Evaluation of variants in the ENTPD1 and ENTPD2 genes in athletic horses with exercise-induced pulmonary haemorrhage.

BACKGROUND: Exercise-induced pulmonary haemorrhage (EIPH) in athletic horses is characterized by the presence of blood from the lungs in the tracheobronchial tree after intense exercise. Despite the high prevalence of EIPH in horses, the primary aetiology remains unknown. Variants in the genes encoding CD39 and CD39L1 (ENTPD1 and ENTPD2, respectively) were previously reported as potential genetic causes involved in EIPH pathogenesis. However, the role of these variants in haemostatic functions is unknown. RESULTS: To investigate the association between EIPH and missense variants in the ENTPD1 (rs1152296272, rs68621348, and rs68621347) and ENTPD2 genes (rs782872967), 76 Thoroughbred horses diagnosed with EIPH and 56 without clinical signs of EIPH (control group) by trachea-bronchial endoscopy were genotyped. The rs1152296272 and rs68621347 variants were linked, which explained why the same results were found in all horses. Approximately 96% and 95% of the EIPH and control horses, respectively, carried at least one nonreference allele for these variants. In contrast, 100% of the control horses and 96% of the EIPH horses were homozygous for the reference allele for the rs68621348 variant. In the EIPH group, 1.5% of the horses were homozygotes and 24% were heterozygous for the nonreference allele of the rs782872967 variant. In the control group, the nonreference allele of this variant was observed only in heterozygotes (16%). There were no significant differences between groups for any of the variants. CONCLUSIONS: The variants previously described in the genes encoding the CD39 and CD39L1 enzymes were highly present in the studied population. However, no association was found between the occurrence of EIPH and the presence of these variants in Thoroughbred horses in this study.

Fecal shedding of Clostridioides difficile in calves in Sao Paulo state, Brazil.

OBJECTIVE: This study aimed to evaluate the fecal shedding of C. difficile in calves on farms in Sao Paulo State, Brazil. MATERIALS AND METHODS: Fecal samples (n = 300) were collected from diarrheic (n = 78) and nondiarrheic (n = 222) calves less than 60 days of age from 20 farms. Fecal samples were inoculated into enrichment broth supplemented with taurocholate and cultured under anaerobic conditions. Colonies suspected to be C. difficile were harvested for DNA extraction and then multiplex PCR for the detection of genes encoding toxins A and B and binary toxins. All toxigenic isolates were ribotyped and tested for antimicrobial susceptibility, and five selected strains were subjected to whole-genome sequencing to determine their sequence type. RESULTS AND DISCUSSION: C. difficile was isolated from 29.3 % (88/300) of the samples. All toxigenic isolates (17/88, 19.3 %) were classified as ribotypes RT046 (13/17-79.47 %, A+B+ CDT-) and RT126 (4/17 = 20.53 %, A+B+ CDT+). The sequenced strains from RT046 were classified as ST35 (Clade 1), while those from RT126 were classified as ST11 (Clade 5). No associations between the epidemiological factors in any of the groups and C. difficile isolation were observed. Most of the toxigenic isolates (16/17 = 94.41 %) were classified as multidrug-resistant. Calves can be an important source of toxigenic C. difficile strains, including multidrug-resistant isolates from ribotypes commonly observed in humans.

Solar radiation leads to increased collagenase gene expression (MMP1) in HERDA (Hereditary equine regional dermal asthenia) affected horses and may explain the typical dorsal distribution of their lesions.

BACKGROUND: Hereditary equine regional dermal asthenia (HERDA) is a genetic disease that alters collagen biosynthesis. Affected horses exhibit fragile, hyperextensible skin, especially over the dorsal region. Although ultraviolet (UV) radiation seems to contribute to the regional distribution of lesions and worsening of clinical signs, the molecular mechanisms involved are largely unknown. OBJECTIVES: To evaluate the effect of solar radiation on matrix metalloproteinase MMP1, MMP8 and MMP13 gene expression in the dorsal and ventral skin of HERDA-affected and HERDA-unaffected horses [wild-type (WT) horses]. ANIMALS: Six HERDA-affected and six unaffected Quarter horses (WT) were paired according to age, sex and coat colour. MATERIALS AND METHODS: Horses were submitted to 30 day sunlight restriction, followed by 15 day sunlight exposure. Dorsal and ventral skin biopsies were obtained at six sampling times over 45 days. The expression of MMP1, MMP8 and MMP13 genes was measured by quantitative PCR. RESULTS: Although solar radiation modulated MMP1, MMP8 and MMP13 expression, the effects were more pronounced on MMP1. Sun exposure for three days significantly upregulated MMP1 in the dorsal region when compared to the ventral skin in both unaffected and HERDA-affected horses. CONCLUSIONS AND CLINICAL RELEVANCE: This study shows that solar irradiation leads to upregulation of skin collagenase genes particularly MMP1 in the dorsal, sun-exposed skin of horses. Furthermore, this was more marked in HERDA-affected horses. The increased activity of collagenases on the disorganised collagen present in HERDA affected horses would explain why UV radiation leads to deterioration of clinical signs in affected individuals.

Validation of high-resolution melting analysis as a diagnostic tool for endothelin receptor B mutation in American Paint horses and allele frequency estimation.

Overo lethal white foal syndrome (OLWFS) is a genetic disorder caused by a dinucleotide mutation in the endothelin receptor type B (EDNRB) gene leading to the death of affected foals shortly after birth. The use of rapid and reliable genetic testing is imperative for the early diagnosis of the mutation avoiding, therefore, either additional suffering or the production of affected animals. In the present study, we developed and validated a high-resolution melting (HRM) genotyping assay to detect the OLWFS causative mutation, and we also determined the frequency of heterozygotes among American Paint horses in Brazil. The HRM genotyping assay resulted in a high sensitivity, specificity, and positive and negative predictive values. The overall estimated frequency of heterozygotes was 21.6%; however, this frequency increased to 89.5% when considering only overo horses. The HRM assay optimized here was a reliable and suitable method for the detection of the dinucleotide mutation observed in the EDNRB gene resulting in a fast, accurate, and precise diagnostic tool. The causative gene mutation of OLWFS is present in heterozygosity in the American Paint Horse population in Brazil and is highly frequent among overo horses.

Methicillin-resistant Staphylococcus spp. investigation in hospitalized horses and contacting personnel in a teaching veterinary hospital.

Staphylococci are well-known opportunistic pathogens associated with suppurative diseases in humans and animals. Antimicrobial resistance is an emergent threat to humans and animals worldwide. This study investigated the prevalence of methicillin-resistant Staphylococcus spp. (MRS) in hospitalized horses and contacting personnel (veterinarians and staff), and assessed possible interspecies transmission in a teaching veterinary hospital. Nasal swabs from horses (n = 131) and humans (n = 35) were collected. The microorganisms were identified by traditional biochemical tests and genotypic methods, i.e., PCR, internal transcript spacer PCR (ITS-PCR), and gene sequencing. Staphylococcal species were isolated in 18% (23/131) of the horses, of which 8% (11/131) were S. hyicus, 4 % (5/131) were S. aureus, 4% (5/131) were S. pseudintermedius, and 2% (2/131) were S. schleiferi subsp. coagulans. The mecA gene was detected in an S. pseudintermedius isolate. Staphylococcus spp. was isolated in 40% (14/35) of the human samples, all of which were S. aureus. In four samples of S. aureus, the clonal profile ST398 was identified; among them, a clonal similarity of 98.1% was observed between a horse and a contacting human. This finding supports the need for biosecurity measures to avoid the spread of multidrug-resistant staphylococci in humans and horses.

Brain Abscesses in Domestic Ruminants: Clinicopathological and Bacteriological Approaches.

Brain abscesses in ruminants often arise from primary infection foci, leading to an unfavorable prognosis for affected animals. This highlights the need for comprehensive studies on brain abscesses across different ruminant species. We retrospectively investigated medical records of epidemiological, clinical, neuroimaging, anatomopathological, and bacteriological findings in six ruminants (three goats, two cows, and one sheep) diagnosed with brain abscesses. All animals studied were female. Apathy (50%), compulsive walking (33%), decreased facial sensitivity (33%), head pressing (33%), seizures (33%), semicomatous mental status (33%), strabismus (33%), unilateral blindness (33%), and circling (33%) represented the most common neurologic signs. Leukocytosis and neutrophilia were the main findings in the hematological evaluation. Cerebrospinal fluid (CSF) analysis revealed predominant hyperproteinorrachia and pleocytosis. In three cases, computed tomography or magnetic resonance imaging were used, enabling the identification of typical abscess lesions, which were subsequently confirmed during postmortem examination. Microbiological culture of the abscess samples and/or CSF revealed bacterial coinfections in most cases. Advanced imaging examinations, combined with CSF analysis, can aid in diagnosis, although confirmation typically relies on postmortem evaluation and isolation of the causative agent. This study contributes to clinicopathological aspects, neuroimages, and bacteriological diagnosis of brain abscesses in domestic ruminants.

Clinical and pathological findings of Africanized bee (Apis mellifera) envenomation in horses.

Africanized crossbred bees (Apis mellifera) originated in Brazil in 1957, and since then, the number of accidents involving humans and animals has significantly increased. Although they are considered clinical emergencies, there are few reports describing the clinical and pathological aspects of bee envenomation in horses. In this context, this report aims to describe the clinical and pathological features of bee toxicity after massive bee envenomation in three horses. The horses were referred to the veterinary hospital the day following the attack, and after clinical and laboratory examination, they presented with vascular, muscular, pulmonary, hepatic, and renal impairment. Even after the initiation of therapy immediately upon admission, the clinical condition of the two horses worsened, and they died within two days of hospitalization, with pathological analysis confirming the previously observed clinical alterations of generalized vasculopathy, liver degeneration, pulmonary edema, and renal tubular necrosis. Many cases of massive bee envenomation have been documented in both humans and animals, particularly in dogs. Understanding the mechanism of action of apitoxin, its effects on various tissues, and the ideal therapy for each patient has proven crucial for improving survival rates.

Effect of dietary iron supplementation on the equine fecal microbiome.

Iron is an essential element for all living organisms, including bacteria, as several virulence factors and replication components are influenced by iron concentration. The objective of this study was to determine whether the composition and diversity of the fecal microbiota of adult horses are affected by supplemental dietary iron. Ten clinically healthy horses were randomly divided into a control and an iron-supplemented group (n = 5). The treated group was supplemented with oral ferrous sulphate monohydrate (720 ppm of iron), whereas the control group received 320 ppm of iron daily for 15 d. Fecal samples were collected before and 5, 10, 15, and 30 d after supplementation and frozen at -80°C. DNA was sequenced using an Illumina MiSeq platform and data were analyzed using the software Mothur and linear discriminant analysis (LDA) effect size (LEfSe). Iron supplementation caused no change in the overall composition of the fecal microbiota, but some minor changes were observed in the low-abundant bacteria, as well as an increased alpha diversity after 15 d of supplementation. Significant differences in community composition of the fecal microbiota over time were observed in both groups, highlighting the importance of a control group, as there are variables that cannot be controlled in microbiome studies.

Le fer est un élément essentiel pour tous les organismes vivants, y compris les bactéries, car plusieurs facteurs de virulence et composants de réplication sont influencés par la concentration en fer. L'objectif de cette étude était de déterminer si la composition et la diversité du microbiote fécal des chevaux adultes sont affectées par la supplémentation en fer alimentaire. Dix chevaux cliniquement sains ont été divisés au hasard en un groupe témoin et un groupe supplémenté en fer, n = 5 par groupe. Le groupe traité a reçu un supplément oral de sulfate ferreux monohydraté (720 ppm de fer) et le groupe témoin a reçu 320 ppm de fer par jour pendant 15 jours. Des échantillons fécaux ont été prélevés avant la supplémentation et 5, 10, 15 et 30 jours après la supplémentation puis congelés à −80 °C. L'ADN a été séquencé à l'aide de la plateforme Illumina MiSeq et les données ont été analysées à l'aide des logiciels Mothur et analyse de la fonction discriminante linéaire taille de l'effet LefSe. La supplémentation en fer n'a provoqué aucun changement dans la composition du microbiote fécal, mais certains changements ont été observés chez les bactéries peu abondantes, ainsi qu'une augmentation de la diversité alpha après 15 jours de supplémentation. Au fil du temps, des différences significatives dans la composition de la communauté bactérienne ont été observées dans les deux groupes, soulignant l'importance d'un groupe témoin, car il existe des variables qui ne peuvent être contrôlées dans les études sur le microbiome.(Traduit par les auteurs).

Hereditary myotonia in cats associated with a new homozygous missense variant p.Ala331Pro in the muscle chloride channel ClC-1.

Three-related cats were evaluated for a history of short-strided gait and temporary recumbency after startle. Neurological examination, electromyography (EMG), muscle biopsies, and a chloride voltage-gated channel 1 (CLCN1) molecular study were performed. Clinically, all 3 cats presented myotonia with warm-up phenomenon and myotonic discharges during EMG examination. Muscle biopsies showed normal muscle architecture and variation in the diameter of myofiber size with the presence of numerous hypertrophic fibers. The molecular study revealed a missense variant (c.991G>C, p.Ala331Pro) in exon 9 of the CLCN1 gene, responsible for the first chloride channel extracellular loop. This mutation was screened in 104 control phenotypically normal unrelated cats, and all were wildtype. The alanine at this position is conserved in ClC-1 (chloride channel protein 1) in different species, and 2 mutations at this amino acid position are associated with human myotonia. This is the third CLCN1 mutation described in the literature associated with hereditary myotonia in cats and the first in domestic animals located in an extracellular muscle ClC-1 loop.

Detection and genomic characterization of a multidrug-resistant Salmonella Newport co-harbouring blaCMY-2, qnrB19 and mcr-9 from the diarrheic faeces of a foal.

OBJECTIVES: This study reports the genomic characterization of the multidrug resistant Salmonella Newport strain 195_20 recovered from the diarrheic faeces of a foal in Brazil and co-harbouring the mcr-9, blaCMY-2 and qnrB19 antibiotic resistance genes. METHODS: Bacterial isolate positive for mobile colistin resistance gene (mcr-9) was submitted to antimicrobial susceptibility testing by disk diffusion and broth microdilution for colistin and polymyxin B. The isolate was submitted to whole genome sequencing by Illumina technology and Nanopore Sequencing. Conjugation assays, plasmid sizes determined by S1-PFGE and plasmid content were investigated by hybrid assembly after MinIon long reads sequencing. RESULTS: Isolate 195_20 was identified as sequence type ST45, resistant to penicillin and cephalosporins (ampicillin, ceftazidime, ceftriaxone and cefotaxime), aminoglycosides (streptomycin and gentamicin), phenicol (chloramphenicol), quinolones and fluoroquinolones (nalidixic acid, ciprofloxacin, and pefloxacin), folate pathway antagonists (sulfonamides and trimethoprim-sulfamethoxazole), and tetracycline. A transferable IncHI2/IncHI2A plasmid sized ca. 262kb was found to carry the mcr-9 gene in a module consisting of IS903-mcr-9-wbuC-IS26. In addition, an 174kb IncC and a 48kb IncN plasmid were also identified in the 195_20 isolate, carrying blaCMY-2 and qnrB19, respectively. CONCLUSIONS: Not surprisingly, isolate 195_20 was susceptible to polymyxins, possibly due to absence of qseBC regulatory operon. Presence of mobile colistin resistance (mcr-9), third-generation cephalosporins (blaCMY-2) and quinolone (qnrB19) resistance determinants in zoonotic pathogens from animals in close contact with humans alerts for the possible route of transmission between these different reservoirs.

Potential in vitro action of an adenosine analog and synergism with penicillin against Corynebacterium pseudotuberculosis.

Caseous lymphadenitis is a well-known disease caused by Corynebacterium pseudotuberculosis affecting small ruminants with small significance to human health because of its minor zoonotic potential. In both cases, few treatment options are available and conventional antimicrobial therapy is commonly refractory due to development of pyogranulomatous reactions, bringing great interest in discovering novel therapeutics for more suitable approaches. Dideoxynucleotides presented antibacterial action against various bacteria but were never described for C. pseudotuberculosis. Hypothesizing the antimicrobial action of 2',3'-dideoxiadenosine (ddATP) against C. pseudotuberculosis, we performed for the first time an investigation of its minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) in the ATCC® 19,410 strain and a well-characterized clinical isolate of C. pseudotuberculosis. We also assessed potential synergism with penicillin. ddATP showed a growth delay effect for C. pseudotuberculosis at 2 µmol/mL and a MIC and MBC of 4 µmol/mL against the ATCC® 19,410 strain, but not for the clinical strain. An antimicrobial effect was observed when using concentrations lower than the MIC of ddATP associated with penicillin for both strains tested. Our data suggest the potential of nucleotide analogs, especially adenosine, and its combination with penicillin, as a possible novel treatment for C. pseudotuberculosis-induced infections, and contributes with knowledge regarding alternative drugs to treat C. pseudotuberculosis infections.

Pleural Empyema in Six Horses: A Retrospective Case Series.

The clinical findings of pleural empyema in six horses were retrospectively studied using epidemiological, clinical pathology, microbiological, ultrasound, and post-mortem data. The clinical findings included tachycardia (n = 3/6), tachypnea (n = 6/6), cyanotic mucosa (n = 2/6), hyperthermia (n = 4/6), inspiratory or mixed dyspnea (n = 6/6), presence of fluid and/or pleural rubbing (n = 2/6) and coarse crackling on auscultation (n = 4/6). Horses demonstrated leukocytosis (16.22 × 103/µL) with neutrophilia (12.32 × 103/µL) and hyperfibrinogenemia (633.33 mg/dL) and an increase in urea (69.80 mg/kg) and globulins (5.22 g/dL). The pleural fluid exhibited exudate (n = 5/6). The pathogens isolated from transtracheal wash (TTW) and/or pleural effusion included Aspergillus fumigatus, Enterobacter cloacae, alpha and beta hemolytic Streptococcus, Pseudomonas aeruginosa, Salmonella sp., Streptococcus equi subsp. zooepidemicus, and beta hemolytic Staphylococcus. The in vitro tests of microbial sensitivity of the isolates revealed that ceftiofur (5/6) and penicillin (3/6) were the most effective drugs. The fatality rate was 83% (5/6). The main post-mortem finding was the presence of fibrin in the pleural cavity with adhesion between the parietal and visceral pleura. These results show that pleural empyema is a complex disease pathophysiology that is refractory to conventional treatment.

Bacterial identification in cerebrospinal fluid of domestic species with neurologic signs: a retrospective case-series study in 136 animals (2005-2021).

Central nervous system (CNS) infections comprise life-threatening clinical conditions in domestic species, and are commonly related to severe sequelae, disability, or high fatality rates. A set of bacterial pathogens have been identified in central nervous infections in livestock and companion animals, although the most of descriptions are restricted to case reports and a lack of comprehensive studies involving CNS-related bacterial infections have been focused on a great number of domestic species. In this scenario, we retrospectively investigated selected epidemiological data, clinical findings, bacteriological culture, and in vitro susceptibility patterns of 136 nonrepetitive neurologic cases in domestic species (2005-2021). Bacterial isolates were recovered from 25% (34/136) of the cerebrospinal fluid (CSF) sampled. The isolates were obtained from cattle (9/136 = 6.6%), dogs (7/136 = 5.1%), horses (6/136 = 4.4%), goats (3/136 = 2.2%), pigs (3/136 = 2.2%), sheep (3/136 = 2.2%), cats (2/136 = 1.5%), and asinine (1/136 = 0.7%). Among animals with bacterial isolation, Staphylococcus aureus (6/34 = 17.6%), Escherichia coli (5/34 = 14.7%), Staphylococcus beta-hemolytic (5/34 = 14.7%), and Trueperella pyogenes (3/34 = 8.8%) were predominant, in addition to a miscellaneous of other bacteria isolated in minor frequency, e.g., Corynebacterium pseudotuberculosis, Enterobacter cloacae, Mannheimia haemolytica, Pseudomonas aeruginosa, and Streptococcus equi subsp. equi. In vitro susceptibility tests of isolates revealed that amoxicillin/clavulanic acid (11/13 = 84.6%), cephalexin (9/11 = 81.8%), and florfenicol (9/12 = 75%) were the most effective antimicrobials. Conversely, isolates exhibited resistance mainly to tetracycline (6/10 = 60%), penicillin (6/11 = 54.5%), and trimethoprim/sulfamethoxazole (5/11 = 45.5%). Also, multidrug resistance to ≥ 3 classes of antimicrobials was found in 23.5% (8/34) strains. Data relative to the outcome was available in 79.4% (27/34) of animals that had bacterial isolation, and from these, the lethality rate was 92.6% (25/27). Incoordination (14/34 = 41.2%), recumbency (11/34 = 32.4%), apathy (10/34 = 29.4%), anorexia (9/34 = 26.5%), blindness (7/34 = 20.6%), seizure (6/34 = 17.6%), limb paresis (5/34 = 14.7%), head-pressing (4/34 = 11.8%), and nystagmus (3/34 = 8.8%) were the most frequent clinical signs. A variety of bacterial pathogens were identified in the CSF of domestic species showing neurologic signs, with a predominance of staphylococci, streptococci, and enterobacteria. High lethality of cases, poor in vitro efficacy of conventional antimicrobials, and a high in vitro multidrug resistance pattern of isolates were seen. Our results contribute to etiological characterization, antimicrobial resistance patterns, and clinical-epidemiological findings of bacterial infections in domestic species with neurological signs.

A nonsense mutation in the tyrosinase gene causes albinism in water buffalo.

BACKGROUND: Oculocutaneous albinism (OCA) is an autosomal recessive hereditary pigmentation disorder affecting humans and several other animal species. Oculocutaneous albinism was studied in a herd of Murrah buffalo to determine the clinical presentation and genetic basis of albinism in this species. RESULTS: Clinical examinations and pedigree analysis were performed in an affected herd, and wild-type and OCA tyrosinase mRNA sequences were obtained. The main clinical findings were photophobia and a lack of pigmentation of the hair, skin, horns, hooves, mucosa, and iris. The results of segregation analysis suggest that this disease is acquired through recessive inheritance. In the OCA buffalo, a single-base substitution was detected at nucleotide 1,431 (G to A), which leads to the conversion of tryptophan into a stop codon at residue 477. CONCLUSION: This premature stop codon produces an inactive protein, which is responsible for the OCA buffalo phenotype. These findings will be useful for future studies of albinism in buffalo and as a possible model to study diseases caused by a premature stop codon.

Flash Visual Evoked Potentials in Conscious Horses: A Preliminary Study.

The visual evoked potential (VEP) has many applications in veterinary neurology, but the test is not routinely used in a clinical setting. The aim of this study was to describe a reliable method for recording flash visual evoked potentials (F-VEPs) in nonsedated horses. F-VEPs were recorded from both eyes in 20 healthy and calm, adult horses. Recordings were accomplished without sedation, anaesthesia, or the use of mydriatic drugs. The mean and standard deviation of the latency of the most evident positive peak was 52.76±2.37 ms (P53). The mean latencies of the preceding and following negative peaks were 38.14±4.62 (N38) and 72.35±5.33 ms (N72), respectively. There were 2 mean peak-to-peak amplitudes (N38 - P53 and P53 - N72), and they were 11.85±6.21 and 22.81±11.50 µV, respectively. F-VEP was also recorded from 3 horses (6 eyes) before and during sedation with 2 doses each of xylazine (0.4 and 1.1 mg/kg) or detomidine (0.005 and 0.014 mg/kg). It was possible to obtain a reliable F-VEP with a P53 latency in horses without sedation that was similar to the P2 peak described in previous studies, and these data can be used in the future as a normal reference for comparisons in horses with different diseases using a similar methodology. Sedation affected the results by depressing peak amplitudes and increasing latencies or by completely obscuring any response. The exact impact of sedation on VEPs must be evaluated with much caution due to the small sample size.

Allele frequency of SLC4A3 (PRA1), TTC8 (PRA2), and PRA-prcd mutations in golden retrievers in Brazil.

Progressive retinal atrophy (PRA) is a term used in veterinary medicine to describe inherited and progressive retinal diseases characterized by progressive retinal degeneration and loss of vision. In the Golden Retriever (GR) breed, the mutations associated with PRA have an autosomal recessive inheritance pattern. This study aimed to verify the allele frequencies of PRA1, PRA2, and PRA-prcd in the GR breed in Brazil. A total of 121 GR DNA samples (n = 66 females and n = 55 males) were analyzed. All animals assessed in this study were identified as wild-type (121/121 animals; 100%) for PRA1 and PRA2 mutations; therefore, no carrier or homozygous animals were identified in this population. For the PRA-prcd mutation, 118 animals (118/121 animals; 97.52%) were wild-type. Three animals were genotyped as heterozygous for PRA-prcd (3/121 animals; 2.47%), demonstrating that this mutation is still present in some bloodlines and animals in Brazil, even with a rare prevalence. Five animals (5/121 animals, 4.2%) had a previous eye disease, which was diagnosed by a veterinarian as entropion (2 animals), keratoconjunctivitis sicca (1 animal), corneal ulcer (1 animal), and bilateral blindness (1 animal). This dog with bilateral blindness was identified as wild type homozygous for three mutations assessed in this study; therefore, blindness was not associated with the investigated mutations. In addition, the vast majority (98.3%) of Brazilian breeders assessed in this study were unaware of these mutations as a cause of blindness in the Golden Retriever. Therefore, the present study will serve to disseminate knowledge about PRA and its genetic etiologies, as well as to support future studies with other Brazilian GR populations.

Sequential Cerebrospinal Fluid Sampling in Horses: Comparison of Sampling Times and Two Different Collection Sites.

Analysis of the cerebrospinal fluid (CSF) is important as a complementary test in horses with neurologic diseases, and sequential analysis may provide information about the treatment response or evolution and quantitative measures of the CSF drug concentration during treatment. The aim of this study was to compare erythrocyte and nucleated cell counts and protein concentration in multiple CSF samples obtained sequentially from two different puncture sites in clinically healthy horses. Eight and 12 horses, with no evidence of neurologic disease, were subjected to CSF collection from the atlanto-occipital (AO) and C1-C2 spaces, respectively. Cytologic and chemical analyses were performed on the CSF obtained at five sampling times (T1, T2, T3, T4, and T5). Repeated measures models were used to compare the mean erythrocyte count, nucleated cell count, and total protein concentration between the AO and C1-C2 groups at each sampling time. C1-C2 CSF had a significantly higher total protein concentration at T1 and T4 than that of AO CSF. All total protein concentration values remained within the reference interval (<90 mg/dL) for all sampling times and groups. No statistical difference was present between results at T2, T3, T4, and T5 and at T1 in both groups for all analyses. In conclusion, five consecutive AO or C1-C2 CSF collections with at least a 7-d interval did not result in alterations in the CSF erythrocyte and nucleated cell counts and total protein concentrations and did not interfere with the CSF analysis results.

Alobar Holoprosencephaly in an Aborted American Quarter Horse Fetus.

Holoprosencephaly is a central nervous system malformation, characterized by incomplete or total lack of division of prosencephalon hemispheres, which is commonly accompanied by craniofacial malformations. A 9-month-gestation aborted American Quarter Horse fetus was submitted for postmortem examination. The fetus lacked haircoat and had severe facial malformations including marked shortening/absence of the maxillary, incisive and nasal bones, bilateral anophthalmia, and pre-maxillary agenesis. The prosencephalon was small and nearly spherical, represented by a single lobe, with no visible separation between cerebral hemispheres. The olfactory bulbs, piriform lobes, and the optic chiasm were absent. At cross sectioning of the prosencephalon, the inner structures of the brain were completely absent, and replaced by a monoventricle lined by the remaining compressed cortex, and the thalami were fused. Since mutations in the sonic hedgehog (SHH) gene have been associated with human holoprosencephaly, the three coding SHH exons were sequenced using liver DNA of the aborted foal. The obtained SHH sequence was similar to the Equus caballus SHH mRNA sequence deposited in Genbank (XM_023640069.1); therefore, no polymorphism in the coding region of this gene justifying the phenotype was observed. This is the first report of alobar holoprosencephaly in horses.

Allele Frequency of APAF1 Mutation in Holstein Cattle in Brazil.

APAF1 is an autosomal recessive inherited mutation, associated with Holstein haplotype 1 (HH1) and characterized by a substitution of cytosine for a thymine (c.1741C>T) in chromosome 5. The mutation causes fetal and embryonic loss, between 60 and 200 days of gestation, and reduced conception rate. The ARMS-PCR is considered a simple and low-cost method to determine single nucleotide polymorphism (SNP) with no need for genetic sequencing of the animal genome. This study aimed to verify the allelic frequency of APAF1 mutation in Brazilian Holstein cattle. A total of 248 Holstein DNA samples (210 cows and 38 bulls) were analyzed, and synthetic genes were manufactured to validate the primers developed by the authors. All animals assessed in this study were classified as wild-type for APAF1 mutation. The primers and protocol developed for the ARMS-PCR technique work with 100% specificity and efficiency since the amplicon formations are as expected according to the genotypes. In conclusion, the mutation responsible for APAF1 was not detected in the Brazilian Holstein cattle population assessed in this prevalence study, although it is not possible to affirm that APAF1 does not occur in Brazilian Holstein animals. The tetra-primer ARMS-PCR protocol for APAF1 mutation that has been validated here may be a relatively simple and economical method to determine the animals' genotype.

Epidemiology, clinical signs, histopathology and molecular characterization of canine leproid granuloma: a retrospective study of cases from Brazil.

Thirty-eight cases of canine leproid granuloma were diagnosed between 2000 and 2008. Diagnosis was based upon clinical and histopathological findings and the presence of acid-fast bacilli in skin sections. The clinical lesions were localized predominantly on the pinnae and included papules, plaques and nodules, with or without ulceration. Boxer dogs were the breed most affected. Histopathological findings included nodular to diffuse pyogranulomatous, lymphoplasmocytic inflammatory infiltrates, with or without necrosis, localized in the dermis or subcutaneous tissue. The bacillary loading and morphology were variable among the lesions analysed. There was no significant correlation between bacterial load and histopathological pattern, dominant type of inflammatory infiltration or the amount of necrosis or giant cells. No correlation was observed between giant cells and histopathological pattern. In the majority of cases where a PCR-based assay was done, a novel mycobacterium species as the main aetiological agent was identified, as reported in previous studies.