Colistin Selection of the Mcr-1 Gene in Broiler Chicken Intestinal Microbiota.

Colistin has a long story of safe use in animals for the treatment and prevention of certain bacterial diseases. Nevertheless, the first description of the mcr-1 gene showed that colistin resistance can spread by horizontal gene transfer and changed the landscape. This study aimed to assess the effect of colistin administration on the dispersion of resistance in the microbiota of day-old broiler chicks and how the presence of mcr-1 genes influences the spread of colistin resistance determinants. In this study, 100 one-day-old chicks were divided into four groups of 25 animals (G1, G2, G3, and G4). Animals from G3/G4 were challenged with mcr-1-carrying Salmonella (day 7), while colistin (600 mg/L) was administered daily to G2/G4 animals through drinking water (from day 8 to day 15). Two quantitative PCR assays were performed to compare the amount of Salmonella and mcr-1 that were present in the caecal samples. We observed that levels of mcr-1 were higher in G3/G4 animals, especially G4, due to the spread of mcr-1-carrying Salmonella. On day 21, Salmonella levels decreased in G4, reaching similar values as those for G3, but mcr-1 levels remained significantly higher, suggesting that colistin may accelerate the spreading process when mcr-1-carrying bacteria reach the gut.

Evaluation of PCR assays for Campylobacter fetus detection and discrimination between C. fetus subspecies in bovine preputial wash samples.

Campylobacter fetus is a zoonotic pathogen found in cattle, in which it is one of the main causes of infectious infertility. Most diagnostic laboratories use PCR as quick easy tool for C. fetus identification. However, there is no standardized PCR assay for C. fetus detection and subspecies differentiation, hindering the comparison of results. In this study, we evaluated selected PCR assays targeting the 16S rRNA, gyrB, cpn60, cstA, cdtB and nahE genes for C. fetus identification and ISCfe1, sapB2, parA and virB11 for subspecies differentiation. Analytical sensitivity and specificity were assessed for each PCR assay, and the assays were then tested on 289 bull preputial samples that had also been analysed by 16S rRNA barcode metagenomics. In total, 41 C. fetus-positive samples were included. The P12 PCR assay targeting the gyrB gene performed best, detecting the pathogen in 95.1% of positive samples. For the discrimination of C. fetus subspecies, we were able to identify a proportion (85.4%) of the C. fetus-positive samples correctly as C. fetus venerealis with at least one subspecies-specific PCR, but C. fetus fetus was not detected in any of the samples tested. Remarkably, C. fetus subspecies amplification was observed following PCR on some samples (33.1%) considered C. fetus-negative, highlighting the need for rigorous criteria for discriminating between C. fetus subspecies, to improve understanding of the role of the two C. fetus subspecies in the epidemiology and pathogenesis of bovine infectious infertility.

Liver Transudate, a Potential Alternative to Detect Anti-Hepatitis E Virus Antibodies in Pigs and Wild Boars (Sus scrofa).

In recent years, cases of hepatitis E virus (HEV) infection have increased in Europe in association with the consumption of contaminated food, mainly from pork products but also from wild boars. The animal's serum is usually tested for the presence of anti-HEV antibodies and viral RNA but, in many cases such as during hunting, an adequate serum sample cannot be obtained. In the present study, liver transudate was evaluated as an alternative matrix to serum for HEV detection. A total of 125 sera and liver transudates were tested by enzyme-linked immunosorbent assay at different dilutions (1:2, 1:10, 1:20), while 58 samples of serum and liver transudate were checked for the presence of HEV RNA by RT-qPCR. Anti- HEV antibodies were detected by ELISA in 68.0% of the serum samples, and in 61.6% of the undiluted transudate, and in 70.4%, 56.8%, and 44.8% of 1:2, 1:10, or 1:20 diluted transudate, respectively. The best results were obtained for the liver transudate at 1:10 dilution, based on the Kappa statistic (0.630) and intraclass correlation coefficient (0.841). HEV RNA was detected by RT-qPCR in 22.4% of the serum samples and 6.9% of the transudate samples, all samples used for RT-qPCR were positive by ELISA. Our results indicate that liver transudate may be an alternative matrix to serum for the detection of anti-HEV antibodies.

High Load of Deformed Wing Virus and Varroa destructor Infestation Are Related to Weakness of Honey Bee Colonies in Southern Spain.

Many factors, including pathogens, contribute to the continuing losses of colonies of the honey bee Apis mellifera, which has led to steady population decline. In particular, colony losses have been linked to deformed wing virus (DWV) and the Varroa destructor mite. To clarify the potential role of these two pathogens in honey bee colony weakening and loss, we sampled colonies in southern Spain during a 21-month period and analyzed the samples for loads of four viruses and varroa. Loads of DWV and black queen cell virus as well as varroa infestation negatively correlated with colony vigor as measured using the subjective colony strength method. Logistic regression identified varroa and DWV as the main factors involved in colony weakening. Our results confirm that varroa and DWV play a key role in triggering colony weakening in southern Spain and provide evidence that experienced beekeepers' and technicians' assessments of colony vigor can accurately estimate colony strength.

Occurrence of Hepatitis E Virus in Pigs and Pork Cuts and Organs at the Time of Slaughter, Spain, 2017.

Zoonotic hepatitis E, mainly caused by hepatitis E virus (HEV) genotype (gt) 3, is a foodborne disease that has emerged in Europe in recent decades. The main animal reservoir for genotype 3 is domestic pigs. Pig liver and liver derivates are considered the major risk products, and studies focused on the presence of HEV in pig muscles are scarce. The objective of the present study was to evaluate the presence of HEV in different organs and tissues of 45 apparently healthy pigs from nine Spanish slaughterhouses (50% national production) that could enter into the food supply chain. Anti-HEV antibodies were evaluated in serum by an ELISA test. Ten samples from each animal were analyzed for the presence of HEV RNA by reverse transcription real-time PCR (RT-qPCR). The overall seroprevalence obtained was 73.3% (33/45). From the 450 samples analyzed, a total of 26 RT-qPCR positive samples were identified in the liver (7/45), feces (6/45), kidney (5/45), heart (4/45), serum (3/45), and diaphragm (1/45). This is the first report on detection of HEV RNA in kidney and heart samples of naturally infected pigs. HEV RNA detection was negative for rib, bacon, lean ham, and loin samples. These findings indicate that pig meat could be considered as a low risk material for foodborne HEV infection.

Application of a specific quantitative real-time PCR (qPCR) to identify Leishmania infantum DNA in spleen, skin and hair samples of wild Leporidae.

The aim of this study was to compare a quantitative real-time PCR (qPCR) validated for the detection of Leishmania infantum in dogs with a nested PCR but in wild Leporidae. Additionally, L. infantum results from indirect immunofluorescent antibody test (IFAT) and in vitro culture were also compared with qPCR. Different samples (spleen, skin and hair) recovered from 224 European rabbits and 70 Iberian hares from two green areas of Madrid Council were analysed for the detection of L. infantum. The presence of Leishmania kDNA was detected by qPCR in 58 out of 221 (26.24%), 162 out of 203 (79.8%) and 22 out of 33 (66.67%) analysed rabbits on spleen, skin and hair samples, respectively; and in 7 out of 69 (10.14%), 39 out of 70 (55.71%) and 17 out of 32 (53.13%) test hares on spleen, skin and hair samples, respectively. The qPCR in all test samples resulted to be more sensitive than nested PCR, with a limit of detection of 1.43 fg/reaction (0.039 parasites) for L. infantum genomic DNA. Additionally, the percentage of positive animals detected by qPCR in at least two out of three samples (n=221 rabbits and 70 hares) tested was higher than those detected by IFAT (n=190 rabbits and 61 hares) and isolation (n=75 rabbits and 20 hares). The highest level of agreement was obtained by nested PCR on spleen/skin (89%/83%) samples and qPCR on spleen samples (81%), followed by IFAT (48%) and qPCR on skin (32%) samples. Our results demonstrate this qPCR is a suitable method for detecting L. infantum DNA in different samples suggesting hair could be considered an adequate sample for direct, reliable and non-invasive diagnosis of L. infantum in wild animals.

DETECTION OF COXIELLA BURNETII INFECTION IN A SAHARAWI DORCAS GAZELLE (GAZELLA DORCAS NEGLECTA).

Coxiella burnetii, the causative agent of Q fever, can infect a wide range of host species, but limited information exists on the occurrence and implications of infection in wild species. This study describes a natural infection in a population of dorcas gazelles ( Gazella dorcas ) from a zoo. A 9-yr-old male Saharawi dorcas gazelle ( Gazella dorcas neglecta) tested positive on enzyme-linked immunosorbent assay (ELISA) and fecal polymerase chain reaction (PCR). Despite treatment with oxytetracycline, the animal did not clear the infection after 6 mo, as confirmed by a PCR test on a semen sample. This is the first report of a Saharawi dorcas gazelle infection with C. burnetii and the first time that C. burnetii was detected in semen from a zoo animal, suggesting the possibility of venereal transmission in captive wild species. This may have major implications for management of zoo populations, particularly in endangered species.

Effect of Preventive Chlamydia abortus Vaccination in Offspring Development in Sheep Challenged Experimentally.

Ovine enzootic abortion, caused by Chlamydia abortus, leads to important economic losses worldwide. In addition to reproductive failures, infection may impact lamb growth during the first weeks after birth, yet this effect has not been well characterized. Vaccination can help to control the disease but variable efficacy values have been described, possibly related with factors associated with the host, the vaccine, the parameter used for efficacy determination, and the challenge conditions. In this context, we evaluated the efficacy of an inactivated standard commercial vaccine and a 1/2 diluted dose in pregnant sheep challenged with C. abortus by examining multiple indicators of vaccine effect (including incidence of reproductive failures, bacterial excretion, and evolution of weight gain of viable lambs during the first month of life). Three groups of ewes [control non-vaccinated, C (n = 18); vaccinated with standard dose, SV (n = 16); and vaccinated with 1/2 dose, DV (n = 17)], were challenged approximately 90 days post-mating and tested using direct PCR (tissue samples and vaginal swabs) and ELISA (serum) until 31 days post-reproductive outcome. There were not significant differences in the proportions of reproductive failures or bacterial shedding after birth/abortion regardless the vaccination protocol. However, a beneficial effect of vaccination on offspring growth was detected in both vaccinated groups compared with the controls, with a mean increase in weight measured at 30 days of life of 1.5 and 2.5 kg (p = 0.056) and an increase in the geometric mean of the daily gain of 8.4 and 9.7% in lambs born from DV and SV ewes compared with controls, respectively. Our results demonstrate the effect of an inactivated vaccine in the development of the offspring of C. abortus-infected ewes at a standard and a diluted dose, an interesting finding given the difficulty in achieving sufficient antigen concentration in the production of enzootic abortion of ewes-commercial vaccines.

Assessment of genetic diversity of zoonotic Brucella spp. recovered from livestock in Egypt using multiple locus VNTR analysis.

Brucellosis is endemic in most parts of Egypt, where it is caused mainly by Brucella melitensis biovar 3, and affects cattle and small ruminants in spite of ongoing efforts devoted to its control. Knowledge of the predominant Brucella species/strains circulating in a region is a prerequisite of a brucellosis control strategy. For this reason a study aiming at the evaluation of the phenotypic and genetic heterogeneity of a panel of 17 Brucella spp. isolates recovered from domestic ruminants (cattle, buffalo, sheep, and goat) from four governorates during a period of five years (2002-2007) was carried out using microbiological tests and molecular biology techniques (PCR, MLVA-15, and sequencing). Thirteen strains were identified as B. melitensis biovar 3 while all phenotypic and genetic techniques classified the remaining isolates as B. abortus (n = 2) and B. suis biovar 1 (n = 2). MLVA-15 yielded a high discriminatory power (h = 0.801), indicating a high genetic diversity among the B. melitensis strains circulating among domestic ruminants in Egypt. This is the first report of the isolation of B. suis from cattle in Egypt which, coupled with the finding of B. abortus, suggests a potential role of livestock as reservoirs of several zoonotic Brucella species in the region.

Detection of anti-Leishmania infantum antibodies in sylvatic lagomorphs from an epidemic area of Madrid using the indirect immunofluorescence antibody test.

An outbreak of human leishmaniasis was confirmed in the southwest of the province of Madrid, Spain, between July 2009 and December 2012. Incidence of Leishmania infection in dogs was unchanged in this period, prompting a search for alternative sylvatic infection reservoirs. We evaluated exposure to Leishmania in serum samples from animals in the area with an indirect immunofluorescence test (IFAT). Using promastigotes from six culture passages and a 1/25 threshold titer, we found anti-Leishmania infantum seroreactivity in 9.3% of cats (4 of 43), 45.7% of rabbits (16/35) and 74.1% of hares (63/85). Use of promastigotes from >10 in vitro passages resulted in a notably IFAT lower titer, suggesting antigenic changes during extended culture. Postmortem inspection of seropositive animals showed no clinical signs of infection. The results clearly suggest that asymptomatic hares were the main reservoir in the outbreak, and corroborate IFAT as a sensitive serological surveillance method to detect such cryptic Leishmania infections.

Management of an outbreak of brucellosis due to B. melitensis in dairy cattle in Spain.

Brucella melitensis is a major human and animal pathogen, with a wide host range that includes all domestic ruminant species, although small ruminants are its preferred hosts. Outbreaks in cattle due to B. melitensis have become a worldwide emerging problem particularly difficult to control due to the lack of knowledge on the epidemiology in this host species and of an effective vaccine. However, combination of molecular tools and strict biosecurity measures can help to solve these difficulties and eventually eradicate the disease from infected herds. In the present report, management of an outbreak in Spain involving four farms, more than 2000 cattle and several human cases is described. Application of Multiple Locus VNTR Analysis (MLVA) allowed identifying the most likely source of infection. Stamping out and test-and-slaughter strategies were applied, proving their usefulness to control the outbreak depending on infection level, and without the need of other alternative measures.

Unexpected inefficiency of the European pharmacopoeia sterility test for detecting contamination in clostridial vaccines.

Three outbreaks of clostridial disease in ruminants were reported in Spain. Out of 202,525 animals in affected herds, 41,767 were infected and 22,189 died. Epidemiological investigation linked these outbreaks with vaccination with three different commercial anti-clostridial vaccines contaminated with Clostridium sordellii. Vaccines were produced by the same manufacturer. Microbiological and molecular genetic analyses confirmed this association, and isolates of C. sordellii with identical biochemical and pulsed-field gel electrophoresis (PFGE) macrorestriction patterns were isolated from both diseased animals and the epidemiologically related vaccines. Contamination of the commercial vaccines was not detected by the sterility test proposed by the European (EU) Pharmacopoeia. However, growth was obtained when the commercial vaccines were inoculated into specific culture media for Clostridium and incubated for up to 60 days.

Salmonella diversity associated with wild reptiles and amphibians in Spain.

During the spring and summer of 2001, faeces from 166 wild reptiles (94 individuals) and amphibians (72 individuals) from 21 different species found in central Spain were examined for the presence of Salmonella. Thirty-nine reptiles (41.5%) yielded 48 Salmonella isolates, whereas all the amphibians examined were negative. Subspecies Salmonella enterica enterica (I) accounted for up to 50% of isolates. Fourteen isolates (29.2%) belonged to subspecies diarizonae (IIIb), six isolates (12.5%) to subspecies salamae (II), and four isolates (8.3%) to subspecies arizonae (IIIa). Twenty-seven different serotypes were identified. Serotypes Anatum (12.5%), Herzliya (8.3%), Abony, 18:l,v:z, 9,12:z29:1,5 and 38:z10:z53 (6.2%/each) were the most frequently isolated. A high percentage (39.6%) of isolates belonged to serotypes previously associated with environmental sources. Also, 37.5% of isolates belonged to serotypes which had been related to human cases of salmonellosis. From these data, it is concluded that wild reptiles, but apparently not amphibians, may represent an important reservoir of Salmonella in nature and have potential implications for public health.

Analysis of genetic diversity of Streptococcus suis clinical isolates from pigs in Spain by pulsed-field gel electrophoresis.

Pulsed-field gel electrophoresis (PFGE) was used to investigate the diversity of Streptococcus suis isolates of various serotypes recovered from swine clinical samples in Spain. Capsular types 9 (64.9%) and 2 (14.8%) were the most frequently isolated serotypes followed by serotype 7 (5.9%) and serotype 8 (4.3%). The PFGE results of this study with 60 different pulsotypes indicate a great genetic diversity among the S. suis isolates, which is consistent with the broad distribution of S. suis in the swine population. Forty-five percent of the pulsotypes corresponded to single isolates, no pulsotype was common to all farms, and at least 3 different pulsotypes were isolated in 56% of herds in which more than 3 clinical isolates were analyzed. These results reveal a great diversity both between and within herds throughout the strains of S. suis studied, demonstrating that different strains of S. suis are associated with infection in pigs. Some pulsotypes were more frequently isolated and exhibited a wider distribution over herds than others, and were the unique or predominant strains in several herds, suggesting the existence of a prevalent or a few prevalent clones responsible for a large proportion of clinical cases. Overall, the great genetic heterogeneity of the clinical strains of S. suis, the isolation of different strains within the same herd, and the predominance of particular strains in some herds are evidence that infection by S. suis is a dynamic process and reinforce the idea that the epidemiology of S. suis infection is very complex.

Influence of Temperature of Incubation on Staphylococcus aureus Growth and Enterotoxin Production in Homemade Mayonnaise.

Homemade mayonnaise, in which pH had been adjusted to a range between 5.0 and 5.8 by the addition of vinegar, was inoculated with eight Staphylococcus aureus strains known to be enterotoxigenic. They were incubated for a maximum of 7 days at 22, 28, 37, and 44°C. Periodically, staphylococcal growth and pH were determined. Mayonnaise samples were examined on d 7 for the presence of enterotoxins A, B, C, and D. Staphylococcal growth was higher at 22°C (average log10 7.21 cfu/g), than at the other temperatures tested (log10 7.15, 6.77, and 5.93 cfu/g, respectively for 28, 37, and 44°C), suggesting a better growth in mayonnaise at low room temperature. Enterotoxin synthesis took place mainly at 28°C, as 33.3% of the total enterotoxins produced were detected at this temperature. However, some strains synthesized high amounts of enterotoxin even at 22°C.

Growth of Staphylococcus aureus and Enterotoxin Production in Homemade Mayonnaise Prepared with Different pH Values.

The ability of Staphylococcus aureus to grow and produce enterotoxins in homemade mayonnaise prepared at different pH values was studied. Ten enterotoxigenic strains, producing one or two enterotoxin types (A, B, C, or D) were inoculated into mayonnaise samples with pH adjusted to values ranging between 4.0 and 5.8, and incubated at 37°C for 7 d. Counts were made on days 1, 3, 5, and 7 and extracts were prepared on day 7 to detect enterotoxin by ELISA. An important difference was seen between those samples prepared with pH below or equal to 4.9 and those over or equal to 5.0; in the range of pH between 4.0 and 4.9 the average of staphylococcal population was 100 CFU/g; at pH 5.0 it was 1.6 × 105, and at pH 5.15 and above it was at least 8 × 106 CFU/g. Enterotoxin was detected only at initial pH over 5.15 and when final pH was not less than 4.7. The highest amount of enterotoxin corresponded to 157.8 ng of SEB/100 g of mayonnaise.