Signal transducers and activators of transcription (STAT) genes are involved in signal mediation of various hormones and cytokines. STAT1 located on chromosome number 2 is involved in mammary gland development and is associated with milk composition traits in bovines. This study aimed to find any relationship and impact of STAT1/BspHI gene with milk fat and protein yields in a herd of Holstein Friesian (HF) crossbred cattle of sub-tropical climate of Northern India. Milk composition data of 535 adult HF crossbred cows for a period of 12 years was collected from the records maintained at Livestock Farm, Guru Angad Dev Veterinary and Animal Sciences University. First lactation data of 222 animals was chosen for further analysis. After data correction for non-genetic factors (season of calving, period of calving, interaction effect of season and period of calving and age at first calving) these animals were categorised into two groups based on corrected high and low milk fat and protein yields. Forty animals were then selected for blood collection and further laboratory analysis. Amplified using PCR-RFLP technique, the 314 bp STAT1 gene was digested using BspHI restriction enzyme. C-T polymorphism at nucleotide position 201 and 260 of the STAT1 amplicon was observed. At 201, for genotype AA and Aa, the genotypic frequencies were 0.80 and 0.20%. At 260, for genotype BB and Bb, the genotypic frequencies were 0.25 and 0.75%. Least square analysis showed a significant association of all genotypes with milk fat and protein yields. Hence, STAT1 can be used as a potential candidate gene to aid in better animal selection in breeding programmes.

Toxoplasma gondii has at least 318 genotypes distributed worldwide, and tropical regions usually have greater genetic diversity. Campeche is a state located in the southeastern region of México and has favourable climate conditions for the replication and dissemination of this protozoan, similar to those in South American countries where broad genetic diversity has been described. Thus, in this study, 4 T. gondii isolates were obtained from tissues of stray dogs and free-range chickens in Campeche, México, and were genotyped by Mn-PCR-RFLP with 10 typing markers (SAG1, altSAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico) and 5 virulence markers (CS3, ROP16, ROP17, ROP18 and ROP5) to provide new information about the distribution and virulence prediction of T. gondii genotypes. Two isolates of T. gondii genotype #116 and 2 of genotype #38 were obtained from stray dogs and chickens, respectively. The parasite load found in these species was between <50 and more than 35 000 tachyzoites per mg of tissue. Virulence marker genotyping revealed a recombinant 1&3 ROP5 RFLP pattern in 2 ToxoDB #116 isolates with no prediction of virulence in a murine model, while in the 2 ToxoDB #38 isolates, the ROP18/ROP5 combination predicted high virulence. Considering all the typed markers, there is a predominance of type I and III alleles, as constantly reported for the isolates characterized in various regions of México. It is crucial to determine their phenotype to corroborate the genetic virulence profile of the T. gondii isolates obtained in this study.

We present the comparative characterisation of 195 non-aureus staphylococci (NAS) isolates obtained from sheep (n = 125) and humans (n = 70) in Sardinia, Italy, identified at the species level by gap gene polymerase chain reaction (PCR) followed by restriction fragment length polymorphism analysis with AluI. Isolates were tested phenotypically with a disc diffusion method and genotypically by PCR, for resistance to 11 antimicrobial agents including cationic antiseptic agents. Among the ovine isolates, Staphylococcus epidermidis (n = 57), S. chromogenes (n = 29), S. haemolyticus (n = 17), S. simulans (n = 8) and S. caprae (n = 6) were the most prevalent species, while among human isolates, S. haemolyticus (n = 28) and S. epidermidis (n = 26) were predominant, followed by S. lugdunensis and S. hominis (n = 4). Of the 125 ovine isolates, 79 (63.2%) did not carry any of the resistance genes tested, while the remainder carried resistance genes for at least one antibiotic. The highest resistance rates among ovine isolates were recorded against tetracycline (20.8%), and penicillin (15.2%); none was resistant to methicillin and two exhibited multidrug resistance (MDR); one of which was positive for the antiseptic resistance smr gene. By contrast, most human isolates (59/70, 84.3%) were resistant to ⩾1 antimicrobials, and 41 (58.6%) were MDR. All 52 (74.3%) penicillin-resistant isolates possessed the blaZ gene, and 33 of 70 (47.1%) harboured the mec gene; of these, seven were characterised by the Staphylococcal Chromosomal Cassette (SCCmec) type IV, 6 the type V, 5 of type III and one representative each of type I and type II. The majority (57.1%) was erythromycin-resistant and 17 isolates carried only the efflux msrA gene, 11 the methylase ermC gene and an equal number harboured both of the latter genes. Moreover, 23 (32.8%) were tetracycline-resistant and all but one possessed only the efflux tetK gene. qacA/B and smr genes were detected in 27 (38.6%) and 18 (25.7%) human NAS, respectively. These results underline a marked difference in species distribution and antimicrobial resistance between ovine and human-derived NAS.

There is a paucity of information on hookworm species in humans, domestic animals and wildlife in southern Africa. Our study aimed to identify hookworm species from stray dogs, humans, and selected wildlife from South Africa. A total of 356 faecal samples were screened for the presence of hookworm-like eggs and subsequently coproculture from the positive samples was carried out to obtain larvae. Hookworm-like eggs were detected in 23.03% (82/356) of samples. Of these samples, 78/296 were from dogs, 3/50 from humans and 1/10 from wildlife. DNA was then isolated from the larvae of 55 positive samples, which were subjected to polymerase chain reaction (PCR), polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequencing of the nuclear ribosomal internal transcribed spacer (ITS1) and 5.8S rRNA region. Presence of Ancylostoma caninum, A. braziliense and A. ceylanicum-like species was recorded in stray dogs and A. caninum was recorded in wildlife and humans, using PCR-RFLP. Although PCR-RFLP results pointed to the presence of A. ceylanicum, we did not get a sequence that matched with A. ceylanicum from GenBank. This may have been due to the low proportion of A. ceylanicum larvae in our samples. Twenty-two of the 27 positive amplicons from stray dogs matched with A. caninum, three with A. braziliense and two had mixed infections of A. braziliense and A. caninum. Sequences from a lion and three humans matched with A. caninum. This is the first confirmation of a patent A. caninum infection in humans as evidenced by the presence of eggs in faeces, with the subsequent larvae from coproculture being identified as A. caninum.

Molecular techniques are useful tools for solving taxonomic confusion among species. Polymerase chain reaction (PCR) and polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) methods were applied for the identification of barnyardgrass, early watergrass, and late watergrass. Total DNA was extracted from 266 accessions, which were collected from different rice growing areas of Turkey. The two primer sets (trn-a and trn-b1, and trn-c and trn-d) specific to a target region of the intergenic spacer between trnT (UGU) and trnL (UAA) and the entire intron region of trnL (UAA), respectively, were used in PCR amplifications. Of the 266 accessions of Echinochloa spp., only eight accessions gave a similar fragment size, which was slightly shorter than 495 bp. The PCR product obtained with the primers trn-a and trn-b1 gave two fragments when EcoRI restriction enzyme was used in barnyardgrass and early watergrass. However, not all accessions of late watergrass were digested with this enzyme. In contrast to EcoRI, the PCR product obtained using the trn-c and trn-d primer set was digested into two fragments by using AluI restriction enzyme in all accessions of late watergrass; whereas, it was not digested in barnyardgrass and early watergrass. This molecular differentiation among barnyardgrass, early watergrass, and late watergrass supports the hypothesis that late watergrass is not a synonym of early watergrass in Turkish accessions.

The objective of this study was to determine the genetic diversity of creole cattle in northwestern Mexico using the BoLA-DRB3.2 locus of the Major Histocompatibility Complex (MHC). A total of 56 creole cattle were sampled from five communities; in the state of Chihuahua (Cerocahui, Guadalupe y Calvo and Cuauhtémoc) and in the state of Baja California Sur (La Paz and Mulegé). The BoLA-DRB3.2 locus was genotyped by PCR-RFLP assay. Thirty-nine alleles were identified, out of which 14 had not been previously reported. The average level of inbreeding in all populations analyzed was FIS = 0.09 (P < 0.0001), but only two populations (Cerocahui and Guadalupe y Calvo) showed an excess of homozygotes (P < 0.05). The breed differentiation in all populations studied was FSC = 0.068 (P < 0.0001). The smallest genetic distance was between La Paz and Mulegé (0.022); but Mulegé presented smaller distances (0.028–0.053) with the populations of La Paz (0.071–0.083) and with Chihuahua. Baja California Sur populations are grouped in a separate branch than Chihuahua populations. We conclude that creole cattle from Baja California Sur and Chihuahua show high genetic diversity in the locus BoLA-DRB3.2.

This study compared genetic diversity of Toxoplasma gondii isolates from Portugal, Austria and Israel. For this, we genotyped 90 T. gondii isolates (16 from Portugal, 67 from Austria and 7 from Israel) using 10 nested PCR-restriction length polymorphism (RFLP) genetic markers and 15 microsatellite (MS) markers. By PCR-RFLP typing, 7 isolates from Portugal chickens were identified as type II (ToxoDB #1 or #3), 4 were type III (ToxoDB #2) and the remaining 4 isolates have unique genotype pattern were designated as ToxoDB #254. One mouse virulent isolate from a bovine fetus (Bos taurus) in Portugal was type I (ToxoDB #10) at all loci and designated as TgCowPr1. All 67 isolates from Austria and 7 from Israel were type II (ToxoDB #1 or #3). By MS typing, many additional genetic variations were revealed among the type II and type III isolates. Phylogenetic analysis showed that isolates from the same geographical locations tend to cluster together, and there is little overlapping of genotypes among different locations. This study demonstrated that the MS markers can provide higher discriminatory power to reveal association of genotypes with geographical locations. Future studies of the type II strains in Europe by these MS markers will be useful to reveal transmission patterns of the parasite.

Transmission of pathogens between domestic and wild life animals plays an important role in epidemiology. Feral pig populations are increasing and expanding in the USA, and may constitute a risk to non-biosecure domestic pig facilities by serving as reservoirs for pathogens. We surveyed, for Sarcocystis infection, the myocardium of 1006 feral pigs (Sus scrofa) trapped or hunted in 29 states during the Comprehensive Feral Swine Disease Surveillance Program of the USDA's Animal and Plant Health Inspection Service, Wildlife Services unit during 2012–2014. Sarcocysts were detected in histological sections of 25% (251/1006) of myocardium with an average parasitic load/intensity of infection of 3·03 sarcocysts/section (1·5×0·7 cm), and higher prevalence of myocarditis in severe infections. Microscopic examination of pepsin digests of 147 hearts revealed a higher prevalence of Sarcocystis bradyzoites (49%, 72/147) than when diagnosed by histology. A fragment of Sarcocystis 18S rRNA was amplified and digested with a restriction endonuclease, revealing a pattern consistent with Sarcocystis miescheriana in all 44 selected samples. Sequencing 31 of these 44 isolates confirmed their correspondence to S. miescheriana. Thus, S. miescheriana infection, but not the zoonotic parasite Sarcocystis suihominis, appears to be prevalent and widespread in feral pigs in the USA.

In recent years, an extensive collection of Toxoplasma gondii samples have been typed using a set of 10 PCR-RFLP genetic markers. Here we summarize the data reported until the end of 2012. A total of 1457 samples were typed into 189 genotypes. Overall, only a few genotypes dominate in the northern hemisphere, which is in stark contrast to the southern hemisphere where hundreds of genotypes coexist with none being notably dominant. PCR-RFLP genotype #1 (Type II clonal), #2 (Type III), #3 (Type II variant) and #10 (Type I) are identified globally. Genotypes #2 and #3 dominate in Africa, genotypes #9 (Chinese 1) and #10 are prevalent in Asia, genotypes #1, #2 and #3 are prevalent in Europe, genotypes #1, #2, #3, #4 and #5 dominate in North America (#4 and #5 are collectively known as Type 12). In Central and South America, there is no clear dominance of any genotype even though a few have relatively higher frequencies. Statistical analysis indicates significant differences among populations in Africa, Asia, Europe, North America, and Central and South America, with only Europe and North America exhibiting similar diversity. Collectively, the results revealed distinct population structures and geographical patterns of diversity in T. gondii.

Molecular approaches for identifying aquatic macroinvertebrate species are increasingly being used but there is ongoing debate about the number of DNA markers needed to differentiate species accurately. Here, we use two mitochondrial genes (cytochrome oxidase I, cytochrome b) and a nuclear gene (carbamoylphosphate synthetase) to differentiate species variation within the taxonomically challenging chironomid genus Procladius from southern Australia, a genus which is important for pollution monitoring. The mitochondrial genes indicated cryptic species that were subsequently linked to morphological variation at the larval and pupal stage. Two species previously described based on morphological criteria were linked to molecular markers, and there was evidence for additional cryptic species. Each genetic marker provided different information, highlighting the importance of considering multiple genes when dissecting taxonomically difficult groups, particularly those used in pollution monitoring.

Introduction. Species typing in leishmaniasis gains importance in diagnostics, epidemiology, and clinical studies. A restriction fragment length polymorphism (RFLP) assay of PCR amplicons from a partial heat-shock protein 70 gene (hsp70) had been described for the New World, allowing identification of some species. Methods. Based on an initial in silico analysis of 51 hsp70 sequences, most of which we recently determined in the frame of a phylogenetic study, species-specific restriction sites were identified. These were tested by PCR-RFLP on 139 strains from 14 species, thereby documenting both inter- and intra-species variability. Results. Our assay could identify Leishmania infantum, L. donovani, L. tropica, L. aethiopica, L. major, L. lainsoni, L. naiffi, L. braziliensis, L. peruviana, L. guyanensis, and L. panamensis by applying 2 subsequent digests. L. mexicana, L. amazonensis, and L. garnhami did not generate species-specific restriction fragment patterns. Conclusion. Currently no assay is available for global Leishmania species discrimination. We present a universal PCR-RFLP method allowing identification of most medically relevant Old and New World Leishmania species on the basis of a single PCR, obviating the need to perform separate PCRs. The technique is simple to perform and can be implemented in all settings where PCR is available.

Faecal specimens from diarrhoeic pre-weaned calves (n=61) and lambs (n=127) collected over a 1-year period (2008–2009) at 27 cattle and 28 sheep farms in Galicia (NW Spain) were examined for the presence of Cryptosporidium oocysts and positive specimens were selected for molecular examination. Overall, 30 calves (49·2%) and 39 lambs (30·7%) tested positive for Cryptosporidium by microscopy. PCR products of the SSU rRNA locus were obtained for 27 Cryptosporidium positive calf isolates and 23 lamb isolates. Restriction analyses generated profiles of C. parvum in all isolates except for 9 lamb specimens from 5 farms that yielded banding patterns and sequences indicative of the Cryptosporidium cervine genotype. Sequence analyses of the glycoprotein (GP60) gene revealed that all but 1 C. parvum isolate from calves belonged to the subtype IIaA15G2R1 and 1 isolate was identified as the novel subtype IIaA13G1R1. Two different subtypes were identified in sheep flocks including IIaA16G3R1, which was seen in 7 lamb isolates from a single farm and subtype IIaA15G2R1, identified in 3 isolates from 2 farms. These findings suggest a limited genetic diversity within C. parvum in ruminant livestock from this geographical area, although both calves and lambs should be considered as a reservoir for zoonotic subtypes.

The cixiid species Reptalus quinquecostatus, R. cuspidatus, R. panzeri and R. melanochaetus are widely distributed in Europe and are receiving growing attention because of their potential role as phytoplasma vectors. Identifying the Reptalus species is restricted to a few specialist entomologists and relies on the morphology of the male genitalia, hampering the identification of juveniles and females. This study provides the tools for species discrimination by integrating the morphological description, which is primarily for the genus identification, with new molecular assays, based on both ribosomal and mitochondrial DNA. PCR-RFLP assays carried out on the mitochondrial cytochrome oxidase I gene (COI) with AluI provided species-specific profiles for the four Reptalus species. Amplification of a ribosomal internal transcribed spacer (ITS2) region produced species-specific fragments of different sizes for R. quinquecostatus, R. melanochaetus, R. cuspidatus and R. panzeri. The digestion of the ITS2 PCR product with TaqI allowed the discrimination of these latter two species. This molecular identification key ensures reliable results and can be successfully applied not only to adults, but also to the nymphs feeding on the roots. The identification of the nymphs (i) extends the collection period of these monovoltine species to the whole year (adults are present for a short summer period) and (ii) allows the unambiguous identification of their actual host plants because nymphs are steady on the root system while adults tend to disperse onto other plants. Fast and reliable identification of the Reptalus species provides useful help in monitoring activities and, therefore, in designing rational control strategies to protect crops from phytoplasma infection.

Introduction. Leishmania donovani, the causative agent of visceral leishmaniasis in the Indian subcontinent, has been reported to be genetically homogeneous. In order to support ongoing initiatives to eliminate the disease, highly discriminative tools are required for documenting the parasite population and dynamics. Methods. Thirty-four clinical isolates of L. donovani from Nepal were analysed on the basis of size and restriction endonuclease polymorphisms of PCR amplicons from kinetoplast minicircle DNA, 5 nuclear microsatellites, and nuclear loci encoding glycoprotein 63, cysteine proteinase B, and hydrophilic acylated surface protein B. We present and validate a procedure allowing standardized analysis of kDNA fingerprint patterns. Results. Our results show that parasites are best discriminated on the basis of kinetoplast minicircle DNA (14 genotypes) and 1 microsatellite defining 7 genotypes, while the remaining markers discriminated 2 groups or were monomorphic. Combination of all nuclear markers revealed 8 genotypes, while extension with kDNA data yielded 18 genotypes. Conclusion. We present tools that allow discrimination of closely related L. donovani strains circulating in the Terai region of Nepal. These can be used to study the micro-epidemiology of parasite populations, determine the geographical origin of infections, distinguish relapses from re-infection, and monitor the spread of particular variants.

Melanocortin-4 receptor (MC4R) is a member of the superfamily of G-protein-coupled receptors (GPCRs), which affects body weight, energy homeostasis and food intake in humans and mice. In this study, the Asp298Asn polymorphism of the MC4R gene was investigated in Laiwu, Yorkshire×Laiwu and commercial cross-bred pig populations using polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP), and the relationship of this mutation with backfat thickness was analysed. The results indicated that only genotype 11 exists in 33 individuals of Laiwu pigs, and three genotypes (11, 12 and 22) were detected in Yorkshire×Laiwu and commercial cross-bred populations. The distributions of allele and genotype frequencies in Yorkshire×Laiwu and commercial cross-bred populations were similar, with the frequency of allele 1 being higher than that of allele 2. In commercial cross-bred pigs, the mean backfat thickness of individuals with genotype 22 was significantly higher than that of individuals with genotypes 12 (P<0.01) and 11 (P<0.05). This study provides evidence that the Asp298Asn polymorphism of the MC4R gene is associated with backfat thickness in commercial cross-bred pigs with Western pigs as parental lines and, therefore, can be used as a DNA marker for breeding in such populations of pigs.

Faecal specimens from 287 diarrhoeic calves younger than 21 days, collected over a 2-year period (2006–2007) from 82 dairy cattle farms in 14 provinces across the north of Spain, were examined for the presence of Cryptosporidium oocysts. Overall, 63 farms (76·8%) and 166 calves (57·8%) tested positive by microscopy. In order to elucidate the genetic diversity, selected positive specimens from 149 calves originating from 61 farms in the 14 provinces were examined by genotyping and subtyping techniques. Cryptosporidium parvum was the only species identified by PCR-RFLP of SSU rDNA from all 149 isolates and sequencing of a subset of 50 isolates, except for 2 specimens that were identified as C. bovis. Sequence analyses of the glycoprotein (GP60) gene revealed that most C. parvum isolates (98%) belonged to the subtype family IIa and 2 isolates were identified as the novel subtype IIdA23G1. Subtype IIaA15G2R1 was the most common and widely distributed (80·3% of the 61 farms), followed by subtype IIaA16G3R1 (14·7%), whereas the remaining IIa subtypes (IIaA16G2R1, IIaA17G2R1, IIaA18G3R1, IIaA19G3R1) were restricted to 1–3 farms. All these C. parvum IIa subtypes have previously been described in human patients, indicating that most isolates from diarrhoeic calves in northern Spain have zoonotic potential.

Trypanosoma cruzi, the agent of Chagas disease is associated with a very high clinical and epidemiological pleomorphism. This might be better understood through studies on the evolutionary history of the parasite. We explored here the value of antigen genes for the understanding of the evolution within T. cruzi. We selected 11 genes and 12 loci associated with different functions and considered to be involved in host-parasite interaction (cell adhesion, infection, molecular mimicry). The polymorphism of the respective genes in a sample representative of the diversity of T. cruzi was screened by PCR-RFLP and evolutionary relationships were inferred by phenetic analysis. Our results support the classification of T. cruzi in 2 major lineages and 6 discrete typing units (DTUs). The topology of the PCR-RFLP tree was the one that better fitted with the epidemiological features of the different DTUs: (i) lineage I, being encountered in sylvatic as well as domestic transmission cycles, (ii) IIa/c being associated with a sylvatic transmission cycle and (iii) IIb/d/e being associated with a domestic transmission cycle. Our study also supported the hypothesis that the evolutionary history of T. cruzi has been shaped by a series of hybridization events in the framework of a predominant clonal evolution pattern.

The four significant pest species in the Helicoverpa genus (H. armigera, H. assulta, H. punctigera and H. zea) are morphologically similar and can only be reliably distinguished through dissection of adult genitalia. Two partial regions of the mitochondrial DNA (mtDNA), the cytochrome oxidase subunit I (COI) and the cytochrome b (Cyt b) genes were amplified by PCR and digested with restriction endonucleases. The restriction patterns, generated by the endonucleases BstZ17I and HphI, demonstrated reliable differentiation of the four Helicoverpa pest species. This technique is fast, reliable and effective at distinguishing specimens irrespective of their life stages and offers support to conventional taxonomic differentiation based on morphological characters.

Natural Cryptosporidium infections in pigs are widespread but generally apathogenic. This study was undertaken to determine the prevalence of zoonotic Cryptosporidium spp. in piggeries in Ireland, where the drinking water supply is particularly vulnerable to contamination with zoonotic species. Overall, infections were detected in 39 out of 342 animals (11·4%), with highest infection rates among weaners (15%) and sows (13·3%). Twenty-nine positive samples were genotyped based on SSU rRNA sequence analysis. Infections with Cryptosporidium parvum, the most important zoonotic species were rare and are likely to be of greater concern to animal handlers than suppliers of drinking water. In addition to C. parvum, Cryptosporidium suis, Cryptosporidium pig genotype II, Cryptosporidium muris and a previously undescribed genotype were identified. ABI-profiles indicated the presence of different alleles in at least 40% of all genotyped isolates. This was confirmed in 3 isolates by cloning of the PCR products. Since chronic mixed infections appear to be quite common in pigs they could be considered as models for mixed infections in immunocompromised humans.