Salmonella and Campylobacter in broilers at slaughter age : a possible source for carcasses contamination in Ecuador

Salmonella and Campylobacter are major foodborne pathogens worldwide and are especially prevalent in the poultry meat chain. Although poultry meat is a principal component of the diet in Ecuador, little is known about the presence of these pathogens in this food chain. The aim of this thesis was to collect data about Campylobacter and Salmonella in the broiler meat chain in Ecuador. To fulfill this objective, we assessed the prevalence and characterized these pathogens in broiler batches at slaughter age and studied the contamination dynamics during the slaughter of Campylobacter positive batches in commercial slaughterhouses. Chapter 1 and Chapter 2 focus on Salmonella and Campylobacter, for which samples from 388 and 379 broiler batches respectively were collected and analyzed by genotypic and phenotypic techniques. Salmonella prevalence at batch level was 16.0%. The most common serotype was S. Infantis (83.9%). S. Infantis isolates showed high resistance rates to 12 antibiotics ranging from 57.7% (kanamycin) up to 98.1% (nalidixic acid and sulfamethoxazole). The prevalence of Campylobacter at batch level was 64.1% and C. coli (68.7%) was the most common species. MIC values showed resistance rates above 67% for tetracycline, ciprofloxacin and nalidixic acid. Genotypic characterization of both pathogens demonstrated that they are widely distributed in farms. In Chapter 3, the dynamics of Campylobacter contamination were studied in three slaughterhouses. The impact of evisceration, final washing and water chilling was evaluated by quantifying Campylobacter contamination. No significant differences were found between Campylobacter counts after evisceration and after final washing, denoting a lack of efficiency of this step. In all slaughterhouses, a significant reduction of Campylobacter counts was found after the chilling step. We reported that S. Infantis and C. coli were the most prevalent ones, which differs from what is reported in other Latin American countries. High resistance rates to antibiotics used in human medicine were found in these pathogens. This could be provoked by the extensive use of antimicrobials in poultry production and might pose a human health concern. The attribution of S. Infantis and C. coli to human infections need further research. Evaluation of Campylobacter counts at slaughterhouses showed that final washing and addition of chlorine in chilling water may be used to decrease Campylobacter numbers on carcasses. This thesis represents the first study on the epidemiology of Campylobacter and Salmonella in the poultry meat chain in Ecuador

Prevalence, antimicrobial resistance and genetic diversity of Campylobacter coli and Campylobacter jejuni in Ecuadorian broilers at slaughter age

Thermotolerant Campylobacter spp. are a major cause of foodborne gastrointestinal infections worldwide. The linkage of human campylobacteriosis and poultry has been widely described. In this study we aimed to investigate the prevalence, antimicrobial resistance and genetic diversity of C. coli and C. jejuni in broilers from Ecuador. Caecal content from 379 randomly selected broiler batches originating from 115 farms were collected from 6 slaughterhouses located in the province of Pichincha during 1 year. Microbiological isolation was performed by direct plating on mCCDA agar. Identification of Campylobacter species was done by PCR. Minimum inhibitory concentration (MIC) values for gentamicin, ciprofloxacin, nalidixic acid, tetracycline, streptomycin, and erythromycin were obtained. Genetic variation was assessed by RFLP-flaA typing and Multilocus Sequence Typing (MLST) of selected isolates. Prevalence at batch level was 64.1%. Of the positive batches 68.7% were positive for C. coli, 18.9% for C. jejuni, and 12.4% for C. coli and C. jejuni. Resistance rates above 67% were shown for tetracycline, ciprofloxacin, and nalidixic acid. The resistance pattern tetracycline, ciprofloxin, and nalidixic acid was the dominant one in both Campylobacter species. RFLP-flaA typing analysis showed that C. coli and C. jejuni strains belonged to 38 and 26 profiles respectively. On the other hand MLST typing revealed that C. coli except one strain belonged to CC-828, while C. jejuni except 2 strains belonged to 12 assigned clonal complexes (CCs). Furthermore 4 new sequence types (STs) for both species were described, whereby 2 new STs for C. coli were based on new allele sequences. Further research is necessary to estimate the impact of the slaughter of Campylobacter positive broiler batches on the contamination level of carcasses in slaughterhouses and at retail in Ecuador

Characterization of cefotaxime resistant Escherichia coli isolated from broiler farms in Ecuador

Antimicrobial resistance (AR) is a worldwide concern. Up to a 160% increase in antibiotic usage in food animals is expected in Latin American countries. The poultry industry is an increasingly important segment of food production and contributor to AR. The objective of this study was to evaluate the prevalence, AR patterns and the characterization of relevant resistance genes in Extended Spectrum beta-lactamases (ESBL) and AmpC-producing E. coli from large poultry farms in Ecuador. Sampling was performed from June 2013 to July 2014 in 6 slaughterhouses that slaughter broilers from 115 farms totaling 384 flocks. Each sample of collected caeca was streaked onto TBX agar supplemented with cefotaxime (3 mg/l). In total, 176 isolates were analyzed for AR patterns by the disk diffusion method and for bla(CTX-M), bla(TEM), bla(CMY), bla(SHV), bla(KPC), and mcr-1 by PCR and sequencing. ESBL and AmpC E. coli were found in 362 flocks (94.3%) from 112 farms (97.4%). We found that 98.3% of the cefotaxime-resistant isolates were multi-resistant to antibiotics. Low resistance was observed for ertapenem and nitrofurantoin. The most prevalent ESBL genes were the ones belonging to the bla(CTX-M) group (90.9%), specifically the bla(CTX-M-65), bla(CTX-M-55) and bla(CTX-M-3) alleles. Most of the AmpC strains presented the bla(CMY-2) gene. Three isolates showed the mcr-1 gene. Poultry production systems represent a hotspot for AR in Ecuador, possibly mediated by the extensive use of antibiotics. Monitoring this sector in national and regional plans of AR surveillance should therefore be considered

Antibiotic prescription patterns and non-clinical factors influencing antibiotic use by Ecuadorian veterinarians working on cattle and poultry farms: A cross-sectional study

Understanding antibiotic prescription patterns and non-clinical factors influencing antibiotic use is essential for implementing strategies to promote appropriate antibiotic use. There is, however, limited research exploring these issues with Ecuadorian veterinarians. Therefore, a questionnaire was developed and applied cross-sectionally to veterinarians (n = 173) from two professional organizations to explore the antibiotic prescription patterns and non-clinical factors (e.g., attitudes and perceptions) influencing antibiotic use, and to identify strategies to reduce antibiotic use. The response rate was 78.4%. Responses were compared between veterinarians working mainly on cattle and poultry farms using Mann-Whitney U tests. The most important attitudes, beliefs and perceptions towards antimicrobial resistance (AMR) and antibiotic use were identified with the Relative Importance Index (RII). Veterinarians showed high awareness of AMR and its implications for public health, as well as the necessity of reducing antibiotic use. However, some veterinarians appear to underestimate the potential contribution of veterinary antibiotic use on AMR in humans. Veterinarians self-reported high prescription (> 20%) of antibiotics for cattle and poultry that are critically important for human medicine, such as 3rd and 4th generation cephalosporins, polymyxins and quinolones. Further, antibiotic therapy was not tailored to disease type. Cattle and poultry veterinarians perceived similar barriers to increasing antibiotic stewardship including: poor biosecurity measures, animal confinement, low feed quality, farmers’ behaviors (such as stopping antibiotic treatment, storing antibiotics on farms, buying antibiotics in veterinary supply stores), and sales agents’ roles as non-professional prescribers of antibiotics. Overall, veterinarians were broadly supportive (>90%) of most strategies to promote appropriate antibiotic use. They saw more merit in improving biosecurity of farms and implementing educational programs for farmers and veterinarians. This study provides insight into the complexity of antibiotic use on Ecuadorian farms and the need for holistic strategies in a One Health context, to achieve antibiotic stewardship

Molecular characterization of infectious bronchitis virus in laying hen farms located in Tungurahua province, Ecuador

La bronquitis infecciosa aviar genera importantes pérdidas económicas y, si bien la vacunación disminuye dichas mermas, el surgimiento continuo de nuevas cepas virales complica el control de la infección. El objetivo de esta investigación fue caracterizar, mediante técnicas moleculares, las cepas del virus de la bronquitis infecciosa aviar circulantes en explotaciones de gallinas ponedoras de la provincia de Tungurahua, Ecuador. Se trabajó con muestras de hisopados y de órganos de 47 granjas y las secuencias obtenidas fueron comparadas con las correspondientes a 17 vacunas a virus vivo empleadas en la zona. Se encontraron 16 granjas positivas al amplificar la región 5’UTR y un segmento de S1. La construcción de un árbol filogenético mostró que cinco de las secuencias se ubicaron en el mismo clado de las cepas de tipo Massachusetts, linaje GI-1; siete se encontraron en el clado del linaje GI-13 o cepas tipo 793B y cuatro secuencias se agruparon en un clado de cepas tipo Q1, linaje G1-16. Si bien se requieren estudios moleculares más completos en los que se amplifique la totalidad del gen S a fin de obtener datos más concluyentes, se logró determinar que existen tres tipos de cepas circulando en la provincia de Tungurahua, dos posiblemente vacunales y otra de origen desconocido.Avian infectious bronchitis (IBV) generates significant economic losses. While vaccination decreases these losses, new viral strains are continually emerging. The objective of this research was to perform molecular characterization of IBV strains circulating in laying hen farms in Tungurahua province, Ecuador. Swabs and organ samples from 47 farms were collected and sequenced, and results were compared to 17 live vaccines used in the area Sixteen farms were positive for IBV as determined by amplification of the 5'UTR region and a S1 segment. The construction of a phylogenetic tree showed that 5 samples were in the same clade as the Massachusetts-type strains, linage GI-1; 7 samples were in the GI-13 lineage clade of vaccine type 793B strains, and 4 samples were into a clade of Q1 type strains. In conclusion, although studies that include the entire protein S sequence are necessary, we were able to detect three types of strains circulating in the province of Tungurahua, two of which are probably vaccine-derived and one has an unknown origin.Facultad de Ciencias Veterinaria

Molecular characterization of infectious bronchitis virus in laying hen farms located in Tungurahua province, Ecuador

La bronquitis infecciosa aviar genera importantes pérdidas económicas y, si bien la vacunación disminuye dichas mermas, el surgimiento continuo de nuevas cepas virales complica el control de la infección. El objetivo de esta investigación fue caracterizar, mediante técnicas moleculares, las cepas del virus de la bronquitis infecciosa aviar circulantes en explotaciones de gallinas ponedoras de la provincia de Tungurahua, Ecuador. Se trabajó con muestras de hisopados y de órganos de 47 granjas y las secuencias obtenidas fueron comparadas con las correspondientes a 17 vacunas a virus vivo empleadas en la zona. Se encontraron 16 granjas positivas al amplificar la región 5’UTR y un segmento de S1. La construcción de un árbol filogenético mostró que cinco de las secuencias se ubicaron en el mismo clado de las cepas de tipo Massachusetts, linaje GI-1; siete se encontraron en el clado del linaje GI-13 o cepas tipo 793B y cuatro secuencias se agruparon en un clado de cepas tipo Q1, linaje G1-16. Si bien se requieren estudios moleculares más completos en los que se amplifique la totalidad del gen S a fin de obtener datos más concluyentes, se logró determinar que existen tres tipos de cepas circulando en la provincia de Tungurahua, dos posiblemente vacunales y otra de origen desconocido.Avian infectious bronchitis (IBV) generates significant economic losses. While vaccination decreases these losses, new viral strains are continually emerging. The objective of this research was to perform molecular characterization of IBV strains circulating in laying hen farms in Tungurahua province, Ecuador. Swabs and organ samples from 47 farms were collected and sequenced, and results were compared to 17 live vaccines used in the area Sixteen farms were positive for IBV as determined by amplification of the 5'UTR region and a S1 segment. The construction of a phylogenetic tree showed that 5 samples were in the same clade as the Massachusetts-type strains, linage GI-1; 7 samples were in the GI-13 lineage clade of vaccine type 793B strains, and 4 samples were into a clade of Q1 type strains. In conclusion, although studies that include the entire protein S sequence are necessary, we were able to detect three types of strains circulating in the province of Tungurahua, two of which are probably vaccine-derived and one has an unknown origin.Fil: Revelo Cueva, María Del Carmen. Universidad Central del Ecuador; EcuadorFil: Vinueza Burgos, Christian Vinicio. Universidad Central del Ecuador; EcuadorFil: Metz, German Ernesto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Microbiología. Cátedra de Virología; Argentina. Centro de Microbiologia Basica y Aplicada (cemiba) ; Facultad de Cs.veterinarias ; Universidad Nacional de la Plata;Fil: Toapanta, Ricardo Lenin. Universidad Central del Ecuador; EcuadorFil: Echeverria, Maria Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Microbiología. Cátedra de Virología; Argentina. Centro de Microbiologia Basica y Aplicada (cemiba) ; Facultad de Cs.veterinarias ; Universidad Nacional de la Plata

Perfil fenotípico de resistencia a los antimicrobianos en pinzones terrestres de tres zonas de la isla Santa Cruz en Galápagos

La resistencia a los antimicrobianos (RAM) representa un problema importante en la salud global. Bacterias, tales como los Enterococcus spp., son empleados como microorganismos centinela, para la determinación de la ocurrencia de la RAM. A su vez, se han aislado bacterias RAM en diversas especies de aves silvestres, siendo estas propuestas como potenciales reservorios y dispersores de genes de resistencia en el ambiente. El objetivo de esta investigación fue determinar los perfiles fenotípicos de RAM en Enterococcus spp., en la Isla Santa Cruz, Galápagos.Trabajo publicado en Cagliada, Maria del Pilar Lilia y Galosi, Cecilia Mónica (comps.). I Congreso de Microbiología Veterinaria. Libro de resúmenes. La Plata: Facultad de Ciencias Veterinarias, 2021.Facultad de Ciencias Veterinaria

Resistencia a los antimicrobianos en aislados de Escherichia coli y Enterobacter spp. aisladas de pinzones terrestres de la isla Santa Cruz

El incremento de resistencia antimicrobiana es un desafío para la salud global. La vida silvestre juega un papel importante para entender la dinámica de resistencia. Se ha reportado resistencia antimicrobiana en aves encontradas en hábitats remotos. Existe la posibilidad de estudiar aves silvestres como centinelas para vigilar la propagación de cepas resistentes. Los pinzones están expuestos a factores para adquirir y esparcir cepas resistentes; por lo que resultan de interés dentro del campo. El objetivo de este trabajo es determinar resistencia fenotípica en aislados de Escherichia coli y Enterobacter spp. procedentes de pinzones terrestres de Santa Cruz.Facultad de Ciencias Veterinaria

Presencia y Resistencia a los Antimicrobianos de serovariedades de Salmonella enterica aisladas en una empresa avícola integrada del Ecuador

Salmonella is the cause of millions of cases of human gastroenteritis worldwide and over one hun-dred thousand deaths each year. A major source of this bacteria are poultry products. The widespread use of antibio-tics in poultry industry has favored the emergence of multidrug-resistant strains of Salmonella. Such strains could be transmitted to humans through consumption of contaminated meat products. The objective of this research was to identify serotypes and antimicrobial resistance patterns of Salmonella strains isolated from an integrated poultry company in Ecuador. A total of 289 samples were analyzed with the ISO 6579 Annex D protocol. The results showed 20.1% of positive samples. Four serotypes were found from which S. Infantis (86.2%) was the most frequent serotype. Most Salmonella strains were resistant to nitrofurantoin (94.8 %), tetracycline (82.8%), chloramphenicol (79.3%) and trimetopin-sulfamethoxazole (81%). The results shown in this study indicate that Salmonella may be present in di-fferent processes of Ecuador’s poultry industry and that antibacterial resistance strains isolated from chicken meat may represent a risk to public health.A nivel mundial Salmonella es la responsable de causar millones de casos de gastroenteritis humana y más de cien mil defunciones cada año. Una de las principales fuentes de contagio esta bacteria son los productos de origen aviar. La utilización generalizada de antimicrobianos en la industria avícola ha favorecido el surgimiento de cepas de Salmonella multirresistentes. Dichas cepas podrían transmitirse al ser humano a través del consumo de productos cárnicos contaminados. El objetivo de esta investigación fue identificar serotipos y patrones de resistencia antimicrobiana de cepas de Salmonella aisladas en una empresa avícola integrada en Ecuador. Al final del período de muestreo se recolectaron 289 muestras que fueron analizadas en base a la norma NTE INEN-ISO 6579 Anexo D. El 20.1% de las muestras fueron positivas a Salmonella, identificándose 4 diferentes serovariedades, siendo S. Infantis (74.1%) el serotipo más frecuente. La mayoría de cepas de Salmonella presentaron resistencia a nitrofurantoína (94.8 %), tetraciclina (82.8%), cloranfenicol (79.3%) y trimetopin-sulfametoxazol (81%). Los resultados de este estudio indican que Salmonella puede estar presente en los diferentes procesos de la industria avícola ecuatoriana, además la resistencia antibacteriana de cepas aisladas en carne de pollo puede representar un riesgo para la salud pública