Dog Serum Samples Research Articles

A Novel Immunochromatographic Test Strip Using Lanthanide-Labeled Fluorescent Nanoparticles for the Serological Detection of Toxoplasma gondii in Dogs and Cats

Toxoplasma gondii (T. gondii) is an important zoonotic pathogen which induces both acute and chronic toxoplasmosis. Timely diagnosis of T. gondii is crucial for effective disease management. Here, we present a pioneering approach using europium (III)-chelated nanoparticles (EuNPs) in a rapid lateral flow immunochromatographic test strip (ICTS) for detecting T. gondii antibodies in serum samples. By conjugating EuNPs with Staphylococcus aureus protein A, we efficiently captured T. gondii-specific antibodies, which bound to T. gondii antigens on the test line (T-line), generating a distinct fluorescent signal. Employing this novel method, we conducted an extensive epidemiological investigation of T. gondii infections among dogs and cats in Shanghai, China. This innovative ICTS allows for rapid results within 25 min, which include a qualitative result through naked-eye observation under an ultraviolet lamp and a quantitative one derived using a strip reader. With a detection limit of 1:6400 for dog positive serum and no cross-reactivity with other canine and feline pathogens, the EuNPs-ICTS demonstrated excellent consistency with standard enzyme-linked immunosorbent assay results for dogs (κ = 0.91) and cats (κ = 0.92). In addition, 20.38% of 996 dog serum samples and 14.18% of 416 cat serum samples revealed T. gondii antibodies, highlighting the efficacy of this approach. Our study presents a rapid, sensitive, specific, and reproducible EuNPs-ICTS, serving as a promising tool for on-the-spot diagnosis of T. gondii infections in dogs and cats.

NMR-based metabolomic investigation of dogs with acute flaccid paralysis due to tick paralysis.

Acute flaccid paralysis (AFP) is a complex clinical syndrome with various aetiologies. If untreated, AFP may lead to death due to failure of respiratory muscles. Tick paralysis, which is a noninfectious neurologic syndrome of AFP, occurs following tick attachment, engorgement, and injection of tick saliva toxins. There is no specific diagnostic test for tick paralysis, and mortality increases as definitive diagnosis is delayed. Although metabolomic investigation of tick saliva was conducted, there is a lack of research on metabolomic evaluation of hosts affected by tick paralysis. Thus, the aim of this study is to investigate metabolomic changes in serum samples of dogs with tick paralysis due to Rhipicephalus sanguineus using NMR-based metabolomics and to identify potential diagnostic/prognostic markers. Forty dogs infested with R. sanguineus, with clinical findings compatible with AFP and with a confirmed tick paralysis diagnosis ex juvantibus, constituted the Paralysis Group. Ten healthy dogs, which were admitted either for vaccination and/or check-up purposes, constituted the Control Group. After the confirmation tick paralysis, medical history, vaccination and nutritional status, body surface area and estimated tick numbers of all the dogs were noted. Physical examination included body temperature, heart and respiratory rate, capillary refill time evaluation and Modified Glasgow Coma Scale calculation. Serum samples were extracted from venous blood samples of all the dogs and were prepared for NMR analysis, and NMR-based metabolomics identification and quantification were performed. NMR-based serum metabolomics of the present study revealed distinct up/down-regulated expressions, presenting a promising avenue. Moreover, it was observed that energy metabolism and especially liver functions were impaired in dogs with tick paralysis, and not only the respiratory system but also the kidneys were affected. It was concluded that the present approach may help to better understand the pathological mechanisms developing in cases of AFP due to tick paralysis.

Synthetic peptides-based SPR biosensor evaluation towards canine Visceral leishmaniasis diagnosis: A simple and effective approach

A vital component of surface plasmon resonance (SPR) biosensors is the recognition element. It generates the sensor selectivity towards the target biomarker and plays an important role in defining the sensor’s analytical sensitivity. In serological diagnostics, recombinant proteins (purified antigens) have presented better selectivity compared to soluble crude antigens in SPR immunosensors. However, recombinant DNA technology to obtain proteins is only explored by specialized laboratories, and commercial recombinant proteins still have a high cost. Thus, with the aim of developing a much simpler and lower-cost method, we investigated the prospect of using synthetic peptides as recognition elements for constructing an SPR biosensor. Two synthetic peptides, named PEP13 and PEP16, were tested for the serological diagnosis of Canine Visceral Leishmaniasis (CVL), an endemic neglected disease that affects humans and dogs worldwide. Between the two peptides tested, PEP13 was more sensitive when evaluating its responses against purified antibodies in buffer solution (LOD = 1.05 nmol L-1), and it also discriminated the response better when applied in diluted serum samples of infected dogs compared to diluted healthy dogs’ samples. For this reason, the PEP13 immunosensor was used to analyze canine serum samples, precisely identifying all the positive (n = 7) and negative (n = 7) CVL cases (p = 0.00136) in less than 12 min. In brief, this study explored promising biostructures through a simple and fast methodology for serological diagnosis, addressing the suitability of synthetic peptides for use in biosensors in the urgent field of neglected diseases.

Incidence of canine viral diseases and prevalence of virus neutralization antibodies of canine distemper virus, adenovirus type 2, parvovirus, and parainfluenza virus type 5 in Korean dogs

Canine distemper virus (CDV), canine adenovirus type 2 (CAV-2), canine parvovirus (CPV), and canine parainfluenza virus 5 (CPIV-5) are the major viral pathogens in dogs. Despite the availability of vaccines for dogs against these four viral pathogens, investigations of antibodies against these pathogens have rarely been reported in South Korea. In this study, we investigated the recent incidence of viral diseases in dogs and conducted sero-surveillance for CDV, CAV-2, CPV, and CPIV-5 in Korean dogs. The most frequently diagnosed canine viral disease in Korean dog samples from 2000 to 2022 was CPV infection, which accounted for 48.7% (464/953) of the cases. A total of 400 dog serum samples collected between 2019 and 2022 were screened for the presence of virus-neutralizing antibodies against CDV, CAV-2, CPV, and CPIV-5. The overall seropositivity rates for CDV, CAV-2, CPV, and CPIV-5 were 83.8%, 77.8%, 99.3%, and 82.0%, respectively. The protection rate against CPV was the highest (98.3%) and that against CAV-2 was the lowest (44.8%) in dog sera. Male and female dogs showed no significant differences in seropositivity rates. CDV and CPIV-5 seropositivity increased with age in dogs, and the highest incidence and seropositivity rates of CPV indicated that Korean dogs have been continuously exposed to wild CPV, and that CPV is a pathogen that urgently requires attention among canine viral diseases.

Development of Colloidal Gold-Based Immunochromatographic Strips for Rapid Detection and Surveillance of Japanese Encephalitis Virus in Dogs across Shanghai, China.

Japanese encephalitis virus (JEV) causes acute encephalitis in humans and is of major public health concern in most Asian regions. Dogs are suitable sentinels for assessing the risk of JEV infection in humans. A neutralization test (NT) or an enzyme-linked immunosorbent assay (ELISA) is used for the serological detection of JEV in dogs; however, these tests have several limitations, and, thus, a more convenient and reliable alternative test is needed. In this study, a colloidal gold immunochromatographic strip (ICS), using a purified recombinant EDIII protein, was established for the serological survey of JEV infection in dogs. The results show that the ICSs could specifically detect JEV antibodies within 10 min without cross-reactions with antibodies against other canine viruses. The test strips could detect anti-JEV in serum with dilution up to 640 times, showing high sensitivity. The coincidence rate with the NT test was higher than 96.6%. Among 586 serum samples from dogs in Shanghai examined using the ICS test, 179 (29.98%) were found to be positive for JEV antibodies, and the high seropositivity of JEV in dogs in China was significantly correlated with the season and living environment. In summary, we developed an accurate and economical ICS for the rapid detection of anti-JEV in dog serum samples with great potential for the surveillance of JEV in dogs.

Neutrophil extracellular traps in CSF and serum of dogs with steroid-responsive meningitis-arteritis.

In steroid-responsive meningitis-arteritis (SRMA), inflammatory dysregulation is driven by neutrophilic granulocytes resulting in purulent leptomeningitis. Neutrophils can generate neutrophil extracellular traps (NET). Uncontrolled NET-formation or impaired NET-clearance evidently cause tissue and organ damage resulting in immune-mediated diseases. The aim of the study was to verify that NET-formation is detectable in ex vivo samples of acute diseased dogs with SRMA by visualizing and measuring NET-markers in serum and cerebrospinal fluid (CSF) samples. CSF-samples of dogs with acute SRMA (n = 5) and in remission (n = 4) were examined using immunofluorescence (IF)-staining of DNA-histone-1-complexes, myeloperoxidase and citrullinated Histone H3 (H3Cit). Immunogold-labeling of H3Cit and neutrophil elastase followed by transmission electron microscopy (TEM) were used to determine ultrastructural NET-formation in the CSF of one exemplary dog. H3Cit-levels and DNase-activity were measured in CSF and serum samples using an H3Cit-ELISA and a DNase-activity-assay, respectively in patients with the following diseases: acute SRMA (n = 34), SRMA in remission (n = 4), bacterial encephalitis (n = 3), meningioma with neutrophilic inflammation (n = 4), healthy dogs (n = 6). NET-formation was detectable with IF-staining in n = 3/5 CSF samples of dogs with acute SRMA but were not detectable during remission. Vesicular NET-formation was detectable in one exemplary dog using TEM. DNase-activity was significantly reduced in dogs suffering from acute SRMA compared to healthy control group (p < 0.0001). There were no statistical differences of H3Cit levels in CSF or serum samples of acute diseased dogs compared to dogs under treatment, dogs suffering from meningioma or bacterial encephalitis or the healthy control group. Our findings demonstrate that NET-formation and insufficient NET-clearance possibly drive the immunologic dysregulation and complement the pathogenesis of SRMA. The detection of NETs in SRMA offers many possibilities to explore the aetiopathogenetic influence of this defence mechanism of the innate immune system in infectious and non-infectious canine neuropathies.

Human and animal exposure to newly discovered sand fly viruses, China.

The Hedi virus (HEDV) and Wuxiang virus (WUXV) are newly discovered Bunyaviruses transmitted by sandflies. The geographical distribution of isolation of these two viruses continues to expand and it has been reported that WUXV causes neurological symptoms and even death in suckling mice. However, little is known about the prevalence of the two viruses in mammalian infections. In order to understand the infection status of HEDV and WUXV in humans and animals from regions where the viruses have been isolated, this study used Western blotting to detect the positive rates of HEDV and WUXV IgG antibodies in serum samples from febrile patients, dogs, and chickens in the forementioned regions. The results showed that of the 29 human serum samples, 17.24% (5/29) tested positive for HEDV, while 68.96% (20/29) were positive for WUXV. In the 31 dog serum samples, 87.10% (27/31) were positive for HEDV and 70.97% (22/31) were positive for WUXV, while in the 36 chicken serum samples, 47.22% (17/36) were positive for HEDV, and 52.78% (19/36) were positive for WUXV. These findings suggest there are widespread infections of HEDV and WUXV in mammals (dogs, chickens) and humans from the regions where these viruses have been isolated. Moreover, the positive rate of HEDV infections was higher in local animals compared to that measured in human specimens. This is the first seroepidemiological study of these two sandfly-transmitted viruses. The findings of the study have practical implications for vector-borne viral infections and related zoonotic infections in China, as well as providing an important reference for studies on the relationship between sandfly-transmitted viruses and zoonotic infections outside of China.

Evaluation of hemagglutination inhibition test for canine respiratory coronavirus antibodies and seroprevalence in Korean dogs

Canine respiratory coronavirus (CRCoV) is a significant pathogen that causes respiratory diseases in dogs, collectively known as a canine infectious respiratory disease. The virus is highly contagious and exhibits high seroprevalence worldwide. Currently, bovine coronavirus (BCoV) enzyme-linked immunosorbent assay (ELISA) kits are used to detect CRCoV antibodies. However, BCoV-ELISA kits cannot differentiate between infections caused by BCoV and those caused by CRCoV. In this study, we evaluated the hemagglutination inhibition (HI) test for CRCoV by comparing it with the virus neutralization (VN) test. Subsequently, we evaluated the seroprevalence of CRCoV in 383 dog serum samples collected from South Korea utilizing the HI test. The HI test for CRCoV showed a strong correlation with the VN test (R = 0.83, p &lt; 0.001). The analysis of seroprevalence revealed that 52.2% (95% confidence interval [CI], 47.2%–57.1%) of the Korean dog serum samples were positive. The seroprevalence exhibited varied with age, with a positivity rate of 43.9% in dogs under 1 year of age and 66.7% in dogs aged 3 to 5 years (odds ratio [OR], 2.54; 95% CI, 1.43–4.59). In conclusion, the HI test to monitor CRCoV antibody proved to be closely related to the VN test. Furthermore, over half of the dogs in Korea tested positive for CRCoV antibodies. These findings contribute to a better understanding of the sero-epidemiology of CRCoV.

Seroprevalence of SARS-CoV-2 antibodies in dogs and cats during the early and mid-pandemic periods in Japan

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has continued to circulate in humans since its emergence in 2019. While infection in humans continues, numerous spillover events to at least 32 animal species, including companion and zoo animals, have been reported. Since dogs and cats are highly susceptible to SARS-CoV-2 and have direct contact with their owners and other household members, it is important to know the prevalence of SARS-CoV-2 in dogs and cats. Here, we established an ELISA to detect serum antibodies against the receptor-binding domain and the ectodomain of the SARS-CoV-2 spike and nucleocapsid proteins. Using this ELISA, we assessed seroprevalence in 488 dog serum samples and 355 cat serum samples that were collected during the early pandemic period (between May and June of 2020) and 312 dog serum samples and 251 cat serum samples that were collected during the mid-pandemic period (between October 2021 and January 2022). We found that two dog serum samples (0.41%) collected in 2020, one cat serum sample (0.28%) collected in 2020, and four cat serum samples (1.6%) collected in 2021 were positive for antibodies against SARS-CoV-2. No dog serum samples collected in 2021 were positive for these antibodies. We conclude that the seroprevalence of SARS-CoV-2 antibodies in dogs and cats in Japan is low, suggesting that these animals are not a major SARS-CoV-2 reservoir.

Toxoplasma gondii exposure in Brazilian indigenous populations, their dogs, environment, and healthcare professionals

Although Toxoplasma gondii exposure has been reported in indigenous populations worldwide, a One Health approach has not been applied to date. This study concurrently assessed T. gondii exposure in indigenous populations, and their dogs, environment, and indigenous or non-indigenous healthcare professionals (HPs). Human and dog serum samples from 9 indigenous communities in Brazil were assessed by indirect immunofluorescence antibody test for anti-T. gondii antibodies. Soil samples (30 per community) were processed with PCR to amplify T. gondii DNA. Associated risk factors and seroprevalence were analyzed using logistic regression models. Human seropositivity and type of water source were assessed by generalized linear mixed model (GLMM) with binomial error distribution, and game meat consumption with chi-squared test. Overall, 225/463 (49%) indigenous persons were seropositive for anti-T. gondii antibodies. Of all the HPs, 67/168 (40%) were positive, and included 54/147 (37%) positive non-indigenous HPs. Indigenous persons more likely to be seropositive compared with non-indigenous HPs (OR: 1.63; 95% CI: 1.11–2.39). A total of 97/253 (38%) dogs were seropositive and highly associated with seropositive owners (p < 0.001). Based on univariate analysis for indigenous individuals, state location of community (p < 0.001), ethnicity (p < 0.001), consumption of game meat (p < 0.001), type of water source (p < 0.001), and educational level (p = 0.026) were associated with seropositivity. Logistic regression showed that indigenous seropositivity was associated with eating game meat (p = 0.002), drinking water from rivers (p < 0.001), and inversely proportional to the educational level. According to univariate analysis for non-indigenous HP, age (p = 0.005), frequency of visits to the indigenous populations (p < 0.001), consumption of water at the indigenous communities (p < 0.001), and ingestion of raw meat (p = 0.023) were associated with T. gondii seropositivity. Logistic regression revealed living outdoors (p = 0.042), habit of hunting (p = 0.008), and drinking river water (p = 0.007) as risk factors associated to seropositivity in dogs. In addition, indigenous communities lacking water treatment had higher seroprevalence for all groups including indigenous persons (GLMM; z = −7.153; p < 0.001), their dogs (GLMM; z = −2.405; p = 0.0162), and all HPs (GLMM; z = −2.420; p = 0.0155). Human seropositivity was associated with that of their dogs (p < 0.001). A single soil sample, out of 270 (0.37%), was positive for T. gondii by PCR. Our results indicate water source is a risk for human and dog toxoplasmosis in indigenous communities; both share similar exposure. Moreover, quality water access was shown to be crucial to prevent toxoplasmosis in both total and non-indigenous HPs who work in these indigenous communities.

Seroprevalence of Toxoplasma gondii in domestic cats, dogs and rabbits from Poland

The seroprevalence of Toxoplasma gondii in domestic cats, dogs and rabbits was evaluated. Samples from cats and dogs were collected from five veterinary practices from various parts of Poland - Poznan (wielkopolskie voivodeship), Przemysl (podkarpackie voivodeship), Kluczbork (opolskie voivodeship), Lublin (lubelskie voivodeship) and Deblin (lubelskie voivodeship). Moreover, the samples from rabbits were collected in Poznan. In total, serum samples from 193 cats, 204 dogs and 71 rabbits were randomly selected and tested for specific antibodies against T. gondii using a commercial ELISA test. Pathogen seroprevalence among cats and dogs was calculated at a 95% confidence interval (CI) for each sex and age category (up to 12 months, 1–3 years, 4–7 years and over 8 years) and compared with a chi-squared test. The highest seroprevalence of T. gondii was noted in cats − 49.74% (96/193; 95% CI: 42.76–56.73). In dogs, it reached 28.92% (59/204; 95% CI: 23.13–35.49). Only 1 rabbit (3-year-old male) was seropositive, and the seroprevalence in rabbits was 1.41% (1/71; 95% CI: 0.25–7.56). A statistically significant correlation between seropositivity and age (p < 0.05) was observed in cats and dogs. No statistically significant difference in seroprevalence concerning gender or location was found in cats and dogs. Our findings indicate that cat and dog serum samples had a high frequency of anti-T. gondii antibodies, while rabbit serum samples had low frequency and that these species are exposed to T. gondii in Poland and develop humoral response due to infection.

Detection of neutralising antibodies against SARS-CoV-2 in companion animals in Istanbul

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in late 2019 and rapidly spread throughout the world. After the susceptibility of various animals to SARS-CoV-2 infection was reported, the prevalence of COVID-19 infection in pet animals and their role in virus transmission became a source of concern. Seroprevalence of SARS-CoV-2 in pet animals has been reported from various parts of the world to date. In this study, 206 serum samples of household dogs and cats were obtained from a diagnostic laboratory in Istanbul. Ninety-nine of the samples were collected during the second wave (November and December 2020) of the pandemic, and 107 were collected between August and September 2021. Neutralizing antibodies against SARS-CoV-2 in companion animals was investigated by virus neutralization test. Among the 99 serum samples which were collected during the second wave (November and December 2020) of the pandemic, while no neutralizing antibodies were found in 61 sera of cats, one of 38 dogs was positive with a titer of 1/256. Similarly, 0/54 cats and 1/53 dogs were tested positive for the neutralizing antibodies among the samples collected between August and September 2021. This study is demonstrating the evidence of SARS-CoV-2 infection in dogs.

Toxoplasma gondii and Neospora caninum Antibodies in Dogs and Cats from Egypt and Risk Factor Analysis.

Toxoplasma gondii and Neospora caninum are major protozoan parasites of worldwide distribution and significance in veterinary medicine and, for T. gondii, in public health. Cats and dogs, as final hosts for T. gondii and N. caninum, respectively, have a key function in environmental contamination with oocysts and, thus, in parasite transmission. Very little is known about the prevalence of T. gondii infections in dogs and cats in Egypt, and even less about the prevalence of N. caninum in the same hosts. In the current study, 223 serum samples of both dogs (n = 172) and cats (n = 51) were investigated for specific antibodies to T. gondii and N. caninum using commercially available ELISAs. A risk factor analysis was conducted to identify factors associated with seropositivity. Exposure to T. gondii was reported in 23.3% of the dogs and in 9.8% of the cats, respectively. In addition, N. caninum-specific antibodies were recorded in 5.8% of dogs and in 3.4% of cats. A mixed infection was found in two dogs (1.2%) and in one cat (2%). Antibodies to T. gondii in dogs were significantly more frequent in dogs aged 3 years or more and in male German Shepherds. As this breed is often used as watchdogs and was the most sampled breed in Alexandria governorate, the purpose "watchdog" (compared to "stray" or "companion"), the male sex, and the governorate "Alexandria" also had a significantly higher seroprevalence for T. gondii. No factors associated with antibodies to N. caninum could be identified in dogs, and no significant factors were determined in cats for either T. gondii or N. caninum infection. Our study substantially adds to the knowledge of T. gondii infection in dogs and cats and presents data on N. caninum infection in cats for the first and in dogs in Egypt for the second time.

Laboratory validation of an ELISA method to measure circulating immune complexes levels in canine visceral leishmaniasis

Susceptible dogs suffering from canine leishmaniasis (CanL) develop an ineffective humoral immune response that leads to the formation of circulating immune complexes (CIC). These CIC are aggregates of Leishmania proteins and anti-Leishmania immunoglobulins. Their deposition in different tissues is considered the main cause of mortality. For this reason, CIC have been suggested as an excellent CanL biomarker for measuring the progression of the disease and the effectiveness of specific treatments. The present study aims to perform a laboratory validation of a Leishmania-specific method to isolate and quantify CIC in dog serum samples. CIC isolated from serum samples of infected dogs, grouped according to the LeishVet classification, were quantified following a PEG-ELISA procedure. The validation established a cut-off of 0.274 OD. All the parameters analyzed (including linearity, specificity, precision, and robustness) fulfilled the defined criteria, confirmed by statistical analyses. The results also proved the reproducibility and reliability of the method when samples were tested under the same conditions, and the consistency and usefulness of the method for an optimal staging of infected dogs. In conclusion, the laboratory validated method offers a potent tool to clinicians for a proper CanL management and to measure the progression of the disease.

Evaluation of stress response in black-tailed prairie dogs (Cynomys ludovicianus) in arid regions from colonies in Chihuahua Mexico

Among all the regulatory homeostatic networks in vertebrates, the activation of the hypothalamic–pituitaryadrenal axis during the stress response, has gained considerable attention, and the measurement of fecal glucocorticoids (FGC) has become an invaluable tool to assess adrenocortical activity related to stressful events in wild and captive animals. However, the use of FGC requires the validation of measurement techniques and the proper selection of the specific hormone according to the study species. The main objective of this study was to identify the dominant glucocorticoid (GC) hormone in the stress response of black-tailed prairie dogs (Cynomys ludovicianus) in an arid grassland of Chihuahua, Mexico. A capture stress challenge in the field was developed to determine if the levels of glucocorticoids (cortisol and corticosterone) both in serum and fecal samples could be attributed to stress in Cynomys ludovicianus. The samples were analysed with the technique of liquid phase radioimmunoassay , and this study showed that both cortisol and corticosterone are present at measurable levels in serum and fecal samples of black-tailed prairie dogs. We found that both GCs were present in similar concentrations in serum, however, corticosterone concentration in fecal samples was higher than cortisol. Likewise, biochemical validations performed in this study to test the assay reached acceptable levels of reliability. Therefore, we confirm that fecal analysis can be implemented as a method to measure stress responses in wild prairie dogs.

Serum and urine profiling by high-throughput TMT-based proteomics for the investigation of renal dysfunction in canine babesiosis

Canine babesiosis is a tick-borne disease caused by Babesia canis, with acute kidney injury as one of the common complications. In the study 8 healthy control dogs and 22 dogs with naturally occurring babesiosis were enrolled, with the aim to analyse differences in serum and urinary proteomes between healthy dogs and dogs with different degree of renal dysfunction in babesiosis using a label-based high-throughput quantitative proteomic approach. In serum, 58 proteins were found differentially abundant between healthy controls and groups of dogs with different degrees of renal dysfunction in babesiosis, while in urine there were 259 differentially abundant proteins. In addition, altered biological pathways were detected in the diseased dogs using bioinformatics tools and validation of several candidate biomarkers was performed. SignificanceThe main aim of this comprehensive study was to perform analyses of serum and urinary proteomes of dogs with renal dysfunction in babesiosis compared to healthy dogs using, for the first time, a high-throughput proteomic method and functional enrichment analyses. Serum and urine samples of the same dogs were investigated in order to gain a more complete picture of pathologic changes taking place in renal dysfunction in babesiosis. We highlighted two putative biomarkers validated herein which could be of importance for early diagnosis of renal dysfunction in canine babesiosis, as they are easily accessible from urine and their concentration rises before the appearance of azotaemia: urinary neutrophil gelatinase-associated lipocalin (NGAL) and urinary liver-type fatty acid-binding protein (L-FABP).

Rabies Virus Seroprevalence among Dogs in Limpopo National Park and the Phylogenetic Analyses of Rabies Viruses in Mozambique.

Rabies is considered a neglected disease among many developing Asian and African countries, including Mozambique, where its re-emergence is often attributed to low dog parenteral vaccination coverage. The objectives of this study were two-fold: (1) to assess the level of antibodies against rabies virus in dogs (n = 418) in Limpopo National Park (LNP), and (2) to genetically characterise selected rabies viruses from brain tissue samples collected in 2017 and 2018. To meet the first objective, we used the BioProTM Rabies blocking ELISA antibody kit, and the results were expressed as the percentage of blocking (%PB). Dog sera with PB ≥ 40% were considered positive for antibodies to rabies virus, whereas sera with PB < 40% were negative. Just under ninety percent (89.2%; n = 373) of dogs were seronegative, and the rest (10.8%; n = 45) had detectable levels of rabies virus-specific antibodies. All eight brain tissue samples were positive for rabies virus antigen using a direct fluorescent antibody test and amplified in a quantitative real-time PCR, but only five (n = 4 from dogs and n = 1 from a cat) were amplified in a conventional reverse-transcription PCR targeting partial regions of the nucleoprotein (N) and the glycoprotein (G) genes. All samples were successfully sequenced. Phylogenetically, the rabies viruses were all of dog origin and were very closely related to each other (Africa 1b rabies virus lineage). Furthermore, the sequences had a common progenitor with other rabies viruses from southern Africa, confirming the transboundary nature of rabies and the pivotal role of dogs in maintaining rabies cycles. The study demonstrates the principal application of the BioProTM rabies ELISA antibody for the detection of anti-lyssavirus-specific antibodies in the serum samples of dogs, and most importantly, it highlights the low levels of antibodies against rabies virus in this dog population.

Comparison of Immunodiagnostic Assays for the Rapid Diagnosis of Coccidioidomycosis in Dogs.

Coccidioidomycosis is a disease caused by the dimorphic fungi Coccidioides spp., which affects humans and a variety of animal species, including domestic dogs. In dogs, accurate diagnosis could provide a substantial improvement on the quality of canine life, as well as an advancement in the mapping of regions endemic for coccidioidomycosis. The purpose of this study was to compare immunodiagnostic assays for anti-Coccidioides antibody (Ab) detection in dogs’ serum. Three commercially available immunodiagnostic assays (IMMY®; Norman, OK, USA) were evaluated, including the sōna Coccidioides Ab Lateral Flow Assay (LFA), Coccidioides IDCF immunodiffusion assay (IDCF), and the Clarus Coccidioides Ab Enzyme Immunoassay (EIA). Assays were evaluated using 98 dog serum samples: 29 from dogs with coccidioidomycosis, 15 from dogs diagnosed with histoplasmosis, 10 from dogs diagnosed with blastomycosis, and 44 from dogs without a fungal disease. Using specimens from dogs with coccidioidomycosis, the IDCF had an accuracy of 92% (95% confidence interval [95% CI] = 85–96%), the EIA had an accuracy of 91% (95% CI = 83–96%), and the LFA displayed an accuracy of 82% (95% CI = 73–89%). Using Kappa analysis, the agreement between LFA and EIA was 0.59 (95% CI = 0.42–0.75), that between LFA and IDCF was 0.64 (95% CI = 0.48–0.79), and that between EIA and IDCF was 0.79 (95% CI = 0.64–0.90). Most cross-reactions were observed in dogs with histoplasmosis. Compared with EIA and IDCF, the LFA requires substantially less laboratory equipment and infrastructure and rapidly produces results, offering a substantial improvement for the initial screening of coccidioidomycosis in dogs.

Validation of a mixture of rK26 and rK39 antigens from Iranian strain of Leishmania infantum to detect anti-Leishmania antibodies in human and reservoir hosts

Mediterranean type of visceral leishmaniasis (VL) is a zoonotic parasitic infection. Some provinces of Iran are endemic for VL while other parts are considered as sporadic areas. This study aimed to assess a combination of recombinant K26 and rK39 antigens as well as crude antigen (CA), derived from an Iranian strain of L. infantum, compared to direct agglutination test (DAT) for the detection of VL in humans and domestic dogs as animal reservoir hosts of the disease. A combination of rK26 and rK39 antigens and also CA was evaluated using indirect ELISA on serum samples of 171 VL confirmed humans (n = 84) and domestic dogs (n = 87) as well as 176 healthy humans (n = 86) and domestic dogs (n = 90). Moreover, 36 serum samples of humans (n = 20) and canines (n = 16) with other potentially infectious diseases were collected and tested for finding cross- reactivity. The results of ELISA were compared to DAT, currently considered as gold standard for the serodiagnosis of VL. The sensitivity and specificity, positive predictive and negative predictive values were calculated compared to DAT. The positive sera had previously shown a positive DAT titer ≥ 1:800 for humans and ≥ 1:80 for dogs. Analysis was done by MedCalc and SPSS softwares. Using the combination of rK26 and rK39 in ELISA, a sensitivity of 95.2% and a specificity of 93.0% % were found in human sera at a 1:800 (cut-off) titer when DAT-confirmed cases were compared with healthy controls; a sensitivity of 98.9% and specificity of 96.7%% were found at a 1:80 (cut-off) titer compared with DAT. A good degree of agreement was found between the combined rK39 and rK26-ELISA with DAT in human (0.882) and dog serum samples (0.955) by kappa analysis (p < 0.05). The ELISA using the CA test showed 75% sensitivity in human and 93.1% in dog serum samples as well as 53.5% specificity in human and 83.3% in dog,s sera, respectively. The combination of rK26 and rK39 recombinant antigen prepared from Iranian strain of Leishmania infantum showed high accuracy for the serodiagnosis of VL in human and domestic dogs. Further extended field trial with a larger sample size is recommended.

Risk Factors for SARS-CoV-2 Infection and Illness in Cats and Dogs1.

We tested swab specimens from pets in households in Ontario, Canada, with human COVID-19 cases by quantitative PCR for SARS-CoV-2 and surveyed pet owners for risk factors associated with infection and seropositivity. We tested serum samples for spike protein IgG and IgM in household pets and also in animals from shelters and low-cost neuter clinics. Among household pets, 2% (1/49) of swab specimens from dogs and 7.7% (5/65) from cats were PCR positive, but 41% of dog serum samples and 52% of cat serum samples were positive for SARS-CoV-2 IgG or IgM. The likelihood of SARS-CoV-2 seropositivity in pet samples was higher for cats but not dogs that slept on owners’ beds and for dogs and cats that contracted a new illness. Seropositivity in neuter-clinic samples was 16% (35/221); in shelter samples, 9.3% (7/75). Our findings indicate a high likelihood for pets in households of humans with COVID-19 to seroconvert and become ill.