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The new pandemic, COVID-19, highlighted the importance of the animal-human-environment interface in the emergence of zoonoses. The jump of the species barrier is considered a rare event although the number of emerging infectious diseases increased significantly in the second half of the 20th century. Most of them transmitted from wildlife to humans. Several elements can contribute to the emergence of zoonoses: human interaction on ecosystems, biodiversity loss, changes in land use, climate change, trade in or consumption of wildlife. There are different stages in the adaptation of an animal pathogen into a specialized human pathogen, varying from its presence in animals without human infection to a pathogen exclusive to humans. The history of zoonoses allows the identification of critical points for their control, as well as the identification of probable virus or bacteria involved in future pandemics. Concerning COVID-19, the knowledge of the possible reservoirs of SARS-CoV-2 contributed to decision-making during the pandemic. Therefore, and given the variety of scenarios that allow the jump of species barrier or the evolution of pathogens in a new host, One Health strategies for zoonosis emergence surveillance should be implemented.

Mycoplasma capricolum subsp. capricolum (Mcc) is one of the causative agents of contagious agalactia, and antimicrobial therapy is the most commonly applied measure to treat outbreaks of this disease. Macrolides and lincosamides bind specifically to nucleotides at domains II and V of the 23S rRNA. Furthermore, rplD and rplV genes encode ribosomal proteins L4 and L22, which are also implicated in the macrolide binding site. The aim of this work was to study the relationship between mutations in these genes and the acquisition of macrolide and lincosamide resistance in Mcc. For this purpose, in vitro selected resistant mutants and field isolates were studied. This study demonstrates the appearance of DNA point mutations at the 23S rRNA encoding genes (A2058G, A2059G and A2062C) and rplV gene (Ala89Asp) in association to high minimum inhibitory concentration values. Hence, it proves the importance of alterations in 23S rRNA domain V and ribosomal protein L22 as molecular mechanisms responsible for the acquisition of macrolide and lincosamide resistance in both field isolates and in vitro selected mutants. Moreover, these mutations enable us to provide an interpretative breakpoint of antimicrobial resistance for Mcc at MIC 0.8 μg/ml.

The extensive use of antimicrobials for disease control has caused a remarkable decrease in antimicrobial susceptibility of different animal mycoplasma species, including Mycoplasma agalactiae (M. agalactiae), the main causative agent of contagious agalactia. However, the molecular mechanisms behind M. agalactiae resistance to macrolides and lincomycin have not yet been elucidated. The aim of the present study was to investigate the association between minimum inhibitory concentration (MIC) values of different antimicrobials and mutations in the 23S rRNA gene and ribosomal proteins L4 and L22, analysing both field isolates (n=50) and in vitro selected resistant mutants of M. agalactiae. The obtained MIC results of the studied field isolates demonstrate an increasing development of tylosin resistance in this bacterium, in comparison to previous studies. Interestingly, predicted amino acid changes in L22 (Ser89Leu and Gln90Lys/His) were the first variations observed when MICs of M. agalactiae started to increase (tylosin MIC ≥0.8μg/ml), whereas mutations at positions 2058 or 2059 of domain V of the 23S rRNA gene appeared from MIC values of 1.6μg/ml. These results were consistent in both field isolates and in vitro selected mutants of M. agalactiae. Thus, although in other mycoplasma species resistance to macrolides and lincosamides had been mainly related to mutations in the 23S rRNA gene, this work demonstrates the role of alterations in ribosomal protein L22 in decreased susceptibility of M. agalactiae. Moreover, these mutations can be used as molecular markers to set an interpretative breakpoint of antimicrobial resistance for M. agalactiae.

M. agalactiae is the main causative agent of contagious agalactia, against which antimicrobial treatment is the main applied control measure. Quinolones are an effective group of antimicrobials inhibiting the growth of M. agalactiae, but in the last years, various reports have demonstrated an increase of resistance in field isolates due to its massive use. Nevertheless, the molecular mechanisms involved in the acquisition of fluoroquinolones resistance in M. agalactiae have not been elucidated yet. Therefore, the aim of this work was to analyze the presence of DNA variations that could be related to changes in fluoroquinolone susceptibility. For this purpose, three M. agalactiae strains were selected to obtain in vitro resistant mutants against enrofloxacin, marbofloxacin and moxifloxacin and afterwards, partial sequences of their gyrA, gyrB, parC and parE genes were analyzed. In addition, a set of field isolates with different MIC values were also studied. Changes related to variations in fluoroquinolones susceptibility were found in gyrB, parC and parE. Specifically, gyrB genes were affected at the predicted amino acid position 424, four amino acid changes were detected in parC (positions 78, 79, 80 and 84) and two substitutions were reported in parE (amino acid positions 429 and 459). Mutations at predicted positions 424 of gyrB and 429 of parE are novel DNA changes which had not been previously described and, on the whole, parC was the first gene showing alterations when changes in susceptibility to fluoroquinolones occurred. Thus, this gene is the most suitable target for a rapid study of fluoroquinolone resistance in field isolates of M. agalactiae.

Quinolones interact with bacterial DNA gyrase and topoisomerase IV, the subunits of which are encoded by gyrA/gyrB and parC/parE, respectively. The aim of this study was to evaluate the relationship between changes in these genes and quinolone susceptibility of Mycoplasma capricolum subsp. capricolum (Mcc). Using in vitro selected resistant mutants and field isolates from goats, predicted amino acid changes in gyrA, gyrB and parC were associated with higher minimum inhibitory concentration values for quinolones. Alterations in parC predicted amino acid sequences were most frequently associated with quinolone resistance in Mcc.

Este artículo se encuentra disponible en la siguiente URL: https://www.mscbs.gob.es/biblioPublic/publicaciones/recursos_propios/resp/revista_cdrom/VOL94/C_ESPECIALES/RS94C_202003022.pdf Las aves acuáticas silvestres representan el principal reservorio natural de los virus influenza y han participado en el reordenamiento tanto de virus pandémicos como de los virus responsables de los brotes de gripe aviar en las especies domésticas y silvestres. Con el objetivo de conocer los determinantes implicados, en el presente trabajo se revisaron los casos humanos de influenza aviar asociados al manejo de aves silvestres y la utilización de los equipos de protección personal, así como las bases de la vigilancia de la influencia aviar altamente patógena en aves silvestres en España. Las evidencias existentes permiten concluir que la transmisión directa de virus influenza desde las aves silvestres al ser humano es un evento raro. No obstante, nuestro contexto epidemiológico se encuentra influido por el cambio climático y queda marcado por la presencia de rutas migratorias desde territorios donde la infección puede estar presente. Por ello, y ante las implicaciones clínicas, económicas y para la salud pública que dichas infecciones pueden tener, los diferentes colectivos expuestos a las aves silvestres (veterinarios, biólogos, ornitólogos, conservacionistas, técnicos de campo, agentes medioambientales, cetreros, cazadores, etc.) deberían conocer las posibles fuentes de contagio y manejar correctamente los equipos de protección personal. Al mismo tiempo, es importante que dichos grupos conozcan la situación sanitaria actualizada respecto a la influenza aviar, para adaptar sus actividades en consecuencia y poder aplicar las medidas de protección de forma proporcionada a la misma, amén de aportar una valiosa información para los programas de vigilancia. / Wild waterfowl are considered the main natural reservoir of influenza viruses and they have contributed to the reassortment of both pandemic viruses and viruses responsible for outbreaks of avian influenza in wild and domestic species. In order to determinate the factors involved, we reviewed the human cases of avian influenza related to the management of wild birds, the use of personal protective equipment, as well as the basis of surveillance programs of highly pathogenic avian influenza in wild birds in Spain. The direct transmission of influenza virus from wild birds to humans is a rare event. However, our epidemiological context is influenced by climate change and marked by the presence of migratory routes from territories where infection may be present. Thus, and due to the clinical, economical and public health implications that such infections may have, the different groups exposed to wild birds (veterinarians, biologists, ornithologists, conservationists, field technicians, environmental officers, falconers, hunters, etc.) should know which are the possible sources of infection and how to handle the personal protective equipment. Besides, it is important that those groups know the current sanitary situation regarding avian influenza so they can consequently adapt their activities and employ proper protective measures, in addition to providing valuable information for surveillance programs.

En las sociedades occidentales, la tenencia de mascotas en el hogar se incrementa anualmente debido a los beneficios emocionales, físicos y psíquicos que aportan. Junto a estos beneficios, debe considerarse también la existencia de diferentes enfermedades que pueden transmitirse desde los animales al resto de convivientes en el hogar, con especial importancia para aquellas personas pertenecientes a grupos de riesgo. El aparente desafío riesgo-beneficio que provoca la tenencia de mascotas deberá solventarse considerando el estado de salud de los miembros del hogar para la selección de la especie y la recomendación de medidas higiénicas y de manejo específicas. De forma general, el conocimiento y la preocupación de los propietarios por las zoonosis asociadas a sus mascotas son escasos, incluso en los hogares donde hay personas de alto riesgo. Además, las limitaciones en los procedimientos de comunicación con los diferentes profesionales sanitarios pueden ocasionar que las situaciones de riesgo pasen desapercibidas. Por todo ello, se impone la necesidad de establecer protocolos, tanto intra como interprofesionales, para proporcionar un adecuado flujo de información que asegure la comunicación con el propietario de acuerdo con las particularidades sanitarias de los convivientes de cada hogar.

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.

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.

The unexpected presence of Mycoplasma auris was evidenced in mastitis samples collected from a dairy goat herd under surveillance programmes of contagious agalactia (CA), a serious disease affecting Mediterranean small ruminant herds. Thus, control programs based on permanent analysis of milk from bulk tank and clinical mastitis, as well as ear swabs, are currently conducted in affected areas. In order to analyze the presence of CA- causing species, three, 11 and 18 of these samples were respectively collected from the same goat herd. Mycoplasmas were isolated in four milk samples from clinical mastitis and two ear swabs samples and no other pathogen was detected. The presence of CA-causing mycoplasmas was discarded by PCR. However, M. auris was identified in all the positive samples after the amplification and posterior PCR product sequencing of a partial fragment of their 16S-ribosomal ARN gene. As far as we know, these are the first isolations of M. auris from milk of goats with clinical mastitis.

The veterinary profession implies a greater risk of infection by zoonotic pathogens than the overall population. The aim of the present work was to evaluate the impact of zoonoses on the occupational health of veterinarians reviewing the published surveys addressing this subject. Following these inquiries, between 4% and 64.3% of the surveyed professionals acknowledge to have suffered at least one zoonotic disease, and dermatophytosis is the most frequently described zoonosis. In addition, from a qualitative point of view, it is necessary to highlight the occurrence of different diseases of important clinical seriousness and the existence of infections by antimicrobial-resistant pathogens. Due to the absence of updated studies in Spain, the present work reflects on the need to recognize the health determinants associated to the veterinary profession within the characteristics of our labour market. The available data suggest that the occurrence of zoonoses as occupational diseases is underestimated in Spain. Therefore, performing surveys on occupational health, documenting and publishing cases and reviewing the risks and the impact of zoonotic diseases on the veterinary profession would contribute to the description and notification of such diseases on behalf of the healthcare administration and would also become an essential tool in occupational risk prevention.La profesión veterinaria presenta mayor riesgo de infección frente a agentes patógenos de carácter zoonósico que la población general. Con el objetivo de valorar el impacto de las zoonosis, el presente trabajo revisa las encuestas publicadas sobre salud laboral en la profesión veterinaria. En dichas encuestas, el rango de profesionales que refieren haber padecido al menos una zoonosis oscila entre el 4% y el 64,3%, siendo la dermatofitosis la zoonosis comunicada con mayor frecuencia. Además, desde el punto de vista cualitativo, cabe destacar la comunicación de diferentes procesos de especial gravedad clínica, así como la existencia documentada de infecciones por agentes patógenos resistentes a los antimicrobianos. Dada la ausencia de estudios actualizados en España, se reflexiona sobre la necesidad de conocer los determinantes de salud asociados a las características del mercado laboral de la profesión en nuestro entorno. Los datos existentes sugieren que la consideración legal de enfermedad profesional para las zoonosis se encuentra infraestimada en nuestro país. Por todo ello, la realización de encuestas de salud laboral, la documentación y publicación de los casos, así como la revisión de los riesgos y el impacto de las zoonosis en la profesión veterinaria, pueden contribuir a la calificación y notificación de dichas enfermedades por parte de la administración sanitaria, al tiempo que suponen una herramienta fundamental en el ámbito de la prevención de riesgos laborales.

This article reviews the diagnosis of mastitis in dairy goats. The benefits and shortcomings are discussed of both indirect diagnostic methods, such as the California mastitis test and somatic cell counts, and direct strategies, such as bacteriologic isolation and PCR identification. The important role of mycoplasmas in causing mastitis in regions where contagious agalactia is endemic makes their detection mandatory in routine diagnostic practice, despite complicating and lengthening protocols. In this context, we discuss both the conditions to be met prior to sampling and the analytical conditions of the sample along with the features of each diagnostic method.

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.

The presence of goat males carriers of the main causal agents of contagious agalactia, Mycoplasma agalactiae (Ma) and Mycoplasma mycoides subsp. capri (Mmc), is a high risk factor for the spread of the infection in artificial insemination centres. The present study was designed to assess the efficacy of a 5 day course of marbofloxacin given intramuscularly (2 mg kg−1 day−1) in terms of its capacity to reverse the carrier state of goat buck populations naturally infected with one or both mycoplasmas (Mmc, n = 25; Mmc + Ma, n = 2). Before and after treatment, ear swabs and semen samples were culture- and PCR-checked for both mycoplasmas. We also evaluated the impact of this treatment on semen quality (sperm count, motility and daily sperm production) by monitoring these variables of 594 semen samples collected during pre- and post-treatment periods of 48 days each. All bucks examined tested positive for the presence of Ma and Mmc before and after treatment though Mycoplasma spp. never was identified in semen. Sperm motility was significantly reduced in response to treatment (from 72.9% to 56.9%), although some recovery was observed at the end of the study (40 days after treatment). Ejaculate volume and concentration remained unchanged throughout the study. Our findings indicate that systemic marbofloxacin therapy is unable to eliminate mycoplasmas from the external auditory canal of male goats, and that it also causes a transient detrimental effect on sperm motility. Other strategies to control the presence of mycoplasma carriers in goat artificial insemination centres need to be assessed.

Background Veterinary students face diverse potential sources of zoonotic pathogens since the first years of their academic degree. Such sources include different animal species and pathologic materials which are used at university facilities as well as commercial clinics, farms and other external facilities. Objectives The present study utilizes a systematic review of the literature to identify zoonoses described in veterinary students. Data sources Web of Science and PubMed. Results Of the 1,254 titles produced by the bibliographic search, 62 were included in this review. Whereas 28 of these articles (45.2%) described individual cases or outbreaks, the remaining 34 (54.8%) reported serological results. The zoonotic etiological agents described were bacteria, in 39 studies (62.9%), parasites, in 12 works (19.4%), virus, in 9 studies (14.5%) and fungi, in 2 (3.2%) of the selected articles. The selected literature included references from 24 different countries and covered the time period of the last 55 years. Limitations The fact that common cases of disease or cases of little clinical importance without collective repercussions are not usually published in peer-reviewed journals limits the possibility to reach conclusions from a quantitative point of view. Furthermore, most of the selected works (66.1%) refer to European or North American countries, and thus, the number of cases due to pathogens which could appear more frequently in non-occidental countries might be underestimated. Conclusions/implications The results of the present systematic review highlight the need of including training in zoonotic diseases since the first years of Veterinary Science degrees, especially focusing on biosecurity measures (hygienic measures and the utilization of the personal protective equipment), as a way of protecting students, and on monitoring programs, so as to adequately advise affected students or students suspicious of enduring zoonoses.

Mycoplasma mycoides subsp. capri (Mmc) is one of the main causative agents of caprine contagious agalactia. Besides, the absence of accurate control methods eases its dispersion between different herds within endemic areas of this disease. In this context, there is a need to implement molecular typing schemes which offer valuable information useful to establish control measures and enables the surveillance of this pathogen. The aim of this study was to assess the genetic variability of different strains of Mmc from a contagious agalactia endemic area through multilocus sequence typing (MLST). For this purpose, five house-keeping genes (fusA, glpQ, gyrB, lepA, rpoB) from 39 field isolates were analysed. These isolates were obtained from different geographic areas of Spain, between the years 2004 and 2015. The results obtained in this study suggest that the selected MLST scheme could be a useful technique to monitor the genetic variability of Mmc in endemic areas. Despite the significant differences found between the assessed field isolates, they could be classified according to their geographical origin. Moreover, it was also possible to detect genetic differences between Mmc strains coming from the same herd at the same sampling time, which may need to be taken into consideration when designing or arranging prophylactic strategies.

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.

This study was designed to monitor the presence of Mycoplasma agalactiae and Mycoplasma mycoides subsp. capri (Mmc) in 66 dairy goat herds of a genetic improvement programme in a region of Spain where contagious agalactia is endemic. Over a whole lactation period, 300 bulk tank milk and 381 milk samples from goats with clinical mastitis were subjected to polymerase chain reaction (PCR) to detect the two mycoplasma species. The presence of mycoplasmas (either species or both) was detected in 66.7% of the herds and M. agalactiae was identified in 95.45% of these positives herds. In a given infected herd, mycoplasmas were not continuously detected over the whole study period. Our findings indicate that in an endemic area, M. agalactiae and Mmc can be monitored through PCR analysis of mastitic milk and bulk tank milk (BTM) samples. Over a lactation period we recommend testing multiple BTM samples on a herd. No relationship was observed between the use of inactivated mycoplasma vaccines and the PCR detection of both mycoplasmas.

Goat milk samples routinely obtained by milk distributing companies and quality control laboratories are preserved using azidiol (AZ) or bronopol (BR). This study was designed to determine if these treated goat milk samples are suitable for Mycoplasma ( M. ) agalactiae detection by PCR. The effects of these preservatives on the limits of M. agalactiae direct PCR detection were established using samples inoculated with mycoplasma cultures. Samples inoculated with a determined concentration of M. agalactiae showed no differences among the 3 preservation methods and in all cases the limits of detection of the PCR assays were 10 3 CFU ml. In addition, 300 bulk tank milk (BTM) samples from 45 herds from an infected area were each divided into three aliquots subjected to different preservation treatments (no preservative, NP, AZ or, BR) and then PCR tested. Of the 300 BTM samples, 43 different ones scored a positive result for M. agalactiae in at least one condition distributed in this way, 32 NP aliquots (74.4% of the positive samples), 27 BP aliquots (62.8%) and 27 AZ aliquots (62.8%). Among the three preservation methods, no significant differences were found ( p > 0.05) and the difference in results was attributed to the sensitivity increasement when analyzing more than one time a BTM sample. These results suggest that the PCR technique can be used as a rapid diagnostic method to detect M. agalactiae in AZ or BR preserved goat milk samples. The possible use of such samples for health programmes would have considerable cost benefits.

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.

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.

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.

This study was designed to compare the ELISA method of diagnosing caprine arthritis encephalitis virus (CAEV) using two different samples: milk whey or blood samples. A commercial CAEV diagnosis kit (ELISA Kit MVV/CAEV serum monocupule, Institut Pourquier, France), was used and determination was carried out on paired samples of blood and milk from 66 lactating dairy goats of known CAEV infection status belonging to two herds. Serum samples were pre-diluted 1:20 and whey samples 1:2. All animals whose sera tested positive for the virus (37/66) also showed a positive whey sample result, and the same agreement was obtained for a negative result (29/66). In no sample was the outcome of the test dubious. Through ANOVA, it was determined that mean S/P ratios for whey (1.551 ± 1.55) and serum (1.719 ± 1.38) did not significantly differ (P > 0.05). Our findings indicate full agreement (Kappa = 1) in terms of the two sample types producing a positive or negative result and a high correlation coefficient (r = 0.92) between the mean S/P ratios recorded for the two data sets. In conclusion, the use of ELISA on whey samples is an appropriate method of diagnosing CAEV with its obvious benefits of non-invasive sample collection and reduced costs over traditional serological diagnosis methods. This simpler method will be especially useful for CAEV surveillance in dairy goats.

A longitudinal study was conducted on two dairy farms to investigate the pattern of shedding of verotoxin-producing Escherichia coli (VTEC) in goats. Faecal samples were taken from 20 goat kids once weekly during the first 4 weeks of life and then once every month for the next 5 months of life, and from 18 replacement animals and 15 adults once every month for 12 months. The proportion of samples containing VTEC was higher for replacement animals and adults (85.7% and 78.7%, respectively) than for goat kids (25.4%). About 90% of the VTEC colonies isolated from healthy goats belonged to five serogroups (O33, O76, O126, O146 and O166) but the most frequent serogroups of these isolates, except one, were different in the two herds studied. E. coli O157:H7 was found in three goat kids on only one occasion. None of the VTEC isolates, except the three E. coli O157:H7 isolates, was eae-positive. The patterns of shedding of VTEC in goat kids were variable, but, in contrast, most of the replacement animals and adults were persistent VTEC shedders. Our results show that isolates of VTEC O33, O76, O126, O146 and O166 are adapted for colonising the intestine of goats but that, in contrast, infection with VTEC O157:H7 in goats seems to be transient.

A 7 month prospective cohort study was designed to determine if feeding bromelain to dairy goats influenced the MSCC, milk yield, milk composition and the incidence of IMI. Forty-four clinically normal goats from 2nd to 6th parities were studied. Daily bromelain dosage was 7.4 grams/animal (185-mg/Kg weight). Samples for diagnostic bacteriology were collected from each udder half every 2 weeks. Samples for MSCC and composition were obtained every 42 days. Milk yield was also recorded every 42 days. Bromelain affected milk protein and fat but not MSCC, milk yield or milk lactose. Bromelain did not decrease the MSCC in healthy goats. Milk protein and fat increased in the bromelain treated group (P0.01), which is important for dairymen because premiums are paid milk fat and protein content. No clinical mastitis was detected in the goats for the total study period and incidence rate of subclinical IMI was 5.7%. Relative risk was 1.50 (0.28RR8.12) which means that the bromelain had no significant effect on IMI (P0.05). In addition, the use of pineapple by-products could be especially important in tropical countries were pineapple waste seems to be a pollution problem.

This cross-sectional study performed on dairy goat herds was designed to establish the relationship between the presence of Mycoplasma species in bulk-tank milk samples from different farms and the bulk-tank milk somatic cell count (BTMSCC) in an area where contagious agalactia (CA) is endemic. Three BTMSCC thresholds, used in payment schemes or as legal requirements for milk quality in Europe and the USA, were considered: (1) 2,000,000 cells/ml; (2) 1,500,000 cells/ml and (3) 1,000,000 cells/ml. Of the 1068 milk samples tested, 7.9% (n = 84) showed the presence of Mycoplasma spp. (Mycoplasma agalactiae 82% and Mycoplasma mycoides subsp. mycoides large colony 17%). Somatic cell counts for bulk-tank samples containing mycoplasmas were higher than those recorded for negative samples (1,176,000 cells/ml vs. 875,000 cells/ml; P

Contagious agalactia (CA) is an infectious syndrome caused by several species of mycoplasma and classically characterized by the triad of mammary, joint and eye symptoms, although further symptoms may also appear. CA is the most serious disease of whatever they affect in small dairy ruminants and so there is an urgent need for administrative bodies and farmers to join forces and design serious strategies with the final aim of completely eradicating the disease. CA is endemic in the Mediterranean and certain Asian and African regions, while in America it is described as sporadic. The emergence of CA in a non-infected herd is almost always linked to the introduction of carriers of CA or to contact with infected herds. It normally implies the rapid spread of clinical problems in the herd coinciding with the period of parturitions and the start of lactation. In endemic areas, this disease provokes subclinical or mild clinical symptoms, and the lack of an established, systemized diagnostic procedure implies, that many infected herds pass unnoticed and may act as a permanent source of infection. Enzyme-linked immunosorbent assay (ELISA) techniques are recommended for the detection of antibodies, but the systematic vaccination of herds in endemic areas prevents the use of serology to detect infected herds. Then, culture and polymerase chain reaction (PCR) techniques must be used. In clinical outbreaks, control measures should be directed towards limiting the economic losses of the herd and avoiding the spread of the disease by isolating the infected herd, antibiotic treatment, selective culling, disinfection measures and the classic prevention measures for mastitis (correct functioning and maintenance of milking equipment, the milking routine, teat dipping, etc.). In chronically infected herds, control measures should be directed to restrict disease spread by way of prevention measures for mastitis, detection and culling of animals with lesions, the administration of antimycoplasma drugs to all animals at the end of lactation, adequate management of replacement animals and, perhaps, vaccination.

This manuscript reviews the most recent knowledge about small ruminant mastitis, pointing out the etiological, epidemiological and control aspects of mastitis. The prevalence of subclinical mastitis in small ruminants averages 5–30%, but the annual incidence of clinical mastitis is generally lower than 5%. Staphylococcus spp., are the most prevalent pathogens responsible for intramammary infection in small ruminants. Mastitis caused by Staphylococcus aureus should be eliminated because of the severity of the clinical symptoms and also because of the risk of contamination of milk products by thermostable toxins. The public health impact of other pathogens causing mastitis is also emphasized in this review, and the efficacy of diagnostic tools is discussed, especially diagnostic bacteriological tests and determination of milk somatic cell counts (MSCC). Several mastitis control strategies are discussed, such

The purpose of the present study was to assess the presence of Mycoplasma agalactiae (Ma), the main causative agent of ovine contagious agalactia (CA), in semen of naturally infected rams. Therefore, semen samples from 167 rams residing in three different artificial insemination (AI) centers of a CA-endemic area were studied by microbiological and molecular techniques. In addition, serial ejaculates from the same rams were evaluated to determine the excretion dynamics of Ma. Of the 384 samples studied, Ma was detected in 56 (14.58%) which belonged to 44 different rams (26.35%). These findings confirm the ability of Ma to be excreted in semen of asymptomatic rams. Furthermore, these results also evidence the presence of these asymptomatic carriers of Ma in ovine AI centers, representing a serious health risk regarding the spread and maintenance of CA, especially in endemic areas. Moreover, the excretion of Ma in semen also points to the risk of venereal transmission of this disease. The current results highlight the need to implement control measures to prevent the admission of infected rams in AI centers and the necessity to continuously monitor semen samples to effectively detect infected individuals.

The viability of Mycoplasma agalactiae and Mycoplasma mycoides subsp. capri (Mmc) was assessed in goat colostrum treated at different temperatures. Samples of colostrum were inoculated with reference strains of M. agalactiae (PG2) and Mmc (PG3) and heated at 56 °C or 60 °C for 0, 30, 60, 90 or 120 min. Viable colonies of M. agalactiae were recovered after all treatments and there was a significant reduction in the concentration of viable M. agalactiae after 30 min at 56 °C and 60 °C. No viable colonies of Mmc were observed after 60 min at 60 °C.

Intramammary infection in seven commercial goat herds was studied using premilking and postmilking samples for purposes as bacteriological diagnosis. Using a positive result on both premilking and postmilking samples as the definitive diagnosis, we compared the efficacy of single samples collected either premilking or postmilking. With this aim, 2268 bacteriological culture results were compared. The κ values (0.60) showed moderate agreement between the two samples. Specificity and positive predictive value were higher for postmilking samples than for premilking samples. Specificity of postmilking samples was 99.4% for coagulase-negative staphylococci, 99.9% for Gram-negative bacilli, 100% for streptococci, 99.9% for corynebacteria and 100% for mixed cultures. Premilking samples specificity was 96.6, 99.5, 99.7, 99.8 and 99.8%, respectively. False positive diagnoses were more frequent for premilking samples. The results suggest that postmilking samples should be used to diagnosis of goat intramammary infection.

With the aim of evaluating the effect of freezing goat milk samples on recovery of intramammary pathogens, 1200 milk samples from udder halves with subclinical intramammary infection were studied. Samples (20 ml) were frozen at -20 and at -80 degrees C. Thawing was carried out at room temperature at 7, 14, 21, 28, 58, 118, 178, 236 and 730 days after collection and bacteriological analyses were carried out to determine the number of colony forming units/ml (CFU/ml). Mixed model statistical analysis showed that bacterial group, temperature of storage, interaction of bacterial group and temperature of storage and the interaction of bacterial group, time and temperature of storage were statistically significant effects. For coagulase negative staphylococci (CNS), least squares means of log CFU/ml recovered at -20 and -80 degrees C were not different. Nevertheless, for Gram negative bacilli (GNB) a significant decrease was detected in samples frozen at -20 vs. -80 degrees C. At both temperatures and at different times of storage, significant increases were detected between log CFU/ml of CNS and values on day zero. At -20 degrees C, a significant decrease in GNB recovery was detected between freezing days zero and 730. This difference was not detected when goat milk samples infected by GNB were frozen at -80 degrees C. The results show that frozen milk samples can be useful in goat subclinical mastitis control programs.

Pathogens involved in goats mastitis and the strategies for their prevention and control are revised. Staphylococcus aureus is an important clinical pathogen but coagulase negative staphylococci (CNS) are the most prevalent in subclinical mastitis (more than 50% in most of the studies). Incorrect milking machine routines are usually involved in high prevalence of CNS. Contagious agalactia (CA) is an important disease affecting goats and antibiotic treatment and vaccination are controversial. Streptococci and Gram negative bacilli (GNB) are major intramammary pathogens but not very frequent in goats and are associated with poor hygienic conditions in housing and the milking parlour. Because of the lower prevalence of GNB mastitis in goats than cows, physiological and environmental factors protecting goats from GNB infection are discussed. Other pathogens affecting the caprine mammary gland are also discussed, such as Arcanobacterium pyogenes, Bacillus spp., Clostridium perfringens, Nocardia asteroides and lentiviruses (caprine arthritis-encephalitis virus).

Mycoplasma capricolum subsp. capricolum is one of the causative agents of contagious agalactia (CA). Nevertheless, there is still a lack of information about its antimicrobial susceptibility and genetic characteristics. Therefore, the aim of this work was to study the antimicrobial and genetic variability of different Mycoplasma capricolum subsp. capricolum field isolates. For this purpose, the growth inhibition effect of 18 antimicrobials and a multilocus sequence typing (MLST) scheme based on five housekeeping genes (fusA, glpQ, gyrB, lepA and rpoB) were performed on 32 selected field isolates from Italy and Spain.The results showed a wide range of growth inhibitory effects for almost all the antimicrobials studied. Macrolides presented lower efficacy inhibiting Mcc growth than in previous works performed on other CA-causative mycoplasmas. Erythromycin was not able to inhibit the growth of any of the studied strains, contrary to doxycycline, which inhibited the growth of all of them from low concentrations. On the other hand, the study of the concatenated genes revealed a high genetic variability among the different Mcc isolates. Hence, these genetic variations were greater than the ones reported in prior works on other mycoplasma species.

Mycoplasma agalactiae (Ma) is the main causative agent of ovine contagious agalactia, which is a serious disease of small ruminants. In endemic areas, its most common clinical situation consists of chronically infected herds, and asymptomatic infected individuals represent an epidemiological risk regarding the transmission of this disease. The aim of this work was to detect the presence of asymptomatic rams infected with Ma in different artificial insemination centers, and to determine the most effective way to identify these individuals so as to implement adequate surveillance protocols. For this purpose, 215 rams and 14 teaser sheep were sampled taking auricular, nasal, and vaginal swabs and serum samples. In addition, ejaculates from 147 rams were analyzed. These samples were subjected to specific culture and molecular techniques to isolate and identify mycoplasmas, and to a serological test to detect antibodies against Ma. Mycoplasma agalactiae was detected in 47 (4.4%) of the 1077 samples analyzed, and also one individual resulted seropositive. Thus, 37 (17.2%) of the 215 studied rams were infected with Ma. The specimens which proportionally yielded the greatest number of positive results for this pathogen were semen samples (13.6%), followed by nasal swabs (5.8%). In contrast, the sampling of the external auricular canal and the serological analyses resulted insufficient to effectively detect infected individuals. Asymptomatic rams infected with Ma were detected in all the analyzed artificial insemination centers, highlighting the need to implement adequate surveillance protocols to prevent the presence of these individuals in these centers, reducing the risk of transmitting contagious agalactia.

Somatic cell counts, the bacteriological condition of the milk and antibodies against caprine arthritis encephalitis virus (cAEv) were measured monthly throughout lactation in 121 lactating goats of the Murcia- Granada breed in four commercial dairy goat herds. The prevalence of bacterial intramammary infection was 5.6 per cent and the prevalence of cAEv infection was 20.6 per cent. An analysis of variance revealed a significant effect of herd, intramammary infection and the interaction between intramammary infection and CAEV on the somatic cell count. In udder halves free of intramammary infection, the somatic cell counts were significantly lower in seronegative goats than in seropositive goats (P

The antibiotic susceptibility of 32 strains of Staphylococcus aureus isolated from the mastitic milk of dairy goats was evaluated. The antibiotics tested were 3 fluoroquinolones that have been developed especially for use in veterinary medicine: danofloxacin, marbofloxacin, and orbifloxacin. Minimum inhibitory concentration (MIC) tests were performed according to the microdilution broth method. The MIC(90) (concentration at which 90% inhibition is achieved) values obtained were 0.5, 1, and 1 mg/L for danofloxacin, marbofloxacin, and orbifloxacin, respectively. Danofloxacin was the most active fluoroquinolone tested against Staph. aureus strains isolated from milk; however, specific testing is required before using these drugs as therapy for the control of clinical mammary infections in goats.

Contagious agalactia affects goats and is caused by several species of mycoplasma including Mycoplasma agalactiae and Mycoplasma mycoides subsp. capri (Mmc). Male goats, latently infected with M. agalactiae and Mmc, were identified at a dairy goat breeding artificial insemination centre. In three samplings, conducted over 1 year, ear swabs were assessed for both of the above organisms using culture and PCR techniques. Serological examination for antibodies against these organisms was performed at each time-point and conjunctival, nasal, rectal and preputial swabs were taken from a sub-sample of animals. Mycoplasma mycoides subsp. capri and M. agalactiae were detected in 80 and four ear swabs, respectively and serology confirmed the presence of both agents. A point prevalence of 0.06 goats infected with Mmc at the first sampling point increased to 0.97 at the last sampling, suggesting spread of infection. Both organisms were also detected in preputial and conjunctival swabs suggesting the shedding of these pathogens by other routes. These findings should inform World Organisation for Animal Health (OIE) guidelines on avoiding the introduction of such pathogens into artificial insemination centres and suggest the need to review current recommendations.

In a cross-sectional study, 324 lactating goats of the Murciano–Granadina breed were randomly selected from 18 herds. Animals were in late lactation and bacteriological analyses including mycoplasmas were made from aseptic milk samples individualy taken from udder halves. The presence of intramammary infection (IMI) was studied in relation to goat parity. Three parity groups were considered: first and second; third, fourth and fifth; and six and greater parities. Prevalence of IMI was 22% of halves and 34% of animals. Most of isolates were Staphylococci (70%). Using the prevalence ratio (PR) and confidence interval (CI), we saw a positive statistical association between subclinical intramammary infection and greater than fifth parity (PR=1.80; 95% CI=1.21, 2.68).

In cows milk, analytical temperature has been identified as one of the possible factors affecting the somatic cell count (SCC). To establish the effect of the temperature used for SCC in goats milk, counts were performed on 45 goats milk samples using the Fossomatic counter. Tests were performed at two temperatures (40° and 60 °C) on preservative-free samples 3 h after collection, and on samples preserved with bronopol (BR) at 3 h, 1, 2, 3, and 4 d post-collection. The test temperature did not modify the SCC of the milk samples analyzed. Incubating samples 3 h post-collection at 60 °C failed to improve the SCC results. Similar counts were obtained for BR-preserved samples stored for 1–4 d at refrigeration temperature, suggesting the possibility of performing Fossomatic SCC in goats milk samples stored for this length of time.

Three hundred and sixty-nine milk samples from half udders of 188 Murciano-Granadina goats in the second and third month of lactation, from ten commercial dairy goat herds were studied. Bacteriological analyses, California Mastitis Test (CMT) and somatic cell count (SCO by Fossomatic Counter were performed. The best SCC threshold for defining subclinical mastitis was 500 × 103 cells ml−1, but this threshold had a poor positive predictive value (28.5%) and only 62.3% of samples were correctly classified. Arithmetical and geometrical means of SCC showed statistical differences between infected and uninfected glands. Arithmetical and geometrical means of SCC per microlitre for each CMT score were, respectively: 312 × 103 and 172 × 103 for Score 0 and traces; 1014 × 103 and 531 × 103 for Score 1; 2912 × 103 and 2051 × 103 for Score 2; and 4950 × 103 and 4436 × 103 for Score 3. The CMT Scores 2 and 3 discriminated between infected and uninfected udder glands. The results show that both tests could be used in the period of study to detect a high percentage of true uninfected glands but the percentage of false positives was high.

Ten herds of Murciano-Granadina goats were examined to determine prevalence and aetiology of subclinical mastitis in Murcia (Spain). In the 369 milk samples analysed from 188 animals, intramammary infection was present in 18% of the glands and 30% of the goats. Prevalence of infection in the different herds ranged from 7% to 34% of the glands and 17% to 44% of the goats. Sixty-nine bacterial isolates were found, comprising staphylococci (71%), corynebacteria (12%), mycoplasmas (9%), enterobacteria (3%), pasteurellas (3%), streptococci (1%) and yeast (1%). Among the 49 staphylococcus isolates, Staphylococcus caprae (22%), Staphylococcus epidermidis (20%) and Staphylococcus chromogenes (12%) were the most frequent of the 12 species identified. Staphylococcus aureus (6%) had a low prevalence, and only one isolate was coagulase positive. S. caprae showed the highest prevalence among the herds, and mycoplasmas and S. epidermidis were most responsible for bilateral infections (60% of those detected).

Staphylococcus aureus is one of the most important pathogenic Staphylococcus species in veterinary medicine. In rabbits, it mainly causes skin infections, pododermatitis and mastitis with subsequent economic losses in industrial rabbit farms (Hermans and others 1999). Fluoroquinolones are antimicrobial drugs which generally have very good activities against a broad spectrum of aerobic bacteria, including Pasteurella species, and mycoplasma (Walker 2000). The main target site for its bactericidal action is the deoxyribonucleic acid (DNA)-gyrase. This enzyme is required for supercoiling of DNA to provide spatial arrangement of DNA in the bacterial cell. Furthermore, fluoroquinolones have other beneficial characteristics such as large volumes of distribution, low plasma protein binding and relatively low minimum inhibitory concentrations (MICs) against susceptible target microorganisms (Brown 1996). Marbofloxacin (MAR) and enrofloxacin (ENR), are relatively new synthetic fluoroquinolone antimicrobial drugs which have been developed especially for using in veterinary medicine, and they could be potentially useful in rabbits. Therefore, this study was designed to evaluate the degree of in vitro activity of MAR and ENR against 27 S aureus strains isolated from rabbits with chronic problems of staphylococcosis in Spain. A total of 27 S aureus strains were isolated from commercial rabbit farms …

This study sought to determine whether male goat auricular carriers of mycoplasmas known to cause contagious agalactia could harbour these microorganisms at anatomical sites other than the ears. A microbiological study was conducted in 6 naturally infected bucks that had been diagnosed as chronic auricular asymptomatic carriers of Mycoplasma (M.) mycoides subsp. capri (Mmc) more than one year previously. To detect mycoplasmas, cultures and PCR were performed on 46 samples taken from each goat from the cardio-respiratory, digestive, nervous, lymph and genitourinary systems and several joints. Of a total of 274 samples analyzed, 28 were positive for mycoplasmas (10.1%): Mmc was detected in 17 (6.1%), Mycoplasma (M.) agalactiae in 12 (4.3%) and both microorganisms were identified in one of the samples. In all 6 goats, mixed infection was observed despite none being auricular carriers of M. agalactiae. Mycoplasma spp. were identified at 15 different sites; the most frequent sites being the joints (31.2%, 5 positive samples), lymph nodes (25%, 4 positive samples) and respiratory tract (25%, 4 positive samples). Positive results were also obtained in three brain tissue (18.7%), two cardiac tissue (12.5%) and one ileum, urethra, testicle and bulbourethral gland (6.25%) samples. The histopathological findings may suggest the presence of mild chronic conditions in some of the organs where the bacteria were found. Our findings reveal for the first time the capacity of Mmc and M. agalactiae to colonize several other organ systems in chronically naturally infected auricular carriers, possibly representing an added risk factor for the spread of these microorganisms. In the case of M. agalactiae, colonization seemed to be independent of the animal's auricular carrier state.

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.