Microbiological safety of popular recreation swimming sites in Central California.

The objective of the study was to assess the microbiological safety of popular recreational swimming sites in Central California. Water samples were collected from eleven monitoring sites across the lower reaches of two watersheds for two consecutive swimming seasons (2012-2013), and levels of indicator and pathogenic microorganisms were determined. Data on ambient weather and water chemistry were collected for analyzing their associations with microorganisms in water. All water samples were positive for indicator E. coli with mean concentrations per site ranging from 3.07 to 216.11 MPN/100 ml in 2012 and 13.4 to 226.97 MPN/100 ml in 2013. Mean E. coli concentrations in 27% and 36% samplings sites exceeded the EPA 2012 Recreational Water Quality Criteria recommended mean concentration of ≤ 126 CFU/100 ml of E. coli, in 2012 and 2013, respectively. Cryptosporidium spp. oocysts were detected in all water samples from all sampling sites, with an overall prevalence of 50% and mean concentrations of 0.08 oocysts/l in 2012 and 0.19 oocysts/l in 2013. Giardia spp. cysts were detected at eight sites, with an overall prevalence of 28.8% and mean concentration of 0.2 cysts/l in both years. The majority of the detected Cryptosporidium spp. oocysts and Giardia spp. cysts appeared damaged under microscopy. E. coli O157:H7 was detected in 9% of water samples, with positive samples limited to three sites. Salmonella spp. were detected in all but one site across the two years, with mean concentrations of 0.94 MPN/l in 2012 and 1.85 MPN/l in 2013. Cryptosporidium spp. oocyst concentrations were negatively associated with 30-day mean wind speed and cumulative precipitation and dissolved oxygen in water. Giardia spp. cyst concentrations were positively associated with turbidity and pH of water and negatively associated with E. coli concentrations and 24-h mean air temperature. Salmonella spp. concentrations were positively associated with 30-day mean air temperature. The occurrence of E. coli O157:H7 was positively associated with previous 30-day cumulative precipitation.

Expression of toll-like receptor 4 and its associated cytokines from peripheral blood mononuclear cells in Leghorn chickens.

Background and Aim: Immune cells require toll-like receptor 4 (TLR4) to respond to lipopolysaccharides (LPS) by releasing pro-inflammatory cytokines. Peripheral blood mononuclear cells (PBMCs) are used to assess changes in cytokines released in response to diseases or pathogens. This study aimed to assess TLR4 gene expression in PBMCs from Leghorn chicken and the release of related cytokines. Materials and Methods: Peripheral blood mononuclear cells were isolated from blood samples obtained from Leghorn chicks. The PBMC cultures were stimulated with various concentrations of LPS (0.01-1 µg/ml). Polymerase chain reaction was used to detect TLR4 expression. The production of tumor necrosis factor-alpha (TNF-α) and interleukins (IL-1ß and IL-6) was quantified using an enzyme-linked immunosorbent assay. Results: We found that TLR4 was expressed in both non-stimulated and stimulated Leghorn chicken PBMCs. In addition, the release of TNF-α, IL-1ß, and IL-6 levels in Leghorn chicken PBMCs increased significantly with an increase in LPS concentration (0.01-1 µg/mL) (p < 0.05). Conclusion: Although TLR4 was expressed in both non-stimulated and stimulated Leghorn chicken PBMCs, its expression was significantly higher in LPS-stimulated PBMCs Therefore, the chicken's endotoxin response can be assessed by evaluating the pro-inflammatory cytokine production from PBMCs.

Economic and value chain analysis to support an investigation and risk mitigation efforts on Marek's disease in layers in the southern part of Thailand.

Background and Aim: Marek's disease (MD) is a common lymphoproliferative disease affecting chickens and causing economic losses in commercial poultry. The MD outbreak was noticed in the southern part of Thailand in 2019. The suspected cases were found with an abnormal number of cases of layers dying with clinical signs, for example, weakness and emaciation, with evidence of MD gross lesions. This study aimed to raise awareness of the MD outbreak through value chain analysis (VCA), identifying associated possible risk factors, and estimating the associated economic impact. Materials and Methods: Value chain analysis, including seasonal calendar, value chain diagram, and layer movement mapping of the layer industry, was conducted. High-risk stakeholders were identified on the basis of risk practices and interactions between stakeholders. A case-control study was conducted to determine risk factors associated with the MD outbreak on layer farms, and partial budget analysis was used to estimate economic losses associated with MD. Results: The value chain diagram showed the linkages between stakeholders, including estimation of the percentage of products moved from one stakeholder group to another and the negotiated price. Fourteen out of 35 layer farms were case farms. Farm size and source of birds were significantly associated with the MD outbreak. The MD outbreak caused total economic losses of 295,823 USD. Farms that slaughtered infected birds with additional revenues incurred losses of 140,930 USD, whereas farms that culled infected birds without additional revenue returned incurred losses of 1995 USD. Conclusion: The VCA provided a better understanding of the layer and egg businesses in South Thailand and guided the development of questionnaires for outbreak investigation. The potential risk factor findings suggested the need for further exploration of the source of the MD outbreak.

Pro-inflammatory cytokine release from chicken peripheral blood mononuclear cells stimulated with lipopolysaccharide.

Background and Aim: The principal cytokines released by the host on infection include pro-inflammatory cytokines such as interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF). These cytokines were regarded as regulators of the host's response to infection. This study aimed to determine the release of pro-inflammatory cytokines from chicken peripheral blood mononuclear cells (PBMCs) following lipopolysaccharide (LPS) stimulation. Materials and Methods: Blood samples were collected from six Betong chickens. To isolate PBMCs, density gradient centrifugation was utilized. PBMC culture in RPMI1640 with 10% fetal bovine serum was stimulated with various concentrations of LPS (0, 0.01, 0.1, and 1.0 µg/mL). The production of TNF-α, IL-1ß, and IL-6 was determined using an enzyme-linked immunosorbent assay. Results: When the PBMCs were cultured for 24 h with varying doses of LPS, there was no significant variation in cell viability. TNF-α, IL-1ß, and IL-6 levels were measured in Betong chicken PBMC. The release of these cytokines increased considerably as LPS concentration (0.01-1 µg/mL) increased (p<0.05). Conclusion: In vitro studies of the chicken immune response, notably the release of pro-inflammatory cytokines, can be conducted using PBMCs obtained from chicken blood.

Development of a novel multiplex PCR assay for the detection and differentiation of Plasmodium caprae from Theileria luwenshuni and Babesia spp. in goats.

Intraerythrocytic parasites are traditionally identified by the microscopic examination of Giemsa-stained blood smears. However, this method does not always allow for the identification of individual species in goat's RBCs. Moreover, its unreliability in detecting low levels of parasitemia makes it unsuitable for epidemiological investigations and leaves goat farms vulnerable to potential outbreaks. In the present study, a novel multiplex PCR (mPCR) targeting the cytochrome c oxidase subunit I (COI) gene was developed to detect and subsequently differentiate Plasmodium caprae, Theileria luwenshuni, and Babesia spp. The specificity of each primer set was assessed both in silico and with a panel of DNA samples from the hosts themselves and other goat hemoparasites. Amplicons generated from each pair of primers were 664, 555, and 320-bp for P. caprae, Babesia spp., and T. luwenshuni, respectively. These products were further confirmed by sequencing. Our novel mPCR reactions successfully demonstrated the accurate and simultaneous amplification of the three parasites' DNA samples. The current mPCR method showed no cross-amplification with unintended targets. The detection limit of the mPCR in this study was 108 parasites' DNA copies per reaction. The current mPCR was able to detect the minimum parasitemia of approximately 0.001%, 0.000005%, 0.00001% for P. caprae, Babesia spp. and T. luwenshuni, respectively. The diagnostic specificity in the detection of P. caprae and T. luwenshuni ranged from 94.9 to 100 %. The mPCR was further applied to a collection of field blood samples from five provinces in Thailand to validate its reliability and applicability. The results demonstrated the successful detection of P. caprae, Babesia spp. and T. luwenshuni in goat samples with the same sensitivity levels as conventional PCR methods. This study also confirmed the presence of T. luwenshuni and Babesia spp. in Thai goats. The current mPCR method offers an alternative for the diagnosis of P. caprae, T. luwenshuni, and Babesia spp., either single or under co-infection conditions, and for large-scale surveillance.

Toxoplasmosis in piglets.

Seventeen-day-old piglets in a small holding farm in southern Thailand manifested signs of convulsion, fever, and death. The morbidity and mortality rate were approximately 26.09% (6/23) and 4.35% (1/23), respectively. Impression smear from lungs demonstrated tachyzoite stage of Toxoplasma gondii. Histopathological investigation revealed interstitial pneumonia. Further investigation, blood collection, educating the farmer, and prescribed affected herd with sulfa-trimethoprim were performed soon after Toxoplasma infection was demonstrated. A serological detection of T. gondii infection among affected herds using latex agglutination test was conducted on 14 serum samples. The overall seroprevalence was 71.43% (10/14). The titers were 1:64 in 3 pigs (21.43%), 1:128 in 4 (28.57%), 1:256 in 2 (14.29%), and 1:512 in 1 (7.14%). A cat in this farm showed antibody titer 1:32 while a dog was seronegative. Data derived from this case indicated two possible routes of transmission: transplacental infection and ingested food or water contaminated with oocysts shed by cat in this farm. However, rodents can not exclude as a role of toxoplasmosis transmission. Serological monitoring at slaughter houses combine with good sanitary practices, rodent and cat control in the farm are important measures to prevent toxoplasma infection and improve human health in the future.

Cross-sectional survey of indicator and pathogenic bacteria on vegetables sold from Asian vendors at farmers' markets in northern California.

A cross-sectional survey was conducted during summer 2013 to determine the occurrence of Escherichia coli, fecal coliforms (FCs), E. coli O157:H7, and Salmonella on raw vegetable commodities common to Asian cuisine from 21 vendors or farmers at six farmers' markets in northern California. Based on 242 samples from six commodities (basil, yardlong beans, bitter squash, okra, squash stems and leaves, cilantro), 100% of samples had detectable FCs and 20% had detectable E. coli. The mean concentrations were 0.67 log CFU/g and 1.26 log CFU per bundle for E. coli and 4.00 log CFU/g and 6.26 log CFU per bundle for FCs. Vegetables irrigated with ground versus surface water contained lower concentrations of FCs, but this difference was not observed for E. coli. Yardlong beans, bitter squash, and okra had lower levels of FCs compared with basil, cilantro, and squash stems and leaves. Sixteen (6.6%) samples had detectable levels of Salmonella serovars (Newport, Enteritidis, Agona, and Worthington), with the majority of positives found in cilantro and squash stems and leaves. There was a twofold higher probability of Salmonella contamination in samples from growers or vendors who stated that they used organic farming practices compared with samples from those using conventional farming practices. Lastly, the concentrations of FC and E. coli bacteria were significantly associated with Salmonella contamination: for each additional 100 CFU/g or bundle, the probability of Salmonella contamination increased by ∼15 and ∼30%, respectively. None of the samples had detectable E. coli O157:H7.

Vaccination coverage and epidemiological parameters of the owned-dog population in Thungsong District, Thailand.

Canine rabies vaccination is delivered in Thungsong District, Thailand, as an annual campaign between March 1 and 31, and also at other times through private veterinary clinics, para-veterinarians and health-care staff residing in the villages. The current questionnaire-interview survey was conducted between June 23 and July 18, 2002 to determine: rabies-vaccination coverage amongst the owned-dog population; basic dog-population information; and community awareness about rabies. The modified expand programme on immunization cluster-survey method was used to collect information about dog demography and management characteristics. Household knowledge about rabies and sources of rabies information were assessed. Vaccinated dogs were identified from vaccine certificates or owner reports confirmed by vaccinators. Seventy percent (95%CI 62-78) of 364 eligible owned dogs were vaccinated within the 6 months prior to data collection. Of these 255 vaccinated dogs, 44, 21, 13, 17 and 5% were vaccinated through the annual vaccination campaign, veterinary clinics, para-veterinarians, other vaccinators and owners, respectively. Fifty-four percent of households owned dogs. The sex ratio in dogs was 2 males per female; the dog: human ratio was 1: 4.6 with an average of 0.9 dogs per household (1.7 dogs per dog-owning household). Most dogs roamed freely and these were less likely to be vaccinated compared to dogs being kept on premises or on a leash. Almost all households were aware of rabies and the need for dog rabies vaccination as a control method. Seventy-six percent believed that rabies only occurred in summer. There was little awareness about cat rabies amongst households. Vaccination coverage in the total dog population clearly has not yet reached the 80% target level set by Thailand's official rabies-control programme. Improved effectiveness of the owned-dog rabies-vaccination campaigns in each community is needed-perhaps by more community education about dog management or by better management of ownerless dogs.

Prevalence and characterization of Escherichia coli and Salmonella strains isolated from stray dog and coyote feces in a major leafy greens production region at the United States-Mexico border.

In 2010, Romaine lettuce grown in southern Arizona was implicated in a multi-state outbreak of Escherichia coli O145:H28 infections. This was the first known Shiga toxin-producing E. coli (STEC) outbreak traced to the southwest desert leafy green vegetable production region along the United States-Mexico border. Limited information exists on sources of STEC and other enteric zoonotic pathogens in domestic and wild animals in this region. According to local vegetable growers, unleashed or stray domestic dogs and free-roaming coyotes are a significant problem due to intrusions into their crop fields. During the 2010-2011 leafy greens growing season, we conducted a prevalence survey of STEC and Salmonella presence in stray dog and coyote feces. Fresh fecal samples from impounded dogs and coyotes from lands near produce fields were collected and cultured using extended enrichment and serogroup-specific immunomagnetic separation (IMS) followed by serotyping, pulsed-field gel electrophoresis (PFGE), and antimicrobial susceptibility testing. A total of 461 fecal samples were analyzed including 358 domestic dog and 103 coyote fecals. STEC was not detected, but atypical enteropathogenic E. coli (aEPEC) strains comprising 14 different serotypes were isolated from 13 (3.6%) dog and 5 (4.9%) coyote samples. Salmonella was cultured from 33 (9.2%) dog and 33 (32%) coyote samples comprising 29 serovars with 58% from dogs belonging to Senftenberg or Typhimurium. PFGE analysis revealed 17 aEPEC and 27 Salmonella distinct pulsotypes. Four (22.2%) of 18 aEPEC and 4 (6.1%) of 66 Salmonella isolates were resistant to two or more antibiotic classes. Our findings suggest that stray dogs and coyotes in the desert southwest may not be significant sources of STEC, but are potential reservoirs of other pathogenic E. coli and Salmonella. These results underscore the importance of good agriculture practices relating to mitigation of microbial risks from animal fecal deposits in the produce production area.

Epidemiologic trends of rabies in domestic animals in southern Thailand, 1994-2008.

Rabies and associated risk factors in dogs, cats and cattle (n = 3,454) in southern Thailand during 1994-2008 were evaluated by using a mixed-effect logistic regression model. Overall prevalence was 48%. In dogs, odds of being rabid were 1.7 times higher in unvaccinated dogs than in vaccinated dogs and two times higher in dogs with bite history than in dogs with no known bite history. Similarly, aggressive dogs were more likely to be rabid than non-aggressive dogs. In cattle, aggression, pharyngeal paralysis, hyperactivity, and depression were clinical signs associated with being rabid. Annual fluctuations of the species-specific prevalence of rabies is suggestive of a positive correlation between canine and either feline (r = 0.60, P = 0.05) or bovine rabies (r = 0.78, P = 0.004). Insufficient vaccination coverage led to maintenance of rabies, which could be easily controlled by increased vaccine coverage and public education.