Phenotypic and genotypic antimicrobial resistance of Listeria spp. in Spain.

Listeriosis is a zoonotic disease caused by Listeria monocytogenes and Listeria ivanovii. The genus Listeria currently includes 27 recognized species and is found throughout the environment. The number of systematic studies on antimicrobial resistance in L. monocytogenes isolates from domestic farms using antimicrobial substances is limited. Importantly, dairy ruminant farms are reservoir of hypervirulent lineage I L. monocytogenes isolates, previously associated with human clinical cases. Considering that the classes of antibiotics used in food-producing domestic animals are frequently the same or closely related to those used in human medicine, studies about the impact of antibiotic use on the acquisition of antibiotic resistance in Listeria spp. in domestic animal farms are, therefore, of high importance. Here, susceptibility to 25 antibiotics was determined. Eighty-one animal-related, 35 food and 21 human pathogenic Listeria spp. isolates and 114 animal-related non-pathogenic Listeria spp. isolates were tested. Whole genome sequencing data was used for molecular characterization. Regarding L. monocytogenes, 2 strains from the clinical-associated linage I showed resistance to erythromycin, both related to dairy ruminants. Acquired resistance to one antibiotic was exhibited in 1.5% of L. monocytogenes isolates compared with 14% of non-pathogenic Listeria spp. isolates. Resistance to tetracycline (7.9%), doxycycline (7.9%), penicillin (4.4%), and ampicillin (4.4%) were the most frequently observed in non-pathogenic Listeria spp. While resistance to two or more antibiotics (5.6%) was most common in Listeria spp., isolates, resistance to one antibiotic was also observed (1.6%). The present results show that non-pathogenic Listeria spp. harbour antimicrobial resistance genes.

Controlling Coxiella burnetii in naturally infected sheep, goats and cows, and public health implications: a scoping review.

Q fever is a worldwide zoonotic disease which domestic ruminants are the main source of infection for humans. This scoping review summarizes the control measures currently available to reduce Coxiella burnetii (Cb) infection in naturally infected sheep, goat and cattle herds. A total of 28 articles were included in the review. A lack of methodological standardization was noted in the articles analyzed. The results indicated that long-term vaccination in cows reduces bacterial excretion in milk and environmental contamination. In small ruminants, the results of vaccination in terms of efficacy are variable. In goats, there is a reduction in bacterial excretion, unlike in sheep, where a long-term vaccination program is necessary to reduce bacterial excretion. Moreover, the high persistence of viable Cb in the environment means that control measures for sheep are needed for several years. The use of antibiotics as a control measure in cows and sheep was not found to reduce excretion. However, the combination of vaccination with antibiotic therapy appears to have positive effects in small ruminants in terms of controlling outbreaks of Q fever. Hygiene and biosecurity measures are the basic means for controlling Cb infection on ruminant farms and ensuring public health.

In Vitro Interaction between Mycoplasma agalactiae and Small Ruminants' Endogenous Bacterial Strains of Enterococcus spp. and Coagulase-Negative Staphylococcus.

Recently, an antimicrobial effect on Mycoplasma agalactiae (Ma), the main etiological agent of contagious agalactia (CA), was reported in vitro with strains of Enterococcus spp. from ovine and caprine milk. The aim of this work was to evaluate the interaction of Ma with the same Enterococcus spp. isolated from other anatomical locations (vagina) and other bacterial populations present in milk, such as coagulase-negative staphylococci (CNS). The vaginal Enterococcus strains and the raw milk CNS were isolated from sheep and goats. Experimental in vitro conditions were prepared to assess the growth of Ma with and without the presence of these strains. The selected vaginal strains were identified as Enterococcus (E.) hirae and E. mundtii, and the strains of CNS were identified as Staphylococcus petrasii. Different interactions of Ma with ovine and caprine wild vaginal strains of Enterococcus and dairy strains of CNS are described for the first time: Ma can grow exponentially during 15 h with the selected strains, although with certain strains, its optimal growth can be negatively affected (p < 0.05). The colonization and/or excretion of Ma could, therefore, be influenced by certain endogenous bacterial strains. Our results increase the knowledge about possible bacterial ecology dynamics surrounding CA.

Antibacterial potential of commercial and wild lactic acid bacteria strains isolated from ovine and caprine raw milk against Mycoplasma agalactiae.

Introduction: The complexity of fighting contagious agalactia (CA) has raised the necessity of alternative antimicrobial therapies, such as probiotics. Lactic acid bacteria (LAB) are present in the mammary gland of small ruminants and their antimicrobial effect have been previously described against species like Mycoplasma bovis but never against Mycoplasma agalactiae (Ma). This in vitro study aims to evaluate the antimicrobial activity against Ma of ovine and caprine LAB strains and a human commercial probiotic (L2) of Lactobacillus spp. Methods: A total of 63 possible LAB strains were isolated from nine ovine and caprine farms in Spain, three isolates (33B, 248D, and 120B) from the 63 strains were selected, based on their capacity to grow in a specific medium in vitro, for an in vitro experiment to assess their antimicrobial activity against Ma in Ultra High Temperature (UHT) processed goat milk (GM). A women commercial vaginal probiotic was also included in the study. The inoculum of L2 was prepared at a concentration of 3.24 × 108 CFU/mL and the average concentration of the inoculum of the wild LAB varied from 7.9 × 107 to 8.4 × 108 CFU/mL. Results: The commercial probiotic L2 significantly reduced the concentration of Ma to 0.000 log CFU/mL (p < 0.001), strain 33B reduced it from 7.185 to 1.279 log CFU/mL (p < 0.001), and 120B from 6.825 to 6.466 log CFU/mL (p < 0.05). Strain 248D presented a bacteriostatic effect in GM. Moreover, the three wild strains and the commercial probiotic produced a significative reduction of the pH (p < 0.001). Discussion: This is the first in vivo report of the antimicrobial potential of LAB strains against Ma and its interaction. Our results support possible future alternative strategies to antibiotic therapy, previously not contemplated, to fight CA in small ruminants. Further studies are necessary to elucidate the action mechanisms through which these LAB are able to inhibit Ma and to assess the safety of using these strains in possible in vivo studies.

Genomic characterization of Listeria spp. isolated from tonsils, udder and feces of domestic dairy ruminants in Spain.

Two species of Listeria are pathogenic, Listeria monocytogenes and Listeria ivanovii. Although studies have shown that dairy ruminants shed Listeria spp. in feces, there is little information about ruminants that do not shed Listeria spp. in their feces but asymptomatically carry them in organs. We evidence that ruminants can asymptomatically carry L. ivanovii in udders and L. monocytogenes and L. ivanovii in tonsils without fecal shedding. Whole-genome sequence of L. monocytogenes and L. ivanovii contained known core genes involved in virulence and antibiotic resistance. This work highlights tonsils and udders as a Listeria intra-host site of colonization.

Description of the vaginal microbiota in nulliparous ewes during natural mating and pregnancy: preliminary signs of the male preputial microbiota modulation.

The vaginal microbiota plays a key role in animals' health. Understanding its diversity and composition and associated changes occurring through the reproductive cycle represents valuable knowledge to disclose the mechanisms leading to dysbiosis and eventually to infection. Even if the human vaginal microbiota has been thoroughly studied, scarce research has been conducted on the vaginal microbiota of livestock. In this study, 16S rRNA gene-based sequencing was performed on vaginal samples of ten nulliparous ewes at three different sampling points: before the estrus synchronization protocol (T0), at the time of estrus before mating (Testrus), and the day of the pregnancy diagnosis (Tpreg). Preputial samples from the three males collected pre and post-mating were also analyzed. Firmicutes, Proteobacteria, Bacteroidetes, and Actinobacteria were the most abundant phyla in vaginal samples. The most abundant genera were Porphyromonas, Anaerococcus, and Peptinophilius. Vaginal microbiota biodiversity decreased during pregnancy. Tenericutes (Ureaplasma spp.) increased significantly at Tpreg in both pregnant and non-pregnant ewes. Differences were observed between pregnant and non-pregnant ewes at Tpreg where pregnant ewes had a significantly higher abundance of Actinobacillus spp. and Ureaplasma spp. Ewes that were diagnosed with pregnancy at Tpreg showed a decreased abundance of gram-negative bacteria such as Bacteroidales, Campylobacterales, and Enterobacteriales. In addition, a significant decrease in the relative abundances of genera within Firmicutes, such as Alloicoccus (Lactobacillales), Atopostipes (Lactobacillales), and an uncultured bacteria W5053 from Family XI (Firmicutes, Clostridiales) was observed in non-pregnant ewes at Tpreg. The four most abundant phyla in the rams' prepuce were the same as in the ewes' vagina. The most abundant genus was Corynebacterium. No major differences were observed in the ram's preputial microbiota between pre and post-mating samples. Nevertheless, the differences in the taxonomic composition of ewes' vaginal microbiota between Testrus and Tpreg could be explained by the exposure to the preputial microbiota. This study offers new insights into the effects of several key steps of the ewe's reproductive cycle such as estrus-synchronization protocol, mating, and pregnancy on ovine vaginal microbiota. The knowledge of the microbiota dynamics during the reproductive cycle can help improve the reproductive outcomes of dams by identifying biomarkers and putative probiotics.

Search of antimicrobial lactic acid bacteria from Salmonella-negative dogs.

BACKGROUND: Salmonellosis is one of the most important food-borne zoonotic disease affecting both animals and humans. The objective of the present study was to identify gastrointestinal (GI) lactic acid bacteria (LAB) of canine-origin from Salmonella-negative dogs' faeces able to inhibit monophasic Salmonella Typhimurium previously isolated from dogs' faeces, in order to be used as a potential probiotic in pet nutrition. RESULTS: Accordingly, 37 LAB were isolated from Salmonella-negative dogs' faeces and tested against monophasic S. Typhimurium using the spot on lawn method out of which 7 strains showed an inhibition halo higher than 2.5 cm. These 7 strains were also tested with the co-culture method and one showed the greatest inhibition value (p < 0.05). Subsequently, the isolate was identified through 16S rRNA sequencing and sequence homology and designated as Ligilactobacillus salivarius (L. salivarius). LAB from Salmonella-positive dogs were also identified and none was the selected strain. Finally, to identify the mechanism of inhibition of L. salivarius, the supernatant was analyzed, and a dose response effect was observed. CONCLUSIONS: It is concluded that the canine-origin L. salivarius, could possess some in vitro functional attributes of a candidate probiotic and could prevent monophasic S. Typhimurium colonization or inhibit its activity if the infection occurs.

Ruminant-associated Listeria monocytogenes isolates belong preferentially to dairy-associated hypervirulent clones: a longitudinal study in 19 farms.

Studies have shown that ruminants constitute reservoirs of Listeria monocytogenes, but little is known about the epidemiology and genetic diversity of this pathogen within farms. Here we conducted a large-scale longitudinal study to monitor Listeria spp. in 19 dairy farms during three consecutive seasons (N = 3251 samples). L. innocua was the most prevalent species, followed by L. monocytogenes. Listeria monocytogenes was detected in 52.6% of farms and more frequently in cattle (4.1%) and sheep (4.5%) than in goat farms (0.2%). Lineage I accounted for 69% of L. monocytogenes isolates. Among animal samples, the most prevalent sublineages (SL) and clonal complexes (CC) were SL1/CC1, SL219/CC4, SL26/CC26 and SL87/CC87, whereas SL666/CC666 was most prevalent in environmental samples. Sixty-one different L. monocytogenes cgMLST types were found, 28% common to different animals and/or surfaces within the same farm and 21% previously reported elsewhere in the context of food and human surveillance. Listeria monocytogenes prevalence was not affected by farm hygiene but by season: higher prevalence was observed during winter in cattle, and during winter and spring in sheep farms. Cows in their second lactation had a higher probability of L. monocytogenes faecal shedding. This study highlights dairy farms as a reservoir for hypervirulent L. monocytogenes.

Review of the bacterial composition of healthy milk, mastitis milk and colostrum in small ruminants.

Bacterial infections are the cause of many reproductive disorders of economic importance, such as mastitis, in livestock. Unfortunately, very little is known about the microbiota and the changes occurring during an infection state in small ruminants. The sequencing of regions of the 16S rRNA gene, is the useful tool to describe the whole dairy microbiome. Using this technique, studies have identified various phyla such as Firmicutes, Actinobacteria, Proteobacteria, Bacteroidetes, Fusobacteria and Cyanobacteria; and also several genera from raw milk of small ruminants. Nevertheless, there does not seem to be a consensus on the predominant genera nor phyla, even within the same breed. There is a lack of information about the mammary microflora in meat-oriented breeds, and about the microflora of colostrum and mastitis milk. Further studies comparing the microbiota between artificial and natural lactations and between healthy and mastitis milk are necessary. Considering the concerns arising from the use overuse of antibiotic therapy in Veterinary Medicine, it would be interesting to develop alternative strategies for the control of mastitis. Probiotics, such as lactic acid bacteria (LAB), have proven to be an interesting antibiotic-free strategy. Therefore, their presence in the dairy microflora of small ruminants and their interactions with other bacteria, such as mastitis-causing pathogens, should be scrutinized, given that the efficacy of probiotics increase when the bacterial strains used are specific to their host.

Ultrasound-Guided Fine-Needle Aspiration of Superficial Lymphadenopathy Performed by Interventional Pathologists: The Applicability of the Sydney System from 2 Years of Experience and 363 Cases.

INTRODUCTION: The Sydney system proposal for the study and reporting of lymphadenopathy by fine-needle aspiration (FNA) constitutes one of the first attempts to standardize this procedure. Here, we review its applicability. MATERIALS AND METHODS: A retrospective study in which all ultrasound-guided FNAs (USFNAs) of superficial lymphadenopathy (palpable or not) performed by interventional pathologists in 2 specialized hospital centers were quantified over 2 years. The procedure was systematized, and the diagnoses were reclassified according to the Sydney system categories. RESULTS: We analyzed 363 USFNAs of lymphadenopathies. The distribution of cases by categories was as follows: insufficient (n = 13; 3.58%), benign (n = 208; 57.30%), atypia of uncertain significance (n = 7; 1.93%), suspicious (n = 21; 5.79), and malignant (n = 114; 31.40%). The risks of malignancy calculated for categories I, II, III, IV, and V were 27%, 3%, 50%, 100%, and 100%, respectively. CONCLUSION: The implementation of the Sydney system allows the systematization and standardization of the lymph node FNA methodology, with increased efficacy and efficiency. Assimilating the recommendations enables the qualification of the diagnostic procedure.

Importance and Antimicrobial Resistance of Mycoplasma bovis in Clinical Respiratory Disease in Feedlot Calves.

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.

[Minimally invasive percutaneous musculoskeletal biopsy performed by Interventional Pathologist for myopathy study. Twenty years of experience]. / Biopsia percutánea muscular mínimamente invasiva realizada por patólogo intervencionista en el estudio de miopatías. Veinte años de experiencia.

INTRODUCTION: Muscle biopsy plays a major role in the final diagnosis of myopathies. Open muscle biopsy is the benchmark procedure, although minimally invasive percutaneous muscle biopsy (MIPMB) has demonstrated comparable diagnostic performance at a lower cost and can be carried out by interventional pathologists. MATERIALS AND METHODS: Muscle biopsies performed from 1997 to 2017 were reviewed and classified according to the type of procedure, whether carried out by an interventional pathologist or another specialist, the diagnosis and the effectiveness of the procedure. RESULTS: 738 muscle biopsies were performed; 32% were open biopsies and 68% MIPMB carried out by pathologist. The muscle most often biopsied was the femoral quadriceps and the most frequent diagnosis was inflammatory myopathies. In only 39 cases (20 open biopsies and 19 MIPMB) was there insufficient tissue for diagnosis. CONCLUSIONS: Muscle biopsy proved highly effective as a diagnostic tool as 90% yielded adequate tissue samples. The results obtained with MIPMB performed by interventionist pathologists were comparable to those of open muscle biopsy.

Listeria spp. Isolated from Tonsils of Wild Deer and Boars: Genomic Characterization.

Listeria monocytogenes is a major human and animal foodborne pathogen. However, data from environmental reservoirs remain scarce. Here, we used whole-genome sequencing to characterize Listeria species isolates recovered over 1 year from wild animals in their natural habitats in Spain. Three different Listeria spp. (L. monocytogenes [n = 19], Listeria ivanovii subsp. londoniensis [n = 4], and Listeria innocua [n = 3]) were detected in 23 animal tonsils (9 deer, 14 wild boars) and 2 feeding troughs. No Listeria species was detected in feces. L. monocytogenes was detected in tonsils of 44.4% (8 out of 18) of deer and 40.7% (11 out of 27) of wild boars. L. monocytogenes isolates belonged to 3 different core genome multilocus sequence typing (cgMLST) types (CTs) of 3 distinct sublineages (SL1, SL387, and SL155) from lineages I and II. While cgMLST type L1-SL1-ST1-CT5279 (IVb; clonal complex 1 [CC1]) occurred only in one animal, types L1-SL387-ST388-CT5239 (IVb; CC388) and L2-SL155-ST155-CT1170 (IIa; CC155) were retrieved from multiple animals. In addition, L1-SL387-ST388-CT5239 (IVb; CC388) isolates were collected 1 year apart, revealing their long-term occurrence within the animal population and/or environmental reservoir. The presence of identical L. monocytogenes strains in deer and wild boars suggests contamination from a common food or environmental source, although interhost transmission cannot be excluded. Pathogenicity islands LIPI-1, LIPI-3, and LIPI-4 were present in 100%, 5%, and 79% of the L. monocytogenes isolates, respectively, and all L. monocytogenes lineage II isolates (n = 3) carried SSI-1 stress islands. This study highlights the need for monitoring L. monocytogenes environmental contamination and the importance of tonsils as a possible L. monocytogenes intrahost reservoir.IMPORTANCEListeria monocytogenes is a foodborne bacterial pathogen responsible for listeriosis. Whole-genome sequencing has been extensively used in public health and food industries to characterize circulating Listeria isolates, but genomic data on isolates occurring in natural environments and wild animals are still scarce. Here, we show that wild animals carry pathogenic Listeria and that the same genotypes can be found at different time points in different host species. This work highlights the need of Listeria species monitoring of environmental contamination and the importance of tonsils as a possible L. monocytogenes intrahost reservoir.

Price Fluctuation, Protected Geographical Indications and Employment in the Spanish Small Ruminant Sector during the COVID-19 Crisis.

Official milk prices in the Spanish small ruminant sector were used for 5 years (2015-2019) to analyze the effect caused by the coronavirus disease (COVID-19) crisis in 2020. Meat price fluctuations were also studied using the weekly prices officially provided by some of the main agrarian markets of the country (n = 6) in 2019 and 2020. Moreover, the sales and prices of three protected geographical indications (PGI) of lamb meat served to study the marketability when the products are sold or not under these quality labels in a crisis context. According to Spanish Government's official communications, 2020 was divided in three periods of study (pre-COVID-19, total confinement and post-confinement). The evolution of employment in this subsector in 2020, as a direct consequence of this crisis, was also analyzed considering data provided by producers. Results showed an intra-annual seasonal effect for milk prices in 2020 for both livestock species, as observed in previous years. However, a negative economic impact on goat milk prices due to the pandemic was checked during the confinement and post-confinement months. Sheep milk prices remained stable. Lamb and goat kid meat prices showed a similar trend in comparison with 2019 during the pre-COVID-19 period. The total confinement period recorded a short interval of 1-2 weeks in which the prices declined, before the suspension of quotations in many markets. In contrast, once confinement was completed, meat prices for both ruminant species rapidly reached levels that existed before the coronavirus crisis. Overall data suggested the protective effect of the PGI marks on lamb meat. Lambs with a PGI had better 2020 prices than non-PGI lambs (+8%), regardless of the period analyzed. Moreover, with fewer lambs sold in 2020, there was a relevant drop in sales of non-PGI lambs vs. PGI (-19% vs. -2%) during the first 7 months. Finally, there was little or no readjustment of the workforce in the small ruminant flocks.

Vaginal Microbiota Is Stable throughout the Estrous Cycle in Arabian Maress.

Lactic acid bacteria (LAB) dominate human vaginal microbiota and inhibit pathogen proliferation. In other mammals, LAB do not dominate vaginal microbiota, however shifts of dominant microorganisms occur during ovarian cycle. The study objectives were to characterize equine vaginal microbiota in mares by culture-dependent and independent methods and to describe its variation in estrus and diestrus. Vaginal swabs from 8 healthy adult Arabian mares were obtained in estrus and diestrus. For culture-dependent processing, bacteria were isolated on Columbia blood agar (BA) and Man Rogosa Sharpe (MRS) agar. LAB comprised only 2% of total bacterial isolates and were not related to ovarian phases. For culture-independent processing, V3/V4 variable regions of the 16S ribosomal RNA gene were amplified and sequenced using Illumina Miseq. The diversity and composition of the vaginal microbiota did not change during the estrous cycle. Core equine vaginal microbiome consisted of Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria at the phylum level. At the genus level it was defined by Porphyromonas, Campylobacter, Arcanobacterium, Corynebacterium, Streptococcus, Fusobacterium, uncultured Kiritimatiaellae and Akkermansia. Lactobacillus comprised only 0.18% of the taxonomic composition in estrus and 0.37% in diestrus. No differences in the relative abundance of the most abundant phylum or genera were observed between estrus and diestrus samples.

Listeria valentina sp. nov., isolated from a water trough and the faeces of healthy sheep.

In the context of a study on the occurrence of Listeria species in an animal farm environment in Valencia, Spain, six Listeria-like isolates could not be assigned to any known species. Phylogenetic analysis based on the 16S rRNA gene and on 231 Listeria core genes grouped these isolates in a monophyletic clade within the genus Listeria, with highest similarity to Listeria thailandensis. Whole-genome sequence analyses based on in silico DNA-DNA hybridization, the average nucleotide blast and the pairwise amino acid identities against all currently known Listeria species confirmed that these isolates constituted a new taxon within the genus Listeria. Phenotypically, these isolates differed from other Listeria species mainly by the production of acid from inositol, the absence of acidification in presence of methyl α-d-glucoside, and the absence of α-mannosidase and nitrate reductase activities. The name Listeria valentina sp. nov. is proposed for this novel species, and the type strain is CLIP 2019/00642T (=CIP 111799T=DSM 110544T).

Short-Term Economic Impact of COVID-19 on Spanish Small Ruminant Flocks.

The human pandemic COVID-19 caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) started in China in 2019 and has rapidly spread around the world, leading to extreme control measures such as population confinement and industry activity closure. Although small ruminants are not sanitary affected by this virus, the short-term economic impact derived by COVID-19 on Spanish flocks is estimated in this study, using data provided by producers and two major slaughterhouses. Milk prices of dairy goat flocks suffered a substantial drop in April 2020, close to 4.5 cts EUR/liter compared to the previous month. In contrast, the monthly milk prices in sheep remained almost stable during this period, and even increases of more than EUR 6 cts were reported in comparison with the previous year. Nevertheless, economical differences are reported by areas where producers could receive a higher income, close to EUR 0.3 per liter of milk. Global data provided by feedlots affecting 2750 Spanish flocks evidenced a lamb price drop ranging from 16.8% to 26.9% after the pandemic arrival; in line with the data directly reported by a limited sample of producers (ranging from 11.0% to 23.7%). The goat kid meat market also suffered a reduction in prices per kg, near 12.5%; although, for some flocks, losses reached up to 40%. In the same line, 2 slaughterhouses reported a sudden sacrifice drop around 27% for lambs and goat kids sacrifices in April, in contrast with the usual sacrifice figures from the beginning of 2020. Moreover, our study showed a temporary and unexpected retention of lambs and goat kids at farms due to a reduction in animals slaughtered during this period. In conclusion, data evidenced a considerable negative economic impact on Spanish small ruminant flocks, throughout the first 60 days after COVID-19's pandemic declaration. Further studies are needed to evaluate the long-term economic consequences, in order to establish contingency plans and avoid the collapse of small ruminant industries when a crisis of these characteristics occurs.

Mycoplasma bovis in Spanish Cattle Herds: Two Groups of Multiresistant Isolates Predominate, with One Remaining Susceptible to Fluoroquinolones.

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 efficient 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 in a 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.

Vaginal Microbiota Changes During Estrous Cycle in Dairy Heifers.

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.

The Addition of Lactobacillus spp., Enrofloxacin or Doxycycline Negatively Affects the Viability of Mycoplasma bovis in Diluted Bovine Semen.

Mycoplasma bovis is an important etiologic agent of bovine mycoplasmosis in cattle. Different transmission routes have been described, including those related to reproduction. The presence of mycoplasma in semen has led to its appearance in infection-free areas through artificial insemination (AI). Semen was recently reported to be the initial source of two M. bovis mastitis outbreaks in two closed dairy herds in Finland. This questions the effectiveness of the antimicrobials currently used in semen extenders to control the pathogens in contaminated semen. They should be re-evaluated, or alternative measures to antimicrobials should be tested to obtain M. bovis-free semen. This in vitro study aimed to assess different strategies to reduce the risk of transmission of M. bovis through AI technologies. The viability of M. bovis (PG45, NCTC 10131) in bull semen diluted (DS) in a Tris-citrate-fructose solution was tested, after the addition of enrofloxacin, doxycycline or a Lactobacillus spp.-based probiotic. The data show the susceptibility of the pathogen to the addition of 0.125 µg/mL of enrofloxacin or 0.0625 µg/mL of doxycycline and to the addition of the probiotic at a concentration of 3.24 × 106 colony forming units (CFU)/mL or 3.24 × 108 CFU/mL in DS. The Tris-citrate-fructose medium negatively affected the viability of M. bovis, although this effect was lower than that observed after the addition of the probiotic and antimicrobials (p < 0.05). Our results may support new strategies for reducing the risk of M. bovis transmission through AI.