The addition of Lactobacillus spp. negatively affects Mycoplasma bovis viability in bovine cervical mucus.

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.

Assessment of in vitro growth characteristics of Mycoplasma ovipneumoniae.

Mycoplasma ovipneumoniae (Movp) is an emerging pathogen that causes respiratory disease in small ruminants worldwide. It is considered to be difficult and time consuming to grow, which complicates diagnostic and control measures including isolation (an essential step required prior to the characterisation of strains), antimicrobial susceptibility testing and the development of vaccines. The objectives of this study were to analyse in vitro growth patterns of Movp strains, and the effects of different media used to support their growth. The study was conducted on 20 ovine and caprine Movp strains, isolated using Thiaucourt's medium. The rapid growth phase varied among the strains from 24 h to 72 h, although 60% of strains (12 of 20) reached a peak at 48 h. All strains were viable at 72 h after incubation, and declining viability was observed at 96 h (13 of 20 remained viable; 65%), 120 h (9 of 20; 45%) and 144 h (4 of 20; 20%). Growth was not detected at 168 h. All strains were able to grow in modified tryptone soy broth, while PH mycoplasma medium-Hayflick modified medium supported the growth of only two strains. Improved techniques of Movp cultivation require consideration of the growth variability among strains, the time of subculturing (during the first three days of incubation) and selection of appropriate media.

Mycoplasma ovipneumoniae (Movp) est un agent pathogène émergent qui provoque une maladie respiratoire chez les petits ruminants du monde entier. Sa croissance est lente et jugée difficile à obtenir, ce qui complique le diagnostic et les mesures de lutte, en particulier l'isolement (étape indispensable pour caractériser les souches), le recours aux antibiogrammes et le développement de vaccins. L'étude présentée par les auteurs a pour objectifs d'analyser in vitro les profils de croissance de différentes souches de Movp et d'observer l'effet sur cette croissance des milieux utilisés. L'étude a porté sur 20 souches de Movp provenant d'ovins et de caprins isolées sur milieu de Thiaucourt. La phase de croissance rapide variait de 24 h à 72 h suivant les souches, avec néanmoins un pic de croissance atteint en 48 h pour 12 des 20 souches (soit 60 %). La totalité des souches étaient viables 72 h après l'incubation ; une perte de la viabilité a été observée à 96 h (13 souches sur 20 demeurant viables, soit 65 %), à 120 h (9 souches viables sur 20, soit 45 %) et à 144 h (4 souches viables sur 20, soit 20 %). Aucun signe de croissance n'était détecté à 168 h. L'ensemble des souches ont présenté des signes de croissance sur bouillon tryptone soja modifié, tandis que deux souches seulement ont présenté des signes de croissance sur milieu PH mycoplasma-milieu de Hayflick modifié. L'amélioration des techniques de mise en culture des Movp passe par la prise en compte de la variabilité de la croissance suivant les souches, par un repiquage réalisé au bon moment (dans les trois premiers jours d'incubation) et par le choix approprié du milieu.

Mycoplasma ovipneumoniae (Movp) es un patógeno emergente que afecta a pequeños rumiantes del mundo entero, en los que provoca una enfermedad respiratoria. Su cultivo está considerado difícil y exige tiempo, lo que complica su diagnóstico y las medidas de lucha, en particular el aislamiento (paso previo indispensable para la caracterización de las cepas), la realización de pruebas de sensibilidad a los antimicrobianos y la obtención de vacunas. Los autores describen un estudio encaminado a analizar las modalidades de crecimiento in vitro de cepas de Movp y los efectos del uso de diferentes medios de cultivo, utilizando para ello 20 cepas ovinas y caprinas de Movp aisladas con empleo de medio de Thiaucourt. La fase de crecimiento rápido, variable en función de la cepa, iba de 24 a 72 horas, aunque un 60% de las cepas (12 de 20) alcanzaba el pico de crecimiento a las 48 horas. Todas las cepas eran viables a las 72 horas de incubación. Se observó asimismo que la viabilidad menguaba al cabo de 96 horas (seguían siendo viables 13 de las 20 cepas, un 65%), 120 horas (9 de 20, un 45%) y 144 horas (4 de 20, un 20%). Al cabo de 168 horas no se detectaba crecimiento alguno. Todas las cepas podían crecer en caldo de triptona de soja modificado, mientras que solo dos cepas crecían en un medio PH mycoplasma-medio Hayflick modificado. Para poder perfeccionar las técnicas de cultivo de Movp es preciso tener en cuenta la variabilidad entre las cepas por lo que respecta a las características de crecimiento, así como el tiempo de subcultivo (durante los tres primeros días de incubación) y la selección del medio apropiado.

Effect of temperature and cell concentration on dolphin (Tursiops truncatus) spermatozoa quality evaluated at different days of refrigeration.

The use of cryopreserved dolphin spermatozoa facilitates the exchange of genetic material between aquatic parks and makes spermatozoa accessible to laboratories for studies to further our understanding of marine mammal reproduction. Sperm cryopreservation in the bottlenose dolphin (Tursiops truncatus) has been developed for the exchange of gametes within the ex situ population. The aim of this study was to develop an effective method for refrigeration of bottlenose dolphin spermatozoa diluted in a commercial extender (BTS). In Experiment 1, the effect of temperature (5 compared with 15 °C) on sperm quality was evaluated during 7 days of storage at 100 × 106 spermatozoa/ml. In Experiment 2, the effect of the storage concentration (100 × 106 compared with 20 × 106 spermatozoa/ml) on sperm quality was assessed during 7 days of storage at 5 °C. In Experiment 1, total motility (including % of rapid sperm) was greater at 5 than 15 °C. When the effect of storage concentration was evaluated (Experiment 2), total motility and ALH were greater at the higher storage concentration (100 × 106 spermatozoa/ml). For both experiments, values for viability, acrosome integrity, and normal morphology variables were consistent throughout the 7 days of refrigeration. In Experiment 3, a microbiological study was performed to evaluate the effect of the refrigeration temperature and days of storage on bacterial growth. The results of microbiological analysis indicated there was Staphylococcus aureus in some samples, however, there was no effect of temperature or days of refrigeration. In conclusion, bottlenose dolphin semen can be refrigerated for a short to medium period of storage and there is maintenance of functionality of sperm when stored at 100 × 106 spermatozoa/ml at 5 °C.

The moderate drift towards less tetracycline-susceptible isolates of contagious agalactia causative agents might result from different molecular mechanisms.

Contagious agalactia is a mycoplasmosis that affects small ruminants, is associated with loss of milk production and high morbidity rates, and is highly deleterious to dairy industries. The etiological agents are four mycoplasma (sub)species, of which the relative importance depends on the countries and the animal host. Tetracyclines are non-expensive, broad-spectrum antimicrobials and are often used to control mastitis in dairy herds. However, the in vitro efficiency of tetracyclines against each of the etiological agents of contagious agalactia has been poorly assessed. The aims of this study were i) to compare the tetracycline susceptibilities of various field isolates, belonging to different mycoplasma (sub)species and subtypes, collected over the years from different clinical contexts in France or Spain, and ii) to investigate the molecular mechanisms behind the decreased susceptibility of some isolates to tetracyclines. The Minimum Inhibitory Concentrations (MICs) of tetracyclines were determined in vitro on a set of 120 isolates. Statistical analyses were run to define the significance of any observed differences in MICs distribution. As mutations in the genes encoding the tetracycline targets (rrs loci) are most often associated with increased tetracycline MICs in animal mycoplasmas, these genes were sequenced. The loss of susceptibility to tetracyclines after year 2010 is not significant and recent MICs are higher in M. agalactiae, especially isolates from mastitis cases, than in other etiological agents of contagious agalactia. The observed increases in MICs were not always associated with mutations in the rrs alleles which suggests the existence of other resistance mechanisms yet to be deciphered.

Comparison of phenotypic and genotypic profiles among caprine and ovine Mycoplasma ovipneumoniae strains.

Mycoplasma ovipneumoniae (Movp) is considered to be one of the most important mycoplasmas causing respiratory disease in small ruminants. Most epidemiologic and characterisation studies have been conducted on strains collected from sheep. Information on the presence and characteristics of Movp in healthy and pneumonic goats is limited. Phenotypic or genotypic differences between sheep and goat isolates have never been studied. The objective of our study was to characterise and compare the similarities and differences between caprine and ovine Movp strains isolated from affected and asymptomatic animals in order to elucidate phenotypic and genotypic variability. Four different techniques were used on a set of 23 Movp isolates. These included SDS-PAGE, Western blotting, random amplified polymorphic DNA and the heat shock protein 70 gene sequence-based method. A high degree of phenotypic and genotypic heterogeneity among Movp strains was demonstrated in this study. Our results demonstrated differences between goat and sheep strains, revealing not only a link between strains and host ruminant species, but by geographical origin as well. However, the finding of immunodominant antigens of molecular masses 36, 38, 40 and 70 kDa (±3 kDa) in Movp isolates from sheep and goats foretells their potential use in the development of serological diagnostic tests and vaccines.

In vitro assessment of the antimicrobial susceptibility of caprine isolates of Mycoplasma mycoides subsp. capri.

The minimum inhibitory concentration (MIC) and minimum mycoplasmacidal concentration (MMC) of 17 antimicrobials against 41 Spanish caprine isolates of Mycoplasma mycoides subsp. capri (Mmc) obtained from different specimens (milk, external auricular canal and semen) were determined using a liquid microdilution method. For half of the isolates, the MIC was also estimated for seven of the antimicrobials using an epsilometric test (ET), in order to compare both methods and assess the validity of ET. Mutations in genes gyrA, gyrB, parC and parE conferring fluoroquinolone resistance, which have been recently described in Mmc, were investigated using PCR. The anatomical origin of the isolate had no effect on its antimicrobial susceptibility. Moxifloxacin and doxycycline had the lowest MIC values. The rest of the fluoroquinolones studied (except norfloxacin), together with tylosin and clindamycin, also had low MIC values, although the MMC obtained for clindamycin was higher than for the other antimicrobials. For all the aminoglycosides, spiramycin and erythromycin, a notable level of resistance was observed. The ET was in close agreement with broth microdilution at low MICs, but not at intermediate or high MICs. The analysis of the genomic sequences revealed the presence of an amino acid substitution in codon 83 of the gene gyrA, which has not been described previously in Mmc.

Epidemiological role of birds in the transmission and maintenance of zoonoses. / Papel epidemiológico de las aves en la transmisión y mantenimiento de zoonosis.

The risk of zoonoses spreading from birds to humans is lower, quantitatively speaking, than the risk of transmission between other host groups, because the two taxonomic groups share fewer pathogens. Nevertheless, birds have a number of epidemiological characteristics that make them extremely important hosts in the transmission and maintenance of zoonoses, including their susceptibility to pathogens that are extremely hazardous to humans (such as highly pathogenic avian influenza virus, West Nile virus and Chlamydia psittaci) and their ability to travel long distances, especially in the case of migratory birds. The fact that the human diet includes poultry products (meat, eggs and their by-products) also means that most human cases of foodborne zoonoses are infections of avian origin. Lastly, close contact between humans and pet birds or urban birds leads to interactions of public health concern. This article sets out to describe the main factors that determine the role of birds in the epidemiology of zoonotic infections.

Le risque que les oiseaux transmettent des zoonoses à l'homme est moins élevé, au plan quantitatif, qu'entre hôtes d'autres catégories, car le nombre d'agents pathogènes affectant à la fois ces deux groupes taxonomiques est moindre. Cependant, certaines particularités épidémiologiques des oiseaux leur font jouer un rôle d'hôtes importants dans la persistance et la transmission de zoonoses : d'une part, leur sensibilité à des agents pathogènes dangereux pour l'homme (par exemple, le virus de l'influenza aviaire hautement pathogène, le virus de West Nile, Chlamydia psittaci) et, d'autre part, leur capacité à se déplacer sur de longues distances, notamment dans le cas des oiseaux migrateurs. En outre, les produits avicoles faisant partie des denrées alimentaires consommées par l'homme (viande de volaille, oeufs et produits dérivés), la majorité des cas de zoonoses d'origine alimentaire diagnostiqués chez l'homme sont d'origine aviaire. Enfin, les contacts étroits entre les humains et leurs oiseaux de compagnie ou avec des oiseaux des villes entraînent des interactions qui sont à prendre en compte en santé publique. Les auteurs décrivent les principales caractéristiques épidémiologiques des oiseaux jugées déterminantes par rapport aux infections zoonotiques.

El riesgo de transmisión de zoonosis de aves a humanos es menor, cuantitativamente hablando, que el que tiene lugar entre otros grupos de hospedadores, debido a que estos dos grupos taxonómicos comparten un menor número de agentes patógenos. No obstante, algunas particularidades epidemiológicas de las aves las convierten en hospedadores de gran importancia en el mantenimiento y la transmisión de zoonosis, como su capacidad de contraer infecciones por agentes patógenos peligrosos para los humanos (como el virus de la influenza aviar altamente patógena, el virus del Nilo Occidental o Chlamydia psittaci, entre otros) así como su gran capacidad de desplazamiento, especialmente en el caso de las aves migratorias. Además, el hecho de que la alimentación humana incluya productos avícolas (carne y huevos y productos derivados) hace que la mayoría de casos de zoonosis de origen alimentario diagnosticados en humanos sean infecciones de origen aviar. Por último, el estrecho contacto entre humanos y mascotas aviares o aves urbanas conlleva interacciones de interés para la salud pública. Este trabajo pretende describir los principales determinantes epidemiológicos de las aves en relación con las infecciones zoonósicas.

Sensitivity of two methods to detect Mycoplasma agalactiae in goat milk.

BACKGROUND: Laboratory diagnostic techniques able to detect Mycoplasma agalactiae are essential in contagious agalactia in dairy goats. This study was designed: 1) to determine the detection limits of PCR and culture in goat milk samples, 2) to examine the effects of experimental conditions including the DNA extraction method, PCR technique and storage conditions (fresh versus frozen stored milk samples) on these methods and 3), to establish agreement between PCR and culture techniques using milk samples from goats with mastitis in commercial dairy herds. The study was conducted both on artificially inoculated and field samples. RESULTS: Our findings indicate that culture is able to detect M. agalactiae in goat milk at lower concentrations than PCR. Qualitative detection of M.agalactiae by culture and PCR was not affected by sample freezing, though the DNA extraction method used significantly affected the results of the different PCR protocols. When clinical samples were used, both techniques showed good agreement. CONCLUSIONS: The results from this study indicate that both culture and PCR are able to detect M. agalactiae in clinical goat mastitis samples. However, in bulk tank milk samples with presumably lower M. agalactiae concentrations, culture is recommended within the first 24 h of sample collection due to its lower limit of detection. To improve the diagnostic sensitivity of PCR in milk samples, there is a need to increase the efficiency of extracting DNA from milk samples using protocols including a previous step of enzymatic digestion.

Survival capacity of Mycoplasma agalactiae and Mycoplasma mycoides subsp capri in the diluted semen of goat bucks and their effects on sperm quality.

This study examines the viability of Mycoplasma agalactiae (Ma) and Mycoplasma mycoides subsp capri (Mmc) during 150 minutes of incubation at 37 °C in contaminated diluted semen (DS) doses. The effects of the presence of both microorganisms on sperm viability, motility, and morphology were also examined. In a second experiment, the viability of Ma and its effects on sperm viability were determined in ejaculate samples and skimmed milk semen extender samples. Ma and Mmc were able to survive in DS at concentrations considered infectious, and no significant differences in mean concentrations were detected (7.1 log colony-forming units [CFU]/mL). However, initial concentration of Ma declined (P < 0.05) from 7.5 to 6.9 log CFU/mL and Mmc declined (P < 0.05) from 7.7 to 7.1 log CFU/mL after incubation. Conversely, ejaculate concentrations of Ma increased significantly (from 7.1 to 7.4 log CFU/mL, P < 0.05). These observations suggest that the natural breeding medium is more suitable for Ma than the medium used for artificial insemination (AI). The presence of Mmc slightly reduced sperm viability in the DS (from 21.7% to 16.6%, P < 0.05). The absence of major effects on sperm quality could lead to the unnoticed use of semen contaminated with Ma and Mmc for AI. As both bacteria were able to survive the conditions of ejaculates and semen doses, these findings suggest a risk of venereal transmission of contagious agalactia and support the use of mycoplasma-free semen samples for (AI).

Short communication: In vitro antimicrobial susceptibility of Mycoplasma agalactiae strains isolated from dairy goats.

This study examined the susceptibility to several antimicrobials of 28 isolates of Mycoplasma agalactiae obtained from goats in a region (southeastern Spain) where contagious agalactia is endemic. For each isolate, the minimum inhibitory concentration (MIC) against 12 antimicrobials of the quinolone, macrolide, aminoglycoside, and tetracycline families was determined. The antimicrobials with the lowest MIC were enrofloxacin, ciprofloxacin, tylosin, and doxycycline, all with MIC90 (concentration at which growth of 90% of the isolates is inhibited) <1 µg/mL. Norfloxacin (a quinolone) showed a wide MIC range (0.1-12.8 µg/mL), suggesting a resistance mechanism toward this antimicrobial that was not elicited by enrofloxacin or ciprofloxacin (the other quinolones tested). Erythromycin showed the highest MIC90 such that its use against Mycoplasma agalactiae is not recommended. Finally, Mycoplasma agalactiae isolates obtained from goat herds with clinical symptoms of contagious agalactia featured higher MIC90 and MIC50 (concentration at which growth of 50% of the isolates is inhibited) values for many of the antimicrobials compared with isolates from asymptomatic animals. The relationship between the extensive use of antimicrobials in herds with clinical contagious agalactia and variations in MIC requires further study.

Controlling contagious agalactia in artificial insemination centers for goats and detection of Mycoplasma mycoides subspecies capri in semen.

Many goat artificial insemination (AI) centers in Spain have adopted new measures to control contagious agalactia (CA). To avoid the introduction of male goats carrying mycoplasma organisms subclinically in their external ear canal (auricular carriers) in these centers, two ear swabs and a blood sample are obtained from all candidate animals for polymerase chain reaction (PCR), culture (swabs) and serologic tests to detect the presence of mycoplasmas. In addition, the semen produced at these centers is routinely cultured and PCR tested also to detect the presence of mycoplasmas. One y after the introduction of this program, we tested 48 ear swabs and 24 blood samples from 24 candidates for admission to these AI Centers. Three of these ear swab samples (3/48, 6.25%) scored positive for the presence of mycoplasmas; Mycoplasma agalactiae (Ma) was detected in two samples and Mycoplasma mycoides subsp. capri (Mmc) in one. All animals were serologically negative for Ma. Also, out of 173 semen samples obtained from 137 admitted animals (2 and 3 samples were obtained in 16 and 10 bucks, respectively), one (1/173, 0.56%) was positive for Mmc. Our findings suggest that ear swab and semen samples are useful tools to control CA at AI Centers. The introduction of this program has also resulted in the first detection of Mmc in semen from a naturally infected goat, confirming the ability of this mycoplasma to colonize the reproductive tract of male goats. These results highlight the need to improve control measures in semen producing centers to minimize the risk of CA transmission.

Presence of contagious agalactia causing mycoplasmas in Spanish goat artificial insemination centres.

Male goats admitted to artificial insemination centres come from herds that have shown no clinical symptoms of contagious agalactia (CA) for the last 6 mo. However, prior reports suggest that this control measure may not be completely effective. This study was designed to detect the presence of CA-causing mycoplasmas in 9 Spanish centres, comprising 159 goats (147 males and 12 teaser does) of 8 different breeds. A microbiological study was conducted during 8 mo on 448 samples (318 ear swabs, 119 semen samples and 11 milk samples). In 86 samples (84 swabs, 1 semen sample and 1 milk sample), CA-causative mycoplasmas were detected by PCR or culture, and 52 animals (49 goat males and 3 teaser does) tested positive. Most of these positive animals were auricular carriers (n = 50), mainly of Mycoplasma mycoides subsp. capri (Mmc), although some M. agalactiae (Ma) and, interestingly, M. capricolum subsp. capricolum (Mcc) carriers were also identified. At least 1 animal infected by CA-causing mycoplasmas was detected in 8 of the 9 centres (88.8%) although in most (66.7%) no infected animals or only 1 or 2 positive animals were identified. Our results indicate the presence of CA carriers as asymptomatic animals in reproductive programmes. These findings have already prompted efficient measures to detect and avoid the entry of these carriers in Spanish centres. We recommend similar measures for all centres in areas where CA is endemic.

Mycoplasma agalactiae detected in the semen of goat bucks.

Contagious agalactia (CA) is among the most significant diseases affecting small ruminant populations in Mediterranean countries. This study was designed to detect the excretion in semen of CA-causing mycoplasmas in goats (Capra hircus) reared in Spain, where the disease is considered endemic. Culture techniques and PCR were conducted on 147 semen samples collected from 113 goat bucks to detect mycoplasmas. No animal showed clinical symptoms of CA at the moment of the screening. M. agalactiae was identified using both diagnostic methods in three semen samples collected from three different bucks. These animals belonged to a group of animals in which semen had been analyzed twice and only the second sample proved positive, suggesting the possibility of intermittent excretion. This is the first report of the isolation of M. agalactiae from semen collected from naturally infected goats. Future studies should investigate whether semen could be a real source of CA infection by determining if the agent may be transmitted during natural service or when semen is used for artificial insemination.

Short communication: effect of storage and preservation on total bacterial counts determined by automated flow cytometry in bulk tank goat milk.

This study was designed to evaluate the effects of different storage conditions on total bacterial count (TBC) determinations made in goat bulk tank milk using an automated flow cytometry method. The storage conditions tested were storage temperature (refrigeration at 4 and 10 degrees C or freezing at -20 degrees C), the use of a preservative (no preservative, NP; azidiol, AZ; or bronopol, BR), and the age of the milk samples for each analytical condition (storage times at 4 degrees C: from 0 h to 5 d for NP; and from 0 h to 22 d for AZ and BR; storage times at 10 degrees C: from 24 h to 2 d for NP and from 24 h to 22 for AZ and BR; storage times at -20 degrees C: from 24 h to 22 d for NP, AZ, and BR). Significant effects on individual bacterial count (IBC) variation were shown by the bulk tank milk sample, preservative, storage temperature, interaction preservative x storage temperature, and milk age within the interaction preservative x storage temperature. In preserved samples, the highest IBC were obtained for AZ and the lowest counts were obtained in samples preserved with BR. Because of the variation in IBC recorded in BR-preserved samples, we recommend that BR should not be used for TBC determinations using the automated flow cytometry method. The NP samples stored at 4 and 10 degrees C showed significantly higher IBC at 24 h postcollection, also invalidating these analytical conditions for TBC analyses. The practical implications of our findings are that goat milk samples preserved with AZ and stored at 10 or 4 degrees C are appropriate for TBC by the BactoScan flow cytometry method for up to 24 h and 11 d postcollection, respectively.

Short communication: Detection limits of non-beta-lactam antibiotics in goat's milk by microbiological residues screening tests.

This study compares the performance of 4 antimicrobial residue screening tests [brilliant black reduction test AiM (Analytik in Milch Produktions- und Vertriebs GmbH, München, Germany), Delvotest MCS (DSM Food Specialties, Delft, the Netherlands), Eclipse 100 test (ZEU-Inmunotec SL, Zaragoza, Spain), and Copan Milk Test (Copan Italia S.p.a., Brescia, Italy)] used to detect 20 antimicrobial agents (aminoglycosides, macrolides, tetracyclines, sulfonamides, and quinolones) in goat's milk, according to International Dairy Federation guidelines. Composite milk samples from 30 antibiotic-free goats were used to prepare spiked milk samples and 11,520 analytical determinations were carried out. According to a logistic regression model, agreement coefficients were greater than 98% for most of the antibiotics, with higher b values obtained for macrolides. Neither tetracyclines nor quinolones were detected at European Union maximum residue limits. Only the Copan Milk Test and the Delvotest MCS were able to detect 3 antimicrobials below their maximum residue limits (neomycin, tylosin, and sulfadimethoxine). Given that these tests are used in control programs for goat's milk, our results indicate their sensitivity would need to be improved to guarantee safety for consumers.

Detection limits of four antimicrobial residue screening tests for beta-lactams in goat's milk.

This study was conducted to compare the detection limits (DL) of several antibiotic residue screening tests with the maximum residue limits (MRL) authorized by the EU according to the guidance for the standardized evaluation of microbial inhibitor tests of the International Dairy Federation. Composite antibiotic-free milk samples from 30 primiparous Murciano-Granadina goats in good health condition were used to prepare test samples spiked with different concentrations of each antimicrobial. In total, 5,760 analytical determinations of 10 beta-lactam antibiotics (penicillin-G, ampicillin, amoxicillin, cloxacillin, oxacillin, dicloxacillin, cefadroxyl, cefalexin, cefoperazone, and cefuroxime) were performed using 4 antibiotic residue screening tests: the brilliant black reduction test BRT AiM (AiM-Analytik in Milch Produktions-und Vertriebs GmbH, München, Germany), Delvotest MCS (DSM Food Specialties, Delft, the Netherlands), Eclipse 100 (ZEU-Inmunotec SL, Zaragoza, Spain), and the Copan Milk Test (CMT; Copan Italia SpA, Brescia, Italy). For each method, we estimated the detection limits of the antimicrobial agents using a logistic regression model. Using the CMT and Delvotest on samples spiked with the 8 antibiotics for which MRL were available, DL were at or below the MRL. The BRT test provided DL at or below the MRL for all of the agents except cefalexin, whereas the Eclipse 100 method failed to detect 4 antibiotics (ampicillin, amoxicillin, cloxacillin, and cefoperazone) at MRL or below. Logistic regression-determined levels of agreement were highest for the CMT method (98.6 to 100%) and lowest for Eclipse 100 (66.3 to 100%). In general, agreement levels indicated good correlation between observed results and those predicted by logistic regression. The lowest b values (closely related to test sensitivity) were recorded for the cephalosporins (0.074 to 0.430) and highest for penicillin G, ampicillin, and amoxicillin (11.270 to 11.504). Delvotest and CMT best fulfilled IDF criteria for the ideal test for detecting antibiotic residues in milk.

Correlating the immune response with the clinical-pathological course of persistent mastitis experimentally induced by Mycoplasma agalactiae in dairy goats.

To correlate the clinical course of mycoplasma mastitis with its immune response, right mammary glands of 15 lactating goats were inoculating with 10(10) colony-forming units (cfu) of Mycoplasma agalactiae (Ma). Before sacrificing the animals at 5, 15 or 45 days post-inoculation (dpi), blood Ma antibody titres and milk mycoplasma colony and somatic cell counts were monitored. Ma colonised the mammary gland and milk counts increased to over 10(12)cfu/ml within 5 dpi. During this period, an innate immune response involving neutrophils and macrophages was observed, and Ma antigen appeared in the degenerated acinar epithelium. From 7 dpi, a specific antibody response coincided with reduced viable mycoplasmas in milk. The humoral immune response was limited; by 37 dpi, all animals scored negative for anti-Ma antibodies, and around 10(8)cfu/ml were shed. Results indicate an early immune response to Ma inoculation unable to control mycoplasmal invasion. An ensuing humoral response, despite reducing the mycoplasma burden, leads to chronic, persistent infection.

In vitro susceptibilities of field isolates of Mycoplasma agalactiae.

In order to determine how widespread antibiotic resistance has become to standard treatments, the in vitro susceptibilities of 28 Mycoplasma agalactiae Spanish field isolates to 16 antimicrobial agents were determined using a broth microdilution method. The most effective antimicrobials based on minimum inhibitory concentration (MIC)(90) values were fluoroquinolones, tetracyclines and macrolides. Two strains were tetracycline resistant. Streptomycin, erythromycin and nalidixic acid resistance was observed in all strains.

Evaluation of the MilkoScan FT 6000 milk analyzer for determining the freezing point of goat's milk under different analytical conditions.

The aim of this research was to evaluate the Milko-Scan FT 6000 (Foss Electric, Hillerød, Denmark) for determining the freezing point (FP) of goat's milk under different analytical conditions. The FP was determined in duplicate in 1,800 milk aliquots obtained from 45 bulk tank milk samples from 10 Murciano-Granadina goat herds, using the MilkoScan method and a reference thermistor cryoscopy method (Advanced Instrument Inc., Norwood, MA). Five different preservation strategies--no preservative, preservation with azidiol (0.006 or 0.018 g of sodium azide/100 mL), and preservation with bronopol (0.020 or 0.040 g/100 mL)--were then used to preserve the milk. For each preservation strategy, 8 different amounts of water were added (0, 1, 2, 3, 4, 5, 6, or 7% total volume). The results obtained with each method under these 40 analytical conditions were examined by comparison of means, comparison of the standard deviations of repeatability (s(r) and its relative value s(r)%), and a regression analysis. Under most analytical conditions, the FP was recorded as lower by the MilkoScan method, with a mean difference of 1.5 m degrees C compared with the reference method. Both methods showed similar repeatabilities (the overall s(r)% was 0.22% for the MilkoScan method and 0.20% for the reference method). In comparisons of the 2 methods, the highest regression coefficients were obtained with aliquots containing >3% added water. The best regression coefficients (0.85 to 1.02) were obtained for milk samples preserved with bronopol at 0.020 g/100 mL. These results allow the MilkoScan method to be used with goat's milk for screening purposes. The factors of added water, preservative, analytical method, lactose concentration, and the effect of the bulk tank milk sample within each lactose group contributed significantly to the observed variation in FP. For practical purposes, either of the bronopol concentrations could be used when determining the FP of goat's milk with the methods tested. However, the increase in the concentration of sodium azide in the azidiol formula contributed to an important reduction in the FP recorded. Thus, the type and concentration of preservative should be taken into account when interpreting FP values.