EXPRESS: First molecular detection of Leishmania infantum in a domestic cat from Costa Rica: a case report

Sperm cryopreservation offers a long-term preservation of male genetic materials for future assisted reproductive technologies. However, dramatic changes in temperature during freezing and thawing injure sperm cells. While motility is essential for AI and membrane integrity is crucial for in vitro fertilization (IVF), sperm DNA integrity is a common index of fertilizing capability required for AI, IVF and intracytoplasmic sperm injection (ICSI). In endangered felids died unexpectedly, attempts have been made to recover as many DNA intact spermatozoa as possible from epididymis and testis to increase the opportunity to produce offspring in future. Although sperm from caudal epididymis has shown retained fertilizing capability after freezing and thawing (27.3% conception rate after unilateral intrauterine insemination), sperm recovery from the corpus epididymis has been suggested as an alternative to increase the amount of preserved genetic materials. To improve epididymal sperm quality, pre-treatment with single-layer centrifugation resulted in selection of sperm cells with intact DNA while post-thaw treatment with extracellular ATP incubation promoted the blastocyst rate. Cold storage of domestic cat testis for 7days at 4°C demonstrated <1% of sperm cells with fragmented DNA. Moreover, isolated testicular sperm cells, stored for 7days at 4°C, produced after ICSI poorer percentages of cleavage, morula and blastocyst than the fresh control. In wild felids, a death-to-necropsy time of 2hr after a jungle cat (Felis chaus) aged 10years died during anaesthesia plus another necropsy-to-sperm recovery time of 25hr has been reported to yield the post-thawed testicular sperm with 22.2% intact DNA. In summary, the chromatin structure of feline ejaculated and epididymal sperm seems to be tolerated to cold storage and cryopreservation; thus, fertilizing capability is well protected. In contrast, the cat testicular sperm DNA is generally damaged through the cryopreservation.

BackgroundThe EMERGEncy ID Net Study Group is investigating whether advanced molecular tests (AMT) increase the detection of causative agents in the CSF of patients presenting with meningoencephalitis (ME). We report findings from a pilot study using AMT on 18 CSF samples from 10 US Urban Emergency Departments. The purpose of the pilot was to compare the performance of these four AMT to established clinical laboratory methods.MethodsWe investigated four AMT: (1) BioFire FilmArray ME Panel targeting 14 causative agents; (2) an in-house target-directed next generation sequencing assay targeting 25 agents; (3) a microarray capable of detecting >2,500 agents; and (4) deep metagenomic next generation sequencing. For targeted sequencing, loci from 12 DNA-based and 13 RNA-based pathogens were amplified from the extracts by multiplex PCR. All sequencing was performed on an Illumina MiSeq using 500 cycle v2 Reagent Kits. Reads from the targeted sequencing were aligned to the 25 specific reference target sequences using Bowtie2 while metagenomics reads were processed with the taxonomic sequence classifying software Kraken. For microarray analysis, Lawrence Livermore Microbial Detection Array v2 arrays were hybridized with Cy3-labeled DNA or cDNA. Scanned images of arrays were analyzed by CLiMax 3.1.ResultsEight CSF samples had results positive for well-established causes of ME from prior testing (Table). The pilot study demonstrated none of the four AMT detected all causative agents in the eight CSF samples known to have well-established causes of ME. BioFire and targeted sequencing performed best, both detecting 6/8, metagenomics deep sequencing detected 3/8, and microarray detected 1/8.ConclusionDespite the sophistication of AMT, they cannot detect pathogens they do not target, that are present in small numbers, or that have been eliminated from the CSF by the immune response. Despite the theoretical potential for microarray and metagenomic sequencing to detect thousands of different agents, the agents probably must be present at high levels for detection.Clinical laboratory evaluationBioFire Film ArrayTargeted sequencingMicroarrayMetagenomic sequencing Cryptococcus spp.43302 Streptococcus pneumoniae 11111Enterovirus2220West Nile Virus1N/Aa000 aWest Nile Virus is not on the BioFire Panel.Disclosures All authors: No reported disclosures.

Arterial thromboembolism is more common in cats. Generally, it develops without premonitory signs. When the thrombus lodged in aortic trifurcation posterior paresis develops. This paper documents one such case and its successful management. A four-year-old male domestic short-haired cat was presented with a history of being unable to stand and walk for the past three hours. Clinical examination revealed that there was paraparesis on hind limbs, pulselessness in both the hind limbs, cold extremist and digital paws that looked cyanotic. Echocardiographic, radiographic and hemato-biochemical analyses were inconclusive. The absence of femoral pulse, pain and absence of blood flow in the Doppler blood pressure crystal and ultrasonographic exam confirmed the aortic thromboembolism. The animal was treated with buprenorphine @ 0.02 mg/kg every 6 hours, injection enoxaparin @ 1 mg/kg every 8 hours and tablet clopidogrel 75 mg total dose (1/4 -0 – 0). On the fourth day of therapy, the animal showed deep pain, improvement in the digital pad (Fig 3) and mild voluntary motor activity. On the seventh day of therapy, the animal showed significant improvement and after 10 days of therapy, the animal showed an uneventful recovery. The paper presents paraparesis due to aortic thromboembolism in a domestic cat and its management.

Roads severely affect the health of ecosystems across the globe by fragmenting and diminishing habitats, reducing population connectivity, and increasing animal mortality. Wildlife underpasses allow for increased road permeability–the ability for animals to safely cross the road. Despite growing success in other regions, little is known about underpass usage in Central America. In this study, I monitored two dry circular culverts and two unfenced tunnels with barbed wire partially blocking their entrances on Route 606 in Guacimal, Costa Rica, from 14 November to 6 December 2021 using 15 camera traps to assess which species used them to cross. Twelve species used the culverts and tunnels for a total of 108 individual crossings. The tunnels were used, in descending order, by agouti (Dasyprocta punctata), common opossum (Didelphis marsupialis), dog (Canis familiaris), nine-banded armadillo (Dasyous novemcinctus), cat (Felis catus), Norway rat (Rattus norvegicus), ocelot (Leopardus pardalis), squirrel (Sciurus variegatoides), northern tamandua (Tamandua mexicana), and coati (Nasua narica). The circular tunnel, Tunnel 1, was used more frequently and by a greater diversity of species than observed in the square tunnel, Tunnel 2. The two smaller culverts were used by common opossum (Didelphis marsupialis), cat (Felis catus), rat opossum (Micoureus alstoni), and Watson’s climbing rat (Tylomus watsoni). Culvert 2 was used more frequently; however, Culvert 1 was used by a greater diversity of species. This study highlights wildlife underpasses as a critical strategy for biological conservation in Costa Rica through improved road safety and habitat connectivity.

Lynxacarus radovskyi Tenorio, 1974, is a mite that infests domestic and wild cats. We report a case in domestic cat, Felis catus Linnaeus, 1758. The cat was remitted to the Veterinary Hospital of the Universidad Juarez Autonoma de Tabasco because it presented alopecia on the sacrum area and small yellowish white dots in its hair. During clinical evaluation, the coat was observed to be in poor condition, and alopecia was observed on the sacrum area, perineum, tail, hind legs, and inguinal area, as well as on the face to a lesser extent. Ectoparasites were observed in the hair and subsequently collected and taxonomically identified as L. radovskyi. The clinical confirmation of the presence of the mite species in Mexico can impact differential diagnosis of feline dermatological diseases in Mexico, especially when clinical signs such as poor coat condition and alopecia are present on the sacrum area, perineum, tail, hind legs, and inguinal area.

Emerging Lagochilascaris minor infections in domestic cats from Costa Rica: A zoonotic threat for the region.

Simple SummaryIn view of the current global situation due to the COVID-19 pandemic and considering the evidence that SARS-CoV-2 has the ability to infect multiple species, this work was aimed at identifying the clinical signs of SARS-CoV-2 infection in domestic and wild felids. Our results evince that the signs developed in felids show similar progression to those occurring in humans, suggesting a relationship between the viral cycle and target tissues of the virus in different species. Recently, it has been proved that SARS-CoV-2 has the ability to infect multiple species. This work was aimed at identifying the clinical signs of SARS-CoV-2 infection in domestic and wild felids. A PRISMA-based systematic review was performed on case reports on domestic and wild cats, reports on experimental infections, case reports in databases, preprints and published press releases. Descriptive statistical analysis of the data was performed. A total of 256 articles, 63 detailed official reports and 2 press articles on SARS-CoV-2 infection in domestic and wild cats were analyzed, of which 19 articles and 65 reports were finally included. In domestic cats, most cats’ infections are likely to be asymptomatic, and 46% of the reported infected animals were symptomatic and predominantly presented respiratory signs such as sneezing and coughing. In wild felines, respiratory clinical signs were most frequent, and up to 96.5% of the reported affected animals presented coughing. It is noteworthy that, to date, symptomatic animals with SARS-CoV-2 infection have been reported to belong to two different subfamilies (Phanterinae and Felinae), with up to five different felid species affected within the Felidae family. Reported results evince that the signs developed in felids show similar progression to those occurring in humans, suggesting a relationship between the viral cycle and target tissues of the virus in different species. While viral transmission to humans in contact with animal populations has not been reported, spill-back could result in the emergence of immune-escape mutants that might pose a risk to public health. Despite the clear results in the identification of the typical clinical picture of SARS-CoV-2 infection in felines, the number of detailed academic reports and papers on the subject is scarce. Therefore, further description of these cases will allow for more accurate and statistically robust clinical approaches in the future.

A culture of coexistence

This study aimed to describe the successful identification and treatment of severe hyperkalemia, cardiac arrhythmia, rhabdomyolysis, and acute kidney injury (AKI) in a domestic cat that underwent general anesthesia for abdominal exploratory surgery. The definitive underlying cause remains unknown; however, a reaction to propofol is suspected. A 6-month-old intact male domestic short-hair cat underwent general anesthesia and developed severe intraoperative rhabdomyolysis, hyperkalemia, ventricular fibrillation, and AKI during surgery despite a documented mild hypokalemia and normal creatinine before inducing anesthesia. Propofol was administered as part of the anesthetic protocol. The patient was resuscitated successfully and responded well to advanced medical intervention. The hyperkalemia and AKI were resolved within less than 24 h from surgery and rhabdomyolysis was resolved at the time of recheck 5 days later. While previously suspected in dogs, to the authors' knowledge, propofol-related infusion syndrome (PRIS) has not been reported in domestic cats. Veterinary professionals should be aware that drug-induced intraoperative rhabdomyolysis and hyperkalemia can develop unexpectedly and should remain a differential for acute cardiac arrhythmias or cardiac arrest and AKI.

Diaphragmatic hernia and congenital anomalies of the axial skeleton are alterations frequently identified in domestic cats. The objective of this case report was to present the electrocardiographic, radiographic, and tomographic findings observed in a 5-year-old neutered domestic shorthair cat, with skeletal malformations simultaneously with chronic diaphragmatic hernia. The cat lived well until he was 10-years old, when he developed progressive respiratory compromise leading to death. To our knowledge, occurrence of combined diaphragmatic hernia and congenital skeletal anomalies in domestic cats is very rare.

The advent of advanced molecular diagnostic techniques has revealed plenty of information about signaling pathways and gene regulation in cancer, as well as new inputs for the classification of cancer subtypes, diagnosis, prognosis, and prediction of response to therapy. However, in most cases we do not have single biomarkers yet and, therefore, the final diagnosis is often rendered by the combination of multiple results by means of complex algorithms, eventually leading to an increase in their costs. The problem of the costs of such tests is particularly relevant in the case of thyroid cancer (TC), because of the observed increase in the number of patients affected by thyroid nodules (TN)s, in what is considered a global pandemic. High-income countries can afford the cost of the advanced molecular tests for such a multitude of TNs, since they are covered by private insurances. People living in upper-middle, lower-middle, and especially in low-income countries, where the costs for these advanced molecular tests are supported by general taxation and out-of-pocket payments, are exposed as a personal financial burden. Immunohistochemistry in cancer management represents an extremely cost-effective method in different clinical scenarios. In the preoperative recognition of TC, the use of such method, based on Galectin-3 and others protein markers, such as HMBE1, proved to be effective in diagnosing TC in TNs indeterminate at conventional cytology (Bethesda classification III or IV), with an extremely low cost. Moreover, Galectin-3 fulfills one of the major criteria of an ideal marker, being involved in the thyroid cell transformation. Despite this evidence, Galectin-3 ThyroTest is not considered and not even mentioned in many reviews, focused on the appropriate identification of TC, as well as in studies where the cost-effectiveness of the different approaches is comparatively evaluated. The aim of this review is to emphasize the value of the Galectin-3 based immunohistochemistry as a cheap and "good enough" method in the preoperative diagnosis of TC especially in, but not limited to, low-middle income countries.

Many mammalian species have recently been successfully cloned using somatic cells. We have produced cloned kittens by somatic cell nuclear transfer (SCNT). The cloning of mammals by SCNT requires epigenetic reprogramming of the differentiated state of donor cells. We recently observed that feline catus satellite DNA regions exhibit DNA methylation in the donor and somatic cells of a cloned cat. The cause of variation could lie in alterations occurring at different steps of the cloning procedure or incomplete reestablishment of DNA methylation patterns. This study was conducted to determine the methylation status of the feline catus satellite DNA region in the somatic cells of cloned cats and domestic normal cats by using a bisulfite sequencing method. Satellite sequences are heavily methylated in somatic cell chromosomes and are associated with dense heterochromatin where bundles of silent genes are wrapped up together. For the analysis of the cat satellite sequence, a 400-bp segment of the satellite genomic region, which has highly conserved 22 CpG sites, was amplified by PCR from bisulfite-treated genomic DNA, and the resulting PCR products were individually cloned and sequenced. For methylation analysis, genomic DNA was first isolated from somatic and placental cells of cloned and domestic normal cats. The DNA methylation status of the satellite DNA region was not significantly different among donor (using NT) cells (70.6, 77.0, and 79.1% in Donors A, B, and C, respectively), somatic cells of cloned cats (88.9, 82.2, and 85.9% in cloned A-1, 2, and 3; 78.0% in cloned B-1; 88.1, 78.9, 80.3, 83.2, 74.6, and 82.3% in cloned C-1, 2, 3, 4, 5, and 6, all respectively), and domestic cats (88.0, 81.6, and 81.8% in NC 1, 2, and 3, respectively). Also, the DNA methylation status of satellite DNA regions in the placenta was significantly different between cloned cats (80.5, 76.7, and 76.8% in cloned C-2, 5, and 7, respectively) and normal domestic cats (64.2, 66.7, and 74.9% in NC 1, 2, and 3, respectively). In conclusion, all of the somatic cells were highly methylated. The methylation status of the somatic cells was not different among groups, but that of placental cells was significantly different. This work was supported by KOSEF (Grant ? M10525010001-05N2501-00110).

The European Union (EU) has recently implemented annual surveys in its member States, in order to prevent further introductions of the harmful organism Xylella fastidiosa, within its territory, upon an outbreak in southern Italy where this quarantine pathogen has been identified as the putative agent of the Olive quick decline syndrome (OQDS), (Boscia, 2014; EFSA, 2015). X. fastidiosa affects several economically important plants reported mainly from the Americas, including grapevine, stone fruits, coffee and citrus and is the causal agent of leaf scorch disease of many ornamentals, shade and forest trees. X. fastidiosa subsp. pauca has also been associated in Costa Rica with a coffee disease known as “crespera”, characterized by irregular leaf growth, atypical curling of the leaf margins, reduced leaf size, shortening of the internodes and severe chlorotic mosaic of the leaves (Montero-Astua et al., 2008). X. fastidiosa subsp. pauca and X. fastidiosa subsp. fastidiosa were recently intercepted and diagnosed in imported Coffea arabica plants from Equador and C. canephora from Mexico. These plant consignements have been destroyed (EPPO, 2012; Legendre et al., 2014). X. fastidiosa was presumptively diagnosed during a survey performed on ornamental C. arabica plants imported from Costa Rica and Honduras into the Netherlands in the autumn of 2014, as part of the recent EU implementing decision (Commission Implementing Decision 2014/87/EU). These plants showed either mild leaf scorch symptoms, or a range of “crespera”- like symptoms, or no symptoms. This presumptive diagnosis of X. fastidiosa was in agreement with EPPO’s (2004) recommendations and was carried out with molecular and serological methods. Total genomic DNA was extracted from leaf veins and petioles of symptomatic and latently infected C. arabica plants with the QuickPic SML plant DNA kit (Bio-Nobile, Finland), using a KingFisher isolation robot (Thermo Scientific, The Netherlands). Real-time PCR on purified genomic DNA using Premix ExTaq (TaKaRa, France) gave positive reactions using the X. fastidiosa gene-specific primers targeted to 16S rRNA-processing gene rimM (Harper et al., 2010, erratum 2013). Conventional PCR using GoTaq polymerase (Promega, The Netherlands) and primer set RST 31/RST 33 (Minsavage et al., 1994) confirmed the presumptive presence of X. fastidiosa in these samples. Sequence analysis of the ca. 700 bp amplicon of the RNA polymerase sigma 70 factor showed 96 to 100% identity with the comparable genomic regions of X. fastidiosa strain sequences from GenBank, which indicated the presence of X. fastidiosa DNA in the plant extract. Remarkably, three different sequences with 97-98% identity among each other (GenBank accession Nos. KP769842-KP769844) were recovered from imported C. arabica plants. Two of these sequences were related to C. arabica plants imported from Costa Rica, whereas the third sequence was only present in plants imported from Honduras. Extracts from leaf veins and petioles were exposed to indirect immunofluorescence (IF), using a commercially available antiserum (Loewe 07319, Germany). IF ascertained the presence of X. fastidiosa, thus confirming the molecular tests. Final diagnosis, based on the recovery of the C. arabica infecting isolate(s) of X. fastidiosa in axenic culture, for evaluation of its pathogenicity and subspecies identification is currently in progress.

An unusual case of Brugia sp. infection in a dog from Alberta, Canada

Hepatic trematode Platynosomum sp. (Dicrocoeliidae) from a domestic cat in Colombo, Sri Lanka: Case report and molecular identification