Mapping Genetic Markers Associated with Antigenicity and Host Range in H9N2 Influenza A Viruses Infecting Poultry in Pakistan.

The aim of the current study was to map the genetic diversity in the haemagglutinin (HA) glycoprotein of influenza A viruses (IAVs) of the H9N2 subtype. Twenty-five H9N2 IAVs were isolated from broiler chickens from March to July 2019. The HA gene was amplified, and phylogenetic analysis was performed to determine the evolutionary relationship. Important antigenic amino acid residues of HA attributed to immune escape and zoonotic potential were compared among H9N2 IAVs. Phylogenetic analysis revealed that sublineage B2 under the G1 lineage in Pakistan was found to be diversified, and newly sequenced H9N2 isolates were nested into two clades (A and B). Mutations linked to the antigenic variation and potential immune escape were observed as G72E (1/25, 4%), A180T (3/25, 12%), and A180V (1/25, 4%). A twofold significant reduction (P < 0.01) in log2 hemagglutination inhibition titers was observed with H9N2 IAV naturally harboring amino acid V180 instead of A180 in HA protein. Moreover, in the last 20 years, complete substitution at residues (T127D, D135N, and L150N) and partial substitution at residues (72, 74, 131, 148, 180, 183, 188, 216, 217, and 249, mature H9 HA numbering) associated with changes in antigenicity were observed. The presence of L216 in all H9N2 IAV isolates and T/V180 in four isolates in the receptor-binding site reveals the potential of these viruses to cross the species barrier to infect human or mammals. The current study observed the circulation of antigenically diverse H9N2 IAV variants that possess potential mutations that can escape the host immune system.

Nota de investigación- Mapeo de marcadores genéticos asociados con la antigenicidad y el rango de huéspedes en los virus de la influenza tipo A subtipo H9N2 que infectan a la avicultura en Pakistán. El objetivo del presente estudio fue mapear la diversidad genética en la glicoproteína hemaglutinina (HA) de los virus de la influenza A (IAV) del subtipo H9N2. Se aislaron veinticinco virus de influenza H9N2 de pollos de engorde de marzo a julio del 2019. Se amplificó el gene HA y se realizó un análisis filogenético para determinar la relación evolutiva. Se compararon importantes residuos de aminoácidos antigénicos de la hemaglutinina atribuidos al escape inmunológico y al potencial zoonótico entre los virus de la influenza aviar H9N2. El análisis filogenético reveló que el sublinaje B2 bajo el linaje G1 en Pakistán estaba diversificado, y los aislados de H9N2 recién secuenciados se agruparon en dos clados (A y B). Se observaron mutaciones relacionadas con la variación antigénica y el posible escape inmunológico como los residuos de aminoácidos G72E (1/25, 4%), A180T (3/25, 12%) y A180V (1/25, 4%). Se observó una reducción significativa al doble (P < 0.01) en los títulos de inhibición de la hemaglutinación log2 cuando el virus de la influenza aviar H9N2 albergaba naturalmente el aminoácido V180 en lugar del A180 en la proteína HA. Además, en los últimos 20 años, sustitución completa en los residuos (T127D, D135N y L150N) y sustitución parcial en los residuos (72, 74, 131, 148, 180, 183, 188, 216, 217 y 249, de acuerdo con la numeración de la HA subtipo madura) asociados con cambios en la antigenicidad. La presencia del residuo L216 en todos los aislados de influenza aviar H9N2 y T/V180 en cuatro aislados en el sitio de unión al receptor revela el potencial de estos virus para cruzar la barrera de las especies para infectar a humanos o mamíferos. El estudio actual observó la circulación de variantes antigénicamente diversas del virus de influenza aviar H9N2 que poseen mutaciones potenciales que pueden escapar del sistema inmunológico del huésped.

Molecular identification and infection pathology of Mycobacterium spp. in captive wild animals in Pakistan.

INTRODUCTION: Animal tuberculosis is an infectious, chronic, granulomatous, and debilitating disease affecting animals as well as humans. However, in recent decades, there have been many endemic geographic localities where animal tuberculosis has been identified in wildlife reservoirs, limiting the eradication program in cattle. This study aimed to identify animal tuberculosis in captive zoo animals in Pakistan. METHODOLOGY: In total, 185 morbid zoo animals were brought for postmortem examination at a veterinary postmortem facility. During the macroscopic examination, these animals were thoroughly examined for the presence of suggestive gross lesions of animal tuberculosis (granulomas/tubercles), and the pattern and distribution of these lesions in different organs. The Ziehl-Neelsen (ZN) staining was performed on smears prepared from granulomatous lesions of lung tissue followed by molecular identification of M. bovis and M. tuberculosis DNA using polymerase chain reaction (PCR). RESULTS: The postmortem examination revealed that 8.1% (15/185) of animals had gross tuberculosis lesions on the lungs and lymph nodes. The ZN staining of tissue smears showed 5.40% positivity while M. bovis and M. tuberculosis DNA was identified in 3.78 % and 1.1% of investigated animals, respectively. CONCLUSIONS: The study showed that animal tuberculosis is prevalent among wildlife in Pakistan and it may pose serious public health concerns to the people visiting these zoos and wildlife parks.

Evidence of Salmonella enterica in Pakistani backyard poultry breeds: isolation, molecular characterization, and pathology.

The present study evaluated the presence of Salmonella enterica in Pakistani backyard poultry. A total 48 chickens from 4 backyard poultry breeds with the clinical presentation of S. enterica infection were randomly selected from villages in the Punjab Province. Cloacal swabs from live poultry and liver samples from the dead birds were collected for bacterial culture and biochemical identification. Liver and spleen samples from dead birds were evaluated for gross and histopathological changes. Bacterial isolates were subjected to PCR and sequencing of ratA gene. Biochemical identification revealed 5/48 (10.42%) chickens positive for S. enterica. Gross pathology included enlarged, discoloured and congested liver and congested spleen. Histopathology demonstrated congestion of sinusoidal capillaries, cellular swelling and cellular/ballooning degeneration, congestion of central hepatic vein, granular hepatocytic cytoplasm and the presence of variable-sized vacuoles in hepatocytes. The PCR yielded a S. enterica specific amplicon (1047 bp). All liver samples that were positive for S. enterica by biochemical tests, were also positive by PCR. The ratA gene sequencing revealed a close resemblance with S. enteritidis isolates from humans. The present study highlights zoonotic risk from backyard poultry and suggests that PCR can be used as an alternate method for rapid detection of Salmonella serovars.

Identification, molecular characterization, and pathological features of orf virus in sheep and goats in Punjab province, Pakistan.

Orf virus (ORFV) causes an acute, contagious, skin disease of sheep and goats which is economically important. The objectives of this study were to identify ORFV and to explore its pathological and phylogenetic profiles in 350 goats and 91 sheep of 14 districts of Punjab, Pakistan, from July 2020 to July 2021. Skin scrapings (total no. of samples = 441) of suspected animals were subjected to polymerase chain reactions, phylogenetic analysis, and pathological observations. The partial length of GIF/IL-2 gene (408 bp) was successfully amplified in 58/441 samples. Phylogenetic analysis of GIF/IL2 gene showed that the study isolates belonged to ORFV-cluster I, together with the viruses reported in India and China. Pakistan ORFV isolates were shared 97.6-98.7% nucleotide and 97.6-100% amino acid identities with the reference strain (NC_005336). Moreover, Chinese ORFV-isolates were detected unique multiple amino acid substitutions (F11L, Q21H, D27N, I46V, N49S, N82D, D103N, S129G) with study isolates. Naturally infected animals were anorexic, emaciated, dull, and depressed. The macroscopic lesions included multifocal to coalescing, ulceration followed by proliferative papules, pustules, and crust formation on the epidermis of gums, lips, mouth commissure, muzzles, nose, and udder. Histopathological examination revealed hyperplasia, anastomosing rete ridges formation and degenerative changes, including spongiosis and vacuolation of epidermal cells. Keratinocytes exhibited eosinophilic intracytoplasmic inclusion bodies with pyknotic and karyorrhexis nuclei. This is the first report on molecular characterization of ORFV from Pakistan, with insight into its pathogenesis and comparative analysis of pathological alterations and genetic diversity between ORFV strains reported in different geographical areas.

Rate of Multiple Viral and Bacterial CoInfection(s) in Influenza A/H9N2-Infected Broiler Flocks.

Repeated cases of low pathogenic influenza A/H9N2 virus (IAV/H9N2) have been reported in commercial chickens since its emergence in 1998 in Pakistan. However, recently increased mortality and severe respiratory complications under field conditions have been noticed, suggesting concomitant influenza infections with respiratory viral and/or bacterial pathogens. Therefore, the present study aimed to investigate the presence of IAV/H9N2 coinfecting with multiple viral and bacterial pathogens in broiler chicken flocks. We surveyed 60 broiler flocks with respiratory signs from March through July 2019 in Punjab, Pakistan. Suspected flocks were screened for the presence of IAV using a lateral-flow device. Tracheal, cloacal, and bone marrow samples were collected and further tested for seven viral agents (chicken anemia; Newcastle disease; infectious bronchitis; infectious laryngeotracheitis [ILT]; and IAV subtypes H9, H7, and H5) and three bacterial agents (Mycoplasma gallisepticum; Mycoplasma synovae; Ornithobacterium rhinotracheale [ORT]) using PCR assays. Upon initial screening for IAV, 35/60 (58.3%) flocks tested positive. The coinfection of IAV/H9N2 with other pathogens was detected in 25 (71.4%) flocks and only IAV/H9N2 was detected in 10 (28.6%) flocks out of total positive IAV flocks (n = 35). IAV subtypes H5 and H7, ILT, and ORT were not detected throughout the study period. The detection rate of double, triple, and quadruple combinations of coinfections with IAV/H9N2 were 37% (13 flocks), 26% (9 flocks), 9% (3 flocks), respectively. Higher average mortality (28.5%) was found in broiler chicken flocks coinfected with viral and/or bacterial pathogens than in flocks where only H9 low pathogenic IAV/H9N2 was detected (20.8%). In conclusion, higher circulation of IAV/H9N2 with other viral and bacterial pathogens may contribute to higher production and economic losses at the farm level.

Nota de investigación- Tasa de coinfecciones virales y bacterianas múltiples en parvadas de pollos de engorde infectadas con virus influenza A/H9N2. Se han reportado varios casos del virus de influenza A de baja patogenicidad H9N2 (IAV/H9N2) en pollos comerciales desde su aparición en 1998 en Pakistán. Sin embargo, recientemente se ha observado un aumento de la mortalidad y complicaciones respiratorias graves en condiciones de campo, lo que sugiere infecciones concomitantes de influenza con patógenos respiratorios virales y/o bacterianos. Por lo tanto, el presente estudio tuvo como objetivo investigar la presencia del virus de influenza aviar H9N2 coinfectando con múltiples patógenos virales y bacterianos en parvadas de pollos de engorde. Se evaluaron 60 parvadas de pollos de engorde con signos respiratorios desde marzo hasta julio del año 2019 en Punjab, Pakistán. Las parvadas sospechosas fueron analizadas para detectar la presencia del virus de influenza aviar utilizando un dispositivo de flujo lateral. Se recolectaron muestras traqueales, cloacales y de médula ósea y se analizaron para detectar siete agentes virales (anemia infecciosa aviar, enfermedad de Newcastle, bronquitis infecciosa, laringeotraqueítis infecciosa [ILT] y subtipos H9, H7 y H5 de influenza aviar) y tres agentes bacterianos (Mycoplasma gallisepticum ; Mycoplasma sinovae; Ornithobacterium rhinotracheale [ORT]) utilizando ensayos de PCR. Tras la detección inicial del virus de la influenza aviar, 35/60 (58.3 %) parvadas resultaron positivas. La coinfección del virus de la influenza H9N2 con otros patógenos se detectó en 25 (71.4 %) parvadas y el virus de influenza aviar H9N2 fue detectado solo en 10 (28.6 %) parvadas del total de parvadas positivas (n = 35). Los subtipos H5 y H7 del virus de influenza, ILT y ORT no se detectaron durante el período de estudio. La tasa de detección de combinaciones dobles, triples y cuádruples de coinfecciones con el virus de influenza H9N2 fue del 37 % (13 parvadas), del 26% (9 parvadas), del 9 % (3 parvadas), respectivamente. Se encontró una mortalidad promedio más alta (28.5 %) en lotes de pollos de engorde coinfectados con patógenos virales y/o bacterianos que en lotes donde solo se detectó al virus de influenza H9 de baja patogenicidad (20.8%). En conclusión, una mayor circulación del virus de influenza aviar H9N2 con otros patógenos virales y bacterianos puede contribuir a mayores pérdidas en la producción y económicas a nivel de granja.

Toxico-pathological effects of heavy metals from industrial drainage wastewater on vital organs of small ruminants in Lahore.

Considering one health concept, human health is thought to be affected by many factors. Heavy metal toxicity is now gaining its place as one of the major factors contributing to detrimental outcomes for human health. The study encompassed to target sites close to the industrial area of Lahore where heavy metal levels are believed to be higher, as industrial waste is drained into the two main drains. Sheep and goats (n = 5 from each species) reared in the locality were included in the study, and effects of heavy metal toxicity were evaluated in the selected organs (intestine, kidneys, liver, and muscles) via histopathological examination along with residual concentration of these heavy metals in the aforementioned organs. Heavy metals chromium, copper, zinc, lead, iron, magnesium, manganese, and nickel were detected in sample of selected organs by atomic absorption spectrometry (AAS) along with digestion method. The findings of the study indicated a statistically significant difference of residual concentrations of almost all the selected elements in almost all the tissue samples between the two sites where the values of site 1 (close to the drain) were higher compared with site 2 (away from the drain). Similar trend was depicted in histopathological examination where a higher degree of tissue degeneration, necrosis, and hence organ damage was observed in tissue samples collected from site 1 compared with site 2.

A study on prevalence and molecular characterization of trypanosomal species infecting equines in Lahore region, Pakistan.

Trypanosomiasis is an important protozoal disease with a diverse range of susceptible host including human. In the current study, molecular characterization of prevalent species was done through a pan-trypanosome polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP). A total of three hundred (n = 300) equines including horses, donkeys and mules (100 each) were randomly selected and the equine blood samples were subjected to screening for trypanosomes through microhaematocrit centrifuge technique (MHCT), conventional PCR, semi-nested PCR and RFLP. Overall prevalence of trypanosomal species was 8% (24/300) as revealed by MHCT and species wise prevalence in horses, donkeys and mules was 4.33% (13/300), 1.33% (4/300) and 2.33% (7/300), respectively. Conventional and semi-nested PCR depicted an overall prevalence of 21% (63/300) and species wise prevalence in horses, donkeys and mules was 12% (36/300), 3.67% (11/300) and 5.33% (16/300), respectively. RFLP analysis of the semi-nested products, using Msp1 and Eco571 enzymes, negated the presence of T. congolense, T. brucei, T. vivax, T. theileri, and T. vivax in the positive samples and revealed that the animals might be suffering from T. evansi infection as the enzymes used were not able to detect this species. This hypothesis was further confirmed by using T. evansi specific primers which depicted all of the 63 samples were positive for T. evansi. It is inferred that T. evansi is the major trypanosome species prevalent in equines. Furthermore, PCR is more sensitive as compared to microscopic examination and the pan-trypanosome PCR-RFLP assay is suitable for carrying out laboratory diagnosis of field samples and epidemiological studies. Further studies on the possibilities of use of other restriction enzymes may help to improve the species specificity of the assay.

Genetic analysis of fructose-1,6-bisphosphatase (FBPase) deficiency in nine consanguineous Pakistani families.

BACKGROUND: Fructose-1,6-bisphosphatase (FBPase) deficiency is a rare inherited metabolic disorder characterized by recurrent episodes of hypoglycemia, ketosis and lactic acidosis. FBPase is encoded by FBP1 gene and catalyzes the hydrolysis of fructose-1,6-bisphosphate to fructose-6-phosphate in the last step of gluconeogenesis. We report here FBP1 mutations in nine consanguineous Pakistani families affected with FBPase deficiency. METHODS: Nine families having one or two individuals affected with FBPase deficiency were enrolled over a period of 3 years. All FBP1 exonic regions including splicing sites were PCR-amplified and sequenced bidirectionally. Familial cosegregation of mutations with disease was confirmed by direct sequencing and PCR-RFLP analysis. RESULTS: Three different FBP1 mutations were identified. Each of two previously reported mutations (c.472C>T (p.Arg158Trp) and c.841G>A (p.Glu281Lys)) was carried by four different families. The ninth family carried a novel 4-bp deletion (c.609_612delAAAA), which is predicted to result in frameshift (p.Lys204Argfs*72) and loss of FBPase function. The novel variant was not detected in any of 120 chromosomes from normal ethnically matched individuals. CONCLUSIONS: FBPase deficiency is often fatal in the infancy and early childhood. Early diagnosis and prompt treatment is therefore crucial to preventing early mortality. We recommend the use of c.472C>T and c.841G>A mutations as first choice genetic markers for molecular diagnosis of FBPase deficiency in Pakistan.

Anti-avian influenza virus H9N2 activity of aqueous extracts of Zingiber officinalis (Ginger) and Allium sativum (Garlic) in chick embryos.

In the present study, anti-Avian influenza virus H9N2 activity of aqueous extracts (5, 10, 15, 20, 25%) of Zingiber officinalis and Allium sativum was evaluated. Embryo-toxicity was evaluated by histopathological scoring of Chorio-allantoic membrane of chick embryos. Cytotoxicity of extracts was determined by MTT assay on Vero cells. Aqueous extract of ginger had antiviral activity at 10, 15, 20 and 25% while garlic had activity at 15, 20 and 25%. Histopathological scoring of chorio-allantoic membrane for aqueous extracts (5, 10, 15, 20, 25%) of ginger (0.66±0.57, 1.33±0.57, 1.66±0.57, 2.66±0.57, 3.66±0.57, respectively) and garlic (1.00±0.00, 1.33±0.57, 2.00±0.00, 2.33±0.57, 3.66±0.57, respectively) was concentration dependant. MTT assay revealed cytotoxicity of both plants was also concentration dependent. Extracts of ginger (5, 10, 15, 20, 25%) had lower cytotoxicity (71, 59, 28, 22, 0 % cell survival, respectively) as compared to garlic (61, 36. 20, 11, 3% cell survival, respectively). Overall results revealed that concentration of aqueous extract of ginger (10%), showing antiviral activity against H9N2, was less toxic to vero cells (> 50% cell survival). It is insinuated that ginger may have anti- Avian influenza virus H9N2 potential and its active compounds needs further investigations.

Effect of sodium butyrate on performance, immune status, microarchitecture of small intestinal mucosa and lymphoid organs in broiler chickens.

OBJECTIVE: This study aimed to examine the effect of sodium butyrate (SB) on growth performance, immune status, organs weights, and microarchitecture of lymphoid organs and small intestine. METHODS: A total of 120, 1-d-old broiler chicks were distributed into the following four treatment groups: corn-soy based basal diet (BD) without supplement (control), or the same BD supplemented with 0.1 g/kg zinc bacitracin (ZnB), 0.5 g/kg SB (SB-0.5), or 1.0 g/kg SB (SB-1), respectively. Six birds/group were killed on d-21 and d-35, and samples were collected. RESULTS: Cell-mediated immune response at 48 h post-Phytohemagglutinin-P injection, and antibody titer against Newcastle disease vaccine and sheep red blood cells on d-35 was noted higher (p<0.05) in SB-1 compared to ZnB and control. Lower (p<0.05) feed conversion ratio (FCR) was attained by the supplemented groups. Thymus and spleen weighed more (p<0.05) in SB-1, and bursa registered more (p<0.05) weight in both SB groups compared to control. On d-21, areas of thymus medulla and spleen germinal centers were noted higher (p<0.05) in SB-1 group. The villus height and villus surface area increased (p<0.05) in duodenum and jejunum in both SB groups on d-21, and in SB-1 on d-35, respectively compared to ZnB and control. On d-21, number of goblet cells containing mucins of acidic nature increased (p<0.05) in all the segments of small intestines in SB-1 group compared to control, and on d-35 in ileum compared to other groups. CONCLUSION: In conclusion, SB improved growth performance and immunity as well as modulated morphology of lymphoid organs and gut mucosa in broiler chickens.

Differential antibrucella activity of bovine and murine macrophages.

Brucella abortus is. an intracellular pathogen affecting macrophages. Macrophages release some antibrucella componen such as lysozymes (LZ), reactive-oxygen species (ROS) and reactive nitrite intermediates (RNI) which prevent intracellul survival of Brucella. The present study compared the antibrucella activity of bovine and murine macrophages followir stimulation with B. abortus lipopolysaccharides. Our results revealed increased production of these antibrucella substanci in murine macrophages as compared to bovine macrophages. The differential production of these antibrucella componen explained the differential B. abortus killing ability of these species (bovine and mice) that was measured in terms intramacrophagic survival of Brucellae in murine and bovine macrophages.

Antibacterial activity of herbal extracts against multi-drug resistant Escherichia coli recovered from retail chicken meat.

Increasing incidence rate of multiple drug resistance in Escherichia coli (E. coli) due to extensive uses of antibiotics is a serious challenge to disease treatment. Contaminated retail chicken meat is one of the major sources of spread of multi drug resistant (MDR) E. coli. Current study has been conducted to study the prevalence of MDR E. coli in retail chicken meat samples from Lahore city of Pakistan and it was found that 73.86% of E. coli isolates have MDR pattern. In vitro evaluation of antibacterial activity of crude ethanolic extracts of six herbs against MDR E. coli phenotypes has revealed that clove and cinnamon have maximum zones of inhibition as compared to other herbal extracts. Mint and coriander gave the intermediate results while garlic and kalonji showed the least antibacterial activity against the MDR E. coli phenotypes using the agar well diffusion technique. Average Minimum Inhibitory Concentrations (MICs) for clove, mint, cinnamon, coriander, kalonji and garlic extracts were 1.15, 1.38, 0.5, 1.99, 2.41, 8.60 mg/mL respectively using the broth micro dilution method. The results obtained in present study were revealed that crude ethanol extracts of selected herbs have had significant antibacterial activity. Hence they can be used as promising alternatives of antimicrobials against MDR E. coli species and can be used for cooked food preservation.

Role of aflatoxin toxicity on transmissibility and pathogenicity of H9N2 avian influenza virus in turkeys.

The study was conducted to investigate the role of aflatoxin on the infectivity and transmissibility of H9N2 AI virus. The experiment was performed on 80 non-vaccinated turkeys, divided into 4 groups of 20 birds each. Group A was kept as non-infected and a non-treated negative control; Group B was inoculated intratracheally with H9N2 AI virus (1 × 10(7) EID50) at 4 weeks of age; Group C was fed on a diet containing 0.5 ppm aflatoxin from Day 1 through the entire experiment period and Group D was fed on diet containing 0.5 ppm aflatoxin as for Group C but inoculated intratracheally with H9N2 AI virus (1 × 10(7) EID50) at the fourth week of age and then mixed with naïve birds. Infected and contact birds showed clinical signs of different severity, showing the most prominent disease signs in birds of the aflatoxin + H9N2 group. All infected birds showed virus shedding, however, the pattern of virus shedding was different for birds of the aflatoxin + H9N2 group showing pronounced virus secretion. Similarly, efficient transmission of virus was observed between infected and contact birds, but more prominent virus transmission was seen in those birds inoculated and fed aflatoxin-treated diet. Moreover, significantly lower antibody titres against H9N2 AIV were observed in birds fed aflatoxin-treated diet, indicating an immunotoxic nature of aflatoxin as the reason for poor seroconversion. Similarly, decreased IFNγ mRNA expression and higher mortality (35%) suggest an immunotoxic and immunosuppressive effect of aflatoxin leading to enhanced pathogenesis of H9N2 viruses in aflatoxin-fed birds. The immunosuppressive nature of aflatoxin might delay influenza virus clearance and this may be one of the reasons for increased pathogenicity of H9N2 LPAI viruses in turkeys under field conditions.

Port catheter placement by nephrologists in an interventional nephrology training program.

We retrospectively reviewed all subcutaneous single- and double-lumen port catheters (PCs) inserted by interventional nephrologists at our institution to determine the success rate, immediate and late complications, and functional life. From January 2000 to August 2002, 187 PCs were placed in 187 patients (42% males, 51% Caucasians, mean age 50 +/- 14 years). There were no immediate complications related to the procedure such as hemorrhage, pulmonary embolism, or pneumothorax. There were a total of 35,078 catheter-days of follow-up. Sixteen catheters were removed during the observation period: three because of infection, seven after completion of chemotherapy, and six for other reasons. The remaining PCs are either functioning or the patients have died. The initial success rate was 100%. Kaplan-Meier analysis showed a 30-day survival of 97% and a 1-year survival of 92%. Interventional nephrologists, who have adequate training in central venous tunneled cuffed catheter placements, can successfully place PCs, with excellent success and minimal complications.