Background and Aim: The diagnosis of bovine brucellosis in animals vaccinated with strain-19 (S19) and Rose Bengal (RB)-51 strain vaccines can be misinterpreted due to false positives. This study aimed to compare diagnostic tests for detecting bovine brucellosis in animals vaccinated with S19 and RB51 vaccine strains. Materials and Methods: Two groups of 12 crossbred Holstein calves between 6 and 8 months of age were used. On day 0, blood samples were collected from the animals, and the competitive enzyme-linked immunosorbent assay was used for serological diagnosis of bovine Brucellosis. All animals tested negative. After the first blood collection, the animals were subcutaneously vaccinated: one group received the S19 vaccine and the other received the RB51 vaccine. From the 3rd month after vaccination, all animals were sampled. Sampling was repeated every 2 months until the 7th month. Serological diagnosis of bovine brucellosis was performed using RB, tube serum agglutination test (SAT), SAT with 2-mercaptoethanol (SAT-2Me), and fluorescence polarization assay (FPA). Results: Animals vaccinated with S19 showed positive results with the RB, SAT, and SAT-2Me tests in all months of post-vaccination diagnosis. In animals vaccinated with S19, FPA showed positive results at months 3 and 5 and negative results at month 7, indicating that this test discriminates vaccinated animals from infected animals 7 months after vaccination. Rose Bengal, SAT, SAT-2Me, and FPA tests showed negative results in animals vaccinated with RB51 in all months of diagnosis. Conclusion: Animals vaccinated with S19 may test positive for brucellosis using RB, SAT, or SAT-2Me tests 7 months later. Fluorescence polarization assay is an optimal alternative for diagnosing animals in the field, thereby preventing false positives, and consequently, unnecessary confiscations of animals. Animals vaccinated with RB51 tested negative with RB, SAT, SAT-2Me, and FPA tests in all months of diagnosis, confirming that the tests are ineffective for diagnosing brucellosis caused by rough strains. Keywords: agglutination, bovine, brucellosis, vaccination.

Escherichia coli (E. coli), especially the Shiga toxin-producing O157:H7 strain, poses severe health risks. In rural Ecuador, raw milk consumption heightens contamination risks. This study analyzed 633 raw milk samples from Pichincha and Manabí to assess E. coli O157:H7 prevalence. The samples were enriched using BHI broth, and then specific culture media were used to isolate E. coli O157:H7. The pathogen in the enriched raw milk was identified, and the isolates were specifically confirmed through the application of a newly designed qPCR assay. The novel qPCR assay demonstrated remarkable sensitivity, capable of detecting up to one copy of genetic material, and specificity (no amplification of other bacteria). An extremely high E. coli O157:H7 prevalence of 0.63 (n = 401) was detected, where the province with the highest number of positive samples was Manabí with 72.8% (n = 225/309) and 54.3% (n = 179/324) for Pichincha. In both provinces, the presence of E. coli O157:H7 contamination exhibited a favorable correlation with small-scale farms and elevated temperatures. This research provides valuable data on the microbiological contamination of E. coli O157:H7 present in raw milk, in addition to an improved method that has been demonstrated to be faster, more sensitive, and more specific than conventional and previously published methods, highlighting the associated risk of food-borne infections and pointing out potential shortcomings in the regulation of agricultural practices and the need for periodic monitoring of bacterial contamination levels with updated methods.

Background and Aim: Viral gastroenteritis in canines is primarily caused by the canine parvovirus 2 (CPV-2). Infections by this virus can cause severe consequences in dogs, such as fever, vomiting, diarrhea, septicemia, systemic inflammation, and immunosuppression. Therefore, the mortality rate of persistent infections caused by this virus is significantly high. The capsid protein VP2 genome of canine parvovirus has undergone many changes, resulting in the emergence of different genotypes, including CPV-2a, CPV-2b, and CPV-2c. Diagnostic procedures often lack the necessary specificity for early infection diagnosis. Early detection of the infection enhances the likelihood of canine survival because the canine will receive prompt therapy. Hence, this study aimed to develop a quantitative polymerase chain reaction (qPCR)-based diagnostic technique using SYBR Green for the rapid and accurate detection and quantification of CPV-2. Materials and Methods: The assay was specifically designed to identify a portion of the conserved NS gene using primers that amplify a 125-bp fragment. The qPCR method was executed in the fast mode to expedite the process using Power up SYBR Green Master Mix reagent. A standard curve was constructed using the amplified and purified PCR product of the NS gene. Results: The limit of detection and quantification were determined in the one amplified-DNA copy. The standard curve showed an efficiency of 99.5% and inter- and intra-assay coefficients of variation of 0.387%–0.976% and 0.085%–0.430%, respectively. The assay was specific for the amplification of CPV-2, as no amplification was observed for other viral genomes (canine adenovirus II, canine distemper virus, canine coronavirus, and canine astrovirus) or from the negative controls. Inter- and intra-tests for repeatability showed low test variability around the run time. To validate the present assay, 200 samples of fezzes from canines with gastroenteritis and symptoms associated with enteric infection were tested using the qPCR protocol. From the analyzed samples, 136 were positive for CPV-2 by qPCR assay, of which 110 were before diagnostic positive for the virus by endpoint PCR, showing high sensitivity of the current assay. CPV-2 was detected in dogs over 2 weeks old up to dogs 9 years old, where the highest viral concentration found was 16429595 gene copies in dogs aged 2 weeks. Conclusion: In the present study, a rapid, specific, repeatable, and sensitive assay was developed for the detection and quantification of CPV-2. Furthermore, it was demonstrated that in the population of domestic dogs in Ecuador affected with gastrointestinal disease, the virus is presented in dogs of different ages and not only in young dogs. Keywords: canine parvovirus, gastroenteritis, quantitative polymerase chain reaction, SYBR green.

Canine parvovirus (CPV-2) is a highly contagious virus in canines, and it is mostly spread by touching infected feces. Dogs of all ages can catch it, but puppies are more likely to suffer from it. Severe signs include vomiting, diarrhea with blood, feeling tired, and not drinking enough water. There are three different types of the original CPV-2 that have been found so far, which are CPV-2a, 2b, and 2c. The genome of CPV-2 is about 5.2 kb long and has two open reading frames (ORFs), namely the VP region and the NS region. Based on changes in amino acids at position 426, the VP2 protein distinguishes the gene apart in the VP region. Using a molecular method, this study contemplated the presence of CPV-2 and its variants in dogs that had gastroenteritis, as well as other infections. There were 511 samples tested, and 401 (78.47%) of them were positive for CPV-2. Of these, 144 (25.91%) were positive for the original genotype, 258 (64.34%) for genotype 2a, 343 (85.54%) for genotype 2b, and 167 (41.65%) for genotype 2c. Using the multiplex qPCR for genotyping, CPV-2a and CPV-2b were determined as the most frequent co-infections (16.45%). The three genotypes (2a, 2b, and 2c) were found in the samples examined based on the amino acids at position 426 of the VP2 protein, as demonstrated by the VP2 gene sequencing. Furthermore, it was discovered that in certain samples, a genetic modification at position 297 was connected to the virus’s pathogenicity.

Background and Aim: Fowl adenovirus (FAdV) is the etiological agent of inclusion body hepatitis (IBH) and hepatitis-hydropericardium syndrome (HHS) in poultry. It is also detected in chickens with runting and stunting syndrome (RSS). FAdV has been detected worldwide, and genotypes 8a, 8b, and 11 have been identified in chickens with enteric problems in Brazil. Nevertheless, none of them have been isolated; therefore, these viruses propagate; thus, the viral behavior and pathogenicity are unknown in Brazil. This study aimed to isolate FAdV from the enteric content of chickens affected by RSS. Materials and Methods: Enteric content samples from chickens affected with RSS and a positive polymerase chain reaction (PCR) test for FAdV were inoculated into specific pathogen-free chicken embryonated eggs (CEEs) through the yolk and chorioallantoic membrane routes on 8 and 10 days of age, respectively and the eggs were incubated for five days for viral isolation. Results: The embryos exhibited dwarfism, beak atrophy, and pale claws. In addition, some embryos displayed edema and gelatin-like characteristics. The liver exhibited hepatomegaly and multiple necrotic foci, resembling the appearance of nutmeg. In addition, the kidneys appeared enlarged and pale. After 8 days of incubation, the hearts of the inoculated embryos showed hydropericardium. Microscopic evaluation revealed the presence of hepatitis, which was characterized by the presence of intranuclear inclusion bodies and cellular necrosis. Viral FAdV particles were observed in kidney cells using electron microscopy. Viral DNA was detected in the embryos in all three passages, and viral gene copies were also measured in some organs, with high FAdV gene copies detected in the spleen and bursa beyond the liver. Conclusion: The molecular characterization of FAdV revealed that the isolated strain belonged to genotype 8a of FAdV. Here, FAdV-8a from chickens infected with RSSs produced IBH/HHS in CEE, and FAdV-8a detected in RSS outbreaks, in addition to producing IBH/HHS in chicken embryos, could be a possible viral agent that causes IBH/HHS in chickens. Keywords: chicken embryonated eggs, fowl adenovirus, hepatitis-hydropericardium syndrome, inclusion body hepatitis, runting and stunting syndrome.

Background: Milk is a highly nutritious food, but its composition makes it an ideal medium for microbial growth, particularly for bacteria like Staphylococcus aureus (S. aureus). In Ecuador, raw milk consumption is culturally rooted, and contamination risks are heightened, especially in informal markets. Staphylococcus aureus, a Gram-positive, coagulase-positive bacterium, commonly colonizes mucous membranes and can cause a range of infections due to its production of thermostable toxins. Its impact extends to bovine mastitis, severely affecting dairy production. Of particular concern is the emergence of methicillin-resistant S. aureus (MRSA) strains, associated with the acquisition of the mecA gene located on the “staphylococcal chromosomal cassette mec” (SCCmec) element and identification of a mecA homologue, mecC, further complicates detection and monitoring efforts. Objectives: This study evaluated the prevalence of S. aureus and MRSA strains in raw milk from Ecuadorian provinces Pichincha and Manabí. Methods: A total of 633 samples were collected and analyzed via real-time PCR (qPCR) and bacterial isolation methods, complemented by endpoint PCR assays for mecA and mecC genes detection. Results: A high prevalence of S. aureus (84%) was observed, with significant differences between regions. MRSA was detected in 23% of all samples, with mecA being more prevalent than mecC among isolates. Sequencing of 16S rDNA confirmed the identity of isolates, while phylogenetic analysis of mecA and mecC genes validated their presence. The findings suggest that suboptimal hygiene practices and varied biosecurity protocols, especially among small and medium dairy producers, may contribute to the persistence of resistant strains. Conclusions: This study highlights the presence of S. aureus and MRSA in raw milk, underscoring the need for strengthened surveillance, improved hygiene practices, the use of molecular diagnostic tools, and proper heat treatments to reduce the public health risks associated with contaminated milk and its derivatives.