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<jats:p> The confirmatory diagnosis of Mycobacterium bovis ( M. bovis) in animal samples is carried out by culture in Stonebrink media. However, culture is very slow because of the extremely long duplication time of the bacillus and difficult because of the scarcity of bacilli in diagnostic samples. This study describes the development of a single-tube touch-down polymerase chain reaction (PCR) protocol for the detection of M. bovis using primers that target the IS 6110 element. Spiked water and milk as well as routine diagnostic samples (milk and nasal swabs) from M. bovis–positive cattle were tested. This protocol allows the rapid and sensitive detection of M. bovis in bovine samples by enhancing the sensitivity of standard PCR amplification. </jats:p>

<jats:p> Brucella canis was recovered from dogs that were canine brucellosis suspect by blood culture using a modified lysis method. Organism identity was established by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The instrument-provided security library identified the isolates as Brucella species. The isolates were further identified as B. canis with the help of phenotypic and genotypic characteristics. The mass spectral profiles from characterized B. canis isolates, when added to the MALDI-TOF MS standard reference library, allowed successful presumptive identification of B. canis. </jats:p>

<jats:p> Mycobacterioses can produce nonspecific clinical signs in dogs and cats that make diagnosis difficult. Furthermore, the full characterization of mycobacterial agents is not always possible or practical. We characterized mycobacteria detected through cytology in 12 dogs and 7 cats with generalized clinical signs from the province of Buenos Aires in Argentina. In dogs, molecular testing confirmed the presence of Mycobacterium avium subsp. hominissuis (MAH) in 8 cases and M. fortuitum in 1 case. All dogs were Miniature Schnauzers, suggesting that this breed may be more susceptible to M. avium than other dog breeds. The cat isolates were 2 M. bovis, 1 M. fortuitum, and 1 MAH. Mycobacterial interspersed repetitive unit–variable-number tandem repeat patterns suggested possible links with cattle, swine, and humans studied previously in Argentina. The results show that pets may act as susceptible hosts with the potential risk of transmitting the infection to humans and other animals. </jats:p>

<jats:p> Canine anaplasmosis is a tick-borne disease of dogs that results following infection with Anaplasma phagocytophilum or Anaplasma platys. The SNAP 4Dx Plus test (IDEXX Laboratories) and the VetScan Canine Anaplasma Rapid test (Abaxis) are commercial in-house rapid tests for the detection of antibody to these 2 antigenically related Anaplasma species. We evaluated 2 tests using serum and whole blood samples obtained from reference laboratories and veterinary hospitals. Samples were obtained from regions of the country known to be habitats of the primary tick vectors. The A. phagocytophilum sample set comprised 236 dog sera from the northeastern and midwestern United States; the A. platys sample set comprised 179 sera from dogs living in the southwestern United States. An indirect immunofluorescent antibody (IFA) test and an A. platys species-specific ELISA were used as reference assays for the A. phagocytophilum and A. platys samples, respectively. The SNAP test demonstrated significantly higher sensitivity (84.7% for A. phagocytophilum and 83.1% for A. platys), compared to the VetScan test (39.0% for A. phagocytophilum and 57.6% for A. platys). The specificity of the SNAP test (95.8% for A. phagocytophilum and 99.2% for A. platys) was significantly greater than the VetScan test (85.6% for A. phagocytophilum and 82.5% for A. platys). In a separate clinic study, conducted within an A. phagocytophilum–endemic state (Minnesota) using 154 whole blood samples from client-owned dogs, the VetScan test was negative for 22 of 39 SNAP and IFA seropositive samples. </jats:p>

<jats:p> Real-time PCR (rtPCR) tests have become a method of choice in many diagnostic settings, both animal and human. A concern remains, however, regarding rtPCR assay inhibition during nucleic acid extraction and/or rtPCR reaction process that may result in false-negative results. The use of an internal positive control, either endogenous or exogenous, to mitigate this issue has become more commonplace. We identified and standardized an endogenous internal positive control that can be utilized in rtPCR assays targeting canine-specific pathogens in either a singleplex or multiplex format. The target chosen for the endogenous internal positive control (EIPC-K9) was a highly conserved region in canine mitochondrial DNA. Samples from 240 dogs and 11 other species were screened with EIPC-K9; all canine samples were detected, and no cross-amplification with other species tested was observed. Additionally, no inhibition was noted when comparing singleplex to multiplex rtPCR formats. </jats:p>

<jats:p> Canine brucellosis is an infectious and contagious disease associated with reproductive losses in breeding kennels. As a zoonotic disease, it poses a risk to human health, especially for veterinarians and breeders who handle materials potentially contaminated with Brucella canis. However, canine brucellosis is a neglected and underestimated disease given the difficulties in establishing a definitive diagnosis. We evaluated the frequency of detection of B. canis in 5 breeding kennels by using various serologic methods and PCR. Circulation of B. canis in these kennels was confirmed by bacterial isolation. The frequency of positive serologic results varied from 6.3% by AGID to 16.5% by dot-ELISA. There was no positive serology for smooth Brucella. PCR testing was positive in 13.9% of samples. The only detection tests with reasonable agreement were PCR and 2ME-MAT. The diagnosis of canine brucellosis remains challenging. The use of a single laboratory method, or even the use of different laboratory methods, may not be sufficient to reach a definitive diagnosis. </jats:p>

<jats:p> Fetal bovine serum (FBS) used in cell culture may be contaminated with adventitious agents, which can affect the production of biologicals and the results of clinical laboratory tests. We carried out a retrospective study to determine the incidence of adventitious agent contamination of Argentinean irradiated FBS dating from 2015 to 2019. We analyzed FBS batches for mycoplasma and adventitious viruses (bovine pestiviruses, bovine adenovirus, bluetongue virus, bovine parainfluenza virus 3, rabies virus, bovine parvovirus, bovine herpesvirus 1, bovine respiratory syncytial virus, and reovirus). Cell passages followed by direct immunofluorescence were carried out to check viability of the mentioned adventitious agents. Also, molecular detection of mycoplasma and pestiviruses was performed on the FBS samples. The presence of neutralizing antibodies against pestiviruses was determined. Molecular analyses indicated that frequencies of mycoplasma and pestiviruses in FBS were 14% and 84%, respectively. All of the batches were seronegative for pestiviral antibodies. After cell passages, all FBS samples were negative for hemadsorbent agents and by immunofluorescence for all of the viral species analyzed; PCR assays were negative for mycoplasma and pestiviruses. Our results demonstrate that, of all adventitious agents tested, local FBS batches only had traces of mycoplasma and pestiviruses; gamma irradiation was effective in inactivating them. </jats:p>

<jats:p> The Kenney–Doig scale is a histopathology categorization (grading) system often used as the standard for assessing endometrial disease and communicating prognostic fertility information for equine breeding prospects. We investigated how Kenney–Doig categories compared within the same institution and across different institutions to determine if observer variability may contribute to category frequencies. We conducted a retrospective analysis of all equine endometrial submission records between 1998 and 2018 at the Western College of Veterinary Medicine (WCVM) and Prairie Diagnostic Services (PDS). Of 726 biopsies, we found the following category distribution: 46 of 726 (6.3%) I, 307 of 726 (42.3%) IIA, 326 of 726 (44.9%) IIB, and 47 of 726 (6.5%) III. We also conducted a review of the literature and included 6 studies reporting Kenney–Doig category distributions. Chi-square analysis showed significant differences between the category distribution found at WCVM and PDS and the category distribution reported in the 6 studies. To account for differences in mare populations, individual category distributions were generated for 5 pathologists at the WCVM and PDS. The Fisher exact test among these 5 Kenney–Doig categories revealed significant differences in category tendencies, suggesting that observer variation affects the use of the scale. Our results suggest that there is a need for prospective inter-rater and intra-rater agreement studies of the repeatability of the Kenney–Doig scale. </jats:p>

<jats:p> Inter- and intra-rater variability negatively affects the reliability of various histopathology grading scales used as prognostic aids in human and veterinary medicine. The Kenney–Doig categorization (grading) scale, which is used to associate equine endometrial histologic lesions with prognostic estimation of a broodmare’s reproductive potential, has not been evaluated for inter- or intra-rater variability, to our knowledge. To assess whether the Kenney–Doig system produces reliable results among observers, 8 pathologists, all with American College of Veterinary Pathologists certification, were recruited to blindly categorize the same set of 63 digital equine endometrial biopsy slides as well as to re-evaluate anonymously 21 of 63 of these slides at a later time. Cohen kappa values for pairwise comparison of final Kenney–Doig categories were −0.05 to 0.46 (unweighted) and 0.08–0.64 (weighted), with an average Light kappa of 0.19 (unweighted) and 0.36 (weighted) across all 8 pathologists, 0.14 (unweighted) and 0.33 (weighted) for pathologists at different institutions, and 0.22 (unweighted) and 0.46 (weighted) for pathologists at the same institution. Intra-class correlations measuring intra-rater agreement were 0.12–0.77 with an average of 0.55 for all 8 pathologists. We found that only slight-to-moderate inter-rater agreement and poor-to-good intra-rater agreement was produced by 8 pathologists using the Kenney–Doig scale, suggesting that the system is subject to significant observer variability and care should be taken when communicating Kenney–Doig categories to submitting clinicians with emphasis on the quality of endometrial lesions present instead of the category and associated expected foaling rate. </jats:p>