Determination of the oxidative burst chemiluminescent response of avian and murine-derived macrophages versus corresponding cell lines in relation to stimulation with Salmonella serotypes.

In contrast to mammalian systems, avian species lack a resident or harvestable macrophage population in the abdominal exudate. Peritoneal macrophages in the chicken can be elicited if an inflammatory agent such as sephadex is injected. This study examines the kinetics of different macrophage populations, derived by different methods of isolation and from different hosts, with respect to the elicited oxidative burst upon infection with host-adapted Salmonella serotypes.

The nature of the oxidative burst elicited by murine and avian-derived and cell line macrophages was determined after stimulation with phorbol myristate (PMA), zymosan A, and Salmonella serotypes. Both murine and chicken peritoneal macrophages, chicken blood monocytes and corresponding cell lines, J774A.1 and HD-11, were unable to produce a detectable chemiluminescent (CL) response after interaction with Salmonella using the luminescent probe luminol. However, both PMA and zymosan A induced a CL response in all cell types, with PMA eliciting a higher and earlier peak response (pkH) than zymosan A. Lucigenin-enhanced CL in both murine and chicken macrophages was achieved with PMA, zymosan A and Salmonella serotypes. In this case, zymosan A induced higher responses than PMA. In the peritoneal macrophages of both hosts, there were no significant differences in the oxidative burst induced by the different Salmonella serotypes. However, the J774A.1 (murine) cells demonstrated significant differences, with S. enterica serotype Choleraesuis (S. choleraesuis and S. gallinarum producing the highest response. In the HD-11 (chicken) cells, S. choleraesuis and S. dublin elicited the higher CL. With both cell lines, S. abortusovis failed to induce an appreciable CL response.

In these experiments it was demonstrated that oxidative burst was not detectable in monocytes/macrophage populations using luminol, which suggests a link to the lack of a myeloperoxidase system in these cells. Lucigenin-enhanced CL appeared independent from the myeloperoxidase system, indicating production of another oxidative species compared with luminol. No discernable effect of host specificity with regard to Salmonella serotype and respective host was seen in host-derived or cell line macrophages, and cell line macrophages displayed altered functional characteristics with regard to oxidative burst in comparison with their primary counterparts.     

Molecular and virulence characteristics of multi-drug resistant Salmonella Enteritidis strains isolated from poultry

Forty-six Salmonella enterica subspecies enterica serovar Enteritidis (S. Enteritidis) strains were isolated from chicken meat, faeces, and eggshells collected from hatcheries throughout Korea. The strains were examined for the presence of antimicrobial resistance and virulence genes. All 46 isolates were resistant to at least one of 21 antibiotics used in this study, 30 (65.2%) were resistant to three or more antimicrobials, and a single remarkable isolate was resistant to 15 antimicrobials. The isolates were primarily resistant to penicillins, sulfisoxazole, streptomycin, tetracycline and quinolones.The high rate of resistance in S. Enteritidis strains, sometimes to multiple drugs, may complicate future options for treating human infections. Nineteen of the 21 penicillin resistant isolates carried the bla_TEM gene, while one strain, resistant both to penicillins and ceftriaxone, carried the bla_CTX-M gene. Thirty-seven of the 45 sulfisoxazole resistant isolates carried sul2, and 23/24 streptomycin resistant isolates carried both strA and strB. All 10 tetracycline resistant isolates carried the tet(A) gene. Most isolates harboured both SPI-1 and SPI-2-associated genes, and the spv operon, which are known to be associated with human infections. The presence of these genes suggests that these strains could give rise to public health problems if dispersed in the general human population.     

Risk factors for Salmonella enterica subsp. enterica contamination in French broiler-chicken flocks at the end of the rearing period

Broiler-chicken are often Salmonella carriers. However, these bacteria are responsible for major food-borne human infection, in which poultry-meat products are frequently implicated. In order to prevent Salmonella spread during the slaughtering process, control measures should be implemented at the farm level to reduce the prevalence before slaughtering. The objective of this study was to identify the risk factors for Salmonella contamination in French commercial broiler flocks at the end of the rearing period. A prospective study was carried out in 1996 and 1997 on 86 broiler flocks located in western France. The Salmonella status of the flocks was assessed by means of litter swabs and dust samples analyzed with classical bacteriological methods. Sixty flocks (70%) had at least one contaminated environmental sample and were classified as Salmonella-contaminated flocks. Logistic regression was used to assess association of managerial practices, general hygiene and results of environmental Salmonella recovery, with the odds that the flock itself would be Salmonella-contaminated at the end of the rearing period. Salmonella contamination of the house before placing day-old chicks and the Salmonella contamination of day-old chicks were significantly related to Salmonella contamination of the flock at the end of the rearing period. The risk for Salmonella contamination of the flock was increased when feed trucks parked near the entrance of the change room and when feed meal, instead of small pellets, was provided at the start.     

Preliminary findings of Salmonella spp. in captive green iguanas (Iguana iguana) and their environment

Captive reptiles are routinely identified as reservoirs of Salmonella spp. and reports of reptile-associated salmonellosis are increasing. Unfortunately, little is known about the epidemiology of Salmonella spp. and green iguanas. We did a limited survey of a green-iguana farm in El Salvador to identify sources of Salmonella spp. in green iguanas and their environment. A limited number of samples for microbiological culture were collected from iguanas (adult, hatchling, and embryos) and their environment (food, water, soil, shelter, insects, and wild-caught lizards). Salmonella spp. was isolated from the intestine of both adult (3/20) and hatchling iguanas (8/20). There was no evidence of Salmonella spp. in the reproductive tracts of female iguanas (0/10). Salmonella spp. was isolated from the surface of 40% (7/16) of the egg surfaces tested. Salmonella spp. was not identified from the externalized yolk-sac of the iguana embryos tested. Soil samples from a breeding pen and a nest were both positive for Salmonella spp. Eight different Salmonella spp. serotypes were identified in this survey. These results suggest that horizontal transmission of Salmonella spp. is a potential source of exposure to hatchling iguanas at this facility.     

Effect of bacitracin on tetracycline resistance in Escherichia coli and Salmonella

Tetracyclines and bacitracin are used extensively as a growth promotant and a therapeutic, respectively in livestock. In this study, we test whether there is an interaction between bacitracin and tetracycline that can select for an increase in microbial tetracycline sensitivity, using Escherichia coli and Salmonella as a model. We found via in vitro studies that bacitracin at sublethal concentrations preferentially selects for outgrowth of tetracycline-sensitive bacteria from among a population of tetracycline-resistant and tetracycline-sensitive E. coli and Salmonella strains. Most of the bacterial strains employed in our study were shown by PCR to possess the tetracycline resistance gene tet(A) or tet(C), either of whose activity is associated with tetracycline efflux. Bacitracin could be substituted for the lipophylic chelator, fusaric acid, used as a positive selective agent for tetracycline sensitivity. Together, these observations suggest that bacitracin-mediated selection for tetracycline sensitivity alters the function of tetracycline efflux proteins. Using bacitracin and tetracycline simultaneously may improve the effectiveness of the antibiotics, while decreasing the risk of selecting for a population of tetracycline-resistant microorganisms.     

Feeding management practices and feed characteristics associated with Salmonella prevalence in live and slaughtered market-weight finisher swine: A systematic review and summation of evidence from 1950 to 2005

This review summarizes evidence for associations between Salmonella prevalence in market-weight swine and changes in feeding management practices or feed characteristics. A systematic review of the topic was conducted with the goal of minimizing the impact of bias on the review conclusions. Potential interventions included feed withdrawal from swine prior to slaughter, feed acidification, heat treatment of feed, pelletized feed versus mash, course versus fine grind, and wet versus dry feeds. In the reviewed literature, Salmonella prevalence was measured either by culture or by the presence of antibodies to Salmonella. The evidentiary value of studies was assessed, and studies that failed to meet predetermined standards were excluded. 7694 potentially relevant references were identified by an extensive literature search; however, 2623 references that were not published in English were excluded, because funds for translation were not available. Of the remaining references, only 277 were considered relevant to the review topic by two independent reviewers, and assessed for methodological quality. During quality assessment, 233 references were excluded because they failed to report design features that limit the introduction of bias or were conducted in a non-target population such as gnotobiotic, neonatal, nursery, or recently weaned pigs and sows. Forty-four publications passed the quality assessment conducted by 2 independent reviewers, but only 15 of the 44 publications reported studies that tested hypotheses associated with feeding management practices and feed characteristics and Salmonella prevalence in market-weight swine. The most common study design was cross-sectional (7/15). The included studies failed to provide strong evidence of an association between any of the potential interventions and Salmonella prevalence, due to the potential for confounding, and the failure to document temporal association between the intervention and Salmonella prevalence. The strongest evidence of an association was found for feed form, i.e. the use of non-pelleted may be potential interventions associated with reduced Salmonella prevalence. The uncertainty is primarily based on studies containing moderate to low evidentiary value or insufficient numbers of tested individuals, resulting in a low degree of confidence that results could be extrapolated to the target population. Therefore, the conclusion of the review is that there should be a low level of comfort among qualified scientists that the claimed association between non-pelleted feed and reduced Salmonella prevalence is scientifically valid. There is no strong evidence regarding associations between presence of Salmonella and the other feed characteristics examined.     

四川省部分地区山羊沙门氏菌健康带菌率及耐药性调查

为了解四川省山羊沙门氏菌带菌情况,本试验采集了四川省部分地区5个规模化山羊养殖场表观健康山羊粪便196份,经BPW预增菌、TTB选择性增菌后,采用靶向invA基因的PCR方法检测沙门氏菌的带菌率;对阳性样本进行细菌分离鉴定,随机选择25株沙门氏菌分离株测定其对15种抗菌药物的敏感性。结果显示,山羊沙门氏菌的平均带菌率为54.59%。药敏试验结果显示,分离株均对丁胺卡那敏感,而对其余14种抗菌药物呈不同程度的耐药,多重耐药菌株占88%,其中耐2~7种药物的占52%,耐9~13种药物的占36%。结果表明,四川省部分地区山羊的沙门氏菌健康带菌率较高,且多重耐药性普遍,其公共卫生意义值得进一步研究。     

Deletion of sodCI and spvBC in Salmonella enterica serovar Enteritidis reduced its virulence to the natural virulence of serovars Agona, Hadar and Infantis for mice but not for chickens early after infection

Salmonella enterica serovars Typhimurium, Enteritidis, Dublin, Choleraesuis or Gallinarum can colonise liver and spleen in particular hosts while infections with serovars Infantis, Agona, Hadar, etc. are usually limited to gastrointestinal tract. Reasons for this behavior are unknown, although it has been shown that sodCI and spv genes exhibit a strict distribution between more and less virulent serovars and they influence Salmonella virulence. However to what extent the presence or absence of these genes is associated with the increased virulence of serovars which possess them has never been addressed experimentally. In this study we therefore first confirmed the exclusive association of spvB and sodCI genes with the former group of serovars. In the next step we removed these two genes from S. Enteritidis genome and compared the virulence of such a mutant with the virulence of S. Infantis, S. Agona and S. Hadar for chickens and highly sensitive Balb/C mice. Single strain infection showed that the deletion of these two genes from S. Enteritidis resulted in the reduction of its virulence for mice but not for chickens. Mixed infection further confirmed these observations and indicated that in mice but not in chickens the virulence of sodCI and spv mutant was reduced to the natural virulence of serovars Infantis, Agona and Hadar. Although sodCI and spv genes do not influence S. Enteritidis virulence for chickens directly, they may be of an indirect effect through the increased persistence of S. Enteritidis in mice and increased probability of the reintroduction of S. Enteritidis into poultry flocks.     

Natural infection by Cryptosporidium sp., Giardia sp. and Eimeria leuckarti in three groups of equines with different handlings in Rio de Janeiro, Brazil

To detect Cryptosporidium sp., Giardia sp. and Eimeria leuckarti in horses, fecal samples were collected from three different handling horse groups from the state of Rio de Janeiro, Brazil. Group A was composed of “Mangalarga Marchador” pure breed horses, Group B was formed by horses of a Military Corporation and Group C by stray horses captured by the Center of Zoonosis Control Paulo Dacorso Filho. A total of 396 fecal samples were collected, 212 samples from Group A, 154 samples from Group B and 30 from Group C. The material was submitted to the centrifugation - flotation technique and staining by the safranin-methylene blue technique and analyzed. Oocysts of Cryptosporidium sp. were identified in 0.75% of the samples (n = 3); cysts of Giardia sp. in 0.5% (n = 2) and oocysts of E. leuckarti in 0.5% (n = 2). One case of E. leuckarti in group A and one of Cryptosporidium sp. in group B were observed. In group C were observed two cases of Cryptosporidium, two of Giardia and one of E. leuckarti,. Horses of group C were more parasitized by the three protozoans than animals from the other groups (p < 0.01). It was possible to verify that factors related to the animals, like host individual susceptibility and sanitary factors may influence the occurrence of natural infections by gastrointestinal protozoans, although the age did not have influence. This study reports, for the first time, the occurrence of Cryptosporidium sp., Giardia sp. and E. leuckarti in equines of the State of Rio de Janeiro.     

Molecular screening and risk factors of enterotoxigenic Escherichia coli and Salmonella spp. in diarrheic neonatal calves in Egypt

The aim of the present study was to carry out molecular epidemiological investigation on enterotoxigenic Escherichia coli (ETEC) K99 and Salmonella spp. in diarrheic neonatal calves. Fecal samples were obtained from 220 diarrheic calves at 9 farms related to four governorates in central and northern Egypt. E. coli and Salmonella spp. isolates were examined for E. coli K99 and Salmonella spp. using PCR. ETEC K99 was recovered from 20 (10.36 %) out of 193 isolates, whereas Salmonella spp. was recovered from nine calves (4.09%).Multivariable logistic regression was used to evaluate the risk factors associated with both infections. ETEC K99 was significantly affected by age (P < 0.01; OR: 1.812; CI 95%: 0.566–1.769), colostrum feeding practice (P < 0.01; OR: 5.525; CI 95%: 2.025–15.076), rotavirus infection (P < 0.001; OR: 2.220; CI 95%: 0.273–1.251), vaccination of pregnant dams with combined vaccine against rotavirus, coronavirus and E. coli (K99) (P < 0.001; OR: 4.753; CI 95%: 2.124–10.641), and vitamin E and selenium administration to the pregnant dam (P < 0.01; OR: 3.933; CI 95%: 0.703–1.248).Infection with Salmonella spp. was found to be significantly affected by the animal age (P < 0.05; OR: 0.376; CI 95%: 0.511–1.369), Hygiene (P < 0.05; OR: 0.628; CI 95%: 1.729–5.612), and region (P < 0. 01; OR: 0.970; CI 95%: 0.841–1.624).The results of the present study indicate the importance of PCR as rapid, effective and reliable tool for screening of ETEC and Salmonella spp. when confronted with cases of undifferentiated calf diarrhea. Moreover, identification of the risk factors associated with the spreading of bacteria causing diarrhea may be helpful for construction of suitable methods for prevention and control.     

Prevalence of Multi-Drug-Resistant Salmonella in Equids Maintained by Low Income Individuals and on Designated Equine Farms in India

We examined 872 equids (445 maintained by low-income individuals and 427 maintained on nine designated equine farms) and, using previously described methods for bacteria, isolated Salmonella from fecal samples of 59 (6.77%) animals. Of the 646 horses, 183 donkeys, and 43 mules that had feces cultured for Salmonella, 42 (6.5%), 7 (3.8%), and 10 (23.3%), respectively, were excreting Salmonella strains in feces. Six horse mares were excreting Salmonella enterica of two different serovars simultaneously. A total of 65 Salmonella enterica isolates belonged to 13 serovars, namely S. paratyphi B var Java (14), S. I. 4, 5, 12, 27: r, i: 1, 5 (11), S. Drogana (8), S. Newport (7), S. Saintpaul (5), S. Lagos (4), S. Typhimurium (5), S. Kottbus (3), S. Bovismorbificans (3), S. Dumfries (2), S. Tshiongwe (1) S. Weltevreden (monophasic) (1), and S. enterica ssp salamae (1). With Salmonella-specific polymerase chain reaction (PCR) using hisJ gene primers, 107 (12.3) fecal samples yielded a specific amplicon of 496 bp. On using PCR, prevalence of Salmonella in donkeys, horses, and mules was 4.9%, 10.8%, and 65.1%, respectively. With both methods of Salmonella detection in feces, prevalence was significantly higher in female than in male donkeys and horses. Salmonella shedding in feces was significantly higher in equids maintained by low-income people than those at designated equine farms. Almost all Salmonella isolates (63 of 65) had multiple-drug-resistance (MDR, resistance to three or more drugs). Salmonella isolates were commonly resistant to sulfamethoxazole (90.8%), tetracycline (70.8%), doxycycline (67.7%), furazolidone (66.2%), and colistin (55.4%). A few isolates had resistance to trimethoprim (3.1%), ciprofloxacin (3.1%), ceftriaxone (3.1%), ceftazidime (3.1%), cefoperazone (3.1%), chloramphenicol (4.6%), cefotaxime (6.2%), gentamicin (9.2%), ampicillin + cloxacillin (9.2%), cotrimoxazole (13.8%), kanamycin (13.8%), amoxicillin + clavulanic acid (16.9%), imipenem (16.9%), ampicillin (18.5%), amikacin (23.1%), neomycin (27.7%), nalidixic acid (33.8%), and streptomycin (36.9%). With the exception of 13 Salmonella isolates of S. Drogana (4), S. Newport (4), S. I. 4, 5, 12, 27: r, i: 1, 5 (4) and S. Kottbus (1) serovars, all had one or more than one plasmid. Molecular weight of plasmids ranged between 3 kDa and >87 kDa. One heavy plasmid (≥87 kda) was present in all the 52 plasmid-positive strains. Presence of plasmid could not be correlated with MDR in Salmonella isolates from equids.     

Choice of the optimal method for the isolation of Salmonella from meat- and bone powder designed for industrial feed mixtures

The purpose of this paper was (1) comparison of four multi-step methods used for Salmonella isolation from meat- and bone powder; (2) elaboration of a new sensitive method of Salmonella isolation from this product; (3) evaluation of a new solid selective medium (BxLH) described by the authors for Salmonella isolation in comparison to brilliant green agar (BGE) according to Edel and Kampelmacher. The study was carried out on 173 meat- and bone powder samples naturally contaminated with Salmonella oranienburg. The samples were examined for the Salmonella presence by means of four compared methods (Methods 1 to 4). The new method of isolation proposed by the authors (Method 3) proved to be the most effective among all compared for Salmonella recovery. It seems that the superiority of Method 3 in comparison to the other applied was a result of, (1) homogenization of the investigated samples in distilled water before preincubation followed by maintenance at room temperature for 2–4 h; (2) the use of a new selective BxLH agar; (3) the use of multiple plating after selective enrichment. The BxLH medium was shown to be more suitable for Salmonella isolation than BGE agar because of more efficient inhibition of other bacterial growth with simultaneously abundant growth of the Salmonella organisms. The additional advantage offered by BxLH agar was the fact that lactose-positive salmonellas grow as typical representatives of this genus. This enables their identification, in contrast to the situation when lactose containing media are used, where the colonies of such salmonellas are similar to the colonies of, for example, Escherichia coli.     

Identification of risk factors for the prevalence and persistence of Salmonella in Belgian broiler chicken flocks

According to the European Food Safety Authority, salmonellosis is still one of the main causes of infectious foodborne gastroenteritis in humans. Broilers are an important source of salmonellosis after eggs and pork. Between 1987 and 1999 the trend of human salmonellosis incidence in Belgium increased constantly. However, from 2000 until 2005 a decrease in human cases was observed, probably following the sanitary measures implemented in the poultry breeder and laying sector. In order to decrease human infections it is essential to tackle the problem at the farm level to minimize cross-contamination from farm to fork. This paper seeks to answer two questions: (i) given the Salmonella status of the farm at a certain occasion (equal to the sampling time of the flock), what are the risk factors that the farm will be Salmonella positive at a following occasion? And (ii) what are the risk factors for a farm to be persistently positive for two consecutive flocks? We used surveillance data on 6824 broiler flocks studied for Salmonella infectivity from 2005 to 2006 in Belgium. The farms were tested regularly (3 weeks before slaughter of each broiler flock) for the presence of Salmonella based on multiple faecal samples per flock on a farm yielding clustered data. Generalized estimating equations, alternating logistic regression models, and random-intercept logistic regression models were employed to analyse these correlated binary data. Our results indicated that there are many factors that influence Salmonella risk in broiler flocks, and that they interact. Accounting for interactions between risk factors leads to an improved determination of those risk factors that increase infection with Salmonella. For the conditional analysis, the risk factors found to increase the risk of Salmonella infection on a farm at a current occasion given the previous Salmonella status included: Salmonella infection of day-old chicks (of the current flock); a previously infected flock even though the farm was equipped with a hygiene place to change clothes prior to entering the broiler house; having temporary workmen when there was a separation between birds of different species; and separating birds of different species in the Walloon region relative to the Flanders region. Sanitary measures such as a cleaning and disinfecting procedure conducted by an external cleaning firm, applying the all-in all-out procedure, and hand washing decreased the risk despite their interaction with other factors. From the joint analysis, the most important factors identified for increased risk for persistent Salmonella on a farm involved the interaction between having temporary workmen when there were poultry or farmers in contact with foreign poultry or persons, and the interaction between having temporary workmen when there were poultry or farmers in contact with external poultry or persons.     

Expression of cytokines and respiratory burst activity of milk cells in response to Azadirachta indica during bovine mastitis

The immuno therapeutic potential of hydro-methanolic extract of Azadirachta indica (A. indica) was studied during bovine clinical mastitis (CM). The somatic cell count (SCC), total bacterial count (TBC), milk differential leukocyte count (DLC), hydrogen peroxide (H₂O₂), superoxide anion (O₂ ⁻) production and interleukin- 2 (IL-2) and gamma interferon (IFN-γ) cytokines expression were studied before and after intramammary infusion of A. indica extract in diseased cows. The results revealed that A. indica treatment significantly (P < 0.05) decreased the SCC, TBC, milk neutrophil percent and significantly (P < 0.05) enhanced milk lymphocyte percent, H₂O₂ and O₂ ⁻ production by milk cells. The IL-2 and IFN-γ were expressed in normal healthy cows and diseased cows after A. indica treatment, whereas both the cytokines could not be expressed in cows treated with antibiotic and in untreated diseased cows. The results of the present study indicated anti inflammatory, antibacterial and immunomodulatory potential of the herb, these activities could be due to the presence of bioactive principle in the extract. This is a preliminary trial indicated beneficial effect of the herb against bovine mastitis it can be developed as an alternative therapy where the use of antibiotics is normally restricted.     

Characterization of the immune response of domestic fowl following immunization with proteins extracted from Dermanyssus gallinae

Dermanyssus gallinae is the most significant ectoparasite of European poultry egg laying production systems due to high costs of control and associated production losses as well as adverse effects on bird welfare. In this study, soluble proteins were extracted from unfed D. gallinae (DGE) using a urea-based detergent and ultra-filtration, passed through a 0.22 μm filter and blended aseptically with adjuvant. One group of laying hens was immunized with DGE and adjuvant (Montanide ISA 50 V) whilst another group (Control) received physiological saline and adjuvant. All birds were immunized on two occasions, 21 days apart. Antibody response to immunization was determined by ELISA and western blotting using immunoglobulins (Igs) extracted from egg yolk. DGE immunization of hens resulted in a significant (P < 0.05) IgY response compared to controls, although there was no significant difference in IgM response between treatments. A number of proteins were identified by western blotting using IgY antibodies from DGE immunized birds, most prominently at 40 and 230 kDa. Analysis of proteins from approximately corresponding bands on SDS-PAGE confirmed the identity of tropomyosin, whilst other proteins showed high sequence homology with myosin and actin from other arachnid and insect species. Immunization of hens with DGE resulted in a 50.6% increase in mite mortality (P < 0.001) 17 h after feeding when tested by an in vitro mite feeding model. Data in this study demonstrate that somatic antigens from D. gallinae can be used to stimulate a protective immune response in laying hens. Further work is needed to identify other proteins of interest that could confer higher protection against D. gallinae, as well as optimization of the vaccination and in vitro testing protocol.     

Age at primary infection with Salmonella enterica serovar Typhimurium in the chicken influences persistence of infection and subsequent immunity to re-challenge

Salmonella enterica remains one of the most important food-borne pathogens of humans and is often acquired through consumption of infected poultry meat or eggs. Control of Salmonella infections in chicken is therefore an important public health issue. Infection with S. enterica serovar Typhimurium results in a persistent enteric infection without clinical disease in chickens of more than 3 days of age, and represents a source for contamination of carcass at slaughter and entry into the human food chain. Data presented indicate a profound effect of age at initial exposure on the persistence of infection and a lesser effect on the development of effective immunity to re-challenge. The percentage of birds positive for Salmonella was high until 8–9 weeks of age, regardless of the age at which the birds were infected (1, 3 or 6 weeks). The birds infected at 3 and 6 weeks of age produced a more rapid and higher antibody response (IgY and IgA) than those infected at 1 week of age, but in all cases infection persisted for a considerable period despite the presence of high antibody levels. Following a re-challenge infection with S. Typhimurium, all three previously-infected groups had fewer bacteria in the gut, spleen and liver compared with age-matched birds receiving a parallel primary infection. However, the birds primary infected at 3 and 6 weeks of age cleared infection more rapidly than those infected at a younger age. Interestingly older-primed birds had higher specific T lymphocyte proliferative responses and specific circulating levels of IgY antibody at time of re-challenge. Although birds initially infected at 1 week of age and those that were previously uninfected produced a stronger antibody response following re-challenge, they were slower to clear Salmonella from the gut than the older-primed groups which expressed a stronger T lymphocyte response. The data presented indicate that clearance of Salmonella from the gut is age-dependent and we propose that this relates to the increased competence of the enteric T cell response. The findings that Salmonella persists beyond 8–9 weeks, irrespective of age at exposure, has implications for the broiler sector and indicates the need to remain Salmonella free throughout the rearing period. Moreover, the re-challenge data demonstrates that infection at a young age is less effective in producing protective immunity than in older chickens. This feature of the development of protective immunity needs to be considered when developing vaccines for the broiler sector of the poultry industry.     

The range of influence between cattle herds is of importance for the local spread of Salmonella Dublin in Denmark

The objective of the study was to estimate the range of influence between cattle herds with positive Salmonella Dublin herd status. Herd status was a binary outcome of high/low antibody levels to Salmonella Dublin in bulk-tank milk and blood samples collected from all cattle herds in Denmark for surveillance purposes. Two methods were used. Initially, a spatial generalised linear mixed model was developed with an exponential correlation function to estimate the range of influence simultaneously with the effect of potential risk factors. An iteratively reweighted generalised least squares procedure was used as a second method for verifying the range of influence estimates. With this iterative procedure, deviance residuals were calculated based on a generalised linear model and the range of influence was estimated based on the residuals using an exponential semivariogram. The range of influence was estimated for six different regions in Denmark using both methods. The analyses were performed on data collected during 1 year after initiation of the Salmonella Dublin surveillance program providing herd classifications for the 4th year-quarter of 2003 and 2 years later for the 4th year-quarter of 2005. The prevalence of dairy herds with a positive Salmonella Dublin herd classification status in this period had decreased from 22.1 to 17.0%. In non-dairy herds, the prevalence was nearly unchanged during the same period (3.4 and 3.7% in 4th quarter of 2003 and 2005, respectively). For all cattle herds, the range of influence was 2.3–6.4 km in 2003 and 1.5–8.3 km in 2005. There seemed to be no association between the range of influence and the density of herds in the different regions.     

Assessment of swine farms in Greece in relation to the risk of exposure of pigs to Trichinella

Commission Regulation (EC) No. 2075/2005 of 5 December 2005 lays down detailed conditions for Trichinella-free farms. The objectives of the present study were: (1) to assess the current state of swine farms in Greece in relation to the risk of exposure of pigs to Trichinella as a first step towards certification of Trichinella-free farms in Greece and (2) to identify a profile and characteristics of swine farms and farmers which can be used to formulate appropriate measures to promote good production practices for reducing risk of exposure of pigs to Trichinella. A total of 70 swine farms were inspected using audit components similar to those used by the U.S. Trichinae Certification Programme. Only four farms (6%) complied with all the criteria used for assessing the good production practices for reducing risk of exposure of pigs to Trichinella. A common characteristic of these four farms was that they had a large size (>300 sows) and produced their own feed. The most common issues among the farms that did not meet audit criteria in the present study were related to proper rodent control and accessibility of other animals in the feed storage area and the facility. In addition, the predominant significant differentiating characteristic for the farms not meeting the criteria was their small (<150 sows) size, while for the farmers it was their low educational level. These characteristics, which are linked to poor infrastructure and reluctance for improvements, respectively, will be difficulties that must be dealt with in the promotion of good production practices for Trichinella-free farms in Greece.     

Cross-sectional study of the seroprevalence to Borrelia burgdorferi sensu lato and granulocytic Ehrlichia spp. and demographic, clinical and tick-exposure factors in Swedish horses

A cross-sectional study of the seroprevalence to Borrelia burgdorferi sensu lato and granulocytic Ehrlichia spp. in Swedish horses was conducted to evaluate associations with demographic, clinical and tick-exposure factors. From September 1997–1998, blood samples from 2018 horses were collected from the animals presented to veterinary clinics affiliated with the Swedish Horserace Totalizator Board (regardless of the primary cause for consultation). Standardized questionnaires with information both from owners and attending veterinarians accompanied each blood sample. The apparent seroprevalences to B. burgdorferi s. l. and granulocytic Ehrlichia spp. were 16.8 and 16.7%, respectively. The northern region had the lowest seroprevalences. Four logistic models were developed (controlling for demographic variables). In the disease model of seropositivity to B. burgdorferi s. l., age, breed, geographic region, the serologic titer to granulocytic Ehrlichia spp., season and the diagnosis coffin-joint arthritis were significant. In the tick-exposure model of B. burgdorferi s. l., pasture access the previous year and gender were significant. Age, racing activity, geographic region, season and the serologic titer to B. burgdorferi s. l. were associated with positivity to granulocytic Ehrlichia spp. In the tick-exposure model of granulocytic Ehrlichia spp., pasture access was a risk factor. An interaction between racing activity and geographic region showed that the risk of positive serologic reactions to Ehrlichia spp. was increased in the horse population in the south and middle of Sweden, but only among horses not used for racing. Except for the positive association between coffin-joint arthritis and serologic reactions to B. burgdorferi s. l., there were no significant associations in the multivariable models between non-specific or specific clinical sign or disease with seropositivity to either of these agents.     

A recombinant chimera composed of repeat region RR1 of Mycoplasma hyopneumoniae adhesin with Pseudomonas exotoxin: in vivo evaluation of specific IgG response in mice and pigs

Using the binding and translocation domain of Pseudomonas exotoxin A [domain III deleted PE termed PE(ΔIII)] as a vehicle, this study characterized and evaluated a novel application of PE toxin in Mycoplasma hyopneumoniae adhesin used as an immunogen. PCR and sequence analysis revealed that 16 copies of AAKPV(E) in tandem repeat region 1 (RR1) of M. hyopneumoniae 97 kDa adhesion were successfully fused to the downstream of PE(ΔIII) to create a subunit vaccine, i.e. PE(ΔIII)-RR1. This chimeric protein, over-expressed in inclusion bodies of E. coli BL21(DE3)pLysS, was characterized by a monoclonal antibody (MAb) F2G5 prepared against RR1 of the 97 kDa adhesin and was readily purified. The data indicated that the epitope recognized by MAb F2G5 was located in the structure of PE(ΔIII)-RR1. Using ELISA and Western blot analyses, the specific IgG immune response against RR1 and whole adhesin in mice immunized with PE(ΔIII)-RR1 was found more marked than that in mice immunized with the M. hyopneumoniae whole cells. Similarly, PE(ΔIII)-RR1 also stimulated a remarkable IgG response against RR1 in pigs compared to that in pigs immunized with the conventional M. hyopneumoniae vaccine. The PE(ΔIII)-RR1 would be potentially useful for the future development of a M. hyopneumoniae adhesin vaccine.