Experimental oral administration of pollen beetle (Astylus atromaculatus) to cattle results in an acute lethal gastrointestinal disease.

In the summer of 2023, ingestion of Astylus atromaculatus (pollen beetle) was linked to spontaneous fatal disease in grazing cattle and sheep in Argentina and Uruguay. While the disease was experimentally reproduced in sheep and guinea pigs in the 1970's, no experimental reproductions have been attempted in cattle, and controversy exists as to whether this insect is indeed noxious to cattle and at which dose. Here, we demonstrate that A. atromaculatus causes acute fatal disease in Hereford calves at single oral dosages of 2.5, 4.5, 10.0, and 15.0 g of insect/kg body weight. Death or severe disease necessitating euthanasia occurred at 38 to 48 hours postinoculation regardless of the dose, suggesting that the single fatal dosage is likely <2.5 g/kg body weight (this dose representing approximately 850 mL of intact beetles in a 100 kg calf). Clinically, the disease was characterized by acute anorexia, prolonged recumbency, reluctance to move, listlessness/apathy, depression, ruminal hypomotility and tympany, hypothermia, bruxism with frothing at the mouth, and mucoid diarrhea progressing to death. Hematologic and biochemical alterations included hemoconcentration, stress/acute inflammatory leukogram, negative energy balance, and ketosis. The pathological hallmark of this experimental disease is acute necrotizing omaso-reticulo-rumenitis, fibrinohemorrhagic enteritis, and exfoliative colitis with intralesional chitinous insect fragments. While A. atromaculatus might contain a gastrointestinal toxin or pathogen, extensive toxicological testing failed to identify a causative toxin. Other pathomechanisms such as direct physical damage caused by insect fragments on the alimentary tract seem plausible, although further studies are needed to elucidate the pathogenesis of A. atromaculatus-associated disease.

Pathologic and Immunohistochemical Evidence of Possible Francisellaceae among Aborted Ovine Fetuses, Uruguay.

The only genus of the Francisellaceae family known to contain species pathogenic to mammals is Francisella, for which reported cases in the Southern Hemisphere have been limited to Australia. We describe severe necrotizing and inflammatory lesions and intralesional immunohistochemical identification of Francisella sp. lipopolysaccharide among aborted ovine fetuses in Uruguay.

Experimental acute Clostridium perfringens type D enterotoxemia in sheep is not characterized by specific renal lesions.

Type D enterotoxemia, caused by Clostridium perfringens epsilon toxin (ETX), is one of the most economically important clostridial diseases of sheep. Acute type D enterotoxemia is characterized by well-documented lesions in the nervous, cardiocirculatory, and pulmonary systems. However, discrepancies and confusion exist as to whether renal lesions are part of the spectrum of lesions of this condition, which is controversial considering that for many decades it has been colloquially referred to as "pulpy kidney disease." Here, the authors assess renal changes in an experimental model of acute type D enterotoxemia in sheep and evaluate the possible role of ETX in their genesis. Four groups of 6 sheep each were intraduodenally inoculated with either a wild-type virulent C. perfringens type D strain, an etx knockout mutant unable to produce ETX, the etx mutant strain complemented with the wild-type etx gene that regains the ETX toxin production, or sterile culture medium (control group). All sheep were autopsied less than 24 hours after inoculation; none of them developed gross lesions in the kidneys. Ten predefined histologic renal changes were scored in each sheep. The proportion of sheep with microscopic changes and their severity scores did not differ significantly between groups. Mild intratubular medullary hemorrhage was observed in only 2 of the 12 sheep inoculated with the wild-type or etx-complemented bacterial strains, but not in the 12 sheep of the other 2 groups. The authors conclude that no specific gross or histologic renal lesions are observed in sheep with experimental acute type D enterotoxemia.

Molecular characterization of multidrug-resistant Escherichia coli of the phylogroups A and C in dairy calves with meningitis and septicemia.

Escherichia coli is an important cause of septicemia (SEPEC) and neonatal meningitis (NMEC) in dairy calves. However, the diversity of virulence profiles, phylogroups, antimicrobial resistance patterns, carriage of integron structures, and fluoroquinolone (FQ) resistance mechanisms have not been fully investigated. Also, there is a paucity of knowledge about the virulence profiles and frequency of potential SEPEC in feces from calves with or without diarrhea. This study aimed to characterize the virulence potential, phylogroups, antimicrobial susceptibility, integron content, and FQ-resistance mechanisms in Escherichia coli isolated from calves with meningitis and septicemia. Additionally, the virulence genes (VGs) and profiles of E. coli isolated from diarrheic and non-diarrheic calves were compared between them and together with NMEC and SEPEC in order to identify shared profiles. Tissue and fluid samples from eight dairy calves with septicemia, four of which had concurrent meningitis, were processed for bacteriology and histopathology. Typing of VGs was assessed in 166 isolates from diverse samples of each calf. Selected isolates were evaluated for antimicrobial susceptibility by the disk diffusion test. Phylogroups, integron gene cassettes cartography, and FQ-resistance determinants were analyzed by PCR, sequencing, and bioinformatic tools. Furthermore, 109 fecal samples and 700 fecal isolates from dairy calves with or without diarrhea were evaluated to detect 19 VGs by uniplex PCR. Highly diverse VG profiles were characterized among NMEC and SEPEC isolates, but iucD was the predominant virulence marker. Histologic lesions in all calves supported their pathogenicity. Selected isolates mainly belonged to phylogroups A and C and showed multidrug resistance. Classic (dfrA17 and arr3-dfrA27) and complex (dfrA17-aadA5::ISCR1::blaCTX-M-2) class 1 integrons were identified. Target-site mutations in GyrA (S83L and D87N) and ParC (S80I) encoding genes were associated with FQ resistance. The VGs detected more frequently in fecal samples included f17G (50%), papC (30%), iucD (20%), clpG (19%), eae (16%), and afaE-8 (13%). Fecal isolates displaying the profiles of f17 or potential SEPEC were found in 25% of calves with and without diarrhea. The frequency of E. coli VGs and profiles did not differ between both groups (p > 0.05) and were identical or similar to those found in NMEC and SEPEC. Overall, multidrug-resistant E. coli isolates with diverse VG profiles and belonging to phylogroups A and C can be implicated in natural cases of meningitis and septicemia. Their resistance phenotypes can be partially explained by class 1 integron gene cassettes and target-site mutations in gyrA and parC. These results highlight the value of antimicrobial resistance surveillance in pathogenic bacteria isolated from food-producing animals. Besides, calves frequently shed potential SEPEC in their feces as commensals ("Trojan horse"). Thus, these bacteria may be disseminated in the farm environment, causing septicemia and meningitis under predisposing factors.

[Neurotropic bovine astrovirus-associated encephalitis: An underdiagnosed disease in South America?] / Encefalitis asociada a astrovirus bovino neurotrópico, ¿una enfermedad subdiagnosticada en Sudamérica?

We describe a case of neurotropic bovine astrovirus-associated encephalitis in a Jersey dairy cow from the department of San José, Uruguay. This represents the second case of this condition reported in the Southern Hemisphere. The cow was the only one affected in a herd of 70 cows, showing neurological signs with a 2-day clinical course, before dying spontaneously. Histopathological examination revealed lymphocytic, histiocytic, and plasmacytic meningoencephalitis with neuronal necrosis, without detectable inclusion bodies. Other infectious agents, including Rabies virus(Lyssavirus), Bovine alphaherpesvirus-1 and Bovine alphaherpesvirus-5(Varicellovirus), Bovine viral diarrhea virus(Pestivirus), West Nile virus(Flavivirus), Listeria monocytogenes, Histophilus somni and other bacteria, were not detected in the brain. We propose that given the recent discovery of neurotropic astroviruses in various mammalian species, including humans, cases of astrovirus encephalitis may have gone undetected in South America. We briefly discuss the differential pathologic diagnosis of infectious bovine encephalitis.

Placentitis and abortion caused by a multidrug resistant strain of Campylobacter fetus subspecies fetus in a sheep in Uruguay.

Campylobacter fetusfetus (Cff) is a major infectious cause of abortion in sheep worldwide, and an opportunistic human pathogen. Information on Cff as an ovine abortifacient in South America is limited. We describe a case of abortion caused by a multidrug resistant strain of Cff in a sheep in Uruguay. In August 2017, 3/57 pregnant ewes (5.3%) aborted whithin one week. Histopathologic examination of the placenta of an aborted ewe revealed severe neutrophilic and fibrinonecrotizing placentitis with vasculitis and thrombosis of the chorionic arterioles. Cff was isolated on microaerobic culture in Skirrow agar, and further confirmed by 16S rDNA PCR amplification and sequencing, and endpoint and real time PCR assays. Antimicrobial sensitivity testing revealed resistance to tetracyclines, nalidixic acid, telithromycin and clindamycin. Other abortifacients were not detected. Further studies are necessary to determine the geographic distribution, ecology, epidemiology, economic impact, and antimicrobial resistance of Cff in sheep flocks in Uruguay.

Molecular detection of coccidian Apicomplexa Parasites isolated from wild crab-eating and pampas foxes through novel TaqMan™ probes: a contribution to their molecular epidemiology.

Neospora caninum, Toxoplasma gondii and Hammondia spp. are coccidian parasites similar in morphology. Molecular techniques are necessary to detect parasite DNA isolated from stool samples in wild canids because they were reported as definitive hosts of N. caninum life cycle. The objective of this study was to develop a highly sensitive and accurate molecular method for the identification of coccidian Apicomplexa parasites in crab-eating fox (Cerdocyon thous) and pampas fox (Lycalopex gymnocercus). Tissue samples from road-killed animals (pampas fox = 46, crab-eating fox = 55) and feces (pampas fox = 84, crab-eating fox = 2) were collected, and species were diagnosed through molecular assay. PCR was used for the amplification of a fragment of the coccidian Apicomplexa nss-rRNA gene. Additionally, we developed a novel real-time PCR TaqMan™ probe approach to detect T. gondii- Hammondia spp. and N. caninum. This is the first report of N. caninum DNA in pampas fox feces (n = 1), thus it was also detected from pampas fox tissues (n = 1). Meanwhile, T. gondii was found in tissues of pampas (n = 1) and crab-eating (n = 1) foxes and H. triffittae in one crab-eating fox tissue. Despite the low percentage (2.5%) of positive samples, the molecular method developed in this study proved to be highly sensitive and accurate allowing to conduct an extensive monitoring analysis for these parasites in wildlife.

Cardiopulmonary Lesions in Sheep Produced by Experimental Acute Clostridium Perfringens Type D Enterotoxemia.

Enterotoxemia caused by Clostridium perfringens type D is one of the most prevalent clostridial diseases of sheep. The lesions of the acute form of this disease, particularly the cerebral lesions, are well characterized; however, detailed descriptions of the cardiac and pulmonary lesions are lacking. Here we describe cardiopulmonary lesions in experimental acute type D enterotoxemia in sheep and determine the role of epsilon toxin (ETX) in the development of these lesions. Four groups of 6 sheep were intraduodenally inoculated with either a wild-type C. perfringens type D strain; its etx knockout mutant, which is unable to produce ETX; the etx mutant complemented with the wild-type etx gene, which regains the ETX toxigenic ability; or sterile culture medium as a control. All sheep were subjected to postmortem examination within 24 hours of inoculation. Lesion scores were compared between groups for pulmonary edema; hydrothorax; ascites; hydropericardium; endocardial, myocardial and epicardial hemorrhages; microscopic lesions of acute myocardial degeneration and necrosis; and myocardial, endocardial, and epicardial edema, hemorrhage, and inflammation. Only sheep inoculated with the wild-type and complemented ETX-toxigenic bacterial strains developed cardiopulmonary lesions, which were present in varying degrees of severity and proportions. These lesions were not present in sheep inoculated with the etx mutant or in the negative control. We conclude that severe acute cardiopulmonary lesions in sheep with experimental enterotoxemia are associated with the capacity of the strains to produce ETX. These changes are likely contributors to the clinical signs and even death of affected animals.

[Outbreak of bovine paralytic rabies in a disease-free region of Argentina]. / Brote de rabia paresiante bovina en la región libre de enfermedad en la Argentina.

We report an outbreak of bovine paralytic rabies in a feed lot in a disease-free area in southern Santa Fe, Argentina. Six of 122 unvaccinated steers died after showing neurological signs (morbimortality = 4.9%) for 24-72 hours, 40-75 days after being transported to the feed lot from a farm located in Formosa. Pathologic examination of the brain in 3 steers revealed gliosis, lymphoplasmacytic meningoencephalitis, and intracytoplasmic inclusion bodies in neurons, along with intralesional detection of the rabies virus antigen by immunohistochemistry in 2 cases. Rabies virus was confirmed by direct immunofluorescence in the brain, and further identified as variant 3a, typical of hematogenous bats, by inoculation in mice followed by indirect immunofluorescence. This represents the first communication of bovine paralytic rabies described in the Pampas plain in Argentina, and evidences that the transport of cattle from endemic to disease-free areas could represent a mechanism of dissemination of this communicable zoonotic disease.

Virulence genes of Escherichia coli in diarrheic and healthy calves.

Escherichia coli ETEC, EPEC, NTEC and STEC/EHEC pathotypes are often isolated from bovine feces. The objective of this study was to detect 21 E. coli virulence genes in feces from 252 dairy calves in Uruguay (149 with neonatal diarrhea - NCD - and 103 asymptomatic). Genes iucD, f17A, afa8E, papC, clpG and f17G(II) were the most prevalent (81.3%; 48.4%; 37.3%; 35.7%; 34.1%; 31.3%, respectively). Genes eae, stx1and stx2 were poorly represented; 13/252 animals harbored one or a combination of these genes. The prevalence of the cnf gene was 4.4%, while that of cdt-IV and cdt-III genes was 24.2% and 12.7% respectively. This study reports updated data about the virulence profiles of E. coli in dairy calves in Uruguay. A large number of adhesins and toxin genes were detected. Our results demonstrate that E. coli from bovine feces has diarrheagenic and extraintestinal profiles although other NCD risks factors may contribute to the disease outcome.

A survey of management practices that influence calf welfare and an estimation of the annual calf mortality risk in pastured dairy herds in Uruguay.

Despite the importance of dairy farming in Uruguay, little information on dairy systems in this country is available in the scientific literature, and management practices that influence calf welfare at the herd level have not been explored. The aims of this study were to (1) describe the prepartum and calf-rearing systems, as well as the management practices that may influence calf welfare in pastured dairy herds in Uruguay, (2) estimate the annual calf mortality risk from birth to weaning, and (3) identify the primary clinical disease syndromes shown by the calves before death. A survey comprising a farm visit and a questionnaire was conducted on 225 randomly selected dairies with >30 milking cows, in 3 strata (31-99, 100-299, and ≥300 milking cows) of 6 Uruguayan departments where dairies are concentrated. Retrospective information from July 2013 to June 2014 was collected. A descriptive analysis was performed and results were inferred into the national dairy cattle population. Several management practices that could contribute to poor calf welfare were identified in a large proportion of farms. The annual calf mortality risk (calves that died between birth and weaning/calves born death or alive × 100, n = 149 farms) was 15.2%. Age at weaning averaged 75 d. Farmers reported that the most common clinical syndromes were diarrhea and respiratory disease in 85.2% and 47.5% of the farms, respectively. There was no continuous veterinary advice in 61.3% of the farms, 20.0% lacked data records, 38.5% had poor drainage in the prepartum area with waterlogging after rainfall, 52.1% monitored the prepartum area ≤2 times per day during the calving season, 65.1% did not perform navel antisepsis on newborns, 62.3% separated the calves from their dams at >24 h postpartum, 95.2% did not have a colostrum management program, 72.4% did not rotate the calf-rearing areas, 59.0% did not disinfect the calf feeders, 85.7% did not have staff dedicated exclusively to calf rearing, and 39.8% did not separate sick from healthy calves. The average volume of milk or milk replacer offered per calf was 4.5 L/day. Several of the identified management practices that affect calf welfare in the prepartum and calf-rearing periods could explain the high mortality risk. An effort should be made to conduct extension work focusing on the dissemination of good management practices to improve calf welfare and reduce calf mortality in Uruguayan dairy farms.

[A comparative study of commercial ELISAs for antibody detection in the diagnostic investigation of Neospora caninum-associated abortion in dairy cattle herds in Uruguay]. / Comparación de ELISAs comerciales para la detección de anticuerpos en la investigación diagnóstica del aborto asociado a Neospora caninum en rodeos lecheros de Uruguay.

Bovine abortion causes considerable economic losses to the livestock industry worldwide and is of concern for public health and food safety, given that many abortigenic infectious agents of cattle are zoonotic. Despite its importance, the etiological diagnosis of abortion in cattle is challenging both for veterinary practitioners and laboratory technicians, partly due to the difficulty in recovering aborted fetuses under extensive field conditions for pathological and microbiological diagnostic investigation, and in the early identification of aborted dams. Neospora caninum is a cosmopolitan protozoon identified as one of the main abortigenic agents in cattle worldwide. In this study we propose a comparative seroepidemiological approach for the diagnosis of abortion by N. caninum in dairy cattle. Samples from 12 to 93 cows/heifers with and without recent history of abortion (cases and controls) in four commercial dairy farms were tested. The ratio of controls to cases tested varied from 1:1 to 4.6:1. All samples (n=230) were analyzed by three commercial ELISA kits for the detection of anti-N. caninum antibodies. In all four dairy farms, the proportion of seropositive cows and/or heifers per kit was significantly higher in the cases than in the controls (Odds Ratios=5.13 to 36, p=0.0002 to 0.0485). The agreement among the three kits varied from weak to strong (Cohens kappa coefficients=0.58 to 0.83). We conclude that, despite the imperfect agreement between these kits, all of them allowed to arrive at similar conclusions regarding the statistical association between N. caninum seropositivity and abortion, thus representing a useful tool for the diagnostic approach at the population level under field conditions.

Bovine coronavirus in Uruguay: genetic diversity, risk factors and transboundary introductions from neighboring countries.

Bovine coronavirus (BCoV) is a recognized cause of severe neonatal calf diarrhea, with a negative impact on animal welfare, leading to economic losses to the livestock industry. Cattle production is one of the most important economic sectors in Uruguay. The aim of this study was to determine the frequency of BCoV infections and their genetic diversity in Uruguayan calves and to describe the evolutionary history of the virus in South America. The overall detection rate of BCoV in Uruguay was 7.8% (64/824): 7.7% (60/782) in dairy cattle and 9.5% (4/42) in beef cattle. The detection rate of BCoV in samples from deceased and live calves was 10.0% (6/60) and 7.6% (58/763), respectively. Interestingly, there was a lower frequency of BCoV detection in calves born to vaccinated dams (3.3%, 8/240) than in calves born to unvaccinated dams (12.2%, 32/263) (OR: 4.02, 95%CI: 1.81-8.90; p = 0.00026). The frequency of BCoV detection was higher in colder months (11.8%, 44/373) than in warmer months (1.5%, 3/206) (OR: 9.05, 95%CI: 2.77-29.53, p = 0.000013). Uruguayan strains grouped together in two different lineages: one with Argentinean strains and the other with Brazilian strains. Both BCoV lineages were estimated to have entered Uruguay in 2013: one of them from Brazil (95%HPD interval: 2011-2014) and the other from Argentina (95%HPD interval: 2010-2014). The lineages differed by four amino acid changes, and both were divergent from the Mebus reference strain. Surveillance should be maintained to detect possible emerging strains that can clearly diverge at the antigenic level from vaccine strains.

Fetal Pathology in an Aborted Holstein Fetus Infected With Bovine Parainfluenza Virus-3 Genotype A.

Bovine parainfluenza virus-3 (BPIV-3) is a recognized respiratory pathogen of cattle, and it has also been identified in aborted fetuses. However, little is known of this agent as a reproductive pathogen and detailed descriptions of fetal pathology on natural cases are lacking in the scientific literature. This article describes and illustrates lesions in a fetus spontaneously aborted by a first-calving Holstein heifer, naturally infected with BPIV-3 genotype A, broadening the current knowledge on fetal pathology by this virus. Fetal autopsy revealed diffusely reddened, rubbery and unexpanded lungs. Histologically, there was necrotizing bronchiolitis/alveolitis with intraluminal fibrin exudate and syncytial cells in the bronchiolar/alveolar spaces, and non-suppurative peribronchiolitis and perivascular interstitial pneumonia. In the small intestine there was multifocal necrotizing cryptitis and occasional necrotic syncytial enterocytes. Intralesional and extralesional BPIV-3 antigen was detected by immunohistochemistry in the lung and small intestine, and BPIV-3a was identified in fetal tissues by RT-PCR and sequencing.

Isolation and molecular characterization of four novel Neospora caninum strains.

Neospora caninum causes neosporosis, a leading cause of bovine abortion worldwide. Uruguay is a developing economy in South America that produces milk to feed seven times its population annually. Naturally, dairy production is paramount to the country's economy, and bovine reproductive failure impacts it profoundly. Recent studies demonstrated that the vast majority of infectious abortions in dairy cows are caused by N. caninum. To delve into the local situation and contextualize it within the international standing, we set out to characterize the Uruguayan N. caninum strains. For this, we isolated four distinct strains and determined by microsatellite typing that these represent three unique genetic lineages, distinct from those reported previously in the region or elsewhere. An unbiased analysis of the current worldwide genetic diversity of N. caninum strains known, whereby six typing clusters can be resolved, revealed that three of the four Uruguayan strains group closely with regional strains from Argentina and Brazil. The remaining strain groups in an unrelated genetic cluster, suggesting multiple origins of the local strains. Microsatellite typing of N. caninum DNA from fetuses opportunistically collected from local dairy farms correlated more often with one of the isolates. Overall, our results contribute to further understanding of genetic diversity among strains of N. caninum both regionally and worldwide.

A Case of Prototheca zopfii Genotype 1 Infection in a Dog (Canis lupus familiaris).

Protothecosis is a rare disease caused by environmental algae of the genus Prototheca. These are saprophytic, non-photosynthetic, aerobic, colorless algae that belong to the Chlorellaceae family. Seven different species have been described. Prototheca zopfii genotype 2 and P. wickerhamii are most commonly involved in pathogenic infections in humans and animals. The objective of this work is to describe, for the first time, a case of protothecosis caused by P. zopfii genotype 1 in a dog. The dog, a 4-year-old mix bred male, was presented to a veterinary clinic in Montevideo, Uruguay, with multiple skin nodules, one of which was excised by surgical biopsy. The sample was examined histologically and processed by PCR, DNA sequencing, and restriction fragments length polymorphisms for the detection and genotyping of P. zopfii. In addition, transmission electron microscopy and scanning electron microscopy were performed. Histology showed severe ulcerative granulomatous dermatitis and panniculitis with myriads of pleomorphic algae. Algal cells were 4-17 µm in size, with an amphophilic, 2-4-µm-thick wall frequently surrounded by a clear halo, contained flocculant material and a deeply basophilic nucleus, and internal septae with daughter cells (endospores) consistent with endosporulation. Ultrastructurally, algal cells/endospores at different stages of development were found within parasitophorous vacuoles in macrophages. Prototheca zopfii genotype 1 was identified by molecular testing, confirming the etiologic diagnosis of protothecosis.

Protein profiling of isolated uterine AA amyloidosis causing fetal death in goats.

Pathologic amyloid accumulates in the CNS or in peripheral organs, yet the mechanism underlying the targeting of systemic amyloid deposits is unclear. Serum amyloid A (SAA) 1 and 2 are produced predominantly by the liver and form amyloid most commonly in the spleen, liver, and kidney. In contrast, SAA3 is produced primarily extrahepatically and has no causal link to amyloid formation. Here, we identified 8 amyloidosis cases with amyloid composed of SAA3 expanding the uterine wall of goats with near-term fetuses. Uterine amyloid accumulated in the endometrium, only at the site of placental attachment, compromising maternal-fetal gas and nutrient exchange and leading to fetal ischemia and death. No other organ contained amyloid. SAA3 mRNA levels in the uterine endometrium were as high as SAA2 in the liver, yet mass spectrometry of the insoluble uterine peptides identified SAA3 as the predominant protein, and not SAA1 or SAA2. These findings suggest that high local SAA3 production led to deposition at this unusual site. Although amyloid A (AA) amyloid deposits typically consist of an N-terminal fragment of SAA1 or SAA2, here, abundant C-terminal peptides indicated that the uterine amyloid was largely composed of full-length SAA3. The exclusive deposition of SAA3 amyloid in the uterus, together with elevated uterine SAA3 transcripts, suggests that the uterine amyloid deposits were due to locally produced SAA3. This is the first report of SAA3 as a cause of amyloidosis and of AA amyloid deposited exclusively in the uterus.

Detection of a novel circovirus PCV3 in pigs with cardiac and multi-systemic inflammation.

BACKGROUND: Porcine circovirus 2 causes different clinical syndromes resulting in a significant economic loss in the pork industry. Three pigs with unexplained cardiac and multi-organ inflammation that tested negative for PCV2 and other known porcine pathogens were further analyzed. METHODS: Histology was used to identify microscopic lesions in multiple tissues. Metagenomics was used to detect viral sequences in tissue homogenates. In situ hybridization was used to detect viral RNA expression in cardiac tissue. RESULTS: In all three cases we characterized the genome of a new circovirus we called PCV3 with a replicase and capsid proteins showing 55 and 35 % identities to the genetically-closest proteins from a bat-feces associated circovirus and were even more distant to those of porcine circovirus 1 and 2. Common microscopic lesions included non-suppurative myocarditis and/or cardiac arteriolitis. Viral mRNA was detected intralesionally in cardiac cells. Deep sequencing in tissues also revealed the presence of porcine astrovirus 4 in all three animals as well as rotavirus A, porcine cytomegalovirus and porcine hemagglutinating encephalomyelitis virus in individual cases. CONCLUSION: The pathogenicity and molecular epidemiology of this new circovirus, alone or in the context of co-infections, warrants further investigations.

A method to preserve low parasitaemia Plasmodium-infected avian blood for host and vector infectivity assays.

BACKGROUND: Avian malaria vector competence studies are needed to understand more succinctly complex avian parasite-vector-relations. The lack of vector competence trials may be attributed to the difficulty of obtaining gametocytes for the majority of Plasmodium species and lineages. To conduct avian malaria infectivity assays for those Plasmodium spp. and lineages that are refractory to in vitro cultivation, it is necessary to obtain and preserve for short periods sufficient viable merozoites to infect naïve donor birds to be used as gametocyte donors to infect mosquitoes. Currently, there is only one described method for long-term storage of Plasmodium spp.-infected wild avian blood and it is reliable at a parasitaemia of at least 1%. However, most naturally infected wild-caught birds have a parasitaemia of much less that 1%. To address this problem, a method for short-term storage of infected wild avian blood with low parasitaemia (even ≤ 0.0005%) has been explored and validated. METHODS: To obtain viable infective merozoites, blood was collected from wild birds using a syringe containing the anticoagulant and the red blood cell preservative citrate phosphate dextrose adenine solution (CPDA). Each blood sample was stored at 4 °C for up to 48 h providing sufficient time to determine the species and parasitaemia of Plasmodium spp. in the blood by morphological examination before injecting into donor canaries. Plasmodium spp.--infected blood was inoculated intravenously into canaries and once infection was established, Culex stigmatosoma, Cx. pipiens and Cx. quinquefasciatus mosquitoes were then allowed to feed on the infected canaries to validate the efficacy of this method for mosquito vector competence assays. RESULTS: Storage of Plasmodium spp.--infected donor blood at 4 °C yielded viable parasites for 48 h. All five experimentally-infected canaries developed clinical signs and were infectious. Pathologic examination of three canaries that later died revealed splenic lesions typical of avian malaria infection. Mosquito infectivity assays demonstrated that Cx. stigmatosoma and Cx. pipiens were competent vectors for Plasmodium cathemerium. CONCLUSIONS: A simple method of collecting and preserving avian whole blood with malaria parasites of low parasitaemia (≤ 0.0005%) was developed that remained viable for further experimental bird and mosquito infectivity assays. This method allows researchers interested in conducting infectivity assays on target Plasmodium spp. to collect these parasites directly from nature with minimal impact on wild birds.