Infected Pups Research Articles

Development of bioluminescent Group B streptococcal strains for longitudinal infection studies.

Group B Streptococcus (GBS) remains the leading bacterial cause of invasive neonatal disease, resulting in substantial morbidity and mortality. New therapeutic approaches beyond antibacterial treatment to prevent neonatal disease outcomes are urgent. One significant limitation in studying GBS disease and progression is the lack of non-invasive technologies for longitudinal studies. Here, we develop and compare three bioluminescent GBS strains for in vivo pathogenic analysis. Bioluminescence is based on the luxABCDE operon on a replicative vector (luxGBS-CC17), and the red-shifted firefly luciferase on a replicative vector (fflucGBS-CC17) or integrated in the genome (glucGBS-CC17). We show that luxGBS-CC17 is suitable for in vitro analysis but does not produce a significant bioluminescent signal in infected pups. In contrast, the fflucGBS-CC17 results in a strong bioluminescent signal proportional to the organ colonisation level. However, the stability of the replicative vector depends on the route of infection, especially when pups acquire the bacteria from infected vaginal mucosa. Stable chromosomal integration of luciferase in glucGBS-CC17 leads to significant bioluminescence in both haematological and vertical infection models associated with high systemic colonisation. These strains will allow the preclinical evaluation of treatment efficacy against GBS invasive disease using whole-mouse bioluminescence imaging.

Purinergic Signaling Drives Multiple Aspects of Rotavirus Pathophysiology

Background: Rotavirus causes life-threatening diarrhea in children, resulting in ~200,000 deaths each year. Rotavirus infects a limited number of cells at the tips of the villi in the small intestine; however, rotavirus is known to dysregulate cells distant from the site of infection through paracrine signaling. We recently identified that rotavirus infected cells release the purinergic signaling molecule ADP, which binds to the P2Y1 receptor on nearby uninfected cells; causing a signaling cascade that affects distant uninfected cells. In vitro P2Y1 was found to contribute to chloride and serotonin secretion; suggesting P2Y1 may activate pathways responsible for diarrhea. Hypothesis: We hypothesize that P2Y1 orchestrates cell-cell communication involved in diarrhea and infection severity. Methods: To elucidate the role of purinergic signaling via P2Y1 receptors during rotavirus infection, we used the mouse-like rotavirus (rD6/2) to investigate the effects of purinergic signaling in the context of homologous murine rotavirus infection in vivo. C57Black6 mouse pups were orally gavaged rD6/2 rotavirus at day 4-6 of age and assessed over the course of 5 days. Beginning at day 1 post infection, infected pups were treated daily by oral gavage with saline or 4mg/kg MRS2500, a P2Y1-selective, competitive antagonist. Stool was collected and scored for diarrhea daily prior to each treatment. Pups were euthanized and small intestine tissue was collected at 3- and 5-days post infection for immunostaining, qRT-PCR and luminal contents. Intestinal organoids derived from Balb/C pup jejunum were inoculated with rD6/2 rotavirus, followed by supplementation with vehicle or MRS2500 to assess fluid secretion by organoid swelling over time. Results: Rotavirus-infected pups exhibited significant diarrhea incidence and high diarrhea scores on days 2, 3, and 4 post infection. Treatment of rotavirus-infected mouse pups with MRS2500 resulted in decreased severity and incidence of diarrhea compared to vehicle treated controls. This was consistent with in vitro, rD6/2-infected organoids exhibiting a significant increase in organoid swelling compared to uninfected organoids, which was significantly prevented by inhibition of P2Y1 signaling. Viral stool shedding from MRS2500 treated pups was significantly lower than saline-treated pups starting at day 4 post infection, which suggests P2Y1 signaling may amplify rotavirus replication in vivo. Finally, transmission experiments in litters with infected (but not treated) and pretreated MRS2500 uninfected pups show a decreased incidence in diarrhea suggesting that P2Y1 inhibition has a potential protective role in reducing the effects of transmission. Conclusion: Collectively these findings establish a conserved role of purinergic signaling in the pathophysiology of rotavirus infection. Additionally, our data indicates P2Y1 as a new candidate for host-targeted therapeutics that could have both antiviral and antidiarrheal effects against rotavirus pathophysiology. R01 DK115507 (Hyser) R01 AI158683 (Hyser)APS Postdoctoral Fellowship (Engevik) F32 DK130288 (Engevik). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Rhesus rotavirus receptor-binding site affects high mobility group box 1 release, altering the pathogenesis ofexperimental biliary atresia.

Biliary atresia (BA) is a neonatal inflammatory cholangiopathy that requires surgical intervention by Kasai portoenterostomy to restore biliary drainage. Even with successful portoenterostomy, most patients diagnosed with BA progress to end‐stage liver disease, necessitating a liver transplantation for survival. In the murine model of BA, rhesus rotavirus (RRV) infection of neonatal mice induces an inflammatory obstructive cholangiopathy that parallels human BA. The model is triggered by RRV viral protein (VP)4 binding to cholangiocyte cell‐surface proteins. High mobility group box 1 (HMGB1) protein is a danger‐associated molecular pattern that when released extracellularly moderates innate and adaptive immune response. In this study, we investigated how mutations in three RRV VP4‐binding sites, RRVVP4‐K187R (sialic acid‐binding site), RRVVP4‐D308A (integrin α2β1‐binding site), and RRVVP4‐R446G (heat shock cognate 70 [Hsc70]‐binding site), affects infection, HMGB1 release, and the murine model of BA. Newborn pups injected with RRVVP4‐K187R and RRVVP4‐D308A developed an obstruction within the extrahepatic bile duct similar to wild‐type RRV, while those infected with RRVVP4‐R446G remained patent. Infection with RRVVP4‐R446G induced a lower level of HMGB1 release from cholangiocytes and in the serum of infected pups. RRV infection of HeLa cells lacking Hsc70 resulted in no HMGB1 release, while transfection with wild‐type Hsc70 into HeLa Hsc70‐deficient cells reestablished HMGB1 release, indicating a mechanistic role for Hsc70 in its release. Conclusion: Binding to Hsc70 contributes to HMGB1 release; therefore, Hsc70 potentially serves as a therapeutic target for BA.

Rotavirus infection elicits host responses and amplifies viral replication via P2Y1 purinergic signaling

Rotavirus causes life‐threatening diarrhea in children, resulting in ~130,000 deaths each year. Rotavirus infects a limited number of cells at the tips of the villi in the small intestine. Yet, rotavirus dysregulates cells far away from the site of infection. We recently identified, using simian (SA11) and rhesus rotavirus (RRV), that infected cells release the purinergic signaling molecule ADP, which binds to the P2Y1 receptor on nearby uninfected cells. Furthermore, using the in vivo mouse model, mild rotavirus diarrhea in mouse pups was alleviated by daily treatment with a P2Y1 inhibitor. To elucidate the role of purinergic signaling via P2Y1 receptors during rotavirus infection, we used the mouse‐like rotavirus (D6/2) to investigate the effects of purinergic signaling in the context of severe rotavirus infection in vivo. C57Black6 mouse pups were orally gavaged D6/2 rotavirus at day 4‐6 of age and assessed over the course of 5 days. Beginning at day 1 post infection, infected pups were treated daily by oral gavage with saline or 4mg/kg MRS2500, a selective P2Y1 antagonist. Stool was collected and scored for diarrhea daily prior to each treatment. Pups were euthanized and small intestine tissue was collected at 3 and 5 days post infection for immunostaining, qRT‐PCR and luminal contents. Similar to mild rotavirus infection, treatment of D6/2‐infected mouse pups with MRS2500 results in decreased severity and incidence of diarrhea. MRS2500 treated pups also exhibit decreased luminal serotonin and chloride content compared to control infected pups. Together, these results confirm that P2Y1 signaling is also involved in the pathogenesis of a homologous murine rotavirus strain. Viral stool shedding, assessed by qRT‐PCR for rotavirus gene 11 levels, revealed that MRS2500 treated pups had significantly lower viral shedding starting at day 4 post infection compared to saline treated pups, which suggests P2Y1 signaling may amplify rotavirus replication. Collectively these findings point to the conserved role of purinergic signaling in the pathophysiology of rotavirus infection, and indicate P2Y1 is a new candidate for host‐targeted therapeutics that could have both antiviral and antidiarrheal effects against rotavirus pathophysiology.

Using Forward and Reverse Genetics to Understand Calcium Dysregulation in Enteric Viral Virulence

Acute gastroenteritis (AGE) remains the second leading cause of death among children under the age of 5 worldwide. AGE becomes fatal when pathogen‐associated upregulation of secretory activity across the intestinal epithelium causes severe volume depletion, hypovolemic shock, and multi‐system failure. While enteric viruses remain the most common cause of fatal AGE in kids, the drivers of their virulence (i.e. how they increase secretory activity) remain poorly understood. We recently found that cells infected with rotavirus, the most prevalent enteric virus in kids, release adenosine diphosphate (ADP) to induce coordinated signals known as “intercellular calcium waves,” which spread through uninfected neighboring cells. This dysregulates calcium signaling pathways, which enhances fluid secretion from uninfected cells and thereby contributes to volume depletion. Pharmacological blockade of calcium waves in infected mouse pups decreases disease severity, suggesting they are integral to rotavirus virulence. Understanding how rotavirus triggers intercellular calcium waves may allow us to design safer, more effective vaccines and therapeutics, but we still lack a mechanistic understanding of this process. Here, we report data showing that recombinant expression of a single rotavirus protein, non‐structural protein 4 (NSP4), is sufficient to activate intercellular calcium waves. Using the rotavirus reverse genetics system, we show that the diminished calcium wave phenotype associated with an attenuated strain of porcine rotavirus segregates with the NSP4 gene upon reassortment. Furthermore, we show that this attenuation is attributable to a single amino acid polymorphism at the c‐terminal end of NSP4. This implicates the c‐terminal end and a putative PDZ‐binding motif therein as key upstream mediators of intercellular calcium waves. These findings deepen our understanding of enteric virus pathogenesis, characterize a new role for rotavirus non‐structural protein 4, offer novel targets for anti‐secretory therapeutics, and expand foundational knowledge to support the development of improved live‐attenuated vaccines.

Inclusion of the Guinea Pig Cytomegalovirus Pentameric Complex in a Live Virus Vaccine Aids Efficacy against Congenital Infection but Is Not Essential for Improving Maternal and Neonatal Outcomes.

The development of a vaccine against congenital human cytomegalovirus (HCMV) infection is a major priority. The pentameric complex (PC) of virion envelope proteins gH, gL, UL128, UL130, and UL131A is a key vaccine target. To determine the importance of immunity to the homologous PC encoded by guinea pig cytomegalovirus (GPCMV) in preventing congenital CMV, PC-intact and PC-deficient live-attenuated vaccines were generated and directly compared for immunogenicity and efficacy against vertical transmission in a vertical transmission model. A virulent PC-intact GPCMV (PC/intact) was modified by galK mutagenesis either to abrogate PC expression (PC/null; containing a frame-shift mutation in GP129, homolog of UL128) or to delete genes encoding three MHC Class I homologs and a protein kinase R (PKR) evasin while retaining the PC (3DX/Δ145). Attenuated vaccines were compared to sham immunization in a two-dose preconception subcutaneous inoculation regimen in GPCMV seronegative Hartley guinea pigs. Vaccines induced transient, low-grade viremia in 5/12 PC/intact-, 2/12 PC/null-, and 1/11 3DX/Δ145-vaccinated animals. Upon completion of the two-dose vaccine series, ELISA titers for the PC/intact group (geometic mean titer (GMT) 13,669) were not significantly different from PC/null (GMT 8127) but were significantly higher than for the 3DX/Δ145 group (GMT 6185; p < 0.01). Dams were challenged with salivary gland-adapted GPCMV in the second trimester. All vaccines conferred protection against maternal viremia. Newborn weights were significantly lower in sham-immunized controls (84.5 ± 2.4 g) compared to PC/intact (96 ± 2.3 g), PC/null (97.6 ± 1.9 g), or 3DX/Δ145 (93 ± 1.7) pups (p < 0.01). Pup mortality in sham-immunized controls was 29/40 (73%) and decreased to 1/44 (2.3%), 2/46 (4.3%), or 4/40 (10%) in PC/intact, PC/null, or 3DX/Δ145 groups, respectively (all p < 0.001 compared to control). Congenital GPCMV transmission occurred in 5/44 (11%), 16/46 (35%), or 29/38 (76%) of pups in PC/intact, PC/null, or 3DX/Δ145 groups, versus 36/40 (90%) in controls. For infected pups, viral loads were lower in pups born to vaccinated dams compared to controls. Sequence analysis demonstrated that infected pups in the vaccine groups had salivary gland-adapted GPCMV and not vaccine strain-specific sequences, indicating that congenital transmission was due to the challenge virus and not vaccine virus. We conclude that inclusion of the PC in a live, attenuated preconception vaccine improves immunogenicity and reduces vertical transmission, but PC-null vaccines are equal to PC-intact vaccines in reducing maternal viremia and protecting against GPCMV-related pup mortality.

Atorvastatin ameliorates viral burden and neural stem/progenitor cell (NSPC) death in an experimental model of Japanese encephalitis

Japanese encephalitis virus, a neurotropic flavivirus, causes sporadic encephalitis with nearly 25% fatal case reports. JEV infects neural stem/progenitor cells (NSPCs) and decreases their proliferation. Statin, a commonly used class of cholesterol lowering drug, has been shown to possess potent anti-inflammatory and neuroprotective effects in acute brain injury and chronic neurodegenerative conditions. Here, we aimed to check the efficacy of atorvastatin in alleviating the symptoms of Japanese encephalitis (JE). Using BALB/c mouse model of JEV infection, we observed that atorvastatin effectively reduces viral load in the subventricular zone (SVZ) of infected pups and decreases the resultant cell death. Furthermore, atorvastatin abrogates microglial activation and production of proinflammatory cyto/chemokine production post JEV infection in vivo. It also reduced interferon-β response in the neurogenic environs. The neuroprotective role of atorvastatin is again evident from the rescued neurosphere size and decreased cell death in vitro. It has also been observed that upon atorvastatin administration, cell cycle regulatory proteins and cell survival proteins are also restored to their respective expression level as observed in uninfected animals. Thus the antiviral, immunomodulatory and neuroprotective roles of atorvastatin reflect in our experimental observations. Therefore, this drug broadens a path for future therapeutic measures against JEV infection.

Heterozygosity at neutral and immune loci is not associated with neonatal mortality due to microbial infection in Antarctic fur seals.

Numerous studies have reported correlations between the heterozygosity of genetic markers and fitness. These heterozygosity–fitness correlations (HFCs) play a central role in evolutionary and conservation biology, yet their mechanistic basis remains open to debate. For example, fitness associations have been widely reported at both neutral and functional loci, yet few studies have directly compared the two, making it difficult to gauge the relative contributions of genome‐wide inbreeding and specific functional genes to fitness. Here, we compared the effects of neutral and immune gene heterozygosity on death from bacterial infection in Antarctic fur seal (Arctocephalus gazella) pups. We specifically developed a panel of 13 microsatellites from expressed immune genes and genotyped these together with 48 neutral loci in 234 individuals, comprising 39 pups that were classified at necropsy as having most likely died of bacterial infection together with a five times larger matched sample of healthy surviving pups. Identity disequilibrium quantified from the neutral markers was positive and significant, indicative of variance in inbreeding within the study population. However, multilocus heterozygosity did not differ significantly between healthy and infected pups at either class of marker, and little evidence was found for fitness associations at individual loci. These results support a previous study of Antarctic fur seals that found no effects of heterozygosity at nine neutral microsatellites on neonatal survival and thereby help to refine our understanding of how HFCs vary across the life cycle. Given that nonsignificant HFCs are underreported in the literature, we also hope that our study will contribute toward a more balanced understanding of the wider importance of this phenomenon.

OPN receptor expression on immune effector cells in RSV infection is regulated by NKT cells

Abstract RSV infection in early life predisposes to asthma later in life. Role of aberrant immune responses to childhood viral infections is well documented. Involvement of myeloid (mDC) and plasmacytoid (pDC) Dendritic Cells (DC) and Natural Killer T-cells (NKT) has been reported. Osteopontin (OPN) modulates the immune responses to viral infections. The current studies looked into the role of NKT cells in regulating the OPN receptor expression in RSV infection on DC, T and B cells, which will elucidate the role of OPN in preventing RSV-induced hyper-reactive airway and asthma. Five-day-old BALB/C and NKT−/−(CD1d−/−) mice were infected intranasally with RSV; and expression of OPN receptors (CD44 and CD51/61) were examined at designated post-infection days on lung mDC (CD11blo/−CD11chiCD45Rlo/−), pDC (CD11blo/−CD11clo/−CD45Rhi), T (CD3+) and B (CD19+) cells by flow cytometry. Expression of CD44 and CD51/61 were decreased on mDC, pDC, T and B cells in RSV infected wild pups compared to the control pups. On the other hand, OPN receptor expression was enhanced in the RSV-infected NKT−/− pups compared to the infected wild pups and uninfected NKT−/− pups suggesting a regulatory role of NKT cells on expression of OPN receptors, as RSV-infected NKT−/− pups had significantly increased CD44 and CD51/CD61 expression on DC and lymphocytes. Thus, decreased OPN receptor expression during RSV infection very likely diminishes the action of endogenous OPN allowing skewed polarization of DC and subsequent T cell responses. Therefore, abolishing the action of NKT cells to augment the action of endogenous OPN; and/or administration of exogenous OPN warrant further investigation as a new therapeutic strategy against RSV-associated asthma.

Parasitism Elicits a Stress Response That Allocates Resources for Immune Function in South American Fur Seals (Arctocephalus australis).

Parasites can cause chronic stress in some animal species, and this type of stress response has been associated with adverse consequences for the host. In order to know whether parasitism elicited a stress response associated with decreased host fitness, hookworm (Uncinaria sp.) infection was studied in a colony of South American fur seals (Arctocephalus australis) in which hookworms infect nearly all pups born in a reproductive season. A parasite-free group was generated by treating a subset of pups with an antiparasitic drug before they developed patent hookworm infection. Stress and metabolic hormones, energy balance, and humoral and cellular immune parameters were measured in this group and hookworm-infected pups. Hookworms elicited a marked increase in plasma cortisol levels in fur seal pups. These hookworm-infected pups were able to maintain constant glucose levels, despite losing body mass over the course of infection potentially because of increased protein catabolism. Infected pups were able to mount an effective immune response against the parasite and eliminated hookworms from the intestine, recovering partial body mass lost as a result of hookworm infection at the end of the study period. As shown in previous studies, adequate glucose levels are critical for proper T lymphocyte reactivity, and it is possible that, through activation of a stress response, energy can be readily available for immune response against the parasite contributing to early recovery from infection. Although there are potential fitness costs to mounting a sustained stress response, these could also be adaptive and promote survival during critical life-history stages.

Changes in Myeloid-Derived Suppressor Cell function during neonatal sepsis

Abstract Infants are more susceptible to infectious disease due to immaturity of the immune system. Our laboratory has shown that the immune suppressive cytokine, interleukin (IL)-27, is elevated in neonates. We and others have also shown that myeloid-derived suppressor cells (MDSCs) are more abundant in neonates, and these cells are a source of IL-27 in neonates. MDSCs suppress immunity with additional effector functions such as nitric oxide synthase (NOS2), arginase-1 (ARG1), and production of oxidative radicals to. We hypothesized that MDSCs gain enhanced immune suppressive function during neonatal sepsis. We utilized a murine model of neonatal Escherichia coli-induced sepsis that that demonstrates reduced weight gain in infected animals compared with controls, and high levels of bacteria in the blood and peripheral tissues. MDSCs were isolated from control or infected pups and the expression of effector function genes evaluated. Gene expression analysis revealed that NOS2, ARG1, and IL-27 gene expression was increased in MDSCs from infected mice. To understand whether or not these changes manifest at the protein/functional level and to further understand if they could be replicated in vitro, MDSCs isolated from control mice were cultured with E. coli. A similar pattern of changes in expression was observed with NOS2, ARG1, and IL-27 genes in vitro when the MDSCs were cultured with bacteria. This was followed by increased arginase activity, elevated nitrite levels, and secreted IL-27. These results suggest that gram-negative bacteria can directly act on MDSCs to enhance effector function associated with immune suppression. They also support further study of MDSC activity in regulating host control of infection early in life.

Neonatal infection leads to increased susceptibility to A\u03b2 oligomer-induced brain inflammation, synapse loss and cognitive impairment in mice

Harmful environmental stimuli during critical stages of development can profoundly affect behavior and susceptibility to diseases. Alzheimer disease (AD) is the most frequent neurodegenerative disease, and evidence suggest that inflammatory conditions act cumulatively, contributing to disease onset. Here we investigated whether infection early in life can contribute to synapse damage and cognitive impairment induced by amyloid-β oligomers (AβOs), neurotoxins found in AD brains. To this end, wild-type mice were subjected to neonatal (post-natal day 4) infection by Escherichia coli (1 × 104 CFU/g), the main cause of infection in low-birth-weight premature infants in the US. E. coli infection caused a transient inflammatory response in the mouse brain starting shortly after infection. Although infected mice performed normally in behavioral tasks in adulthood, they showed increased susceptibility to synapse damage and memory impairment induced by low doses of AβOs (1 pmol; intracerebroventricular) in the novel object recognition paradigm. Using in vitro and in vivo approaches, we show that microglial cells from E. coli-infected mice undergo exacerbated activation when exposed to low doses of AβOs. In addition, treatment of infected pups with minocycline, an antibiotic that inhibits microglial pro-inflammatory polarization, normalized microglial response to AβOs and restored normal susceptibility of mice to oligomer-induced cognitive impairment. Interestingly, mice infected with by E. coli (1 × 104 CFU/g) during adolescence (post-natal day 21) or adulthood (post-natal day 60) showed normal cognitive performance even in the presence of AβOs (1 pmol), suggesting that only infections at critical stages of development may lead to increased susceptibility to amyloid-β-induced toxicity. Altogether, our findings suggest that neonatal infections can modulate microglial response to AβOs into adulthood, thus contributing to amyloid-β-induced synapse damage and cognitive impairment.

Use of recombinant canine granulocyte-colony stimulating factor to increase leukocyte count in dogs naturally infected by canine parvovirus

Canine parvovirus (CPV) is one of the most important cause of mortality in young dogs and no specific treatment exists. Since prolonged leukopenia greatly increases the risk of death in infected pups, strategies to counteract this decline were investigated. The outcomes of CPV naturally infected pups treated with the recombinant canine granulocyte-colony stimulating factor (rcG-CSF), in combination with the routine therapy, were compared with similarly-managed infected pups not treated with rcG-CSF. A non-randomized prospective clinical trial was performed on 62 CPV infected pups with WBC counts <3000 cells/μL and two different groups were selected based on a non-randomized approach. Group A dogs (31/62) received 5 μg/Kg of rcG-CSF daily from the hospitalization day until WBC reached the reference range (3–5 days) and group B (31/62) received 1 ml of placebo injection. All dogs in group A recovered, while five dogs in group B died. The rcG-CSF treatment demonstrated a statistically significant effect on WBC counts (p < 0.0001) and, surprisingly, also on lymphocytes and monocytes counts (p < 0.0001). There was no significant effect of treatment on neutrophil count (p = 0.5502). Although lymphocytes and monocytes are not a specific target for rcG-CSF, our study highlights that rcG-CSF is able to improve haematological parameters compared to untreated dogs and a clear increase in their number was detected, as previously described for humans treated with the homologous molecule.

The life history strategy of a fur seal hookworm in relation to pathogenicity and host health status.

The strategies that parasites use to exploit their hosts can lead to adverse effects on human and animal populations. Here, we describe the life cycle, epidemiology, and consequences of hookworm (Uncinaria sp.) disease in South American fur seals (Arctocephalus australis), and propose that hookworm adaptation to fur seal life history traits has led to maximizing transmission at high levels of parasite-induced anemia and mortality. Fur seal pups acquire hookworms during their first days of life through their mothers' colostrum and most adult hookworms are expelled from the pups' intestine 30–65 days later. This gives hookworms little time to feed and reproduce. However, despite reaching high within-host densities, female hookworms do not decrease egg output, therefore pups with high hookworm burden contribute disproportionately to parasite egg shedding. These heavily infected pups also suffer severe anemia and high levels of hookworm-induced mortality. Alternative strategies to maximize total egg shedding and/or transmission, such as increased environmental survival of larval stages or avoidance of clearance, have not been developed by this hookworm. We propose that fur seal hookworms exploit a live fast-die young life history strategy, which translates to the highest levels of host anemia and mortality recorded among hookworms.

Long-term shedding of Canine alphaherpesvirus 1 in naturally infected newborn pups

The long-term shedding of Canine alphaherpesvirus 1 (CaHV-1) by neonatal pups with natural infection is reported. The pups belonged to a litter of 11 pointers of a breeding kennel in southern Italy, 9 of which developed a fatal form of systemic infection, as resulted by the detection of CaHV-1 in internal organs (kidney, liver, lung and brain) of one of this dogs and in the vaginal swab of their mother. The two remaining animals displayed a milder form of disease, with one pup showing ocular involvement, and underwent a progressive recovery. These pups were monitored from 11 to 36 days of age, showing a long-term shedding of the virus through the nasal and ocular secretions and the faeces. CaHV-1 shedding, as assessed by means of a specific and sensitive real-time PCR assay, occurred mainly through the nasal secretions, although the pup displaying ocular disease shed the virus at high titres and for a long period even in the ocular secretions.

Tissue distribution and cell tropism of Brucella canis in naturally infected canine foetuses and neonates

Brucella canis infection is an underdiagnosed zoonotic disease. Knowledge about perinatal brucellosis in dogs is extremely limited, although foetuses and neonates are under risk of infection due to vertical transmission. In this study, immunohistochemistry was used to determine tissue distribution and cell tropism of B. canis in canine foetuses and neonates. Diagnosis of B. canis in tissues of naturally infected pups was based on PCR and sequencing of amplicons, bacterial isolation, and immunohistochemistry, whose specificity was confirmed by laser capture microdissection. PCR positivity among 200 puppies was 21%, and nine isolates of B. canis were obtained. Tissues from 13 PCR-positive puppies (4 stillborn and 9 neonates) presented widespread immunolabeling. Stomach, intestines, kidney, nervous system, and umbilicus were positive in all animals tested. Other frequently infected organs included the liver (92%), lungs (85%), lymph nodes (69%), and spleen (62%). Immunolabeled coccobacilli occurred mostly in macrophages, but they were also observed in erythrocytes, epithelial cells of gastrointestinal mucosa, renal tubules, epidermis, adipocytes, choroid plexus, ependyma, neuroblasts, blood vessels endothelium, muscle cells, and in the intestinal lumen. These results largely expand our knowledge about perinatal brucellosis in the dog, clearly demonstrating a pantropic distribution of B. canis in naturally infected foetuses and neonates.

Experimental bovine rotavirus-A (RV-A)infection causes intestinal and extra-intestinal pathology in suckling mice

We describe here the intestinal and extra-intestinal spread of the species A rotavirus (RV-A) and associated lesions thereof in Swiss albino suckling mice pups, inoculated with a bovine-origin RV-A strain. In total, 35 suckling pups were used, wherein 20 pups received cell culture isolated RV-A @ 160 μL (TCID50/ml, 5 × 106.5) per pup [oral 80 μL and intra peritoneal (IP) 80 μL] and served as an infected group, while 15 pups were kept in the control group and inoculated the same volume of phosphate buffered saline (PBS) of neutral pH orally and IP. Four pups from the infected group and 3 from control group were sacrificed at 3, 5, 7, 9 and 12 day post infection (DPI). Of note, infected pups exhibited signs of dullness and restlessness till 5DPI, but none showed diarrhea at any point of time. No appreciable gross lesions were evident in any of the organs, except for mild congestion of the small intestine and yellowish catarrhal smearing over the luminal surface. However, light microscopic lesions in hematoxylin and eosin (H&E) stained sections of jejunum and ileum revealed vacuolation and pyknosis of nuclei of the mature enterocytes, their lysis and detachment, constriction and detachment of villi, mild mononuclear cells (MNCs) infiltration in the lamina propria and mildcell depletion of Peyer's patches and mesenteric lymph nodes (MLN). The extra-intestinal lesions of the cellular degeneration and mild MNCs infiltration were identified in the liver and kidneys from 3 to 7 DPI, but no lesion was seen in the brain. Interstitial thickening with MNCs of lung parenchyma was visible from 3 to 7 DPI. The lesions in the intestine, lymphoid tissues and lungs resolved after 7 DPI. The presence of viral nucleic acid was seen in the intestinal contents from 3 to 5 DPI by using a RV-A specific reverse-transcription polymerase chain reaction (RT-PCR), while in the MLNs and the lungs it could be detected till 5 DPI by both the RT-PCR and direct fluorescent antigen test (dFAT). However, liver, spleen and brain were tested negative for the presence of RV-A by any of these tests. Nonetheless, the persistence of the RV-A was seen in the MLNs even after the absence of virus from the small intestines. Findings here conclusively indicates that heterologous host origin RV-A has an affinity not only to the intestine but also to extra-intestinal tissues like MLNs and lung tissues.

Substitutions at residues 300 and 389 of the VP2 capsid protein serve as the minimal determinant of attenuation for canine parvovirus vaccine strain 9985-46.

Identifying molecular determinants of virulence attenuation in live attenuated canine parvovirus (CPV) vaccines is important for assuring their safety. To this end, we identified mutations in the attenuated CPV 9985-46 vaccine strain that arose during serial passage in Crandell-Rees feline kidney cells by comparison with the wild-type counterpart, as well as minimal determinants of the loss of virulence. Four amino acid substitutions (N93K, G300V, T389N and V562L) in VP2 of strain 9985-46 significantly restricted infection in canine A72 cells. Using an infectious molecular clone system, we constructed isogenic CPVs of the parental virulent 9985 strain carrying single or double mutations. We observed that only a single amino acid substitution in VP2, G300V or T389N, attenuated the virulent parental virus. Combinations of these mutations further attenuated CPV to a level comparable to that of 9985-46. Strains with G300V/T389N substitutions did not induce clinical symptoms in experimentally infected pups, and their ability to infect canine cells was highly restricted. We found that another G300V/V562L double mutation decreased affinity of the virus for canine cells, although its pathogenicity to dogs was maintained. These results indicate that mutation of residue 300, which plays a critical role in host tropism, is not sufficient for viral attenuation in vivo, and that attenuation of 9985-46 strain is defined by at least two mutations in residues 300 and 389 of the VP2 capsid protein. This finding is relevant for quality control of the vaccine and provides insight into the rational design of second-generation live attenuated vaccine candidates.

Streptococcus pneumoniae Transmission Is Blocked by Type-Specific Immunity in an Infant Mouse Model.

ABSTRACTEpidemiological studies on Streptococcus pneumoniae show that rates of carriage are highest in early childhood and that the major benefit of the pneumococcal conjugate vaccine (PCV) is a reduction in the incidence of nasopharyngeal colonization through decreased transmission within a population. In this study, we sought to understand how anti-S. pneumoniae immunity affects nasal shedding of bacteria, the limiting step in experimental pneumococcal transmission. Using an infant mouse model, we examined the role of immunity (passed from mother to pup) on shedding and within-litter transmission of S. pneumoniae by pups infected at 4 days of life. Pups from both previously colonized immune and PCV-vaccinated mothers had higher levels of anti-S. pneumoniae IgG than pups from non-immune or non-vaccinated mothers and shed significantly fewer S. pneumoniae over the first 5 days of infection. By setting up cross-foster experiments, we demonstrated that maternal passage of antibody to pups either in utero or post-natally decreases S. pneumoniae shedding. Passive immunization experiments showed that type-specific antibody to capsular polysaccharide is sufficient to decrease shedding and that the agglutinating function of immunoglobulin is required for this effect. Finally, we established that anti-pneumococcal immunity and anti-PCV vaccination block host-to-host transmission of S. pneumoniae. Moreover, immunity in either the donor or recipient pups alone was sufficient to reduce rates of transmission, indicating that decreased shedding and protection from acquisition of colonization are both contributing factors. Our findings provide a mechanistic explanation for the reduced levels of S. pneumoniae transmission between hosts immune from prior exposure and among vaccinated children.

Hookworms (Uncinaria lyonsi) in Dead California Sea Lion (Zalophus californianus) Pups and Sand in Winter (2014–2015) on San Miguel Island, California

ABSTRACT: Necropsy of dead California sea lion (CSL) (Zalophus californianus) pups (n = 20) born in 2014 was done on San Miguel Island, California, in December 2014 and February 2015. The main objective was to obtain data on prevalence/intensity of hookworms (Uncinaria lyonsi) in pups at that time of the year and to compare the results with data from previous studies conducted in December, January, and February. Fourteen dead pups were examined for adult hookworms in December 2014; all pups were infected (prevalence = 100%) with intensity from 6 to 140 (average 58.9 ± 38.9 SD) nematodes per host. Six dead pups were examined in February 2015. Hookworms were present in the intestines of 2 pups (prevalence=33%); two adult female worms were found in each infected pup. From 1 to 23 (average 7.5) parasitic larvae (L3) were found in the blubber of 4 pups. The findings in this study (2014–2015) verify, from earlier observation, that adult U. lyonsi are shed by pups by late February. The prevalence of parasitic L...