Species-specific PCR Research Articles

Distribution of Vibrionaceae in farmed Asian sea bass, Lates calcarifer in Thailand and their high prevalence of antimicrobial resistance

This study describes the etiological agent of Vibriosis along with its distribution and antimicrobial resistance profiles among farmed Asian sea bass (Lates calcarifer) in Thailand. The study isolated 283 Vibrionaceae from 15 Asian sea bass farms located around the provinces of the Andaman Sea and Gulf of Thailand coasts to uncover the distribution and antimicrobial resistance profiles. Bacterial identification based on a combination of the biochemical characteristics, Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) analysis, and the species-specific PCR demonstrated the predominant Vibrionaceae were Vibrio harveyi (n=56), Photobacterium damselae (n=35), and V. vulnificus (n=31), respectively. According to a laboratory challenge experiment, among the six isolates, only V. harveyi was found to cause clinical signs of muscle necrosis and scale loss in Asian sea bass. Antibiotics resistance test results exhibited high resistance to antibiotics such as metronidazole (100%), streptomycin (97%), clindamycin (96%), colistin sulphate (70%) and amoxicillin (59%). Remarkably, 100% of Vibrionaceae isolates are susceptible to florfenicol. The 28 of 29 resistance profiles were multidrug resistances (MDR), with V. vulnificus having the highest MAR value (0.66). The findings of this study advise that a surveillance program, as well as preventive and control measures, be developed for Vibrionaceae to reduce production loss, pathogen proliferation, and antibiotic abuse, whereas AMR data indicate substantial health problems for aquatic animals and humans.

Beyond Domestic Cats: Environmental Detection of Sporothrix brasiliensis DNA in a Hyperendemic Area of Sporotrichosis in Rio de Janeiro State, Brazil.

In Brazil, sporotrichosis has transitioned from a rural to urban disease, driven by a shift in the initiation of infection from the accidental inoculation of organic matter to the traumatic implantation of the fungus by cats. Since the emergence of zoonotic sporotrichosis caused by Sporothrix brasiliensis, investigations have largely ignored the environmental habitat of the pathogen due to its association with domestic cats. Therefore, we investigated 18 environmental samples collected from rural areas of two cities where zoonotic sporotrichosis is endemic, but where domestic cats are scarce. We utilized traditional culture methods, and samples were also examined with two molecular methods used for the clinical diagnosis of sporotrichosis: a nested-PCR targeting the ITS region and a species-specific PCR targeting the calmodulin gene. No Sporothrix colonies were identified by traditional culture methods. However, the nested-PCR and the species-specific PCR for S. brasiliensis were positive for 18 and 5 samples, respectively. Sequencing revealed that positive results with the nested-PCR were due to non-specific amplification of other Ophiostomatales DNA, rather than Sporothrix spp. Three of the five amplicons from the species-specific PCR were suitable for sequencing and confirmed the presence of S. brasiliensis DNA. Hence, we confirmed that S. brasiliensis, as with other Sporothrix species, has an environmental habitat. Our findings underscore the challenges of nested-PCR for Sporothrix environmental studies and highlight that sequencing must follow PCR protocols to definitively identify Sporothrix spp. in environmental samples.

Multiplex PCR assay for correct identification of the fish pathogenic species of Edwardsiella genus reveals the presence of E. anguillarum in South America in strains previously characterized as E. tarda.

Develop a species-specific multiplex PCR to correctly identify Edwardsiella species in routine diagnostic for fish bacterial diseases. The genomes of 62 Edwardsiella spp. isolates available from the National Center for Biotechnology Information (NCBI) database were subjected to taxonomic and pan-genomic analyses to identify unique regions that could be exploited by species-specific PCR. The designed primers were tested against isolated Edwardsiella spp. strains, revealing errors in commercial biochemical tests for bacterial classification regarding Edwardsiella species. Some of the genomes of Edwardsiella spp. in the NCBI platform were incorrectly classified, which can lead to errors in some research. A functional mPCR was developed to differentiate between phenotypically and genetically ambiguous Edwardsiella, with which, we detected the presence of Edwardsiella anguillarum affecting fish in Brazil. This study shows that the misclassification of Edwardsiella spp in Brazil concealed the presence of E. anguillarum in South America. Also, this review of the taxonomic classification of the Edwardsiella genus is a contribution to the field to help researchers with their sequencing and identification of genomes, showing some misclassifications in online databases that must be corrected, as well as developing an easy assay to characterize Edwardsiella species in an end-point mPCR.

Identification of novel molecular targets for Weissella species-specific real-time PCR based on pangenome analysis.

Some Weissella species are used in probiotic products because of their beneficial effects in humans, whereas some species are considered as opportunistic pathogens that cause infections in humans. Therefore, an accurate and rapid identification of Weissella species is essential to control pathogenic Weissella species or isolate new functional strains with probiotic effects from their habitat. The objective of our study was to extract novel molecular targets using pangenome analysis for the identification of major Weissella species present in food. With 50 genomes representing 11 Weissella species, novel molecular targets were mined based on their 100% presence in the respective strains of the target species and absence in the strains of non-target bacteria. Primers based on molecular targets showed positive results for the corresponding species, whereas 79 non-target strains showed negative results. Standard curves revealed good linearity in the range of 103-108 colony-forming units per reaction. Our method was successfully applied to 74 Weissella strains isolated from food samples to demonstrate that the molecular targets provided a viable alternative to the 16S rRNA sequence. Furthermore, it was possible to identify and quantify Weissella communities in fermented foods. These results demonstrate that our method can be used for effective and accurate screening for the presence of Weissella species in foods. KEY POINTS: • This is first study to mine novel targets for differentiating 11 Weissella species. • The novel targets showed higher resolution than the 16S rRNA gene sequence. • The PCR method effectively detected Weissella species with opposing properties.

MOLECULAR ASSESSMENT OF FECAL LACTOBACILLI POPULATIONS IN CHILDREN WITH FUNCTIONAL CONSTIPATION.

Investigation of the gut-specific bacterial strains including lactobacilli is essential for understanding the bacterial etiology of constipation. This study aimed to compare the prevalence and quantity of intestinal lactobacilli in constipated children and healthy controls. Forty children fulfilling Rome IV criteria for functional constipation and 40 healthy controls were recruited. Fecal samples were analyzed using species-specific polymerase chain reaction followed by random amplified polymorphic DNA-PCR and quantitative real-time PCR. Totally, seven different species of lactobacilli were detected. Out of 80 volunteers, 65 (81.3%) were culture and species-specific PCR positive from which 25 (38.46%) constipated children and 40 (61.54%) healthy subjects. The most prevalent species were L. paracasei 21 (32.3%) followed by L. plantarum 18 (27.7%) among both healthy and patient groups. Analysis of the RAPD dendrograms displayed that strains isolated from constipated and non-constipated children have similarity coefficients of more than 90%. The qPCR assays demonstrated constipated children had a lower amount of total lactobacilli population (per gram of feces) than healthy controls. Our findings showed that the mere existence of various species of Lactobacillus in the gut does not enough to prevent some gastrointestinal disorders such as functional constipation, and their quantity plays a more important role.

Development of multiplex species-specific PCR for the simultaneous identification of three closely related species in the genera Misgurnus and Paramisgurnus

We aimed to develop a multiplex PCR method for the simultaneous detection of the three most consumed commercial loaches in East Asia: Misgurnus mizolepis, Misgurnus anguillicaudatus, and Paramisgurnus dabryanus. Phylogenetic analysis of genetic divergence and a phylogenetic tree based on the cytochrome oxidase I gene enabled successful genetic identification of the 3 species of loach. Multiplex species-specific PCR (MSS-PCR) primers designed based on single nucleotide polymorphisms were able to differentiate the species and generate PCR products among species that differed in size by at least 150 bp. The lengths of the amplified fragments were 818 bp for P. dabryanus, 669 bp for M. mizolepis, and 486 bp for M. anguillicaudatus, which could be clearly distinguished by gel electrophoresis. The three sets of PCR primers were mixed and applied to simultaneously detect the three loaches. The MSS-PCR markers designed in this study will be useful for the detection and identification of M. mizolepis, M. anguillicaudatus, and P. dabryanus.

Environmental Isolation of Sporothrix brasiliensis in an Area With Recurrent Feline Sporotrichosis Cases.

Sporotrichosis has been expanding throughout the Brazilian territory in recent years. New outbreaks have emerged, and consequently, the sporotrichosis agents, mainly Sporothrix brasiliensis, should remain in the environment somehow. Therefore, the aim of this study was to investigate the presence of Sporothrix spp. in the environment from an area of ​​the Rio de Janeiro state, Brazil, with recurrent cases of human and animal sporotrichosis. Abandoned demolition timber wood samples were collected in the garden of a house where the cases of human and feline sporotrichosis have occurred in the last 10 years. The environmental survey revealed a Sporothrix spp. colony from the serial dilution cultures of one abandoned demolition wood sample. In addition, a fungal strain isolated from a cat with skin lesions that lived in the house was also included in the study. The species-specific PCR, and calmodulin partial sequencing identified the environmental and cat isolates as S. brasiliensis. Furthermore, the phylogenetic analysis performed with the partial sequences of internal transcribed spacer region and constitutive genes (calmodulin, β-tubulin, and chitin synthase) showed high similarity between environmental and cat isolates from the same geographic region. Moreover, the antifungal susceptibility test revealed that the minimal inhibitory concentration of itraconazole from the environment isolate was lower than the cat isolate, while amphotericin B and terbinafine were similar. Our results show that S. brasiliensis is able to maintain itself in the environmental material for years. With this, we corroborate that the eco-epidemiology of sporotrichosis is not well understood, and despite the major occurrence of S. brasiliensis in Brazil, it is rarely isolated from the environment.

Molecular survey of Babesia parasites in Kenya: first detailed report on occurrence of Babesia bovis in cattle

BackgroundAmong protozoan parasites in the genus Babesia, Babesia bigemina is endemic and widespread in the East African region while the status of the more pathogenic Babesia bovis remains unclear despite the presence of the tick vector, Rhipicephalus microplus, which transmits both species. Recent studies have confirmed the occurrence of R. microplus in coastal Kenya, and although B. bovis DNA has previously been detected in cattle blood in Kenya, no surveillance has been done to establish its prevalence. This study therefore investigated the occurrence of B. bovis in cattle in Kwale County, Kenya, where R. microplus is present in large numbers.MethodsA species-specific multiplex TaqMan real-time PCR assay targeting two Babesia bovis genes, 18S ribosomal RNA and mitochondrially-encoded cytochrome b and B. bigemina cytochrome b gene was used to screen 506 cattle blood DNA samples collected from Kwale County for presence of Babesia parasite DNA. A sub-set of 29 B. bovis real-time PCR-positive samples were further amplified using a B. bovis-specific spherical body protein-4 (SBP-4) nested PCR and the resulting products sequenced to confirm the presence of B. bovis.ResultsA total of 131 animals (25.8%) were found to have bovine babesiosis based on real-time PCR. Twenty-four SBP4 nucleotide sequences obtained matched to B. bovis with a similarity of 97–100%. Of 131 infected animals, 87 (17.2%) were positive for B. bovis while 70 (13.8%) had B. bigemina and 26 (5.1%) were observed to be co-infected with both Babesia species. A total of 61 animals (12.1%) were found to be infected with B. bovis parasites only, while 44 animals (8.7%) had B. bigemina only. Babesia bovis and B. bigemina infections were detected in the three Kwale sub-counties.ConclusionThese findings reveal high prevalence of pathogenic B. bovis in a Kenyan area cutting across a busy transboundary livestock trade route with neighbouring Tanzania. The Babesia multiplex real-time PCR assay used in this study is specific and can detect and differentiate the two Babesia species and should be used for routine B. bovis surveillance to monitor the spread and establishment of the pathogen in other African countries where B. bigemina is endemic. Moreover, these findings highlight the threat of fatal babesiosis caused by B. bovis, whose endemic status is yet to be established.Graphical Abtract

The probiotic properties and potential of vaginal Lactobacillus spp. isolated from healthy women against some vaginal pathogens.

During the last decade, probiotic research has progressed considerably and significant advances have been made in the selection and characterization of specific probiotic strains. The most studied probiotics belong to the genus Lactobacillus. In this study, 80 Lactobacillus spp. isolated from healthy women tolerated low pH and were able to grow in the presence of bile salts. RAPD PCR technique resulted in the identification of 38 different types. These isolates were then evaluated based on adhesion capacity, antibiotic susceptibility and tolerance in simulated gastrointestinal tract. Species-specific PCR and detection of bacteriocin-related genes were also surveyed. Among the isolates, five strains-Lacticaseibacillus rhamnosus NO21, Lacticaseibacillus casei NO1, Lactiplantibacillus plantarum NO4, Lactobacillus acidophilus NO7 and Lactobacillus gasseri NO38-presented acceptable antibiotic susceptibility pattern. Further analysis showed antimicrobial activity of Lacticaseibacillus culture against various bacterial pathogens and real-time PCR showed all five strains were able to prevent the colonization of bacterial pathogens. All five selected strains produced organic acids, hydrogen peroxide and were resistant to the spermicide. In addition, they lacked haemolytic activity with the ability of hydrophobicity, auto-aggregation and co-aggregation with pathogens. These results suggest that the vaginal microbiome could be a good source for the isolation of probiotics and the strains of this study may be considered as good probiotic candidates.

Isolation and Characterization of High-Ethanol-Tolerance Lactic Acid Bacteria from Australian Wine.

Lactic acid bacteria are very important in winemaking. In this study, 108 lactic acid bacteria isolates were obtained from high-ethanol-content (~17% (v/v)) Grenache wines during uninoculated malolactic fermentation (MLF). The 16S rRNA and species-specific PCR showed that 104 of these were Oenococcus oeni, three were Lactobacillus hilgardii, and one was Staphylococcus pasteuri. AFLP of HindIII and MseI digests of the genomic DNA of the O. oeni strains was developed for the first time to discriminate the strains. The results showed that the method was a suitable technique for discriminating the O. oeni strains. Based on the cluster analysis, nine O. oeni strains were chosen for inclusion in an ethanol tolerance assay involving monitoring of optical density (ABS600nm) and viable plating. Several O. oeni strains (G63, G46, G71, G39) survived and grew well in MRS-AJ with 17% (v/v) ethanol, while the commercial O. oeni reference strain did not. Strain G63 could also survive and grow for 168 h after inoculation in MRS-AJ medium with 19% (v/v) ethanol. These results suggest that O. oeni G63, G46, G71, and G39 could potentially be used as MLF starters for high-ethanol-content wines. All three L. hilgardii strains could survive and grow in MRS-AJ with 19% (v/v) ethanol, perhaps also indicating their suitability as next-generation MLF starter cultures.

Isolation of Biotype 1 Serotype 12 and Detection of Actinobacillus pleuropneumoniae from Wild Boars.

Actinobacillus pleuropneumoniae is a major pathogen of swine, which can cause severe pleuropneumonia in pigs, but sometimes the disease can be generalized. Diseases caused by A. pleuropneumoniae are frequent all over the world, resulting in high losses among domestic pigs. However, our knowledge on the occurrence of A. pleuropneumoniae in wild boars and feral pigs is limited. We aimed to examine the carriage of A. pleuropneumoniae by hunted wild boars. The presence of A. pleuropneumoniae was examined in tonsils of 68 hunted wild boars collected at a game processing unit. An in-house designed species-specific PCR test was used to detect the gene of Apx IV toxin, and the samples were inoculated on a modified selective agar. A. pleuropneumoniae was detected in 10 animals (14.7%) by PCR and one A. pleuropneumoniae serotype 12 strain was isolated. The antibiotic resistance pattern of the strain resembled field strains that were isolated from farmed pigs in Hungary. This is the first case for the detection of A. pleuropneumoniae not only using PCR or ELISA, but also its isolation, identification, and serotyping.

Genomic characterisation of an entomopathogenic strain of Serratia ureilytica in the critically endangered phasmid Dryococelus australis.

Between 2014 and 2019, unexpected mortalities were observed in a colony of Dryococelus australis, an endangered stick-insect kept at the Melbourne Zoo for a breeding and conservation program. Pure cultures of Serratia spp. were obtained from the haemolymph of moribund and recently deceased individuals. The combined bacteriological and histopathological observations suggested an infectious cause of these mortalities. Genotyping of Serratia sp. isolated from the insects and their environment revealed a predominant strain profile. A representative isolate, AM923, was entirely sequenced and compared to 616 publicly available Serratia spp. genomes, including 37 associated with insects. The genomes were distributed into 3 distinct groups, with 63% of the insect-associated isolates within a single clade (clade A) containing AM923, separated from most environmental/plant-associated strains (clade B) and human isolates (clade C). Average nucleotide identity and phylogenetic analyses identified AM923 as S. ureilytica and revealed similarities with putatively entomopathogenic strains. An experimental infection model in honey bees (Apis mellifera) confirmed the pathogenic potential of AM923. A urease operon was found in most insect isolates and a PCR assay, based on the ureB gene sequence, was used to confirm the presence of AM923 in experimentally infected bees. This species-specific PCR could be applied to detect entomopathogenic Serratia spp. in infected insects or their environment.

Low-cost, specific PCR assays to identify the main aflatoxigenic species of Aspergillus section Flavi

Aflatoxins are fungal metabolites that are present as contaminants in food globally. Most aflatoxigenic species belong to Aspergillus section Flavi, and the main ones are grouped in the A. flavus clade, where many cryptic species that are difficult to discriminate are found. In this study, we investigated inter- and intraspecific diversity of the A. flavus clade to develop low-cost, species-specific PCR assays for identifying aflatoxigenic species. A total of 269 sequences of the second largest subunit of RNA polymerase II (RPB2) locus were retrieved from GenBank, and primer pairs were designed using data mining to identify A. flavus, A. parasiticus, and A. novoparasiticus. Species-specific amplicons of approximately 620, 350, and 860 bp enabled identification of target species as A. flavus, A. parasiticus, and A. novoparasiticus, respectively.

A Comparison of Copromicroscopic and Molecular Methods for the Diagnosis of Cat Aelurostrongylosis.

Simple SummaryFeline aelurostrongylosis is a worldwide distributed parasitic disease of domestic cats caused by the nematode Aelurostrongylus abstrusus. Cats may have a subclinical infection with this parasite or may show signs that overlap with those of many other respiratory conditions of cats, either parasitic or not. The diagnosis of feline aelurostrongylosis currently relies on fecal examinations, though this approach has some limits that can be overcome by innovative molecular techniques. Therefore, the present study has investigated the diagnostic reliability of a PCR assay performed on different samples collected from cats from Italy and Greece. The results of the study confirmed that PCR is highly sensitive and specific for the diagnosis of A. abstrusus infections in domestic cats.The gold standard method for the diagnosis of cat aelurostrongylosis is the detection of Aelurostrongylus abstrusus first stage larvae with the Baermann’s examination. Nevertheless, molecular assays have shown higher diagnostic performances compared to copromicroscopy. This study evaluated the usefulness of an A. abstrusus species-specific PCR on different biological samples collected in clinical settings from 100 privately-owned cats in Italy (n. 60) and Greece (n. 40). A fecal sample was collected from each animal and a pharyngeal swab was also obtained for cats from Italy. All stool samples were subjected to flotation and Baermann’s test. The cats were categorized in three groups based on the results of copromicroscopy, i.e., Group A (n. 50 cats with A. abstrusus infection regardless of positivity for other helminths), Group B (n. 25 cats negative for A. abstrusus but positive for at least one of any other helminth), Group C (n. 25 cats negative for any helminth). DNA was extracted from individual aliquots of feces, flotation supernatant, Baermann’s sediment and the pharyngeal swab and then subjected to a PCR specific for A. abstrusus. At least one fecal aliquot or the pharyngeal swab scored positive by the A. abstrusus-specific PCR for 48/50 (96%) cats enrolled in Group A; in particular, 38/50 (76%), 35/50 (70%), 41/50 (82%) and 21/25 (84%) DNA extracts from feces, flotation supernatant, Baermann’s sediment and pharyngeal swabs were positive by PCR. These results confirm that molecular tools are highly sensitive and specific and indicate that pharyngeal swabs are the most suitable sample for molecular analysis in clinical settings.

Combining DNA Mini-Barcoding and Species-Specific Primers PCR Technology for Identification of Heosemys grandis

Heosemys grandis, a species of Asian water turtle, that has a wide range of applications in the food and pharmaceutical industries. Since some processed products cannot be identified exclusively by morphological and microscopic identification, a reliable and quick approach to guarantee authenticity is critical. Thus, we fostered an effective and stable molecular identification system to identify Heosemys grandis based on DNA mini-barcoding and species-specific primers PCR technology. A total of 48 turtle samples from 16 different species were collected. To distinguish Heosemys grandis from its counterfeits, DNA mini-barcoding and a pair of species-specific primers were designed and verified by PCR after analyzing the COI sequences of samples. The results showed that only Heosemys grandis samples could generate a single clear band following amplification using species-specific primers. Employing DNA mini-barcoding to amplify samples can verify authenticity by sequence alignment. These findings indicated that species-specific primers PCR technology combined with DNA mini-barcoding could accurately detect the authenticity of Heosemys grandis. This technology broadens the application of molecular biology techniques in the food and pharmaceutical industries. It provides a reliable and convenient method for identifying raw materials to standardize the market and protect customers’ rights and interests.

Molecular identification of Theileria spp. in ruminants and ticks from southern littoral of Caspian Sea, Iran.

The present study aimed at evaluating the presence of tick-borne apicomplexan parasites including Theileria ovis, Theileria lestoquardi, Theileria annulata, and Theileria orientalis in 92 cattle and 105 sheep from 6 different districts of Guilan and Mazandaran Provinces, inthe southern littoral of Caspian Sea. Furthermore, ixodid ticks were collected from the same animals. Stained blood smears were microscopically evaluated for the presence of blood parasites, and a specific PCR was applied for the detection of Theileria species. Besides, ticks were subsequently examined by species-specific PCR. Microscopic examination of blood smears demonstrated no evidence of intraerythrocytic piroplasms. Species-specific diagnostic PCRs demonstrated that 52.17% of sheep blood samples were positive for T. ovis. In addition, 31.03% and 24.13% of cattle blood samples were positive for T. annulata and T. orientalis, respectively. Moreover, 3 species of the ixodid ticks, namely, Rhipicephalus annulatus (58.47%), Ixodes ricinus (29.82%), and Haemaphysalis inermis (11.69%), were identified in Guilan Province, while Hyalomma detritum (73.03%) and Rhipicephalus sanguineus (26.92%) were found in Mazandaran Province. Additionally, by obtaining the data with respect to tick-borne apicomplexan parasites in 122 infected ticks, 35.24%, 22.95%, and 2.45% of tick samples were positive for T. annulata, T. orientalis, and T. ovis, respectively. Species-specific PCR revealed that H. inermis and R. annulatus were positive for T. orientalis. In addition, T. annulata was found in R. annulatus, H. inermis, and H. detritum. Besides, T. ovis was the only species of Theileria found in R. sanguineus. In conclusion, the results revealed that T. annulata infection was prevalent among cattle and ovine theileriosis caused by T. ovis was the only Theileria species found in sheep in the studied areas of the southern littoral of Caspian Sea. R. annulatus, H. inermis, and H. detritum were the main vectors for T. annulata, followed by H. inermis and R. annulatus for T. orientalis, and R. sanguineus for T. ovis.

First detection of Theileria equi in free-roaming donkeys (Equus africanus asinus) in Sri Lanka.

Equine piroplasmosis (EP) is a tick-borne disease caused by Theileria equi and Babesia caballi in equids, including horses, donkeys, zebras, and mules. It is globally endemic with significant economic impact on the equine industry. Infected animals may serve as carriers, and they may be a source of infection for ticks, thereby posing a great challenge for disease management. Sri Lanka is a tropical country, where infections by various tick-borne parasites are common among livestock animals. However, infections by T. equi and B. caballi remain unstudied in Sri Lanka. Therefore, in the present study, we conducted an epidemiological survey to investigate the presence of T. equi and B. caballi in apparently healthy free-roaming donkeys. Blood samples were randomly taken from 111 donkeys in Mannar (n=100) and Kilinochchi (n=11) districts in Sri Lanka. Thin blood smears were prepared from the blood samples and subjected to microscopic examination. Additionally, blood DNA samples were prepared and screened for T. equi and B. caballi infections using species-specific PCR assays. Our results showed that 64 (57.7%) and 95 (85.6%) of the donkeys were positive for T. equi by microscopy and PCR, respectively. However, all samples were negative for B. caballi. Phylogenetic analysis of the T. equi 18S rRNA sequences detected two distinct genotypes, namely C and D. To our knowledge, this is the first report of T. equi in Sri Lanka and of genotype C in donkeys. The present study highlights the importance of monitoring the shrinking donkey population in Sri Lanka owing to EP caused by T. equi.

Molecular Detection and Characterization of Babesia and Theileria in Cattle and Water Buffaloes from Southern Luzon, Philippines

Babesia and Theileria are tick-borne protozoan parasites that can cause significant economic losses in the cattle industry. This study aimed to contribute to the limited epidemiological data on Theileria orientalis as well as Babesia bigemina and B. bovis in large ruminants in the Philippines. Blood samples of 412 cattle and 108 water buffalo collected from four provinces in Southern Luzon, Philippines, were initially tested for the 18S rRNA gene of piroplasms through nested PCR. Positive samples were further subjected to species-specific PCR. The 18s rRNA of piroplasms was detected in 123 (29.9%) cattle and three (2.8%) water buffaloes. Theileria orientalis was found to be the most common piroplasm in cattle with a detection rate of 17.5%, followed by Babesia bovis and B. bigemina. Co-infections were also observed. Two water buffaloes were found infected with B. bovis, while one was positive for B. bigemina. The phylogenetic tree for B. bovis showed clustering of the isolates in two clades together with isolates from other countries, and a third separate clade. Meanwhile, the T. orientalis isolates in this study were distributed in three clades together with reported isolates from other countries. This study confirms the presence of T. orientalis in the Philippines and reports the genetic diversity of B. bovis and T. orientalis.

DETECTION OF FOOD-BORNE PATHOGENS IN CHICKEN MEAT SOLD IN RETAIL OUTLETS OF CHENNAI CITY

Contaminated poultry meat is regarded as a major source of food-borne pathogens, so the present study was under taken to screen the raw broiler chicken breast meat samples for the presence of food-borne pathogens viz. Salmonella spp., Campylobacter jejuni and Staphylococcus aureus collected from different retail outlets in Chennai city, India. The presence of Salmonella spp., C. jejuni and S. aureus was assessed by cultural method and species-specific PCR assay. The polymerase chain reaction was carried out by targeting the invA, flaA and nuc gene for Salmonella spp., C. jejuni and S. aureus respectively. Out of 60 samples, the frequency of isolation of S. aureus and C. jejuni was 16.6 and 11.6% respectively by both the methods. However, in case of Salmonella spp. the isolation rate was noted to be 10% and 15% by cultural method and PCR assay respectively. The PCR assay was found to be more specific for identification of Salmonella spp. Cultural method combined with PCR assay would provide a holistic approach in terms of sensitivity and specificity with regards to identification of these food-borne pathogens. The above findings also indicate the need of creating awareness among general public and butchers operating retail meat shop regarding good hygienic practice before, after cooking and during processing of carcass. Identifying the source of these organisms at the farm level will also help to control the entry of these organisms in food.

Dirofilaria Repens in Dogs and Humans in Slovenia.

IntroductionThe prevalence of Dirofilaria repens in dogs in countries bordering Slovenia ranges from 1.5% to 47.3%. The aim of this study was to estimate its prevalence in Slovenian dogs and to present the cases of dirofilariasis diagnosed in humans from 2010 to 2020.Material and MethodsEpidemiological data were collected and blood samples were taken from 465 dogs older than one year and born in Slovenia. A real-time PCR was performed on all samples to detect filarioid DNA, and a D. repens-and D. immitis-specific real-time PCR was performed on positive samples. Blood samples from 446 dogs were tested for Dirofilaria spp. using a modified Knott’s test. Human cases were diagnosed from histological sections of excised subcutaneous nodules. Descriptive statistics were used to characterise the samples. The one-sample nonparametric chi-squared test was used to assess whether categories of a variable were equally distributed.ResultsThree dogs’ samples tested positive for D. repens using the species-specific real-time PCR, while D. immitis DNA was not detected. The modified Knott’s test was positive in two of the three PCR-positive dogs, two of which had never travelled outside Slovenia’s borders. Four human patients with D. repens dirofilariasis were diagnosed. Since their travel history was unknown, autochthonous transmission could not be confirmed.ConclusionOur study demonstrated a 0.64% prevalence of D. repens infection in dogs in Slovenia. Two cases could be autochthonous.