Epidemiologic investigation and genetic characterization of canine respiratory coronavirus in the Southeastern United States.

Canine respiratory coronavirus (CRCoV) is one of the main causative agents of canine infectious respiratory disease (CIRD), an illness whose epidemiology is poorly understood. We assessed the prevalence, risk factors, and genetic characterization of CRCoV in privately owned dogs in the Southeastern United States. We PCR-screened 189 nasal swabs from dogs with and without CIRD clinical signs for 9 CIRD-related pathogens, including CRCoV; 14% of dogs, all diagnosed with CIRD, were positive for CRCoV, with a significantly higher rate of cases in younger dogs and during warmer weather. Notably, the presence of CRCoV, alone or in coinfection with other CIRD pathogens, was statistically associated with a worse prognosis. We estimated a CRCoV seroprevalence of 23.7% retrospectively from 540 serum samples, with no statistical association to dog age, sex, or season, but with a significantly higher presence in urban counties. Additionally, the genomes of 6 CRCoVs were obtained from positive samples using an in-house developed targeted amplicon-based approach specific to CRCoV. Subsequent phylogeny clustered their genomes in 2 distinct genomic groups, with most isolates sharing a higher similarity with CRCoVs from Sweden and only 1 more closely related to CRCoVs from Asia. We provide new insights into CIRD and CRCoV epidemiology in the Southeastern United States and further support the association of CRCoV with more severe cases of CIRD. Additionally, we developed and successfully tested a new amplicon-based approach for whole-genome sequencing of CRCoV that can be used to further investigate the genetic diversity within CRCoVs.

Adaptive immune protection of the middle ears differs from that of the respiratory tract.

The efficacy of the adaptive immune system in the middle ear (ME) is well established, but the mechanisms are not as well defined as those of gastrointestinal or respiratory tracts. While cellular elements of the adaptive response have been detected in the MEs following infections (or intranasal immunizations), their specific contributions to protecting the organ against reinfections are unknown. How immune protection mechanisms of the MEs compares with those in the adjacent and attached upper and lower respiratory airways remains unclear. To address these knowledge gaps, we used an established mouse respiratory infection model that we recently showed also involves ME infections. Bordetella bronchiseptica delivered to the external nares of mice in tiny numbers very efficiently infects the respiratory tract and ascends the Eustachian tube to colonize and infect the MEs, where it causes severe but acute inflammation resembling human acute otitis media (AOM). Since this AOM naturally resolves, we here examine the immunological mechanisms that clear infection and protect against subsequent infection, to guide efforts to induce protective immunity in the ME. Our results show that once the MEs are cleared of a primary B. bronchiseptica infection, the convalescent organ is strongly protected from reinfection by the pathogen despite its persistence in the upper respiratory tract, suggesting important immunological differences in these adjacent and connected organs. CD4+ and CD8+ T cells trafficked to the MEs following infection and were necessary to robustly protect against secondary challenge. Intranasal vaccination with heat killed B. bronchiseptica conferred robust protection against infection to the MEs, even though the nasopharynx itself was only partially protected. These data establish the MEs as discrete effector sites of adaptive immunity and shows that effective protection in the MEs and the respiratory tract is significantly different. This model system allows the dissection of immunological mechanisms that can prevent bacteria in the nasopharynx from ascending the ET to colonize the ME.

Glioma with cribriform plate involvement in 6 dogs.

Distinct patterns of local infiltration are a common feature of canine oligodendroglioma and astrocytoma, and typically involve the surrounding neuroparenchyma, ventricles, or leptomeninges. Infiltration of adjacent extraneural sites is rare and has not been well documented in veterinary medicine. Here we describe 6 canine gliomas with cribriform plate involvement (compression or infiltration) and caudal nasal invasion confirmed by neuroimaging, autopsy, and/or histology. All affected dogs were adults (9-12-y-old), and 3 were brachycephalic. Clinical signs were associated with the brain tumor, with no respiratory signs reported. Magnetic resonance imaging in 2 patients revealed a rostral intraparenchymal telencephalic mass with extension into the cribriform plate. All dogs were euthanized. Gross changes consisted of poorly demarcated, white or pale-yellow, soft, and, in oligodendrogliomas, gelatinous, intraparenchymal masses that expanded the rostral portions of the telencephalon and adhered firmly to the ethmoid bone and cribriform plate. Gliomas were classified as high-grade oligodendrogliomas (4 cases) and high-grade astrocytomas (2 cases) based on histology and immunohistochemistry for OLIG2 and GFAP. In all cases, there was evidence of cribriform plate invasion and, in one case, additional invasion of the caudal nasal cavity.

Systemic salmonellosis in 4 cats.

Clinical signs in 4 cases of salmonellosis in cats included vomiting, diarrhea (2 cases each), fever, dystocia, icterus, and seizures (1 case each). Three cats died, and one was euthanized. Grossly, all cats were in poor body condition and had yellow-to-dark-red perianal feces (3 cases), oral and ocular pallor (2 cases) or icterus (1 case), fluid or pasty yellow intestinal contents (4 cases), white or dark-red-to-black depressed areas on the hepatic surface (2 cases), yellow abdominal fluid with swollen abdominal lymph nodes (1 case), and fibrin strands on the placental chorionic surface (1 case). Histologically, all cats had necrotizing enterocolitis and random hepatocellular necrosis. Other histologic findings included mesenteric (4 cases) or splenic (2 cases) lymphoid necrosis, and endometrial and chorioallantoic necrosis (1 case). Gram-negative bacilli were observed within neutrophils and macrophages in the intestinal lamina propria (4 cases), liver, spleen, lymph node, endometrium, and placenta (1 case each). Aerobic bacterial culture on frozen samples of small intestine, mesenteric lymph node, lung, and liver yielded Salmonella enterica subsp. enterica. Serotyping was consistent with S. Enteritidis (cases 1, 3) and S. Typhimurium (cases 2, 4).

Novel murine model reveals an early role for pertussis toxin in disrupting neonatal immunity to Bordetella pertussis.

The increased susceptibility of neonates to specific pathogens has previously been attributed to an underdeveloped immune system. More recent data suggest neonates have effective protection against most pathogens but are particularly susceptible to those that target immune functions specific to neonates. Bordetella pertussis (Bp), the causative agent of "whooping cough", causes more serious disease in infants attributed to its production of pertussis toxin (PTx), although the neonate-specific immune functions it targets remain unknown. Problematically, the rapid development of adult immunity in mice has confounded our ability to study interactions of the neonatal immune system and its components, such as virtual memory T cells which are prominent prior to the maturation of the thymus. Here, we examine the rapid change in susceptibility of young mice and define a period from five- to eight-days-old during which mice are much more susceptible to Bp than mice even a couple days older. These more narrowly defined "neonatal" mice display significantly increased susceptibility to wild type Bp but very rapidly and effectively respond to and control Bp lacking PTx, more rapidly even than adult mice. Thus, PTx efficiently blocks some very effective form(s) of neonatal protective immunity, potentially providing a tool to better understand the neonatal immune system. The rapid clearance of the PTx mutant correlates with the early accumulation of neutrophils and T cells and suggests a role for PTx in disrupting their accumulation. These results demonstrate a striking age-dependent response to Bp, define an early age of extreme susceptibility to Bp, and demonstrate that the neonatal response can be more efficient than the adult response in eliminating bacteria from the lungs, but these neonatal functions are substantially blocked by PTx. This refined definition of "neonatal" mice may be useful in the study of other pathogens that primarily infect neonates, and PTx may prove a particularly valuable tool for probing the poorly understood neonatal immune system.

Multi-Influenza HA Subtype Protection of Ferrets Vaccinated with an N1 COBRA-Based Neuraminidase.

The influenza neuraminidase (NA) is a promising target for next-generation vaccines. Protection induced by vaccination with the computationally optimized broadly reactive NA antigen (N1-I COBRA NA) was characterized in both influenza serologically naive and pre-immune ferret models following H1N1 (A/California/07/2009, CA/09) or H5N1 (A/Vietnam/1203/2004, Viet/04) influenza challenges. The N1-I COBRA NA vaccine elicited antibodies with neutralizing ELLA activity against both seasonal and pandemic H1N1 influenza, as well as the H5N1 influenza virus. In both models, N1-I COBRA NA-vaccinated ferrets that were challenged with CA/09 virus had similar morbidity (weight loss and clinical symptoms) as ferrets vaccinated with the CA/09 HA control vaccine. There were significantly reduced viral titers compared to the mock-vaccinated control animals. Ferrets vaccinated with N1-I COBRA NA or Viet/04 NA vaccines were protected against the H5N1 virus infection with minimal clinical symptoms and negligible weight loss. In contrast, ferrets vaccinated with the CA/09 NA vaccine lost ~10% of their original body weight with 25% mortality. Vaccination with either HA or NA vaccines did not inhibit contact transmission of CA/09 virus to naïve cage mates. Overall, the N1-I COBRA vaccine elicited protective immune responses against both H1N1 and H5N1 infections and partially mitigated disease in contact-transmission receiving ferrets. These results indicate that the N1-I COBRA NA performed similarly to the CA/09 HA and NA positive controls. Therefore, the N1-I COBRA NA alone induces protection against viruses from both H5N1 and H1N1 subtypes, indicating its value as a vaccine component in broadly protective influenza vaccines.

Modeling the catarrhal stage of Bordetella pertussis upper respiratory tract infections in mice.

Pertussis (whooping cough) is a highly transmissible human respiratory disease caused by Bordetella pertussis, a human-restricted pathogen. Animal models generally involve pneumonic infections induced by depositing large numbers of bacteria in the lungs of mice. These models have informed us about the molecular pathogenesis of pertussis and guided development of vaccines that successfully protect against severe disease. However, they bypass the catarrhal stage of the disease, when bacteria first colonize and initially grow in the upper respiratory tract. This is a critical and highly transmissible stage of the infection that current vaccines do not prevent. Here, we demonstrate a model system in which B. pertussis robustly and persistently infects the nasopharynx of TLR4-deficient mice, inducing localized inflammation, neutrophil recruitment and mucus production as well as persistent shedding and occasional transmission to cage mates. This novel experimental system will allow the study of the contributions of bacterial factors to colonization of and shedding from the nasopharynx, as occurs during the catarrhal stage of pertussis, and interventions that might better control the ongoing circulation of pertussis.

Pathology in Practice.

In collaboration with the American College of Veterinary Pathologists.

SARS-CoV-2 and Influenza A Virus Coinfections in Ferrets.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and seasonal influenza viruses are cocirculating in the human population. However, only a few cases of viral coinfection with these two viruses have been documented in humans with some people having severe disease and others mild disease. To examine this phenomenon, ferrets were coinfected with SARS-CoV-2 and human seasonal influenza A viruses (IAVs; H1N1 or H3N2) and were compared to animals that received each virus alone. Ferrets were either immunologically naive to both viruses or vaccinated with the 2019 to 2020 split-inactivated influenza virus vaccine. Coinfected naive ferrets lost significantly more body weight than ferrets infected with each virus alone and had more severe inflammation in both the nose and lungs compared to that of ferrets that were single infected with each virus. Coinfected, naive animals had predominantly higher IAV titers than SARS-CoV-2 titers, and IAVs were efficiently transmitted by direct contact to the cohoused ferrets. Comparatively, SARS-CoV-2 failed to transmit to the ferrets that cohoused with coinfected ferrets by direct contact. Moreover, vaccination significantly reduced IAV titers and shortened the viral shedding but did not completely block direct contact transmission of the influenza virus. Notably, vaccination significantly ameliorated influenza-associated disease by protecting vaccinated animals from severe morbidity after IAV single infection or IAV and SARS-CoV-2 coinfection, suggesting that seasonal influenza virus vaccination is pivotal to prevent severe disease induced by IAV and SARS-CoV-2 coinfection during the COVID-19 pandemic. IMPORTANCE Influenza A viruses cause severe morbidity and mortality during each influenza virus season. The emergence of SARS-CoV-2 infection in the human population offers the opportunity to potential coinfections of both viruses. The development of useful animal models to assess the pathogenesis, transmission, and viral evolution of these viruses as they coinfect a host is of critical importance for the development of vaccines and therapeutics. The ability to prevent the most severe effects of viral coinfections can be studied using effect coinfection ferret models described in this report.

Pertactin contributes to shedding and transmission of Bordetella bronchiseptica.

Whooping cough is resurging in the United States despite high vaccine coverage. The rapid rise of Bordetella pertussis isolates lacking pertactin (PRN), a key vaccine antigen, has led to concerns about vaccine-driven evolution. Previous studies showed that pertactin can mediate binding to mammalian cells in vitro and act as an immunomodulatory factor in resisting neutrophil-mediated clearance. To further investigate the role of PRN in vivo, we examined the functions of pertactin in the context of a more naturally low dose inoculation experimental system using C3H/HeJ mice that is more sensitive to effects on colonization, growth and spread within the respiratory tract, as well as an experimental approach to measure shedding and transmission between hosts. A B. bronchiseptica pertactin deletion mutant was found to behave similarly to its wild-type (WT) parental strain in colonization of the nasal cavity, trachea, and lungs of mice. However, the pertactin-deficient strain was shed from the nares of mice in much lower numbers, resulting in a significantly lower rate of transmission between hosts. Histological examination of respiratory epithelia revealed that pertactin-deficient bacteria induced substantially less inflammation and mucus accumulation than the WT strain and in vitro assays verified the effect of PRN on the induction of TNF-α by murine macrophages. Interestingly, only WT B. bronchiseptica could be recovered from the spleen of infected mice and were further observed to be intracellular among isolated splenocytes, indicating that pertactin contributes to systemic dissemination involving intracellular survival. These results suggest that pertactin can mediate interactions with immune cells and augments inflammation that contributes to bacterial shedding and transmission between hosts. Understanding the relative contributions of various factors to inflammation, mucus production, shedding and transmission will guide novel strategies to interfere with the reemergence of pertussis.

The equine navicular apparatus as a premier enthesis organ: Functional implications.

Navicular syndrome has been traditionally characterized by progressive lameness with chronic degeneration of the navicular bone. Advances in imaging techniques have revealed that its associated soft tissue structures are also affected. This distribution of lesions is explained by conceptualizing the equine navicular apparatus as an enthesis organ that facilitates the dissemination of mechanical stress throughout the tissues of the foot. The navicular apparatus has the same structural adaptations to mechanical stress as the human Achilles tendon complex. These adaptations efficiently dissipate mechanical force away from the tendon's bony attachment site, thereby protecting it from failure. The comparison of these two anatomically distinct structural systems demonstrates their similar adaptations to mechanical forces, and illustrates that important functional insights can be gained from studying anatomic convergences and cross-species comparisons of function. Such a functional conceptualization of the equine navicular apparatus resolves confusion about the diagnosis of navicular syndrome and offers insights for the development of mechanically based therapies. Through comparison with the human Achilles complex, this review (1) re-conceptualizes the equine navicular apparatus as an enthesis organ in which mechanical forces are distributed throughout the structures of the organ; (2) describes the relationship between failure of the navicular enthesis organ and lesions of navicular syndrome; (3) considers the therapeutic implications of navicular enthesis organ degeneration as a form of chronic osteoarthritis; and based upon these implications (4) proposes a focus on whole body posture/motion for the development of prehabilitative and rehabilitative therapies similar to those that have already proven effective in humans.

Probing Immune-Mediated Clearance of Acute Middle Ear Infection in Mice.

Acute otitis media (AOM) is commonly caused by bacterial pathobionts of the nasopharynx that ascend the Eustachian tube to cause disease in the middle ears. To model and study the various complexities of AOM, common human otopathogens are injected directly into the middle ear bullae of rodents or are delivered with viral co-infections which contribute to the access to the middle ears in complex and partially understood ways. Here, we present the novel observation that Bordetella bronchiseptica, a well-characterized respiratory commensal/pathogen of mice, also efficiently ascends their Eustachian tubes to colonize their middle ears, providing a flexible mouse model to study naturally occurring AOM. Mice lacking T and/or B cells failed to resolve infections, highlighting the cooperative role of both in clearing middle ear infection. Adoptively transferred antibodies provided complete protection to the lungs but only partially protected the middle ears, highlighting the differences between respiratory and otoimmunology. We present this as a novel experimental system that can capitalize on the strengths of the mouse model to dissect the molecular mechanisms involved in the generation and function of immunity within the middle ear.

Disrupting Bordetella Immunosuppression Reveals a Role for Eosinophils in Coordinating the Adaptive Immune Response in the Respiratory Tract.

Recent findings revealed pivotal roles for eosinophils in protection against parasitic and viral infections, as well as modulation of adaptive immune responses in the gastric mucosa. However, the known effects of eosinophils within the respiratory tract remain predominantly pathological, associated with allergy and asthma. Simulating natural respiratory infections in mice, we examined how efficient and well-adapted pathogens can block eosinophil functions that contribute to the immune response. Bordetella bronchiseptica, a natural pathogen of the mouse, uses the sigma factor btrS to regulate expression of mechanisms that interfere with eosinophil recruitment and function. When btrS is disrupted, immunomodulators are dysregulated, and eosinophils are recruited to the lungs, suggesting they may contribute to much more efficient generation of adaptive immunity induced by this mutant. Eosinophil-deficient mice failed to produce pro-inflammatory cytokines, to recruit lymphocytes, to organize lymphoid aggregates that resemble Bronchus Associated Lymphoid Tissue (BALT), to generate an effective antibody response, and to clear bacterial infection from the respiratory tract. Importantly, the failure of eosinophil-deficient mice to produce these lymphoid aggregates indicates that eosinophils can mediate the generation of an effective lymphoid response in the lungs. These data demonstrate that efficient respiratory pathogens can block eosinophil recruitment, to inhibit the generation of robust adaptive immune responses. They also suggest that some post-infection sequelae involving eosinophils, such as allergy and asthma, might be a consequence of bacterial mechanisms that manipulate their accumulation and/or function within the respiratory tract.

Comparative Effects of 1/4-inch and 1/8-inch Corncob Bedding on Cage Ammonia Levels, Behavior, and Respiratory Pathology of Male C57BL/6 and 129S1/Svlm Mice.

Corncob is a common bedding material used in laboratory rodents, but little is known about differences in the effects of the 2 available sizes on rodent models and health. This study compared the effects of these 2 corncob bedding sizes on cage ammonia levels, behavior, and respiratory pathology in mice. We hypothesized that the beddings would not differ significantly in their effects on these parameters. Two strains of male mice (C57BL/6 and 129S1/Svlm) were housed in static, filter-top cages containing 1 of the 2 bedding types for the duration of the study (12 wk). Intracage ammonia was measured during 1 wk of the study on days 0, 3, 5, and 7. Behavior was evaluated by using circadian rhythm, open field, and Morris water-maze tests. Animals were euthanized with injectable euthanasia solution to collect respiratory and ocular tissues for histopathologic lesion scoring. Animals that were euthanized immediately upon arrival from the vendor served as negative controls. Bedding size did not significantly affect behavior or ammonia levels. Average intracage ammonia levels on day 7 were 525 ppm for 1/4-in. bedding and 533 ppm for 1/8-in. bedding. Regardless of the bedding size, lesions noted in both strains of mice were of similar incidence and severity, were limited to the nose, and consisted of minimal to mild suppurative rhinitis. The eyes, trachea, and lungs were not affected. In conclusion, 1/4-in. and 1/8-in. corncob beddings have comparable effects on cage ammonia levels and the behavior and respiratory pathology in male mice of the strains tested.

Shift work influences the outcomes of Chlamydia infection and pathogenesis.

Shift work, performed by approximately 21 million Americans, is irregular or unusual work schedule hours occurring after 6:00 pm. Shift work has been shown to disrupt circadian rhythms and is associated with several adverse health outcomes and chronic diseases such as cancer, gastrointestinal and psychiatric diseases and disorders. It is unclear if shift work influences the complications associated with certain infectious agents, such as pelvic inflammatory disease, ectopic pregnancy and tubal factor infertility resulting from genital chlamydial infection. We used an Environmental circadian disruption (ECD) model mimicking circadian disruption occurring during shift work, where mice had a 6-h advance in the normal light/dark cycle (LD) every week for a month. Control group mice were housed under normal 12/12 LD cycle. Our hypothesis was that compared to controls, mice that had their circadian rhythms disrupted in this ECD model will have a higher Chlamydia load, more pathology and decreased fertility rate following Chlamydia infection. Results showed that, compared to controls, mice that had their circadian rhythms disrupted (ECD) had higher Chlamydia loads, more tissue alterations or lesions, and lower fertility rate associated with chlamydial infection. Also, infected ECD mice elicited higher proinflammatory cytokines compared to mice under normal 12/12 LD cycle. These results imply that there might be an association between shift work and the increased likelihood of developing more severe disease from Chlamydia infection.

Comparison of endoscopic endotracheal intubation and the v-gel supraglottic airway device for spontaneously ventilating New Zealand white rabbits undergoing ovariohysterectomy.

BACKGROUND: Due to the technical difficulties with endotracheal intubation of rabbits, a prospective, randomised, controlled study was performed to compare a rabbit-specific supraglottic airway device (SGAD), the v-gel, with endoscopic endotracheal intubation (EEI) in spontaneously breathing rabbits undergoing ovariohysterectomy. METHODS: Fourteen adult female New Zealand white rabbits were randomly allocated to one of two groups based on the method of airway establishment: EEI or v-gel SGAD. Anaesthesia was induced with ketamine and xylazine and maintained using isoflurane in 100 per cent oxygen. Comparisons were made between groups based on placement time of endotracheal tube/SGAD, number of attempts and adjustments, the necessity to increase isoflurane concentrations to maintain a surgical plane of anaesthesia, arterial blood gas values, gross laryngeal evaluation, and laryngotracheal histopathology. RESULTS: Both techniques resulted in elevated arterial pCO2 levels, but the v-gel was associated with more elevated pCO2 in comparison with EEI (P=0.045). Airway trauma was histologically present but clinically negligible in both groups, with no statistically significant differences observed between techniques (P>0.05). Placement time of the v-gel was significantly faster (P=0.003) and required less technical skill than EEI, but was more easily displaced when changing the animal's position (P=0.004). CONCLUSION: The v-gel is a practical alternative to EEI for securing the airway of healthy spontaneously ventilating rabbits, provided a capnograph is utilised to ensure continuous placement. Both airway techniques appear safe and effective with few complications, as long as intermittent positive pressure ventilation can be employed to correct hypercapnia.

Effect of Time of Day of Infection on Chlamydia Infectivity and Pathogenesis.

Genital chlamydia infection in women causes complications such as pelvic inflammatory disease and tubal factor infertility, but it is unclear why some women are more susceptible than others. Possible factors, such as time of day of chlamydia infection on chlamydial pathogenesis has not been determined. We hypothesised that infections during the day, will cause increased complications compared to infections at night. Mice placed under normal 12:12 light: dark (LD) cycle were infected intravaginally with Chlamydia muridarum either at zeitgeber time 3, ZT3 and ZT15. Infectivity was monitored by periodic vaginal swabs and chlamydiae isolation. Blood and vaginal washes were collected for host immunologic response assessments. The reproductive tracts of the mice were examined histopathologically, and fertility was determined by embryo enumeration after mating. Mice infected at ZT3 shed significantly more C. muridarum than mice infected at ZT15. This correlated with the increased genital tract pathology observed in mice infected at ZT3. Mice infected at ZT3 were less fertile than mice infected at ZT15. The results suggest that the time of day of infection influences chlamydial pathogenesis, it indicates a possible association between complications from chlamydia infection and host circadian clock, which may lead to a better understanding of chlamydial pathogenesis.

Correction: A model of chronic, transmissible Otitis Media in mice.

[This corrects the article DOI: 10.1371/journal.ppat.1007696.].