Prevalence of thermophilic Campylobacter species in Swedish dogs and characterization of C. jejuni isolates

Background

The aims of this study were to investigate the prevalence of Campylobacter species in Swedish dogs, to identify the species of the Campylobacter isolates and to genotype the C. jejuni isolates. Young and healthy dogs were targeted and the sampling was performed at 11 veterinary clinics throughout Sweden from October 2011 to October 2012. Faecal swab samples were collected and sent to the laboratory at the National Veterinary Institute (SVA) for isolation of Campylobacter, speciation and genotyping.

Results

Campylobacter spp. were isolated from 67 of the 180 sampled dogs which yields an overall prevalence of 37%. The most prevalent species of Campylobacter among the participating dogs was C. upsaliensis with 52 of the 67 identified isolates. A lower prevalence was observed for C. jejuni with seven identified isolates and one isolate was identified as C. helveticus. Multi-locus sequence typing (MLST) was carried out on the seven C. jejuni isolates and all sequence types that were found are also commonly found in humans. The dogs were divided into three age groups; 1) under 12 months, 2) 12 to 23 months and 3) 24 months and older. The highest prevalence was found in the two younger age groups. Dogs shedding C. jejuni were between 3–12 months of age while dogs shedding C. upsaliensis were found in all ages.

Conclusions

The present investigation finds that Campylobacter spp. known to cause campylobacteriosis in humans are present in Swedish dogs. The results suggest an age predisposition where dogs under 2 years of age are more likely to shed Campylobacter spp. than older dogs. The most commonly isolated species was C. upsaliensis followed by C. jejuni, which was only detected in dogs up to 12 months of age. All C. jejuni isolates identified in the present study were of the same MLST types that have previously been described both in humans and in animals. The awareness of the Campylobacter risk of healthy young dogs may be an important way to reduce the transmission from dogs to infants, young children and immunocompromised adults.     

Bacteriological and Molecular Detection of Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus in Equines of Northern India

Present study was undertaken to study the prevalence of β-haemolytic streptococci in equine of northern temperate region of Jammu and Kashmir, India. One hundred and forty one samples were collected in duplicate from nasopharyngeal tract of diseased (53) and apparently healthy equine (88) for isolation and direct PCR. A total of 77 isolates of streptococci were recovered from 141 samples with an overall prevalence rate of 54.60%. Out of these 77 isolates, 52 were from diseased and 25 from apparently healthy animals. Of the 77 isolates, 4 were identified as Streptococcus equi subsp. equi, 56 as S. equi subsp. zooepidemicus and 17 as S. dysgalactiae subsp. equisimilis. Thus the overall prevalence of S. equi subsp. equi, S. equi subsp. zooepidemicus and S. dysgalactiae subsp. equisimilis was 2.83, 39.71 and 12.05% respectively. The sensitivity of the PCR for the detection of S. equi species was found higher when attempted from direct swab samples.     

Seroprevalence against Borrelia burgdorferi sensu lato and occurence of antibody co-expression with Anaplasma phagocytophilum in dogs in Latvia

Background

Lyme disease is commonly diagnosed in humans in Latvia, but up to date no studies have been performed to investigate its prevalence in dogs. The aim of this study was to evaluate if seroprevalence against B. burgdorferi sensu lato (B. burgdorferi s.l.) and co-expression of antibodies against B.burgdorferi s.l. and A. phagocytophilum is higher in dogs with clinical suspicion of tick-borne diseases compared to healthy dogs.

Findings

Venous blood was taken from healthy dogs (n=441) and dogs suspected to have borreliosis and/ or canine granulocytic anaplasmosis (n=29). The presence of antibodies was detected with SNAP 4Dx test (IDEXX, Westbrook, Maine, USA). The seroprevalence against B. burgdorferi s.l. in healthy dogs was 2.49% (11/441) and 36% (4/11) of seropositive dogs had antibodies against both of investigated bacteria. None of the dogs in sick dog group had detectable antibodies against B. burgdorferi s.l.

Conclusions

We conclude that seroprevalence to B. burgdorferi s.l. in dogs in Latvia is low and that dogs with suspicion of tick-borne disease do not have higher B. burgdorferi s.l. seroprevalence than healthy dogs. Dogs that express antibodies against B. burgdorferi s.l. frequently co-express antibodies against A. phagocytophilum.     

Detection of Escherichia coli O157 and Escherichia coli O157:H7 by the immunomagnetic separation technique and stx1 and stx2 genes by multiplex PCR in slaughtered cattle in Samsun Province, Turkey

This study was conducted to investigate the presence of Escherichia (E.) coli O157 and E. coli O157:H7 and stx1 and stx2 genes on cattle carcasses and in rectal samples collected from Samsun Province of Turkey. A total of 200 samples collected from cattle carcasses and the rectal contents of 100 slaughtered cattle from two commercial abattoirs were tested using the immunomagnetic separation technique and multiplex PCR methods. E. coli O157 and E. coli O157:H7 were detected in 52 of the 200 samples (26%) tested. Of the positive samples, 49 were E. coli O157 and three were E. coli O157:H7. The E. coli O157 strain was isolated from 24 carcasses and 25 rectal samples, while E. coli O157:H7 was isolated from two carcasses and one rectal sample. Of the 49 samples positive for E. coli O157, 32 were from the rectal and carcass samples of the same animal, while two E. coli O157:H7 isolates were obtained from rectal swabs and carcasses of the same animal. The stx1 and stx2 genes were both detected in 35 E. coli O157 isolates and one E. coli O157:H7 isolate, but the stx2 gene was only detected alone in two E. coli O157 isolates. Overall, 16 carcasses tested positive for E. coli O157 and one carcass tested positive for E. coli O157:H7 based on both carcass and rectal samples. Overall, the results of this study indicate that cattle carcasses pose a potential risk to human health due to contamination by E. coli O157 and E. coli O157:H7 in the feces.     

Restoring catalase activity in Staphylococcus aureus subsp. anaerobius leads to loss of pathogenicity for lambs

Staphylococcus aureus subsp. anaerobius, a microaerophilic and catalase-negative bacterium, is the etiological agent of abscess disease, a specific chronic condition of sheep and goats, which is characterized by formation of necrotic lesions that are located typically in superficial lymph nodes. We constructed an isogenic mutant of S. aureus subsp. anaerobius (RDKA84) that carried a repaired and functional catalase gene from S. aureus ATCC 12600, to investigate whether the lack of catalase in S. aureus subsp. anaerobius plays a role in its physiological and pathogenic characteristics. The catalase activity had no apparent influence on the in vitro growth characteristics of RDKA84, which, like the wild-type, did not grow on aerobically incubated agar plates. Restoration of catalase activity in RDKA84 substantially increased resistance to H₂O₂ when analyzed in a death assay. The intracellular survival rates of the catalase-positive mutant RDKA84 in polymorphonuclear neutrophils (PMN) isolated from adult sheep were significantly higher than those of the wild-type, while no differences were found with PMN isolated from lambs. RDKA84 showed significantly lower survival rates in murine macrophages (J774A.1 cells) than the wild-type strains did, whereas, in bovine mammary epithelial cells (MAC-T), no differences in intracellular survival were observed. Interestingly, the virulence for lambs, the natural host for abscess disease, of the catalase-positive mutant RDKA84 was reduced dramatically in comparison with wild-type S. aureus subsp. anaerobius in two experimental models of infection.     

Initial adherence of EPEC,EHEC and VTEC to host cells

Initial adherence to host cells is the first step of the infection of enteropathogenic Escherichia coli (EPEC), enterohaemorrhagic Escherichia coli (EHEC) and verotoxigenic Escherichia coli (VTEC) strains. The importance of this step in the infection resides in the fact that (1) adherence is the first contact between bacteria and intestinal cells without which the other steps cannot occur and (2) adherence is the basis of host specificity for a lot of pathogens. This review describes the initial adhesins of the EPEC, EHEC and VTEC strains. During the last few years, several new adhesins and putative colonisation factors have been described, especially in EHEC strains. Only a few adhesins (BfpA, AF/R1, AF/R2, Ral, F18 adhesins) appear to be host and pathotype specific. The others are found in more than one species and/or pathotype (EPEC, EHEC, VTEC). Initial adherence of EPEC, EHEC and VTEC strains to host cells is probably mediated by multiple mechanisms.     

Etiologic and epidemiologic analysis of bacterial infectious upper respiratory disease in Thoroughbred horses at the Seoul Race Park

Infectious upper respiratory disease (IURD) of Thoroughbred racehorses has been a frequent problem (29.6% of incidence) at the Seoul Race Park (Korea). Risk factors for IURD include the season with a high transfer rate (summer and fall), the stabling period (≤ 3 months), and age (2 to 3 years old), suggesting that the movement and new environment may have depressed the immune system of the horses and decreased their ability to respond properly to pathogens. The bacterial strains (n = 98) isolated from IURD horses included Pseudomonas spp., Escherichia coli, Staphylococcus spp., Streptococcus equi subsp. equi and zooepidemicus.     

In vivo morphological and antigenic characteristics of Photobacterium damselae subsp. piscicida

The present study was conducted to examine the morphology and antigenicity of Photobacterium damselae subsp. piscicida by culturing the bacterium in vivo in the peritoneal cavity of sea bass (Dicentrarchus labrax) within dialysis bags with either a low molecular weight (LMW) cut-off of 25 kDa or a high molecular weight (HMW) cut-off of 300 kDa. Differences were observed in the growth rate between the bacteria cultured in vivo or in vitro. Bacteria cultured in vivo were smaller and produced a capsular layer, which was more prominent in bacteria cultured in the HMW bag. Antigenicity was examined by Western blot analysis using sera from sea bass injected with live Ph. d. subsp. piscicida. The sera recognised bands at 45 and 20 kDa in bacteria cultured in vivo in the LMW bag. Bacteria cultured in vivo in the HMW bag did not express the 45 kDa band when whole cell extracts were examined, although the antigen was present in their extracellular products. In addition, these bacteria had a band at 18 kDa rather than 20 kDa. Differences in glycoprotein were also evident between bacteria cultured in vitro and in vivo. Bacteria cultured in vitro in LMW and HMW bags displayed a single 26 kDa band. Bacteria cultured in the LMW bag in vivo displayed bands at 26 and 27 kDa, while bacteria cultured in vivo in the HMW bag possessed only the 27 kDa band. These bands may represent sialic acid. The significance of the changes observed in the bacterium''s structure and antigenicity when cultured in vivo is discussed.     

Development of a multiplex loop-mediated isothermal amplification assay to detect shiga toxin-producing Escherichia coli in cattle

A multiplex loop-mediated isothermal amplification (mLAMP) assay was developed for simultaneous detection of the stx1 and stx2 genes and applied for detection of shiga toxin-producing Escherichia coli (STEC) in cattle farm samples. Two target genes were distinguished based on T_m values of 85.03 ± 0.54℃ for stx1 and 87.47 ± 0.35℃ for stx2. The mLAMP assay was specific (100% inclusivity and exclusivity), sensitive (with a detection limit as low as 10 fg/µL), and quantifiable (R² = 0.9313). The efficacy and sensitivity were measured to evaluate applicability of the mLAMP assay to cattle farm samples. A total of 12 (12/253; 4.7%) and 17 (17/253; 6.7%) STEC O157, and 11 (11/236; 4.7%) non-O157 STEC strains were isolated from cattle farm samples by conventional selective culture, immunomagnetic separation, and PCR-based culture methods, respectively. The coinciding multiplex PCR and mLAMP results for the types of shiga toxin revealed the value of the mLAMP assay in terms of accuracy and rapidity for characterizing shiga toxin genes. Furthermore, the high detection rate of specific genes from enrichment broth samples indicates the potential utility of this assay as a screening method for detecting STEC in cattle farm samples.     

Lineages of Streptococcus equi ssp. equi in the Irish equine industry

Background

Streptococcus equi ssp. equi is the causative agent of ‘Strangles’ in horses. This is a debilitating condition leading to economic loss, yard closures and cancellation of equestrian events. There are multiple genotypes of S. equi ssp. equi which can cause disease, but to date there has been no systematic study of strains which are prevalent in Ireland. This study identified and classified Streptococcus equi ssp. equi strains isolated from within the Irish equine industry.

Results

Two hundred veterinary isolates were subjected to SLST (single locus sequence typing) based on an internal sequence from the seM gene of Streptococcus equi ssp equi. Of the 171 samples which successfully gave an amplicon, 162 samples (137 Irish and 24 UK strains) gave robust DNA sequence information. Analysis of the sequences allowed division of the isolates into 19 groups, 13 of which contain at least 2 isolates and 6 groups containing single isolates. There were 19 positions where a DNA SNP (single nucleotide polymorphism) occurs, and one 3 bp insertion. All groups had multiple (2–8) SNPs. Of the SNPs 17 would result in an amino acid change in the encoded protein. Interestingly, the single isolate EI8, which has 6 SNPs, has the three base pair insertion which is not seen in any other isolate, this would result in the insertion of an Ile residue at position 62 in that protein sequence. Comparison of the relevant region in the determined sequences with the UK Streptococcus equi seM MLST database showed that Group B (15 isolates) and Group I (2 isolates), as well as the individual isolates EI3 and EI8, are unique to Ireland, and some groups are most likely of UK origin (Groups F and M), but many more probably passed back and forth between the two countries.

Conclusions

The strains occurring in Ireland are not clonal and there is a considerable degree of sequence variation seen in the seM gene. There are two major clades causing infection in Ireland and these strains are also common in the UK.     

Geographical, clinical, clinicopathological and radiographic features of canine angiostrongylosis in Irish dogs: a retrospective study

Background

Angiostrongylus vasorum infection is associated with high morbidity and mortality in dogs. Although recognised in Ireland, there are no large series of cases reported. The aim of this retrospective study was to identify pertinent clinical and geographical features in Irish dogs.

Results

The case records of dogs presenting to the University College Dublin Veterinary Hospital (1999-2010) were reviewed. A contemporaneous review of external faecal parasitology and post mortem submissions was also performed. A positive diagnosis of angiostrogylosis was identified in 49 dogs including 24 clinical, 10 post mortem and 15 external faecal sample cases. The majority (n = 44 (90%)) resided on the East Coast.In the clinical cases, the median age was 20 months, 29% of cases were older than 2 years. Clinical features included cardiorespiratory (63%), coagulopathic (71%) and other (63%) signs. Cough (n = 10), dyspnoea (n = 5) and tachypnoea (n = 3) were the most common cardiorespiratory abnormalities. Of animals with evidence of coagulopathy, excessive haemorrhage from a wound (n = 5), airway haemorrhage (n = 9), epistaxis (n = 3), haematoma (n = 4), suspected haemarthrosis (n = 3), neurological signs (n = 2) and haematuria (n = 1) were found. Ten dogs were anaemic, of which two were severe (haematocrit ≤ 0.20 L/L). Ten animals had thrombocytopenia, with four severely affected (≤50 × 10⁹/L). PT and APTT values were prolonged in 4 (24%) of 17 and a BMBT was prolonged in 5 (63%) of 8 cases. Vague signs of exercise intolerance (n = 6), lethargy (n = 6) and weakness (n = 2) were identified, with two (8%) animals having only these signs. In one animal the diagnosis appeared to be incidental. Thoracic radiographs (n = 19) identified abnormalities in 100% of cases. Four (17%) animals died before or within 24 hours of treatment and post mortem examinations confirmed angiostrongylosis. Fenbendazole was administered in 19 cases, 18 (95%) recovered. Two animals were euthanised, one which failed to respond to therapy and another in which an ante mortem diagnosis had not been made.

Conclusions

Angiostrongylosis is not uncommon in Ireland, is not confined to young dogs or the East Coast and can present with a wide variety of signs, particularly coagulopathic, respiratory or neurological signs.     

spa typing and enterotoxin gene profile of Staphylococcus aureus isolated from bovine raw milk in Korea

Staphylococcus aureus is a major etiological pathogen of bovine mastitis, which triggers significant economic losses in dairy herds worldwide. In this study, S. aureus strains isolated from the milk of cows suffering from mastitis in Korea were investigated by spa typing and staphylococcal enterotoxin (SE) gene profiling. Forty-four S. aureus strains were isolated from 26 farms in five provinces. All isolates grouped into five clusters and two singletons based on 14 spa types. Cluster 1 and 2 isolates comprised 38.6% and 36.4% of total isolates, respectively, which were distributed in more than four provinces. SE and SE-like toxin genes were detected in 34 (77.3%) isolates and the most frequently detected SE gene profile was seg, sei, selm, seln, and selo genes (16 isolates, 36.3%), which was comparable to one of the genomic islands, Type I νSaβ. This is a first report of spa types and the prevalence of the recently described SE and SE-like toxin genes among S. aureus isolates from bovine raw milk in Korea. Two predominant spa groups were distributed widely and recently described SE and SE-like toxin genes were detected frequently.     

Virulence factors in Escherichia coli isolated from calves with diarrhea in Vietnam

This study was conducted to determine the prevalence and characteristics of pathogenic Escherichia (E.) coli strains from diarrheic calves in Vietnam. A total of 345 E. coli isolates obtained from 322 diarrheic calves were subjected to PCR and multiplex PCR for detection of the f5, f41, f17, eae, sta, lt, stx1, and stx2 genes. Of the 345 isolates, 108 (31.3%) carried at least one fimbrial gene. Of these 108 isolates, 50 carried genes for Shiga toxin and one possessed genes for both enterotoxin and Shiga toxin. The eae gene was found in 34 isolates (9.8%), 23 of which also carried stx genes. The Shiga toxin genes were detected in 177 isolates (51.3%) and the number of strains that carried stx1, stx2 and stx1/stx2 were 46, 73 and 58, respectively. Among 177 Shiga toxin-producing E. coli isolates, 89 carried the ehxA gene and 87 possessed the saa gene. Further characterization of the stx subtypes showed that among 104 stx1-positive isolates, 58 were the stx1c variant and 46 were the stx1 variant. Of the 131 stx2-positive strains, 48 were stx2, 48 were stx2c, 11 were stx2d, 17 were stx2g, and seven were stx2c/stx2g subtypes. The serogroups most prevalent among the 345 isolates were O15, O20, O103 and O157.     

Dissemination and tracking of Salmonella spp. in integrated broiler operation

Controlling Salmonella in integrated broiler operation is complicated because there are numerous potential sources of Salmonella contamination, including chicks, feed, rodents, wild poultry operations, and the processing plant. The objective of this study was to investigate the distribution of Salmonella through all phases of two integrated broiler operations and to determine the key areas related to the control of all known sources of infection. Two different Salmonella serotypes were observed at integrated broiler chicken company A. S. enteritidis, the predominant company A isolate, was consistently found in the breeder farm, hatcheries, broiler farms, and chicken slaughterhouse. At company B, a total of six different serotypes, S. heidelberg, S. senftenberg, S. enteritidis, S. blockley, S. gallinarum, and S. virchow, were detected. Although S. heidelberg was not found in the broiler farms, it was consistently found in the breeder farm, hatcheries, and chicken slaughterhouse. In addition, S. enteritidis was found in the hatcheries, broiler farm, and chicken slaughterhouse. In order to obtain the genetic clonality, 22 S. enteritidis isolates were digested with XbaI and analyzed by pulsed-field gel electrohporesis (PFGE). A difference in the PFGE pattern was found to be related to the origin of the integrated broiler operation. These data support the critical need to control Salmonella in breeder farms and hatcheries, and demonstrate important points related to the control of infection in large-scale poultry operations of Korea.     

Physiology, pathogenicity and immunogenicity of lon and/or cpxR deleted mutants of Salmonella Gallinarum as vaccine candidates for fowl typhoid

To construct a novel live vaccine candidate for fowl typhoid (FT) caused by Salmonella Gallinarum (SG), the lon and cpxR genes that are related to host-pathogen interaction were deleted from a wild type SG using the allelic exchange method. The mutants were grown normally, as was the wild type. The biochemical properties of the mutants remained very similar to those of the wild-type, while JOL914 (Δlon) and JOL916 (ΔlonΔcpxR) were mucoid. Extracellular polysaccharide increased 30.6-, 1.3-, and 46.2-fold in JOL914, JOL915 (ΔcpxR), and JOL916, respectively. Dot-blot analysis demonstrated significant increases of FimA expression at 6.77-, 2.33-, and 3.90-fold for JOL914, JOL915, and JOL916, respectively. Internalizations of JOL914, JOL915, and JOL916, in chicken abdominal macrophages, were increased at 4.65-, 0.50-, and 2.72-fold, respectively. Virulences of JOL914, JOL915 and JOL916, analyzed by LD₅₀ using 1-week-old chickens, were attenuated approximately at 10¹-, 10¹-, and > 10³-fold, respectively. The oral inoculations of 2 × 10⁷ cfu of the wild type, JOL914, JOL915 and JOL916 caused 55.6, 16.7, 22.2, and 0.0% mortality, respectively. Significantly moderate gross lesions of the liver and spleen were observed in the JOL916 group compared to the other groups. An induced immune response and significant peripheral mononuclear proliferation reaction were observed in the JOL916 group. At the protection against the wild type challenge, JOL916 offered 100% protection. Thus, the results of this study suggest that JOL916 among the mutants studied represented the safest and most effective live vaccine candidate against FT.     

Control of Mycobacterium bovisinfection in two sika deer herds in Ireland

In a number of countries, tuberculosis (due to infection with Mycobacterium bovis) is a significant health problem of captive deer. This paper describes outbreaks of bovine tuberculosis in sika deer (Cervus nippon) on two farms in Ireland and the methods used to control the disease. On Farm A, infection was first detected during 1993. The infection was eradicated using a programme of test and removal, in association with segregation of young animals. A second outbreak (also due to infection with M. bovis, but a different RFLP profile) was detected in 2002. In the latter outbreak, infection was particularly prevalent in two groups of young deer. M. bovis with the same RFLP profile was also isolated in a badger found dead on the farm. Control was achieved by test and removal in association with herd management changes. In Herd B, infection was first detected in 1995, and subsequently eradicated using test and removal alone. In Herd A, re-infection remains an ongoing risk. Control rather than eradication of infection may more realistic in the short-to medium-term.     

Sortase anchored proteins of Streptococcus uberis play major roles in the pathogenesis of bovine mastitis in dairy cattle

Streptococcus uberis, strain 0140J, contains a single copy sortase A (srtA), encoding a transamidase capable of covalently anchoring specific proteins to peptidoglycan. Unlike the wild-type, an isogenic mutant carrying an inactivating ISS1 insertion within srtA was only able to infect the bovine mammary gland in a transient fashion. For the first 24 h post challenge, the srtA mutant colonised at a similar rate and number to the wild type strain, but unlike the wild type did not subsequently colonise in higher numbers. Similar levels of host cell infiltration were detected in response to infection with both strains, but only in those mammary quarters infected with the wild type strain were clinical signs of disease evident. Mutants that failed to express individual sortase substrate proteins (sub0135, sub0145, sub0207, sub0241, sub0826, sub0888, sub1095, sub1154, sub1370, and sub1730) were isolated and their virulence determined in the same challenge model. This revealed that mutants lacking sub0145, sub1095 and sub1154 were attenuated in cattle. These data demonstrate that a number of sortase anchored proteins each play a distinct, non-redundant and important role in pathogenesis of S. uberis infection within the lactating bovine mammary gland.