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Antimicrobial resistance is increasing in both human and veterinary medicine. Bacteria can be part of the etiology of respiratory disorders in horses. Bactericidal activity of silver has been largely described and silver is currently used in veterinary therapeutic applications such as wound dressings. The aim of this study was to assess the in vitro bactericidal effects of nebulized silver nanoparticles (AgNP) on 2 common equine respiratory bacteria, Streptococcus equi subsp. zooepidemicus and Actinobacillus equuli subsp. equuli. Firstly, antimicrobial susceptibility of AgNP was determined over time by turbidity assessment in liquid broth. Secondly, bacterial growth inhibition was tested after instillation or after nebulization of low (100 ppm) and high (500, 1,000 and 2,000 ppm) concentrations of AgNP on agar plate. Both bacteria were susceptible to AgNP, even at dilution 1:4 for A. equuli and 1:8 for S. zooepidemicus after 8 hours of incubation, and 1:256 for both bacteria after 24 hours of incubation. The bacterial growth was partially inhibited at low concentration and completely inhibited at high concentrations of instilled AgNP. The bacterial growth was completely inhibited after nebulization of low concentrations of AgNP for A. equuli and high concentrations of AgNP for S. zooepidemicus. We concluded nebulized AgNP could be a candidate for innovative therapeutic way against bacterial respiratory disorders in horses. Nevertheless, further investigations are required to assess the in vivo potential and toxicity of nebulized AgNP., (Copyright © 2021. Published by Elsevier Inc.)

Objectives: Recently a highly virulent Escherichia coli O80:H2 pathotype carrying Shiga toxin genes, the intimin subtype eaeξ, and genes associated with the extraintestinal pathogenic E. coli (ExPEC) pS88 plasmid was described in France. In this study we examine the relatedness of Belgian E. coli O80:H2 isolated from humans and diarrhoeic calves as well their similarities with the French pathotype., Methods: Eighteen Belgian E. coli O80:H2 strains (nine human Shiga toxin-producing E. coli (STEC) (2008-2016), two bovine STEC (1987) and seven bovine atypical enteropathogenic E. coli (aEPEC) (2009-2015)) were characterized with conventional PCR, disc diffusion susceptibility testing and whole genome sequencing., Results: Only nine sporadic human STEC O80:H2 cases have been detected in Belgium. All patients were female, just two of them suffered from haemolytic uremic syndrome. All studied strains had the eaeξ subtype, belonged to the multi-locus sequence type ST-301, and carried virulence genes associated with the type III secretion system and effectors not encoded by the locus of enterocyte effacement (LEE). Multiple genes of the pS88 plasmid were detected in all but two strains (one human and one calf STEC). The Shiga toxin subtypes stx1a (n = 3; one human, two calf), stx2a (n = 2) and stx2d (n = 6) were detected. All strains were multidrug resistant, two were extended-spectrum β-lactamase positive. Core genome MLST revealed that some human and calf E. coli differed by only 22 loci., Conclusions: The STEC/ExPEC O80:H2 pathotype was present in calves in Belgium as early as 1987, but human infections have been rare and mostly mild. The human STEC and bovine aEPEC cluster together and have the potential to be as virulent as the French isolates, as shown by their similar gene content., (Copyright © 2018 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Milk losses associated with mastitis can be attributed to either effects of pathogens per se (i.e. direct losses) or to effects of the immune response triggered by the presence of mammary pathogens (i.e. indirect losses). Test-day milk somatic cell counts (SCC) and number of bacterial colony forming units (CFU) found in milk samples are putative measures of the level of immune response and of the bacterial load, respectively. Mediation models, in which one independent variable affects a second variable which, in turn, affects a third one, are conceivable models to estimate direct and indirect losses. Here, we evaluated the feasibility of a mediation model in which test-day SCC and milk were regressed toward bacterial CFU measured at three selected sampling dates, 1 week apart. We applied this method on cows free of clinical signs and with records on up to 3 test-days before and after the date of the first bacteriological samples. Most bacteriological cultures were negative (52.38%), others contained either staphylococci (23.08%), streptococci (9.16%), mixed bacteria (8.79%) or were contaminated (6.59%). Only losses mediated by an increase in SCC were significantly different from null. In cows with three consecutive bacteriological positive results, we estimated a decreased milk yield of 0.28 kg per day for each unit increase in log2-transformed CFU that elicited one unit increase in log2-transformed SCC. In cows with one or two bacteriological positive results, indirect milk loss was not significantly different from null although test-day milk decreased by 0.74 kg per day for each unit increase of log2-transformed SCC. These results highlight the importance of milk losses that are mediated by an increase in SCC during mammary infection and the feasibility of decomposing total milk loss into its direct and indirect components.

A significant decrease in adherence rates of Mycoplasma bovis to bovine bronchial epithelial (BBE) cells has been observed after passage of the organism in artificial medium. Analysis of the proteins expressed by M. bovis isolate 2610 by two-dimensional (2-D) electrophoresis demonstrated differences between the cells harvested after the 7th and 116th passage. Three silver-stained prominent spots observed in 2-D electrophoretic separation of protein extracts of the lower-passaged cells were considerably less strongly expressed in the sample from higher-passaged cells. These spots had a molecular mass of approximately 24 kDa and an isoelectric point of about 5. The mass spectrometry analysis of these trypsin-sensitive proteins led to their identification as a unique new member of the Vsps family of membrane-associated proteins. Serum from a mouse immunized with these proteins significantly reduced adherence of M. bovis to BBE cells. This result underlines the function of this new Vsp in adherence of M. bovis to host cells.

Recent studies by DNA-DNA hybridisation assays conducted on a large collection of Escherichia coli strains isolated from chickens, ducks and turkeys suffering from colibacillosis, showed that 76% of the strains were negative for the presence of the pap gene cluster. The objective of this paper was to study the virulence associated with the avian E. coli strains negative for the P fimbriae, but carrying the f17 or the afa-8 gene cluster coding for adhesins associated with strains pathogenic for mammals. Three strains carrying the f17 fimbriae and three carrying the afa-8 adhesin-encoding gene cluster were studied in three in vivo experimental models of avian colibacillosis: subcutaneous inoculation of 1-day-old chicks, inoculation of specific-pathogen-free (SPF) chickens via the intra-thoracic air sac, and intra-tracheal inoculation of axenic chickens. The results showed that the six P-negative E. coli isolates carrying the f17 or the afa-8 gene cluster were lethal for 1-day-old chicks. They were also able to reproduce clinical signs and lesions of colibacillosis (aerosacculitis, pericarditis, perihepathitis), with bacteraemia and septicaemia, in SPF chickens inoculated via the thoracic air sacs as well as in axenic chickens inoculated by the intra-tracheal route. Further studies with f17 and afa-8 allelic mutants constructed by disruption must be performed to confirm a role of F17 fimbrial and Afa-VIII afimbrial adhesins in the pathogenesis of avian colibacillosis.

Newborn colostrum-restricted calves were orally inoculated with an Escherichia coli strain, identified originally as non-pathogenic, and into which the plasmid pVir was conjugally transferred. This resulted in diarrhea, intestinal lesions and extra-intestinal invasion, suggesting that factors affecting these pathogenic properties are located on pVir. In order to analyze the respective roles of the toxins CNF2 and CDTIII in the pathogenesis, colostrum-restricted calves were inoculated with isogenic mutants in the cnf2 and the cdt-III genes. The loss of cnf2 is associated with a reduction in the pathogenicity, since diarrhea does not occur in calves challenged, in spite of successful colonization of the intestine. Nevertheless, the mutant strain remains able to invade the bloodstream and to localize in the internal organs. Conversely, the calves inoculated with mutant in the cdt-III gene evolved in the same way as wild-type strain-inoculated calves with regard to clinical signs and macroscopic or microscopic lesions.

Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC) infections are characterised by the formation of attaching and effacing lesions on intestinal epithelial cells. The first step of EPEC and EHEC pathogenesis involves the initial adherence of the bacterium to the intestinal epithelium. A collection of bovine EPEC and EHEC strains belonging to different serogroups was tested by colony blot hybridization with gene probes for putative adhesins (BFPA, LPFA, IHA, LIFA) of human EPEC and EHEC, and also for fimbrial and afimbrial adhesins (AFA8, F17, Cs31A) of bovine necrotoxigenic E. coli (NTEC). In the bovine EPEC and EHEC strains tested, sequences homologous to lifA, ihA, and lpfA genes were detected, sometimes in association with particular serogroups. Bovine 026 EPEC also possessed a sequence homologous to a gene of the c/p operon, coding for the CS31A adhesin, associated with bovine NTEC. Overall results showed that different genes encoding for putative adhesins of human EHEC strains are present in bovine EPEC and EHEC strains, but not one of them is present in all strains.

Enteropathogenic Escherichia coli (EPEC) are isolated from man and farm animals but also from dogs and cats. They produce typical histological lesions called 'attaching and effacing' lesions. Both plasmid and chromosomal elements are involved in the pathogenesis of EPEC infection. The presence of these genetic elements was investigated in 14 dog and three cat EPEC isolates. A bfpA-related gene was detected in five of the 17 isolates in association with high molecular weight plasmids, and a locus of enterocyte effacement (LEE) was present in all isolates. The LEE was inserted in the selC region in only 12% of the isolates. The eae, tir, espA and espB genes were analyzed by multiplex PCR. The results indicated the presence of those genes in the tested isolates with heterogeneity in the gene subtypes present: eae gamma-tir alpha-espA alpha-espB alpha (65%), eae beta-tir beta-espA beta-espB beta (29%), eae alpha-tir alpha-espA alpha-espB alpha (6%). Moreover, the espD gene was also present in dog and cat EPEC. The DEPEC and CEPEC form a heterogeneous group and five of them are closely related to human EPEC.

Two types of myxomatosis vaccine are available commercially, namely, vaccine prepared from the Shope fibroma virus (SFV) and that prepared from an attenuated myxoma virus (MV) strain, e.gSG33. An experiment was designed to compare two vaccination schemes for their ability to protect rabbits against challenge with either a virulent amyxomatous MV strain or a virulent nodular MV strain. Apart from a difference in the cutaneous expression of the disease, the two challenge strains resembled each other in respect of mortality rate, naso-conjunctival shedding of virus, and tissue infection. Vaccination with SFV alone failed to prevent clinical signs, naso-conjunctival shedding or tissue infection. Vaccination with SFV followed by a booster inoculation with SG33 protected rabbits against the development of clinical signs and significantly reduced both viral shedding in naso-conjunctival exudates and viral infection of eyelids, lungs and testes; virus was, however, isolated from testes of some surviving animals.

The virulence of five amyxomatous myxoma virus (MV) strains, the clinical and pathogenetic effects of which had been studied previously in specific pathogen-free (SPF) rabbits, was determined by inoculation of five groups of 10 crossbred New Zealand White/Californian conventional rabbits. A much more acute myxomatosis syndrome was produced in conventional rabbits than that reproduced previously in SPF animals. However, the main clinical signs were of the respiratory type. The MV strains MYX 254/95 and 801 appeared very virulent, killing all the inoculated animals. The strains MYX 217/95, MYX 555/94 and Saint Benoist were somewhat attenuated, killing only seven, six and six rabbits, respectively. Extensive lung lesions due to supervening bacterial infections were observed in 36 of the 39 rabbits that died. Lethality was found to be a better estimate of virulence than mean survival time. By 98 days after viral inoculation, all the surviving animals had completely recovered. At that time, they were immunosuppressed by treatment with adrenocorticotrophic hormone (ACTH) for 10 days to determine whether they still harboured the virus. After the ACTH treatment, eight of the 11 surviving rabbits showed clinical signs that resembled amyxomatous myxomatosis. All the virological examinations performed on naso-conjunctival exudate, on mononuclear cells, on eyelids and on ovaries remained negative but infectious virus was isolated from the testes of three of six surviving male rabbits.

Attaching and effacing (AE) lesions are produced among others by enteropathogenic Escherichia coli and enterohaemorrhagic E. coli (EHEC), which differs from the former by the production of cytotoxins active on various cell cultures, the verocytotoxins, or shigacytotoxins. EHEC are associated with diarrhoea and dysentery in humans and in ruminants, mainly calves from two to eight weeks of age. Clinical signs and/or lesions have been reproduced experimentally with EHEC strains belonging to serotypes O5:K4/Nm, O26:K-:H11, O111:Nm, and O157:H7 which are isolated from cattle and/or humans. The purpose of this work was to develop an experimental model of infection in newborn calves with a bovine EHEC strain isolated from a calf which of died of diarrhoea, and belonging to the O118:H16 serotype, which is also common to both cattle and humans. The bovine O118:H16 EHEC strain was able to colonize the gut of three newborn calves, and to induce diarrhoea twenty-four hours after challenge and to produce AE lesions in the small and/or large intestines. AE lesions were detected microscopically and ultrastructurally in the small intestine of one calf and in the whole intestinal track of two calves. Internalization of bacteria and also of pedestal-bacteria complex inside of the enterocyte was observed in two of the three calves. The significance of this stage is unknown but may be related to the invasion of the calf by the bacteria. The challenge strain was isolated from the mesenteric lymph nodes of the same two calves but not from other organs or from heart blood. No blood was observed in the faeces of any of the three calves, nor were any lesions in the internal organs, which may have been related to the production of a verotoxin whose role is still unknown in cattle.

Enteropathogenic (EPEC) and enterohaemorrhagic (EHEC) Escherichia coli isolated from cattle were studied by DNA colony hybridization to subtype their intimin-encoding (eae) gene with probes derived from the variable parts of the eae alpha gene of the human EPEC strain E2348/69, the eae gamma gene of the human O157:H7 EHEC strain ATCC43888, and the eae beta gene of the bovine O26:H- EHEC strain 193, whose eae gene was first cloned and sequenced during this work. The EPEC and EHEC had been isolated from diarrhoeic calves (143 EPEC and 48 EHEC) and from healthy animals at the slaughterhouse (10 EPEC and 34 EHEC). The 191 bovine EPEC and EHEC isolated from diseased calves were positive with the Eae beta probe (55 and 27% respectively) and with the Eae gamma probe (9 and 73% respectively), whereas 52 EPEC (36%) were negative with the Eae alpha, Eae beta, and Eae gamma probes. The results were different for the 44 bovine EPEC and EHEC isolated from healthy cattle at slaughterhouses: most tested positive with the Eae gamma probe (80 and 82% respectively) and the remaining (20 and 18% respectively) with the Eae beta probe. Nine O26 human EHEC tested positive with the Eae beta probe and seven O111 with the Eae gamma probe. The bovine and human EPEC and EHEC belonging to these two serogroups gave identical results: the 18 bovine and human O26 isolates tested positive with the Eae beta probe, whereas the 13 O111 isolates were positive with the Eae gamma probe. In contrast, the isolates belonging to other serogroups (O5, O15, O18, O20, and O118) gave more variable results. The eae beta and eae gamma, but not the eae alpha, variants were thus distributed amongst bovine EPEC and EHEC. The eae beta variant seemed to be more frequently associated with the presence of clinical signs in calves, but one third of EPEC from diarrhoeic calves carried an eae gene variant other than the alpha, beta, or gamma variants. In addition, the use of these gene probes did not enable differentiation between bovine and human EHEC belonging to the same O serogroup.