Antimicrobial drug resistance and molecular characterization of Salmonella isolated from domestic animals, humans, and meat products.

OBJECTIVES: 1) To characterize and determine genotypic relatedness of Salmonella serovars commonly isolated from domestic animals and humans in North Dakota, and 2) to assess their role in transferring antimicrobial resistance (AMR) to humans. MATERIALS AND METHODS: A total of 434 Salmonella isolates obtained from 1) feces of apparently healthy feedlot, range, and dairy cattle in North Dakota; 2) clinical samples from sick or dead animals submitted to North Dakota State University-Veterinary Diagnostic Laboratory (2000-2005); 3) previous meat product surveillance studies in North Dakota; and 4) 179 samples from human patients in North Dakota (2000-2005) by the North Dakota Department of Health were studied. The isolates were initially serotyped and later genotyped by pulsed-field gel electrophoresis (PFGE) to investigate their relatedness. The National Antimicrobial Resistance Monitoring Systems panel was used to compare AMR profiles of animal and human isolates to assess a possible role of domestic animals in transfer of AMR to humans. RESULTS: Salmonella Typhimurium was the predominant serotype in both humans (13.4%) and domestic animals (34.3%), followed by Newport in animals (2.6%) and human (3.9%). Salmonella Arizona (0.7%), Salmonella Give (0.9%), and Salmonella Muenster (3.5%) were isolated from sick or dead animals. PFGE results confirmed occurrence of similar Salmonella genotypes in both domestic animals and humans. AMR profiles showed that most animal strains were multidrug resistant. A single human isolate had PFGE and multidrug resistance profiles similar to a major cattle genotype, suggesting a possible AMR transmission from cattle to humans. CONCLUSION AND APPLICATION: Similar Salmonella genotypes were infecting domestic animals and humans in North Dakota. The AMR levels were higher in domestic animal isolates than in humans, implying that the occurrence of AMR in animal isolates may not translate directly into AMR in human isolates in North Dakota. This is helpful in determining future policies regarding antimicrobial drug use in domestic animals and humans.

Antimicrobial resistance and association with class 1 integrons in Escherichia coli isolated from turkey meat products.

The objective of this study was to quantify the role of class 1 integrons in antimicrobial resistance in Escherichia coli isolated from turkey meat products purchased from retail outlets in the Midwestern United States. Of 242 E. coli isolates, 41.3% (102 of 242) tested positive for class 1 integrons. A significant association was shown between presence of class 1 integrons in E. coli isolates and the resistance to tetracycline, ampicillin, streptomycin, gentamicin, sulfisoxazole, and trimethoprim-sulfamethoxazole. Attributable risk analysis revealed that for every 100 E. coli isolates carrying class 1 integrons, resistance was demonstrated for ampicillin (22%), gentamycin (48%), streptomycin (29%), sulfisoxazole (40%), trimethoprim-sulfamethoxazole (7%), and tetracycline (26%). Non-integron-related antimicrobial resistance was demonstrated for ampicillin (65%), gentamycin (16.9%), streptomycin (42.1%), sulfisoxazole (35.8%), and tetracycline (49.7%). Population-attributable fraction analysis showed that class 1 integrons accounted for the following resistances: gentamycin, 71% (50 of 71), amoxicillin-clavulanic acid, 19.6% (6 of 33), nalidixic acid, 34% (7 of 21), streptomycin, 28% (30 of 107), sulfisoxazole, 38% (40 of 106), and tetracycline, 14%, (26 of 185). In conclusion, although class 1 integrons have been implicated in resistance to antimicrobial agents, other non-integron resistance mechanisms seem to play an important part.

Characterisation of mycobacteria isolated from slaughter cattle in pastoral regions of Uganda.

BACKGROUND: Bovine tuberculosis is a zoonotic problem in pastoral cattle and communities in Uganda. Tuberculin tests in pastoral cattle had shown a high herd but low animal prevalence, with a high proportion of avian reactors. No work had been done to identify the mycobacterial species involved. The objective of the study was to isolate and characterise Mycobacterial species causing tuberculous lesions in slaughtered animals. Lesioned organs compatible with bovine tuberculosis in slaughtered cattle from pastoral areas in Uganda were collected and cultured to isolate mycobacteria. AccuProbe culture identification kits for the Mycobacterium tuberculosis complex, M. avium complex and M. avium were used to identify the isolates. Spoligotyping and Insertion Sequence (IS) 1311 and IS1245 Restriction Fragment Length Polymorphism analysis (RFLP) were used to further characterise the isolates. RESULTS: Of the 61 lesioned organs and tissues cultured, 19 isolates were identified as M. bovis, 3 as M. avium subsp.hominissuis, 1 as M. intracellulare, 1 as a mixed culture of M. bovis and M. avium sp. and 1 as M. avium sp. and unidentified mycobacteria. Eleven other mycobacteria outside the tuberculosis and avium complex groups were also isolated. Ten new spoligopatterns grouped into three clusters were identified from M. bovis isolates. Two of the three M. avium subsp.hominissuis isolates showed similar patterns on the IS1311 RFLP but all were different on the IS1245 RFLP. CONCLUSION: The isolation of M. bovis confirms the ongoing infection with spoligotypes unique to Uganda. Isolation of environmental mycobacteria could explain the high avian or non specific tuberculin reactor patterns commonly observed in pastoral cattle and suggests their pathogenic or opportunistic role in the infection of cattle with disseminated bovine tuberculous lesions.

Evaluation of three serological tests for brucellosis in naturally infected cattle using latent class analysis.

Serological methods are traditionally used in diagnosis of brucellosis. However, the comparative performance of these tests and their accuracy under the local environment in Zambia has not been assessed. Thus, the objective of our study was to evaluate the diagnostic performance of three serological tests for brucellosis; Rose Bengal Test (RBT), competitive ELISA (c-ELISA) and Fluorescence Polarisation Assay (FPA) in naturally infected cattle in Zambia without an appropriate reference test to classify animals into truly infected and non-infected. Serological test results from a study to determine sero-prevalence were used to compare the performance of RBT, c-ELISA and FPA in diagnosing brucellosis in traditional cattle. Since none of the tests can be seen as a perfect reference test or gold standard, their performance in a population of naturally infected cattle was evaluated using latent class analysis which allows the sensitivity (Se) and specificity (Sp) to be estimated in the absence of a gold standard. The highest Se was achieved by the c-ELISA (97%; Credible Posterior Interval (CPI)=93-100%) and the highest Sp by the FPA (93%; CPI=85-99%), conversely these tests also had the lowest Sp and Se, respectively, with the RBT performing well in both the Se (93%; CPI=84-98%) and Sp (81%; CPI=61-97).

Risk factors for brucellosis in indigenous cattle reared in livestock-wildlife interface areas of Zambia.

We conducted this cross-sectional study to investigate risk factors of Brucella seropositivity in cattle herds reared in livestock-wildlife interface areas of Blue Lagoon and Lochinvar National Parks in Zambia between August 2003 and September 2004. Sera were collected from cattle aged > or =2 years from 124 herds. Data on husbandry practices, grazing strategies, and herd structure (sex and age composition) were also collected. Sera were screened for anti-Brucella antibodies using the Rose Bengal test (RBT) as a presumptive test and a competitive-ELISA (c-ELISA) as a confirmatory test. A herd was classified as Brucella seropositive if at least one animal tested positive on both RBT and c-ELISA in series testing. Risk factors for herd-level brucellosis seropositivity were tested using multivariable logistic regression; risk factors for increases in the within-herd counts of seropositive cattle were analyzed using the negative binomial regression model with the number of seropositive animals as outcome and total number of cattle tested in a herd as the population at risk (exposure). Of the 110 herds tested, 68 (62; 95% CI: 53, 71% after adjusting for clustering by area) tested seropositive for exposure to Brucella spp. The final logistic-regression model identified geographical area, with Lochinvar (OR=3.4; CI: 0.97, 12) and Kazungula (OR=4.3; CI: 0.91, 20) recording higher odds of Brucella infections compared to Blue Lagoon. Herds coming in contact with wildlife had higher odds compared to those without contact (OR=3.4; CI: 1, 11). Similarly, the odds of Brucella infection were progressively higher in the larger herd categories (26-40 cattle, OR=2.6; CI: 0.70, 10; 41-82 cattle, OR=4.9; CI: 0.93, 26; >82 cattle, OR=9.4; CI: 1.7-51) compared to the smallest herd category (10-25). The negative binomial regression model identified geographical area, contact with wildlife, and herd size as having significant effect on counts of seropositive cattle in a herd.

Risk factors for herd-level bovine-tuberculosis seropositivity in transhumant cattle in Uganda.

We investigated the prevalence and risk factors to positive herd-level tuberculin reactivity between October 2003 to May 2004 to bovine tuberculosis (BTB) in the four transhumant districts of Uganda: three districts (Karamoja region) of nomadic transhumance cattle rearing (30 superherds and 1522 cattle), and one district (Nakasongola) of fixed-transhumance (7 herds and 342 cattle). We used the comparative intradermal skin-test, sampled 50 animals per superherd/herd, and considered herd positive if there was at least one reactor. Of the 30 superherds under nomadic transhumance, 60% (95% CI 41.4, 79) were tuberculin-test positive; of the 7 fixed herds, 14.3% (95% CI -20.7, 49.2) were tuberculin test positive. The true herd prevalence was estimated at 46.6%. Many risk factors were collinear. The final multivariable logistic-regression model included: recent introductions from market (OR=3.4; 95% CI 1.1, 10.3), drinking water form mud holes during dry season (OR=49; 95% CI 9.1, 262), and the presence of monkeys (OR=0.08; 95% CI 0.0, 0.6) or warthogs (OR=0.1; 95% CI 0.0, 0.3). No association was found between herd size or number of herd contacts with reactors; it was probably masked by the effect of high between-herd interactions. Provision of water from mud holes in dry river beds and introductions of new animals are risk factors that might be targeted to control BTB in transhumance areas.

Yield of undetected tuberculosis and human immunodeficiency virus coinfection from active case finding in urban Uganda.

OBJECTIVES: To determine the yield of undetected active tuberculosis (TB), TB and human immunodeficiency virus (HIV) coinfection and the number needed to screen (NNS) to detect a case using active case finding (ACF) in an urban community in Kampala, Uganda. METHODS: In a door-to-door survey conducted in Rubaga community from January 2008 to June 2009, residents aged ≥15 years were screened for chronic cough (≥2 weeks) and tested for TB disease using smear microscopy and/or culture. Rapid testing was used to screen for HIV infection. The NNS to detect one case was calculated based on population screened and undetected cases found. RESULTS: Of 5102 participants, 3868 (75.8%) were females; the median age was 24 years (IQR 20-30). Of 199 (4%) with chronic cough, 160 (80.4%) submitted sputum, of whom 39 (24.4%, 95%CI 17.4-31.5) had undetected active TB and 13 (8.1%, 95%CI 6.7-22.9) were TB-HIV co-infected. The NNS to detect one TB case was 131 in the whole study population, but only five among the subgroup with chronic cough. CONCLUSION: ACF obtained a high yield of previously undetected active TB and TB-HIV cases. The NNS in the general population was 131, but the number needed to test in persons with chronic cough was five. These findings suggest that boosting the identification of persons with chronic cough may increase the overall efficiency of TB case detection at a community level.

Mortality and commercial off-take rates in adult traditional cattle of Zambia.

A cohort study was conducted in the livestock-wildlife interface areas of Zambia to determine cattle mortality and commercial off-take rates among adult cattle as well as factors influencing them. A total of 416 animals from 43 herds were followed up for one year and animals were individually identified and their fate was indicated as sold, slaughtered, dead or present as appropriate. The overall mortality incidence risk was estimated at 7.5%. Cattle in Kazungula were at a greater risk of dying compared to those in Blue Lagoon and Lochnivar. Annual off-take was estimated at 13.7% (8.1-19.3%), unadjusted values, and 16.4% (8.1-24.5%) after adjusting for sampling fraction in primary sampling units (herds) and area stratification. Area variations were observed with Kazungula recording the highest in both instances, which was attributed to a contagious bovine pleural pneumonia (CBPP) outbreak. Herd size and gender were observed to influence cattle mortality rates. Cattle in the middle-sized herds (50-150 cattle) recorded high mortality rates (OR = 3.91) compared to smaller herds (10-50) and so were females compared to males (OR = 4.16). The logistic regression model showed that cattle death was influenced by managerial factors and that off-take rates tend to increase in the face of disease outbreaks.

Prevalence of bovine tuberculosis in Ethiopian slaughter cattle based on post-mortem examination.

A study aimed at describing the magnitude and distribution of gross lesions compatible with bovine tuberculosis (BTB) in Ethiiopian slaughter cattle in five abattoirs (four municipal and one export) located in various cattle husbandry systems in Ethiopia was carried out from July 2006 to January 2007 using detailed meat inspection procedure. Five representative abattoirs (four municipal and one export) located in distinct livestock management systems were selected. A total of 3322 cattle; 2876 (86.6%) male, 446 (13.4%) female; 3094 (93.1%) indigenous zebu, 140 (4.2%) crossbred and 88 (2.7%) pure exotic cattle were included in the study. A nine-year meat inspection record was also analyzed to elucidate the trend of BTB in the local cattle population.Of the carcasses inspected, 337 (10.2%, 95%CI= [9.2-11.2]) had lesions suggestive of tuberculosis, 69 (20.5%) generalized and 268 (79.5%) localized.TB prevalence showed a marked variation between categories of age, breed, class of animals, abattoir, geographic origin and husbandry system. It was higher in old and young animals than middle age group; in exotic than local breed; in calves than other classes of animals. The highest and lowest prevalence of TB was recorded in Adama (24.7%, 95%CI= [21.1-28.7]) and Yabello abattoirs (4.2%, 95%CI= [2.6-6.6]), respectively. Cattle whose origin was from Addis Ababa and its surrounding areas had higher prevalence of TB infection (23.9%, 95%CI= [17.6-31.5]).Cattle maintained in dairy farms had high degree of exposure (23.9%, 95%CI= [16.7-32.9]) to the infection than those maintained in other types of management system. Analysis of meat inspection records also revealed an increasing incidence of TB over the years. Our study demonstrated a high prevalence of tuberculosis in Ethiopian slaughter cattle and this could infer to similar scenario in a reference cattle population in the country. In view of Ethiopia's increasing involvement in livestock export trade, the reported high prevalence of tuberculosis could be a major obstacle, particularly at this moment when sanitary requirements from importing countries are so much strict. Furthermore, the growing concern over increasing incidence of tuberculosis/HIV/AIDS co-infection, the high incidence of extra- pulmonary tuberculosis and a high risk of acquiring zoonotic tuberculosis among the majority of the population emphasize the need for paying the necessary attention towards the control of bovine tuberculosis.

A cross-sectional study of bovine tuberculosis in the transhumant and agro-pastoral cattle herds in the border areas of Katakwi and Moroto districts, Uganda.

A study to determine the prevalence of bovine tuberculosis in the transhumant and agro-pastoral cattle herds in the border areas of Katakwi and Moroto districts in Uganda was carried out from July 2006 to January 2007 using comparative intradermal tuberculin test containing bovine and avian PPDs. A total of 1470 animals, 612 (41.6%) males and 858 (58.4%) females, 883 (60%) young, 555 (37.8%) adult and 32 (2.2%) old animals were included. The study involved a cross-sectional multistage sampling technique with random selection of individual animals from a herd. The results revealed a 1.3% overall prevalence of bovine tuberculosis in cattle herds in the study area, with a marked variation between sub-counties. The highest recorded prevalence was 6.0% in Kapujan, while no cases were recorded in Ongogonja, Magoro and Katakwi sub-counties. Distinctly different patterns in the avian-bovine reactions were also found in different sub-counties. A multivariate logistic regression showed more positive reactions (OR = 6.3; 95%CI (1.4-26.34) in females than males. BTB prevalence did not differ significantly between cattle maintained in pastoral and agro-pastoral production systems. The study demonstrated a relatively low prevalence of bovine tuberculosis in local zebu cattle reared under traditional husbandry systems in Uganda, suggesting low infectiousness of the disease under such mode of production. The risk associated with the consumption of raw milk among the pastoral communities and that, the pooling of milk together from different animals is a common practice, warrants more investigation into the zoonotic transmission of tuberculosis within these communities.

Mycobacteria causing human cervical lymphadenitis in pastoral communities in the Karamoja region of Uganda.

Mycobacteria from lymph node biopsies of patients with cervical lymphadenitis reporting for tuberculosis treatment in Matany and Moroto Hospitals in the transhumant areas of Karamoja, Uganda were isolated and characterized. The AccuProbe culture identification kits for Mycobacterium tuberculosis complex (MTC), M. avium complex (MAC) and M. avium were used to identify the isolates. Spoligotyping, IS901 PCR and IS1311 and IS1245 restriction fragment length polymorphism (RFLP) were used to characterize the isolates. Of the 43 biopsies, ten M. avium, seven M. tuberculosis, three M. bovis, and two M. intracellulare were isolated. Two isolates could not be identified with AccuProbe and from 19 samples no mycobacteria could be isolated. Three isolates with the Beijing spoligotype were identified from the seven M. tuberculosis isolates. The spoligopatterns of the M. bovis isolates had previously been detected in cattle in Uganda. Isolation of members of the MAC group reflects the complex interaction between the transhumant communities, water sources and their cattle. None of the M. avium isolates harboured IS901, and all showed several bands on IS1311 and IS1245 RFLP, in accordance with M. avium subsp. hominissuis. Composite dendrograms of IS1311 and IS1245 RFLP showed that the isolates were similar and identical patterns were found. The isolation of M. bovis confirms the human infection with zoonotic mycobacteria in areas where consumption of raw milk and meat is routine. Isolation of environmental mycobacteria also confirms their increasing role in human disease and the occupational risk of infection in the transhumant ecosystem in the absence of safe drinking water and environmental contamination.

Evaluation of Salmonella occurrence in domestic animals and humans in North Dakota (2000-2005).

OBJECTIVES: To evaluate the occurrence, serotypes, and antimicrobial susceptibility of Salmonellae from domestic animals and humans in North Dakota. MATERIALS AND METHODS: Salmonellosis data (2000-2005) in humans (n = 286) and animals (n = 258) were extracted from the North Dakota Department of Health (NDDoH) and North Dakota State University Veterinary Diagnostic Laboratory (NDSU-VDL), and analyzed for temporal and spatial trends, and for other associations. Additionally, random samples of 35, 30, and 15 Salmonella isolates from NDSU-VDL, NDDoH, and North Dakota healthy cattle, respectively, were tested for antimicrobial susceptibility. RESULTS: Most animal salmonellosis occurred in cattle (64.7%) sheep (12%), pigs (10.9%), and bison (0.4%) with Salmonella Typhimurium (45.7%) as the predominant serotype; Salmonella Arizona (10.9%) and Dublin (10.5%) were host specific in sheep and cattle respectively. In humans, Salmonella Typhimurium (32.5%) and Salmonella Newport (11.2%) were predominant. Season influenced human (p = 0.027) and animal (p = 0.014) salmonellosis with cases peaking in the spring and summer for animals and humans, respectively. Salmonella Typhimurium case reports in humans were not seasonally related to domestic animals (p = 0.001) nor cattle (p = 0.001). Over time, case reports increased in humans but decreased in domestic animals. Most serotypes from domestic animals were multidrug resistant compared to human isolates. CONCLUSIONS AND APPLICATIONS: Many Salmonella serotypes (17) were involved in North Dakota human and animal salmonellosis with case reports closely related in fall and winter, but not during warmer months. Spatial clustering of human and animal cases was similar. Antimicrobial resistance was widespread but lower in human isolates. These data are helpful in determining future policy, research, and control strategies for salmonellosis in humans and domestic animals.

Responses to tuberculin among Zebu cattle in the transhumance regions of Karamoja and Nakasongola district of Uganda.

Responses to tuberculin in Zebu cattle of the transhumant pastoral farming system in Karamoja region and Nakasongola district in the north-eastern and mid-central regions in Uganda, respectively, were investigated using a comparative intradermal tuberculin skin test. Of the 1864 cattle tested from 30 large units (superherds) in Karamoja and 7 herds in Nakasongola, a total of 28 animals from 19 herds (51.4%) tested positive. Inter-district tuberculin reactor prevalence variations seemed to be influenced by climate, with impact on both the management patterns and transmissibility of agent. High herd tuberculin reactor prevalence (51.4%) was attributed to widespread contacts and mixing of animals between herds. Low individual animal tuberculin test positivity (mean = 1.4%) was attributed to low transmissibility of the agent under the Karamoja climate, which is semi-arid, and to increased resistance due to non-specific response to environmental mycobacteria and natural selection, since there was no active control against bovine tuberculosis. Owing to similarities in management practices in Karamoja and widespread risk factors, it was difficult to identify which were more important, but variations in sources of drinking water pointed to provision of lake and borehole water during dry season as reducing the risk. Positive bovine tuberculin reactor prevalence and skin reactor status were related to age.

Prevalence of antibodies to Brucella spp. and individual risk factors of infection in traditional cattle, goats and sheep reared in livestock-wildlife interface areas of Zambia.

A cross-sectional study was performed in the livestock-wildlife interface areas of Lochinvar and Blue Lagoon National Parks and the non-interface area of Kazungula to determine the prevalence of antibodies to Brucella spp. in domestic ruminants and identify individual animal risk factors of infection. A total of 1245 cattle from 124 herds and 280 goats and sheep from 29 flocks were tested sequentially for Brucella antibodies using the Rose Bengal test (RBT) and competitive ELISA. In cattle, individual seroprevalence ranged from 14.1% to 28.1%, while herd sero-prevalence ranged from 46.2% to 74.0% in the three study areas. No goat or sheep tested positive for Brucella antibodies. Three types of cattle grazing strategies were encountered: locally grazed herds (LGH), transhumantly grazed herds (TGH) and river flood plain grazed herds (FGH). Brucella seroprevalence was seen to vary according to area and grazing strategy: Lochinvar and transhumant grazed herds recorded the highest figures, respectively. Age, sex and history of abortion were found to have independent effects on individual seroprevalence. This study establishes that brucellosis is endemic in domestic animals in the livestock-wildlife interface areas of Blue Lagoon and Lochinvar national parks and the disease is also present in Kazungula. We observed that type of grazing strategy had significant impact on cattle Brucella seroprevalence and that transhumant herds were at high risk of being infected.