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1.  Characterization of a Moraxella species that causes epistaxis in macaques 
Veterinary microbiology  2010;147(0):367-375.
Bacteria of the genus Moraxella have been isolated from a variety of mammalian hosts. In a prior survey of bacteria that colonize the rhesus macaque nasopharynx, performed at the Tulane National Primate Research Center, organisms of the Moraxella genus were isolated from animals with epistaxis, or “bloody nose syndrome.” They were biochemically identified as Moraxella catarrhalis, and cryopreserved. Another isolate was obtained from an epistatic cynomolgus macaque at the U.S. Army Medical Research Institute of Infectious Diseases. Based on differences in colony and cell morphologies between rhesus and human M. catarrhalis isolates, we hypothesized that the nonhuman primate Moraxella might instead be a different species. Despite morphological differences, the rhesus isolates, by several biochemical tests, were indistinguishable from M. catarrhalis. Analysis of the cynomolgus isolate by Vitek 2 Compact indicated that it belonged to a Moraxella group, but could not differentiate among species. However, sequencing of the 16S ribosomal RNA gene from four representative rhesus isolates and the cynomolgus isolate showed closest homology to Moraxella lincolnii, a human respiratory tract inhabitant, with 90.16% identity. To examine rhesus macaques as potential hosts for M. catarrhalis, eight animals were inoculated with human M. catarrhalis isolates. Only one of the animals was colonized and showed disease, whereas four of four macaques became epistatic after inoculation with the rhesus Moraxella isolate. The nasopharyngeal isolates in this study appear uniquely adapted to a macaque host and, though they share many of the phenotypic characteristics of M. catarrhalis, appear to form a genotypically distinct species.
doi:10.1016/j.vetmic.2010.06.029
PMCID: PMC3971920  PMID: 20667430
Moraxella; Epistaxis; Nonhuman primate; Macaca
2.  Rabies virus glycoprotein is an important determinant for the induction of innate immune responses and the pathogenic mechanisms 
Veterinary microbiology  2012;162(2-4):601-613.
Our previous studies have suggested that street and fixed rabies viruses (RABV) induce diseases in the mouse model via different mechanisms. In the present study, attempts were made to determine if it is the glycoprotein (G) that is responsible for the observed differences in the pathogenic mechanisms. To this end, an infectious clone from fixed virus B2c was established and used as a backbone for exchange of the G from street viruses. The rate of viral replication, expression of viral proteins, and the induction of innate immune responses were compared in cells or in mice infected with each of the viruses. Furthermore, the infiltration of inflammatory cells into the CNS and the enhancement of blood-brain-barrier (BBB) permeability were also compared. It was found that fixed viruses induced stronger innate immune responses (expression of chemokines, infiltration of inflammatory cells, and enhancement of BBB permeability) than street RABV or recombinant viruses expressing the G from street RABVs. Fixed viruses induce disease via an immune-mediated pathogenic mechanism while street viruses or recombinant viruses expressing the G from street RABVs induce diseases via a mechanism other than immune-mediated pathogenesis. Therefore, RABV G is an important determinant for the induction of innate immune responses and consequently the pathogenic mechanisms.
doi:10.1016/j.vetmic.2012.11.031
PMCID: PMC3568536  PMID: 23265241
Rabies virus; innate immune response; inflammation; blood brain barrier
3.  Multidrug-resistant Escherichia coli from canine urinary tract infections tend to have commensal phylotypes, lower prevalence of virulence determinants and ampC-replicons☆ 
Veterinary Microbiology  2014;169(3-4):171-178.
Multidrug-resistant Escherichia coli is an emerging clinical challenge in domestic species. Treatment options in many cases are limited. This study characterized MDR E. coli isolates from urinary tract infections in dogs, collected between 2002 and 2011. Isolates were evaluated in terms of β-lactamase production, phylogenetic group, ST type, replicon type and virulence marker profile. Comparisons were made with antibiotic susceptible isolates also collected from dogs with urinary tract infections. AmpC β-lactamase was produced in 67% of the MDR isolates (12/18). Of these, 8 could be specifically attributed to the CMY-2 gene. None of the isolates tested in either group expressed ESBLs. Phylo-group distribution was as expected in the susceptible isolates, with an over representation of the pathogenic B2 phylo-group (67%). In contrast, the phylogenetic background for the MDR group was mixed, with representation of commensal phylo-groups A and B1. The B2 phylo-group represented the smallest proportion (A, B1, B2 or D was 28%, 22%, 11% and 33%, respectively). Virulence marker profiles, evaluated using Identibac® microarray, discriminated between the two groups. Marker sequences for a core panel of virulence determinants were identified in most of the susceptible isolates, but not in most of the MDR isolates. These findings indicate that for MDR isolates, plasmid-mediated AmpC is an important resistance mechanism, and while still capable of causing clinical disease, there is evidence for a shift towards phylogenetic groups of reduced inferred virulence potential. There was no evidence of zoonotic potential in either the susceptible or MDR urinary tract isolates in this study.
doi:10.1016/j.vetmic.2014.01.003
PMCID: PMC3969583  PMID: 24485933
Escherichia coli; Plasmid-mediated AmpC; Urinary tract infection; Dog; Multidrug-resistant; β-Lactamase
4.  Correlation of cell surface marker expression with African swine fever virus infection☆ 
Veterinary Microbiology  2014;168(2-4):413-419.
The expression of surface markers on African swine fever virus (ASFV) infected cells was evaluated to assess their involvement in infection. Previous findings indicated CD163 expression was correlated with ASFV susceptibility. However, in this study the expression of porcine CD163 on cell lines did not increase the infection rate of these cells indicating other factors are likely to be important in determining susceptibility to infection. On adherent porcine bone marrow (pBM) cells the expression of CD45 was strongly correlated with infection. CD163 and CD203a expression correlated at intermediate levels with infection, indicating cells expressing these markers could become infected but were not preferentially infected by the virus. Most of the cells expressing MHCII were infected, indicating that they may be preferentially infected although expression of MHCII was not essential for infection and a large percentage of the infected cells were MHCII negative. CD16 showed a marked decrease in expression following infection and significantly lower levels of infected cells were shown to express CD16. Altogether these results suggest CD163 may be involved in ASFV infection but it may not be essential; the results also highlight the importance of other cell markers which requiring further investigation.
doi:10.1016/j.vetmic.2013.12.001
PMCID: PMC3969584  PMID: 24398227
African swine fever virus; Surface markers; CD163; MHCII; CD203a; CD45
5.  Gene expression analysis of Salmonella enterica SPI in macrophages indicates differences between serovars that induce systemic disease from those normally causing enteritis☆ 
Veterinary Microbiology  2013;167(3-4):675-679.
Global gene expression of the invasive Salmonella serovars S. Enteritidis and S. Typhimurium, and the less-invasive S. Infantis and S. Hadar was studied during infection of a chicken macrophage cell line. Major functional gene groups responsible for intracellular physiological changes were regulated similarly in all four serovars. However, SPI1 and SPI4 genes of S. Enteritidis and S. Typhimurium were strongly repressed in the macrophages whereas S. Infantis, S. Hadar and other similar serovars maintained up-regulation of these gene sets. This phenomenon may explain some of the biological differences between invasive and non-invasive Salmonella serovars.
doi:10.1016/j.vetmic.2013.07.034
PMCID: PMC3878769  PMID: 24080352
Salmonella; Macrophage; Gene expression; Microarray; SPI
6.  Molecular detection of murine noroviruses in laboratory and wild mice 
Veterinary microbiology  2012;160(3-4):463-467.
Fecal specimens collected from 121 laboratory mice, 30 striped field mice (Apodemus agrarius), 70 yellow-necked mice (Apodemus flavicollis), and 3 bank voles (Myodes glareolus) were tested in sample pools for the presence of murine noroviruses (MNV). Ten of 41 laboratory mice and 2 of 3 striped field mice pooled samples were positive for MNV. All laboratory mouse MNVs were closely related to previously described MNVs. The complete ORF2 (VP1) of both striped field mouse MNVs identified in this study was 1623 nt (541 aa) long and differed at 12% nt (8% aa) positions from each other, at 22–24% nt (15–18% aa) positions from the laboratory mouse MNVs and at 20–22% nt (13–14% aa) positions from the recently described wood mouse (Apodemus sylvaticus) MNVs. This study provides further evidence for the circulation of novel, genetically diverse MNVs in wild mice.
doi:10.1016/j.vetmic.2012.06.002
PMCID: PMC3469783  PMID: 22748629
mouse; calicivirus; murine norovirus
7.  Co-infection of porcine dendritic cells with porcine circovirus type 2a (PCV2a) and genotype II porcine reproductive and respiratory syndrome virus (PRRSV) induces CD4+CD25+FoxP3+ T cells in vitro 
Veterinary microbiology  2012;160(1-2):233-239.
Porcine circovirus associated disease (PCVAD) is currently one of the most economically important diseases in the global swine industry. Porcine circovirus type 2 (PCV2) is the primary causative agent, however co-infection with other swine pathogens such as porcine reproductive and respiratory syndrome virus (PRRSV) is often required to induce the full spectrum of clinical PCVAD. While the specific mechanisms of viral co-infection that lead to clinical disease are not fully understood, immune modulation by the co-infecting viruses likely plays a critical role. We evaluated the ability of dendritic cells (DC) infected with PRRSV, PCV2 or both to induce regulatory T cells (Tregs) in vitro. DCs infected with PCV2 significantly increased CD4+CD25+FoxP3+ Tregs (p<0.05) and DCs co-infected with PRRSV and PCV2 induced significantly higher numbers of Tregs than with PCV2 alone (p<0.05). Cytokine analysis indicated that the induction of Tregs by co-infected DCs may be dependent on TGF-β and not IL-10. Our data support the immunomodulatory role of PCV2/PRRSV co-infection in the pathogenesis of PCVAD, specifically via Treg mediated immunosuppression.
doi:10.1016/j.vetmic.2012.04.040
PMCID: PMC3443269  PMID: 22633482
porcine reproductive and respiratory syndrome virus (PRRSV); porcine circovirus type 2 (PCV2); dendritic cells; porcine circovirus associated disease (PCVAD); regulatory T cell; co-infection
8.  Bordetella avium Antibiotic Resistance, Novel Enrichment Culture, and Antigenic Characterization 
Veterinary microbiology  2012;160(1-2):189-196.
Bordetella avium continues to be an economic issue in the turkey industry as the causative agent of bordetellosis, which often leads to serious secondary infections. This study presents a broad characterization of the antibiotic resistance patterns in this diverse collection of B. avium strains collected over the past thirty years. In addition, the plasmid basis for the antibiotic resistance was characterized. The antibiotic resistance pattern allowed the development of a novel enrichment culture method that was subsequently employed to gather new isolates from diseased turkeys and a healthy sawhet owl. While a healthy turkey flock was shown to seroconvert by four weeks-of-age, attempts to culture B. avium from healthy turkey poults were unsuccessful. Western blot of B. avium strains using pooled serum from diseased and healthy commercial turkey flocks revealed both antigenic similarities and differences between strains. In sum, the work documents the continued exposure of commercial turkey flocks to B. avium and the need for development of an effective, inexpensive vaccine to control spread of the disease.
doi:10.1016/j.vetmic.2012.05.026
PMCID: PMC3469198  PMID: 22721730
Bordetellosis incidence; serology; antibiotic resistance; Bordetella avium; poultry
9.  Initial sequence characterization of the rhabdoviruses of squamate reptiles, including a novel rhabdovirus from a caiman lizard (Dracaena guianensis) 
Veterinary Microbiology  2012;158(3-4):274-279.
Rhabdoviruses infect a variety of hosts, including non-avian reptiles. Consensus PCR techniques were used to obtain partial RNA-dependent RNA polymerase gene sequence from five rhabdoviruses of South American lizards; Marco, Chaco, Timbo, Sena Madureira, and a rhabdovirus from a caiman lizard (Dracaena guianensis). The caiman lizard rhabdovirus formed inclusions in erythrocytes, which may be a route for infecting hematophagous insects. This is the first information on behavior of a rhabdovirus in squamates. We also obtained sequence from two rhabdoviruses of Australian lizards, confirming previous Charleville virus sequence and finding that, unlike a previous sequence report but in agreement with serologic reports, Almpiwar virus is clearly distinct from Charleville virus. Bayesian and maximum likelihood phylogenetic analysis revealed that most known rhabdoviruses of squamates cluster in the Almpiwar subgroup. The exception is Marco virus, which is found in the Hart Park group.
doi:10.1016/j.vetmic.2012.02.020
PMCID: PMC3371314  PMID: 22397930
Rhabdoviridae; phylogeny; Dracaena guianensis; Marco virus; Chaco virus; Timbo virus; Sena Madureira virus
10.  Detection of classical and newly described staphylococcal superantigen genes in coagulase-negative staphylococci isolated from bovine intramammary infections 
Veterinary microbiology  2010;147(0):149-154.
The coagulase negative staphylococci (CNS) are the most prevalent mastitis pathogen group yet their virulence characteristics have not been well described. We investigated the presence of 19 classical and newly described staphylococcal superantigen (SAg) genes in CNS isolates from bovine intramammary infections (IMI). A total of 263 CNS representing 11 different Staphylococcus spp. were examined, and 31.2% (n = 82) of CNS isolates had one or more SAg genes; there were 21 different SAg gene combinations. The most prevalent combination of SAg genes (seb, seln, and selq; n = 45) was found in S. chromogenes, S. xylosus, S. haemolyticus, S. sciuri subsp. carnaticus, S. simulans and S. succinus. The genes for SAgs appear to be widely distributed amongst CNS isolated from bovine IMI.
doi:10.1016/j.vetmic.2010.06.021
PMCID: PMC3689430  PMID: 20667668
coagulase-negative staphylococci; staphylococcal superantigens; multiplex PCR
11.  Comparison of phenotypic and genotypic methods for the species identification of coagulase-negative staphylococcal isolates from bovine intramammary infections 
Veterinary microbiology  2010;147(0):142-148.
Coagulase-negative staphylococci (CNS) are the most frequently isolated pathogens from cows with intramammary infection (IMI). Although API STAPH ID 20, a commercially available identification system, and PCR-restriction fragment length polymorphism (PCR-RFLP) of the gap gene (gap PCR-RFLP) have been successfully applied for the identification of CNS isolates from human specimens, their accuracy in the identification of veterinary isolates has not been fully established. In this study, we identified 263 CNS isolates from bovine IMI at species level by partial 16S rRNA gene sequence analysis as the definitive test. Species identification obtained using partial 16S rRNA gene sequence analysis was compared to results from the API STAPH ID 20 and gap PCR-RFLP analysis. Eleven different CNS species were identified by partial 16S rRNA gene sequence analysis. Only 76.0 % (200 / 263) of the species identification results obtained by API STAPH ID 20 matched those obtained by partial 16S rRNA gene sequence analysis, whereas 97.0 % (255 / 263) of the species identification results obtained by the gap PCR-RFLP analysis matched those obtained by partial 16S rRNA gene sequence analysis. The gap PCR-RFLP analysis could be a useful and reliable alternative method for the species identification of CNS isolates from bovine IMI and appears to be a more accurate method of species identification than the API STAPH ID 20 system.
doi:10.1016/j.vetmic.2010.06.020
PMCID: PMC3689435  PMID: 20667671
coagulase-negative staphylococci; PCR-RFLP; gap gene
12.  The effect of Clostridium perfringens type C strain CN3685 and its isogenic beta toxin null mutant in goats 
Veterinary Microbiology  2012;157(3-4):412-419.
Clostridium perfringens type C is an important cause of enteritis and/or enterocolitis in several animal species, including pigs, sheep, goats, horses and humans. The disease is a classic enterotoxemia and the enteric lesions and associated systemic effects are thought to be caused primarily by beta toxin (CPB), one of two typing toxins produced by C. perfringens type C. This has been demonstrated recently by fulfilling molecular Koch’s postulates in rabbits and mice. We present here an experimental study to fulfill these postulates in goats, a natural host of C. perfringens type C disease. Nine healthy male or female Anglo Nubian goat kids were inoculated with the virulent C. perfringens type C wild-type strain CN3685, an isogenic CPB null mutant or a strain where the cpb null mutation had been reversed. Three goats inoculated with the wild-type strain presented abdominal pain, hemorrhagic diarrhea, necrotizing enterocolitis, pulmonary edema, hydropericardium and death within 24 h of inoculation. Two goats inoculated with the CPB null mutant and two goats inoculated with sterile culture media (negative controls) remained clinically healthy during 24 h after inoculation and no gross or histological abnormalities were observed in the tissues of any of them. Reversal of the null mutation to partially restore CPB production also increased virulence; 2 goats inoculated with this reversed mutant presented clinical and pathological changes similar to those observed in goats inoculated with the wild-type strain, except that spontaneous death was not observed. These results indicate that CPB is required for C. perfringens type C to induce disease in goats, supporting a key role for this toxin in natural C. perfringens type C disease pathogenesis.
doi:10.1016/j.vetmic.2012.01.005
PMCID: PMC3348370  PMID: 22296994
Beta toxin; Clostridium perfringens type C; enterotoxemia; goats
13.  Expression of the C-type lectins DC-SIGN or L-SIGN alters host cell susceptibility for the avian coronavirus, infectious bronchitis virus 
Veterinary microbiology  2012;157(3-4):285-293.
Infectious bronchitis virus (IBV), an avian coronavirus, is a cause of great economic loss in the poultry industry. The virus mainly infects respiratory epithelium, but can be also detected in other organs. The functional receptor for the virus has not been found and field strains of IBV do not infect conventional cell lines. Recently, it has been shown that the C-type lectins DC-SIGN/L-SIGN can promote entry of several coronaviruses. Here we examine whether DC-SIGN/L-SIGN are entry determinants for IBV. We show that by introducing human DC-SIGN/L-SIGN into non-permissive cells, infection by the IBV is dramatically increased. DC-SIGN mediated infection was inhibited by mannan and anti-lectin antibodies, and was independent of sialic acid levels on the cell. Enhancement of IBV infection also occurred for different serotypes of IBV. Our findings demonstrated that even in the absence of avian-specific receptor, DC-SIGN-like lectins are capable of mediating efficient IBV infection.
doi:10.1016/j.vetmic.2012.01.011
PMCID: PMC3600652  PMID: 22340967
Coronavirus; infectious bronchitis virus; receptor; infectivity; lectin; DC-SIGN
14.  Detergents modify proteinase K resistance of PrPSc in different transmissible spongiform encephalopathies (TSEs) 
Veterinary Microbiology  2011;157(1-2):23-31.
Prion diseases are diagnosed by the detection of their proteinase K-resistant prion protein fragment (PrPSc). Various biochemical protocols use different detergents for the tissue preparation. We found that the resistance of PrPSc against proteinase K may vary strongly with the detergent used. In our study, we investigated the influence of the most commonly used detergents on eight different TSE agents derived from different species and distinct prion disease forms. For a high throughput we used a membrane adsorbtion assay to detect small amounts of prion aggregates, as well as Western blotting. Tissue lysates were prepared using DOC, SLS, SDS or Triton X-100 in different concentrations and these were digested with various amounts of proteinase K. Detergents are able to enhance or diminish the detectability of PrPSc after proteinase K digestion. Depending on the kind of detergent, its concentration - but also on the host species that developed the TSE and the disease form or prion type - the detectability of PrPSc can be very different. The results obtained here may be helpful during the development or improvement of a PrPSc detection method and they point towards a detergent effect that can be additionally used for decontamination purposes. A plausible explanation for the detergent effects described in this article could be an interaction with the lipids associated with PrPSc that may stabilize the aggregates.
doi:10.1016/j.vetmic.2011.12.008
PMCID: PMC3338006  PMID: 22226540
prion protein; scrapie; BSE; chronic wasting disease; proteinase resistance; detergent
15.  Porcine IPEC-J2 Intestinal Epithelial Cells in Microbiological Investigations 
Veterinary Microbiology  2011;156(3-4):229-237.
IPEC-J2 cells are porcine intestinal columnar epithelial cells that were isolated from neonatal piglet mid-jejunum. This cell line forms polarized monolayers with high transepithelial electrical resistance when cultured on 0.4 μm pore-size filters. The cell line is unique in that it is derived from small intestinal tissue (compared to the common human colon-derived lines HT-29, T84, and Caco-2) and is not transformed (compared to the porcine small intestinal line, IPI-2I). Porcine intestinal epithelial cells more closely mimic human physiology than analogous rodent cell lines (e.g. IEC-6 or IEC-18), which is important in studies of zoonotic infections; in addition, they provide specificity to study porcine-derived infections. IPEC-J2 cells are increasingly being used in microbiological studies to examine the interactions of various animal and human pathogens, including Salmonella enterica and pathogenic Escherichia coli, with intestinal epithelial cells. The IPEC-J2 cell line has also been employed in some probiotic studies, in which the cells have been used as an initial screening tool for adhesiveness and anti-inflammatory properties of the potential probiotic microorganisms. The validity of these studies is not clear as follow-up studies to assess the efficacy of the probiotics in vivo have not been published to date. The aims of this review are to provide a comprehensive overview of the microbiological studies that have been conducted with IPEC-J2 cells and a reference guide of key cellular and immune markers that have been identified in this cell line that may prove to be useful in future studies.
doi:10.1016/j.vetmic.2011.10.017
PMCID: PMC3289732  PMID: 22074860
IPEC-J2; porcine intestinal epithelial cell; cell line; Salmonella enterica; Escherichia coli
16.  Pandemic A/H1N1/2009 influenza virus in Swine, Cameroon, 2010 
Veterinary Microbiology  2011;156(1-2):189-192.
Although swine origin A/H1N1/2009 influenza virus (hereafter “pH1N1”) has been detected in swine in 20 countries, there has been no published surveillance of the virus in African livestock. The objective of this study was to assess the circulation of influenza A viruses, including pH1N1 in swine in Cameroon, Central Africa. We collected 108 nasal swabs and 98 sera samples from domestic pigs randomly sampled at 11 herds in villages and farms in Cameroon. pH1N1 was isolated from two swine sampled in northern Cameroon in January 2010. Sera from 28% of these herds were positive for influenza A by competitive ELISA and 92.6% of these swine showed cross reactivity with pandemic A/H1N1/2009 influenza virus isolated from humans. These results provide the first evidence of this virus in the animal population in Africa. In light of the significant role of swine in the ecology of influenza viruses, our results call for greater monitoring and study in Central Africa.
doi:10.1016/j.vetmic.2011.09.003
PMCID: PMC3251638  PMID: 21963416
swine influenza virus; pandemic A/H1N1/2009 influenza virus; Cameroon; Central Africa; agriculture; zoonotic diseases
17.  Modulation of chemokine and chemokine receptor expression following infection of porcine macrophages with African swine fever virus 
Veterinary Microbiology  2013;162(2-4):937-943.
African swine fever virus (ASFV) is the only member of the Asfarviridae, a large DNA virus family which replicates predominantly in the cytoplasm. Most isolates cause a fatal haemorrhagic disease in domestic pigs, although some low virulence isolates cause little or no mortality. The modulation of chemokine responses following infection of porcine macrophages with low and high virulence isolates was studied to indicate how this may be involved in the induction of pathogenesis and of effective immune responses. Infection with both low and high virulence isolates resulted in down-regulation of mRNA levels for chemokines CCL2, CCL3L, CXCL2 and chemokine receptors CCR1, CCR5, CXCR3, CXCR4 and up-regulation in expression of mRNAs for CCL4, CXCL10 and chemokine receptor CCR7. Levels of CCL4, CXCL8, CXCL10 mRNAs were higher in macrophages infected with low virulence isolate OURT88/3 compared to high virulence isolate Benin 97/1. Levels of CXCL8 and CCL2 protein were significantly reduced in supernatants from macrophages infected with Benin 97/1 isolate compared to OURT88/3 and mock-infected macrophages. There was also a decreased chemotactic response of donor cells exposed to supernatants from Benin 97/1 infected macrophages compared to those from OURT88/3 and mock-infected macrophages. The data show that infection of macrophages with the low virulence strain OURT88/3 induces higher expression of key inflammatory chemokines compared to infection with high virulence strain Benin 97/1. This may be important for the induction of effective protective immunity that has been observed in pigs immunised with the OURT88/3 isolate.
doi:10.1016/j.vetmic.2012.11.027
PMCID: PMC3605585  PMID: 23265239
African swine fever; Chemokines; Immune evasion; Transcription
18.  Isolation of canine Anaplasma phagocytophilum strains from clinical blood samples using the Ixodes ricinus cell line IRE/CTVM20 
Veterinary Microbiology  2013;162(2-4):980-986.
Anaplasma phagocytophilum is an intracellular tick-borne rickettsial pathogen, which causes granulocytic anaplasmosis in various species of livestock and companion animals and also in humans. Previously A. phagocytophilum has been isolated and propagated in cell lines derived from the tick Ixodes scapularis and in the human promyelocytic cell line HL60. In this study we used the Ixodes ricinus-derived cell line IRE/CTVM20 to isolate and propagate two new canine strains of A. phagocytophilum.
Blood samples were collected by veterinarians from two dogs, one from Germany and the other from Austria. Suspicion of clinical canine granulocytic anaplasmosis was raised by the treating veterinarians and after confirmation of A. phagocytophilum infection by real-time PCR, buffy coat cells were isolated and co-cultivated with IRE/CTVM20 cells maintained at 28 °C in L15/L15B medium.
In the tick cells, rickettsial inclusions were first recognised after 86 days of incubation. Electron microscopic examination of tick cells infected with one of the isolates revealed cytoplasmic vacuoles containing pleomorphic organisms with individual bacteria enveloped by a bilayer membrane. Sequencing of 16S rRNA genes confirmed the isolation of A. phagocytophilum and showed the highest identity to the A. phagocytophilum human HZ strain. The two A. phagocytophilum isolates were passaged several times in IRE/CTVM20 cells and transferred to the I. scapularis cell line ISE6. This confirms for the first time the successful establishment and continuous cultivation of this pathogen in I. ricinus cells as well as infectivity of these canine strains for I. scapularis cells.
doi:10.1016/j.vetmic.2012.10.021
PMCID: PMC3757156  PMID: 23146170
Tick cell lines; Anaplasma phagocytophilum; IRE/CTVM20; Dog; Electron microscopy
19.  Evidence Against GB virus C Infection in Dromedary Camels 
Veterinary microbiology  2011;154(3-4):403-406.
A recent publication described finding GB virus C (GBV-C) RNA in four of twenty two dromedary camel sera, and sequence analysis found that these viruses were phylogenetically clustered within human GBV-C isolates. Since all other GB viruses to date form monophyletic groups according to their host species, the close relationship between the sequences generated from camel sera and human GBV-C isolates seemed implausible, leading us to conduct an independent analysis of the sequences. Our investigation found three lines of evidence arguing against GBV-C infection in dromedary camels. First, strong evidence of artifactual sequence generation was identified for some of the sequences. Secondly, the sequence diversity within individual camel sera was ten- to one-hundred fifty two-fold greater than that described for GBV-C within a human host. Finally, GBV-C sequences generated from each camel shared near complete identity with human isolates previously described by the same laboratory. Taken together, these data strongly suggest laboratory contamination. We suggest that additional validation experiments are needed before it is possible to conclude that camels are permissive for GBV-C infection.
doi:10.1016/j.vetmic.2011.06.017
PMCID: PMC3210887  PMID: 21757300
GB virus; Flavirirus; Dromedary Camel
20.  Expression patterns of five polymorphic membrane proteins during the Chlamydia abortus developmental cycle 
Veterinary Microbiology  2012;160(3-4):525-529.
It has been suggested that polymorphic membrane proteins (Pmps) belonging to the Type V autotransporter protein family play an important role in the pathogenesis of Chlamydia abortus (C. abortus; formerly Chlamydophila abortus) infection. In a previous study we demonstrated the expression of all the pmps at the transcriptional level. The purpose of this study was to measure the number of Pmp positive inclusions throughout the C. abortus developmental cycle to investigate heterogeneity in expression patterns. McCoy cells were infected with C. abortus and analysed for Pmp expression over a 72 h period by fluorescent immunocytochemistry. Pmp18D could be detected at all analysed time points, and could only be accurately quantified from 36 hpi while Pmp10G positive inclusions could be visualised from 36 hpi. Expression of Pmps 13G, 16G and 17G could only be visualised later in the cycle and within less than half of visualised inclusions. These results indicate that while expression of specific Pmps is constitutive (Pmp18D), the pattern of expression of other Pmps is more variable. This suggests that different members of the Pmp family may play different roles within the developmental cycle of the organism, with some (Pmps10G and 18D) having roles throughout the cycle, while the heterogeneity of expression of others may aid in antigenic variation.
doi:10.1016/j.vetmic.2012.06.017
PMCID: PMC3504296  PMID: 22776512
Chlamydia abortus; Polymorphic membrane protein; Antigenic variation
21.  Recent progress in understanding the pathogenesis of Clostridium perfringens type C infections 
Veterinary microbiology  2011;153(1-2):37-43.
Clostridium perfringens type C causes necrotizing enteritis in humans and several other animal species. Type C isolates must produce at least beta toxin (CPB) and alpha toxin (CPA) and most strains produce several other toxins including perfringolysin O (PFO) and TpeL. However, current evidence indicates that CPB is the main virulence factor for type C infections. Most of this evidence is based upon the loss of virulence shown by isogenic type C CPB knock out mutants on cells, and also in rabbit intestinal loops and in mouse models. This virulence is regained when these mutants are complemented with the wild-type cpb gene. Many type C isolates respond to close contact with enterocyte-like Caco-2 cells by producing all toxins, except TpeL, much more rapidly than occurs during in vitro growth. This in vivo effect involves rapid transcriptional upregulation of the cpb, cpb2, pfoA and plc toxin genes. Rapid Caco-2 cell-induced upregulation of CPB and PFO production involves the VirS/VirR two-component system, since upregulated in vivo transcription of the pfoA and cpb genes was blocked by inactivating the virR gene and was reversible by complementation to restore VirR expression.
doi:10.1016/j.vetmic.2011.02.048
PMCID: PMC3151542  PMID: 21420802
Clostridium perfringens; beta toxin; enterotoxemia; pathogenesis; type C
22.  Impact of early versus later fluoroquinolone treatment on the clinical; microbiological and resistance outcomes in a mouse-lung model of Pasteurella multocida infection 
Veterinary Microbiology  2010;148(2-4):292-297.
The early curative uses of antimicrobial drugs such as fluoroquinolones before the onset of symptoms in veterinary medicine may be regarded as irrational antibiotic consumption. However, it should be stressed that in early curative antimicrobial treatment as in metaphylaxis, the bacterial burden at the infection site is often very low, and so the rapid eradication of the bacterial population could result.
We investigated the impact of early versus later curative administrations of 1 or 40 mg/kg of marbofloxacin on the survival of mice, the eradication of the targeted pathogen and the selection of resistant bacteria in a mouse lung infection with Pasteurella multocida.
In this model, for a given marbofloxacin dose, the clinical and bacteriological outcomes were better, and the selection of resistance less frequent, for the early rather than for the late treatment. Moreover, the early administration of 1 mg/kg led to better clinical and similar bacteriological (eradication and selection of resistance) outcomes than the late administration of 40 mg/kg marbofloxacin. Our results suggest that the optimal doses for the animals’ cure could be lower when administered early during the time course of the infection than when administered after the disease outbreak. As the main argument against early treatments such as metaphylaxis is the possible enhancement of resistance at the gut level, further studies should assess if lower doses of antibiotic administered to all the animals of a herd could have less impact on the commensal digestive flora than higher doses only administered to animals showing clinical symptoms.
doi:10.1016/j.vetmic.2010.09.005
PMCID: PMC3498970  PMID: 20888712
Animals; Anti-Bacterial Agents; administration & dosage; pharmacokinetics; therapeutic use; Drug Resistance, Bacterial; Female; Fluoroquinolones; administration & dosage; pharmacokinetics; therapeutic use; Lung; microbiology; Mice; Microbial Sensitivity Tests; Pasteurella Infections; drug therapy; Pasteurella multocida; drug effects; growth & development; Respiratory Tract Infections; drug therapy; Time Factors; Treatment Outcome; antimicrobial resistance; metaphylaxis; early treatment; fluoroquinolone; marbofloxacin; Pasteurella multocida
23.  Differential effects of clathrin and actin inhibitors on internalization of Escherichia coli and Salmonella choleraesuis in porcine jejunal Peyer’s patches 
Veterinary microbiology  2005;113(1-2):117-122.
Peyer's patches constitute both an inductive immune site and an enteropathogen invasion route. Peyer's patch mucosae from porcine jejunum were mounted in Ussing chambers, and either Salmonella choleraesuis vaccine strain SC-54 or non-pathogenic rodent and porcine E. coli strains contacted the Peyer's patch mucosa for 90 min. Internalized bacteria were quantified by a gentamicin resistance assay. Monodansylcadaverine (300 µM, luminal addition), an inhibitor of clathrin-mediated endocytosis, significantly inhibited internalization of both E. coli strains relative to tissues untreated with the inhibitor; internalization of SC-54 was unaffected. The actin-disrupting agent cytochalasin D (10 µM, luminal addition), inhibited internalization of pig-adapted E. coli but not that of rodent-adapted E. coli or SC-54. Internalization of SC-54 and non-pathogenic E. coli in Peyer's patches appears to occur through different cellular routes.
doi:10.1016/j.vetmic.2005.10.022
PMCID: PMC3437647  PMID: 16326046
Salmonella choleraesuis; Escherichia coli; mucosal immunity; enteropathogen; endocytosis; clathrin
24.  Novel gastric helicobacters and oral campylobacters are present in captive and wild cetaceans 
Veterinary microbiology  2011;152(1-2):138-145.
The mammalian gastric and oral mucosa may be colonized by mixed Helicobacter and Campylobacter species, respectively, in individual animals. To better characterize the presence and distribution of Helicobacter and Campylobacter among marine mammals, we used PCR and 16S rDNA sequence analysis to examine gastric and oral samples from ten dolphins (Tursiops gephyreus), one killer whale (Orcinus orca), one false killer whale (Pseudorca crassidens), and three wild La Plata river dolphins (Pontoporia blainvillei). Helicobacter spp. DNA was widely distributed in gastric and oral samples from both captive and wild cetaceans. Phylogenetic analysis demonstrated two Helicobacter sequence clusters, one closely related to H. cetorum, a species isolated from dolphins and whales in North America. The second related cluster was to sequences obtained from dolphins in Australia and to gastric non-Helicobacter pylori helicobacters, and may represent a novel taxonomic group. Dental plaque sequences from four dolphins formed a third cluster within the Campylobacter genus that likely represents a novel species isolated from marine mammals. Identification of identical Helicobacter spp. DNA sequences from dental plaque, saliva and gastric fluids from the same hosts, suggests that the oral cavity may be involved in transmission. These results demonstrate that Helicobacter and Campylobacter species are commonly distributed in marine mammals, and identify taxonomic clusters that may represent novel species.
doi:10.1016/j.vetmic.2011.04.023
PMCID: PMC3142288  PMID: 21592686
Helicobacter; Campylobacter; marine mammals; cetaceans; gastritis
25.  Salmonella Cerro isolated over the past twenty years from various sources in the US represent a single predominant Pulsed-Field Gel Electrophoresis type 
Veterinary microbiology  2011;150(3-4):389-393.
Salmonella Cerro prevalence in US dairy cattle has increased significantly during the past decade. Comparison of 237 Salmonella isolates collected from various human and animal sources between 1986 and 2009 using pulsed- field gel electrophoresis, antimicrobial resistance typing, and spvA screening, showed very limited genetic diversity, indicating clonality of this serotype. Improved subtyping methods are clearly needed to analyze the potential emergence of this serotype. Our results thus emphasize the critical importance of population-based pathogen surveillance for the detection and characterization of potentially emerging pathogens, and caution to critically evaluate the adequacy of diagnostic tests for a given study population and diagnostic application.
doi:10.1016/j.vetmic.2011.01.026
PMCID: PMC3095722  PMID: 21349663
Salmonella Cerro; molecular epidemiology; PFGE; emerging clone

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