Characterization of Erysipelothrix rhusiopathiae strains isolated from acute swine erysipelas outbreaks in Eastern China

Recently, a series of acute swine erysipelas outbreaks occurred in Eastern China. Eight strains isolated from cases of septicemia were determined as serotype 1a, and 4 of the isolates were resistant to acriflavine. One isolate strain named {"type":"entrez-nucleotide","attrs":{"text":"HX130709","term_id":"383559396","term_text":"HX130709"}}HX130709 was attenuated on agar media containing acriflavine dye. The 432-bp hypervariable region in spaA gene of the field and attenuated strains were amplified and sequenced. It was further compared with the vaccine strain G₄T₁₀, and thus, the eight field strains can be divided into four spaA-types. The partial spaA gene analysis also showed that no point mutations occurred among different archived passages of {"type":"entrez-nucleotide","attrs":{"text":"HX130709","term_id":"383559396","term_text":"HX130709"}}HX130709 during the attenuation. Results of pulsed-field gel electrophoresis showed that eight distinct patterns with 22 to 30 DNA fragment bands were produced from field strains, and twelve distinct patterns with 23 to 27 DNA fragment bands were produced from different passages of the attenuated strains. Mouse pathogenicity test showed that the mortality of the mice infected with 10⁴ CFU field strains was 100% and the attenuation of strain {"type":"entrez-nucleotide","attrs":{"text":"HX130709","term_id":"383559396","term_text":"HX130709"}}HX130709 occurred between 46 and 50 passages. All the field and attenuated strains were highly sensitive to β-lactam antibiotics, tetracyclines and macrolides. So, we can make conclusions that the acute swine erysipelas outbreaks in Eastern China were caused by serotype 1a E. rhusiopathiae strains with different biochemical characteristics, and the virulence of serotype 1a E. rhusiopathiae strains is unrelated with some point mutations in 432-bp hypervariable region of the spaA gene.     

Helicobacter apodemus sp. nov., a new Helicobacter species identified from the gastrointestinal tract of striped field mice in Korea

A novel Helicobacter species was identified from the gastrointestinal tract of the Korean striped field mouse (Apodemus agrarius). Biochemical testing, ultrastructure characterization, and 16S rRNA gene sequence analysis suggested that this bacterium represents a distinct taxon. The bacterium was positive for urease activity, susceptible to cephalothin and nalidixic acid, and weakly positive for oxidase and catalase activity. Electron microscopy revealed that the bacterium has spirally curved rod morphology with singular bipolar nonsheathed flagella. Genotypically, the isolated bacterial strains (YMRC 000215, YMRC 000216, and YMRC 000419) were most closely related to a reference strain of Helicobacter mesocricetorum (97.25%, 97.32%, and 97.03% 16S rRNA sequence similarities, respectively). The 16S rRNA sequences of these strains were deposited into GenBank under accession numbers {"type":"entrez-nucleotide","attrs":{"text":"AF284754","term_id":"18389594","term_text":"AF284754"}}AF284754, {"type":"entrez-nucleotide","attrs":{"text":"AY009129","term_id":"18409527","term_text":"AY009129"}}AY009129, and {"type":"entrez-nucleotide","attrs":{"text":"AY009130","term_id":"18409528","term_text":"AY009130"}}AY009130, respectively. We propose the name Helicobacter apodemus for this novel species.     

Genetic variation analyses of porcine epidemic diarrhea virus isolated in mid-eastern China from 2011 to 2013

Porcine diarrhea outbreaks caused by porcine epidemic diarrhea virus (PEDV) has occurred in China with significant losses of piglets since 2010. In this study, the complete S and ORF3 genes of 15 field PEDV isolates in mid-eastern China from 2011 to 2013 were detected and compared with other reference strains. Based on S gene, all of the PEDV strains could be assigned to 3 genogroups. Only 1 isolate, {"type":"entrez-nucleotide","attrs":{"text":"JS120103","term_id":"364436861","term_text":"JS120103"}}JS120103, belonged to genogroup 1 and showed a close relationship with previous Chinese strains DX and JS-2004-2, European strain CV777, and Korean strain DR13. The other 14 isolates belonged to genogroup 3 and showed a close relationship with other Chinese strains isolated after 2010. The S genes of those isolates were 9 nucleotides longer in length than {"type":"entrez-nucleotide","attrs":{"text":"JS120103","term_id":"364436861","term_text":"JS120103"}}JS120103 and the other reference strains in genogroup 1, with 15 bp insertion and 6 bp deletion. Homology analyses revealed that all of the Chinese field isolates, except {"type":"entrez-nucleotide","attrs":{"text":"JS120103","term_id":"364436861","term_text":"JS120103"}}JS120103, are 97.6% to 100% (95.8% to 100%) identical in nucleotide (deduced amino acid) sequence to each other. Meanwhile, based on the ORF3 gene, all of the PEDV isolates could be separated into 3 genogroups. Eleven of the 15 field isolates in this study belonged to genogroup 3 and were 95.8% to 100% identical in nucleotide sequence or 95.6% to 100% in deduced amino acid sequence to each other. Our results indicate that the variant PEDV strain spread wildly in mid-eastern China. This will be useful to take into consideration in the control and prevention of this disease.     

Variant -and individual dependent nature of persistent Anaplasma phagocytophilum infection

Background

Anaplasma phagocytophilum is the causative agent of tick-borne fever in ruminants and human granulocytotropic anaplasmosis (HGA). The bacterium is able to survive for several months in immune-competent sheep by modifying important cellular and humoral defence mechanisms. Little is known about how different strains of A. phagocytophilum propagate in their natural hosts during persistent infection.

Methods

Two groups of five lambs were infected with each of two 16S rRNA gene variants of A. phagocytophilum, i.e. 16S variant 1 which is identical to GenBank no {"type":"entrez-nucleotide","attrs":{"text":"M73220","term_id":"148293","term_text":"M73220"}}M73220 and 16S variant 2 which is identical to GenBank no {"type":"entrez-nucleotide","attrs":{"text":"AF336220","term_id":"13325085","term_text":"AF336220"}}AF336220, respectively. The lambs were infected intravenously and followed by blood sampling for six months. A. phagocytophilum infection in the peripheral blood was detected by absolute quantitative real-time PCR.

Results

Both 16S rRNA gene variants of A. phagocytophilum established persistent infection for at least six months and showed cyclic bacteraemias, but variant 1 introduced more frequent periods of bacteraemia and higher number of organisms than 16S rRNA gene variant 2 in the peripheral blood.

Conclusion

Organisms were available from blood more or less constantly during the persistent infection and there were individual differences in cyclic activity of A. phagocytophilum in the infected animals. Two 16S rRNA gene variants of A. phagocytophilum show differences in cyclic activity during persistent infection in lambs.     

Molecular detection and characterization of ovine herpesvirus-2 using heminested PCR in Pakistan

BackgroundMalignant catarrhal fever (MCF) is a highly fatal lymphoproliferative disease of cattle, deer, bison, water buffalo, and pigs caused by the gamma-herpesviruses alcelaphine herpesvirus-1 (AlHV-1) and ovine herpesvirus-2 (OvHV-2).ObjectivesThis study aimed to determine the prevalence of OvHV-2 in sheep, goats, cattle, and buffalo in Rawalpindi and Islamabad, Pakistan, by applying molecular and phylogenetic methods.MethodsBlood samples were aspirated from sheep (n = 54), goat (n = 50), cattle (n = 46) and buffalo (n= 50) at a slaughterhouse and several farms. The samples were subjected to heminested polymerase chain reaction (PCR), followed by sequencing and phylogenetic analysis of the OvHV-2 POL gene and the OvHV-2 ORF75 tegument protein gene.ResultsThe highest percentage of MCF positive samples was in sheep (13%), whereas goat, cattle, and buffalo had lower positive percentages, 11%, 9%, and 6.5%, respectively. Four OvHV-2-positive PCR products obtained from sheep samples were sequenced. The sequences obtained were submitted to the NCBI GenBank database ({"type":"entrez-nucleotide","attrs":{"text":"MK852173","term_id":"1694854431","term_text":"MK852173"}}MK852173 for the POL gene; {"type":"entrez-nucleotide","attrs":{"text":"MK840962","term_id":"1686143390","term_text":"MK840962"}}MK840962, {"type":"entrez-nucleotide","attrs":{"text":"MK852171","term_id":"1694854427","term_text":"MK852171"}}MK852171, and {"type":"entrez-nucleotide","attrs":{"text":"MK852172","term_id":"1694854429","term_text":"MK852172"}}MK852172 for the ORF75 tegument protein gene). Phylogenetic analysis revealed a close similarity of study sequences with those of worldwide samples.ConclusionsThis study is the first cross-sectional study on the prevalence and molecular detection of OvHV-2 in apparently healthy cattle and buffalo that could be carrying OvHV-2 acquired from OvHV-2-positive sheep and goats. The results indicate that OvHV-2 is circulating in Pakistan. Further studies are needed to characterize OvHV-2 and elucidate further its prevalence.     

Both flagella and F4 fimbriae from F4ac+ enterotoxigenic Escherichia coli contribute to attachment to IPEC-J2 cells in vitro

The role of flagella in the pathogenesis of F4ac⁺ Enterotoxigenic Escherichia coli (ETEC) mediated neonatal and post-weaning diarrhea (PWD) is not currently understood. We targeted the reference {"type":"entrez-nucleotide","attrs":{"text":"C83902","term_id":"2706834","term_text":"C83902"}}C83902 ETEC strain (O8:H19:F4ac⁺ LT⁺ STa⁺ STb⁺), to construct isogenic mutants in the fliC (encoding the major flagellin protein), motA (encoding the flagella motor), and faeG (encoding the major subunit of F4 fimbriae) genes. Both the ΔfliC and ΔfaeG mutants had a reduced ability to adhere to porcine intestinal epithelial IPEC-J2 cells. F4 fimbriae expression was significantly down-regulated after deleting fliC, which revealed that co-regulation exists between flagella and F4 fimbriae. However, there was no difference in adhesion between the ΔmotA mutant and its parent strain. These data demonstrate that both flagella and F4 fimbriae are required for efficient F4ac⁺ ETEC adhesion in vitro.     

Molecular characterization of two Bangladeshi infectious bursal disease virus isolates using the hypervariable sequence of VP2 as a genetic marker

Two Bangladeshi infectious bursal disease virus (IBDV) isolates collected in 2007, termed GB1 and GB3, were subjected to comparative sequencing and phylogenetic analyses. Sequence analysis of a 474-bp hypervariable region in the VP2 gene revealed that among four major amino acid substitutions observed in the strains, two were unique to GB1 and GB3 (Ser217Leu and Ala270Thr) while one substitution was only found in GB1 (Asn299Ser). Among IBDVs from Bangladesh including GB1 and GB3, the rate of identity and homology was around 97~99%. The amino acid sequences of GB1 and GB3 differ from those of previous Bangladeshi IBDV isolates and contain amino acid substitutions Pro222Ala and Asn299Ser (in GB3 only). Phylogenetic analysis revealed that GB1 and GB3 are grouped with other very virulent IBDVs of European and American origin in contrast to two previously isolated Bangladeshi IBDV strains (GenBank accession Nos. {"type":"entrez-nucleotide","attrs":{"text":"AF362776","term_id":"14582985","term_text":"AF362776"}}AF362776 and {"type":"entrez-nucleotide","attrs":{"text":"AF260317","term_id":"13957666","term_text":"AF260317"}}AF260317), which belong to the Asian group. It was concluded that GB1 and GB3 belong to a very virulent group of IBDVs. However, amino acid sequences of GB1 and GB3 differ from those of the other Bangladeshi IBDVs by one or two amino acids encoded in the hypervariable region of the VP2 gene.     

A comparative study of clinical manifestations,haematological and serological responses after experimental infection with Anaplasma phagocytophilum in two Norwegian sheep breeds

Background

It has been questioned if the old native Norwegian sheep breed, Old Norse Sheep (also called Norwegian Feral Sheep), normally distributed on coastal areas where ticks are abundant, is more protected against tick-borne infections than other Norwegian breeds due to a continuously high selection pressure on pasture. The aim of the present study was to test this hypothesis in an experimental infection study.

Methods

Five-months-old lambs of two Norwegian sheep breeds, Norwegian White (NW) sheep and Old Norse (ON) sheep, were experimentally infected with a 16S rRNA genetic variant of Anaplasma phagocytophilum (similar to GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"M73220","term_id":"148293"}}M73220). The experiment was repeated for two subsequent years, 2008 and 2009, with the use of 16 lambs of each breed annually. Ten lambs of each breed were inoculated intravenously each year with 0.4 ml A. phagocytophilum-infected blood containing approximately 0.5 × 10⁶ infected neutrophils/ml. Six lambs of each breed were used as uninfected controls. Half of the primary inoculated lambs in each breed were re-challenged with the same infectious dose at nine (2008) and twelve (2009) weeks after the first challenge. The clinical, haematological and serological responses to A. phagocytophilum infection were compared in the two sheep breeds.

Results

The present study indicates a difference in fever response and infection rate between breeds of Norwegian sheep after experimental infection with A. phagocytophilum.

Conclusion

Although clinical response seems to be less in ON-lambs compared to NW-lambs, further studies including more animals are needed to evaluate if the ON-breed is more protected against tick-borne infections than other Norwegian breeds.     

Superinfection occurs in Anaplasma phagocytophilum infected sheep irrespective of infection phase and protection status

Background

Anaplasma phagocytophilum infection in domestic ruminants is widespread in the coastal areas of southern Norway. The bacteria may persist in mammalian hosts. Several genetic variants of A. phagocytophilum exist. In the present study, we investigate whether superinfection occurs in the acute and persistent phase of the infection.

Methods

Five-month-old lambs of the Norwegian Dala breed were experimentally infected with two 16S rRNA gene variants of A. phagocytophilum, i.e. A. phagocytophilum variant 1 (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"M73220","term_id":"148293","term_text":"M73220"}}M73220) and variant 2 (GenBank acc. no. {"type":"entrez-nucleotide","attrs":{"text":"AF336220","term_id":"13325085","term_text":"AF336220"}}AF336220). Eighteen lambs were used, two lambs in each group. Eight groups were experimentally inoculated with either variant 1 or 2 on day 0. Six of these groups were then challenged with the other variant on either days 7, 42 or 84, respectively. One group was left uninfected. The occurrence of A. phagocytophilum in blood samples was determined using semi-nested PCR analysis and gene sequencing. Specific antibodies were measured by an indirect immunofluorescence antibody assay (IFA).

Results

A. phagocytophilum variant 1 and 2 differed significantly with regards to clinical reaction and cross-immunity in infected lambs. Both variants were found in the blood after challenge. However, variant 1 was detected most frequently.

Conclusion

The present experiment indicates that superinfection of different genotypes occurs during the acute as well as the persistent phase of an A. phagocytophilum infection, even in lambs protected against the challenged infection.     

An iron-acquisition-deficient mutant of Corynebacterium pseudotuberculosis efficiently protects mice against challenge

Caseous lymphadenitis (CLA) is a chronic disease that affects sheep and goats worldwide, and its etiological agent is Corynebacterium pseudotuberculosis. Despite the economic losses caused by CLA, there is little information about the molecular mechanisms of bacterial pathogenesis, and current immune prophylaxis against infection has been unable to reduce the incidence of CLA in goats. Recently, 21 different mutant strains of C. pseudotuberculosis were identified by random mutagenesis. In this study, these previously generated mutants were used in mice vaccination trials to develop new immunogens against CLA. Based on this analysis, {"type":"entrez-nucleotide","attrs":{"text":"CZ171053","term_id":"58296493","term_text":"CZ171053"}}CZ171053, an iron-acquisition-deficient mutant strain, was selected. After challenge with a virulent strain, 80% of the animals that were immunized with the {"type":"entrez-nucleotide","attrs":{"text":"CZ171053","term_id":"58296493","term_text":"CZ171053"}}CZ171053 strain survived. Furthermore, this vaccination elicited both humoral and cellular responses. Intracellular survival of the bacterium was determined using murine J774 cells; in this assay, the {"type":"entrez-nucleotide","attrs":{"text":"CZ171053","term_id":"58296493","term_text":"CZ171053"}}CZ171053 had reduced intracellular viability. Because iron acquisition in intracellular bacteria is considered one of their most important virulence factors during infection, these results demonstrate the immunogenic potential of this mutant against CLA.     

Inability of NS1 protein from an H5N1 influenza virus to activate PI3K/Akt signaling pathway correlates to the enhanced virus replication upon PI3K inhibition

Background

Phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, activated during influenza A virus infection, can promote viral replication via multiple mechanisms. Direct binding of NS1 protein to p85β subunit of PI3K is required for activation of PI3K/Akt signaling. Binding and subsequent activation of PI3K is believed to be a conserved character of influenza A virus NS1 protein. Sequence variation of NS1 proteins in different influenza A viruses led us to investigate possible deviation from the conservativeness.

Results

In the present study, NS1 proteins from four different influenza A virus subtypes/strains were tested for their ability to bind p85β subunit of PI3K and to activate PI3K/Akt. All NS1 proteins efficiently bound to p85β and activated PI3K/Akt, with the exception of NS1 protein from an H5N1 virus (A/Chicken/Guangdong/1/05, abbreviated as GD05), which bound to p85β but failed to activate PI3K/Akt, implying that as-yet-unidentified domain(s) in NS1 may alternatively mediate the activation of PI3K. Moreover, PI3K inhibitor, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, did not suppress but significantly increased the replication of GD05 virus.

Conclusions

Our study indicates that activation of PI3K/Akt by NS1 protein is not highly conserved among influenza A viruses and inhibition of the PI3K/Akt pathway as an anti-influenza strategy may not work for all influenza A viruses.     

Differences in copper and selenium metabolism between Angus (Bos taurus) and Brahman (Bos indicus) cattle

A 2-yr study was conducted at the Range Cattle Research and Education Center, University of Florida – Institute of Food and Agricultural Sciences (IFAS) (Ona, FL), to evaluate differences in the metabolism of Cu and Se of Angus (Bos taurus) and Brahman (Bos indicus) cattle. Thirty-two pregnant beef cows (n = 8 Brahman and 8 Angus/yr) were enrolled in the study in the first trimester of gestation. This study consisted of three phases: 1) restriction (day 0 to 90), 2) supplementation (day 91 to 150), and 3) calving. During all three phases, cows were individually fed and housed in partially covered drylot pens. During the restriction and supplementation phases, cows were provided a 1.5 kg/d of a grain-based concentrate supplement, which was fortified with flowers of S (50 g of supplemental S/cow daily; restriction phase) or Cu and Se (100 and 3 mg/d of Cu and Se, respectively; supplementation phase). Blood and liver samples were collected from all cows at 30 d intervals and from both cows and calves within 24 h of calving. Colostrum and milk samples were collected at calving and 7 d after birth. All data were analyzed using the MIXED procedure of SAS, where cow and calf were the experimental unit. During the restriction phase, a breed × day effect (P = 0.03) was observed where Brahman had greater liver Cu concentration than Angus cows in all sampling days. For liver Se concentration, a tendency (P = 0.07) for a breed effect was observed where Angus cows tended to have greater liver Se concentration than Brahman. During the supplementation phase, breed (P < 0.001) and day (P < 0.01) effects were observed, where Brahman cows had greater liver Cu concentration than Angus. For liver Se concentration, a day effect (P < 0.001) was observed, where liver Se concentration increased (P < 0.001) from day 90 to 120 and remained unchanged (P = 0.86) until day 150. At calving, no effects of breed (P = 0.34) were observed for liver Cu concentration of cows; however, Brahman calves tended (P = 0.09) to have greater liver Cu concentration than Angus calves. For Se liver concentration at calving, Angus cows tended (P = 0.07) to have greater liver Se concentration than Brahman cows; however, no breed differences (P = 0.70) were observed for liver Se concentration of calves at birth. In summary, substantial differences in multiple indicators of Cu and Se status were observed between Angus and Brahman cattle, implying that Angus and Brahman cattle possibly have different mechanisms to maintain adequate Cu and Se status.     

A Polymorphism in the Melanocortin 4 Receptor Gene (MC4R:c.92C>T) Is Associated with Diabetes Mellitus in Overweight Domestic Shorthaired Cats

Background

Feline diabetes mellitus (DM) shares many pathophysiologic features with human type 2 DM. Human genome‐wide association studies have identified genes associated with obesity and DM, including melanocortin 4 receptor (MC4R), which plays an important role in energy balance and appetite regulation.

Hypothesis/Objectives

To identify single nucleotide polymorphisms (SNPs) in the feline MC4R gene and to determine whether any SNPs are associated with DM or overweight body condition in cats.

Animals

Two‐hundred forty domestic shorthaired (DSH) cats were recruited for the study. Of these, 120 diabetics were selected (60 overweight, 60 lean), along with 120 nondiabetic controls (60 overweight and 60 lean). Males and females were equally represented.

Methods

A prospective case‐control study was performed. Genomic DNA was extracted from blood samples and used as template for PCR amplification of the feline MC4R gene. The coding region of the gene was sequenced in 10 cats to identify polymorphisms. Subsequently, genotyping by restriction fragment length polymorphism (RFLP) analysis assessed MC4R:c.92C > T allele and genotype frequencies in each group of cats.

Results

No significant differences in MC4R:c.92C>T allele or genotype frequencies were identified between nondiabetic overweight and lean cats. In the overweight diabetic group, 55% were homozygous for the MC4R:c.92C allele, compared to 33% of the lean diabetics and 30% of the nondiabetics. The differences between the overweight diabetic and the nondiabetics were significant (P < .01).

Conclusions and Clinical Importance

We identified a polymorphism in the coding sequence of feline MC4R that is associated with DM in overweight DSH cats, similar to the situation in humans.     

Synergistic effect of ERK inhibition on tetrandrine-induced apoptosis in A549 human lung carcinoma cells

Tetrandrine (TET), a bis-benzylisoquinoline alkaloid from the root of Stephania tetrandra, is known to have anti-tumor activity in various malignant neoplasms. However, the precise mechanism by which TET inhibits tumor cell growth remains to be elucidated. The present studies were performed to characterize the potential effects of TET on phosphoinositide 3-kinase/Akt and extracellular signal-regulated kinase (ERK) pathways since these signaling pathways are known to be responsible for cell growth and survival. TET suppressed cell proliferation and induced apoptosis in A549 human lung carcinoma cells. TET treatment resulted in a down-regulation of Akt and ERK phosphorylation in both time-/concentration-dependent manners. The inhibition of ERK using PD98059 synergistically enhanced the TET-induced apoptosis of A549 cells whereas the inhibition of Akt using {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 had a less significant effect. Taken together, our results suggest that TET: i) selectively inhibits the proliferation of lung cancer cells by blocking Akt activation and ii) increases apoptosis by inhibiting ERK. The treatment of lung cancers with TET may enhance the efficacy of chemotherapy and radiotherapy and increase the apoptotic potential of lung cancer cells.     

Inhibitory effects of SKF96365 on the activities of K+ channels in mouse small intestinal smooth muscle cells

In order to investigate the effects of {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365 (SKF), which is a non-selective cationic channel blocker, on K⁺ channel currents, we recorded currents through ATP sensitive K⁺ (I_KATP), voltage-gated K⁺ (I_Kv) and Ca²⁺ activated K⁺ channels (I_BK) in the absence and presence of SKF in single small intestinal myocytes of mice with patch-clamp techniques. SKF (10 µM) reversibly abolished I_KATP that was induced by cromakalim (10 µM), which is a selective ATP sensitive K⁺ channel opener. These inhibitory effects were induced in a concentration-dependent and voltage-independent manner. The 50% inhibitory concentration (IC₅₀) was 0.85 µM, which was obviously lower than that reported for the muscarinic cationic current. In addition, SKF (1 µM ≈ the IC₅₀ value in I_KATP suppression) reversibly inhibited the I_Kv that was induced by repetitive depolarizing pulses from −80 to 20 mV. However, the extent of the inhibitory effects was only ~30%. In contrast, SKF (1 µM) had no significant effects on spontaneous transient I_BK and caffeine-induced I_BK. These results indicated that SKF inhibited ATP sensitive K⁺ channels and voltage-gated K⁺ channels, with the ATP sensitive K⁺ channels being more sensitive than the voltage-gated K⁺ channels. These inhibitory effects on K⁺ channels should be considered when SKF is used as a cationic channel blocker.     

A novel multiplex PCR assay to detect and distinguish between different types of Paenibacillus larvae and Melissococcus plutonius,and a survey of foulbrood pathogen contamination in Japanese honey

Paenibacillus larvae and Melissococcus plutonius are the causative agents of American and European foulbroods of honey bees, respectively. Since their virulence and resistance to disinfectants differ depending on the genotypes/phenotypes of the strains, the discrimination of strain types is important for the effective control of these diseases. Methods to detect and differentiate pathogens in honey are useful for surveying the contamination status of beehives/apiaries. In the present study, we selected a sequence (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"FI763267","term_id":"224923825"}}FI763267) as the specific target for enterobacterial repetitive intergenic consensus (ERIC) II-type P. larvae strains for the first time and developed a novel multiplex PCR assay that precisely distinguishes between the major types of foulbrood pathogens (ERIC I and II P. larvae and typical and atypical M. plutonius) in one reaction. In addition, we found that commercially available kits designed for DNA extraction from Mycobacterium in feces efficiently extracted DNA from foulbrood pathogens in honey. Using the multiplex PCR assay and DNA extraction kits, all the targeted types of P. larvae and M. plutonius were detected in honey spiked with the pathogens at a concentration of 100 bacterial cells/strain/ml. Moreover, 94% of the Japanese honey samples examined in the present study were contaminated with one or more types of the foulbrood pathogens. These results indicate that the newly developed methods are useful for detecting foulbrood pathogens in honey. The epidemiological information obtained by these methods will contribute to the effective control of foulbroods in apiaries.     

Associations between bacterial genotype and outcome of bovine clinical Staphylococcus aureus mastitis

Background

Staphylococcus aureus is an important cause of clinical mastitis in dairy cows worldwide. The cure rate after antimicrobial treatment of clinical S. aureus mastitis is very variable due to both cow and bacterial factors. Studies have shown that bacterial genotype might affect short-term bacteriological and clinical cure, but the long-term outcome has been less studied. The objectives of this study were to investigate associations between bacterial genotype and long-term outcome of veterinary-treated clinical mastitis (VTCM) caused by S. aureus during a follow-up period of 120 days and to study genotype variation among Swedish S. aureus isolates. S. aureus isolates from cases of VTCM were genotyped by pulsed-field gel electrophoresis. Long-term outcome measurements used were somatic cell count (SCC), additional diagnoses of VTCM, milk yield and culling. Isolates were classified into clusters (>80% similarity) and pulsotypes (100% similarity). Clusters and pulsotypes were grouped according to occurrence. Multivariable mixed-effect linear regression models including cow and bacterial factors with possible influence on SCC or milk yield were used to calculate differences in SCC or milk yield between groups. Additional outcome measures were calculated using a test of proportions.

Results

The isolates (n = 185) were divided into 18 clusters and 29 pulsotypes. Two pulsotypes were classified as common, and were found in 64% of the cases of VTCM. Remaining isolates were classified as less common or rare pulsotypes. The distribution was similar at cluster level. Outcome was calculated from follow-up data on 111 cows. Significantly lower SCC during the follow-up period was found in cows infected with common clusters compared to in cows infected with less common/rare clusters. The proportion of cows with SCC <200 000 cells/ml during the whole follow-up period was significantly higher in the group common clusters than in the group less common/rare clusters. Bacterial genotype did not influence the other outcome parameters.

Conclusions

In Sweden, two S. aureus pulsotypes, identified in about 64% of clinical S. aureus cases, were widespread. Cows infected with the common genotypes had significantly lower SCC during 120 days after treatment compared to cows infected with less common or rare genotypes.     

Genotype x environment interactions involving proportion of Brahman breeding and season of birth. II. Postweaning growth, sexual development and reproductive performance of heifers

Postweaning growth, sexual development and reproductive traits were evaluated over a 3-yr period on 201 spring-born and 180 fall-born crossbred heifers with 0, 1/4 or 1/2 Brahman breeding. The proportion of Brahman breeding X season of birth interaction was significant for five traits (average daily gain weaning to yearling, yearling condition score, percentage of heifers detected in estrus, prebreeding condition score and percentage of heifers that became pregnant) and was not significant for six traits (yearling weight, hip height and conformation score, age and weight at puberty and prebreeding weight. Among spring-born heifers, 1/4 and 1/2 Brahman heifers outgained (P less than .05) 0 Brahman heifers from weaning to yearling by 50 and 66 g/d, respectively; among fall-born heifers, 1/4 and 1/2 Brahman heifers outgained (P less than .05) 0 Brahman heifers by 41 and 104 g/d, respectively. Yearling weight of 1/2 Brahman heifers was 13 and 10 kg heavier (P less than .05), respectively, than 0 and 1/4 Brahman heifers. Yearling hip height of 1/4 and 1/2 Brahman heifers were 1.9 and 5.7 cm taller (P less than .05), respectively, than 0 Brahman heifers. Weight prior to the start of the breeding season were similar among crossbred heifer groups and spring-born heifers were 66 kg heavier (P less than .05) than fall-born heifers. The percentage of heifers that became pregnant was similar among spring-born crossbred heifer groups, whereas among fall-born heifer groups 1/4 and 1/2 Brahman heifers were 25.2 and 49.4 percentage points lower (P less than .05), respectively, than 0 Brahman heifers.