Identification of stable normalization genes for quantitative real-time PCR in porcine articular cartilage

Background

Expression levels for genes of interest must be normalized with an appropriate reference, or housekeeping gene, to make accurate comparisons of quantitative real-time PCR results. The purpose of this study was to identify the most stable housekeeping genes in porcine articular cartilage subjected to a mechanical injury from a panel of 10 candidate genes.

Results

Ten candidate housekeeping genes were evaluated in three different treatment groups of mechanically impacted porcine articular cartilage. The genes evaluated were: beta actin, beta-2-microglobulin, glyceraldehyde-3-phosphate dehydrogenase, hydroxymethylbilane synthase, hypoxanthine phosphoribosyl transferase, peptidylprolyl isomerase A (cyclophilin A), ribosomal protein L4, succinate dehydrogenase flavoprotein subunit A, TATA box binding protein, and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein—zeta polypeptide. The stability of the genes was measured using geNorm, BestKeeper, and NormFinder software. The four most stable genes measured via geNorm were (most to least stable) succinate dehydrogenase flavoprotein, subunit A, peptidylprolyl isomerase A, glyceraldehyde-3-phosphate dehydrogenase, beta actin; the four most stable genes measured via BestKeeper were glyceraldehyde-3-phosphate dehydrogenase, peptidylprolyl isomerase A, beta actin, succinate dehydrogenase flavoprotein, subunit A; and the four most stable genes measured via NormFinder were peptidylprolyl isomerase A, succinate dehydrogenase flavoprotein, subunit A, glyceraldehyde-3-phosphate dehydrogenase, beta actin.

Conclusions

BestKeeper, geNorm, and NormFinder all generated similar results for the most stable genes in porcine articular cartilage. The use of these appropriate reference genes will facilitate accurate gene expression studies of porcine articular cartilage and suggest appropriate housekeeping genes for articular cartilage studies in other species.     

Detection of virulence-associated genes in Staphylococcus aureus isolated from bovine clinical mastitis milk samples in Guangxi

Staphylococcus aureus is recognized worldwide as a pathogen causing many serious diseases in humans and animals and is one of the most common etiological agents of clinical and subclinical bovine mastitis. The purpose of this study was to determine the presence of genes encoding clfA, fnbA, fnbB, cap5, cap8, hla, hlb, nuc, sea, and tst of S. aureus strains (n?=?39) isolated from bovine clinical mastitis in Guangxi by polymerase chain reaction amplification. The results of the present study indicated that all isolates were found to contain one or more virulence-associated genes. The most frequently encountered genes were fnbA (97?%) and nuc (90?%), followed by hla (85?%) and hlb (82?%), respectively. None of the investigated S. aureus strains harbored fnbB and sea genes. The data in the present study showed a relatively wide distribution of the genes fnbA and nuc among the investigated isolates, indicating that they play an important role on bovine mastitis pathogenesis. The study provides a valuable insight into the virulence-associated genes of this important pathogen.     

Vibrios as causal agents of zoonoses

Vibrios are Gram-negative rod-shaped bacteria that are widespread in the coastal and estuarine environments. Some species, e.g. Vibrio anguillarum and V. tapetis, comprise serious pathogens of aquatic vertebrates or invertebrates. Other groups, including Grimontia (=Vibrio) hollisae, Photobacterium (=Vibrio) damselae subsp. damselae, V. alginolyticus, V. harveyi (=V. carchariae), V. cholerae, V. fluvialis, V. furnissii, V. metschnikovii, V. mimicus, V. parahaemolyticus and V. vulnificus, may cause disease in both aquatic animals and humans. The human outbreaks, although low in number, typically involve wound infections and gastro-intestinal disease often with watery diarrhoea. In a minority of cases, for example V. vulnificus, there is good evidence to actually associate human infections with diseased animals. In other cases, the link is certainly feasible but hard evidence is mostly lacking.     

Virulence profiles of enterotoxigenic, shiga toxin and enteroaggregative Escherichia coli in South African pigs

Enterotoxigenic Escherichia coli (ETEC) and shiga toxin E. coli (STEC) are important causes of colibacillosis in piglets. Recently, enteroaggregative E. coli heat-stable enterotoxin 1 (EAST-1) has been implicated in pig diarrhoea. This study investigated the prevalence of enterotoxin [heat-labile toxins (LT), heat-stable toxin a (STa), heat-stable toxin b (STb)], shiga toxins (Stx1, Stx2, Stx2e), enteroaggregative heat-stable E. coli (EAST-1), associated fimbriae (F4, F5, F6, F41, F18ab, F18ac) and non-fimbrial adhesins [adhesin involved in diffuse adherence 1 (AIDA-1), attaching and effacing factor, porcine attaching- and effacing-associated factor] in South African pigs. A total of 263 E. coli strains were isolated from Landrace (n?=?24), Large White (n?=?126), Duroc (n?=?28) and indigenous (n?=?85) breeds of piglets aged between 9 and 136 days. PCR was used in the analysis. Virulent genes were detected in 40.3 % of the isolates, of which 18.6, 0.4 and 17.5 % were classified as ETEC, STEC and enteroaggregative E. coli (EAEC), respectively. Individual genes were found in the following proportions: STb (19.01 %), LT (0.4 %), STa (3.4 %), St2xe (1.1 %) and EAST-1 (20.2 %) toxins. None of the tested fimbriae were detected in ETEC and STEC isolates. About one third of the ETEC and STEC isolates was tested negative for both fimbrial and non-fimbrial adhesins. Twenty-five pathotypes from ETEC-, EAEC- and STEC-positive strains were identified. Pathotypes EAST-1 (30.2 %), STb (13.2 %) and STb/AIDA-1 (10.4 %) were most prevalent. The study provided insight on possible causes of colibacillosis in South African pigs.     

Effect of substitution of concentrate mix with Sesbania sesban on feed intake, digestibility, body weight change, and carcass parameters of Arsi-Bale sheep fed a basal diet of native grass hay

An experiment was conducted to assess the effect of substitution of concentrate mix with Sesbania sesban on feed intake, digestibility, average daily gain (ADG), and carcass parameters of Arsi-Bale sheep. The experiment employed 25 male sheep with mean (±standard error) initial body live weight (BLW) of 19.1?±?0.09 kg. The experiment consisted of 7 days of digestibility and 90 days of feeding trials followed by carcass evaluation. The experiment employed a randomized complete block design with five treatments and five blocks. Treatments comprised of grass hay alone fed ad libitum (GHA; control), GHA?+?100 % concentrate mix (CM) consisting of wheat bran and noug seed cake at a ratio of 2:1 (0 S. sesban), GHA?+?67 % CM?+?33 % S. sesban (33 S. sesban), GHA?+?33 % CM?+?67 % S. sesban (67 S. sesban), and GHA?+?100 % S. sesban (100 S. sesban). Total dry matter intake (DMI) was higher (p?<?0.001) for sheep in 0 S. sesban–100 S. sesban (800–821 g/day) compared to sheep in control (611 g/day). However, the effect of S. sesban inclusion (0 S. sesban–100 S. sesban) on total DMI was quadratic, and DMI declined after 67 S. sesban. Digestibility of DM, organic matter (p?<?0.01), and crude protein were higher (p?<?0.001) in supplemented group compared to the control. ADG, feed conversion efficiency (ADG/DMI), slaughter BLW, hot carcass weight, and total edible offals were higher (p?<?0.05–0.001) for sheep in 0 S. sesban–100 S. sesban than those in control. Increased level of S. sesban inclusion, in general, reduced growth and carcass parameters in this study. However, there was no difference between 0 S. sesban and 33 S. sesban in most parameters studied. Thus, it can be concluded that S. sesban could substitute a concentrate when it accounted for up to 33 % of the mix.     

Virulence factors in Escherichia coli isolated from calves with diarrhea in Vietnam

This study was conducted to determine the prevalence and characteristics of pathogenic Escherichia (E.) coli strains from diarrheic calves in Vietnam. A total of 345 E. coli isolates obtained from 322 diarrheic calves were subjected to PCR and multiplex PCR for detection of the f5, f41, f17, eae, sta, lt, stx1, and stx2 genes. Of the 345 isolates, 108 (31.3%) carried at least one fimbrial gene. Of these 108 isolates, 50 carried genes for Shiga toxin and one possessed genes for both enterotoxin and Shiga toxin. The eae gene was found in 34 isolates (9.8%), 23 of which also carried stx genes. The Shiga toxin genes were detected in 177 isolates (51.3%) and the number of strains that carried stx1, stx2 and stx1/stx2 were 46, 73 and 58, respectively. Among 177 Shiga toxin-producing E. coli isolates, 89 carried the ehxA gene and 87 possessed the saa gene. Further characterization of the stx subtypes showed that among 104 stx1-positive isolates, 58 were the stx1c variant and 46 were the stx1 variant. Of the 131 stx2-positive strains, 48 were stx2, 48 were stx2c, 11 were stx2d, 17 were stx2g, and seven were stx2c/stx2g subtypes. The serogroups most prevalent among the 345 isolates were O15, O20, O103 and O157.     

Changes in the Expression Patterns of the Genes Involved in the Segregation and Function of Inner Cell Mass and Trophectoderm Lineages During Porcine Preimplantation Development

In mouse embryos, segregation of the inner cell mass (ICM) and trophectoderm (TE) lineages is regulated by genes, such as OCT-4, CDX2 and TEAD4. However, the molecular mechanisms that regulate the segregation of the ICM and TE lineages in porcine embryos remain unknown. To obtain insights regarding the segregation of the ICM and TE lineages in porcine embryos, we examined the mRNA expression patterns of candidate genes, OCT-4, CDX2, TEAD4, GATA3, NANOG, FGF4, FGFR1-IIIc and FGFR2-IIIc, in blastocyst and elongated stage embryos. In blastocyst embryos, the expression levels of OCT-4, FGF4 and FGFR1-IIIc were significantly higher in the ICM than in the TE, while the CDX2, TEAD4 and GATA3 levels did not differ between the ICM and TE. The expression ratio of CDX2 to OCT-4 (CDX2/OCT-4) also did not differ between the ICM and TE at the blastocyst stage. In elongated embryos, OCT-4, NANOG, FGF4 and FGFR1-IIIc were abundantly expressed in the embryo disc (ED; ICM lineage), but their expression levels were very low in the TE. In contrast, the CDX2, TEAD4 and GATA3 levels were significantly higher in the TE than in the ED. In addition, the CDX2/OCT-4 ratio was markedly higher in the TE than in the ED. We demonstrated that differences in the expression levels of OCT-4, CDX2, TEAD4, GATA3, NANOG, FGF4, FGFR1-IIIc and FGFR2-IIIc genes between ICM and TE lineages cells become more clear during development from porcine blastocyst to elongated embryos, which indicates the possibility that in porcine embryos, functions of ICM and TE lineage cells depend on these gene expressions proceed as transition from blastocyst to elongated stage.     

Molecular detection of Theileria spp. in sheep and vector ticks in the North Khorasan Province, Iran

A survey was carried in North Khorasan Province, Iran in 2010–2011, designed to identify Theileria spp. infections of both sheep and ticks. The tick species were also examined. Ninety sheep from different flocks were clinically examined, and blood samples and ixodid ticks were collected. Light microscopy of blood smears revealed Theileria spp. infection in 37 (41.1 %), while 74 (82.2 %) of blood samples were positive using semi-nested PCR. Theileria ovis, Theileria lestoquardi, and mixed infection were detected in 63/90 (70 %), 5/90 (5.5 %), and 6/90 (6.6 %) of samples, respectively. Of the 434 ticks that were collected, the most prevalent species was Rhipicephalus turanicus (69.3 %) followed by Hyalomma marginatum turanicum (18.4 %), Dermacentor marginatus (6.4 %), and Rhipicephalus bursa (5.7 %). The ticks were separated into 42 tick pools, and the salivary glands were dissected out in 0.85 % (w/v) saline under a stereomicroscope and examined using semi-nested PCR. Three pools of H. marginatum turanicum salivary glands were infected with T. ovis and T. lestoquardi, and one pool of R. turanicus was infected with T. ovis. Based on these results, it is concluded that the prevalence of T. ovis is higher than T. lestoquardi and that H. marginatum turanicum and R. turanicus are likely vectors of T. lestoquardi and T. ovis in this area.     

Flea species infesting dogs in Florida and Bartonella spp. prevalence rates

Several Bartonella spp. associated with fleas can induce a variety of clinical syndromes in both dogs and humans. However, few studies have investigated the prevalence of Bartonella in the blood of dogs and their fleas. The objectives of this study were to determine the genera of fleas infesting shelter dogs in Florida, the prevalence of Bartonella spp. within the fleas, and the prevalence of Bartonella spp. within the blood of healthy dogs from which the fleas were collected. Fleas, serum, and EDTA-anti-coagulated whole blood were collected from 80 healthy dogs, and total DNA was extracted for PCR amplification of Bartonella spp. The genera of fleas infesting 43 of the dogs were determined phenotypically. PCR amplicons from blood and flea pools were sequenced to confirm the Bartonella species. Amplicons for which sequencing revealed homology to Bartonella vinsonii subsp. berkhoffii (Bvb) underwent specific genotyping by targeting the 16S–23S intergenic spacer region.A total of 220 fleas were collected from 80 dogs and pooled by genus (43 dogs) and flea species. Bartonella spp. DNA was amplified from 14 of 80 dog blood samples (17.5%) and from 9 of 80 pooled fleas (11.3%). B. vinsonii subsp. berkhoffii DNA was amplified from nine dogs and five of the flea pools. Bartonella rochalimae (Br) DNA was amplified from six dogs and two flea pools. One of 14 dogs was co-infected with Bvb and Br. The dog was infested with Pulex spp. fleas containing Br DNA and a single Ctenocephalides felis flea. Of the Bvb bacteremic dogs, five and four were infected with genotypes II and I, respectively. Of the Bvb PCR positive flea pools, three were Bvb genotype II and two were Bvb genotype I.Amplification of Bvb DNA from Pulex spp. collected from domestic dogs, suggests that Pulex fleas may be a vector for dogs and a source for zoonotic transfer of this pathogen from dogs to people. The findings of this study provide evidence to support the hypothesis that flea-infested dogs may be a reservoir host for Bvb and Br and that ectoparasite control is an important component of shelter intake protocols.     

Freeze-resistant Trichinella (Trichinella nativa) Established on the Scandinavian Peninsula

Until the 1970’s, Trichinella spiralis (Owen 1835) was considered the only species within the genus Trichinella. Then T. pseudospiralis (Garkavi 1972) was classified as a separate species on the basis of morphological and biological features. The remaining morphologically homogenous “T. spiralis-group” has been split into 4 different species (or subspecies) on the basis of their biological and biochemical characteristics; T. nativa (Britov & Boev 1972), T. nelsoni (Britov & Boev 1972), T. spiralis sensu stricto and T. britovi (Pozio et al. 1992).     

Occurrence of Mycobacterium spp. and other pathogens in lymph nodes of slaughtered swine and wild boars (Sus scrofa)

Mycobacterium spp. and other pathogens were investigated in 258 swine lymph nodes (129 with and 129 without apparent lesions), and 120 lymph nodes (60 with and 60 without lesions) from wild boars (Sus scrofa). A total of lymph nodes from swine and wild boars were collected of different animals. Submaxillar and mesenteric lymph nodes were submitted to microbiological examination and colonies suggestive of Mycobacterium spp. (alcohol-acid bacilli) were submitted to PCR Restriction Assay (PRA). In swine with lymphadenitis, Mycobacterium spp. (24.1%) and Rhodococcus equi (13.2%) were the most prevalent microorganisms, while in lymph nodes without lesions were identified a complex of microorganisms, including of environmental mycobacteria. In wild boars with lymphadenitis, ß-haemolytic Streptococcus (10.0%), Mycobacterium spp (8.4%) and R. equi (6.6%) were the most frequent. Among mycobacterias were identified predominantly Mycobacterium avium subspecies type 1 (48.3%) and M. avium subspecies type 2 (16.1%), followed by Mycobacterium intracellulare, Mycobacterium szulgai,Mycobacterium fortuitum, Mycobacterium gordonae, Mycobacterium simiae, Mycobacterium nonchromogenicum and Mycobacterium intracellulare type 2.     

Phase I biotransformation of albendazole in lancet fluke (Dicrocoelium dendriticum)

Dicroceliosis, a lancet fluke infection, is a frequent parasitosis of small ruminants and the anthelmintic drug albendazole (ABZ) is effective in control of this parasitosis. The aim of our project was to study the metabolism of ABZ and ABZ sulphoxide (ABZ.SO) in lancet fluke. Both invitro (subcellular fractions of fluke homogenates) and exvivo experiments (adult flukes cultivated in medium) were performed for this purpose. ABZ was metabolised invitro by lancet fluke NADPH-dependent enzymes by two oxidative steps (sulphoxidation and sulphonation). The apparent kinetic parameters of these reactions have been determined. In the exvivo experiments, only ABZ sulphoxidation was observed. The stereospecificity in ABZ sulphoxidation invitro was slight, with preferential formation of (+)-ABZ.SO enantiomer. In contrast (−)-ABZ.SO formation predominated in exvivo experiments. Sulphoreduction of ABZ.SO occurred neither invivo nor exvivo. The detection of ABZ oxidative metabolites indicates the presence of drug metabolising oxidases in lancet fluke.     

Recent advances in the understanding of Chlamydophila pecorum infections, sixteen years after it was named as the fourth species of the Chlamydiaceae family

Chlamydophila pecorum found in the intestine and vaginal mucus of asymptomatic ruminants has also been associated with different pathological conditions in ruminants, swine and koalas. Some endangered species such as water buffalos and bandicoots have also been found to be infected by C. pecorum. The persistence of C. pecorum strains in the intestine and vaginal mucus of ruminants could cause long-term sub-clinical infection affecting the animal’s health. C. pecorum strains present many genetic and antigenic variations, but coding tandem repeats have recently been found in some C. pecorum genes, allowing C. pecorum strains isolated from sick animals to be differentiated from those isolated from asymptomatic animals. This review provides an update on C. pecorum infections in different animal hosts and the implications for animal health. The taxonomy, typing and genetic aspects of C. pecorum are also reviewed.     

Molecular detection of piroplasms in ixodid ticks infesting cattle and sheep in western Oromia, Ethiopia

In Ethiopia, ticks and tick-borne diseases are widely distributed and contribute to important economic losses. Several studies investigated the prevalence and species composition of ticks infesting ruminants; however, data on tick-borne pathogens are still scarce. During the study period from October 2010 to April 2011, a total of 1,246 adult ticks and 264 nymphs were collected from 267 cattle and 45 sheep in Bako District, western Oromia, Ethiopia. The study showed infestation of 228/267 (85.4 %) cattle and 35/45 (77.8 %) sheep with adult ticks. Overall, eight tick species, belonging to three genera (Amblyomma, Rhipicephalus, Hyalomma), were identified and Amblyomma cohaerens (n?=?577), Rhipicephalus evertsi evertsi (n?=?290), Rhipicephalus (Boophilus) decoloratus (n?=?287), and Amblyomma variegatum (n?=?85) were the more prevalent species. A statistically significant host preference in A. cohaerens for cattle and R. evertsi evertsi for sheep was noticed. Molecular detection of piroplasms, performed only for adult ticks of two species of the genus Rhipicephalus (R. evertsi evertsi and R. decoloratus), revealed an overall prevalence of 4 % (8/202) Theileria buffeli/sergenti/orientalis, 0.5 % (1/202) Theileria velifera, and 2 % (4/202) Theileria ovis. The study showed that tick infestation prevalence is considerably high in both cattle and sheep of the area, but with a low intensity of tick burden and a moderate circulation of mildly pathogenic piroplasm species.     

Aerobic bacteria from mucous membranes, ear canals, and skin wounds of feral cats in Grenada, and the antimicrobial drug susceptibility of major isolates

In a 2-year period 54 feral cats were captured in Grenada, West Indies, and a total of 383 samples consisting of swabs from rectum, vagina, ears, eyes, mouth, nose and wounds/abscesses, were cultured for aerobic bacteria and campylobacters. A total of 251 bacterial isolates were obtained, of which 205 were identified to species level and 46 to genus level. A commercial bacterial identification system (API/Biomerieux), was used for this purpose. The most common species was Escherichia coli (N = 60), followed by Staphylococcus felis/simulans (40), S. hominis (16), S. haemolyticus (12), Streptococcus canis (9), Proteus mirabilis (8), Pasteurella multocida (7), Streptococcus mitis (7), Staphylococcus xylosus (7), S. capitis (6), S. chromogenes (4), S. sciuri (3), S. auricularis (2), S. lentus (2), S. hyicus (2), Streptococcus suis (2) and Pseudomonas argentinensis (2). Sixteen other isolates were identified to species level. A molecular method using 16S rRNA sequencing was used to confirm/identify 22 isolates. Salmonella or campylobacters were not isolated from rectal swabs. E. coli and S. felis/simulans together constituted 50% of isolates from vagina. S. felis/simulans was the most common species from culture positive ear and eye samples. P. multocida was isolated from 15% of mouth samples. Coagulase-negative staphylococci were the most common isolates from nose and wound swabs. Staphylococcus aureus, or S. intemedius/S. pseudintermedius were not isolated from any sample. Antimicrobial drug resistance was minimal, most isolates being susceptible to all drugs tested against, including tetracycline.     

The use of tick transmission by Boophilus microplus to isolate pure strains of Babesia bovis,Babesia bigemina and Anaplasma marginale from cattle with mixed infections

Pure strains of Babesia bovi, Babesia bigemina and Anaplasma marginale were isolated from cattle infected with all 3 species as well as a Theileria sp. and Eperythrozoon teganodes, using only transmission by the tick, Boophilus microplus. Unengorged adult ticks transferred to susceptible cattle transmitted A. marginale, but not Babesia. Engorged adults gave rise to progeny that transmitted Babesia, B. bovis by larvae and B. bigemina by male ticks. The Theileria and E. teganodes were not transmitted by the ticks and thus did not appear in calves used for isolating the pure strains of Babesia and A. marginale.     

In Vitro Fermentative Capacity of Equine Fecal Inocula of 9 fibrous Forages in the Presence of Different Doses of Saccharomyces cerevisiae

This experiment was conducted to evaluate in vitro effects of equine fecal inocula fermentative capacity on 9 fibrous forages in the presence of Saccharomyces cerevisiae. The fibrous feeds were corn stover (Zea mays), oat straw (Avena sativa), sugarcane bagasse and leaves (Saccharum officinarum), llanero grass leaves (Andropogon gayanus), Taiwan grass leaves (Pennisetum purpureum), sorghum straw (Sorghum vulgare), and steria grass leaves (Cynodon plectostachyus). Fibrous feed samples were incubated with several doses of S. cerevisiae; 0 (control), 1.25 (low), 2.5 (medium) and 5 (high) mg/g dry matter (DM) of a commercial yeast product containing 1 × 10¹⁰/g. Fecal inoculum was collected from 4 adult horses were fed on an amount of commercial concentrate and oat hay ad libitum. Gas production (GP) was recorded at 2, 4, 6, 8, 10, 12, 24, and 48 hours post inoculation. An interaction occurred between feeds and yeast dose for fecal pH (P < .01), asymptotic GP (b, ml/g DM); rate of GP (c, /hr); initial delay before GP began (L, hours), GP at 4 hours and 48 hours (P < .01), and GP at 8 hours (P < .01) and at 24 hours (P < .01). Differences in fecal fermentation capacity between the tropical and template grass (P < .05) occurred for fecal pH, c, and GP during first 12 hours, whereas differences occurred (P < .05) between the agriculture byproducts and the grasses for fecal pH, b, and GP from 8 to 48 hours. Fermentation capacity between straws versus not straws (P < .05) differed for fecal pH, b, and GP after 12 hours between straws versus not straws. Addition of S. cerevisiae to Z. mays stover reduced (P < .01) fecal pH and the c fraction with a higher (P < .01) b fraction versus the other feeds. From 4 to 24 hours, S. officinarum bagasse improved GP to the highest values versusS. officinarum leaves. After 24 hours, Z. mays stover had the highest GP, whereas C. plectostachyus leaves had the lowest. There were no differences among the yeast doses for all measured parameters with the exception of L values (linear effect; P < .01). The Z. mays stover had the highest nutritive compared to the other fibrous feeds. However, addition of S. cerevisiae at 2.5 to 5.0 g/kg DM improved fecal fermentation capacity of low-quality forages.     

Wild Bonelli’s eagles (Aquila fasciata) as carrier of antimicrobial resistant Salmonella and Campylobacter in Eastern Spain

Wild birds have repeatedly been found to be involved in the dissemination of enteric bacterial pathogens in the environment. The aim of this study was to determine the occurrence of Salmonella and Campylobacter as well as the antimicrobial resistance in wild Bonelli’s eagles nestlings in Eastern Spain. In addition, we compared the efficiency of two sampling methods (fresh faecal samples from nest and cloacal swabs from nestlings) for detection of both bacteria. A total of 28 nests with 45 nestlings were analysed. In the nest, Salmonella occurrence was 61 ± 9.2%, while Campylobacter occurrence was 11 ± 5.8% (p < 0.05). In the nestlings, Salmonella occurrence was 36 ± 7.1%, while Campylobacter occurrence was 11 ± 4.7% (p < 0.05). Eight Salmonella serovars were identified, and the most frequently isolated were S. Enteritidis, S. Typhimurium, S. Houston, and S. Cerro. Only one Campylobacter species was identified (C. jejuni). Regarding antimicrobial resistance, the Salmonella strains isolated were found to be most frequently resistant to ampicillin and to tigecycline; however, the sole Campylobacter strain recovered was multidrug resistant. In conclusion, this study demonstrated that wild Bonelli’s eagles nestlings are greater carriers of Salmonella than of Campylobacter. Both Salmonella and Campylobacter isolates exhibited antimicrobial resistance. In addition, faecal samples from nests were most reliable for Salmonella detection, while cloacal swab from nestlings were most reliable for Campylobacter detection.     

Serological epidemiological surveillance for vapN-harboring Rhodococcus equi infection in goats

Rhodococcus equi causes suppurative pneumonia in foals aged 1–3 months; moreover, it has emerged as a pathogenic cause of zoonotic diseases. After the initial report of the ruminant-pathogenic factor VapN encoded by the novel virulence plasmid pVAPN, several reports have described ruminant infections caused by vapN-harboring R. equi. Herein, we conducted a serological epidemiological surveillance in goats at a breeding farm (Farm A) and characterized the vapN-harboring R. equi isolates from this farm. First, we established a simple screening enzyme-linked immunosorbent assay (ELISA) using recombinant glutathione S-transferase–tagged VapN as an immobilized antigen. This method revealed that the VapN antibody titers were elevated in 12 of 42 goats. Subsequently, we attempted to isolate R. equi from the goat feces and soil of Farm A. choE⁺/vapN⁺ R. equi was isolated from the feces of Goat No. 27 and a soil sample near the shed. The pulsed-field gel electrophoresis (PFGE) patterns of five vapN-harboring R. equi strains isolated from Farm A in 2013 and 2019 were investigated and found to be the same except for the strain (OKI2019F1). However, no difference was observed in VapN expression and growth in macrophages among these vapN-harboring R. equi isolates. Our results revealed that some goats had histories of vapN-harboring R. equi infections, and two genomic types of vapN-harboring R. equi were found in isolates from Farm A. Ruminant-specific (pVAPN-carrying) R. equi might be an unrecognized pathogen in Japan and further studies are required to determine its prevalence and distribution.