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Within the field of clinical research, reports on the stability of avian serum amyloid A (SAA) under varying storage conditions are currently scarce. In this study, avian plasma samples were evaluated for SAA, a major acute-phase protein in birds, to assess how varying storage periods and repeated freeze-thaw cycles impact the stability of SAA in the frozen samples. Seven plasma samples from two species and six plasma samples from three species stored at ‒20 °C were used to evaluate the time and temperature effects accordingly. A chicken-specific SAA ELISA kit was used for the measurements. Statistical analysis was performed using SPSS, and the Kruskal-Wallis test and Spearman's correlation coefficient were applied, with statistical significance set at P < 0.05. The SAA concentrations measured daily for 30 days showed no statistically significant differences over time. Freezing-thawing was repeated five times, and a significant negative relationship was confirmed over the cycles (r=‒0.8857, P < 0.05). Although no significance was observed between a decreased concentration and the number of cycles, a decrease in the concentration of > 10% was observed after the fourth cycle in four out of six samples. However, one to three freeze-thaw cycles did not result in a significant decline. Taken together, the results indicate that a negative correlation existed between the mean concentration and multiple freeze-thaw cycles, indicating that these should be avoided where possible., (© 2024. The Author(s).)

Influenza A virus (IAV) is an important pathogen in Brazilian swine herds, and monitoring the viral circulation is essential to control and reduce the transmission. Surveillance programs for IAV are often based on individual piglets level sampling, making the evaluation of the available diagnostic tools crucial to assessing IAV circulation in herds. Thus, two sample collection methodologies were compared in pig herds in southern Brazil to detect IAV by RT-qPCR: nasal swab (NS) and nasal wipe (NW). A Bayesian latent class model (BLCM) was set for two tests and two populations. The NW and NS used are more specific (higher than 95 % for both) than sensitive. The sensitivity for NW was lower than the NS, 84.14 % (70 % - 95 %; posterior probability interval (PPI): 95 %) and 87.15 % (73 % - 97 %; PPI: 95 %), respectively, and the specificity was 95 % (90 % - 99 %; PPI: 95 %) and 99 % (96 % - 100 %; PPI: 95 %), respectively. Although the wipe sample collection loses both sensitivity and specificity compared with nasal swab, differences in test performance were very limited and PPIs largely overlapped. Therefore NW can also be considered a valuable tool. The decision about the use of both techniques should be based on the trade-off between their performance limitations and feasibility in routine monitoring., Competing Interests: Declaration of Competing Interest none., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Consistent information and standardization procedures regarding the time of storage for frozen samples and the effects of storage time on enzyme activity are still missing in the literature. Thus, we evaluated the effects of different storage temperatures (-20 °C and - 80 °C), three repetitive freeze/thaw cycles, and 24-h mimic transportation on the activities of PON1 (paraoxonase and arylesterase), enzymes involved in the protection and detoxification processes of reactive molecules. PON1 enzymes' activity was validated on serum and heparinized plasma in horses. The results revealed that conditions and time of storage of blood samples for PON1 analyses altered the activities of both enzymes in both sample types, evidencing that these conditions can lead to protein degradation or general alteration. Specifically, paraoxonase and arylesterase activities significantly decreased among storage temperatures, with major effects detected at -20 °C. The repeated freeze/thaw cycles at -20 °C and 24-h mimic transport conditions also generated an expected degradation of the arylesterase in both serum and heparinized plasma while freeze/thaw cycles at -80 °C caused an increase of both arylesterase and paraoxonase activities on both sample types. In general, similar enzyme responses were detected between serum and heparinized plasma., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The isolation of Campylobacter fetus subsp. venerealis (Cfv) from clinical samples is the gold standard for confirming cases of bovine genital campylobacteriosis, an important cause of infertility in cattle and a potential public health concern. Furthermore, isolation is also necessary for the development of autologous vaccines, characterization of strains for antimicrobial susceptibility patterns, etc. Nevertheless, the sensitivity of culture methods is usually low, and there is no standardized protocol to maximize the recovery of Cfv from clinical samples. The aim of the current study is to design a protocol for the culture of Cfv from preputial samples by evaluating the combination of different transport, enrichment and culture media considering the impact of certain factors (time between collection and enrichment, temperature, and use of filters). The use of modified Lander's transport medium and storing the sample for 24 h at 21 ± 2 °C led to the highest recovery of Cfv CFUs. In contrast, the storage of the samples during 24-48 h in PBS and Thomann rarely allowed the recovery of Cfv regardless of the temperature. The enrichment medium yielding the best results was Preston (significantly higher recovery than Brucella medium), while Cfv could not be isolated with Bolton. Regarding our diagnostic assay (using Lander as transport medium and Preston as enrichment medium), the best protocol in terms of maximizing Cfv recovery as well as limiting contaminations is to culture the samples in i) solid media Preston or Skirrow, and ii) using 0.65 μm filters and incubating plates at 37 °C in microaerophilic conditions., (© 2024. The Author(s).)

Objectives: To evaluate the prevalence of bacterial presence in free-catch urine samples preceded by either a standardised prepped ("clean-catch") protocol versus unprepped (non-cleaned) voiding., Materials and Methods: The study was a single-centre prospective single-blinded randomised controlled trial. Urine samples were obtained from 100 client-owned dogs presenting for routine evaluation. Dogs were randomly assigned to either the prepped group (preputial or peri-vulvar area cleaned with sterile saline before collection) or the unprepped group (no preliminary cleansing) stratified by sex. Urinalysis and urine culture (blood and MacConkey agar) were performed on all samples. Significant bacterial presence on urine culture was defined as >10 4  colony forming units (CFU)/mL., Results: There were no statistically significant associations between prepped versus unprepped collection method or sex with a urinalysis positive for bacteriuria. However, on culture, significant bacterial growth was almost five times more likely to be associated with males relative to females (odds ratio 4.59, 95% confidence interval 1.61 to 13.10). The probability of finding a positive culture was not statistically associated with prep method (odds ratio 1.43, 95% confidence interval 0.50 to 4.08)., Clinical Significance: For the majority of dogs without clinical signs of urinary tract infection, free-catch urine collection does not result in significant bacteriuria found on analysis or culture. The presence of bacteria found in free-catch samples may be secondary to sample contamination or subclinical bacteriuria. Sample contamination or subclinical bacteriuria may be more prevalent in male dogs., (© 2024 British Small Animal Veterinary Association.)

Introduction: Silica-sprayed tubes (SSTs) are often used to transport synovial fluid samples in equine practice. They promote the coagulation of the sample. The objective of the study is to evaluate the effect of SST on bacterial culture., Materials and Methods: The study was divided into two parts: sterile saline (Part A) and synovial fluid (Part B). Four common bacteria associated with equine synovial sepsis were used: Streptococcus pyogenes, Escherichia coli, Staphylococcus aureus and methicillin-resistant S. aureus (MRSA). Three collection tubes were used: STT, plain (no-additives) and brain and heart infusion (BHI) broth. Bacteria were cultured in horse blood agar plates for 48 h. Outcome variables were negative culture, positive culture and total number of colony-forming units (CFUs). Statistical analysis was performed using Mann-Whitney U test, and significance was set at p < 0.05., Results: The total number of agar plates read was 1557 (779 saline; 778 synovial fluid). Total negative cultures were 25/779 on saline and 3/778 on synovial fluid. In broth, maximum growth CFU was achieved after 8 h for both saline and synovial fluid for all bacteria. S. pyogenesand E. coli produced a significantly lower number of CFU when in SST compared to plain or broth after 4 h, whereas S. aureus (American Type Culture Collection [ATCC] and MRSA) only after 24 h., Discussion: Silica-containing tubes reduced bacterial proliferation, whereas the use of a BHI broth provided the highest bacterial load in the sample. The use of SST may have a negative effect on bacterial proliferation in samples obtained from clinical cases., (© 2024 The Author(s). Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

Mycoplasma bovis is a bacterial pathogen endemic to cattle. In the early 2000s, M. bovis emerged as a cause of respiratory disease in American bison (Bison bison), causing significant morbidity and mortality. Bison herds that experience an outbreak of M. bovis are at higher risk for subsequent outbreaks, suggesting that chronic, subclinical infections can be established. Antemortem testing is therefore crucial to disease management; however, the precise sampling method to maximize detection of M. bovis in bison is unknown. We evaluated two sample types-superficial nasal swabs and deep nasopharyngeal swabs-collected from apparently healthy or symptomatic bison from January 2021 through December 2022. We used real-time PCR to detect M. bovis in 76/938 bison (8.1%) from 11 herds. For bison testing positive on at least one swab type, M. bovis was detected in 63/76 (82.8%) deep nasopharyngeal swabs and 29/73 (38.1%) superficial nasal swabs. Agreement between swabs for positive bison was 21% (n=16, kappa coefficient 0.319). We conclude that deep nasopharyngeal swabbing is more sensitive than superficial nasal swabbing for detection of M. bovis in bison and that low agreement between methods may be related to stage of infection. We further tested pooled samples by PCR and found that pooling of up to five samples can be effective to increase throughput and minimize costs. Management of wild bison relies on the ability to relocate animals to maintain gene flow and healthy populations. Sensitive and specific diagnostic tests are needed to inform decisions and minimize risk of transmission, especially from subclinical carriers. This study provides valuable insight that will inform best practices for M. bovis testing, thereby supporting the conservation of bison as healthy wildlife, which in turn promotes ecological restoration, safeguards cultural practices of Tribal Nations, and upholds the bison as a unique American icon., (© Wildlife Disease Association 2024.)

The aim of the present study was to validate methods of stool sample conservation for the egg hatch test (EHT). This study involved the use of a bovine naturally infected predominantly by Cooperia spp. and one equine naturally infected predominantly by cyathostomins characterized as susceptible to benzimidazoles in the EHT. Fecal samples were submitted to three treatments: aerobic methods (anaerobic storage in plastic bottles, anaerobic storage in vacuum-sealed bags or aerobic storage in plastic bags), under two temperature conditions (room temperature and refrigeration) analyzed at four different assessment times (48, 72, 96 and 120 h). As the standard test, an assay was also performed within 3 h. The tests were performed in triplicate for each drug concentration and with three experimental repetitions at one-week intervals. Two criteria were used for the storage methods: hatchability in the negative control group and sensitivity of the eggs to thiabendazole, comparing the EC50 and 95% confidence interval for each treatment to those of the standard test and the other repetitions. Bovine samples can be stored for up to 96 h and refrigerated vacuum storage can be used, ensuring hatchability of the negative control and sensitivity of the eggs to thiabendazole. For equine samples, no forms of storage were indicated due to the variation among the repetitions and the reduction in the sensitivity of the eggs to thiabendazole, which could result in a false positive detection of resistance., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Mariana Green de Freitas reports financial support was provided by CAPES., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Ornithologic study skins are specimens of avian skins that have been preserved by drying after removing the viscera and muscle. Because of the high value of study skins for scientific studies, specimens are shared among researchers. There is concern that study skins might be contaminated with high-consequence diseases such as highly pathogenic avian influenza virus (HPAIV) or Newcastle disease virus (NDV). To mitigate risk, thermal or chemical treatment of study skins may be required before transfer; however, such treatments might damage the specimens. Therefore, a study was conducted to evaluate the duration of infectivity of HPAIV and NDV in study skins prepared from infected chickens (Gallus gallus). Study skins were prepared from 10 chickens infected with each virus. Skin and feather pulp samples were taken at the time of study skin preparation to establish starting titers. Mean starting titers in the skin was 4.2 log10 and 5.1 log10 50% egg infectious doses (EID50) for HPAIV and NDV groups respectively, and were 6.7 log10 EID50 for HPAIV, and 6.4 log10 EID50 for NDV in feather pulp. Samples were collected at 2 and 4 wk of drying to quantify viable virus. At 2 wk, fewer samples had detectable virus and mean titers were 1.8 log10 (skin) and 2.1 log10 (feathers) EID50 for HPAIV, and 1.7 log10 (skin) and 3.5 log10 (feathers) EID50 for NDV. At 4 wk viable virus could not be detected in either tissue type., (© Wildlife Disease Association 2024.)

Objective: To assess the temperature stability of an autodefrost freezer commonly used in veterinary practices, whether the use of a Styrofoam cooler within the freezer provides temperature stability, and the ease of use of a remote monitoring system for the notification of temperature elevations., Animals: None., Methods: Temperature in the freezer and 2 Styrofoam coolers were assessed with remote monitoring thermometers every 15 minutes. Temperature values were monitored from October 11 to December 18, 2023 (for a 68-day period). Data analysis focused on temperatures for the freezer exceeding 0 °C and the elevations in temperatures within the coolers relative to the freezer., Results: The freezer had an increase in temperature approximately every 16 hours. Over 68 days, the freezer had a temperature greater than 0 °C 27 times, representing 26 separate elevations. The Styrofoam coolers within the freezer never registered a temperature higher than -5 °C. Elevations in temperature within the freezer were larger in magnitude than temperature elevations within the coolers, which showed smaller-magnitude changes in temperature., Clinical Relevance: The temperature stability provided by the Styrofoam cooler would avoid potential freeze-thaw cycles of any stored biological samples. Additionally, the remote temperature monitoring system is easy to install and monitor, providing peace of mind to practice management.

Background: Infectious bovine rhinotracheitis (IBR), caused by Bovine alphaherpesvirus-1 (BoAHV-1), is an acute, highly contagious disease primarily characterized by respiratory tract lesions in infected cattle. Due to its severe pathological damage and extensive transmission, it results in significant economic losses in the cattle industry. Accurate detection of BoAHV-1 is of paramount importance. In this study, we developed a real-time fluorescent quantitative PCR detection method for detecting BoAHV-1 infections. Utilizing this method, we tested clinical samples and successfully identified and isolated a strain of BoAHV-1.1 from positive samples. Subsequently, we conducted a genetic evolution analysis on the isolate strain's gC, TK, gG, gD, and gE genes., Results: The study developed a real-time quantitative PCR detection method using SYBR Green II, achieving a detection limit of 7.8 × 10 1 DNA copies/μL. Specificity and repeatability analyses demonstrated no cross-reactivity with other related pathogens, highlighting excellent repeatability. Using this method, 15 out of 86 clinical nasal swab samples from cattle were found to be positive (17.44%), which was higher than the results obtained from conventional PCR detection (13.95%, 12/86). The homology analysis and phylogenetic tree analysis of the gC, TK, gG, gD, and gE genes of the isolated strain indicate that the JL5 strain shares high homology with the BoAHV-1.1 reference strains. Amino acid sequence analysis revealed that gC, gE, and gG each had two amino acid mutations, while the TK gene had one synonymous mutation and one H to Y mutation, with no amino acid mutations observed in the gD gene. Phylogenetic tree analysis indicated that the JL5 strain belongs to the BoAHV-1.1 genotype and is closely related to American strains such as C33, C14, and C28., Conclusions: The established real-time fluorescent quantitative PCR detection method exhibits good repeatability, specificity, and sensitivity. Furthermore, genetic evolution analysis of the isolated BoAHV-1 JL-5 strain indicates that it belongs to the BoAHV-1.1 subtype. These findings provide a foundation and data for the detection, prevention, and control Infectious Bovine Rhinotracheitis., (© 2024. The Author(s).)

The present study was designed to evaluate equine blastocyst re-expansion rate, quality, and sex following perforation of the blastocoel, collection of blastocoel fluid (BF), and PCR amplification of free DNA. Experiment 1 tested the feasibility of the BF sample collection with a hand-held, small-gauged needle (26g) and subsequent PCR amplification of the TSP-Y gene for males and AMEL-Y gene for males and AMEL-X gene for females. Experiment 2 tested the application of the technique. Equine embryos were collected via uterine flushes 8d after ovulation. Thereafter, embryos (n = 19) were initially assessed and transferred to a 50 μL droplet of holding medium in which the blastocoel was manually perforated as in Experiment 1. Within 1 min of detecting a diameter decrease or collapse, the entire volume of each droplet of medium was collected and stored at -20 °C until PCR. In Experiment 1, amplification of the TSP-Y gene was positive for males at 60% (9/15) and negative for females at 40% (6/15). In Experiment 2, a total of 42 embryos were randomly assigned to a collapsed embryo (CE) or intact embryo (IE) groups and stored at room temperature (RT, 25 °C) or cold temperature (CT, 5 °C) for 24h as follows: 1) CERT, n = 11; 2) CECT n = 11; 3) IERT, n = 10; and 4) IECT, n = 10. After 24h, embryo diameter and quality were reassessed. For all collapsed embryos (n = 19), blastocoel fluid was subjected to double PCR amplification of the TSPY gene with blood from adult male and female horses as controls. Positive gene amplification indicated 57.9% (11/19) of embryos were male and negative amplification indicated 31.6% (6/19) of embryos were female. Relative to the least diameter (0%) after perforation of collapsed embryos or fullest diameter (100%) of intact embryos at T0, percentage change in diameter and quality Grade 1 or 2 embryos after 24h of storage for all groups were, respectively: 31.2% and 54% for CERT group, 28.2% and 0% for CECT group, 25.9% and 100% for IERT group, 4.3% and 80% for IECT group, respectively. Thus, needle-induced leakage and collapse of the blastocoel at T0 resulted in a high rate of blastocyst re-expansion (69%) with many embryos (54%) achieving good quality at T24 with potential for transfer as either male or female embryos. For both collapsed and intact embryos, it was observed that storage for 24h at room temperature (25 °C) was associated with improved embryo growth and morphological quality compared to storage at cold temperature (5 °C)., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Inc. All rights reserved.)

This article is intended to highlight toxicosis-associated pathology in horses that might be observed by a clinician in the living animal and at gross necropsy. When the clinician is aware of these pathologic changes (particularly when coupled with a suggestive environmental or herd history), then collaboration with a diagnostic laboratory can begin to help identify specific toxicants. Proper sampling and communication with the diagnostic laboratory will vastly improve the likelihood of a specific diagnosis; postmortem sampling and specimen submission are reviewed in the last section of this article., Competing Interests: Disclosure None., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Objective: To identify the safest, most efficient method for hair sample collection from companion dogs among clippers, scissors, and razors and to validate obtained samples with cortisol concentration analysis., Animals: 25 healthy, privately owned dogs., Methods: 2 hair samples were collected from each dog's ischiatic region with different implements (scissors, razors, or clippers). The collecting clinician completed a Hair Collection Questionnaire (HCQ) for each sample that compared subjective sample quality, time of collection, restraint needed, and patient experience. Each sample was evaluated by cortisol enzyme immunoassay., Results: Clippers had higher overall HCQ scores than scissors, and scissors had higher HCQ scores than razors. Collection was faster for clippers than scissors, and scissors were faster than razors. There were no differences in sample quality between scissors and clippers, and sample quality was lower with razors. There was no difference in restraint needed or patient experience. Collection of long hair had higher HCQ scores than collection of medium and short hair. Collection of hair from dogs with an undercoat had higher HCQ scores than collection of hair from dogs without an undercoat. Dog size had no effect on HCQ score. Hair cortisol concentration did not vary between scissors or clippers (P = .111). Hair color and age did not affect hair cortisol concentration (P = .966 and P = .676, respectively)., Clinical Relevance: Clippers are recommended for hair sample collection from companion dogs. Scissors are an adequate alternative.

Objectives: To evaluate the effects of a cell-free supernatant from Lactococcus lactis (CFSM) on performance and diarrhearelated parameters and the presence of F4+ enterotoxigenic E. coli (ETEC) in piglets during post-weaning, and to evaluate the in vitro effect of the CFSM on faeG gene expression in an E. coli F4+., Animals and Procedure: In 3 trials with 90 piglets per trial, pigs were assigned to receive a placebo or 1 of 2 CFSM treatments and observed for diarrhea and performance. Fecal swabs were taken to determine the presence of ETEC. Quantitative RT-PCR was used to assess faeG gene expression in E. coli 21259 after treatment with CFSM at 50 mg/mL., Results: The CFSM administered for 14 d at a dose of 24 mg/kg BW (2X) reduced diarrhea-related parameters compared to the placebo. Quantitative RT-PCR showed that, in E. coli 21259 treated with CFSM at 50 mg/mL, expression of the faeG gene was significantly repressed ( P < 0.0001) relative to that in the untreated control., Conclusion: The evaluated CFSM reduced the frequency and prevalence of diarrhea in a field situation. The in vitro treatment had an inhibitory effect on the expression of the faeG gene in F4+ E. coli 21259., (Copyright and/or publishing rights held by the Canadian Veterinary Medical Association.)

Background: Researchers and pig veterinarians are interested in assessing pigs' fecal consistency. This study developed a standardized protocol and scale for the cotton swab method, which is a way of assessing the fecal consistency in pigs. The accuracy of the cotton swab method was evaluated in weaned pigs using fecal dry-matter analysis as a golden standard. The study also proposed fecal dry-matter percentage thresholds for the categorization of fecal consistency on a four-point scale., Results: The thresholds of 10.3%, 16.6%, and 21.9% fecal dry-matter were suggested for categorization of the consistency of fecal samples on a four-point scale. The accuracy of the cotton swab method was high. The agreement to the four-point fecal consistency score derived from the fecal dry-matter percentage was almost perfect (weighted Gwet's agreement coefficient = 0.87 [95% confidence interval: 0.84; 0.91]). The cotton swab method had a sensitivity of 85.0% (95% confidence interval: 76.5; 91.4) and a specificity of 95.2% (95% confidence interval: 92.0; 97.3) when used to diagnose whether pigs had diarrhea or not. For non-diarrheic pigs, the method almost always (n = 287/289) required less handling than the collection of a fecal sample by digital rectal manipulation., Conclusion: The cotton swab method is an accurate way to assess fecal consistency in pigs, both on a four-point scale and as a dichotomous diarrhea score. The method is quick to perform and less invasive than methods relying on the collection of fecal samples. New fecal dry-matter thresholds between feces of different consistencies were proposed., (© 2024. The Author(s).)

Background: The Platelet function analyzer-200 (PFA-200) can determine the effect of clopidogrel in cats, but analysis traditionally must be performed at point-of-care (POC). The ability to ship samples of blood to a laboratory would allow widespread access., Objectives: We aimed to validate the shipping of blood samples for PFA-200 analysis in cats to determine the effect of clopidogrel., Methods: Twenty healthy cats and 10 cats receiving clopidogrel were recruited. Blood was collected from cats and aliquoted into two samples, one was analyzed at POC within 2 hours using the PFA-200, and the other was packaged and transported to a location 4 km away, stored, and transported back to the lab for analysis the following day., Results: Median closure times (CTs) with the collagen/adenosine diphosphate (COL/ADP) cartridge in healthy cats were 51.5 seconds (POC) and 78.8 seconds (shipped), which were significantly different (P < 0.001), and for cats on clopidogrel, median CTs were 147.5 seconds (POC) and 190 seconds (shipped), which were not significantly different (P = 0.131). Median CTs with the P2Y cartridge in healthy cats were 50.5 seconds (POC) and 64.9 seconds (shipped), which were significantly different (P = 0.03), and in cats receiving clopidogrel, median CTs were 300 seconds (POC) and 300 seconds (shipped) which were not significantly different (P = 1.000). Reference intervals for CTs differed for COL/ADP at POC (19.8-89.7 seconds) and shipped (50.9-161.6 seconds) and for P2Y at POC (35.5-118.8 seconds) and shipped (35.1-108.9 seconds). Receiver operating characteristics showed similar areas under the curve (AUCROCs) regarding the effect of clopidogrel for COL/ADP at POC (0.994 seconds) and shipped (0.932) and for P2Y at POC (0.904 seconds) and shipped (0.975 seconds). When classifying for the presence of clopidogrel effects, Cohen's Kappa was 0.62 for COL/ADP and 1.00 for P2Y., Conclusions: Shipping blood samples for PFA analysis are feasible with similar performance to POC analyses for determining the effect of clopidogrel in cats., (© 2023 The Authors. Veterinary Clinical Pathology published by Wiley Periodicals LLC on behalf of the American Society for Veterinary Clinical Pathology.)

Background: Silica from plastic red top sample collection tubes and barium cause recognized artifacts in slide preparations for microscopic examination., Objectives: The objectives of this study were to evaluate and directly compare the microscopic appearance of silica and barium particles and various slide preparation techniques (e.g., use of coverslips, oil immersion, and different stains). A secondary objective of this study was to evaluate the effects of silica particles on cellular morphology after mechanical trauma with cytocentrifugation., Methods: Fluid samples (deionized water, pleural effusion, peritoneal effusion, cerebrospinal fluid, and urine) were collected and evaluated in silica- and non-silica-containing tubes. Barium was added to silica and non-silica samples. Direct and cytocentrifuge preparations were compared to evaluate the effect of silica particles on cellular morphology. Preparations were stained with Wright-Giemsa, rhodizonic acid disodium salt, Alizarin Red, Grocott's methenamine silver, and Prussian blue., Results: Silica and barium particles were identifiable via light microscopy with and without polarized light, although silica particles diminished with immersion oil. Barium particles retained their structure and diminished less under oil. Cytoseal mounting medium for coverslip placement resulted in diminished refractility of silica and some barium particles. Silica particles with mechanical interaction during cytocentrifugation resulted in disrupted cellular morphology with many lysed cells. Silica and barium particles were negative for all special stains tested., Conclusions: Silica from plastic red top tubes adversely affects cell morphology in cytocentrifuge preparations, potentially affecting manual differential cell counts and compromising diagnostic interpretation. Samples intended for microscopic evaluation should not be collected in silica-containing tubes., (© 2023 American Society for Veterinary Clinical Pathology.)

To compare the sensitivity of conjunctival swab (CS) and conventional samples (blood, spleen, liver, lymphoid and cutaneous tissue) in the diagnosis of canine visceral leishmaniasis (CVL) by polymerase chain reaction (PCR), a systematic review and meta-analysis was carried out using PubMed, Science Direct, Scopus, Web of Science, VHL/BVS (Virtual Health Library), CAPES, and Scielo databases. Articles published from 2002 to 2022 were considered and the review was updated in Jul 2023. From the total of 371 identified studies, 8 met all the eligibility criteria and were included in this review. Data from 658 CVL-positive dogs and 2541 PCR results were considered. Using a random effect model, data on the sensitivity of the test was compared between intervention (CS samples) and comparison (all the other samples) groups. Overall, the use of CS in the PCR diagnosis of CVL produced 12% higher sensitivity (p=0.013) in the test than all the other samples in combination. The animals' clinical condition did not influence (p>0.142) this overall result. However, when CS was individually compared to each of the conventional samples, the consistent result was observed (p=0.012) only in the CS versus bone marrow comparison. Given their rapid acquisition, minimal invasiveness, and lower cost relative to conventional samples, CS samples present a promising alternative for the molecular diagnosis of CVL.

Plasma electrophoresis is an ancillary diagnostic tool in avian medicine, with agarose gel electrophoresis (AGE) and capillary zone electrophoresis (CZE) being the most common techniques. Frozen samples can be used for quantitative studies or comparative diagnostic purposes, but stability of avian plasma proteins under freezing is poorly described. To evaluate the influence of plasma freezing on electrophoretograms in white storks ( Ciconia ciconia ), heparin blood was sampled from 30 individuals during annual health examinations. Plasma samples were obtained after centrifugation of fresh samples and divided into aliquots. Both AGE and CZE were performed on fresh aliquots. The remaining aliquots were frozen at -20°C (-4°F) or -180°C (-292°F) and thawed following different protocols: 1 freeze/thaw cycle after 6 months at -20°C; 1, 2, 4, and 7 cycles over 12 months at -20°C; and 1 cycle after 18 months at -180°C. For both techniques, electrophoretic profiles obtained from these thawed aliquots were compared to fresh electrophoretograms. Quantitatively, significant differences ( P < 0.05) in most fractions were seen from 6 months postfreezing at -20°C for both techniques. Fewer statistically significant differences were observed after 18 months under cryogenic preservation (-180°C). Qualitatively, AGE provided more repeatable and stable results than CZE over time on samples stored at -20°C, and electrophoretograms were stable after 18 months of cryogenic storage. An electromigration distortion associated with freezing was seen with CZE only. Plasma samples stored in a conventional freezer (-20°C) should not be compared to fresh plasma. For quantitative studies, cryogenic storage should be privileged.

Background: This study aimed to identify complications associated with cerebrospinal fluid (CSF) collection in dogs., Methods: This was a prospective, observational multicentre study using data collected from 102 dogs undergoing CSF collection for the investigation of neurological disease. CSF was collected from the cerebellomedullary cistern (CMC), lumbar subarachnoid space (LSAS) or both sites. Pre-, intra- and postprocedural data were collected. Descriptive statistics were performed to outline complications associated with CSF collection., Results: CSF sampling was attempted on 108 occasions, and CSF was acquired on 100 occasions (92.6%). Collection from the CMC was more likely to be successful than that from the LSAS. No dogs exhibited neurologic deterioration following CSF collection. There was no significant difference between pre- and post-CSF collection short-form Glasgow composite measure pain scores in ambulatory dogs (p = 0.13)., Limitations: The scarcity of complications limited the ability to quantify the incidence of some potential complications reported elsewhere., Conclusions: Our results may be used to inform clinicians and owners that CSF sampling is associated with a low frequency of complications when performed by trained personnel., (© 2023 The Authors. Veterinary Record published by John Wiley & Sons Ltd on behalf of British Veterinary Association.)

In a traditional blood level bioequivalence (BE) study, every subject provides drug concentrations at each blood sampling time. However, this approach is not suitable for animals whose blood volume limits or prohibits multiple sample collections. In our previous research, we presented an approach that can be applied to studies using a destructive sampling design where each animal provides only 1 blood sample that is then incorporated into a composite profile. Another situation we sometimes face is that of when the animals can contribute more than one sample but are still limited in the number of blood draws (e.g., 3) such that a complete profile per animal is not feasible. Unlike the destructive sampling situation, we cannot combine all blood samples into a single "composite" profile and ignore the correlation of values obtained from the same subject. To avoid the complexities associated with needing to include a covariance component among experimental units into the statistical model, we propose an approach whereby study subjects are randomly assigned to housing unit (e.g., cage or pen) and then randomly assigned to a sampling schedule within each housing unit. In doing so, housing unit rather than the individual subject serves as the experimental unit. This article provides an assessment of this alternative approach to assess product BE when only a limited number of samples can be obtained per study subject., (Published 2023. This article is a U.S. Government work and is in the public domain in the USA.)

Amphibians are susceptible to a multitude of skin disorders, many of which can appear grossly similar. The most common clinical presentations include hyperemia, discoloration, dermal mass, ulceration, and necrosis. Many amphibian skin diseases are related to captive husbandry. The diagnostic process starts with environmental evaluations, a full history, physical examination and sampling for direct observation, histology, polymerase chain reaction testing, and bacterial and fungal culture. This review emphasizes the main conditions encountered in amphibian dermatology., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Creatinine is a urinary marker used widely in ruminant's experimental trials. However, despite its great importance no data were found in the literature about the best way to store bovine urine samples. In the sheep urine, was observed an increase in the urinary concentration of creatinine when it was stored acidified (pH 2.5 to 3.5) at a temperature of 28 to 39 °C for 150 days of storage. Nevertheless, urine should be stored acidified (pH below 3) to avoid purine derivative degradation, So, aimed to evaluate creatinine recovery in bovine urine as a function of storage time and temperature. A total of 25 animals' urine (10 Nellore cattle and 15 Holstein cattle) were collected. The urine (40 mL) was diluted in 160 mL of distilled water and its pH was corrected to a value lower than 3 using sulfuric acid drops. A sample of the diluted urine was analyzed to obtain the creatinine concentration reference value on the collection day. The remaining urine was fractionated and preserved at room temperature, cooled (4 °C) or frozen (-20 °C and -40 °C). In the urine of five Holstein cattle was added creatine solutions (20, 40 and 60 mg/dL) to evaluate the creatine to creatinine conservation. These urine samples were analyzed on different days after collection (1, 3, 7, 15, 30 and 45 days). The urine without any added creatine was analyzed on Days 1, 3, 7, 10, 15, 30, 45, 60, 90, 120, and 150 of storage. The addition of creatine in the urine caused an increase in the creatinine concentration (P < 0.05) after 30 days of storage at room temperature and under refrigeration (4 °C). In frozen samples, there was no change in creatinine concentration (P > 0.05). However, creatinine recovery was constant (P > 0.05) until day 15 of storage, regardless of the temperature used, when creatine was not added. After 30 days of storage, an effect of time and/or temperature was observed on creatinine recovery (P < 0.05). Urine samples can be stored at any temperature for up to 15 days after collection to estimate the creatinine concentration. Samples that need storage times longer than 15 days should be frozen (at -20 °C and -40 °C) to avoid creatinine concentration variation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Silva Júnior et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Biological variation helps determine whether population-based or subject-based reference intervals are more appropriate to assess changes in serial analytical values. Previous studies have investigated the biological variation of biochemical analytes weekly or with variable frequency over 5-14 weeks in cats, but none have considered biological variation at less frequent intervals over 1 year., Objectives: We aimed to evaluate the long-term biological variation of 19 biochemical analytes in clinically healthy cats., Methods: A prospective, observational study in which 15 clinically healthy, client-owned cats were sampled for serum biochemical analyses every 8 weeks for 1 year. Frozen serum samples were single-batch analyzed. Restricted maximum likelihood estimation was used to determine the coefficients of variation (CV), describing variation within each cat, between cats, and the analytical variation. These CVs were used to determine the indices of individuality and reference change values (RCVs)., Results: Albumin, alkaline phosphatase, creatine kinase, and globulin had high indices of individuality, indicating that they are best evaluated by RCVs. Phosphorus, potassium, chloride, sodium, symmetric dimethylarginine, and total CO 2 had low indices of individuality, indicating that population-based reference intervals are appropriate. Alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen, calcium, cholesterol, creatinine, glucose, total bilirubin, and total protein had intermediate indices of individuality, indicating that RCVs may provide additional insight into the interpretation of analyte measurements beyond the population-based reference intervals., Conclusions: For many analytes, the biological variation detected was similar to that reported in prior studies. Clinicians should consider the biological variation of analytes to best interpret clinically relevant changes in serial analyte measurements., (© 2022 American Society for Veterinary Clinical Pathology.)

Designing a disease surveillance program to detect a disease is challenging when animals are organized into herds, in part because disease cases are likely to be clustered. Clustered diseases are often surveilled using two-stage sampling, which allocates tests both among herds and within herds. Finding the optimal allocation of tests is computationally difficult, so some surveillance programs simply seek an approximate solution. We developed a search algorithm to find the optimal allocation of tests by iteratively searching for adjustments to the test allocation that yielded marginal improvements in system sensitivity. We digitally generated 21 herds of various sizes, evenly divided among three regions that differed in relative risk. We then analyzed 29 scenarios that differed in disease and testing characteristics. We also analyzed a Chronic Wasting Disease (CWD) surveillance effort for 23 elk game management units of various sizes that were spread across three regions in Arizona, USA. We compared our marginal sensitivity approach to two other strategies for approximating the optimal distribution of tests: allocating the same number of tests to all herds selected for testing, and allocating tests so that all herds selected for testing achieve the same sensitivity. Across analysis scenarios, we found that low prevalence, high relative risk, a small budget, or high overhead costs were best addressed by concentrating tests in large, high-risk herds. When we expect multiple herds to be infected, the optimal allocation of tests depended on how we expected the cases to be distributed. Across the analyzed scenarios, our marginal sensitivity approach was most efficient, with alternative strategies requiring 0-228 % more tests to achieve the same sensitivity. For CWD in Arizona, we found the potential to double system sensitivity, given a population design prevalence of 0.16 %, from 35.8 % to 70.5 %, although social and budgetary considerations would likely constrain changes to the current allocation of tests. The marginal sensitivity approach we developed has the potential to improve disease surveillance, especially when a population includes a limited number of herds that differ in size. An important limitation of our approach is that computer runtimes could become unacceptably long for a population with many herds., Competing Interests: Declaration of interest None., (Copyright © 2023 Elsevier B.V. All rights reserved.)

In the current article, a developed, patented method denoted the 'Camel Semen Collection Kit-CSCK', was designed to solve the problem of semen collection in dromedary camels. CSCK is composed of three main parts: (1) Semen collection sac: made from supersensitive flexible low-density polyethylene- (LDPE); (2) Metal stainless steel applicator: designed to introduce the collection sac intravaginally and fixate it to the vaginal wall of a female camel through air insufflation; (3) Fixation sticker: a cushion sheet sticker is used to secure the outer portion of the collection sac to the female's perineal area. Semen was collected twice a week from eight dromedary bulls by using electroejaculation (EJ), artificial vagina (AV) and CSCK. Successful semen collections were 81.3%, 84.4% and 43.8% using EJ, CSCK and AV techniques respectively. Semen obtained by EJ technique showed lower semen volume, gross activity, sperm concentration, total sperm motility and percentage of live sperm cells compared to the other two techniques. Semen collected by CSCK showed a longer collection period and higher volume, gross activity, sperm motility and percentage of live spermatozoa and a lower rate of visible contamination compared to AV technique. The advantages and disadvantages of the three techniques were compared and discussed. In conclusion, CSCK represents a practical and easy method to reliably collect high-quality semen from any untrained male dromedary camel and may facilitate the widespread application of assisted reproductive technologies (ARTs) on a large scale in this species., (© 2022 Wiley-VCH GmbH.)

Endometritis is a relevant cause of subfertility in mares. However, the accurate diagnosis, essential for effective treatment, can be difficult due to the variability of results and interpretations resulting from different examination methods and sample collection techniques. The present work compared gynecological evaluation methods and sample collection techniques to diagnose endometritis in subfertile mares. Forty animals with a history of subfertility were selected for gynecological evaluation using clinical methodologies, such as perineal conformation, transrectal palpation and ultrasonography, vaginoscopy, and digital examination of the cervix. In addition, we performed laboratory analyses, including uterine microbiological culture and endometrial cytology and histology, of which the latter is the gold standard for the diagnosis of endometritis. Samples were collected for microbiological culture and endometrial cytological evaluations using three different techniques: a commercial cytobrush/swab collector, low-volume uterine flush, and a new tested technique, by flush the fragment resulting from the endometrial biopsy. Transrectal palpation and ultrasound showed the best results among clinical examinations. However, they were less efficient in laboratory tests of endometrial cytology and uterine microbiological culture, in which the latter showed the highest sensitivity and specificity for endometritis compared with endometrial histology. The use of multiple results from different methods has also proved to be an effective alternative for diagnosis. Among the techniques used to collect endometrial material for cytology and microbiological culture, the most effective and practical in this study was the commercial cytobrush/swab collector., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Adult house flies feed and breed in a variety of microbe-rich habitats and serve as vectors for human and animal pathogens. To better understand their role in harbouring and disseminating bacteria, we characterized the composition and diversity of bacterial communities in the gut of female house flies collected from three different habitats in Kansas: agricultural (dairy farm), urban (business area dumpsters) and mixed (business located between residential and animal agriculture areas). Bacterial community composition and diversity were influenced more by the house flies' habitat than by sampling time. The most abundant taxa were also highly prevalent in the house flies collected from all three habitats, potentially representing a 'core microbiome' attributable to the fly's trophic and reproductive associations with substrates and food sources comprised of decaying matter and/or animal waste. Bacterial taxa associated with vertebrate guts/faeces and potential pathogens were highly abundant in agricultural fly microbial communities. Interestingly, taxa of potential pathogens were highly abundant in flies from the mixed and urban sites. House flies harboured diverse bacterial communities influenced by the habitat in which they reside, including potential human and animal pathogens, further bolstering their role in the dissemination of pathogens, and indicating their utility for pathogen surveillance., (© 2022 Royal Entomological Society. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Objective: To compare: (1) the load and diversity of cultivatable bacterial species isolated from tissue biopsies with cultures from surface swabs, and (2) the ability of each technique to detect methicillin-resistant Staphylococcus aureus (MRSA) in a model of MRSA-infected equine wounds., Study Design: Experimental in vivo study., Animals: Three light-breed adult horses., Methods: Four 2.5 × 2.5 cm full-thickness skin wounds were created on the dorsolateral aspect of each forelimb. Five days later, each wound was inoculated with a pure culture of MRSA (ATCC 43300). One hundred microlitres of 0, 5 × 10 8 , 5 × 10 9 or 5 × 10 10 colony forming units (CFU)/ml was used to inoculate each wound. Surface swabs (Levine technique) and tissue biopsy samples (3 mm punch biopsy) were obtained at 2, 7, 14, and 21 days after inoculation. Quantitative aerobic culture was performed using routine clinical techniques., Results: A similar bacterial profile was identified from the culture of each wound-sampling technique and there was moderate correlation (R = 0.49, P < .001) between the bacterial bioburdens. Agreement was fair (κ = 0.31; 95% CI, 0.129-0.505) between the sampling techniques in identification of MRSA. Methicillin-resistant Staphylococcus aureus was isolated more frequently (P = .016) from cultures of tissue biopsies (79%; 76/96) than from surface swabs (62%; 60/96)., Conclusion: Bacterial load and diversity did not differ between sampling techniques but MRSA was detected more often from the cultures of tissue biopsies., Clinical Significance: Tissue biopsy should be preferred to culture swab in wounds where MRSA is suspected., (© 2022 American College of Veterinary Surgeons.)

Rabies is an encephalitis caused by rabies virus, whose transmission occurs upon contact with infected animals' saliva. The diagnosis is usually performed post-mortem through a direct fluorescent antibody test (DFAT). If the DFAT results are negative, they must be confirmed with an isolation test, usually the mouse inoculation test (MIT), which implies the suffering and death of the animals, high costs and most importantly, up to 28 days to confirm a negative result. Another issue related to rabies diagnosis is the sample collection and storage, which is critical for the rabies virus' RNA genome. Thus, this study aimed to evaluate (i) reverse transcriptase polymerase chain reaction (RT-PCR) and Rabies Tissue Culture Infection Tests (RTCIT) in comparison to DFAT and MIT and (ii) FTA ® cards as an alternative sample collection and preservation method. Eighty animal samples were evaluated through DFAT, RTCIT and RT-PCR; MIT was performed only in DFAT-negative samples. FTA ® cards were evaluated with a subset of 64 samples, with sufficient material for imprinting. Sensitivity, specificity, positive (PPV) and negative predictive values (NPV), agreement and Cohen's kappa were calculated for each test combination. RTCIT had higher sensitivity (92.5%) and RT-PCR had higher specificity (92.3%) compared to DFAT. The combination of tests enhanced sensitivity, NPV and Cohen's kappa (considering positive results by RTCIT or RT-PCR), and specificity and PPV (when both tests were concordant). The PCR based on FTA ® cards as sample source was specific (84.6%-96.2%) but presented lower sensitivity (29.7%-73.0%), although it could detect as positive four DFAT-negative samples. RTCIT and RT-PCR may be used as confirmatory tests in DFAT-negative samples. Moreover, FTA ® cards may be helpful for sample collection in field situations where a long time is needed until the sample undergoes laboratory testing., (© 2022 Wiley-VCH GmbH.)

Widespread surveys of avian haemosporidia (Plasmodium, Haemoproteus, and Leucocytozoon) in wild birds have substantially advanced information on the haemosporidian fauna of Japan. However, many areas and bird species remain insufficiently investigated. Bird carcasses collected for personal specimen collection seldom reach academic audience particularly in the veterinary field. The presence of avian haemosporidia was investigated in these personally collected bird carcasses, in order to better understand the avian haemosporidian fauna in Japan. Bird carcasses were donated through personal contact upon approval of the study. Tissue samples were collected from the birds and examined for haemosporidian parasites using nested-PCR targeting the cytochrome b gene. One hundred and forty-three birds of 85 species were donated, including 34 species and two subspecies that were molecularly or collectively investigated for the first time in Japan. Avian haemosporidian DNA was detected from 37 of the 134 tested birds (27.61%). In 8 bird species, avian haemosporidia was detected for the first time. Twenty-nine lineages were detected, including 8 novel and 9 known lineages detected in Japan for the first time. Furthermore, 16 lineages were detected from novel host species. While information that could be drawn was limited and risk management of zoonotic diseases needs re-consideration, these findings expanded information on the host range and distribution of several lineages. Collectively, this method of investigation using personally collected bird samples can provide important additions to more fully understand the avian haemosporidian fauna of Japan, as well as other areas with limited investigations.

The COVID-19 pandemic has highlighted the critical role that animal models play in elucidating the pathogenesis of emerging diseases and rapidly analyzing potential medical countermeasures. Relevant pathologic outcomes are paramount in evaluating preclinical models and therapeutic outcomes and require careful advance planning. While there are numerous guidelines for attaining high-quality pathology specimens in routine animal studies, preclinical studies using coronaviruses are often conducted under biosafety level-3 (BSL3) conditions, which pose unique challenges and technical limitations. In such settings, rather than foregoing pathologic outcomes because of the inherent constraints of high-containment laboratory protocols, modifications can be made to conventional best practices of specimen collection. Particularly for those unfamiliar with working in a high-containment laboratory, the authors describe the logistics of conducting such work, focusing on animal experiments in BSL3 conditions. To promote scientific rigor and reproducibility and maximize the value of animal use, the authors provide specific points to be considered before, during, and following a high-containment animal study. The authors provide procedural modifications for attaining good quality pathologic assessment of the mouse lung, central nervous system, and blood specimens under high-containment conditions while being conscientious to maximize animal use for other concurrent assays.

Background: Fungal culture is widely used as a diagnostic tool for detecting dermatophytosis. However, the presence of fungal contaminants can influence the culture's performance and compromise the diagnosis., Objective: To verify whether the sample processing time can affect the performance of fungal culture for the diagnosis of Microsporum canis infection in cats., Animals: Forty Persian cats., Methods and Materials: Hair and scale samples were collected by combing the coat using a 5 × 5 cm sterile polyester carpet. The carpets were assigned randomly to four groups based on time point of processing samples after collection (i.e. used for culture on a selective agar medium for dermatophytes): Group 1: 8 h (n = 10); Group 2: 24 h (n = 10); Group 3: 48 h (n = 10); and Group 4: 72 h (n = 10). Cultures were compared regarding the degree of fungal invasion by either M. canis or nondermatophytic contaminant moulds (NDM)., Results: Processing samples after 24 h of storage resulted in increased isolation rates of NDM and decreased isolation rates of M. canis. Samples processed after 48 h and 72 h presented more than half of the plates with a high degree of fungal contamination (i.e. NDM occupying ≥50% of the total fungal mass). However, samples processed after 8 h and 24 h presented a lower degree (P < 0.05) of NDM plate invasion and higher recovery rates of M. canis when compared to samples processed after 48 h and 72 h., Conclusions and Clinical Importance: Delayed processing time is closely associated with the overgrowth of contaminants and with lower recovery rates of M. canis., (© 2021 ESVD and ACVD.)

During the current COVID-19 pandemic, different methods have been used to evaluate patients with suspected SARS-CoV-2 infection. In this study, we experimentally evaluate the ability of spiked saliva-moist swabs and spiked swabs without any transport medium to retain SARS-CoV-2 for storage and transport at different environmental settings during different incubation time periods. Our results show that at ambient temperature of 20°C, SARS-CoV-2 RNA remains stable for up to 9 days allowing a long-time span for transport and storage without compromising clinical results. Additionally, this study demonstrates that saliva-moist swabs can also be stored at -20°C and +4°C for up to 26 days without affecting RT-qPCR results. Our data are relevant for low-and middle-income countries, which have limited access to rapid refrigerated transport and storage of samples representing an economical alternative. Finally, our study demonstrates the practical and economic advantage of using swabs without transport medium., (© 2021 Wiley-VCH GmbH.)

Elephant endotheliotropic herpesviruses (EEHVs) are important causes of death in both captive and wild Asian elephants ( Elephas maximus ). Nothing is known about the prevalence of EEHVs in wild or domestic elephants in China. To determine if EEHVs are present in elephants in China, 126 wild elephants from three populations and 202 captive individuals from zoos ( n = 155) and the Wild Elephant Valley ( n = 47) were screened using semi-nested polymerase chain reaction assays with EEHV-redundant and EEHV1/4/5-specific primers. EEHV1B and EEHV4 were detected in samples from both wild (EEHV1B:8/126; EEHV4:2/126) and captive (EEHV1B:5/155; EEHV4:9/155) elephants, while EEHV1A (six cases) and EEHV5 (one case) were only present in the captive elephants from the Wild Elephant Valley. EEHV1 was detected in blood and trunk and oral swabs; EEHV4 was detected in trunk and oral swabs as well as feces; EEHV5 was found in trunk and oral swabs. No significant age or sex association with EEHV1A, EEHV1B, or EEHV5 positivity was observed. An age association with EEHV4 positivity was found, with all unweaned elephants being EEHV4 positive, but an association with the sex of the elephant was not observed. These findings represent the first documentation of EEHV presence in captive and wild elephants in China. These findings also document EEHV1B and EEHV4 shedding in feces and demonstrate the utility of fecal screening as a tool for investigating EEHV4 infection in wild populations of elephants. It is recommended that EEHV testing be included in surveillance programs for captive and wild elephants in China.

Background: Rigorous testing is a prerequisite to prove freedom of notifiable influenza A virus infections in commercially farmed ostriches, as is the isolation and identification of circulating strains. Pooling 5 ostrich tracheal swabs in a 50 % v/v phosphate-buffered saline (PBS): glycerol transport medium (without antibiotics) is the current standard practice to increase reverse transcription real time PCR (RT-rtPCR) testing throughput and simultaneously reduce the test costs. In this study we investigated whether doubling ostrich tracheal swabs to 10 per pool would affect the sensitivity of detection of H5N8 high pathogenicity avian influenza virus (HPAIV) and H7N1 low pathogenicity avian influenza virus (LPAIV) by quantitative RT-rtPCR, and we also compared the effect of a protein-rich, brain heart infusion broth (BHI) virus transport media containing broad spectrum antimicrobials (VTM) on the efficacy of isolating the H5N8 and H7N1 viruses from ostrich tracheas, since the historical isolation success rate from these birds has been poor., Results: Increasing the ostrich swabs from 5 to 10 per pool in 3 mls of transport medium had no detrimental effect on the sensitivity of the RT-rtPCR assay in detecting H5N8 HPAIV or H7N1 LPAIV; and doubling of the swab pool size even seemed to improve the sensitivity of virus detection at levels that were statistically significant (p less than or equal to 0.05) in medium and low doses of spiked H5N8 HPAIV and at high levels of spiked H7N1 LPAIV. On virus isolation, more samples were positive when swabs were stored in a protein-rich viral transport medium supplemented with antimicrobials in PBS: glycerol (10/18 vs. 7/18 for H5N8 HPAI); although the differences were not statistically significant, overall higher virus titres were detected (10 6.7 - 10 3.0 vs. 10 6.6 - 10 3.1 EID 50 for H5N8 HPAIV and 10 5.5 - 10 1.4 vs. 10 5.1 - 10 1.3 EID 50 for H7N1 LPAIV); and fewer passages were required with less filtration for both H5N8 HPAI and H7N1 LPAI strains., Conclusion: Ostrich tracheal swab pool size could be increased from 5 to 10 in 3mls of VTM with no loss in sensitivity of the RT-rtPCR assay in detecting HPAI or LPAI viruses, and HPAI virus could be isolated from a greater proportion of swabs stored in VTM compared to PBS: glycerol without antibiotics., (© 2022. The Author(s).)

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., (Copyright © 2021. Published by Elsevier Inc.)

Catecholamine release increases in dogs with pheochromocytomas and in situations of stress. Although plasma catecholamines degrade rapidly, their metabolites, normetanephrine (NME) and metanephrine (ME), are stable in acidified urine. Our aim was to verify a human urine ELISA kit for the quantification of NME and ME in canine urine and to determine the effects on metabolite stability of sampling time (morning or midday) and day (ordinary or day spent in a clinic). We analyzed 179 urine samples from 17 healthy dogs. For NME, the mean intra-assay CV was 6.0% for all samples and 4.3% for the canine control; inter-assay CVs were 3.3, 3.8, and 12% for high and low concentration human urine positive controls supplied in the ELISA kit and a positive canine control, respectively; spike-recovery was 90-101%. For ME, mean intra-assay CV was 6.5% for samples and 9.0% for the canine control; inter-assay CVs were 12.7, 7.2, and 22.5% for high and low concentration human urine positive controls supplied in the ELISA kit and a positive canine control, respectively; spike-recovery was 85-89%. Dilution recovery was unsatisfactory for both metabolites. Based on our verification results, NME was selected for remaining analyses. We found no effect on NME concentrations of acidification or room temperature storage for up to 24 h. The NME:creatinine ratio was higher after the first of 3 clinic days compared to the same morning (111.2 ± 5.5 vs. 82.9 ± 5.3; p  < 0.0001), but not on the other days. NME verification results were generally superior to ME. Dilution studies were unsatisfactory for both metabolites. Given that NME was stable without acidification at room temperature, urine samples can be collected at home. The clinic environment can cause higher NME:creatinine ratios, especially in unaccustomed dogs.

Digital microscopy (DM) is increasingly replacing traditional light microscopy (LM) for performing routine diagnostic and research work in human and veterinary pathology. The DM workflow encompasses specimen preparation, whole-slide image acquisition, slide retrieval, and the workstation, each of which has the potential (depending on the technical parameters) to introduce limitations and artifacts into microscopic examination by pathologists. Performing validation studies according to guidelines established in human pathology ensures that the best-practice approaches for patient care are not deteriorated by implementing DM. Whereas current publications on validation studies suggest an overall high reliability of DM, each laboratory is encouraged to perform an individual validation study to ensure that the DM workflow performs as expected in the respective clinical or research environment. With the exception of validation guidelines developed by the College of American Pathologists in 2013 and its update in 2021, there is no current review of the application of methods fundamental to validation. We highlight that there is high methodological variation between published validation studies, each having advantages and limitations. The diagnostic concordance rate between DM and LM is the most relevant outcome measure, which is influenced (regardless of the viewing modality used) by different sources of bias including complexity of the cases examined, diagnostic experience of the study pathologists, and case recall. Here, we review 3 general study designs used for previous publications on DM validation as well as different approaches for avoiding bias.

This study was conducted to evaluate whether there were differences in viability of cryopreserved semen when using two different freezing (Minitube Cryoguard - F1 or Androstar® CryoPlus - F2) and thawing (Minitube Cryoguard Thawing solution - T1 or Androstar® Plus - T2) extenders. Ejaculates were collected, diluted (1:1), and cooled before shipping at 17 °C overnight. Samples were aliquoted in cryopreservation extender F1 or F2. Four straws from each treatment sample were thawed and diluted in T1 or T2, resulting in four treatments (F1-T1, F1-T2, F2-T1, and F2-T2). The sperm in diluted semen were evaluated for motility kinetics at 30, 180, and 360 min after thawing. The integrity assessments of the plasma and acrosomal membranes were performed at 30 and 360 min after thawing. There was no interaction between F × T × Time (P > 0.05), and no interaction between F × T (P > 0.05). The sperm progressive motility (PMOT) as time post-thawing increased was greater (P = 0.015) when dilutions occurred using F1 compared with F2 extender. Sperm thawed in T1 had a greater TMOT (P = 0.008) and PMOT (P = 0.033) at all times evaluated. The sperm plasma and acrosomal membrane integrity (AIMI) were greater (P = 0.009) when samples were preserved in F1 compared to F2 extender. The use of T2, as compared with T1 thawing extender, resulted in an enhanced integrity of the plasma and acrosomal membranes (P = 0.008). It is concluded different combinations of commercial freezing extenders and thawing solutions have effects on the quality of cryopreserved boar semen in vitro., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Objective: To compare bacteriologic culture results for superficial swab and tissue biopsy specimens obtained from dogs with open skin wounds., Animals: 52 client-owned dogs., Procedures: For each dog, 1 wound underwent routine preparation prior to collection of 2 specimens, 1 by superficial swab (Levine) technique and 1 by tissue biopsy. Specimens were processed for bacteriologic culture. Two observers determined whether any detected difference in culture results for the 2 types of specimen would have resulted in differing treatment plans., Results: Culture results of swab and tissue biopsy specimens were identical in 11/52 (21.2%) cases. Tissue biopsy specimen and swab cultures yielded positive results for 44 (84.6%) and 40 (76.9%) wounds, respectively. With regard to mean recovery rates of bacteria from wounds with positive culture results, both the biopsy specimens and swabs yielded 3.4 bacterial species/wound. All wounds for which swab cultures yielded no growth also had negative culture results for biopsy specimens. Biopsy specimen and swab culture results were in agreement with regard to the most common bacteria cultured. In 7/52 (13%) wounds, the observers would have treated the patient differently on the basis of the results of the 2 cultures., Conclusions and Clinical Relevance: Results indicated that culture of a swab collected by the Levine technique is an appropriate noninvasive alternative to culture of a tissue biopsy specimen. A negative result obtained from culture of a swab is likely to be reliable. Disagreement between the results of swab and tissue biopsy specimen cultures is likely of low clinical importance.

Background: Quantitative bacterial culture (QBC) is the gold standard for diagnosing canine urinary tract infection. Current guidelines recommend QBC within 24 h of urine collection and that unpreserved urine is refrigerated until culture. However, temperature-controlled transport is rarely feasible, indicating a need for alternative storage during transport of urine from primary veterinary practices to the microbiology laboratory. The objective was to investigate the effect of storage temperature and boric acid sponge-preservation on quantitative bacterial culture of canine urine., Results: Significant bacteriuria was detected in 72 out of 179 samples (40%) collected from 141 dogs. Overall accuracy was 94-98% for both storage conditions and time points. Non-inferiority (15% margin) to reference quantitative bacterial culture was evident for sensitivity, specificity and predictive values for both storage methods and time points, except for the negative predictive value for 48 h boric acid preservation (NPV: 89, 95% CI [79;95]). There was no significant difference between the sensitivity and specificity for either of the time-points (p-value = 0.07-1)., Conclusions: Boric acid sponge-preservation using Uriswab™ is a useful alternative to refrigeration of urine samples during transport. Reliable quantitative bacterial culture results can be obtained from canine urine up to 48 h after collection if urine is refrigerated, and for at least 24 h if urine is stored using a boric acid-containing urine transport system., (© 2021. The Author(s).)

Interest in the use of assisted reproductive technology in reindeer husbandry has gradually increased during the last decades. This article reviews Western and Russian literature on reindeer semen collection, semen cryopreservation and artificial insemination. In addition, literature on the synchronisation of the stage of reindeer oestrous cycle among females, recovery of in vivo embryos, embryo transfer, the production of in vitro-produced embryos and pregnancy diagnosis is reviewed., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Objectives: To determine if lyophilized canine-specific albumin (CSA) can be stored beyond the manufacturer recommended 6 hours post-reconstitution without significant concern for bacterial growth., Design: Laboratory research., Setting: Large private teaching hospital., Interventions: Six bottles of lyophilized CSA were retrieved from a medical grade (4°C) refrigerator and were reconstituted with 0.9% NaCl to a 5% solution. Time to complete dissolution of all bottles at room temperature without agitation was recorded in minutes. A 1.5-mL sample was collected from each bottle at the following time points: time 0 (at the time of complete dissolution), and then 6, 12, and 24 hours after removal from 4°C. Each aliquot was inoculated into a blood culture tube and was immediately submitted for aerobic and anaerobic bacterial culture. A positive and negative control were cultured at the 24-hour time point. At each time point, an additional 0.5-mL aliquot from each albumin bottle was collected, and these were combined to determine the average albumin concentration (g/L, g/dL) as measured by an in-house analyzer. The albumin was stored at 4°C between sample collection., Measurements and Main Results: The reconstitution of CSA and storage at 4°C for up to 24 hours did not result in anaerobic or aerobic bacterial growth in any of the 24 cultured samples. Storage did not have a clinically significant effect on the albumin concentration of the solution. The average albumin concentration of all samples was 42 g/L (4.2 g/dL). No growth was noted in the negative control, and the positive control grew 4 strains of bacteria., Conclusions: It appears safe to use lyophilized CSA up to 24 hours post-reconstitution without significant risk of bacterial growth or change in albumin concentration., (© Veterinary Emergency and Critical Care Society 2021.)

Between-herd transmission of Mycobacterium avium subsp. paratuberculosis (MAP) by subclinically infected cattle is an important risk which can hamper effective control of paratuberculosis. Knowledge of herd status would substantially reduce this risk; MAP positive farms can be detected with environmental sampling. The objective of this study was to compare cumulative sensitivities of annual environmental sampling with two or four samples per sampling event without knowledge of true herd status and to calculate the number of sampling events to achieve a cumulative sensitivity of at least 0.9. Data from three repeated sampling events in two study populations, one with 55 herds (two samples/event) and another with 30 herds (four samples/event) including test results, herd and sample characteristics and prior prevalence estimates, were derived from the Alberta Johne's Disease Initiative (Alberta, Canada). A recursive Bayesian latent class model was used to predict the cumulative sensitivity of repeated environmental sampling events. A sampling scheme with four samples per sampling event had a higher cumulative sensitivity than an alternative scheme with two samples. To achieve a cumulative sensitivity of at least 0.9 with 95% probability, eight sampling events with two environmental samples per set, or four sampling events with four samples per set were required. Further model assessment demonstrated that these results can only be generalized to cattle populations with a similar within-herd prevalence to those studied here (approximately 0.08). Nonetheless, these results could help predict herd-level prevalence in cattle populations after environmental testing and provide information regarding the uncertainty behind status estimates for herds repeatedly tested using environmental samples., (Copyright © 2021 Bundesinstitut für Risikobewertung. Published by Elsevier Ltd.. All rights reserved.)

Sorbitol dehydrogenase (SDH) activity is one of the most sensitive and specific markers for hepatocellular injury in horses, but its reported lability makes it impractical for use in many clinical settings. To date, stability of SDH in equine samples has only been evaluated in a limited number of studies in serum samples of horses with activities within reference intervals. The objective of the study was to determine pre-analytical stability of equine SDH activity in heparinized plasma stored at different temperatures for up to 72 h. Twenty client-owned horses admitted to a veterinary teaching hospital for any reason were included in the study. Blood samples collected in lithium-heparin tubes were immediately centrifuged and SDH activity was analyzed within 1 h of collection (T0). Aliquots of plasma were stored at room temperature, 4 °C and -20 °C and SDH activity was re-analyzed after 4 h (T4), 24 h (T24) and 72 h (T72). A significant difference from values measured at T0 was found for samples stored at room temperature (P = 0.022) and -20 °C (P < 0.001), but not at 4 °C. The activity of SDH was within ±20% of that measured at T0 for all samples under all temperature conditions stored for 4 h, and for all samples stored at 4 °C for 24 h. Bland-Altman plots revealed narrow limits of agreement at T4 for all storage temperatures and at T24 for samples stored at 4 °C. The mean absolute percentage error and 95th percentile of the absolute percentage error were lower for samples stored at 4 °C than those stored at room temperature or -20 °C. The activity of SDH has adequate stability for 4 h regardless of storage temperature and 24 h if stored at 4 °C across a wide range of values. Knowledge of the pre-analytical stability of SDH may permit its broader use in assessing hepatic disorders in horses., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

An appreciated sampling technique is essential for achieving optimum results from diagnostic tests of cutaneous leishmaniasis (CL); conventional sampling methods, such as skin biopsy and dermal scrapping, are painful for the patients and require qualified staff and hospital facilities, while swabbing is patient-friendly more comfortable than invasive traditional techniques and can be carried out under field conditions. The aim of this study is to evaluate a non-invasive sampling method (swab) in the cutaneous leishmaniasis diagnosis, compared to microscopic examination. The recruitment of 205 patients was done during 3 years in six regions known as endemic CL foci in the south-east, the centre, the south-west and the north of Morocco. The results showed that molecular detection of the nuclear marker ITS1 on swab materials is to be less sensitive than microscopic examination, and the difference was statistically significant (p-value = .036). Thereafter, 55 patients were randomly selected to compare the results of two molecular techniques (ITS1-PCR and nested KDNA-PCR), performed both on swab and on stained smears used for microscopic examination; ITS1-PCR results from stained smears reached 87% positivity against 65,2% for cotton swab; and it was statistically significant (p-value = .019); on the other hand for the KDNA marker, results from cotton swab reached 93% of positivity against 91% for stained smears; and statistically the difference was not significant (p-value = .5113). One can presume that rubbing over the lesion with cotton swab implies a low parasitic load collection, but the choice of the amplification target greatly influences the results obtained from cotton swab; on top of that swab remains useful in the cases of patients with multiples lesions or the latter are located in sensitive places difficult to reach with an invasive method such as the eyelids, the lips or other mucous areas., (© 2020 Wiley-VCH GmbH.)

Measuring enzymatic starch availability is commonly used as a quality control method to ensure steam-flaked corn manufacturing consistency in commercial cattle feeding operations. However, starch availability estimates can be variable. We conducted five experiments to evaluate factors influencing starch availability estimates of steam-flaked corn. In Exp. 1, sample handling methods were evaluated. Sifted flakes were immediately placed into a plastic bag, air equilibrated for 240 min, oven-dried, or freeze-dried. Directly oven-drying samples at 55°C decreased (P < 0.01) starch availability compared to other sample handling methods. In Exp. 2, sifted flakes were air equilibrated for 0, 15, 30, 60, 120, or 240 min. Air equilibration time did not influence (P ≥ 0.54) starch availability. In Exp. 3, samples were evaluated for effects of sifting through a 4-mm screen (flakes + fines vs. sifted flakes) and air equilibration time (0 vs. 240 min). Both sifting steam-flaked corn samples and air equilibration for 240 min increased starch availability (P < 0.01 and P = 0.02, respectively). In Exp. 4, we evaluated the effects of air equilibration time (0 vs. 240 min) on the two sifted portions (sifted flakes vs. sifted fines). There was an air equilibration time × sifted portion interaction for starch availability because air equilibration time increased (P < 0.01) starch availability of sifted fines but did not influence starch availability of sifted flakes. Concentrations of crude protein, soluble crude protein, neutral and acid detergent fiber, ether extract, and acid-hydrolyzed fat, Ca, P, K, Mg, S, Fe, Zn, Mg, and Cu were greater (P < 0.01) for sifted fines compared to sifted flakes. Starch availability and total starch concentration were greater (P < 0.01) for sifted flakes compared to sifted fines. In Exp. 5, effects of air equilibration time (0 vs. 240 min) and storage temperature (23°C vs. 55°ºC) on flakes + fines were evaluated. Storage of flakes + fines in heat-sealed foil bags at 55°C for 3-d decreased (P < 0.01) starch availability by 40.7%. Sifted flakes contained less moisture, greater total starch concentrations, and greater starch availability than sifted fines. Moisture, sifting, air equilibration time, and storage temperature influence starch availability of steam-flaked corn. Adoption of the strategies discussed in the current study will lead to more consistent estimates of starch availability., (© The Author(s) 2021. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Semen collection has an essential role in the initial bacterial load in boar ejaculates and extended semen. The study aimed to explore the efficacy of an adjusted penis fixation in a semi-automatic collection system on reducing bacterial contamination of ejaculates in two-boar studs with different scenarios. Historically, stud A had low levels of bacterial load in raw semen, while stud B had a high level of contamination. A total of 56 mature boars had their semen collected using two methods of penis fixation: (a) Traditional: The penis was fixed directly with the artificial cervix and transferred to the adjustable clamp; (b) Adjusted: The fixation was performed with one gloved-hand, and after exteriorization, the penis was gripped using the artificial cervix with the other gloved-hand and transferred to the adjustable clamp. The bacterial load (p = .0045) and the occurrence of ejaculates >231 CFU/ml (p = .0101) were reduced in the Adjusted compared to the Traditional method. Bacterial load was reduced when using the Adjusted method in stud B (p = .0011), which showed a greater occurrence of critical factors for bacterial contamination (p ≤ .0034). The Adjusted method reduced the occurrence of ejaculates >231 CFU/ml when the preputial ostium was dirty (p = .016) and the duration of semen collection was >7 min (p = .022) compared to the Traditional method. In conclusion, the Adjusted penis fixation was efficient in reducing bacterial load of ejaculates, mainly in boar stud B, which had high contamination challenges., (© 2021 Wiley-VCH GmbH.)