kept fur (Fig. 2B) are often one of the first clinical signs noted in diseased mice. It is non-specific and a result of decreased grooming. Alopecia in mice may present on the head (Fig. 2A) or in a patches (Fig. 2C). Barbering by cage mates may result in areas of hair loss; however, distinctive patterns (Fig. 3A) and the presence of one unaffected (dominant) mouse in the cage will help to rule out dermatitis. Histologically, dermatitis is also variable in presentation and severity, depending on etiology and chronicity. Mild lesions (Fig. 2E) with few scattered inflammatory cells may be noted in regions of clinical alopecia. Severe lesions may occur and are often a combination of ulceration, necrosis, and associated chronic-active inflammation. Healing is by dermal fibrosis and marked epithelial hyperplasia (Fig. 2F). Like rectal prolapses, open skin lesions serve as a nidus for bacterial infection with skin commensals (Staphylococcal species) and result in smoldering inflammation that may affect physiological parameters such as inflammatory cytokines, leukograms, lymphadenopathy, and reactive amyloidosis (31).

Ribavirin was tested in comparison to a placebo group receiving the vehicle (0.9% NaCl solution) (Fig. 5). Both groups of IFNAR−/− mice were infected with 100 FFU CCHFV. Although one animal survived after treatment, ribavirin did not significantly increase the survival rate (p = 0.4). However, the drug prolonged the time to death (median 3 vs. 6 days for placebo vs. ribavirin, p = 0.0007), reduced the levels of AST (p = 0.001) and ALT (p = 0.006) at day 2, reduced the virus titer in blood at day 2 (p = 0.0007), increased the weight at day 2 and 3 (p = 0.03 and p = 0.002, respectively), and reduced the terminal virus concentration in all organs when compared to placebo at day 3 (p<0.001 separately for each organ). Histopathological analysis of organs collected at day 3 from ribavirin-treated mice revealed only small disseminated foci of necrosis; most of the liver parenchyma resembled naïve mice. Markedly reduced hepatocellular necrosis correlated with low numbers of apoptotic hepatocytes (cleaved caspase-3), T-cells (CD3), B-cells (B220), and activated monocyte-derived cells (iNOS). Virus antigen-positive cells (NP) were significantly reduced in liver and spleen compared to untreated or placebo-treated mice (data not shown). However, ribavirin-treated mice that succumbed to infection on days 4–9 showed extensive bridging hepatocellular necrosis at the time of death (Fig. 2). Like in untreated mice, the necrosis was accompanied by presence of numerous Iba-1-positive macrophages (Kupffer cells), showing enlarged cell bodies and focal clustering, and iNOS-expressing activated monocyte-derived cells (Fig. 3). Both alterations are suggestive for strong monocyte/macrophage activation. However, in contrast to untreated mice, virus antigen was hardly detectable in liver tissue of the treated mice (Fig. 2), consistent with the low virus titer in all organs (Fig. 5, bottom). Thus, ribavirin reduces CCHFV load and delays disease progression, but it does not prevent terminal liver necrosis, monocyte/macrophage activation, and lethal outcome in the IFNAR−/− mouse model.

Arbidol hydrochloride [75 and 150 mg/(kg×d)] was tested in comparison to a placebo group receiving the vehicle (0.5% methylcellulose) [Fig. 5 and data not shown for 75 mg/(kg×d)]. Both groups were infected with 1,000 FFU CCHFV. Mice were pretreated one day before inoculation. However, the drug changed neither survival rate and survival time, nor any of the other parameter measured. Even reducing the inoculation dose to 10 FFU had no effect when compared to the historical control group.

T-705 was tested in comparison to a placebo group receiving the vehicle (0.5% methylcellulose) (Fig. 6). All groups were infected with 100 FFU CCHFV. Initially, a high dose of T-705 [300 mg/(kg×d)] was tested. The drug was administered from day 0 to day 8. Placebo-treated animals died between day 3 and 4. At day 3, they showed weight loss of nearly 20%, increase in body temperature up to 40°C, AST values of 1,200–51,000 U/l, and ALT values of 260–6,700 U/l. All animals of the treatment group survived the infection and showed no signs of disease. Virus was detected neither in blood nor in the organs throughout the observation period (Fig. 6 and data not shown). Histopathology and IHC at day 3 revealed largely normal liver tissue with absence of virus antigen and inflammatory cells (Figs. 2 and 3).

To determine the efficacy of the drug at an advanced stage of the infection, time-of-addition experiments were performed (Fig. 6). Treatment with a high dose of the drug was commenced 1 day or 2 days after virus inoculation and continued until day 8. Survival was 100% in both groups and animals showed hardly any signs of disease. Only if treatment started 2 days p.i., minor changes in weight, temperature, and AST were seen at day 3. Virus remained undetectable in blood and organs in both time-of-addition groups throughout the observation period. To provide evidence for infection of the animals in the T-705 treatment groups, the development of CCHFV-specific antibodies was measured 21 days p.i. Only 1/10 (10%) of the animals treated post-exposure, but 10/10 (100%) of the animals treated from day 1 or 2 p.i. developed antibodies, indicating that virus replication under post-exposure treatment with T-705 was even not sufficient to elicit antibodies.

To define the lowest effective dose of T-705, animals received 30, 15, or 7.5 mg/(kg×d) T-705 or 0.5% methylcellulose as a placebo (Fig. 7). Treatment was commenced 1 h p.i. and continued until death or day 8. All animals of the 30 and 15 mg/(kg×d) treatment groups survived and showed hardly any signs of disease. Virus was detected at low level only in blood of one animal of the 15 mg/(kg×d) treatment group at day 11 (Fig. 7). A dose of 7.5 mg/(kg×d) did not prevent a lethal outcome, although it prolonged the time to death (p = 0.0007), and reduced the levels of AST (p = 0.0007) and ALT (p = 0.004) at day 3.Taken together, T-705 is highly efficient against CCHFV in the IFNAR−/− mouse model.

a Ferret Infected by Coronavirus in Japan

A number of different poxviruses can infect both people and domestic animals; with cowpox being the best described and most commonly encountered poxvirus infection of cats. Cowpox virus (CPxV) is a member of Orthopoxviridae family and is endemic in Northern Europe and western areas of the Soviet Union.1

The usual route of infection is via skin inoculation from infected rodent bites, typically voles, or rarely via oronasal infection.2 Reflective of this transmission route, skin lesions are commonly found on the head, neck and forelimbs. However, systemic illness such as pyrexia, anorexia, lethargy and/or pneumonia can occur during the viraemic phase and is usually associated with immune dysfunction and death.3,4

It is important that clinicians recognise the signs of potential CPxV infection and perform appropriate diagnostics early. A significant factor contributing to the prognosis of CPxV infection in cats is the speed at which appropriate therapy is instituted, so rapid recognition is crucial. Cytological analysis of the affected organs can be misleading, with the secondarily dysplastic cells having the potential to mimic neoplasma. Instead, electron microscopy, virus isolation and PCR of tissues (including skin scabs, bronchoalveolar or pleural fluid, or pulmonary aspirates) are the easiest ways to confirm the diagnosis.5 Serum assays (including virus neutralisation, haemagglutination inhibition, complement fixation and ELISA) can be utilised to detect a humoral response to orthopoxvirus, although a rising titre is required to support active infection.4

Treatment includes broad-spectrum antibiotics to control secondary bacterial infection and recombinant feline interferon omega (rFeIFN-ω) to modulate the immune response. Antiviral drugs, such as famciclovir, commonly used for the treatment of feline herpesvirus (FeHV-1) disease in cats,6 could also be considered; however, their efficacy against CPxV is unknown.

Our previous case series detailed cats that had pulmonary CPxV-infection, complicated by concomitant infection with FeHV-1, Bordetella bronchiseptica and/or Mycoplasma species.4 The cases described herein have novel findings which have not previously been described in relation to this infection, including central nervous system (CNS) involvement and presentation as a laryngeal mass.

After the first outbreak in the park, the second one occurred at the farm (Fig 1, enclosure B1) in August 2011, in a family group consisting of a breeding female (Aduke) and two male (Top Cut, Clyde) and one female offspring (Bonnie). The index patient, a 7-month-old male (Top Cut), presented with a poorly healing wound on the front paw. Upon clinical examination of the cheetah under general anesthesia, multiple papular dermal nodules were noticed on the entire body, and poxvirus infection was suspected. Despite antibiotic and anti-inflammatory treatment (initially 8 mg/kg cefovecin i.m. once, from day 2 clindamycin 11 mg/kg BID p.o., and meloxicam 0.05 mg/kg SID p.o.), the animal died 19 days after the disease was first noticed.

Only one of its two siblings (Clyde) developed an ulcer on the tongue on day 14, the other sibling (Bonnie) and the dam did not show any signs of infection at all. The animals remained together in their enclosure throughout the disease process.

Starting in 2013, all cheetahs were vaccinated with Modified Vaccinia Ankara (MVA) smallpox vaccine, following the vaccination regime as recommended by the producer. However, another outbreak occurred in September 2014 in the park (enclosure A1) where three female siblings were living (Nova, Novi, Heidi). The 2.5-year-old Nova showed severe skin lesions consistent with cowpox in the face, on her body, and her legs. She received daily oral antibiotics (5 mg/kg enrofloxacin SID p.o.) and improved after 11 days. Her two sisters did not show any lesions.

The minimum inhibitory concentration (MIC) of each antimicrobial against B. bronchiseptica strains Bb01 and Bb01+ were determined via broth microdilution susceptibility testing according to the guidelines recommended by the Clinical and Laboratory Standards Institute (CLSI, 2018). The broth microdilution susceptibility testing of B. bronchiseptica was tested as described previously (Pruller et al., 2015b). Fourteen antimicrobials, ampicillin, ampicillin/sulbactam, tiamulin, tilmicosin, erythromycin, ofloxacin, ciprofloxacin, ceftiofur sodium, chloramphenicol, gentamicin, tetracycline, trimethoprim, sulfamethoxazole, and polymyxin B, were tested in our current study. E. coli ATCC 25922 was used as a control strain for antimicrobial susceptibility testing.

A 58-year-old male was admitted to the ICU with a two-week history of cough with sputum and sudden deterioration with a fever of 38.1 °C prior to admission. On admission, the patient had paroxysmal ventricular tachycardia, and his mean arterial pressure was maintained at 70 mmHg by administration of norepinephrine at a dose of 0.67 μg/kg·min. Oxygen inhalation through a nasal tube was administered to maintain oxygen saturation at >90%.

The patient had a recent history of contact with live poultry 10 days prior to admission. Respiratory secretions were positive for A(H7N9) H7, N9 and M genes. Chest radiography on admission showed bilateral pulmonary infiltrates (Figure 4A). On admission, an echocardiogram showed a dilated left ventricle with a low LVEF of 37.5% (Figure 4D), together with weakened cardiac wall motion. The serum creatine phosphokinase (CK) level was 799 U/L, and TNI was negative. ECG did not show the evidence of acute myocardial infarction on admission. A second echocardiogram showed recovery of LVEF to 62% without accompanying weakened cardiac wall motion at day 3. The patient had a 30-year history of bronchiectasia that had been progressing to pulmonary heart disease for 10 years.

On admission, severe liver injury was identified. ALT, AST and LDH activities were elevated to 1281, 3029, and 2787 U/L, respectively, the highest levels seen during the patient's hospital stay (Figure 5). Total bilirubin level was 20 μmol/L. INR was prolonged to 1.61 with a D-dimer value of 57872 μg/L. The dynamic changes in liver function and INR are described in Figure 5.

The patient was identified as having a severe A(H7N9) infection with MOF and HH. Antiviral therapy of oral oseltamavir (75 mg) twice daily was administered. Due to the progression of the infiltrates and consolidation (Figure 4B and 4C), the patient was placed on a mechanical ventilator on the third day after admission. Continuous haemofiltration was given when the creatinine level reached 135 µmol/L with oliguria on the third day of admission. Unfortunately, the patient died on day 4 of hospitalization due to deterioration of MOF.