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.

Study of the dynamics of in vitro infection with bovine gammaherpesvirus type 4 and apoptosis markers in susceptible cells.

Bovine gammaherpesvirus type 4 (BoHV-4) shows tropism for the endometrium, in which it causes the death of epithelial and stroma cells. Despite having anti-apoptotic genes in its genome, experiments based on immortalized cell lines have shown that BoHV-4 induces cell death by apoptosis. In the present study, we evaluated BoHV-4 replication, pro-apoptotic (Bax) and anti-apoptotic (Bcl-2) mitochondrial genes expression and chromatin condensation in bovine endometrium primary culture cells (BEC) and in the Madin Darby bovine kidney (MDBK) cell line. Results showed that BoHV-4 has a preference for replication in BEC cells over the MDBK cell line, demonstrated by the high viral titer that is consistent with the tropism of the virus. In BEC cells, chromatin condensation was consistent with the values of viral kinetics at the late stage of infection, accompanied with a balance in the mRNA levels of apoptotic mitochondrial proteins. As a consequence, in those cells viral transmission would be enhanced by inhibiting apoptosis in the early stage of virus proliferation, allowing the complete production of viral progeny, and then, the induction of apoptosis in late stages would allow neighboring cells infection. In MDBK cells replication kinetics was coincident with the up-regulation of Bcl-2, which suggests that the productive infection in MDBK is associated with a lytic phase of the virus or another cell death pathway (probably autophagy mechanism) at the late stage of infection. The results agree with the study of nuclear morphology, where a constant chromatin condensation was observed over time. It is clear that the documented BoHV-4 apoptotic responses observed in the cell lines studied above are not valid in cells from primary cultures. The data presented in this study suggest that BoHV-4 could induce apoptosis in BEC cells without a leading role of the mitochondria pathway. Further studies will be necessary to characterize in detail the programmed cell death pathways involved in BoHV-4 infection in the primary cell cultures evaluated.

Molecular detection of bovine Noroviruses in Argentinean dairy calves: Circulation of a tentative new genotype.

Bovine noroviruses are enteric pathogens detected in fecal samples of both diarrheic and non-diarrheic calves from several countries worldwide. However, epidemiological information regarding bovine noroviruses is still lacking for many important cattle producing countries from South America. In this study, three bovine norovirus genogroup III sequences were determined by conventional RT-PCR and Sanger sequencing in feces from diarrheic dairy calves from Argentina (B4836, B4848, and B4881, all collected in 2012). Phylogenetic studies based on a partial coding region for the RNA-dependent RNA polymerase (RdRp, 503 nucleotides) of these three samples suggested that two of them (B4836 and B4881) belong to genotype 2 (GIII.2) while the third one (B4848) was more closely related to genotype 1 (GIII.1) strains. By deep sequencing, the capsid region from two of these strains could be determined. This confirmed the circulation of genotype 1 (B4848) together with the presence of another sequence (B4881) sharing its highest genetic relatedness with genotype 1, but sufficiently distant to constitute a new genotype. This latter strain was shown in silico to be a recombinant: phylogenetic divergence was detected between its RNA-dependent RNA polymerase coding sequence (genotype GIII.2) and its capsid protein coding sequence (genotype GIII.1 or a potential norovirus genotype). According to this data, this strain could be the second genotype GIII.2_GIII.1 bovine norovirus recombinant described in literature worldwide. Further analysis suggested that this strain could even be a potential norovirus GIII genotype, tentatively named GIII.4. The data provides important epidemiological and evolutionary information on bovine noroviruses circulating in South America.

Molecular characterization of Cryptosporidium isolates from calves in Argentina.

Cryptosporidiosis is responsible for significant fatalities of neonatal calves, resulting in substantial economic loss in dairy farming in several countries. Additionally, the high shedding of environmentally resistant oocysts by calves promotes contamination of drinking water and facilitates outbreaks of cryptosporidiosis in humans. Here we report on the Cryptosporidium species and GP60 subtypes of 45 calves originating from the Humid Pampa, the main productive dairy farming area of Argentina. Polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis of the 18S rRNA gene was done to determine the infecting Cryptosporidium species and only Cryptosporidium parvum was detected. Subtyping by sequence analysis of the GP60 gene revealed 6 different alleles all pertaining to the zoonotic IIa family. Of these, IIaA23G1R1 represents a novel IIa subtype. Other identified subtypes, IIa18G1R1, IIaA20G1R1, IIaA21G1R1, and IIaA22G1R1 have been recognized in very few studies and/or with low frequencies. Interestingly, different alleles prevailed in the provinces of Buenos Aires (IIaA17G1R1 and IIaA21G1R1), Santa Fe (IIaA23G1R1), and Cordoba (IIaA20G1R1 and IIaA21G1R1), and different allele distribution patterns were observed. Subtypes IIaA18G1R1 and IIaA17G1R1, the latter often found in this study, are strongly implicated in zoonotic transmission, suggesting that calves may represent a potential source for human cryptosporidiosis in this region. This is the first published report of a molecular analysis of Cryptosporidium infection in dairy and beef calves from Argentina.