Molecular diagnosis and characterization of Anaplasma marginale and Ehrlichia ruminantium infecting beef cattle of Maputo Province, Mozambique.

BACKGROUND: Members of the Anaplasmataceae family, such as the Anaplasma and Ehrlichia species, cause economic losses and public health risks. However, the exact economic impact has not been comprehensively assessed in Mozambique due to limited data available on its basic epidemiology. Therefore, we investigated the molecular occurrence and identity of Anaplasma and Ehrlichia spp. infecting beef cattle in Maputo province, Mozambique. METHODS: A total of 200 whole blood samples were collected from apparently healthy beef cattle. Whole blood DNA was extracted and tested for presence of Anaplasma spp. and Ehrlichia ruminantium DNA through amplification of the 16S rRNA and map1 genes. Positive samples to Anaplasma spp. were subject to PCR assay targeting the A. marginale-msp5 gene. Amplicons obtained were purified, sequenced and subject to phylogenetic analyses. RESULTS: Anaplasma spp., A. marginale and E. ruminantium were detected in 153 (76.5%), 142 (71%) and 19 (9.5%) of all the samples analyzed, respectively. On this same sample group, 19 (9.5%) were co-infected with A. marginale and E. ruminantium. The 16S rRNA sequences of Anaplasma spp. obtained were phylogenetically related to A. marginale, A. centrale and A. platys. Phylogenetic analysis revealed that A. marginale-msp5 nucleotide sequences were grouped with sequences from Asia, Africa and Latin America, whereas E. ruminantium-map1 DNA nucleotide sequences were positioned in multiple clusters. CONCLUSION: Cattle in Maputo Province are reservoirs for multiple Anaplasma species. A high positivity rate of infection by A. marginale was observed, as well as high genetic diversity of E. ruminantium. Furthermore, five new genotypes of E. ruminantium-map1 were identified.

Diagnostic evaluation of nested PCR and microscopy for Cryptosporidiosis in goats: can COWP gene based qRT-PCR be useful in assessment of active Cryptosporidial infections?

The present research delved into the transmission patterns, diagnostic methods, molecular traits, and phylogenetic analysis of Cryptosporidium species. The research was undertaken to enhance comprehension of the epidemiology and the potential for zoonotic transmission. A total of 80 goat-kid samples were tested, 7 were confirmed positive by mZN microscopy and 12 by nested-PCR. By PCR, 18SSUrRNA, HSP70, and GP60 amplicons were tested for Cryptosporidium. The restriction enzymes viz., SspI, VspI and MboII were used to genotype 12 Cryptosporidium positive samples by which C. parvum and C. bovis mixed infections were detected. Quantitative reverse transcription real-time PCR was used to transcriptionally screen the COWP-subunit genes to assess the severity of the infection in goat-kids, which showed upregulation of COWP6 and COWP4, while COWP9 and COWP3 genes were downregulated. A silent mutation was found at the codon CCA→CCC, which is being reported for the first time in goat field isolates. Phylogenetic and sequencing analyses confirmed the presence of the anthropozoonotic IIe subtype.

Development of PCR-based genetic test for detection of TTF1 mutation causing abortion in Holstein Friesian cattle.

A stop-gain mutation (rs715966442; BTA11: 1,02,463,944 nucleotide position) in transcription termination factor, RNA polymerase I (TTF1) gene causes abortion in Holstein Friesian (HF) cattle. A PCR-restriction fragment length polymorphism (PCR-RFLP)-based genetic test has been developed and validated to screen the TTF1 mutation locus in HF cattle. The mutation locus was screened in 80 HF and HF crossbreds using the protocol, which revealed two animals as carriers of the mutant TTF1 allele. The test employed is cost-effective, rapid and precise and can be utilized as an effective tool for the screening of TTF1 mutation carriers in HF cattle population.

Feline leishmaniasis in an animal shelter in northeastern Brazil: Clinical aspects, coinfections, molecular detection, and serological study of a new recombinant protein.

Infection and clinical cases of leishmaniasis caused by Leishmania infantum in cats have been increasingly reported in several countries, including Brazil. In this study, we used an enzyme-linked immunosorbent assay (ELISA) and an immunochromatographic test (ICT) based on a recombinant antigen (rKDDR-plus) to detect anti-Leishmania antibodies in cats from an animal shelter in northeastern Brazil. We compared the results with an ELISA using L. infantum crude antigen (ELISA-CA). We also investigated the presence of Leishmania DNA in blood or ocular conjunctival samples as well as the association between Leishmania PCR positivity and serological positivity to feline immunodeficiency virus (FIV), feline leukemia virus (FeLV) and Toxoplasma gondii. Concerning serological assays, a higher positivity was detected using the ICT-rKDDR-plus (7.5%; 7/93) as compared to ELISA-rKDDR-plus (5.4%; 5/93) and ELISA-CA (4.3%; 4/93). Upon PCR testing, 52.7% (49/93) of the ocular conjunctival swabs and 48.3% (44/91) of the blood samples were positive. Together, PCR and serological testing revealed overall positivities of 73.1% (68/93) and 12.9% (12/93), respectively. Among PCR-positive samples, 45.5% (31/68) showed co-infection with FIV, 17.6% (12/68) with FeLV, and 82.3% (56/68) with T. gondii. More than half of the PCR-positive cats showed at least one clinical sign suggestive of leishmaniasis (58.8%; 40/68) and dermatological signs were the most frequent ones (45.5%; 31/68). Both tests employing the recombinant antigen rKDDR-plus (i.e., ICT-rKDDR-plus and ELISA-rKDDR-plus) detected more positive cats than the ELISA-CA but presented low overall accuracy. PCR testing using either blood or ocular conjunctival samples detected much more positive cats than serological tests.

Molecular identification of Sarcocystis species in wild boar (Sus scrofa) and pigs (Sus scrofa domesticus) in Brazil.

Sarcocystis spp. are protozoan parasites that form cysts in the organs and musculature of various animal species. The species Sarcocystis miescheriana and Sarcocystis suihominis are pathogenic to pigs and wild boars (Sus scrofa), acting as intermediate hosts, while humans are the definitive host for S. suihominis. To date, there have been no reports of the identification of these coccidian species in Sus scrofa in Brazil. Therefore, in this study, we conducted the first molecular identification of Sarcocystis species using PCR-RFLP and sequencing. A total of 210 samples were analyzed, of this total, 67 tested positive for Sarcocystis spp., representing 31.9% of the total samples assessed. Out of the total positive samples, 55 (82.1%) were identified as S. miescheriana and 8 (11.9%) as S. suihominis, a zoonotic species. Additionally, other species related to bovines, such as S. cruzi and zoonotic S. hominis, were detected in 3.0% of the samples, serving as contaminants in the pork products. The presence of S. suihominis in swine and wild boar samples is concerning due to the zoonotic risk and potential environmental contamination, as humans act as definitive hosts, also for the presence of S. hominis as a bovine contaminant in pork sausages. Furthermore, we confirmed the efficacy of the PCR-RFLP technique as a reliable tool for the identification of Sarcocystis species, demonstrating its potential use in laboratories for molecular diagnosis and rapid identification of these parasites, aiming to protect public health and ensure food safety.

Identification of third stage larvae of strongyles and molecular diagnosis of Strongylus vulgaris in the feces of Thoroughbred horses kept in training centers in Rio de Janeiro, Brazil.

The aims of the present study were to identify strongyles in the feces of Thoroughbred horses based on larval morphology; to detect Strongylus vulgaris using molecular diagnosis and compare results to those of feces culture; and to determine the association between the presence of S. vulgaris with corresponding animal information (age range, gender, and anthelmintic use). Feces of horses kept in six Training Centers in Rio de Janeiro State, that showed the presence of ≥500 eggs per gram of feces (EPG) were subjected to strongyle identification. Of the 520 fecal samples collected, 35 had an EPG ≥ 500. After fecal culture for L3 larvae identification, DNA was extracted, subjected to PCR to amplify the ITS2 region DNA fragment of S. vulgaris, and sequenced. A total of 3500 larvae were analyzed. Most were classified as small strong (99.7%), with an emphasis on the type A subfamily of Cyathostominae. Forms of S. vulgaris only corresponded to 0.2%. In all, 25 samples showed amplified S. vulgaris DNA products and 11 showed nucleotide sequences with high sequence identity. Fecal culture and PCR results showed poor agreement (kappa = 0.105) for S. vulgaris diagnosis. Age, gender, anthelmintic use, and anthelmintic administration interval were not statistically significant. The present study showed the presence of S. vulgaris in the feces of horses kept in Rio de Janeiro Training Centers, mainly seen via PCR, which has emerged as the most effective tool for diagnosis. This study made it possible to identify strongyles that infect horses in the region, emphasizing upon the necessity for constant monitoring of the animals.

Moringa oleifera as a potential antimicrobial against pathogenic Clostridium perfringens isolates in farm animals.

Background: Clostridium perfringens (CP) is an emerging anaerobic pathogen that can aggravate severe fatal infections in different hosts and livestock. Aim: This paper was designed to monitor the antibacterial efficacy of Moringa oleifera (M. oleifera) plant against different CP isolates of variant toxin genotypes comparing that with commercial antibiotics in the veterinary field. Methods: A total of 200 examined fecal, intestinal, and liver samples from cattle, sheep, and goats were investigated bacteriologically and biochemically for CP. Then, the isolates were examined by polymerase chain reaction (PCR) for toxin gene typing. Thereafter, the antimicrobial susceptibility testing as well as the antibacterial efficacy of M. oleifera were evaluated and statistically analyzed against recovered isolates. Results: The prevalence rate of CP was 51% (102/200); of which 54.5% was from cattle, 50% from sheep, and 40% from goat. Moreover, all CP isolates were highly resistant to tetracycline and lincomycin drugs; meanwhile, they were of the least resistance against ciprofloxacin (8.3%-16.7%), cefotaxime (16.7%-25%), and gentamycin (26.7%-33.3%). For M. oleifera, high antibacterial efficacy with greater inhibition zones of the plant was recorded with its oil (20-24 mm) and ethanolic extracts (16-20 mm) against CP than the aqueous extract (≤ 10 mm). A good correlation was stated between M. oleifera oil and toxin type of CP isolates particularly type A followed by D and B types. Interestingly, the oil and ethanolic extracts of M. oleifera gave higher antibacterial efficacy than most commercial antibiotics against the recovered isolates. Conclusion: This study highlighted the potent antibacterial properties of M. oleifera for suppressing CP isolated from farm animals; hence, more investigations on M. oleifera are suggested to support its use as a medical herbal plant substituting antibiotics hazards and resistance problems worldwide.

Implementation of point-of-care platforms for rapid detection of porcine circovirus type 2.

BACKGROUND: Porcine circovirus type 2 (PCV2) infection is ubiquitous around the world. Diagnosis of the porcine circovirus-associated disease requires clinic-pathological elements together with the quantification of viral loads. Furthermore, given pig farms in regions lacking access to sufficient laboratory equipment, developing diagnostic devices with high accuracy, accessibility, and affordability is a necessity. OBJECTIVES: This study aims to investigate two newly developed diagnostic tools that may satisfy these criteria. METHODS: We collected 250 specimens, including 170 PCV2-positive and 80 PCV2-negative samples. The standard diagnosis and cycle threshold (Ct) values were determined by quantitative polymerase chain reaction (qPCR). Then, two point-of-care (POC) diagnostic platforms, convective polymerase chain reaction (cPCR, qualitative assay: positive or negative results are shown) and EZtargex (quantitative assay: Ct values are shown), were examined and analyzed. RESULTS: The sensitivity and specificity of cPCR were 88.23% and 100%, respectively; the sensitivity and specificity of EZtargex were 87.65% and 100%, respectively. These assays also showed excellent concordance compared with the qPCR assay (κ = 0.828 for cPCR and κ = 0.820 for EZtargex). The statistical analysis showed a great diagnostic power of the EZtargex assay to discriminate between samples with different levels of positivity. CONCLUSIONS: The two point-of-care diagnostic platforms are accurate, rapid, convenient and require little training for PCV2 diagnosis. These POC platforms can discriminate viral loads to predict the clinical status of the animals. The current study provided evidence that these diagnostics were applicable with high sensitivity and specificity in the diagnosis of PCV2 infection in the field.

Seroprevalence and molecular detection of Toxoplasma gondii and Neospora caninum in beef cattle and goats in Hunan province, China.

BACKGROUND: Toxoplasma gondii and Neospora caninum are closely related protozoan parasites that are considered important causes of abortion in livestock, causing huge economic losses. Hunan Province ranks 12th in the production of beef and mutton in China. However, limited data are available on the seroprevalence, risk factors and molecular characterization of T. gondii and N. caninum in beef cattle and goats in Hunan province, China. METHODS: Sera of 985 beef cattle and 1147 goats were examined for the presence of specific antibodies against T. gondii using indirect hemagglutination test (IHAT) and anti-N. caninum IgG using competitive-inhibition enzyme-linked immunoassay assay (cELISA). Statistical analysis of possible risk factors was performed using PASW Statistics. Muscle samples of 160 beef cattle and 160 goats were examined for the presence of T. gondii DNA (B1 gene) and N. caninum DNA (Nc-5 gene) by nested PCR. The B1 gene-positive samples were genotyped at 10 genetic markers using the multilocus nested PCR-RFLP (Mn-PCR-RFLP). RESULTS: Specific IgG against T. gondii were detected in 8.3% (82/985) and 13.3% (153/1147) and against N. caninum in 2.1% (21/985) and 2.0% (23/1147) of the beef cattle and goats, respectively. Based on statistical analysis, the presence of cats, semi-intensive management mode and gender were identified as significant risk factors for T. gondii infection in beef cattle. Age was a significant risk factor for T. gondii infection in goats (P < 0.05), and age > 3 years was a significant risk factor for N. caninum infection in beef cattle (P < 0.05). PCR positivity for T. gondii was observed in three beef samples (1.9%; 3/160) and seven chevon samples (4.4%; 7/160). Genotyping of PCR positive samples identified one to be ToxoDB#10. The N. caninum DNA was observed in one beef sample (0.6%; 1/160) but was negative in all chevon samples. CONCLUSIONS: To our knowledge, this is the first large-scale serological and molecular investigation of T. gondii and N. caninum and assessment of related risk factors in beef cattle and goats in Hunan Province, China. The findings provide baseline data for executing prevention and control of these two important parasites in beef cattle and goats in China.

Molecular detection of Toxoplasma gondii and Neospora caninum in seabirds collected along the coast of Santa Catarina, Brazil.

Toxoplasma gondii and Neospora caninum are two closely related protozoans that infect a wide range of animals, including birds. However, the occurrence of N. caninum and T. gondii in seabirds is unknown. Therefore, this study aimed to determine the presence of T. gondii and N. caninum DNA in tissue samples of seabirds. Tissue samples of the pectoral muscles, heart, and brain were collected from 47 birds along the coastline of Santa Catarina State, SC, Brazil. The DNA was extracted from the tissues and screened using nested-PCR (nPCR) targeting internal transcribed spacer 1 (ITS1). T. gondii DNA was detected in tissues from seven seabirds (7/47, 14.8%), kelp gull (Larus dominicanus) (5/21), and Manx shearwater (Puffinus puffinus) (2/8). N. caninum DNA was detected in tissues of nine seabirds (9/47, 19.1%), the kelp gull (L. dominicanus) (4/21), Manx shearwater (P. puffinus) (2/8), neotropic cormorant (Phalacrocorax brasilianus) (1/4), brown booby (Sula leucogaster) (1/5), and white-chinned petrel (Procellaria aequinoctialis) (1/1); however, no co-infection was observed. In conclusion, this study showed the circulation of N. caninum and T. gondii in seabirds along the coastline of Santa Catarina State. Further studies are required to clarify the role of these birds in the epidemiology of neosporosis and toxoplasmosis.

In silico and PCR Screening for a Live Attenuated Salmonella Typhimurium Vaccine Strain.

The application of live attenuated Salmonella Typhimurium vaccines has significantly helped control Salmonella in poultry products. Because the U.S. Department of Agriculture-Food Safety Inspection Service (USDA-FSIS) scores all Salmonella as positive, regardless of serovar, attenuated vaccine strains that are identified at processing contribute negatively toward Salmonella performance standards. This study was designed to determine the incidence of a live attenuated Salmonella serovar Typhimurium vaccine identified in broiler products by FSIS and to develop a PCR assay for screening of isolates. Salmonella Typhimurium short-read sequences from broiler samples uploaded to the National Center for Biotechnology Information (NCBI) Pathogen Detection database by the USDA-FSIS from 2016 to 2022 were downloaded and assembled. These were analyzed using the Basic Local Alignment Search Tool (BLAST) with a sequence unique to field strains, followed by a sequence unique to the vaccine strain. The PCR assays were developed against field and vaccine strains by targeting transposition events in the crp and cya genes and validated by screening Salmonella serovar Typhimurium isolates. Between 2016 and 2022, 1708 Salmonella Typhimurium isolates of chicken origin were found in the NCBI Pathogen Detection database, corresponding to 7.99% of all Salmonella identified. Of these, 104 (5.97%) were identified as the vaccine strain. The PCR assay differentiated field strains from the vaccine strain when applied to isolates and was also able to detect the vaccine strain from DNA isolated from mixed serovar overnight Salmonella enrichment cultures. Live attenuated Salmonella vaccines are a critical preharvest tool for Salmonella control and are widely used in industry. With forthcoming regulations that will likely focus on Salmonella Typhimurium, along with other serovars, there is a need to distinguish between isolates belonging to the vaccine strain and those that are responsible for causing human illness.

Detección in silico y por PCR de una cepa vacunal viva atenuada de Salmonella Typhimurium. La aplicación de vacunas vivas atenuadas contra Salmonella Typhimurium ha ayudado significativamente a controlar Salmonella en productos avícolas. Debido a que el Servicio de Inspección de Seguridad Alimentaria del Departamento de Agricultura de los Estados Unidos. (USDA-FSIS) califica todas las Salmonella como positivas, independientemente del serovar. Las cepas atenuadas de la vacuna que se identifican en el procesamiento contribuyen negativamente a los estándares de desempeño de Salmonella. Este estudio fue diseñado para determinar la incidencia de una vacuna viva atenuada de Salmonella serovar Typhimurium identificada en productos de pollo de engorde por el FSIS y para desarrollar un ensayo de PCR para la detección de aislados. Se recolectaron y ensamblaron secuencias de lectura corta de Salmonella Typhimurium de muestras de pollos de engorde introducidas en la plataforma de detección de patógenos del Centro Nacional de Información Biotecnológica (NCBI) por el USDA-FSIS entre los años 2016 al 2022. Estos se analizaron utilizando la herramienta de búsqueda de alineación local básica con una secuencia exclusiva para las cepas de campo, seguida de una secuencia exclusiva para la cepa vacunal. Los ensayos de PCR se desarrollaron contra cepas de campo y vacunales centrándose en eventos de transposición en los genes crp y cya y se validaron mediante la detección de aislados de Salmonella serovar Typhimurium. Entre 2016 y 2022, se encontraron 1708 aislados de Salmonella Typhimurium de origen avícola en el sistema de detección de patógenos del NCBI, lo que corresponde al 7.99 % de todas las Salmonellas identificadas. De ellas, 104 (5.97%) fueron identificadas como cepa vacunal. El ensayo de PCR diferenció las cepas de campo de la cepa de la vacuna cuando se aplicó a los aislados y también fue capaz de detectar la cepa de la vacuna a partir del ADN aislado de cultivos de enriquecimiento por toda la noche de Salmonella con serovares mixtos. Las vacunas vivas atenuadas contra Salmonella son una herramienta fundamental para el control de Salmonella y se utilizan ampliamente en la industria. Con las próximas regulaciones que probablemente se centrarán en Salmonella Typhimurium, junto con otros serovares, es necesario distinguir entre los aislados que pertenecen a la cepa vacunal y los que son responsables de causar enfermedades humanas.

Multiplex fluorescent amplification-refractory mutation system PCR method for the detection of 10 genetic defects in Holstein cattle and its comparison with the KASP genotyping assay.

The common deleterious genetic defects in Holstein cattle include haplotypes 1-6 (HH1-HH6), haplotypes for cholesterol deficiency (HCD), bovine leukocyte adhesion deficiency (BLAD), complex vertebral malformation (CVM) and brachyspina syndrome (BS). Recessive inheritance patterns of these genetic defects permit the carriers to function normally, but homozygous recessive genotypes cause embryo loss or neonatal death. Therefore, rapid detection of the carriers is essential to manage these genetic defects. This study was conducted to develop a single-tube multiplex fluorescent amplification-refractory mutation system (mf-ARMS) PCR method for efficient genotyping of these 10 genetic defects and to compare its efficiency with the kompetitive allele specific PCR (KASP) genotyping assay. The mf-ARMS PCR method introduced 10 sets of tri-primers optimized with additional mismatches in the 3' end of wild and mutant-specific primers, size differentiation between wild and mutant-specific primers, fluorescent labeling of universal primers, adjustment of annealing temperatures and optimization of primer concentrations. The genotyping of 484 Holstein cows resulted in 16.12% carriers with at least one genetic defect, while no homozygous recessive genotype was detected. This study found carrier frequencies ranging from 0.0% (HH6) to 3.72% (HH3) for individual defects. The mf-ARMS PCR method demonstrated improved detection, time and cost efficiency compared with the KASP method for these defects. Therefore, the application of mf-ARMS PCR for genotyping Holstein cattle is anticipated to decrease the frequency of lethal alleles and limit the transmission of these genetic defects.

Detection of Bartonella schoenbuchensis (sub)species DNA in different louse fly species in Saxony, Germany: The proof of multiple PCR analysis necessity in case of ruminant-associated bartonellae determination.

BACKGROUND: Hippoboscid flies are bloodsucking arthropods that can transmit pathogenic microorganisms and are therefore potential vectors for pathogens such as Bartonella spp. These Gram-negative bacteria can cause mild-to-severe clinical signs in humans and animals; therefore, monitoring Bartonella spp. prevalence in louse fly populations appears to be a useful prerequisite for zoonotic risk assessment. METHODS: Using convenience sampling, we collected 103 adult louse flies from four ked species (Lipoptena cervi, n = 22; Lipoptena fortisetosa, n = 61; Melophagus ovinus, n = 12; Hippobosca equina, n = 8) and the pupae of M. ovinus (n = 10) in the federal state of Saxony, Germany. All the samples were screened by polymerase chain reaction (PCR) for Bartonella spp. DNA, targeting the citrate synthase gene (gltA). Subsequently, PCRs targeting five more genes (16S, ftsZ, nuoG, ribC and rpoB) were performed for representatives of revealed gltA genotypes, and all the PCR products were sequenced to identify the Bartonella (sub)species accurately. RESULTS AND CONCLUSIONS: The overall detection rates for Bartonella spp. were 100.0%, 59.1%, 24.6% and 75.0% in M. ovinus, L. cervi, L. fortisetosa and H. equina, respectively. All the identified bartonellae belong to the Bartonella schoenbuchensis complex. Our data support the proposed reclassification of the (sub)species status of this group, and thus we conclude that several genotypes of B. schoenbuchensis were detected, including Bartonella schoenbuchensis subsp. melophagi and Bartonella schoenbuchensis subsp. schoenbuchensis, both of which have previously validated zoonotic potential. The extensive PCR analysis revealed the necessity of multiple PCR approach for proper identification of the ruminant-associated bartonellae.

Detection, Identification, and Characterization of Eimeria spp. from Commercial Chicken Farms in Different Parts of Iran by Morphometrical and Molecular Techniques.

PURPOSE: Coccidiosis of domestic chicken is an important disease caused by any of seven species of Eimeria which, by developing within the epithelial cells of the intestine, cause lesions therein. We carried out a study on poultry farms located in various regions of Iran to determine the incidence and spread of Eimeria species by employing a single PCR test. METHODS: A total of 64 fully confirmed clinically intestinal tracts were collected from different parts of Iran. From these 64 intestinal tracts, 82 samples were prepared from the different sites involved in the digestive tract. In morphological assessment, 23 samples could not be isolated and its information was not evaluated. RESULTS: Using morphological methods, the following seven species of Eimeria were identified: E. acervulina (15/59; 25.42%), E. tenella (30/59; 50.84%), E. maxima (12/59; 20.33%), E. praecox (1/59; 1.69%), E. necatrix (2/59; 3.38%), E. mitis (5/59; 8.47%), and E. mivati (2/59; 3.38%). Mixed infections were found in eight (13.55%) samples. In molecular assessment, 31 samples could not be isolated and its information was not evaluated. Totally, the following five species were identified using molecular methods: E. acervulina (35/51; 68.62%), E. tenella (33/51; 64.70%), E. maxima (6/51; 11.76%), E. brunetti (5/51; 9.80%), and E. necatrix (2/51; 3.92%). Mixed infections were found in 23 (45.09%) samples. CONCLUSIONS: The present study is an update on the situation of poultry coccidiosis in Iran and provides the first data on the molecular detection, identification, and characterization of Eimeria spp. in the poultry population of this country and confirmed the presence of different species of this parasite in this area. According to the results, E. acervulina and E. tenella, as the main disease-causing species, should be considered in control programs such as treatment and vaccination strategies.

Detection of Mycoplasma hyopneumoniae viability using a PCR-based assay.

Mycoplasma hyopneumoniae detection in clinical specimens is accomplished by PCR targeting bacterial DNA. However, the high stability of DNA and the lack of relationship between bacterial viability and DNA detection by PCR can lead to diagnostic interpretation issues. Bacterial messenger RNA is rapidly degraded after cell death, and consequently, assays targeting mRNA detection can be used for the exclusive detection of viable bacterial cells. Therefore, this study aimed at developing a PCR-based assay for the detection of M. hyopneumoniae mRNA and at validating its applicability to differentiate viable from inert bacteria. Development of the RNA-based PCR encompassed studies to determine its analytical sensitivity, specificity, and repeatability, as well as its diagnostic accuracy. Comparisons between DNA and mRNA detection for the same target gene were performed to evaluate the ability of the RNA-based PCR to detect exclusively viable M. hyopneumoniae after bacterial inactivation using various methods. The RNA-based PCR was also compared to the DNA-based PCR as a tool to monitor the growth of M. hyopneumoniae in vitro. Under the conditions of this study, the developed RNA-based PCR assay detected only viable or very recently inactivated M. hyopneumoniae, while the DNA-based PCR consistently detected cells irrespective of their viability status. Changes in growth activity over time were only observable via RNA-based PCR. This viability PCR assay could be directly applied to evaluate the clearance of M. hyopneumoniae or to determine the viability of the bacterium at late stages of eradication programs.

Analysis of gene and protein expression in the endometrium for validation of an ex vivo model of the equine uterus using PCR, digital and visual histopathology.

In the past, most research in equine reproduction has been performed in vivo but the use of in vitro and ex vivo models has recently increased. This study aimed to evaluate the functional stability of an ex vivo hemoperfused model for equine uteri with molecular characterization of marker genes and their proteins. In addition, the study validated the respective protein expression and the aptness of the software QuPath for identifying and scoring immunohistochemically stained equine endometrium. After collection, uteri (n = 12) were flushed with preservation solution, transported to the laboratory on ice, and perfused with autologous blood for 6 h. Cycle stage was determined by examination of the ovaries for presence of Graafian follicles or corpora lutea and analysis of plasma progesterone concentration (estrus: n = 4; diestrus: n = 4; anestrus: n = 4). Samples were obtained directly after slaughter, after transportation, and during perfusion (240, 300, 360 min). mRNA expression levels of progesterone (PGR), estrogen (ESR1) and oxytocin (OXTR) receptor as well as of MKI67 (marker of cell growth) and CASP3 (marker of apoptosis) were analyzed by RT-qPCR, and correlation to protein abundance was validated by immunohistochemical staining. Endometrial samples were analyzed by visual and computer-assisted evaluation of stained antigens via QuPath. For PGR, effects of the perfusion and cycle stage on expression were found (P < 0.05), while ESR1 was affected only by cycle stage (P < 0.05) and OXTR was unaffected by perfusion and cycle stage. MKI67 was lower after 360 min of perfusion as compared to samples collected before perfusion (P < 0.05). For CASP3, differences in gene expression were found after transport and samples taken after 240 min (P < 0.05). Immunohistochemical staining revealed effects of perfusion on stromal and glandular cells for steroid hormone receptors, but not for Ki-67 and active Caspase 3. OXTR was visualized in all layers of the endometrium and was unaffected by perfusion. Comparison of QuPath and visual analysis resulted in similar results. For most cell types and stained antigens, the correlation coefficient was r > 0.5. In conclusion, the isolated hemoperfused model of the equine uterus was successfully validated at the molecular level, demonstrating stability of key marker gene expression. The utility of computer-assisted immunohistochemical analysis of equine endometrial samples was also confirmed.

Sex identification of birds in Taipei Zoo.

As a conservation and breeding institution for birds, Taipei Zoo plays an important role in restoring endangered species. As approximately half of all bird species are monomorphic, precisely confirming the sex of individuals is critical for the management of ex-situ conservation breeding populations, as well as for understanding the sex ratio of those in the wild. Generally, PCR is used more reliably for sex determination versus traditional methods such as plumage, behavior or hormone levels. Nevertheless, the various primer sets and annealing temperatures vary between species, and so inaccurate sexing can occasionally happen due to inadequate PCR conditions. To reduce the probability of misidentification, and to establish a PCR condition database for sex determination across the diverse range of avian taxa, we tested multiple primer sets and annealing temperatures for amplification of the bird sex-specific gene fragments (CHD1) for each captive or rescued avian species held at Taipei Zoo since 2014. A total of 162 species across 22 orders were tested using one or two primer sets. One hundred and fifty-five species were successfully sexed by the primer set 2550F/2718R and the success rate of sex typing reached over 90% of species tested in each order. Most species have suitable PCR annealing temperatures between 45°C and 55°C, and the species in the same avian taxa showed similar results in temperature. This indicates that it is possible to select the annealing temperature of other species in the same family when the species had not been tested before. We expect this study will improve the success rate of identifying sex by using applicable PCR conditions and reduce the time for searching references every time before attempts to PCR sex birds.

Molecular detection of Coxiella burnetii in tick and blood samples from small ruminants in northwest of Iran.

This survey sought to molecularly detect Coxiella burnetii in Argasidae and Ixodidae ticks attached to small ruminants in the region of West Azerbaijan (Northwest of Iran) and blood samples collected from the same animals. 451 tick samples and 927 blood samples were obtained from sheep (n = 536) and goats (n = 391) and tested by nested PCR for detection of C. burnetii insertion sequence IS1111 or icd gene sequence. The collected ticks were morphologically classified as Rhipicephalus sanguineus, Rhipicephalus turanicus, Hyalomma asiaticum, Hyalomma anatolicum, or Argas reflexus. 14% of ticks (65 in total 43 for IS1111 and 22 for icd gene) tested positive for C. burnetii, none of which were from the Argas genus. Among the 927 blood samples, 218 (23.5%) tested positive for C. burnetii. The positive result from analysis targeting the genes IS1111 and icd were 131 and 87 respectively. As Q fever is a tickborne zoonosis and endemic to Iran, such information is critical for creating effective, coordinated, and strategic tick and pathogen control programs to prevent disease outbreak in domestic animals and humans.

Echinococcus granulosus sheep strain (G1) as the predominant genotype in definitive host (dogs) isolates in northeastern Iran.

Echinococcus granulosus sensu lato (E. granulosus s.l.) is a zoonotic parasite, causing cystic echinococcosis in humans. In the present study, prevalence and genotypes of E. granulosus s.l. was assessed in stools collected from 244 dogs including 138 stray and 106 domestic animals using high resolution melting curve (HRM) method. Initially, to detect taeniid eggs in feces, all samples were examined using the formalin-ether techniques. Genomic DNA was extracted from the positive samples and E. granulosus s.l. was differentiated from other Taeniidae parasites using SSU-rDNA gene and E. granulosus s.l. was analyzed for genotyping using HRM based on the cox1 gene. In total, 12.7% (31/244) of the samples were positive for Taeniidae eggs. In addition, among the positive samples, 77.4% (24/31) were positive for E. granulosus s.l.. In details, 11.3% (12/106) of the domestic dogs and 8.7% (12/138) of the stray dogs were positive for E. granulosus s.l.. The results of HRM analysis showed that all E. granulosus s.l. isolates were G1 strain. Findings of the present study indicated a considerable prevalence of E. granulosus G1 among dogs in the northeast of Iran and imply a serious risk of transmitting to humans and livestock.

Simultaneous detection and differentiation of DuCV-1 and DuCV-2 by high-resolution melting analysis.

Birds infected with duck circovirus (DuCV) can potentially cause immunosuppression by damaging lymphoid tissues, causing great losses in the duck breeding industry. Duck circovirus can be divided into two genotypes (DuCV-1 and DuCV-2), but simultaneous detection and differentiation of DuCV-1 and DuCV-2 by high-resolution melting (HRM) analysis is still lacking. Here, we designed specific primers according to the sequence characteristics of the newly identified ORF3 gene and then established a PCR-HRM method for the simultaneous detection and differentiation of DuCV-1 and DuCV-2 via high-resolution melting analysis. Our data showed that the established PCR-HRM assay had the advantages of specificity, with the lowest detection limits of 61.9 copies/µL (for DuCV-1) and 60.6 copies/µL (for DuCV-2). The melting curve of the PCR-HRM results indicated that the amplification product was specific, with no cross-reaction with common waterfowl origin pathogens and a low coefficient of variation less than 1.50% in both intra-batch and inter-batch repetitions, indicating the advantages of repeatability. We found that the percentage of DuCV-2-positive ducks was higher than that of DuCV-1-positive ducks, with 8.62% rate of DuCV-1 and DuCV-2 coinfection. In addition, we found DuCV-2-positive in geese firstly. In conclusion, this study provides a candidate PCR-HRM assay for the detection and accurate differentiation of DuCV-1 and DuCV-2 infection, which will help us for further epidemiological surveillance of DuCVs.