Catherine G. Lamm

Schmallenberg Virus Pathogenesis, Tropism and Interaction with the Innate Immune System of the Host

Schmallenberg virus (SBV) is an emerging orthobunyavirus of ruminants associated with outbreaks of congenital malformations in aborted and stillborn animals. Since its discovery in November 2011, SBV has spread very rapidly to many European countries. Here, we developed molecular and serological tools, and an experimental in vivo model as a platform to study SBV pathogenesis, tropism and virus-host cell interactions. Using a synthetic biology approach, we developed a reverse genetics system for the rapid rescue and genetic manipulation of SBV. We showed that SBV has a wide tropism in cell culture and “synthetic” SBV replicates in vitro as efficiently as wild type virus. We developed an experimental mouse model to study SBV infection and showed that this virus replicates abundantly in neurons where it causes cerebral malacia and vacuolation of the cerebral cortex. These virus-induced acute lesions are useful in understanding the progression from vacuolation to porencephaly and extensive tissue destruction, often observed in aborted lambs and calves in naturally occurring Schmallenberg cases. Indeed, we detected high levels of SBV antigens in the neurons of the gray matter of brain and spinal cord of naturally affected lambs and calves, suggesting that muscular hypoplasia observed in SBV-infected lambs is mostly secondary to central nervous system damage. Finally, we investigated the molecular determinants of SBV virulence. Interestingly, we found a biological SBV clone that after passage in cell culture displays increased virulence in mice. We also found that a SBV deletion mutant of the non-structural NSs protein (SBVΔNSs) is less virulent in mice than wild type SBV. Attenuation of SBV virulence depends on the inability of SBVΔNSs to block IFN synthesis in virus infected cells. In conclusion, this work provides a useful experimental framework to study the biology and pathogenesis of SBV.

Canine Parvovirus Types 2c and 2b Circulating in North American Dogs in 2006 and 2007

ABSTRACT Parvovirus is the most common viral cause of diarrhea in young puppies. Based on the analysis of a partial VP2 sequence of 54 samples, canine parvovirus type 2c (CPV-2c) (n = 26), CPV-2b (n = 25), and CPV-2 (n = 3) were detected in the United States. The American CPV-2b isolates have unique codons (494 and 572) in VP2.

Classical Swine Fever Virus Can Remain Virulent after Specific Elimination of the Interferon Regulatory Factor 3-Degrading Function of Npro

ABSTRACT Pestiviruses prevent alpha/beta interferon (IFN-α/β) production by promoting proteasomal degradation of interferon regulatory factor 3 (IRF3) by means of the viral Npro nonstructural protein. Npro is also an autoprotease, and its amino-terminal coding sequence is involved in translation initiation. We previously showed with classical swine fever virus (CSFV) that deletion of the entire Npro gene resulted in attenuation in pigs. In order to elaborate on the role of the Npro-mediated IRF3 degradation in classical swine fever pathogenesis, we searched for minimal amino acid substitutions in Npro that would specifically abrogate this function. Our mutational analyses showed that degradation of IRF3 and autoprotease activity are two independent but structurally overlapping functions of Npro. We describe two mutations in Npro that eliminate Npro-mediated IRF3 degradation without affecting the autoprotease activity. We also show that the conserved standard sequence at these particular positions is essential for Npro to interact with IRF3. Surprisingly, when these two mutations are introduced independently in the backbones of highly and moderately virulent CSFV, the resulting viruses are not attenuated, or are only partially attenuated, in 8- to 10-week-old pigs. This contrasts with the fact that these mutant viruses have lost the capacity to degrade IRF3 and to prevent IFN-α/β induction in porcine cell lines and monocyte-derived dendritic cells. Taken together, these results demonstrate that contrary to previous assumptions and to the case for other viral systems, impairment of IRF3-dependent IFN-α/β induction is not a prerequisite for CSFV virulence.

Streptococcal Infection in Dogs A Retrospective Study of 393 Cases

Streptococcus spp are opportunistic pathogens that normally reside in the upper respiratory, intestinal, lower urinary, and genital tracts but can cause localized infection or septicemia in dogs of all ages. A retrospective study of streptococcal infection in 393 dogs was conducted to identify the species of Streptococcus isolated, determine demographics of affected dogs, and characterize the disease processes associated with infection. The major streptococcal species isolated were S canis (88 cases, 22.4%), S dysgalactiae ssp equisimilis (13, 3.3%), and S equi ssp zooepidemicus (4, 1.0%). Sex was not a risk factor (P > .30). Fetuses and neonates were more likely to have streptococcal infection than were other age groups (P < .001). Streptococcal septicemia was considered an important cause of abortion and neonatal death and was isolated from all samples submitted for aerobic culture from dogs in that age group. There was a seasonal trend, with dogs more likely to have streptococcal infection in summer months. In dogs for which a disease process was identified, streptococcal infection was associated with dermatitis (29 dogs), pneumonia (24 dogs), adult septicemia (13 dogs), and fetal/neonatal septicemia leading to abortion or neonatal death (16 dogs). Identification of other clinically significant bacterial, viral, fungal, and parasitic organisms was common (267 of 393 dogs, 68%), especially in dogs with dermatitis or pneumonia. Infection with Streptococcus spp should be considered in the differential diagnosis in cases of abortion, septicemia, dermatitis, and pneumonia in dogs. Clinical significance of isolation of streptococcal organisms should be interpreted in context of clinical signs and pathologic findings.

Activation of Wnt signalling promotes development of dysplasia in Barrett's oesophagus

Barretts oesophagus is a precursor of oesophageal adenocarcinoma, via intestinal metaplasia and dysplasia. Risk of cancer increases substantially with dysplasia, particularly high‐grade dysplasia. Thus, there is a clinical need to identify and treat patients with early‐stage disease (metaplasia and low‐grade dysplasia) that are at high risk of cancer. Activated Wnt signalling is critical for normal intestinal development and homeostasis, but less so for oesophageal development. Therefore, we asked whether abnormally increased Wnt signalling contributes to the development of Barretts oesophagus (intestinal metaplasia) and/or dysplasia. Forty patients with Barretts metaplasia, dysplasia or adenocarcinoma underwent endoscopy and biopsy. Mice with tamoxifen‐ and β‐naphthoflavone‐induced expression of activated β‐catenin were used to up‐regulate Wnt signalling in mouse oesophagus. Immunohistochemistry of β‐catenin, Ki67, a panel of Wnt target genes, and markers of intestinal metaplasia was performed on human and mouse tissues. In human tissues, expression of nuclear activated β‐catenin was found in dysplasia, particularly high grade. Barretts metaplasia did not show high levels of activated β‐catenin. Up‐regulation of Ki67 and Wnt target genes was also mostly associated with high‐grade dysplasia. Aberrant activation of Wnt signalling in mouse oesophagus caused marked tissue disorganization with features of dysplasia, but only selected molecular indicators of metaplasia. Based on these results in human tissues and a mouse model, we conclude that abnormal activation of Wnt signalling likely plays only a minor role in initiation of Barretts metaplasia but a more critical role in progression to dysplasia. Copyright

Bovine Viral Diarrhea Virus Abortion in Goats Housed with Persistently Infected Cattle

Twenty-four border disease virus-seronegative, pregnant, mixed breed goats were experimentally comingled with 3 heifers persistently infected with bovine viral diarrhea virus type 2a (BVDV-2a). Twelve of the 24 exposed does aborted. Twenty-nine fetuses and 16 placentas from affected does were submitted to the Oklahoma Animal Disease Diagnostic Laboratory for a necropsy examination. Infection with BVDV was confirmed with a combination of immunohistochemistry, BVDV-2 polymerase chain reaction, and virus isolation in 19 of the 29 fetuses. On gross examination of the 19 fetuses and placentas in which BVDV-2a infection was confirmed, a mild placentitis (3/19), fetal mummification (1/19), and facial deformities (4/19) were noted. Histologically, placentitis (2/19), myocarditis (4/19), thymic depletion (5/19), choroid plexitis (3/19), encephalitis (2/19), and cerebral gliosis (1/19) were noted. Other causes of abortion in goats, including common bacterial and viral infections, were ruled out with histology, virus isolation, polymerase chain reaction, and aerobic bacteriologic cultures. As supported by the findings in this case, BVDV-2a should be included as a differential for abortion in goats. This is the first report of abortion in goats after exposure to persistently infected cattle.

Comparison of antemortem antimicrobial treatment regimens to antimicrobial susceptibility patterns of postmortem lung isolates from feedlot cattle with bronchopneumonia

A retrospective study was performed to compare the treatment regimens in feedlot cattle that died with bovine respiratory disease (BRD) to the antimicrobial susceptibility patterns of the microorganisms isolated from lungs. Forty-three cattle submitted by the Willard Sparks Beef Research Center (WSBRC) to the Oklahoma Animal Disease Diagnostic Laboratory for postmortem examination during 2007 had bronchopneumonia (acute = 16, subacute = 5, or chronic = 22). Lungs from cattle were cultured aerobically (40 cattle) and for Mycoplasma spp. (34 cattle). Susceptibility panels were performed. At least 1 BRD pathogen (Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, Mycoplasma bovis, or Arcanobacterium pyogenes) was isolated from 39 cattle, and 77% (30/39) had multiple organisms recovered. Mycoplasmal infections were common (25/34) and a major component of mixed infections (24/25). The majority (60%) of the M. haemolytica, P. multocida, and H. somni isolates were resistant to tetracycline. Most of the H. somni isolates (67%) were susceptible to tilmicosin (Ti), enrofloxacin (En), ceftiofur (Ce), and florfenicol, despite extensive treatment with Ti, En, and Ce (75% of isolates were from cattle that received each antimicrobial once). Most of the M. haemolytica (65%) and P. multocida (79%) isolates were susceptible to En and Ce, despite antemortem treatment of cattle with these antimicrobials. Hence, the current study reports a discrepancy between the antemortem treatment of clinical BRD and the susceptibility patterns of the bacteria isolated from lungs postmortem. Based on these findings, factors other than antimicrobial resistance are playing a role in the death of feedlot cattle with BRD.

Evaluation of a modified proportional margins approach for surgical resection of mast cell tumors in dogs: 40 cases (2008–2012)

OBJECTIVE To evaluate a modified proportional margins approach to resection of mast cell tumors (MCTs) in dogs. DESIGN Retrospective case series. ANIMALS 40 dogs with subcutaneous and cutaneous MCTs undergoing curative intent surgery. PROCEDURES Medical records were searched to identify dogs with a cytologically or histologically confirmed diagnosis of MCT that had not previously been treated surgically and that had undergone full oncological staging. In those dogs, tumors were resected with lateral margins equivalent to the widest measured diameter of the tumor and a minimum depth of 1 well-defined fascial plane deep to the tumor. Surgical margins were evaluated histologically. Cutaneous tumors were graded by use of the Patnaik system and the 2-tier system described by Kiupel et al. The prognosis for subcutaneous tumors was assessed in accordance with published recommendations. Follow-up information on dog health status was obtained through clinical examination, the dog owners, and the referring veterinarians. RESULTS The 40 dogs had 47 tumors. Forty-one (87%) tumors were cutaneous, and 6 (13%) were subcutaneous. On the basis of the Patnaik system, 21 (51%) cutaneous tumors were considered grade I, 18 (44%) were considered grade II, and 2 (5%) were considered grade III. On the basis of the Kiupel system, 37 (90%) cutaneous tumors were considered low grade, and 4 (10%) were considered high grade. The prognosis for the 6 subcutaneous tumors was classified as likely resulting in a shorter (2) or longer (4) survival time. Forty tumors were deemed to have been excised with clear margins and 7 with incomplete margins. Local recurrence was not recorded for any dog but was suspected for 1 (2%) tumor, although not confirmed. Interval from tumor excision to follow-up ranged from 30 to 1,140 days (median, 420 days). CONCLUSIONS AND CLINICAL RELEVANCE The modified proportional margins system resulted in satisfactory local disease control in dogs with MCTs.

Systemic coronavirus-associated disease resembling feline infectious peritonitis in ferrets in the UK.

FERRET systemic coronavirus (FRSCV)-associated disease is an emerging fatal disease of ferrets, with confirmed cases in Spain and the USA dating back to 2002 (Garner and others 2008). The clinicopathological characteristics of FRSCV-associated disease are remarkably similar to feline infectious peritonitis (FIP), a fatal systemic disease of cats caused by a virulent variant of feline coronavirus. FRSCV is closely related to ferret enteric coronavirus (FRECV), the cause of epizootic catarrhal enteritis (ECE) (Wise and others 2010). It is unclear whether FRSCV and FRECV are distinct viruses or whether FRSCV arises de novo by mutation of FRECV in vivo. An outbreak of ECE in Yorkshire in 2010 confirmed the presence of FRECV in the UK (Thomas and others 2012). In recent months, we have confirmed four cases of systemic FRSCV-associated disease in ferrets aged between …

Distribution of Bovine Viral Diarrhea Virus Antigen in Aborted Fetal and Neonatal Goats by Immunohistochemistry

Bovine viral diarrhea virus (BVDV) infection in goats can result in severe reproductive losses, with abortion rates reaching 80%. Infection with BVDV in aborted goat fetuses and stillborn kids can result in placentitis, encephalitis, myocarditis, and thymic depletion. This study investigates the distribution of viral antigen within the organ systems of aborted goat fetuses, stillborn kids, and nonviable kids infected with BVDV at various stages of gestation using immunohistochemistry (IHC). Virus antigen was detected within the placenta (8/13), thymus (4/9), heart (4/11), and brain (4/15) of affected goats. Uncommonly, BVDV antigen was detected within the skin (1/14), liver (1/13), kidney (1/12), lung (1/11), and trachea (1/3). BVDV antigen was not detected within the spleen (0/9), nasal turbinate (0/2), or thyroid (0/3). The results of this study indicate that placenta, heart, thymus, and brain are the most reliable tissues for BVDV antigen detection using IHC in aborted goat fetuses.