Evaluation of species-specific polyclonal antibodies to detect and differentiate between Neospora caninum and Toxoplasma gondii.

Neosporosis and toxoplasmosis are major causes of abortion in livestock worldwide, leading to substantial economic losses. Detection tools are fundamental to the diagnosis and management of those diseases. Current immunohistochemistry (IHC) tests, using sera raised against whole parasite lysates, have not been able to distinguish between Toxoplasma gondii and Neospora caninum. We used T. gondii and N. caninum recombinant proteins, expressed in Escherichia coli and purified using insoluble conditions, to produce specific polyclonal rabbit antisera. We aimed to develop species-specific sera that could be used in IHC on formalin-fixed, paraffin-embedded (FFPE) tissue sections to improve the diagnosis of ruminant abortions caused by protozoa. Two polyclonal rabbit sera, raised against recombinant proteins, anti-Neospora-rNcSRS2 and anti-Toxoplasma-rTgSRS2, had specificity for the parasite they were raised against. We tested the specificity for each polyclonal serum using FFPE tissue sections known to be infected with T. gondii and N. caninum. The anti-Neospora-rNcSRS2 serum labeled specifically only N. caninum-infected tissue blocks, and the anti-Toxoplasma-rTgSRS2 serum was specific to only T. gondii-infected tissues. Moreover, tissues from 52 cattle and 19 sheep previously diagnosed by lesion profiles were tested using IHC with our polyclonal sera and PCR. The overall agreement between IHC and PCR was 90.1% for both polyclonal anti-rNcSRS2 and anti-rTgSRS2 sera. The polyclonal antisera were specific and allowed visual confirmation of protozoan parasites by IHC, but they were not as sensitive as PCR testing.

Infectious sporadic bovine abortions: retrospective analysis.

Infectious sporadic abortions in cattle are mainly caused by opportunistic bacteria and fungi usually present in environmental or gastrointestinal and reproductive microbiota of healthy animals. A retrospective analysis was carried out to evaluate the main opportunistic microorganisms involved in bovine abortions recorded at INTA Balcarce (Argentina) from 1997 to 2023, accounting for 2.2% of the total diagnosed etiologies of bovine abortion. The opportunistic agents identified as the cause of abortion in 29 fetuses were bacteria (90%) and fungi (10%). Escherichia coli (n = 8), Trueperella pyogenes (n = 5), and Histophilus somni (n = 4) were the bacterial species most often identified as causing infectious abortions, whereas Aspergillus spp. (n = 3) was implicated in all fungal abortions identified. Pure culture of bacteria or fungus was achieved from abomasal content and/or lung essential. Main microscopic findings were bronchopneumonia, myo- and epicarditis, meningitis, and portal hepatitis. Herein, we highlight the importance of detecting potential infectious bacteria in cultures to improve etiological diagnosis of bovine abortions associated with compatible microscopic findings to confirm the etiology.

Pollen beetle (Astylus atromaculatus)-associated gastroenteric disease in cattle: report of 6 natural outbreaks.

Astylus atromaculatus is a pollen beetle native to South America, commonly found in crop flowers. Experimental intoxication of sheep and guinea pigs by this beetle resulting in fibrinonecrotizing enteritis has been reported. We describe here 6 natural outbreaks of intoxication in cattle associated with consumption of alfalfa (5 of 6) and mixed native (1 of 6) pastures heavily contaminated with A. atromaculatus. The outbreaks occurred during the summer (January-February) of 2023 in Argentina (n = 4) and Uruguay (n = 2), in beef cattle under extensive or semi-extensive rearing systems, with overall cumulative incidence and mortality of 22.3% and 17.8%, respectively. The main clinical signs included acute onset of anorexia, lethargy, hyperthermia, hindlimb weakness, reluctance to move, and diarrhea, for up to 15 d. In 2 outbreaks, sudden death was observed. Eight Hereford, Angus, and/or crossbreed heifers, cows, steers, and/or calves were autopsied. Gross and microscopic findings included multifocal necrosis with fibrinous pseudomembranes in the forestomachs and/or small and large intestines. Fragments or whole specimens of A. atromaculatus were identified in the ruminal content of all animals. Testing for multiple gastroenteric pathogens was negative as was testing of A. atromaculatus for cantharidin and batrachotoxin. GC-MS and LC-MS/MS performed on the beetles did not identify any known toxic compounds. Based on the exposure to A. atromaculatus-contaminated pasture, gross and microscopic lesions, and negative results of all testing for multiple gastroenteric pathogens, a diagnosis of intoxication by A. atromaculatus is proposed. Disease caused by A. atromaculatus consumption has not been reported previously in cattle, to our knowledge.

Amaranthus hybridus (syn. quitensis) intoxication in cattle in Argentina: Case report.

Amaranthus spp. is a nephrotoxic plant with unknown toxic principle, affecting production animals worldwide, mainly in South America. The aim of this paper is to describe 5 spontaneous outbreaks of A. hybridus intoxication in beef cattle, where 7 autopsies were performed. Main gross findings were pale diffuse and enlarged kidneys. Microscopically, kidneys were characterized by severe tubular acute to subacute nephrosis, with dilatated tubules showing different degrees of epithelial degeneration and necrosis, and containing intraluminal eosinophilic hyaline casts. Intratubular birefringent crystals, compatible with oxalate, were observed under polarized light in kidneys from 3 autopsies. Positive von Kossa and red alizarin S staining confirmed the intratubular crystals as calcium deposits. This intoxication occurs mainly in stubble paddocks during summer and early autumn. The data from the present study suggests that oxalates were related to nephrotoxicity due to Amaranthus consumption.

Transplacental infection by bovine alphaherpesvirus type 1 induces protein expression of COX-2, iNOS and inflammatory cytokines in fetal lungs and placentas.

Bovine alphaherpesvirus type 1 (BoAHV-1) is associated with respiratory and reproductive syndromes. Until present the immunologic mechanisms involved in BoAHV-1 abortion are partially known. We studied key elements of the innate immune response in the placentas and fetal lungs from cattle experimentally-inoculated with BoAHV-1. These tissues were analyzed by histopathology. Furthermore, virus identification was performed by qPCR and the expression of the inflammatory cytokines such as tumor necrosis factor-alpha, interleukin 1-alpha and inflammatory mediators like inducible nitric oxide synthase and cyclooxeganse-2 was evaluated by immunohistochemistry. The viral transplacental infection was confirmed by the detection of BoAHV-1 by qPCR in the placenta and fetal organs, which revealed mild inflammatory lesions. Inducible nitric oxide synthase immunolabelling was high in the lungs of infected fetuses and placentas, as well as for tumor necrosis factor-alpha in the pulmonary parenchyma and cyclooxeganse-2 in fetal annexes. However, the expression of interleukin 1-alpha was weak in these organs. To our knowledge, this is the first study that provides strong evidence of an early immune response to BoAHV-1 infection in the conceptus. Advances in the knowledge of the complex immunological interactions at the feto-maternal unit during BoAHV-1 infection are needed to clarify the pathogenesis of abortion.

Identification and molecular characterization of Orfvirus infection in occupationally exposed women inSouth America

Abstract Contagious Ecthyma (CE) is a severe exanthematous dermatitis caused by the Orf virus (ORFV) that mainly affects domestic small ruminants such as sheep and goats. It is a worldwide-distributed occupational zoonosis, particularly infecting those in close contact with animals or animal products such as shepherds, farmers and veterinarians, among others. In the present work, we report the first human CE case confirmed in Argentina. A phylogenetic analysis based on four gene sequences of the isolated strain responsible for the disease showed that this isolate grouped with other ORFV sequences that caused reported CE cases in sheep from the same Argentine province. We also sequenced a sample from a Chilean human case reported in 2017, whose phylogenetic analysis showed that it groups together with other Argentine isolates from locations close to the border with Chile. Keywords: Contagious Ecthyma; Dermatitis; Human Orf; Zoonosis; Molecular characterization.

Resumen El ectima contagioso (EC) es una dermatitis exantemática grave causada por el virus Orf (ORFV), que afecta mayormente a pequeños rumiantes domésticos, como ovinos y caprinos. Es una zoonosis ocupacional con distribución mundial, infecta a humanos en estrecho contacto con animales o sus productos, como granjeros, esquiladores y veterinarios, entre otros. En este trabajo se informa el primer caso humano de EC confirmado en Argentina. Un análisis filogenético basado en cuatro genes de la cepa responsable de este caso mostró que el aislamiento agrupa con otras secuencias de ORFV que causaron casos en ovinos en la misma provincia argentina. También se secuenció una muestra del caso de ectima humano reportado en Chile en 2017 y el análisis filogenético mostró que dicho aislamiento forma un grupo con otros aislamientos argentinos de localidades cercanas a la frontera con Chile. Palabras clave: Ectima contagioso; Dermatitis; Orf en humanos; Zoonosis; Caracterización molecular.

Equine abortion and stillbirth in California: a review of 1,774 cases received at a diagnostic laboratory, 1990-2022.

Reproductive failure represents an important cause of economic loss for the equine industry. We reviewed the cases of equine abortion and stillbirth submitted to the California Animal Health and Food Safety Laboratory System, University of California-Davis from 1990 to 2022. A total of 1,774 cases were reviewed. A confirmed cause of abortion was determined in 29.2% of the cases. Abortion or stillbirth was attributed to infectious agents in 18.7% of the cases, with Streptococcus spp., equine herpesvirus 1, and Leptospira spp. being the most prevalent. Noninfectious causes of abortion were established in 10.5% of the cases, with umbilical cord torsion being the most common. In 70.8% of the cases, a definitive cause of abortion could not be established. Our study demonstrated the difficulties in establishing an etiologic diagnosis, even when following a standard diagnostic work-up. New diagnostic approaches are needed to improve the likelihood of reaching a final diagnosis in cases of equine abortion and stillbirth.

Identification and molecular characterization of Orf virus infection in occupationally exposed women in South America.

Contagious Ecthyma (CE) is a severe exanthematous dermatitis caused by the Orf virus (ORFV) that mainly affects domestic small ruminants such as sheep and goats. It is a worldwide-distributed occupational zoonosis, particularly infecting those in close contact with animals or animal products such as shepherds, farmers and veterinarians, among others. In the present work, we report the first human CE case confirmed in Argentina. A phylogenetic analysis based on four gene sequences of the isolated strain responsible for the disease showed that this isolate grouped with other ORFV sequences that caused reported CE cases in sheep from the same Argentine province. We also sequenced a sample from a Chilean human case reported in 2017, whose phylogenetic analysis showed that it groups together with other Argentine isolates from locations close to the border with Chile.

Mycoplasma bovis-pneumonia and polyarthritis in feedlot calves in Argentina: First local isolation / Neumonía y poliartritis por Mycoplasma bovis en terneros de feedlot: primeraislamiento local

Abstract Bovine respiratory disease (BRD) is one of the most frequent clinical concerns inweaned calves after their arrival at the feedlot. This work reports the first local isolation ofMycoplasma bovis from feedlot calves with pneumonia and polyarthritis in Argentina. Twentyfour out of 545 calves showed progressive, subacute to chronic respiratory distress, coughing,and fever. Thirty percent of the affected calves also showed lameness and swelling of elbowor carpal, and knee or tarsal joints. Five necropsies were performed and severe multifocal tocoalescent pulmonary nodules, containing white-yellowish caseous exudate encircled by fibroustissue, and fibrinonecrotic arthritis and tenosynovitis were detected. Mycoplasma was isolatedfrom lung and joint samples. The 16S-23S rRNA ITS consensus sequence obtained from theseisolates showed 100% similarity with the same region of M. bovis strains. Since there are no com-mercially available vaccines in the region for the prevention and control of M. bovis pneumoniaand arthritis, surveillance is a priority to reduce the source of disease to naïve animals.

Resumen La enfermedad respiratoria bovina es uno de los problemas sanitarios más fre-cuentes en terneros recién destetados luego de su arribo a los corrales de encierre (feedlots).Este trabajo describe el primer aislamiento local de Mycoplasma bovis de terneros de feedlotcon neumonía y poliartritis en Argentina. Se vieron afectados 24 de 545 terneros; mostraron enfermedad respiratoria progresiva, subaguda a crónica, tos y fiebre. De los terneros afecta-dos, el 30% presentó, además, problemas en articulaciones carpales o tarsales. Se realizaron5 necropsias de terneros afectados y se observaron nódulos pulmonares multifocales a coa-lescentes, que contenían un exudado caseoso blanco-amarillento, rodeados de tejido fibroso,artritis y tenosinovitis fibrinonecrótica. Se aisló Mycoplasma de muestras de pulmón y artic-ulación. La secuencia consenso del gen codificante del ARNr 16S-23S rRNA obtenido de estosaislamientos mostró un 100% de similitud con la misma región de cepas de M. bovis. Teniendoen cuenta que no hay vacunas disponibles comercialmente en la región para la prevencióny el control de neumonías y poliartritis por M. bovis, es importante realizar una vigilanciaepidemiológica a fin de reducir las fuentes de infección para animales susceptibles.

A review of cardiac blackleg in cattle, and report of 2 cases without skeletal muscle involvement in Argentina.

Blackleg is an infectious disease caused by Clostridium chauvoei. Cardiac blackleg has been reported in ruminants as an uncommon presentation of the disease; its pathogenesis is not understood completely. We include here a literature review of cardiac blackleg and a description of 2 cases in 12-15-mo-old feedlot steers in Argentina. Fourteen of 1,190 steers died suddenly over a period of 10 d. Postmortem examinations were performed on 5 of these animals. Grossly, severe, diffuse, fibrinous pericarditis and pleuritis, multifocal necrohemorrhagic myocarditis, diffuse pulmonary congestion, mild splenomegaly, and moderate congestion of meningeal vessels were observed. No significant gross lesions were observed in the skeletal muscles of any animal. Histology was performed on 2 of the steers. The main microscopic features were necrotizing myocarditis with myriad intralesional gram-positive rods with subterminal spores plus fibrinosuppurative pericarditis and pleuritis. C. chauvoei was detected by immunohistochemistry and PCR in the myocardium of both animals. These findings confirm a diagnosis of cardiac blackleg in these 2 steers and presumptively in the other affected animals.

Reactivation and Foetal Infection in Pregnant Heifers Infected with Neospora caninum Live Tachyzoites at Prepubertal Age.

Neospora caninum is recognised for causing cattle abortion, provoking severe economic losses in the livestock industry worldwide. The aim of the present study was to evaluate the reactivation and foetal infection in pregnant heifers inoculated with live N. caninum tachyzoites before puberty. A total of 15 30-month-old pregnant heifers were allocated into four groups: animals inoculated with live tachyzoites of NC-Argentina LP1 isolate before puberty and challenged with live tachyzoites of NC-1 strain at 210 days of gestation (DG) (Group A); animals mock inoculated before puberty and challenged with NC-1 strain at 210 DG (Group B), animals inoculated before puberty but not subsequently challenged (Group C); and noninfected and nonchallenged animals (Group D). The results of this study showed that 100% of animals infected before puberty (Groups A and C) suffered reactivation of the infection at the seventh month of gestation. In addition, in three and two calves from Groups A and C, respectively, congenital infection was confirmed. Interestingly, we provide evidence that the use of live N. caninum tachyzoites in young animals as a strategy to induce protection is neither safe nor effective.

Spatial-temporal trends and economic losses associated with bovine abortifacients in central Argentina.

The aims of this work are, firstly, to provide the geolocalization of cases of bovine abortion with definitive diagnosis and, secondly, to estimate the economic losses due to the most frequent abortifacients diagnosed agents in cattle in Buenos Aires province, Argentina. The total beef and dairy cattle population at risk of abortion is 8,358,186 and 538,076, respectively. In beef cattle, the overall risk of abortion was estimated at 4.5% for all pregnancies, where 27.9% are due to Campylobacter fetus, Neospora caninum, Leptospira spp., Brucella abortus, and bovine viral diarrhea virus with economic losses of US$ 440 per abortion, being the annual loss to the beef industry of US$ 50,144,101. In dairy cattle, there was an 8.0% risk of suffering abortion, 26.1% produced by the same abortigenic agents. The economic losses were estimated at US$ 1,415 per abortion, which equals a total loss of US$ 17,298,498 for the dairy industry in the region. The results of this study show that infectious causes are highly prevalent in Buenos Aires province, and they caused severe economic impacts in the dairy and beef industries. Furthermore, changes in temporal trends of infectious abortion occurrence were detected, probably related to the inclusion of molecular diagnostic techniques with more sensitivity or different epidemiological or husbandry conditions in the region analyzed.

Mycoplasma bovis-pneumonia and polyarthritis in feedlot calves in Argentina: First local isolation.

Bovine respiratory disease (BRD) is one of the most frequent clinical concerns in weaned calves after their arrival at the feedlot. This work reports the first local isolation of Mycoplasma bovis from feedlot calves with pneumonia and polyarthritis in Argentina. Twenty four out of 545 calves showed progressive, subacute to chronic respiratory distress, coughing, and fever. Thirty percent of the affected calves also showed lameness and swelling of elbow or carpal, and knee or tarsal joints. Five necropsies were performed and severe multifocal to coalescent pulmonary nodules, containing white-yellowish caseous exudate encircled by fibrous tissue, and fibrinonecrotic arthritis and tenosynovitis were detected. Mycoplasma was isolated from lung and joint samples. The 16S-23S rRNA ITS consensus sequence obtained from these isolates showed 100% similarity with the same region of M. bovis strains. Since there are no commercially available vaccines in the region for the prevention and control of M. bovis pneumonia and arthritis, surveillance is a priority to reduce the source of disease to naïve animals.

Iatrogenic doramectin overdosing causes toxicity in sheep: A case report.

Macrocyclic lactones are widely used endectocides in ruminants, with a high margin of safety for labeled indications. No previous report of iatrogenic doramectin overdosing has been published. We report an outbreak in a sheep flock in Northeast Patagonia, Argentina. Toxicity signs were observed in more than 10% and 59% of ewes and lambs, respectively, particularly those with low body condition, treated with doramectin 3.5% long-acting injectable formulation, presumably at the indicated dose of 700 µg/kg. Clinical signs included lethargy, mydriasis and coma. Doramectin concentration in blood samples was 826.8 (±119.3) ng/ml. Cerebrospinal fluid (CSF) and liver doramectin concentration in euthanized lambs were 3.26-4.28 ng/ml and 8506-8772 ng/g, respectively. Epidemiological and clinical information, and high doramectin concentration were sufficient to confirm the neurotoxicity. Scarce fat deposition could have altered doramectin pharmacokinetic which may have accounted for the observed neurotoxicity. Special care should be taken when animals under similar nutritional conditions are treated with macrocyclic lactones.

Epidemic abortions due to Neospora caninum infection in farmed red deer (Cervus elaphus).

This study describes for the first time an abortion outbreak caused by Neospora caninum in farmed red deer. During a 5-year period, farmed hinds, naturally mated, were regularly ultrasound monitored to detect reproductive losses over their gestation. During the 4 years previous to the outbreak, abortion rates ranged from 4.7 to 8.6% (average 6.5%), and serology for indirect diagnosis of neosporosis and toxoplasmosis was performed. At the fifth year, the abortion rate increased to 25.3%. During this outbreak, three aborted foetuses and their placentas were recovered and submitted to laboratory for etiological diagnosis. Blood samples were collected from the 81 hinds at the end of the gestational period and the seropositivity rate for N. caninum, Toxoplasma gondii, Brucella abortus, bovine viral diarrhoea virus and bovine alphaherpesvirus type 1 was 66.7%, 67.9%, 0.0%, 8.6% and 0.0%, respectively. Neospora caninum-seropositive hinds (OR = 5.7, P = 0.0271) and hinds with high antibody titres to N. caninum (OR = 7.4, P = 0.0130) were more likely to abort than seronegative hinds. In addition, N. caninum seropositivity rate in the aborted hinds was higher (OR = 5.4, P = 0.033) than the non-aborted hinds. No association was found between T. gondii nor BVDV-seropositivity and abortions. Typical protozoal histopathologic findings (necrotizing non suppurative encephalitis, meningitis, myocarditis, hepatitis, among others) were observed in all foetuses. Neospora caninum was immunolabelled by immunohistochemistry in several tissues from two foetuses, and infection was also confirmed in the three foetuses by serology and/or DNA detection. No other abortifacient agent was detected in the foetuses. Their dams showed high N. caninum antibody titres (≥ 6400). Serologic evidence and epidemiological data recorded suggested a point-source of N. caninum infection before the occurrence of the outbreak, probably related with contaminated feedstuff with oocysts. Moreover, the intensive production system with a high stocking rate could be also considered a factor which might have increased the risk of horizontal N. caninum infection in this herd.

Ruminal effects of excessive dietary sulphur in feedlot cattle.

Sulphur (S) dietary excess can limit productive performance and increase polioencephalomalacia (PEM) incidence in feedlot cattle (FC). Sulphur excess ingested is transformed to hydrogen sulphide (H2 S) by sulfo-reducing ruminal bacteria (SRB), being high ruminal H2 S concentration responsible for aforementioned damages. As the ruminal mechanisms involved in H2 S concentrations increase have not been elucidated, this study aimed to evaluate the ruminal environment, and the association between ruminal H2 S and dissimilatory SRB (DSRB) concentration in FC experimentally subjected to S dietary excess. Twelve crossbred steers were randomly assigned to one of two dietary S levels (6 animals per treatment): low (LS, 0.19% S) and high (HS, 0.39% S obtained by sodium sulfate inclusion at 0.86%). The study lasted 38 days, and on days 0, 22 and 38, ruminal gas samples were taken to quantify H2 S concentration, and ruminal fluid to determine total bacteria, DSRB, protozoa, volatile fatty acid and ammonia nitrogen concentration. For ruminal H2 S concentration, S dietary × sampling day interaction was significant (p < 0.001), so that the greater concentration was observed on days 22 and 38 with the HS diet. The remaining ruminal parameters were not affected by dietary S level, and no significant correlation between H2 S and DSRB concentrations was observed. The ruminal adaptation that maximizes H2 S production in FC consuming S excess does not seem to be associated with biological or biochemical alterations, nor DSRB concentration changes. The microbial diversity and ruminal environment were resilient to the S excess evaluated, suggesting that 0.39% of dietary S achieved by 0.86% sodium sulfate addition, could be used without disturbances on digestion nor health of FC.

Morphometric study of encephalic lesions in aborted bovine fetuses naturally infected by two subpopulations of Neospora caninum.

Neospora caninum is a major reproductive disease in cattle worldwide. In the Argentinian Humid Pampa, the seroprevalence, incidence of abortions, and economic losses due to neosporosis are considerably higher in dairy than in beef cattle. Despite this, we recently demonstrated that N. caninum subpopulations are indistinctly distributed in both dairy and beef production systems. The association between genotypic characteristics defined by microsatellite analysis and the virulence of the different strains-particularly with regard to the severity and extension of histological lesions-is largely unknown. Herein, we used a morphometric approach to analyze encephalic lesions in 62 bovine fetuses spontaneously infected by N. caninum. Morphometric parameters (average size of focal lesions, number of foci/cm2 and the percentage of the section affected by lesions) were compared according to the N. caninum subpopulations found in our previous microsatellite genotyping analysis, animal biotype (beef versus dairy), and fetal age (second stage of gestation versus third stage). The average size of the lesions differed significantly among fetuses with different gestational ages; however, no significant differences among animal biotypes or genotypic patterns were found. Further research into the genetic, molecular, and husbandry factors that could account for this greater impact in Argentinian dairy herds is needed.

Retrospective analysis of hairy vetch (Vicia villosa roth) poisoning in Argentina (2004-2019).

Hairy vetch (Vicia villosa) poisoning in cattle is characterized by a systemic granulomatous inflammatory response that resembles a type-IV hypersensitivity reaction. Hairy vetch toxicity has been described in cattle worldwide. The aim of this paper was to describe 10 outbreaks of hairy vetch poisoning in cattle studied at INTA EEA Salta and INTA EEA Balcarce, Argentina, from 2004 to 2019. Clinical signs included weakness, pyrexia, dermatitis, alopecia, and progressive weight loss, which leads to death over a clinical course of approximately two weeks. A total of 12 necropsies were performed and tissue samples were collected for histopathology. The main gross changes were observed in skin, lymph nodes, liver, heart, spleen and kidneys. Other tissues, such as pancreas, thyroid, and adrenal glands, were also affected. Histological lesions consisted of multifocal to diffuse granulomatous inflammation in those organs. The toxicity of hairy vetch has been described in several countries of the world. In Argentina, the use of hairy vetch as a cover crop has become common in some regions during the past years. The data suggest that hairy vetch poisoning is an important disease in cattle. More studies are needed to contribute with further information.