Incidence of Salmonella serovars isolated from commercial animal feed mills in the United States and serovar identification using CRISPR analysis.

AIMS: In this study, we sought to determine the incidence and diversity of Salmonella in a broad collection of commercial animal feeds collected from animal feed mills across the United States over an 11-month period and utilize CRISPR analysis to identify individual serovars. METHODS AND RESULTS: Over two independent trials, 387 feed samples from 135 different animal feed mills in the United States were screened for Salmonella. A total of 6·2% (24/387) of samples were contaminated with Salmonella, which is concordant with similar studies. Clustered regularly interspaced short palindromic repeats (CRISPR)-typing was used to serotype Salmonella isolates, and serovars Infantis and Tennessee were the most common. CONCLUSIONS: Serogroups O:4 and O:7 were enriched in the feed samples, suggesting that these serogroups are better adapted to surviving in low moisture animal feeds. The study supports the utility of CRISPR to determine serovar type since most of the serovars identified in this study have been also isolated and identified in earlier studies using more classical serotyping methods. SIGNIFICANCE AND IMPACT OF THE STUDY: This work contributes to a growing body of literature concerning the Salmonella prevalence in animal feeds and highlights the need to effectively mitigate pathogens in livestock and poultry feed.

Conserved CRISPR arrays in Salmonella enterica serovar Infantis can serve as qPCR targets to detect Infantis in mixed serovar populations.

Salmonellosis is a leading bacterial cause of foodborne illness, and numerous Salmonella enterica serovars have been responsible for foodborne outbreaks. In the United States outbreaks are often linked to poultry and poultry-related products. The prevalence of Salmonella serovar Infantis has been increasing in poultry processing facilities over the past few years and in 2018 was identified as the causative agent for a large multistate outbreak linked to raw chicken. CRISPR-typing is a subtyping approach based on PCR and the sequencing of two Salmonella loci, CRISPR1 and CRISPR2. CRISPR-typing was used to interrogate 138 recent (2018-2019) isolates and genomes of ser. Infantis. Results show that the CRISPR elements are remarkably conserved in this serovar. The most conserved spacers, and those also unique to ser. Infantis, were used as targets to develop a ser. Infantis-specific qPCR assay. This assay was able to detect ser. Infantis in mixed serovar cultures of Salmonella, down to 0·1% of the population, highlighting the utility of this molecular approach in improving surveillance sensitivity for this important food safety pathogen. SIGNIFICANCE AND IMPACT OF THE STUDY: The incidence of human salmonellosis cases caused by Salmonella enterica serovar Infantis (ser. Infantis) has been increasing, as has its prevalence in broiler chickens, which are a frequent reservoir of Salmonella. A cluster of ser. Infantis genetically linked to an outbreak strain have been identified in numerous processing facilities. A qPCR assay targeting CRISPR elements that are unique to ser. Infantis has been developed and can detect this serovar directly from mixed cultures. This assay is sensitive enough to reveal ser. Infantis within a mixed Salmonella population where it constitutes only 0·1% of the population. The rapid nature of qPCR lends this assay to high-throughput screening of poultry samples to detect this important pathogen.

Prevalence and Spatial Distribution of Salmonella Infections in the Pennsylvania Raccoon (Procyon lotor).

A study was conducted to determine the prevalence and spatial distribution of Salmonella infection in Pennsylvania raccoons (Procyon lotor), common wildlife mammals known to occupy overlapping habitats with humans and domestic food animals. The Pennsylvania Game Commission provided a total of 371 raccoon intestinal samples from trapped and road-killed raccoons collected between May and November 2011. Salmonella was isolated from the faeces of 56 (15.1%) of 371 raccoons in 35 (54%) of 65 counties across Pennsylvania. The five most frequently isolated serotypes were Newport (28.6%), Enteritidis (19.6%), Typhimurium (10.7%), Braenderup (8.9%) and Bareilly (7.1%). Pulsed-field gel electrophoresis (PFGE) analysis of the Salmonella isolates and subsequent comparison to the Pennsylvania Department of Health human Salmonella PFGE database revealed 16 different pulsetypes in Salmonella isolates recovered from raccoons that were indistinguishable from pulsetypes of Salmonella collected from clinically ill humans during the study period. The pulsetypes of seven raccoon Salmonella isolates matched those of 56 human Salmonella isolates by month and geographical region of sample collection. Results from Clustered Regularly Interspaced Short Palindromic Repeats and Multi-Virulence Locus Sequence Typing (CRISPR-MVLST) analysis corroborated the PFGE and serotyping data. The findings of this study show that several PFGE pulsetypes of Salmonella were shared between humans and raccoons in Pennsylvania, indicating that raccoons and humans might share the same source of Salmonella.

Laboratory Study on Biological Control of Ticks (Acari: Ixodidae) by Entomopathogenic Indigenous Fungi (Beauveria bassiana).

BACKGROUND: Chemical control method using different acaricides as spray, dipping solution or pour-on is routinely used for controlling ticks. Biological control agents are favorable due to their safety for animals and environment. Entomopathogenic fungi such as Beauveria bassiana are well known for controlling ticks. In this study, two Iranian indigenous strains of B. bassiana (B. bassiana 5197 and B. bassiana Evin) were selected and grown on specific media. The pathogenic effects of these strains were evaluated on adult stages of two Iranian Ixodidae members (H. anatolicum anatolicum Koch 1844, and H. marginatum Koch 1844) by dipping method. METHODS: Two Iranian strains of Beauveria bassiana (Beauveria bassiana 5197 and Beauveria bassiana Evin) were selected and were grown successfully on specific media. The pathogenic effects of these strains were evaluated on adult stages of Iranian Ixodidae members such as, Hyalomma anatolicum anatolicum and H. marginatum by dipping method (these ticks were grown up at laboratory conditions during 2002 up to 2003 and still it is continued) . RESULTS: There was no effect of strain 5197 on mortality or fecundity rates for ticks. There was acute phase sign of paralysis in test group after dipping ticks in suspension made from Evin strain of B. bassiana. In addition, the test groups were totally died after four months, but the control groups survived for six months. CONCLUSION: High concentration of fungal spores is needed for inducing fungal infection. Additional study using different strains and fungi on Iranian ticks is proposed.

Isolated growth hormone deficiency type II caused by a point mutation that alters both splice site strength and splicing enhancer function.

A heterozygous single base mutation in the human growth hormone (GH) gene (GH-1) was identified in a family presenting with isolated GH deficiency type II (IGHD II). Affected individuals have a guanine to adenine transition at the first nucleotide of exon 3 (E3+1 G-->A) that results in exon skipping and production of a dominant-negative 17.5-kDa isoform. We show that the mechanistic basis for exon skipping is due to the unique position of this mutation because it weakens the 3' splice site and simultaneously disrupts a splicing enhancer located within the first seven bases of exon 3. A G-->T mutation at this same position not only affects splicing but also results in a premature stop codon for those transcripts that include exon 3. Thus, mutations that alter the first nucleotide of exon 3 illustrate the various mechanisms by which changes in sequence can cause disease: splice site selection, splicing enhancer function, messenger RNA decay, missense mutations, and nonsense mutations. For IGHD II, only exon skipping leads to production of the dominant-negative isoform, with increasing skipping correlating with increasing disease severity.