2. Universidad Cardenal Herrera-CEU

Campylobacter is recognised as one of the most important foodborne bacteria, with a worldwide health and socioeconomic impact. This bacterium is one of the most important zoonotic players in poultry, where efficient and fast detection methods are required. Current official culture methods for Campylobacter enumeration in poultry usually include >44 h of culture and >72 h for identification, thus requiring at least five working shifts (ISO/TS 10272-2:2017). Here, we have assembled a portable sequencing kit composed of the Bento Lab and the MinION and developed a workflow for on-site farm use that is able to detect and report the presence of Campylobacter from caecal samples in less than five hours from sampling time, as well as the relationship of Campylobacter with other caecal microbes. Beyond that, our workflow may offer a cost-effective and practical method of microbiologically monitoring poultry at the farm. These results would demonstrate the possibility of carrying out rapid on-site screening to monitor the health status of the poultry farm/flock during the production chain.

Background: Campylobacter is the main pathogen involved in zoonotic gastrointestinal diseases. Last year, the European regulation 2017/1495 on Campylobacter in broiler carcasses came into force. In this context, the aim of this study was to assess the potential risk factors associated with exceeding the 1,000 CFU/g criterion set by the European Commission in several slaughterhouses in Spain. Methods: Information relating to 12 factors were collected using questionnaires. Samples were collected from 12 Spanish abattoirs during June, July and August 2017 (n=1,725) and were analysed following ISO/TS 10272-2:2006 method. Results: The proportion of Campylobacter-positive samples was 23.7% (n=409). Analysis of the flock age (41-50 days) revealed a significantly increased OR in Campylobacter enumeration (OR=7.41). Moreover, scalding temperature (51.9.54 ºC) was positively associated with an increase in OR (OR=2.75). Time in transit to slaughter (1-1.5h), showed a significant OR decrease (OR=0.25). However, when processed for more than 2 hours, presented an increase in OR (OR=4.44). Regarding carcass weight, the range from 3.21-3.58 presented a decrease in OR (OR=0.01). Conclusion: The outcomes of this study suggest that although most chickens are contaminated by the bacterium, the prevalence that exceeds the limit of 1,000 CFU/ is not so high as we thought.

Different studies have reported the prevalence and antibiotic resistance of Salmonella in dromedaries’ camels and its role in camelid-associated salmonellosis in humans, but little is known about the epidemiology of Campylobacter in dromedaries. Here we investigate the prevalence, genetic diversity and antibiotic resistance of Campylobacter and Salmonella in dromedary camels (Camelus dromedarius). A total of 54 individuals were sampled from two unique dromedary farms located in Tenerife (Canary Islands, Spain). Whilst all the samples were Campylobacter-negative, Salmonella prevalence was 5.5% (3/54) and the only serovar isolated was S. Frintrop. The pulsed field gel electrophoresis analysis revealed a low genetic diversity, with all isolates showing a nearly identical pulsotype (similarity > 95%). Our results indicate that dromedaries’ camels could not be a risk factor for Campylobacter human infection, but seems to be a reservoir for Salmonella transmission. Since camel ride has become one of the main touristic attractions in several countries and its popularity has considerably risen in the last years, a mandatory control, especially for zoonotic pathogens, such as Campylobacter and Salmonella should be implemented.

Bovine respiratory disease (BRD) is an important viral and/or bacterial disease that mainly affects feedlot calves. The involvement of Mycoplasma bovis in BRD can lead to chronic pneumonia poorly responsive to antimicrobial treatment. Caseonecrotic bronchopneumonia is a pulmonary lesion typically associated with M. bovis. In Spain, M. bovis is widely distributed in the feedlots and circulating isolates are resistant to most antimicrobials in vitro. However, the role of this species in clinical respiratory disease of feedlot calves remains unknown. Furthermore, available data are relative to a fixed panel of antimicrobials commonly used to treat BRD, but not to the specific set of antimicrobials that have been used for treating each animal. This study examined 23 feedlot calves raised in southeast Spain (2016–2019) with clinical signs of respiratory disease unresponsive to treatment. The presence of M. bovis was investigated through bacteriology (culture and subsequent PCR), histopathology and immunohistochemistry. The pathogen was found in 86.9% (20/23) of the calves, mainly in the lungs (78.26%; 18/23). Immunohistochemistry revealed M. bovis antigens in 73.9% (17/23) of the calves in which caseonecrotic bronchopneumonia was the most frequent lesion (16/17). Minimum inhibitory concentration assays confirmed the resistance of a selection of 12 isolates to most of the antimicrobials specifically used for treating the animals in vivo. These results stress the importance of M. bovis in the BRD affecting feedlot calves in Spain.

Staphylococcal mastitis is a major health problem in humans and livestock that leads to economic loss running in millions. This process is currently one of the main reasons for culling adult rabbit does. Surprisingly, the two most prevalent S. aureus lineages isolated from non-differentiable natural clinical mastitis in rabbits (ST121 and ST96) generate different immune responses. This study aimed to genetically compare both types of strains to search for possible dissimilarities to explain differences in immune response, and to check whether they showed similar virulence in in vitro tests as in experimental intramammary in vivo infection. The main differences were observed in the enterotoxin gene cluster (egc) and the immune-evasion-cluster (IEC) genes. While isolate ST121 harboured all six egc cluster members (seg, sei, selm, seln, selo, selu), isolate ST96 lacked the egc cluster. Strain ST96 carried a phage integrase Sa3 (Sa3int), compatible with a phage integrated into the hlb gene (β-haemolysin-converting bacteriophages) with IEC type F, while isolate ST121 lacked IEC genes and the hlb gene was intact. Moreover, the in vitro tests confirmed a different virulence capacity between strains as ST121 showed greater cytotoxicity for erythrocytes, polymorphonuclear leukocytes and macrophages than strain ST96. Differences were also found 7 days after experimental intramammary infection with 100 colony-forming units. The animals inoculated with strain ST121 developed more severe gross and histological mastitis, higher counts of macrophages in tissue and of all the cell populations in peripheral blood, and a significantly larger total number of bacteria than those infected by strain ST96.

Mycoplasma bovis is an important bovine pathogen causing pneumonia, mastitis, and arthritis and is responsible for major economic losses worldwide. In the absence of an effcient vaccine, control of M. bovis infections mainly relies on antimicrobial treatments, but resistance is reported in an increasing number of countries. To address the situation in Spain, M. bovis was searched in 436 samples collected from beef and dairy cattle (2016–2019) and 28% were positive. Single-locus typing using polC sequences further revealed that two subtypes ST2 and ST3, circulate in Spain both in beef and dairy cattle, regardless of the regions or the clinical signs. Monitoring of ST2 and ST3 isolates minimum inhibitory concentration (MIC) to a panel of antimicrobials revealed one major difference when using fluoroquinolones (FQL): ST2 is more susceptible than ST3. Accordingly, whole-genome sequencing (WGS) further identified mutations in the gyrA and parC regions, encoding quinolone resistance-determining regions (QRDR) only in ST3 isolates. This situation shows the capacity of ST3 to accumulate mutations in QRDR and might reflect the selective pressure imposed by the extensive use of these antimicrobials. MIC values and detection of mutations by WGS also showed that most Spanish isolates are resistant to macrolides, lincosamides, and tetracyclines. Valnemulin was the only one effective, at least in vitro, against both STs.

The vaginal microbiota plays an important role in the health of dairy cattle, and it could be manipulated for the prevention and treatment of reproduction-related infections. The present study profiles and compares the vaginal microbiota of healthy dairy heifers during the estrous cycle focusing the results in follicular (estrus) and luteal (diestrus) phases using 16S rRNA sequencing of the V3–V4 hypervariable region. Twenty 13–16-months-old virgin dairy heifers from a single farm were included in this study. Vaginal swabs and blood samples were obtained during estrus (6–8 h before artificial insemination) and diestrus (14 days after insemination). Estrus was evaluated by an activity monitoring system and confirmed with plasma progesterone immunoassay. Results showed that the taxonomic composition of the vaginal microbiota was different during the follicular and luteal phases. At the phylum level, the most abundant bacterial phyla were Tenericutes, Firmicutes, and Bacteroidetes which comprised more than 75%of the vaginal microbiota composition. The next more abundant phyla, in order of decreasing abundance, were Proteobacteria, Actinobacteria, Fusobacteria, Epsilonbacteraeota, and Patescibacteria. Together with Tenericutes, Firmicutes, and Bacteroidetes represented more than 96% of the bacterial composition. Ureaplasma, Histophilus, f_Corynebacteriaceae, Porphyromonas, Mycoplasma, Ruminococcaceae UCG-005, were the most abundant genera or families. The results also showed that the vaginal microbiota of dairy heifers was non-lactobacillus dominant. The genus Lactobacillus was always found at a low relative abundance during the estrous cycle being more abundant in the follicular than in the luteal phase. Despite more research is needed to explore the potential use of native vaginal microbiota members as probiotics in dairy heifers, this study represents an important step forward. Understanding how the microbiota behaves in healthy heifers will help to identify vaginal dysbiosis related to disease.

Background: Mycoplasma bovis is an important pathogen for the cattle industry worldwide causing significant economic losses. Several transmission routes, including those related to reproduction, have been described. Indeed, the pathogen can colonize the female reproductive tract after artificial insemination (AI) with contaminated semen. Lactobacillus spp.-based probiotics have been used for vaginal dysbiosis treatment in women and cows although their role in controlling cervico-vaginal infections due to M. bovis is unknown. The objective of the present work is to assess the viability of M. bovis (PG45, NCTC 10131) in experimentally contaminated cervical mucus after the addition of Lactobacillus spp. at different concentrations as a competing agent and pH acidifier. Results: The addition of probiotic at a concentration higher than 108 colony forming units (CFU/mL had a detrimental effect (P < 0.05) on mycoplasma viability in cervical mucus. This coincided with a significant LAB growth and an important decrease in pH from 8.4 to 5.6 (P < 0.05). However, after the addition of less concentrated probiotic, M. bovis survival was not affected and there was no significant LAB growth despite the drop of pH from 8.4 to 6.73 (P < 0.05). Conclusion: The addition of concentrations higher than 108 CFU/mL of Lactobacillus spp. negatively affects M. bovis viability in bovine cervical mucus under in vitro conditions. Although the effect observed on the pathogen viability seems to be related to the pH decrease after LAB proliferation in cervical mucus, further studies are necessary to elucidate if other factors are implicated. Nevertheless, the administration of Lactobacillus spp.-based probiotics might be used in the future to control M. bovis proliferation in the cervico-vaginal tract of cows.

Sheep estrous synchronization is mainly based on progestagen-impregnated sponges which could cause vaginitis. Several species of Lactobacillus used as probiotics are commonly used in the treatment or prevention of urogenital infections in humans. However, no studies have been performed to analyze the potential use of probiotics to prevent urogenital infections in sheep. A randomized controlled clinical trial was conducted with 21 one-year-old ewes to develop a model of probiotic infusion in vaginal sponges in order to study their influence in ewe’s vaginal microbiota, general health status, fertility and prolificity. Synchronization of estrus was based on intravaginal sponges for 14 days. Bacterial communities (Enterobacteriaceae and lactic acid bacteria) were highly fluctuating over time and between animals. The safety of probiotic infusion (mix of Lactobacillus spp. 60% L. crispatus, 20% L. brevis and 20% L. gasseri) in the vagina of healthy ewes was firstly confirmed. Neutrophils were observed in 80% (8/10) of the control ewes compared to 36% (4/11) of the ewes in the probiotic group 2 days after sponge removal (p = 0.056). Fertility in the control and probiotic groups was 60% (6/10) and 91% (10/11), respectively p = 0.097. These results suggest that Lactobacillus spp. infusion in the ewe’s vagina does not affect general health status or fertility.